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Myeloperoxidase (MPO) is an enzyme secreted by neutrophil granulocytes as a result of phagocytosis during inflammation. In colorectal cancer, tumour infiltration by MPO expressing cells has been shown to be independently associated with a favourable prognosis. In this study, we explored the role of MPO-positive cell infiltration and its prognostic significance in invasive breast cancer. We performed immunohistochemical staining for MPO on multiple tissue microarrays comprising a total of 928 human breast cancer samples with detailed clinical-pathological annotation and outcome data. MPO-positive cell infiltration (≥ 5 cells/tissue punch) was found in 150 (16%) of the 928 evaluable breast cancer cases. In univariate survival analyses, infiltration by MPO-positive cells was associated with a significantly better overall survival (p

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). Materials and Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor&ndash, biological axes of high prognostic relevance, which warrant further investigation and validation.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. Significance: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.

// Andreas Maxeiner 1 , Ergin Kilic 2 , Julia Matalon 1 , Frank Friedersdorff 1 , Kurt Miller 1 , Klaus Jung 1,3 , Carsten Stephan 1,3 and Jonas Busch 1 1 Department of Urology, Charite, Universitaetsmedizin Berlin, Berlin, Germany 2 Department of Pathology, Charite, Universitaetsmedizin Berlin, Berlin, Germany 3 Berlin Institute for Urologic Research, Berlin, Germany Correspondence to: Andreas Maxeiner, email: // Keywords : prostate cancer; biomarker; prostate health index; prostate-specific antigen; biochemical recurrence Received : January 30, 2017 Accepted :April 15, 2017 Published : April 27, 2017 Abstract The purpose of this study was to investigate the Prostate-Health-Index (PHI) for pathological outcome prediction following radical prostatectomy and also for biochemical recurrence prediction in comparison to established parameters such as Gleason-score, pathological tumor stage, resection status (R0/1) and prostate-specific antigen (PSA). Out of a cohort of 460 cases with preoperative PHI-measurements (World Health Organization calibration: Beckman Coulter Access-2-Immunoassay) between 2001 and 2014, 437 patients with complete follow up data were included. From these 437 patients, 87 (19.9%) developed a biochemical recurrence. Patient characteristics were compared by using chi-square test. Predictors were analyzed by multivariate adjusted logistic and Cox regression. The median follow up for a biochemical recurrence was 65 (range 3-161) months. PHI, PSA, [-2]proPSA, PHI- and PSA-density performed as significant variables (p < 0.05) for cancer aggressiveness: Gleason-score

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7&ndash, 20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, profound downregulation of gluconeogenesis and oxidative phosphorylation was observed at the protein level. In contrast, analysis of the The Cancer Genome Atlas (TCGA) papillary RCC cohort revealed no significant change in transcripts encoding oxidative phosphorylation compared to normal kidney tissue, highlighting the importance of proteomic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.

Introduction: Patients with consistent suspicion for prostate cancer (PCa) and multiple negative prebiopsies prior to multiparametric magnetic resonance imaging (mpMRI) are still frequently evaluated for an image-guided biopsy and are reported with heterogeneous detection rates. The inclusion of a systematic biopsy (SB) is also still recommended with predominant sampling within the posterior/peripheral zone of the prostate. The aim of this study was (I) to evaluate PCa detection rates using a modified 10 core SB template including anterior biopsies in combination with mpMRI/ultrasound fusion-guided targeted biopsy (TB) in patients with 3 or more negative prebiopsies and (II) to compare mpMRI index lesion localization with histologically confirmed locali­zation from associated prostatectomy samples. Methods: Overall 1,337 consecutive patients underwent sensor-based registration TB of the prostate and a subsequent 10-core SB between January 2012 and December 2015 at our institution. For this study, 101 patients with ≥3 negative prebiopsies and prostate imaging – reporting data system lesions ≥3 were pooled prospectively and underwent TB and a modified SB including 2 ventral (anterior) biopsies. Detection rates were estimated for the modified SB, TB, and its combination. A subgroup analysis of 35 patients undergoing prostatectomy was performed by a head-to-head comparison of mpMRI index lesion and histologically confirmed PCa index lesion localization. Results: The overall detection rate for PCa was 54.5%. The combination of TB and SB detected 14 (25.4%) more cases missed by TB alone (p < 0.001) and 7 (12.7%) more cases missed by SB alone (p = 0.016), respectively. A postoperative Gleason upgrade was seen in 12/35 (34.3%) cases within the TB group and in 14/35 (40.0%) in the SB group, respectively. The subgroup analysis showed a predominant location of PCa index lesions anteriorly at the level of the midgland. The MRI detection rate of the anteriorly located index lesions was 70.4% (15/21 cases) with a clinically significant Gleason score (≥3 + 4 = 7a [International Society of Urological Pathology grade 2]) in 80.9%. Interestingly a modified SB template detected 90.5% (19/21) of the anteriorly located index lesions. Conclusion: Our data suggest that in patients with multiple prebiopsies PCa seems to be predominantly located anteriorly. We suggest the general integration of anterior biopsies despite TB in repeat biopsy patients.

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increasedde novosynthesis rate of GSH, based on glutamine consumption. Furthermore, rewiring of the main pathways involved in ATP and glucose synthesis was observed at the protein level. In contrast, transcripts encoding for the respiratory chain were not regulated, which prompts for non-genetic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.SIGNIFICANCE STATEMENTWe applied proteome- and metabolome profiling to elucidate molecular features in malign papillary renal cell carcinomas. By this characterization, a reprogramming of the main metabolic pathways, such as gluconeogenesis and fatty acid- and amino acid metabolism were identified. The proteins involved in the respiratory chain and the corresponding enzymatic activities were strongly reduced in pRCC, showing an anti-correlation compared with the transcriptome. Similar to renal oncocytomas, the ROS scavenger glutathione was identified as a hallmark in pRCC. Our results suggest that impaired metabolism and dysfunctional mitochondria determine the fate of pRCC. Furthermore, we propose that the specific regulation of the mitochondrial respiratory chain can differentiate highly similar malignant pRCCs from benign renal oncocytomas.

SummaryChromophobe renal cell carcinomas (chRCC) are derived from intercalated cells of the collecting duct system, and are thought to be the malignant counterpart of benign renal oncocytomas. Here, we report the characterization of nine chRCC with adjacent healthy kidney tissues by applying proteome-, transcriptome (TCGA)-, and metabolome profiling. Most strikingly, the reactive oxygen species scavenger glutathione was significantly elevated in chRCC, caused by down-regulated enzymes involved in glutathione degradation. Metabolic reprogramming including stalled gluconeogenesis, down-regulated fatty acid- and amino acid metabolism was identified, even though, the abundance of amino acids and “energy carrier” molecules were unchanged. A striking anti-correlation of the mitochondrial respiratory chain between the transcriptome and the proteome was discovered, the transcripts coding for the respiratory chain were up-, while corresponding proteins and enzymatic activities were down-regulated. Similar to renal oncocytomas, chRCC exhibited a significant increase in glutathione, but are distinguishable by distinct regulation of the respiratory chain.

Exoskeleton robots are generally used for rehabilitation and load-carrying applications. Stable and flexible walking with minimal energy consumption of the human body is achieved by the compliant operation of the human joints. Essentially, the stiffness of the human ankle joint varies continuously, while the stiffness of the knee and hip joints remains nearly constant during loading phases of the walking cycles. With inspiration from the human leg biomechanics, a new biomimetic compliant lower limb exoskeleton robot (BioComEx) was constructed and its preliminary experimental tests were carried out in this paper. A variable stiffness actuator was developed for the ankle joint of BioComEx, and series elastic actuator designs were employed in the knee and hip joints of the robot. The joint actuators of BioComEx were designed longitudinally in order to be embedded inside of the thigh and shank leg segments for the compactness. Separate force sensors were employed in each segment of the robot for the modularity of the control strategies. Since the exoskeletons can be used for both load-carrying and rehabilitation, the developed robot was tested in both human-in-charge and robot-in-charge modes, respectively. The robot needs to mimic movement of healthy user joints in the human-in-charge mode and maximize the compliance between the user and robot; thus, the interaction forces should be reduced as possible. Hence, a modular closed-loop impedance control algorithm was developed for this mode. On the other hand, in the robot-in-charge mode, the robot joints need to track predefined gait references to make the patient walk. PID position control algorithm was chosen for preliminary test of the robot in this mode. Experimental results show that BioComEx is sufficiently satisfactory for walking applications of healthy and paralyzed users.

Nodal metastasis is a strong prognostic parameter in prostate cancer (PCa). We analysed the detection of micrometastases (miN + ) in initially nodal-negative (pN0) radical prostatectomy specimens from pT2a-c and pT3a PCa patients by immunohistochemistry (IHC). A total of 2352 lymph nodes of 193 PCa patients were centrally re-examined for miN + or miN- status using IHC. Results were correlated with clinical and follow-up data. Recurrence-free survival (RFS) was calculated with the log-rank test using the Kaplan-Meier method. In addition, a logistic regression analysis was performed. IHC detected miN + in a total of 17 patients (8.8 %). miN + seemed to be significantly associated with a higher Gleason score and was detected in more advanced pT stages. A total of 45 patients (23.1 %) had a biochemical recurrence (BCR). BCR was associated with miN +. Patients with miN + had a significantly shorter RFS (22.9 versus 58.7 months; p 0.001). In the univariate (OR: 5.04; 95 % CI: 2.46 - 10.6; p-value: 0.0001) and multivariate (OR: 3.29; 95 % CI: 1.54 - 7.08; p-value: 0.002) regression model, the miN + status was the strongest predictor of a BCR. IHC seems to be of high diagnostic value for the detection of micrometastases in initially nodal-negative PCa patients. IHC should therefore be performed in PCa patients with nodal-negative findings. Lymphknotenmetastasen sind ein wichtiger prognostischer Parameter im Rahmen eines Prostatakarzinoms (PCa). Innerhalb einer Kohorte von Prostatektomie-Patienten mit pT2a-c- und pT3a-Tumoren und initial postoperativ nodal negativem (pN0) Status wurde die Detektion von Mikrometastasen (miN + ) mittels Immunhistochemie (IHC) ermittelt. Retrospektiv wurden 2352 Lymphknoten von 193 Patienten zentral auf das Vorliegen eines miN + Status untersucht. Die Ergebnisse wurden mit klinischen und postoperativen Verlaufsparametern korreliert. Das Rezidiv-freie-Überleben (RFS) wurde mittels Log-rank-Test und Kaplan-Meier-Methode ermittelt. Ferner wurde eine logistische Regression durchgeführt. IHC detektierte miN + in insgesamt 17 (8,8 %) Patienten. Eine signifikante Assoziation von miN + zeigte sich mit höheren Gleason-Scores als auch fortgeschrittenen Tumorstadien. Insgesamt hatten 45 (23,1 %) Patienten ein biochemisches Rezidiv (BCR). Das Vorliegen eines BCR war ebenfalls mit dem miN + assoziiert: miN + Patienten wiesen ein signifikant kürzeres RFS (22,9 versus 58,7 Monate; p 0,001) auf. In der Regressionsanalyse zeigte sich sowohl univariat (OR: 5,04; 95 % CI: 2.46 – 10,6; p-value: 0.0001) als auch multivariat der miN + Status als stärkster Prädiktor für ein BCR. IHC scheint von hohem diagnostischen Wert in der Detektion von Mikrometastasen in initial nodal negativen PCa-Patienten zu sein. Daher sollte die Option einer IHC stets in Patienten mit nodal negativem Befund evaluiert werden.

Background The Rearranged during Transfection (RET) protein is overexpressed in a subset of Estrogen Receptor (ER) positive breast cancer, with both signalling pathways functionally interacting. This cross-talk plays a pivotal role in the resistance of breast cancer cells to anti-endocrine therapies, and RET expression is assumed to correlate with poor prognosis based on findings in small patient cohorts. The aim of our study was to investigate the impact of RET expression on patient outcome in human breast cancer. Methods We performed an immunohistochemical analysis of RET protein expression on a tissue microarray encompassing 990 breast cancer patients and correlated its expression with clinicopathological parameters and survival data. Results Expression of RET was detected in 409 out of 990 cases (41.3%). RET and ER expression significantly correlated (p

e16533 Background: The objective of the present study was to identify molecular charactersitics associated with FGFR positive tumors in matched urine and TUR biopsy samples from patients being suspicious of bladder cancer and undergoing first TURB at the pilot center of the multicentric BRIDGister Real World Experience trial that are helpful for patient management and prognosis. Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 28 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). Relative gene expression of subtyping markers (KRT5, KRT0) as well as FGFR1, FGFR2, FGFR3, FGFR4, ERBB2 was centrally tested by standardized test systems (STRATIFYER Molecular Pathology GmbH, Cologne). In addition urine samples were analyzed by commercially available urine tests (UBC Rapid, BTA stat, NMP22). Spearman correlation, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were done by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 28 patients (median age: 73, male 71% vs female 29%) of diverse clinical stages (Benign lesions/no tumor 21%, pTa 32%, pT1 21%, pT2 21%) and WHO 1973 grade (G1 7%, G2 43%, G3 21%). Based on FFPE tissue testing using Therascreen FGFR IVD kit 9 out of 28 patients exhibited FGFR alterations (32%). FGFR positive tumors were associated with high expression of FGFR3 mRNA (r = 5.951, p = 0.0011) but low FGFR1 mRNA as well as FGFR4 mRNA (r = -0.3882, p = 0.0412 and r = -0.6305, p = 0.0004, respectively). Interestingly, FGFR alteration was positively associated with the basal marker KRT5 (r = 0.3929, p = 0.0386), but not with luminal KRT20 mRNA expression (r = -0.0208, p = 0.9179). Moreover FGFR3 altered bladder cancer was associated with elevated NMP22 levels in pretreatment urine (r = 0.3978, p = 0.0361). Conclusions: In early bladder cancer FGFR3 alterations are tightly associated with a characteristic FGFR mRNA signature. Mutation/Fusion of FGFR3 results in high FGFR3 but low FGFR1 and FGFR4 mRNA expression, which might be i.a. relevant for the response to FGFR inhibition and important to predict outcome of FGFR inhibitors. Morever FGFR alteration was associated with elevated NMP22 urine levels, which might be helpful for detecting and monitoring FGFR altered bladder cancer in a non invasive fashion. These results warrant further investigation and its impact on outcome prediction (BCG responsiveness, recurrence, proegression, etc) will be prospectively analyzed in the framework of the ongoing multicenter BRIDGister Real World Experience trial.

Aim: To evaluate the expression and prognostic value of RARRES1 at protein level in renal cell carcinoma (RCC). Materials & methods: Expression profile of RARRES1 was analyzed in 903 documented RCC followed by clinicopathological correlations and survival analysis. Results: RARRES1 expression was seen in 72.5% of RCC. A stronger RARRES1 expression was seen in high grade compared with low grade RCC (p < 0.001). Logrank tests revealed shorter overall survival in RARRES1 positive RCC (p = 0.006) and in pT1/2 tumors with RARRES1 expression (p = 0.002). Conclusion: The variable expression profile in low and high grade RCC may reflect and confirm the differences of previous gene expression analysis. There was a significant prognostic value of RARRES1 expression in patients with RCC, especially in pT1/2 tumors.

SALL4 is a transcription factor originally identified as a homeotic gene essential for organ development. Early studies suggested that SALL4 is a useful marker to identify testicular and ovarian germ cell tumors. The aim of the study was to evaluate the diagnostic potential of SALL4 immunohistochemistry. Immunohistochemical staining was performed on a tissue microarray (TMA) with 3966 samples from 94 different tumor types and on a further TMA with 492 esophagus carcinomas. SALL4 immunostaining was by far most prevalent and most intensive in testicular tumors with a positivity rate of 93.1% in seminomas, 80% in mixed germ cell tumors (embryonic carcinomas/yolk sac tumors), and 18.5% in teratomas, respectively. However, SALL4 expression is not specific to germ cell tumors. We observed SALL4 positivity in non-germ cell tumors as carcinomas of the kidney (28.9% of chromophobe, 34.4% of clear cell carcinoma), in intestinal type adenocarcinoma of the stomach (10.9%), in adenocarcinoma (10.5%) and squamous cell carcinoma (7.2%) of the esophagus, and in malignant melanoma (8.1%) and invasive urothelial bladder carcinoma (20%). SALL4 expression was not found in lymphomas, in soft tissue tumors or breast tumors. At analysis of esophagus carcinoma TMA, no significant association was seen between SALL4 expression and overall survival in adenocarcinoma. However, SALL4 expression was strongly associated with worse overall survival in squamous cell carcinoma. SALL4 expression can be found at relevant frequencies in various tumors of different primary sites. SALL4 expression in squamous cell carcinoma of the esophagus may constitute a sign of dedifferentiation leading to poor patient prognosis.

Background There is inconsistent information about the clinical usefulness of circulating cell-free DNA (cfDNA) in plasma from clear cell renal cell cancer (RCC) patients. This is attributed to preanalytical, analytical, and clinical factors that were considered as far as possible in this study. Methods cfDNA was extracted from EDTA plasma of healthy people (n = 40), non-metastatic (n = 145) and metastatic (n = 84) RCC patients using the QIAamp Circulating Nucleic Acid Kit. Genomic and mitochondrial cfDNA concentrations were determined using qPCR of different cfDNA fragments (67–306 bp). Their diagnostic and prognostic potential was estimated using receiver operating characteristics (ROC) and Cox regression analyses. Results The 67 bp and 180 bp genomic cfDNA fragments did not differ between the three study groups while the 306 bp fragment was lower in RCC patients than in controls. The mitochondrial cfDNA was higher in metastatic than in non-metastatic patients and controls. The cfDNA integrity indices decreased from controls to metastatic patients. Models built by logistic regression and Cox regression resulted in area under the ROC curves > 0.75 and concordance indices of > 0.800 in predicting recurrence-free survival and overall survival. Conclusion The study suggests that combinations of cfDNA markers have promising diagnostic and prognostic potential in RCC patients and are worth for further validation in future prospective multicenter studies.

// Gerrit Kurschner 1, 2, * , Qingzhou Zhang 3, * , Rosanna Clima 4, 5 , Yi Xiao 1, 6 , Jonas Felix Busch 7 , Ergin Kilic 8 , Klaus Jung 7, 9 , Nikolaus Berndt 10 , Sascha Bulik 10 , Hermann-Georg Holzhutter 10 , Giuseppe Gasparre 5 , Marcella Attimonelli 4 , Mohan Babu 3 and David Meierhofer 1 1 Max Planck Institute for Molecular Genetics, Mass Spectrometry Facility, Berlin, Germany 2 Technical University of Berlin, Institute of Bioanalytics, Department of Biotechnology, Berlin, Germany 3 University of Regina, Department of Biochemistry, Regina, Canada 4 University of Bari, Department of Biosciences, Biotechnology and Biopharmaceutics, Bari, Italy 5 Department of Medical and Surgical Sciences-DIMEC, Medical Genetics Unit, University of Bologna, Bologna, Italy 6 Freie Universitat Berlin, Fachbereich Biologie, Chemie, Pharmazie, Berlin, Germany 7 University Hospital Charite, Department of Urology, Berlin, Germany 8 University Hospital Charite, Institute of Pathology, Berlin, Germany 9 Berlin Institute for Urologic Research, Berlin, Germany 10 Charite University Medicine Berlin, Institute of Biochemistry Computational Systems Biochemistry Group, Berlin, Germany * These authors have contributed equally to this work Correspondence to: David Meierhofer, email: Meierhof@molgen.mpg.de Mohan Babu, email: mohan.babu@uregina.ca Keywords: renal oncocytoma; complex I deficiency; glutathione metabolism; mtDNA mutation Received: July 10, 2017 Accepted: September 21, 2017 Published: November 11, 2017 ABSTRACT Renal oncocytomas are rare benign tumors of the kidney and characterized by a deficient complex I (CI) enzyme activity of the oxidative phosphorylation (OXPHOS) system caused by mitochondrial DNA (mtDNA) mutations. Yet, little is known about the underlying molecular mechanisms and alterations of metabolic pathways in this tumor. We compared renal oncocytomas with adjacent matched normal kidney tissues on a global scale by multi-omics approaches, including whole exome sequencing (WES), proteomics, metabolomics, and metabolic pathway simulation. The abundance of proteins localized to mitochondria increased more than 2-fold, the only exception was a strong decrease in the abundance for CI subunits that revealed several pathogenic heteroplasmic mtDNA mutations by WES. We also observed renal oncocytomas to dysregulate main metabolic pathways, shunting away from gluconeogenesis and lipid metabolism. Nevertheless, the abundance of energy carrier molecules such as NAD + , NADH, NADP, ATP, and ADP were significantly higher in renal oncocytomas. Finally, a substantial 5000-fold increase of the reactive oxygen species scavenger glutathione can be regarded as a new hallmark of renal oncocytoma. Our findings demonstrate that renal oncocytomas undergo a metabolic switch to eliminate ATP consuming processes to ensure a sufficient energy supply for the tumor.

This study proposes an Electromyography (EMG) based neural network and admittance control strategy for an Active wrist orthosis (AWO) that is mobile, powerful and lightweight in order to avoid the occurrence and/or for the treatment of repetitive strain injuries. The device has an EMG based control strategy so that it can track the contraction of the forearm muscles of interest in real time to assist the device user during the extension and flexion wrist movements. Indeed, time-delayed artificial neural networks were trained offline to predict the forearm muscle forces based on features extracted from raw EMG signals. The predicted force values were used to calculate a reference velocity command by using an admittance model with properly adjusted parameters. A closed loop velocity control system was used to perform the desired wrist motion. Some experimental studies were performed to evaluate the assistive performance of the AWO device with the proposed control system under various disturbance loads. The experimental results show that the activation levels of the forearm muscles were considerably reduced when the AWO device was enabled.

People who perform excessive wrist movements throughout the day because of their professions have a higher risk of developing lateral and medial epicondylitis. If proper precautions are not taken against these diseases, serious consequences such as job loss and early retirement can occur. In this study, the design and control of an active wrist orthosis that is mobile, powerful and lightweight is presented as a means to avoid the occurrence and/or for the treatment of repetitive strain injuries in an effective manner. The device has an electromyography-based control strategy so that the user’s intention always comes first. In fact, the device–user interaction is mainly activated by the electromyography signals measured from the forearm muscles that are responsible for the extension and flexion wrist movements. Contractions of the muscles are detected using surface electromyography sensors, and the desired quantity of the velocity value of the wrist is extracted from a fuzzy logic controller. Then, the actuator system of the device comes into play by conveying the necessary motion support to the wrist. Experimental studies show that the presented device actually reduces the demand on the muscles involved in repetitive strain injuries while performing challenging daily life activities including extension and flexion wrist motions.

To improve the clinical decision‐making regarding further treatment management and follow‐up scheduling for patients with muscle‐invasive bladder cancer (MIBC) after radical cystectomy (RC), a better prediction accuracy of prognosis for these patients is urgently needed. The objective of this study was to evaluate the validity of differentially expressed microRNAs (miRNAs) based on a previous study as prognostic markers for overall survival (OS) after RC in models combined with clinicopathological data. The expression of six miRNAs (miR‐100‐5p, miR‐130b‐3p, miR‐141‐3p, miR‐199a‐3p, miR‐205‐5p, and miR‐214‐3p) was measured by RT‐qPCR in formalin‐fixed, paraffin‐embedded tissue samples from 156 MIBC patients who received RC in three urological centers. Samples from 2000 to 2013 were used according to their tissue availability, with follow‐up until June 2016. The patient cohort was randomly divided into a training (n = 100) and test set (n = 56). Seventy‐three samples from adjacent normal tissue were used as controls. Kaplan–Meier, univariate and multivariate Cox regression, and decision curve analyses were carried out to assess the association of clinicopathological variables and miRNAs to OS. Both increased (miR‐130b‐3p and miR‐141‐3p) and reduced (miR‐100‐5p, miR‐199a‐3p, and miR‐214‐3p) miRNA expressions were found in MIBC samples in comparison to nonmalignant tissue samples (P

Introduction C-kit overexpression has previously been described in chromophobe renal cell carcinoma (cpRCC) and renal oncocytoma (RO). However, so far no KIT mutations have been found. The objective of our study was to analyse c-kit in a large cohort of renal tumors and to perform KIT mutation analysis in a subset cpRCC and RO cases with overexpression of c-kit. Materials and methods We studied the immunohistochemical expression of c-kit on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 948 patients with renal tumors. CpRCC and RO cases with c-kit overexpression ( n = 23) were analyzed for KIT mutations in exons 9, 11, 13, 14, 15, and 17. Results Expression of c-kit was found in 6/642 (0.9%) clear cell RCC, 3/154 (1.9%) papillary RCC, 54/69 (78.3%) cpRCC, 37/45 (82.2%) RO and 2/30 (6.7%) of other unclassified tumor types. In none of the RO and cpRCC cases analyzed, a KIT gene mutation was found. Conclusion C-kit expression is found in the majority of cpRCC and RO, but these tumors do not harbor the usual c-kit activating mutations. This may have implications for the use of tyrosine kinase inhibitors in patients with advanced cpRCC and c-kit expression.

Hintergrund: Patienten mit steigenden PSA-Werten oder positivem Tastbefund in der digital rektalen Untersuchung (DRU) oder einer atypischen mikroazinaren Proliferation nach einer negativen Prostatabiopsie erhalten nach den Leitlinien der Europaischen Gesellschaft fur Urologie eine Wiederholungsbiopsie. Die geringen Detektionsraten bei der transrektal Ultraschall (TRUS) gestutzten Wiederholungsbiopsie zeigen das Dilemma in der Prostatakarzinom (PCa) Diagnostik. Die Kombination des dynamischen TRUS mit den hohen Spezifitaten und Sensitivitaten der Prostata Magnet Resonanz Tomografie (MRT) im Rahmen der sogenannten Echtzeit MRT/US-Fusion, wurde fur die Detektion klinisch signifikanter Karzinome evaluiert. Material und Methoden: Es wurden 128 konsekutive Patienten in der Zeit von Januar 2012 bis August 2013 in die Studie eingeschlossen. Alle Patienten hatten mindestens eine vorausgegangene negative Prostatabiopsie und es bestand weiterhin der klinische Verdacht auf ein PCa. Alle Patienten erhielten zuvor eine multiparametrische 3 Tesla MRT ohne Endorektalspule. Es folgte die Datenfusion der MR-Sequenzen mit folgenden US-Techniken: B-Bild, Farbdoppler, Elastografie, CEUS zur Charakterisierung der MR-suspekten Herde. Diese Herde wurden mittels der genannten US-Techniken mit einem US-Summen-Score bewertet und anschliesend gezielt biopsiert. Unmittelbar im Anschluss daran folgte eine systematische 10-fach Biopsie. Ergebnisse: Unter 128 Patienten konnte in 51 Fallen ein PCa diagnostiziert werden; dies entspricht einer Rate von 39,8%. Von den 51 PCa Patienten konnten klinisch signifikante PCa haufiger durch die MRT/US-Fusion nachgewiesen werden: Gleason Score >7 in 9 von 10 Fallen; Gleason Score=7 in 14 von 20 Fallen und Gleason Score Schlussfolgerung: Die MRT/US-Fusion erhoht die Detektionsrate in zuvor negativ biopsierter Patienten. Insbesondere Tumoren mit einem Gleason Score ≥ 7 sind nahezu ausschlieslich durch die MRT/US-Fusionsbiopsie detektiert und biopsiert worden.

The purpose of this study was to evaluate the magnetic resonance prostate imaging reporting and data system (PI-RADS) for the detection of prostate cancer by the results of magnetic resonance imaging (MRI)-guided biopsy of the prostate as a reference standard.In 55 patients who had undergone MRI-guided biopsy of the prostate, we retrospectively matched every biopsy core with the corresponding lesion in previously acquired endorectal multiparametric MRI including T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and dynamic contrast-enhanced MRI (DCE-MRI) at 1.5 T. Two readers blinded to the results of the biopsy evaluated each biopsied lesion according to the PI-RADS scoring system. The results of the targeted biopsy were used as a reference standard. Receiver operating characteristic analysis was performed for statistical analysis.A total of 113 lesions in the 55 patients were evaluated; 30 lesions were malignant. When evaluated according to the criteria of the PI-RADS scoring system, DCE-MRI revealed a lower area under the receiver operating characteristic curve (AUC) (0.76) compared with T2WI (0.88; P=0.06) and DWI (0.93; P=0.004). A sum score combining T2WI, DWI, and DCE-MRI yielded an AUC of 0.93, whereas a sum score combining only T2WI and DWI yielded an AUC of 0.95. In central gland lesions, T2WI showed a numerically higher AUC compared with DWI (0.98 and 0.95), whereas, in peripheral zone lesions, DWI was superior (AUC of 0.93 and 0.73; P=0.04). An approach assigning a PI-RADS score for T2WI to central gland lesions and for DWI to peripheral zone lesions yielded an AUC of 0.96 and was numerically superior compared with any sequence alone and sum scores combining T2WI and DWI as well as T2WI, DWI, and DCE-MRI.The PI-RADS scoring system shows a good diagnostic performance for the detection of prostate cancer when using a sum score. However, DCE-MRI does not seem to add significant value when evaluated according to the recommended criteria. Assigning a score for T2WI to central gland lesions and for DWI to peripheral zone lesions might be sufficient for stratification of patients for further diagnostic workup.

Bladder cancer is a common cancer in the Western world. The current prognosticators such as tumor grade, stage, size, and multifocality do not accurately reflect the clinical outcome. It is of clinical interest to identify biomarkers that could improve diagnostic and/or prognostic predictions. The objectives of this study were to identify deregulated miRNAs in bladder cancer samples and evaluate their potential as diagnostic and prognostic biomarkers. We screened 723 miRNAs by microarray and selected a subset of 15 distinctively deregulated miRNAs for further validation by real-time quantitative RT-(q)PCR. Seven miRNAs (miR-20a, miR-106b, miR-130b, miR-141, miR-200a, miR-200a*, and miR-205) were found to be up-regulated and eight miRNAs (miR-100, miR-125b, miR-130a, miR-139-5p, miR-145*, miR-199a-3p, miR-214, and miR-222) were found to be down-regulated in malignant bladder tissue samples compared to healthy tissue. Four miRNAs that have already been described in the literature (miR-141, miR-199a-3p, miR-205, and miR-214) were significantly differentially expressed between nonmuscle-invasive and muscle-invasive bladder cancer. Furthermore, real-time RT-qPCR of all miRNAs provided high overall correct classification (>75%) of bladder cancer diagnosis. Two miRNAs (miR-141 and miR-205) were associated with overall survival time. The verification of tumor-specific miRNA expression profile, together with the observed association of miR-141 and miR-205 expression with overall survival, underline the potential of miRNAs to function as diagnostic and/or prognostic markers of bladder cancer.

Background MicroRNAs are promising diagnostic and prognostic biomarkers in oncology. We aimed to evaluate the prognostic potential of selected microRNAs in primary clear cell renal cell carcinomas (ccRCC) as predictors of tumor recurrence after radical nephrectomy. Methods miR-122, miR-141, miR-155, miR-184, miR-200c, miR-210, miR-224, and miR-514, validated as differentially expressed in a previous study, were measured by RT-PCR in matched malignant and non-malignant tumor samples after nephrectomy from 111 patients (89 without, 22 with metastases) and clinicopathological and outcome data were collected. Non-parametric statistical tests, receiver-operating characteristics, Kaplan–Meier-, and univariate as well as multivariate Cox regression analyses were performed. Results Downregulation of miR-141/-184/-200c/-514 and upregulation of miR-122/-155/-210/-224 were not different between samples of non-metastatic and metastatic tumors except for miR-122 and miR-514. miR-514 was further downregulated in metastatic compared with non-metastatic tumors while the upregulation of miR-122 was significantly reduced in metastatic carcinomas. All miRNAs were suitable to discriminate malignant from non-malignant tissue. miR-122 and miR-514 were significantly related to the recurrence risk but only miR-514 provided independent prognostic information in the final model including relevant clinicopathological variables. Conclusions MiR-122 and miR-514 play a role in tumor recurrence after nephrectomy. Expression of miR-514 was particularly downregulated in primary metastatic tumor and those that recur and might be a suitable adjunct marker for predicting tumor recurrence.

Objectives To verify retrospectively the margin status and analyse the location and characteristics of positive surgical margins (PSMs) in patients undergoing radical prostatectomy (RP), by a central pathology review, based on the consensus conference 2009 updated margin criteria from the International Society of Urological Pathology (ISUP). Patients and methods The detailed PSM characteristics of 441 patients who underwent laparoscopic RP (LRP) between 1999 and 2007 were centrally reviewed with regard to location, number, Gleason score at the PSM and tumour width. Predictors of PSMs and the impact of several PSM characteristics on clinical outcomes were examined. Patient characteristics were compared using the chi-squared test. Differences in recurrence-free survival (RFS) rates were analysed using the log-rank test and presented as Kaplan-Meier survival curves. Univariable and multivariable Cox regression analysis for the prediction of RFS was performed. Results Central pathology review using the updated PSM definition according to ISUP 2009, resulted in reclassification of a substantial number of patients with PSMs (n = 113, 26.6%) as R0. Several PSM characteristics with a higher risk of biochemical recurrence (BCR) were identified as the strongest independent predictors of RFS: pathological stage; Gleason score; and the presence of multiple PSMs (hazard ratio [HR] 1.78; 95% confidence interval [CI] 1.08-2.96; P = 0.025). Further analysis replacing the location of PSM by the width categories of PSM showed that a PSM >3 mm was an independent predictor of RFS (HR 1.72; 95% CI 1.08-2.72; P = 0.022). Conclusions The impact of PSMs after LRP for prostate cancer remains unclear. PSMs in the present cohort of patients undergoing LRP had different characteristics and conferred different risks of BCR. A better understanding of PSM characteristics and a careful standardized pathological evaluation is needed to adequately counsel patients with respect to prognosis and adjuvant therapy after LRP.

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

Exoskeleton robots are wearable electromechanical structures interacting with human limbs. They are used for extending or replacing human performance in power augmentation and rehabilitation applications. The neuro-muscular system of the human body provides flexible and stable movement with minimum energy consumption by means of the compliant actuation of human joints. Similar to human body, compliant actuation can be used to maximize the performance in exoskeleton robots. In the present study, we designed a new biomimetic compliant lower limb exoskeleton robot (BioComEx). Firstly, the current exoskeleton robot designs and biomechanics of the human body joints are reviewed. Then, according to the inferences of human joint biomechanics review, ankle joint is designed as variable stiffness actuator, knee and hip joints are designed as series elastic actuators. Kinetostatic analysis of these joint mechanisms is conducted, and finally the design details of each joint and complete exoskeleton structure adapted to a human dummy model are explained.

We evaluated the usefulness of serum 25-hydroxyvitamin D as a marker of aggressive prostate cancer and for active surveillance compared to PHI (Prostate Health Index).Of 480 prospectively biopsied men 222 had prostate cancer and 258 had no evidence of malignancy. In all men prostate specific antigen was less than 20 ng/ml. We measured 25-hydroxyvitamin D, prostate specific antigen, free prostate specific antigen and -2proPSA using a commercially available immunoassay system. PHI was calculated according to the equation, -2proPSA/free prostate specific antigen × √PSA. We determined 25-hydroxyvitamin D using a 2-step competitive binding immunoenzymatic vitamin D assay.The 25-hydroxyvitamin D concentrations were not associated with Gleason grade according to the 2014 ISUP (International Society of Urological Pathology) consensus conference Gleason grading system. PHI values were higher with increasing Gleason grade. Median 25-hydroxyvitamin D did not differ between men with prostate cancer vs no evidence of malignancy (50.6 vs 48.2 nmol/l, p = 0.192) or in ISUP Gleason subgroups despite seasonal variations of 25-hydroxyvitamin D. However, PHI values significantly differed between the subgroup with no evidence of malignancy and all Gleason subgroups (p0.0001). The ROCs of all men revealed an advantage of PHI over 25-hydroxyvitamin D (AUC 0.78 vs 0.535, p0.0001). PHI could also significantly better separate patients with no evidence of malignancy from those with nonaggressive disease (ISUP Gleason grade 1) from those with aggressive prostate cancer (ISUP Gleason grades 2-5).It remains highly improbable that 25-hydroxyvitamin D could be used as decision or selection marker for aggressive prostate cancer or for active surveillance compared to accepted markers, as recently suggested.

Background Due to the poor prognosis for advanced renal cell carcinoma (RCC), there is an urgent need for new therapeutic targets and for prognostic markers to identify high risk tumors. MicroRNAs (miRNAs) are frequently dysregulated in tumors, play a crucial role during carcinogenesis and therefore might be promising new biomarkers. In previous studies, we identified miR-141-3p and miR-145-5p to be downregulated in clear cell RCC (ccRCC). Our objective was to investigate the functional association of these miRNAs, focusing on the cooperative regulation of new specific targets and their role in ccRCC progression. Methods The effect of miR-141-3p and miR-145-5p on cell migration was examined by overexpression in 786-O cells. New targets of both miRNAs were identified by miRWalk, validated in 786-O and ACHN cells and additionally characterized in ccRCC tissue on mRNA and protein level. Results In functional analysis, a tumor suppressive effect of miR-141-3p and miR-145-5p by decreasing migration and invasion of RCC cells could be shown. Furthermore, co-overexpression of the miRNAs seemed to result in an increased inhibition of cell migration. Both miRNAs were recognized as post-transcriptional regulators of the targets EAPP, HS6ST2, LOX, TGFB2 and VRK2. Additionally, they showed a cooperative effect again as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression after simultaneous overexpression of both miRNAs. In ccRCC tissue, LOX mRNA expression was strongly increased compared to normal tissue, allowing also to distinguish between non-metastatic and already metastasized primary tumors. Finally, in subsequent tissue microarray analysis LOX protein expression showed a prognostic relevance for the overall survival of ccRCC patients. Conclusion These results illustrate a jointly strengthening effect of the dysregulated miR-141-3p and miR-145-5p in various tumor associated processes. Focusing on the cooperative effect of miRNAs provides new opportunities for the development of therapeutic strategies and offers novel prognostic and diagnostic capabilities.

Urological cancers are a very common type of cancer worldwide and have alarming high incidence and mortality rates, especially in kidney cancers, illustrate the urgent need for new therapeutic targets. Recent publications point to a deregulated COP9 signalosome (CSN)-cullin-RING ubiquitin-ligase (CRL) pathway which is here considered and investigated as potential target in urological cancers with strong focus on renal cell carcinomas (RCC). The CSN forms supercomplexes with CRLs in order to preserve protein homeostasis and was found deregulated in several cancer types. Examination of selected CSN-CRL pathway components in RCC patient samples and four RCC cell lines revealed an interesting deregulated p27(Kip1)-Skp2-CAND1 axis and two p27(Kip1) point mutations in 786-O cells; p27(Kip1)V109G and p27(Kip1)I119T. The p27(Kip1) mutants were detected in patients with RCC and appear to be responsible for an accelerated growth rate in 786-O cells. The occurrence of p27(Kip1)V109G and p27(Kip1)I119T in RCC makes the CSN-CRL pathway an attractive therapeutic target.

Background MicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis. Material and Methods miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines. Results Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments. Conclusions We identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.

Activated leukocyte cell adhesion molecule (CD166) is a member of the immunoglobulin superfamily and is aberrantly expressed in different tumors, including prostate cancer. To learn more on the prevalence and clinical significance of activated leukocyte cell adhesion molecule expression in prostate cancer, a tissue microarray containing 3261 primary prostate cancers treated by radical prostatectomy was used. A total of 2390 different prostate cancers were analyzed by immunohistochemistry in a tissue microarray format. Activated leukocyte cell adhesion molecule immunostaining in cancers was compared with clinical follow-up, which was available for 1746 patients. Membranous activated leukocyte cell adhesion molecule immunostaining was recorded in 1663 (69.6%) of cases. High activated leukocyte cell adhesion molecule expression levels were significantly associated with favorable tumor features (pT: P = .0015; pN: P = .0008; preoperative prostate-specific antigen: P = .0057) and a lower risk of a biochemical recurrence (P = .0067). Cytoplasmatic activated leukocyte cell adhesion molecule staining was usually associated with membranous staining. The small number of cancers with pure cytoplasmatic staining did not reveal any particularities with respect to clinical outcome or tumor phenotype. It is concluded that activated leukocyte cell adhesion molecule protein is almost always expressed in prostate cancer and that decreased levels of activated leukocyte cell adhesion molecule expression may lead to an aggressive behavior of tumor cells. The abundant presence of activated leukocyte cell adhesion molecule and its membranous localization in prostate cancer epithelium make activated leukocyte cell adhesion molecule a potentially attractive structure for targeted therapy.

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor gene that was shown to be deleted in 10% to 20% of breast cancers by array comparative genome hybridization analysis. We developed fluorescent in situ hybridization to detect the ING4 gene directly in the tissue samples on tumor tissue microarrays. We evaluated the ING4 gene status in 1033 breast cancer tissue samples and observed that ING4 was deleted in 16.5% (170/1033) of all breast cancers. ING4 deletion was significantly associated with Her2 overexpression: of the tumors with ING4 deletion, 23.8% (39/164) were human epidermal growth factor 2 (HER2) positive, as compared with 14.1% (115/814) of the tumors without ING4 deletion (P = .002). In addition, the tumors with ING4 deletion were more likely to belong to the HER2 molecular subtype (estrogen receptor negative/progesterone receptor negative/human epidermal growth factor positive) of breast cancer, compared with the other subtypes (28.4% HER2 versus 15.7% all, P = .002). ING4 deletion did not affect survival outcome of all patients with breast cancer (P = .797) or of the patients with HER2-positive tumors (P = .792). We conclude that ING4 deletion in breast cancer is relatively common, as 1 in 6 breast cancer harbors ING4 deletion. Furthermore, ING4 deletion is more prevalent in HER2-positive tumors, suggesting a functional antagonistic relationship between the ING4 tumor suppressor and the HER2 oncogene. These results sustain the view that ING4 is a tumor suppressor in breast cancer and suggest that ING4 deletion may contribute to the pathogenesis of HER2-positive breast cancer.

Although survival rates of colon cancer patients diagnosed at an early stage (T1-2N0M0; Dukes A) vary considerably according to the studies cited, several studies indicate development of distant metastases already occurring in a considerable percentage of these patients leading to the death of the patients. This particular high risk group cannot be identified properly as no marker exists to identify these patients. As the Wnt/Win pathway plays a crucial role in metastasis formation in colorectal carcinoma, we analysed whether the transcription factor brachyury critically involved in this pathway may predict metastasis formation in these patients. The expression of brachyury-homologous (T) was immunohistochemically analysed in 748 patients and the data were correlated with classical and newer prognostic markers in colorectal cancer. Early stages colorectal cancer patients (T1-2N0M0, Dukes A) showed a significantly decreased survival when brachyury was expressed in the tumour tissue while no correlation was observed in later tumour stages. Hence a subset of colorectal cancers exists in which the ability to metastasise is already present at early stages of tumour growth and this high risk group can now be detected by immunohistochemistry.

BACKGROUND Prostate specific membrane antigen (PSMA) is a suggested target for antibody-based therapy of prostate cancer potentially involved in the regulation of cell migration. This study was undertaken, to gain more insight on the role of PSMA in early prostate cancer and its distribution in various normal tissues. METHODS A total of 1,700 different prostate cancers treated by radical prostatectomy and 612 samples from 76 different normal tissue types were successfully analyzed by immunohistochemistry (IHC) in a tissue microarray (TMA) format. PSMA immunostaining in cancers was also compared with clinical follow-up, preexisting HER2 expression and Ki67 labeling index data. RESULTS PSMA staining was only found in prostate epithelium and expression was higher in cancer cells than in benign tissue. PSMA staining was found in 94.1% of cancers and was significantly associated with tumor stage, high Gleason grade, preoperative PSA, and HER2 expression (P

To identify the clinicopathological association of PBRM1 (Polybromo-1 gene) and VHL (von Hippel-Lindau gene) expression at mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) and its role in tumor progression.Immunohistochemical analysis, Western blotting and qPCR analysis of PBRM1 and VHL were performed on fresh-frozen ccRCC and adjacent normal tissue obtained from 70 patients who underwent radical nephrectomy. In addition, a tissue microarray (TMA) from specimens of 326 ccRCC patients was used to evaluate the effect of loss of PBRM1 and VHL immunohistological expression on clinicopathological features as well as patient survival.In frozen tissue, PBRM1 and VHL mRNA were significantly down-regulated in most ccRCC tumors (77.6%/80.6%). Simultaneous weak PBRM1 and VHL protein expression was observed in 21.4% of frozen tumors. In the TMA samples, weak PBRM1 and VHL immunohistochemical staining was observed in 60.4% of the cases and was correlated (P0.001). The association of PBRM1 and VHL immunohistochemical expression with clinicopathological parameters depicts a variable picture: predominantly weak PBRM1 and VHL expression were significantly associated with higher Fuhrman grade (P = 0.012 and 0.024, respectively) but only weak VHL expression was associated with a higher pT stage (P = 0.023). PBRM1 expression did not affect the overall survival, whereas weak VHL expression was associated with decreased patient overall survival (P = 0.013).Our data suggest that reduced expression of PBRM1 and VHL is correlated with an increased tumor aggressiveness. Low VHL expression was identified as a risk factor for worse patient overall survival, independently from PBRM1 expression pattern.

SummaryCongenital testicular tumours are seldom reported in bovine species. This case report describes the clinical, sonographical, haematological, pathomorphological and immunohistological features of a Sertoli cell tumour in a neonatal German Holstein calf. Microscopically, the enlarged testicle was composed of neoplastic cells, which were packed in well-formed tubules. The mostly polygonal shaped cells had round to elongated nuclei and a scanty eosinophilic cytoplasm. Some cells were arranged perpendicularly to the light PAS-positive basement membrane. These cells were packed in broad sheets separated by dense fibrous stroma. Mitotic figures were present. The features described above are indicative of a Sertoli cell tumour. The contralateral testicle showed a well formed rete testis, fusiform cells and a dense central capillary convolute and haemorrhagic foci. The features are indicative of an extensive fibrosis and older haemorrhage. The neoplasia was immunopositive for vimentin, α-oestrogen receptor, α-inhibin and S-100 protein, but immunonegative for cytokeratine, CD30, progesterone receptor, α-fetoprotein, SALL4, OCT4 and glypican-3. The myco - toxicological investigations revealed the presence of residues of zea - ra lenone, deoxynivalenol, ochratoxin, HT2 toxin and their metabolites in feeds and urine of heavily pregnant cows of the herd. Furthermore, information is provided about oestrogen and testosterone levels of the affected and healthy neonatal calves. A possible influence of mycotoxins on the cancerogenesis is discussed.

Grainyhead transcription factors control epithelial barriers, tissue morphogenesis, and differentiation, but their role in the kidney is poorly understood. Here, we report that nephric duct, ureteric bud, and collecting duct epithelia express high levels of grainyhead-like homolog 2 (Grhl2) and that nephric duct lumen expansion is defective in Grhl2-deficient mice. In collecting duct epithelial cells, Grhl2 inactivation impaired epithelial barrier formation and inhibited lumen expansion. Molecular analyses showed that GRHL2 acts as a transcriptional activator and strongly associates with histone H3 lysine 4 trimethylation. Integrating genome-wide GRHL2 binding as well as H3 lysine 4 trimethylation chromatin immunoprecipitation sequencing and gene expression data allowed us to derive a high-confidence GRHL2 target set. GRHL2 transactivated a group of genes including Ovol2, encoding the ovo-like 2 zinc finger transcription factor, as well as E-cadherin, claudin 4 (Cldn4), and the small GTPase Rab25. Ovol2 induction alone was sufficient to bypass the requirement of Grhl2 for E-cadherin, Cldn4, and Rab25 expression. Re-expression of either Ovol2 or a combination of Cldn4 and Rab25 was sufficient to rescue lumen expansion and barrier formation in Grhl2-deficient collecting duct cells. Hence, we identified a Grhl2/Ovol2 network controlling Cldn4 and Rab25 expression that facilitates lumen expansion and barrier formation in subtypes of renal epithelia.

Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in pre-eclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branching morphogenesis. Selective Grhl2 inactivation only in epiblast-derived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIP-seq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction.

Mastopathy is a common disease of the breast likely associated with elevated estrogen levels and a putative risk factor for breast cancer. The role of estrogen receptor alpha (ESR1) in mastopathy has not been investigated previously. Here, we investigated the prevalence of ESR1 gene amplification in mastopathy and its prediction for breast cancer. Paraffin-embedded tissues from 58 women with invasive breast cancer were analyzed. For all women, tissues with mastopathy taken at least 1.5 years before first diagnosis of breast cancer were available. Tissue from 46 women with mastopathy without a diagnosis of breast carcinoma in the observed time frame (12–18 years) was used as control. Fluorescence in situ hybridization analysis revealed that ESR1 was amplified in nine of 58 (15.5 %) breast cancers. All ESR1-amplified breast cancers were strongly positive for estrogen receptor with ER immunohistochemistry. Interestingly, in women with ESR1 amplification in breast cancer, the amplification was detectable in mastopathic tissues prior to the first diagnosis of breast cancer but was absent in tissues from women with mastopathy who did not develop breast cancer. Our study suggests that ESR1 gene amplification is an early event in breast pathology and might be a helpful predictive marker to identify patients at high risk of developing breast cancer.

MicroRNAs (miRNAs) have shown to be promising novel biomarkers in various cancers. We aimed to translate the results of an own previous tissue-based miRNA profile of prostate carcinoma (PCa) with upregulated miR-183 and downregulated miR-205 into a urine-based testing procedure for diagnosis of PCa.Urine sediments were prepared from urine samples collected after a standardized digital-rectal examination (DRE) of patients undergoing prostate biopsy with PSA (prostate-specific antigen) values Both miRNAs were correlated to the mRNA PSA concentrations in the sediments indicating a relationship to the released prostate cells after DRE. However, they had no discriminating capacity between patients with and without PCa. In contrast, PCA3 clearly differentiated between these two patients groups. There was also no significant correlation between miRNAs and standard clinicopathologic variables like Gleason score and serum PSA.The data of our study show that miR-183 and miR-205 failed to detect early and aggressive PCa despite their highly dysregulated expression in cancer tissue. Our results and the critical evaluation of the few data of other studies raise serious doubts concerning the capability of urinary miRNAs to replace or improve PCA3 as predictive marker for prostate biopsy outcome.

We demonstrate here that epithelial carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) downregulation in prostate intraepithelial neoplasia (PIN) is inversely correlated with its upregulation in adjacent blood vessels. CEACAM1 silencing in prostate cancer cell line DU-145 via small interfering ribonucleic acid (siRNA) increased but its overexpression suppressed the expression of angiogenic/lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, -C and -D, and angiogenic inhibitor collagen 18/endostatin. Furthermore, CEACAM1 overexpression in DU-145 cells increased but CEACAM1 silencing reduced angiopoietin-1 expression. Inverse relation was found for angiopoietin-2. Supernatant of CEACAM1-overexpressing DU-145 suppressed but that of CEACAM1-silenced increased the VEGF-induced endothelial tubes. Electron microscopically the majority of PIN-associated blood vessels was structurally destabilized exhibiting endothelial fenestration, trans- and inter-endothelial gaps. In some PIN areas, invasion of single tumor cells into the destabilized blood vessels was observed. These data show that disappearance of epithelial CEACAM1 in PIN is accompanied by its upregulation in adjacent vasculature which apparently correlates with vascular destabilization and increased vascularization of prostate cancer. Strategies to either conserve the epithelial CEACAM1 or to target endothelial CEACAM1 might be useful for an anti-angiogenic therapy of prostate cancer.