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1.  Staging lymphadenectomy in patients with localized high risk prostate cancer: comparison of the laparoendoscopic single site (LESS) technique with conventional multiport laparoscopy 
BMC Urology  2014;14(1):92.
In patients with localized high-risk prostate cancer awaiting radiation therapy, pelvic lymphadenectomy (PL) is a reliable minimally invasive staging procedure. We compared outcomes after laparoendoscopic single site PL (LESSPL) with those after conventional multiport laparoscopic PL (MLPL).
A retrospective case-control study was carried out at the authors’ center. For LESSPL the reusable X-Cone single port was combined with straight and prebent laparoscopic instruments and an additional 3 mm needlescopic grasper. MLPL was performed via four trocars of different sizes using standard laparoscopic instruments.
Patients who underwent either LESSPL (n = 20) or MLPL (n = 97) between January 2008 and July 2013, were included in the study. Demographic data were comparable between groups. Patients in the LESSPL group tended to be older and had a significantly higher ASA-score. The mean operating time was 172.4 ± 34.1 min for LESSPL and 116.6 ± 40.1 min for MLPL (P < .001). During LESSPL, no conversion to MLPL was necessary. An average of 12 lymph nodes per patient was retrieved, with no significant difference between study groups. Postoperative pain scores were similar between groups. The hospital stay was 2.3 ± 0.7 days after LESSPL and 3.1 ± 1.2 days after MLPL (P = .01). Two days postoperatively, significantly more patients after LESSPL than after MLPL recovered their normal physical activity (P < .001). Six months postoperatively, no complications were registered in the LESSPL group and cosmetic results were excellent.
In the present study, shorter hospitalization and quicker postoperative recovery were major benefits of LESSPL over MLPL. In patients with localized prostate cancer, staging LESS pelvic lymphadenectomy may be a safe alternative to conventional multiport laparoscopy.
PMCID: PMC4247718  PMID: 25412566
Laparoscopy; LESS; Single port; Prostate cancer staging
2.  Denosumab and Bone Metastasis–Free Survival in Men With Nonmetastatic Castration-Resistant Prostate Cancer: Exploratory Analyses by Baseline Prostate-Specific Antigen Doubling Time 
Journal of Clinical Oncology  2013;31(30):3800-3806.
Denosumab, an anti–RANK ligand monoclonal antibody, significantly increases bone metastasis–free survival (BMFS; hazard ratio [HR], 0.85; P = .028) and delays time to first bone metastasis in men with nonmetastatic castration-resistant prostate cancer (CRPC) and baseline prostate-specific antigen (PSA) ≥ 8.0 ng/mL and/or PSA doubling time (PSADT) ≤ 10.0 months. To identify men at greatest risk for bone metastasis or death, we evaluated relationships between PSA and PSADT with BMFS in the placebo group and the efficacy and safety of denosumab in men with PSADT ≤ 10, ≤ 6, and ≤ 4 months.
Patients and Methods
A total of 1,432 men with nonmetastatic CRPC were randomly assigned 1:1 to monthly subcutaneous denosumab 120 mg or placebo. Enrollment began February 2006; primary analysis cutoff was July 2010, when approximately 660 men were anticipated to have developed bone metastases or died.
In the placebo group, shorter BMFS was observed as PSADT decreased below 8 months. In analyses by shorter baseline PSADT, denosumab consistently increased BMFS by a median of 6.0, 7.2, and 7.5 months among men with PSADT ≤ 10 (HR, 0.84; P = .042), ≤ 6 (HR, 0.77; P = .006), and ≤ 4 months (HR, 0.71; P = .004), respectively. Denosumab also consistently increased time to bone metastasis by PSADT subset. No difference in survival was observed between treatment groups for the overall study population or PSADT subsets.
Patients with shorter PSADT are at greater risk for bone metastasis or death. Denosumab consistently improves BMFS in men with shorter PSADT and seems to have the greatest treatment effects in men at high risk for progression.
PMCID: PMC3795889  PMID: 24043751
3.  Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes 
PLoS Pathogens  2014;10(8):e1004301.
We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.
Author Summary
Listeria monocytogenes is ubiquitously present in the environment, highly adaptable and tolerant to various stresses. L. monocytogenes is also a foodborne pathogen associated with the largest foodborne outbreaks in recent US history. Signaling pathways involving the second messenger c-di-GMP play important roles in increased stress survival of proteobacteria and mycobacteria, yet roles of c-di-GMP signaling pathways in L. monocytogenes have remained unexplored. Here, we identified and systematically characterized functions of the proteins involved in c-di-GMP synthesis, degradation and sensing. We show that elevated c-di-GMP levels in L. monocytogenes result in synthesis of a previously unknown exopolysaccharide that promotes cell aggregation, inhibits motility in semi-solid media, and importantly, enhances bacterial tolerance to commonly used disinfectants as well as desiccation. These properties of the exopolysaccharide may increase listerial survival in food processing plants as well as on produce during transportation and storage. Elevated c-di-GMP levels also grossly diminish listerial invasiveness in enterocytes in vitro, and impair bacterial accumulation in selected mouse organs during oral infection.
PMCID: PMC4125290  PMID: 25101646
4.  Comparison of surgical technique (Open vs. Laparoscopic) on pathological and long term functional outcomes following radical prostatectomy 
BMC Urology  2014;14:18.
Few studies to date have directly compared outcomes of retropubic (RRP) and laparoscopic (LRP) radical prostatectomy. We investigated a single institution experience with RRP and LRP with respect to functional and pathological outcomes.
168 patients who underwent RRP were compared to 171 patients who underwent LRP at our institution. Pathological and functional outcomes including postoperative urinary incontinence and erectile dysfunction (ED) of the two cohorts were examined.
Patients had bilateral, unilateral and no nerve sparing technique performed in 83.3%, 1.8% and 14.9% of cases for RRP and 23.4%, 22.8% and 53.8% of cases for LRP, respectively (p < 0.001). Overall positive surgical margin rates were 22.2% among patients who underwent RRP compared to 26.5% of patients who underwent LRP (p = 0.435). Based upon pads/day, urinary continence postoperatively was achieved in 83.2% and 82.8% for RRP and LRP, respectively (p = 0.872). Analysis on postoperative ED was limited due to lack of information on the preoperative erectile status. However, postoperatively there were no differences with respect to ED between the two cohorts (p = 0.151). Based on ICIQ-scores, surgeons with more experience had lower rates of postoperative incontinence irrespective of surgical technique (p = 0.001 and p < 0.001 for continuous and stratified data, respectively).
RRP and LRP represent effective surgical approaches for the treatment of clinically localized prostate cancer. Pathological outcomes are excellent for both surgical techniques. Functional outcomes including postoperative urinary incontinence and ED are comparable between the cohorts. Surgeon experience is more relevant than surgical technique applied.
PMCID: PMC3922887  PMID: 24506815
Prostate cancer; Erectile dysfunction; Incontinence; Radical prostatectomy; Laparoscopic prostatectomy
5.  Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis 
Matrix metalloproteinases can promote invasion and metastasis, which are very frequent in renal cell carcinoma even at the time of diagnosis. Knowing the reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as an inhibitor of matrix metalloproteinases and the extracellular matrix metalloproteinase inducer (EMMPRIN) protein as inducer, we aimed to determine their expression, localization and possible antagonistic action in the pathogenesis and progression of renal cell tumors in a retrospective study.
Tumor and adjacent normal tissues of 395 nephrectomized patients were immunostained for RECK and EMMPRIN on a tissue microarray.
RECK strongly decreased in renal cell carcinoma compared to normal counterparts (Wilcoxon signed rank test, P < 0.001), and it discriminated tumor entities showing the highest expression in oncocytomas. EMMPRIN, however, could be significantly correlated to pT stage and Fuhrman grading (Spearman’s correlation coefficient rs = 0.289 and rs = 0.382, respectively). Higher expression of EMMPRIN was associated with decreased overall survival in Kaplan-Meier analysis (P < 0.001), and the EMMPRIN level could independently predict survival for cases without metastasis and involvement of lymph nodes. Decreased RECK expression was confirmed by Western blotting in tissue of eight normal/tumor matches of patients after radical nephrectomy, whereas the EMMPRIN pattern appeared to be heterogeneous.
We propose RECK down regulation in renal cell carcinoma to be an early event that facilitates tumor formation and progression. EMMPRIN, however, as a prognostic tumor marker, increases only when aggressiveness is proceeding and could add an additional step to invasive properties of renal cell carcinoma.
PMCID: PMC3853196  PMID: 24131772
RECK; EMMPRIN; Renal cell carcinoma; TMA
6.  Interdisciplinary decision making in prostate cancer therapy – 5-years’ time trends at the Interdisciplinary Prostate Cancer Center (IPC) of the Charité Berlin 
Patients with prostate cancer face the difficult decision between a wide range of therapeutic options. These men require elaborate information about their individual risk profile and the therapeutic strategies´ risks and benefits to choose the best possible option. In order to detect time trends and quality improvements between an early patient population (2003/2004) and a later reference group (2007/2008) data was analysed with regards to epidemiologic parameters, differences in diagnostics and the type and ranking of the recommended therapies taking into account changes to Gleason Grading System and implementation of new therapeutic strategies, particularly Active surveillance, in 2005.
Data from all 496 consecutive patients who received consultation in 2003/2004 (n = 280) and 2007/2008 (n = 216) was retrospectively evaluated. Categorical variables were compared using the Chi-square test. Dependent variables were analysed using the unpaired Students´ t-test and the Mann–Whitney U-test.
The cohorts were comparable concerning clinical stage, initial PSA, prostate volume, comorbidities and organ confined disease. Patients in Cohort I were younger (66.44 vs. 69.31y; p < .001) and had a longer life expectancy (17.22 vs. 14.75y; p < .001). 50.9%, 28.2% and 20.9% in Cohort I and 37.2%, 39.6% and 23.2% in Cohort II showed low-, intermediate- and high-risk disease (D´Amico) with a trend towards an increased risk profile in Cohort II (p = .066). The risk-adapted therapy recommended as first option was radical prostatectomy for 91.5% in Cohort I and 69.7% in Cohort II, radiation therapy for 83.7% in Cohort I and 50.7% in Cohort II, and other therapies (brachytherapy, Active surveillance, Watchful waiting, high-intensity focused ultrasound) for 6.5% in Cohort I and 6.9% in Cohort II (p < .001). Radiation therapy was predominant in both cohorts as second treatment option (p < .001). Time trends showing quality improvement involved an increase in biopsy cores (9.95 ± 2.38 vs. 8.43 ± 2.29; p < .001) and an increased recommendation for bilateral nerve sparing (p < .001).
In the earlier years, younger patients with a more favourable risk profile presented for interdisciplinary consultation. A unilateral recommendation for radical prostatectomy and radiation therapy was predominant. In the later years, the patient population was considerably older. However, this group may have benefitted from optimised diagnostic possibilities and a wider range of treatment options.
PMCID: PMC3751298  PMID: 23915212
Prostate cancer; Interdisciplinary consultation; Medical decision making; Time trends
7.  Detection of Previously Unidentified Metastatic Disease as a Leading Cause of Screening Failure in a Phase III Trial of Zibotentan Versus Placebo in Patients with Nonmetastatic, Castration Resistant Prostate Cancer 
The Journal of urology  2012;188(1):103-109.
Understanding the extent of disease in asymptomatic patients with castration resistant prostate cancer is important when making treatment decisions and designing clinical trials. The ENTHUSE M0 (ENdoTHelin A USE) trial (NCT00626548) was a large phase III study comparing the endothelin A receptor antagonist zibotentan with placebo in patients with nonmetastatic, castration resistant prostate cancer. The study was stopped prematurely after early efficacy review indicated that it was unlikely to meet its co-primary objectives of improved overall and progression-free survival vs placebo. Screening failed in an unexpectedly high number of patients. We investigated this screening failure rate to promote better classification of patients thought to have nonmetastatic castration resistant prostate cancer and inform the design of future clinical trials in this setting.
Materials and Methods
The number of patients enrolled in and subsequently excluded from study was analyzed by geographic region and by the specialty of the investigating clinician (oncology or urology) who enrolled the study patients.
Of 2,577 patients enrolled in a total of 350 hospital based centers in 39 countries screening failed in 1,155 (45%). The most common reason for screening failure was the detection of metastatic disease in 32% of all screened patients and in 71% of those in whom screening failed. The leading reasons for failed screening did not differ between investigator specialties overall or by geographic region.
The high frequency of asymptomatic metastasis in men thought to have nonmetastatic, castration resistant prostate cancer highlights the importance of periodic staging assessments for the condition. Optimal treatment modalities may differ for metastatic and nonmetastatic disease.
PMCID: PMC3473078  PMID: 22583636
prostate; prostatic neoplasms; neoplasm metastasis; mass screening; ZD4054
8.  Denosumab and Bone Metastasis-Free Survival in Men With Castration-Resistant Prostate Cancer: Results of a Global Phase 3, Randomised, Placebo-Controlled Trial 
Lancet  2011;379(9810):39-46.
Bone metastases are a major cause of morbidity and mortality in men with prostate cancer. Preclinical studies suggest that osteoclast inhibition may prevent bone metastases. This phase 3 study evaluated denosumab, a fully human anti-RANKL monoclonal antibody, to prevent bone metastasis or death from any cause in men with non-metastatic castration-resistant prostate cancer (CRPC).
Men with non-metastatic CRPC at high risk for bone metastasis (PSA ≥8.0 ng/mL and/or PSA doubling time ≤10.0 months) were enrolled in 319 centers from 30 countries. Patients were randomised 1:1 in blinded fashion using an interactive voice response system to receive monthly subcutaneous denosumab 120 mg or placebo. The primary endpoint was bone metastasis-free survival, a composite endpoint determined by time to first occurrence of bone metastasis (symptomatic or asymptomatic) or death.
1432 patients were randomised, 716 to receive denosumab and 716 to receive placebo. Denosumab significantly increased bone metastasis-free survival by a median of 4.2 months over placebo (hazard ratio 0.85 [0.73–0.98]; P=0.028). Denosumab also significantly delayed time to first bone metastasis (hazard ratio 0.84 [0.71–0.98]; P=0.032). Overall survival was similar between groups (hazard ratio 1.01 [0.85–1.20]; P=0.91). Rates of adverse events (AEs) and serious AEs were generally similar between groups, except for osteonecrosis of jaw (ONJ) and hypocalcemia. Yearly cumulative incidence of ONJ for denosumab was: 1%, 3%, 4% in years 1, 2, 3, respectively; overall, less than 5% (n=33). Hypocalcemia occurred in under 2% (n=12) of denosumab and under 1% (n=2) of placebo patients. The blinded treatment phase has been completed.
In men with CRPC, denosumab significantly prolonged bone metastasis-free survival and delayed time to bone metastasis. This is the first large randomised study to demonstrate that targeting the bone microenvironment prevents bone metastasis in men with prostate cancer.
PMCID: PMC3671878  PMID: 22093187
urology/prostate disease; denosumab; prostate cancer; prevention; bone metastasis; survival; hormone refractory; castration-resistant
9.  External Validation of an Artificial Neural Network and Two Nomograms for Prostate Cancer Detection 
ISRN Urology  2012;2012:643181.
Background. Multivariate models are used to increase prostate cancer (PCa) detection rate and to reduce unnecessary biopsies. An external validation of the artificial neural network (ANN) “ProstataClass” (ANN-Charité) was performed with daily routine data. Materials and Methods. The individual ANN predictions were generated with the use of the ANN application for PSA and free PSA assays, which rely on age, tPSA, %fPSA, prostate volume, and DRE (ANN-Charité). Diagnostic validity of tPSA, %fPSA, and the ANN was evaluated by ROC curve analysis and comparisons of observed versus predicted probabilities. Results. Overall, 101 (35.8%) PCa were detected. The areas under the ROC curve (AUCs) were 0.501 for tPSA, 0.669 for %fPSA, 0.694 for ANN-Charité, 0.713 for nomogram I, and 0.742 for nomogram II, showing a significant advantage for nomogram II (P = 0.009) compared with %fPSA while the other model did not differ from %fPSA (P = 0.15 and P = 0.41). All models overestimated the predicted PCa probability. Conclusions. Beside ROC analysis, calibration is an important tool to determine the true value of using a model in clinical practice. The worth of multivariate models is limited when external validations were performed without knowledge of the circumstances of the model's development.
PMCID: PMC3399415  PMID: 22830050
10.  Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas 
PLoS ONE  2012;7(6):e39309.
Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma.
Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper.
Principal Findings
Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization.
The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.
PMCID: PMC3380005  PMID: 22745731
11.  Laparoscopic and open postchemotherapy retroperitoneal lymph node dissection in patients with advanced testicular cancer – a single center analysis 
BMC Urology  2012;12:15.
The open approach represents the gold standard for postchemotherapy retroperitoneal lymph node dissection (O-PCLND) in patients with residual testicular cancer. We analyzed laparoscopic postchemotherapy retroperitoneal lymph node dissection (L-PCLND) and O-PCLND at our institution.
Patients underwent either L-PCLND (n = 43) or O-PCLND (n = 24). Categorical and continuous variables were compared using the Fisher exact test and Mann–Whitney U test respectively. Overall survival was evaluated with the log-rank test.
Primary histology was embryonal cell carcinomas (18 patients), pure seminoma (2 cases) and mixed NSGCTs (47 patients). According to the IGCCCG patients were categorized into “good”, “intermediate” and “poor prognosis” disease in 55.2%, 14.9% and 20.8%, respectively. Median operative time for L-PCLND was 212 min and 232 min for O-PCLND (p = 0.256). Median postoperative duration of drainage and hospital stay was shorter after L-PCLND (0.0 vs. 3.5 days; p < 0.001 and 6.0 vs. 11.5 days; p = 0.002). Intraoperative complications occurred in 21.7% (L-PCLND) and 38.0% (O-PCLND) of cases with 19.5% and 28.5% of Clavien Grade III complications for L-PCLND and O-PCLND, respectively (p = 0.224). Significant blood loss (>500 ml) was almost equally distributed (8.6% vs. 14.2%: p = 0.076). No significant differences were observed for injuries of major vessels and postoperative complications (p = 0.758; p = 0.370). Tumor recurrence occurred in 8.6% following L-PCLND and in 14.2% following O-PCLND with a mean disease-free survival of 76.6 and 89.2 months, respectively. Overall survival was 83.3 and 95.0 months for L-PCNLD and O-PCLND, respectively (p = 0.447).
L-PCLND represents a safe surgical option for well selected patients at an experienced center.
PMCID: PMC3431976  PMID: 22651395
Advanced testicular cancer; Postchemotherapy; Retroperitoneal lymph node dissection; Laparoscopy; Metastasis
12.  Morbidity and Quality of Life in Bladder Cancer Patients following Cystectomy and Urinary Diversion: A Single-Institution Comparison of Ileal Conduit versus Orthotopic Neobladder 
ISRN Urology  2012;2012:342796.
Objective. To evaluate and compare noncontinent and continent urinary diversion after radical cystectomy in patients with bladder cancer. Methods. A total of 301 patients submitted to radical cystectomy at the Charité-University Hospital Berlin from 1993 to 2007 including 146 with an ileal conduit and 115 with an ileal neobladder. Clinical and pathological data as well as oncological outcome were retrospectively analyzed and compared. Quality of life was analyzed using the EORTC QLQ-C30 and BLM30 questionnaires. Results. 69.1% and 69.6% of all patients who received an ileal conduit and ileal neobladder, respectively, developed early complications. The two groups differed significantly concerning the occurrence of postoperative ileus (P = 0.02) favoring patients who received an ileal conduit but not with regard to any other early-onset complication evaluated. Patients with ileal neobladder had a significantly better global health status and quality of life (P = 0.02), better physical functioning (P = 0.02), but also a higher rate of diarrhoea (P = 0.004). Conclusion. Cystectomy with any type of diversion remains a complication-prone surgery. Even if the patient groups are not homogeneous in all respects, there are many arguments in favor of the ileal neobladder as the urinary diversion of choice.
PMCID: PMC3302022  PMID: 22523713
13.  Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas 
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.
PMCID: PMC3492794  PMID: 23139634
Renal cell carcinoma; Microarray; RT-qPCR; microRNAs; Metastasis.
14.  Intrinsic resistance to tyrosine kinase inhibitors is associated with poor clinical outcome in metastatic renal cell carcinoma 
BMC Cancer  2011;11:295.
Data on sequential therapy in patients with metastatic renal cell carcinoma (mRCC) and intrinsic resistance to receptor tyrosine kinase inhibitor (rTKI) treatment remains vague.
We retrospectively studied treatment characteristics and outcome of mRCC patients refractory to first rTKI therapy.
Thirty-five mRCC patients (male, 18; female, 11) with primary resistance to first rTKI therapy (sunitinib, n = 28; sorafenib, n = 7) and a median treatment interval of 2.4 months (1 - 4.6) were identified. In 22 patients, progressive disease (PD) was determined by a new metastatic lesion. Of these, 16 patients received subsequent therapy with 12 patients remaining refractory and 4 patients achieving disease stabilization. In 13 patients continuous growth of existing metastatic lesions determined PD. Of these, 9 received sequential therapy with 6 achieving disease stabilization. Altogether, 25 patients were treated sequentially (rTKI: n = 15; mTOR-inhibitor: n = 10) and achieved a median PFS of 3.2 months (range, 1-16.6). Fifteen patients failed to respond to either line of therapy. Disease control was not associated with type of subsequent therapy. Median OS was 14.9 months (CI: 5.5-24.4).
Intrinsic resistance to rTKI is associated with a low chance of response to sequential therapy and a poor prognosis in mRCC patients.
PMCID: PMC3154177  PMID: 21756335
15.  Suitable reference genes for relative quantification of miRNA expression in prostate cancer 
Experimental & Molecular Medicine  2010;42(11):749-758.
Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa-miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa-miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.
PMCID: PMC2992854  PMID: 20890088
gene expression profiling; microRNA; polymerase chain reaction; prostate neoplasms
16.  Regulation of Mannose Phosphotransferase System Permease and Virulence Gene Expression in Listeria monocytogenes by the EIItMan Transporter ▿  
Applied and Environmental Microbiology  2009;75(21):6671-6678.
The EIItMan phosphotransferase system (PTS) permease encoded by the mpt operon is the principal glucose transporter in Listeria monocytogenes. EIItMan participates in glucose-mediated carbon catabolite repression (CCR) and downregulation of virulence gene expression, and it is the receptor for class IIa bacteriocins. The regulation of this important protein and its roles in gene control were examined using derivatives of strain EGD-e in which the mpt operon or its regulatory genes, manR and lmo0095, were deleted. Real-time reverse transcription-PCR analysis showed that the mpt mRNA level was 10- and 100-fold lower in the lmo0095 and manR deletion strains, respectively. The manR mRNA level was higher in the mpt deletion mutant in medium lacking glucose, possibly due to disruption of a regulatory process that normally downregulates manR transcription in the absence of this sugar. Analysis of the mpt deletion mutant also showed that EIItMan participates to various degrees in glucose-mediated CCR of PTS operons. CCR of the lmo0027 gene, which encodes a β-glucoside PTS transporter, required expression of EIItMan. In contrast, genes in two mannose PTS operons (lmo0024, lmo1997, and lmo2002) were repressed by glucose even when EIItMan was not synthesized. A third mannose PTS operon, mpo, was not regulated by glucose or by the level of EIItMan. Finally, the mRNA levels for five genes in the prfA virulence gene cluster were two- to fourfold higher in the mpt deletion mutant. The results show that EIItMan participates to various extents in glucose-mediated CCR of PTS operons and makes a small, albeit significant, contribution to downregulation of virulence gene transcription by glucose in strain EGD-e.
PMCID: PMC2772439  PMID: 19734332
17.  Metastasizing pleomorphic adenoma presenting as an asymptomatic kidney tumor twenty-nine years after parotidectomy – urological viewpoint and overview of the literature to date 
Rare Tumors  2009;1(2):e54.
Pleomorphic adenomas (benign mixed tumors) are the most common tumors of glandular origin in the head and neck and are one of the few benign neoplasms that can undergo malignant transformation.1 Mixed tumors that are seemingly benign at the microscopic level but metastasize have been termed metastasizing mixed tumors (MZMTs). The entity of metastasizing benign mixed tumors has been reported since the early 1940s, with up to approximately 50 cases described in the literature to date. Despite their bland morphologic appearance, MZMTs have been associated with an overall mortality rate of about 20–40%. We report the case of a MZMT of the kidney almost 30 years after lateral parotidectomy owing to the same tumor entity. For benign mixed tumors, we are unaware of more than two other cases of metastasis to the kidney that have been published, whereas metastases to the bone, lung, and lymph nodes are more common. Parotidectomy is widely accepted as the first choice of treatment,13 but once metastases have occurred the therapeutic strategy is uncertain with surgery being the only curative option in cases with resectable disease. This case report provides information about the rare event of metastatic disease to the kidney and points out therapeutic strategies. However, in view of the general lack of adequate information in the literature, the best therapy for systemic disease still remains unresolved.
PMCID: PMC2994456  PMID: 21139933
carcinoma ex-mixed tumor; metastasizing mixed tumor; metastasizing pleomorphic adenoma; kidney metastasis; kidney tumor.
18.  Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients 
Retrovirology  2009;6:92.
A novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) has been recently identified and found to have a prevalence of 40% in prostate tumor samples from American patients carrying a homozygous R462Q mutation in the RNaseL gene. This mutation impairs the function of the innate antiviral type I interferon pathway and is a known susceptibility factor for prostate cancer. Here, we attempt to measure the prevalence of XMRV in prostate cancer cases in Germany and determine whether an analogous association with the R462Q polymorphism exists.
589 prostate tumor samples were genotyped by real-time PCR with regard to the RNaseL mutation. DNA and RNA samples from these patients were screened for the presence of XMRV-specific gag sequences using a highly sensitive nested PCR and RT-PCR approach. Furthermore, 146 sera samples from prostate tumor patients were tested for XMRV Gag and Env antibodies using a newly developed ELISA assay. In agreement with earlier data, 12.9% (76 samples) were shown to be of the QQ genotype. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies.
Our results indicate a much lower prevalence (or even complete absence) of XMRV in prostate tumor patients in Germany. One possible reason for this could be a geographically restricted incidence of XMRV infections.
PMCID: PMC2770519  PMID: 19835577
19.  Down-regulation of the pro-apoptotic XIAP associated factor-1 (XAF1) during progression of clear-cell renal cancer 
BMC Cancer  2009;9:276.
Decreased expression of the interferon-stimulated, putative tumour suppressor gene XAF1 has been shown to play a role during the onset, progression and treatment failure in various malignancies. However, little is yet known about its potential implication in the tumour biology of clear-cell renal cell cancer (ccRCC).
This study assessed the expression of XAF1 protein in tumour tissue obtained from 291 ccRCC patients and 68 normal renal tissue samples, utilizing immunohistochemistry on a tissue-micro-array. XAF1 expression was correlated to clinico-pathological tumour features and prognosis.
Nuclear XAF1 expression was commonly detected in normal renal- (94.1%) and ccRCC (91.8%) samples, without significant differences of expression levels. Low XAF1 expression in ccRCC tissue, however, was associated with progression of tumour stage (p = 0.040) and grade (p < 0.001). Low XAF1 tumour levels were also prognostic of significantly shortened overall survival times in univariate analysis (p = 0.018), but did not provide independent prognostic information.
These data suggest down-regulation of XAF1 expression to be implicated in ccRCC progression and implies that its re-induction may provide a therapeutic approach. Although the prognostic value of XAF1 in ccRCC appears to be limited, its predictive value remains to be determined, especially in patients with metastatic disease undergoing novel combination therapies of targeted agents with Interferon-alpha.
PMCID: PMC3087333  PMID: 19664236
20.  Brain-type and liver-type fatty acid-binding proteins: new tumor markers for renal cancer? 
BMC Cancer  2009;9:248.
Renal cell carcinoma (RCC) is the most common renal neoplasm. Cancer tissue is often characterized by altered energy regulation. Fatty acid-binding proteins (FABP) are involved in the intracellular transport of fatty acids (FA). We examined the level of brain-type (B) and liver-type (L) FABP mRNA and the protein expression profiles of both FABPs in renal cell carcinoma.
Paired tissue samples of cancerous and noncancerous kidney parts were investigated. Quantitative RT-PCR, immunohistochemistry and western blotting were used to determine B- and L-FABP in tumor and normal tissues. The tissue microarray (TMA) contained 272 clinico-pathologically characterized renal cell carcinomas of the clear cell, papillary and chromophobe subtype. SPSS 17.0 was used to apply crosstables (χ2-test), correlations and survival analyses.
B-FABP mRNA was significantly up-regulated in renal cell carcinoma. In normal tissue B-FABP mRNA was very low or often not detectable. RCC with a high tumor grading (G3 + G4) showed significantly lower B-FABP mRNA compared with those with a low grading (G1 + G2). Western blotting analysis detected B-FABP in 78% of the cases with a very strong band but in the corresponding normal tissue it was weak or not detectable. L-FABP showed an inverse relationship for mRNA quantification and western blotting. A strong B-FABP staining was present in 52% of the tumor tissues contained in the TMA. In normal renal tissue, L-FABP showed a moderate to strong immunoreactivity in proximal tubuli. L-FABP was expressed at lower rates compared with the normal tissues in 30.5% of all tumors. There was no correlation between patient survival times and the staining intensity of both FABPs.
While B-FABP is over expressed in renal cell carcinoma in comparison to normal renal tissues L-FABP appears to be reduced in tumor tissue. Although the expression behavior was not related to the survival outcome of the RCC patients, it can be assumed that these changes indicate fundamental alterations in the fatty metabolism in the RCC carcinogenesis. Further studies should identify the role of both FABPs in carcinogenesis, progression and with regard to a potential target in RCC.
PMCID: PMC2732640  PMID: 19622156
21.  Exogenous or l-Rhamnose-Derived 1,2-Propanediol Is Metabolized via a pduD-Dependent Pathway in Listeria innocua▿  
Applied and Environmental Microbiology  2008;74(22):7073-7079.
1,2-Propanediol (1,2-PD) added exogenously to cultures or produced endogenously from l-rhamnose is metabolized to n-propanol and propionate in Listeria innocua Lin11. The pduD gene, which encodes a diol dehydratase ß subunit homolog, is required for 1,2-PD catabolism. pduD and 16 other genes within the pduA-to-pduF region of a large gene cluster are induced in medium containing 1,2-PD.
PMCID: PMC2583486  PMID: 18805996
22.  Regulation of the mpt Operon in Listeria innocua by the ManR Protein▿  
Applied and Environmental Microbiology  2007;73(17):5648-5652.
The phosphotransferase system regulation domain (PRD)-containing activator, ManR, is required for glucose-controlled transcription of the mannose permease two (mpt) operon in Listeria innocua. His-871 in ManR PRD-II is needed for mpt repression in glucose-free media. His-506 in PRD-I is needed for mpt induction by glucose.
PMCID: PMC2042094  PMID: 17616620
23.  Impact of hormonal therapy on the detection of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1) in prostate cancer 
BMC Urology  2006;6:15.
In spite of excellent cure rates for prostate cancer patients with favorable tumor characteristics, patients with unfavorable characteristics after radical prostatectomy are still at a significantly increased risk of tumor progression. Early adjuvant hormonal therapy (AHT) has been shown to be of prognostic benefit in these patients. Unfortunately initiation and duration of early AHT in the individual patient is based on statistic data. PSA, as the standard prostate marker is neither able to reliably indicate minimal residual tumor disease in the early postoperative phase, nor can it be used for therapy monitoring due to the suppressive effect of hormonal therapy on PSA production. Promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1-HM) has been shown to be the most common DNA alteration of primary prostatic carcinoma which, when used as a marker, is supposed to be able to overcome some of the disadvantages of PSA. However until now information on the impact of hormonal therapy on the detection of GSTP1-HM is lacking. The purpose of our study was to assess the impact of endocrine therapy on the detection of GSTP1-HM by methylation-specific PCR (MSP) in prostate cancer.
Paraffin embedded tumor samples from the radical prostatectomy (RP) specimens from 15 patients after hormonal therapy (HT) (mean 8 months) were assessed by MSP. In 8 of the patients the GSTP-1 status of the tumors before HT was assessed on the corresponding initial diagnostic biopsies.
Following HT MSP showed GSTP1-HM in 13/15 of the RP specimens. In two patients analysis of the RP specimens failed to show GSTP1-HM. All initial tumor samples (8/8 biopsy specimens) showed GSTP1-HM, including both patients negative for GSTP1 HM in the corresponding RP specimen.
In most cases hormonal therapy appears to not alter GSTP1 HM detection. However the change from a positive to a negative GSTP1 HM status in a subset of the patients may point to an, at least partial androgen dependency. Further studies on a larger cohort of patients are necessary to assess its frequency and the exact hormonal interactions.
PMCID: PMC1540432  PMID: 16803634
24.  Relationship between cytokines and the embryotoxicity of hydrosalpingeal fluid 
Objective: The exact chemical composition of hydrosalpingeal fluid is unknown. The objective of this study was to characterize cytokines in hydrosalpingeal fluid (HSF) and examine their possible role in the embryo development.
Study Design: HSF was aspirated at laparoscopic salpingectomy in eight infertile women. Levels of IL-1β, IL-13, IL-8, IL-6 and TNF-α in the HSF were determined by quantitative immunoassay kits. Two-cell mouse embryos were incubated with 0, 25, 50 and 75% concentrations of HSF. The blastocyst development rate (BDR) of mouse embryos was measured at each HSF concentration.
Result(s): The embryotoxicity of HSF was concentration dependent. An increase in the HSF concentration resulted in significant decrease in % BDR (p < 0.01). IL-1β was present in six of the eight HSF samples with a mean (± SD) concentration of 0.9 ± 0.8 pg/mL. IL-13 was not detected in any of the HSF samples. IL-8, IL-6 and TNF-α were detected in all samples with a mean (± SD) concentration of 4741.2 ± 6554.4 pg/mL, 204.8± 132.8 pg/ml and 12 ± 12.8 pg/mL respectively. IL-6 was positively correlated with BDR (r = 0.53; p < 0.04).
Conclusion(s): We demonstrated for the first time the presence of IL-1β, IL-8, IL-6 and TNF-α and the absence of IL-13 in human hydrosalpingeal fluid. IL-6 was positively related to the BDR.
PMCID: PMC3455287  PMID: 16021860
Cytokines; embryotoxicity; Hydrosalpinx
25.  Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance 
To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 μg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EIItMan, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by σ54 factor and ManR (a σ54-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (σ54) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.
PMCID: PMC1065182  PMID: 15746330

Results 1-25 (32)