Publications by authors named "Jörg Hennenlotter"

145 Publications

Evaluation of carbonic anhydrase IX as a potential therapeutic target in urothelial carcinoma.

Urol Oncol 2021 May 31. Epub 2021 May 31.

Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada.

Objective: Carbonic anhydrase IX (CA9) is important in the regulation of intra- and extracellular pH in solid tumors, contributing to cell growth and invasion. In urothelial carcinoma (UC), CA9 has been identified as a urinary marker for disease detection, but its biologic role is unknown. To date, differential gene expression patterns of CA9 in various molecular subtypes and potential effects of CA9 inhibition in UC cells are unknown. We aimed to investigate the function of CA9 and the effects of CA9 inhibition in invasive UC.

Methods: Immunohistochemistry was used to assess CA9 expression in a cohort of 153 patients undergoing radical cystectomy. CA9 expression was correlated with molecular subtype by analysis of the TCGA data and of our own cohort of 223 patients with invasive UC receiving neoadjuvant chemotherapy. CA9 expression was assessed in a panel of 12 UC cell lines by Western Blot and qPCR, and multiple siRNAs were used to silence CA9 in 2 cell lines. Effects of CA9 silencing on cell growth, migration, and invasion were assessed. We also used the small molecule inhibitor U-104 to inhibit CA9 in vitro and in an orthotopic xenograft model.

Results: CA9 expression was higher in cancer tissue compared to benign urothelium and was particularly highly expressed in luminal papillary and basal squamous tumors. CA9 expression did not correlate with outcome after neoadjuvant chemotherapy and/or radical cystectomy. Silencing of CA9 by siRNA diminished invasion but did not induce a consistent change of cell growth and migration. Treatment with U-104 led to cell growth reduction only at high concentrations in vitro and failed to have a significant effect on tumor growth in vivo.

Conclusions: The present study confirms over-expression of CA9 in UC and for the first time shows a correlation with molecular subtypes. However, CA9 expression showed no association with the outcome of patients with muscle invasive bladder cancer and inhibition of CA9 did not lead to a consistent inhibition of tumor growth. Based on these data, CA9 exhibits a role neither as a predictive or prognostic marker nor as a therapeutic target in invasive UC.
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http://dx.doi.org/10.1016/j.urolonc.2021.04.011DOI Listing
May 2021

Evaluation of split-filter dual-energy CT for characterization of urinary stones.

Br J Radiol 2021 May 14:20210084. Epub 2021 May 14.

Radiologie Münster MVZ, Von-Steuben-Str. 10a, 48143 Münster, Germany.

Objective: To assess accuracy of dual-energy computed tomography (DECT) to differentiate uric acid from calcium urinary stones in dual-energy split filter sequential-spiral dual-source acquisition.

Methods: Thirty-four urinary stones (volume 89.0 ± 77.4 mm³; 17 calcium stones, 17 uric acid stones) were scanned in a water-filled phantom using a split-filter equipped CT scanner (SOMATOM Definition Edge, Siemens Healthineers, Forchheim, Germany) in split-filter mode at 120kVp and sequential-spiral mode at 80 and 140kVp. Additional DE scans were acquired at 80 and 140kVp (tin filter) with a dual-source CT scanner (SOMATOM Definition FLASH, Siemens Healthineers). Scans were performed with a CTDIvol of 7.3mGy in all protocols. Urinary stone categorization was based on dual energy ratio (DER) using an automated 3D segmentation. As reference standard, infrared spectroscopy was used to determine urinary stone composition.

Results: All three DECT techniques significantly differentiated between uric acid and calcium stones by attenuation values and DERs ( < 0.001 for all). Split-filter DECT provided higher DERs for uric acid stones, when compared with dual-source and sequential-spiral DECT, and lower DERs for calcified stones when compared with dual-source DECT ( < 0.001 for both), leading to a decreased accuracy for material differentiation.

Conclusion: Split-filter DECT, sequential-spiral DECT and dual-source DECT all allow for the acquisition of DER to classify urinary stones.

Advances In Knowledge: Split-filter DECT enables the differentiation between uric acid and calcium stones despite decreased spectral separation when compared with dual-source and dual-spiral DECT.
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http://dx.doi.org/10.1259/bjr.20210084DOI Listing
May 2021

Receptor Activator of NF Kappa B (RANK) Expression Indicates Favorable Prognosis in Patients with Muscle-invasive Bladder Cancer.

Eur Urol Focus 2021 May 4. Epub 2021 May 4.

Department of Urology, University Hospital, Tübingen, Germany; Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada; Clinical Trials Unit, Studienpraxis Urologie, Nürtingen, Germany. Electronic address:

Background: Receptor activator of NF kappa B (RANK) and its ligand have an essential role in T-cell regulation and the development of bone metastases. The role of RANK expression in muscle-invasive bladder cancer (MIBC) is unknown.

Objective: To assess the relevance of RANK expression in patients with MIBC.

Design, Setting, And Participants: Expression of RANK was assessed via immunohistochemistry of benign urothelium, MIBC tissue, and lymph node metastases from 153 patients undergoing radical cystectomy. Expression data from The Cancer Genome Atlas (TCGA) cohort were analyzed for potential associations with molecular subtypes and outcome.

Outcome Measurements And Statistical Analysis: RANK expression was correlated with clinical and pathological parameters and to individual data for the clinical course of MIBC.

Results And Limitations: Expression of RANK was significantly higher in both primary tumors (p = 0.02) and lymph node metastases (p = 0.01) compared to normal urothelium. In tumor tissue, RANK expression was significantly lower in patients with locally advanced disease and lymph node involvement compared to those with organ-confined disease (p = 0.0009) and node-negative MIBC (p = 0.0002). In univariable and multivariable analyses, high expression of RANK was associated with a longer time to recurrence (p = 0.0005 and 0.01) and better cancer-specific (p = 0.0004 and 0.007) and overall survival (p = 0.002 and 0.04). High expression of RANK was associated with better outcome for patients with luminal infiltrated tumors in the TCGA cohort.

Conclusions: RANK expression is increased in bladder cancer tissue compared to benign urothelium, with higher expression in organ-defined compared to locally advanced disease. High RANK expression indicates a favorable prognosis in MIBC. The prognostic role differs in tumors of different molecular subtypes.

Patient Summary: Expression of a protein involved in bone turnover regulation (RANK) is higher in bladder cancer tissue than in benign bladder tissue. However, high levels of RANK on tumor cells indicate favorable prognosis for patients with bladder cancer that invades the muscle layer of the bladder.
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http://dx.doi.org/10.1016/j.euf.2021.04.015DOI Listing
May 2021

DNA methylation of tumor associated calcium signal transducer 2 (TACSTD2) loci shows association with clinically aggressive renal cell cancers.

BMC Cancer 2021 Apr 21;21(1):444. Epub 2021 Apr 21.

Department of Urology and Urologic Oncology, Hannover Medical School, 30625, Hannover, Germany.

Background: DNA methylation is frequently observed in the development and progression of many human tumors as well as renal cell cancer (RCC). Tumor Associated Calcium Signal Transducer 2 (TACSTD2) participates in cell cycle progression through MAPK signalling pathway activation. Moreover, tumor-specific hypermethylation and association with aggressive cancer characteristics has been found for lung adenocarcinoma, hepatocellular carcinoma and cholangiocarcinoma. Whether TACSTD2 is tumor specifically hypermethylated in RCC or shows association of methylation with adverse clinicopathological parameters and survival of patients has not been investigated at yet.

Methods: Quantitative methylation-specific PCR (qMSP) analysis of a locus in the intron 1 region of TACSTD2 gene was carried out in a cross-sectional study of 127 paired RCC and normal samples. In silico analysis of TACSTD2 methylation in the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) dataset of 280 patients served as validation cohort. Statistical analyses were carried out using the two-sided paired t-test for matched tumor and normal sample comparisons, logistic regression for subgroup comparisons, Cox regression for analysis of recurrence free survival (RFS) and Pearson correlation analysis for correlation of TACSTD2 methylation and TACSTD2 mRNA in KIRC data.

Results: Higher methylation levels in RCC were significantly associated with advanced disease (p < 0.001), high tumor stage (p = 0.003), tumor differentiation (p = 0.033) and presence of lymph node (p = 0.021) or distant metastases (p = 0.008). TACSTD2 hypermethylation was associated with a shorter RFS of patients and demonstrate statistical independency from clinical parameters as state of metastasis, tumor stage, grade and state of advanced disease. In silico validation using TCGA KIRC data also demonstrated association of TACSTD2 loci with adverse clinicopathology and shortened RFS of patients. In addition, in silico analyses of TCGA KIRC data showed an inverse correlation between DNA methylation levels of TACSTD2 and mRNA expression.

Conclusions: Our results suggest an association between TACSTD2 methylation and disease progression and clinical course of RCC.
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http://dx.doi.org/10.1186/s12885-021-08172-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061065PMC
April 2021

Increased Expressions of Matrix Metalloproteinases (MMPs) in Prostate Cancer Tissues of Men with Type 2 Diabetes.

Biomedicines 2020 Nov 16;8(11). Epub 2020 Nov 16.

Department of Internal Medicine IV, Division of Diabetology, Endocrinology and Nephrology, University Hospital Tübingen, 72076 Tübingen, Germany.

Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E-cadherin/N-cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial-mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases () and CC chemokine ligands () were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of and , which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase and gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa.
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http://dx.doi.org/10.3390/biomedicines8110507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696165PMC
November 2020

Optimized protocol for metabolomic and lipidomic profiling in formalin-fixed paraffin-embedded kidney tissue by LC-MS.

Anal Chim Acta 2020 Oct 13;1134:125-135. Epub 2020 Aug 13.

Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany and University of Tübingen, Tübingen, Germany. Electronic address:

Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines [LPE]) remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.
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http://dx.doi.org/10.1016/j.aca.2020.08.005DOI Listing
October 2020

Characterization of Hormone-Dependent Pathways in Six Human Prostate-Cancer Cell Lines: A Gene-Expression Study.

Genes (Basel) 2020 10 7;11(10). Epub 2020 Oct 7.

Department of Internal Medicine IV, Division of Diabetology, Endocrinology, and Nephrology, University Hospital Tübingen, 72076 Tübingen, Germany.

Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.
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http://dx.doi.org/10.3390/genes11101174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599530PMC
October 2020

Transcript Levels of Aldo-Keto Reductase Family 1 Subfamily C (AKR1C) Are Increased in Prostate Tissue of Patients with Type 2 Diabetes.

J Pers Med 2020 Sep 12;10(3). Epub 2020 Sep 12.

Department of Internal Medicine IV, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, 72076 Tübingen, Germany.

Aldo-keto reductase family 1 (AKR1) enzymes play a crucial role in diabetic complications. Since type 2 diabetes (T2D) is associated with cancer progression, we investigated the impact of diabetes on gene expression in the context of prostate cancer (PCa) development. In this study, we analyzed benign (BEN) prostate and PCa tissue of patients with and without T2D. Furthermore, to replicate hyperglycemia in vitro, we treated the prostate adenocarcinoma cell line PC3 with increasing glucose concentrations. Gene expression was quantified using real-time qPCR. In the prostate tissue of patients with T2D, and transcripts were higher compared to samples of patients without diabetes. In PC3 cells, high glucose treatment induced the gene expression levels of , and . Furthermore, both in human tissue and in PC3 cells, the transcript levels of and showed positive associations with oncogenes, which are involved in proliferation processes and HIF1α and NFκB pathways. These results indicate that in the prostate glands of patients with T2D, hyperglycemia could play a pivotal role by inducing the expression of and . The higher transcript level of was furthermore associated with upregulated HIF1α and NFκB pathways, which are major drivers of PCa carcinogenesis.
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http://dx.doi.org/10.3390/jpm10030124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564141PMC
September 2020

Prognostic impact of somatostatin receptor expression in advanced bladder cancer.

Urol Oncol 2020 12 7;38(12):935.e17-935.e28. Epub 2020 Aug 7.

Clinical Trial Unit, Studienpraxis Urologie, Nürtingen, Germany; Medical School, Eberhard-Karls-University Tübingen, Tübingen, Germany. Electronic address:

Introduction And Objectives: Somatostatin receptors (SSTR) recently have been identified as potential targets for treatment of solid tumors. Furthermore, they have been shown to be of high relevance for tumor biology and prognosis in various types of cancer. However, there is a lack of clinical data for SSTR in bladder cancer (BC). Aim of this study was to determine the expression of all relevant somatostatin receptor subtypes in benign urothelium and tumor tissue of patients with muscle invasive BC. Furthermore, their potential role as prognostic factor for cancer-specific survival (CSS) and overall survival (OS) was evaluated.

Methods: The collective included BC and benign urothelium tissue of 103 patients (Median age 69; range 32-84, 79 male, 24 female) who underwent a radical cystectomy. A tissue microarray with subsequent immunohistochemical staining was used to assess membranous expression of SSTR1-5. Results were correlated to clinical and histopathological data as well as CSS and OS.

Results: Expressions of SSTR1-4 were significantly decreased in BC compared to benign urothelium (P < 0.002 each), whereas SSTR5 expression was increased (P = 0.0017). Expression of SSTR1 was associated with organ-confined disease (≤pT2) (P = 0.0477). No correlation between SSTR1-5 expression and N- and M-stage was observed. Univariate analyses showed a significantly longer CSS and OS in patients with high expression of SSTR3 (P = 0.0316 and 0.0044). Multivariate analyses confirmed SSTR3 expression as independent marker of improved CSS and OS (P = 0.0324 and 0.0076).

Conclusions: The majority of somatostatin receptor subtypes exhibit decreased expression in BC compared to benign bladder tissue. Expression of SSTR3 is an indicator for favorable prognosis in patients with muscle-invasive BC. These results support preclinical investigations using somatostatin receptor analogues such as octreotide to influence BC growth.
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http://dx.doi.org/10.1016/j.urolonc.2020.07.005DOI Listing
December 2020

Human Prostate Cancer is Characterized by an Increase in Urea Cycle Metabolites.

Cancers (Basel) 2020 07 6;12(7). Epub 2020 Jul 6.

Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, 72076 Tübingen, Germany.

Despite it being the most common incident of cancer among men, the pathophysiological mechanisms contributing to prostate cancer (PCa) are still poorly understood. Altered mitochondrial metabolism is postulated to play a role in the development of PCa. To determine the key metabolites (which included mitochondrial oncometabolites), benign prostatic and cancer tissues of patients with PCa were analyzed using capillary electrophoresis and liquid chromatography coupled with mass spectrometry. Gene expression was studied using real-time PCR. In PCa tissues, we found reduced levels of early tricarboxylic acid cycle metabolites, whereas the contents of urea cycle metabolites including aspartate, argininosuccinate, arginine, proline, and the oncometabolite fumarate were higher than that in benign controls. Fumarate content correlated positively with the gene expression of oncogenic HIF1α and NFκB pathways, which were significantly higher in the PCa samples than in the benign controls. Furthermore, data from the TCGA database demonstrated that prostate cancer patients with activated NFκB pathway had a lower survival rate. In summary, our data showed that fumarate content was positively associated with carcinogenic genes.
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http://dx.doi.org/10.3390/cancers12071814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408908PMC
July 2020

Impact of Histopathological Prostate Inflammation on Urine-Based Prostate Cancer Prediction Using the Prostate Cancer Gene 3 Score.

Urol Int 2020 8;104(5-6):483-488. Epub 2020 May 8.

Department of Urology, Eberhard Karls University of Tübingen, Tübingen, Germany.

Introduction: The Prostate Cancer gene 3 (PCA3) urine test has gained importance in the diagnostic workup of prostate cancer (PC). Limited evidence suggests that PCA3 is not altered in the presence of inflammation.

Objective: To assess the impact of histological inflammation on PCA3.

Methods: PCA3 was evaluated in patients prior to prostate biopsy (n = 193) and to radical prostatectomy (n = 197). In patients without PC, inflammation was assessed and quantified by individual scores integrating grade and extent. Uni- and multivariate analyses were performed to assess the impact of inflammation grade on PCA3.

Results: The PCA3 scores prior to prostatectomy were lower (median 45) than those before positive biopsy (57; p = 0.008). Of 101 negative biopsies, 78% showed inflammation. The median PCA3 scores in the groups with no inflammation and with maximum grade 1 (n = 22), 2 (n = 38), and 3 (n = 19) inflammation were 45, 38, 27, and 25 (p = 0.016). The multivariate models revealed a decrease in PCA3 proportional to the grade and extent of inflammation (p < 0.04 each).

Conclusions: The present data imply that the PCA3 score decreases in the presence of inflammation, which is relevant, for instance, to testing after a recently performed biopsy. In general, inflammation should be regarded as a factor putatively influencing PCA3 and other available and upcoming PC tests.
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http://dx.doi.org/10.1159/000506885DOI Listing
March 2021

Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy.

Genome Med 2020 03 30;12(1):32. Epub 2020 Mar 30.

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.

Background: Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy.

Methods: We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n = 452). DNA methylation (TCGA KIRC, n = 273), somatic mutations (TCGA KIRC, n = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n = 30) and immune-oncological markers (cohort 1, n = 37) were analyzed to characterize regulatory and functional involvements. CD8 T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture.

Results: A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8 T cells, including peptides derived from EGLN3. Functional analysis of EGLN3 revealed possible tumor-promoting functions.

Conclusions: Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC.
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http://dx.doi.org/10.1186/s13073-020-00731-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106651PMC
March 2020

Age-Adapted Prostate Cancer Gene 3 Score Interpretation - Suggestions for Clinical Use.

Clin Lab 2020 Mar;66(3)

Background: The prostate cancer antigen 3 (PCA3) gene urine assay is established for biopsy decision in case of prostate cancer (PC) suspicion. Recent findings pointed to an age dependence of PCA3, with putative impact on test interpretation. However, to date no experience has been reported with regard to the extent age might modify the score in certain age ranges. Therefore, the aim of the present study was to re-evaluate the age dependency and, moreover, give suggestions for interpretation of the PCA3 score in dependence of patient's age in daily routine.

Methods: The study comprised 684 patients before prostate biopsy or prostatectomy. Post-massage voided urine samples were assessed by PCA3 measurement. PCA3 scores were correlated to patient's age. The collective was divided into four subcollectives by quartiles of age distribution. For every subcollective the cutoff value at specificity of ≥ 60 was determined. Results were classified by age-class specific cutoff values and test qualities were compared at different cutoffs.

Results: In the collective, 59.1% of patients had a positive biopsy. PCA3 correlated to patient's age in univariate and multivariate analysis (p < 0.001 each). The division into age subcollectives revealed groups < 60, 60 - 65, 66 - 69 and > 69 years. Median PCA3 values of patients without/with PC were 17/32, 27/42, 34/55 and 52/68 in the four age classes. Cutoff values for which specificity was determined with ≥ 60 were 23, 39, 42, and 65. Constant cutoff values showed lower sensitivities in younger and lower specificities in older patients. Only the age adjusted values revealed an improved performance with PPV 68.7, accuracy 59.5 and sensitivity 57.7 at specificity of 62.1% in the whole cohort.

Conclusions: The study confirms that the PCA3 score increases with age. The recommended cutoff score of 35 is suitable especially for patients aged in their sixties. Lower reference values between 20 and 30 have to be taken into account in patients aged < 60 years and higher values around 40 to 50 may point to suspicion for PC in patients > 69 years. These results may further improve the diagnostic performance of the PCA3 test and keep the PCA3 test as a significant test in PC diagnostics along with new upcoming urine markers.
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http://dx.doi.org/10.7754/Clin.Lab.2019.190714DOI Listing
March 2020

Age-, tumor-, and metastatic tissue-associated DNA hypermethylation of a T-box brain 1 locus in human kidney tissue.

Clin Epigenetics 2020 02 18;12(1):33. Epub 2020 Feb 18.

Klinik für Urologie und urologische Onkologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str.1, D-30625, Hannover, Germany.

Background: While a considerable number of tumor-specific hypermethylated loci have been identified in renal cell cancer (RCC), DNA methylation of loci showing successive increases in normal, tumoral, and metastatic tissues could point to genes with high relevance both for the process of tumor development and progression. Here, we report that DNA methylation of a locus in a genomic region corresponding to the 3'UTR of the transcription factor T-box brain 1 (TBR1) mRNA accumulates in normal renal tissues with age and possibly increased body mass index. Moreover, a further tissue-specific increase of methylation was observed for tumor and metastatic tissue samples.

Results: Biometric analyses of the TCGA KIRC methylation data revealed candidate loci for age-dependent and tumor-specific DNA methylation within the last exon and in a genomic region corresponding to the 3'UTR TBR1 mRNA. To evaluate whether methylation of TBR1 shows association with RCC carcinogenesis, we measured 15 tumor cell lines and 907 renal tissue samples including 355 normal tissues, 175 tissue pairs of normal tumor adjacent and corresponding tumor tissue as well 202 metastatic tissues samples of lung, bone, and brain metastases by the use of pyrosequencing. Statistical evaluation demonstrated age-dependent methylation in normal tissue (R = 0.72, p < 2 × 10), association with adiposity (P = 0.019) and tumor-specific hypermethylation (P = 6.1 × 10) for RCC tissues. Comparison of tumor and metastatic tissues revealed higher methylation in renal cancer metastases (P = 2.65 × 10).

Conclusions: Our analyses provide statistical evidence of association between methylation of TBR1 and RCC development and disease progression.
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http://dx.doi.org/10.1186/s13148-020-0823-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029553PMC
February 2020

Simultaneous whole-body PET/MRI with integrated multiparametric MRI for primary staging of high-risk prostate cancer.

World J Urol 2020 Oct 6;38(10):2513-2521. Epub 2020 Jan 6.

Department of Urology, University Hospital Tuebingen, Hoppe-Seyler-Str. 3, 72076, Tübingen, Germany.

Purpose: Whole-body positron emission tomography/magnetic resonance imaging (wbPET/MRI) is a promising diagnostic tool of recurrent prostate cancer (PC), but its role in primary staging of high-risk PC (hrPC) is not well defined. Thus, the aim was to compare the diagnostic accuracy for T-staging of PET-blinded reading (PBR) and PET/MRI.

Methods: In this prospective study, hrPC patients scheduled to radical prostatectomy (RPx) with extended lymphadenectomy (eLND) were staged with wbPET/MRI and either Ga-PSMA-11 or C-choline including simultaneous multiparametric MRI (mpMRI). Images were assessed in two sessions, first as PBR (mpMRI and wbMRI) and second as wbPET/MRI. Prostate Imaging Reporting and Data System criteria (PIRADS v2) were used for T-staging. Results were correlated with the exact anatomical localization and extension as defined by histopathology. Diagnostic accuracy of cTNM stage according to PBR was compared to pathological pTNM stage as reference standard.

Results: Thirty-four patients underwent wbPET/MRI of Ga-PSMA-11 (n = 17) or C-choline (n = 17). Twenty-four patients meeting the inclusion criteria of localized disease ± nodal disease based on imaging results underwent RPx and eLND, whereas ten patients were excluded from analysis due to metastatic disease. T-stage was best defined by mpMRI with underestimation of tumor lesion size by PET for both tracers. N-stage yielded a per patient sensitivity/specificity comparable to PBR.

Conclusion: MpMRI is the primary modality for T-staging in hrPC as PET underestimated T-stage in direct comparison to final pathology. In this selected study, cohort MRI shows no inferiority compared to wbPET/MRI considering N-staging.
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http://dx.doi.org/10.1007/s00345-019-03066-1DOI Listing
October 2020

Intratumoral Heterogeneity Determines the Expression of mTOR-pathway Proteins in Prostate Cancer.

Dis Markers 2019 11;2019:1296865. Epub 2019 Dec 11.

Department of Urology, Eberhard Karls University of Tübingen, Tübingen, Germany.

The aim of this study was to evaluate the expression of mammalian target of rapamycin (mTOR), phosphorylated-mTOR (p-mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in prostate cancer (PCa) in order to assess intratumoral heterogeneity and correlation with clinicopathological parameters. Tissue samples from 115 patients undergoing radical prostatectomy were included in a tissue microarray comprising (A) tissue from the tumor center, (B) malignant border of the tumor, (C) tumor-adjacent benign tissue, and (D) tumor-distant benign prostatic tissue. Immune reactive scores 0-12 were correlated with clinical data in reference to localization. A meta-analysis of studies investigating the association between biochemical recurrence (BCR) and parameters of the mTOR pathway was conducted. Regardless of the location within the tumor, cancer tissue showed higher expression of mTOR, p-mTOR, and 4EB-P1 compared to benign tissue ( < 0.01). Significant differences in expression between tissue samples from groups C and D were observed for mTOR and p-mTOR. When considering expression according to the pathological stage, we observed lower p-mTOR expression in pT3 vs. pT2 (7.9 and 6.3; = 0.01). After a median follow-up of 74.5 months (IQR 65.0-84.0), 27 patients (23.47%) developed BCR. Weak staining of mTOR was associated with shorter time to BCR (HR: 2.0; = 0.049) after correcting for PSA and T stage. However, a significant association of mTOR expression with BCR was found for specimens from the malignant border of the tumor (B) but not the tumor center (A) ( = 0.0034 log rank). In a meta-analysis, we found that the expressions of mTOR ((RR) = 0.70; 95% CI 0.43-1.12; = 0.13) and 4E-BP1 ((RR) = 0.86; = 0.53) were not statistically associated with BCR, while strong staining of p-mTOR was associated with a lower risk of BCR ((RR) = 0.57; = 0.002). All 3 markers showed stronger expression in PCa and exhibited local gradients in relation to the border of tumor and healthy tissue. Our results suggest an important role of intratumor heterogeneity for the use of mTOR parameters as biomarkers in PCa.
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http://dx.doi.org/10.1155/2019/1296865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927025PMC
May 2020

DNA methylation of sarcosine dehydrogenase (SARDH) loci as a prognosticator for renal cell carcinoma.

Oncol Rep 2019 Nov 9;42(5):2159-2168. Epub 2019 Sep 9.

Department of Urology, Hannover Medical School (MHH), D‑30625 Hannover, Germany.

DNA methylation plays an important role in the genesis and progression of tumor diseases. To identify new DNA methylation markers possibly associated with the clinical characteristics of renal cell carcinoma (RCC), we investigated loci in the sarcosine dehydrogenase (SARDH) gene. SARDH is involved in the metabolism of the glycine‑derivative sarcosine and is closely linked through a functional control loop. Statistical evaluation of methylation data and clinical characteristics of patients showed that kidney tumors with clinically aggressive features such as a high tumor stage, positive lymph nodes, distant metastases or a previously advanced tumor status exhibited significantly lower methylation of a locus in the SARDH gene. Moreover, SARDH methylation was found to be a significant prognostic factor for recurrence‑free survival in RCC patients showing statistical independence from the clinical prognosticators, grade, stage and state of metastasis. In conclusion, the methylation status of the SARDH‑CGI was identified as an independent prognostic candidate marker for RCC.
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http://dx.doi.org/10.3892/or.2019.7305DOI Listing
November 2019

PD-1 and LAG-3 Dominate Checkpoint Receptor-Mediated T-cell Inhibition in Renal Cell Carcinoma.

Cancer Immunol Res 2019 Nov 4;7(11):1891-1899. Epub 2019 Sep 4.

Department of Immunology, University of Tübingen, Tübingen, Germany.

Drugs targeting the programmed cell death protein 1 (PD-1) pathway are approved as therapies for an increasing number of cancer entities, including renal cell carcinoma. Despite a significant increase in overall survival, most treated patients do not show durable clinical responses. A combination of checkpoint inhibitors could provide a promising improvement. The aim of the study was to determine the most promising checkpoint blockade combination for renal cell carcinoma patients. Tumor-infiltrating lymphocytes (TIL) and autologous peripheral blood mononuclear cells (PBMC) were isolated from patients undergoing surgery for primary tumors. Cells were stained for multicolor flow cytometry to determine the (co)expression of five inhibitory receptors (iR), PD-1, LAG-3, Tim-3, BTLA, and CTLA-4, on T-cell populations. The function of these TILs was assessed by intracellular cytokine staining after stimulation in the presence or absence of PD-1 ± LAG-3 or Tim-3-specific antibodies. Although the percentage of iR T cells was low in PBMCs, both CD4 and CD8 T cells showed increased frequencies of PD-1, LAG-3, and Tim-3 cells on TILs. The most frequent iR combination was PD-1 and LAG-3 on both CD4 and CD8 TILs. Blockade of PD-1 resulted in significant LAG-3, but not Tim-3, upregulation. The dual blockade of PD-1 and LAG-3, but not PD-1 and Tim-3, led to increased IFNγ release upon stimulation. Together, these data suggest that dual blockade of PD-1 and LAG-3 is a promising checkpoint blockade combination for renal cell carcinoma.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0146DOI Listing
November 2019

Assessment of concomitant non-oncologic medication in patients with surgically treated renal cell carcinoma: impact on prognosis, cell-cycle progression and proliferation.

J Cancer Res Clin Oncol 2019 Jul 22;145(7):1835-1843. Epub 2019 Apr 22.

Department of Urology, Eberhard Karls University, Hoppe-Seyler- Strasse 3, 72076, Tuebingen, Germany.

Introduction: Based on the observation of beneficial effects on cancer metabolism, microenvironment, or VEGF-signaling, several non-anticancer drugs have been discussed as useful in renal cell carcinoma (RCC). In the present study, we investigated the prognostic impact of concomitant medication in RCC and correlated comedication with cell-cycle and proliferation activity in corresponding surgical specimen.

Methods: A total of 388 patients who underwent surgery for localized RCC were included. The individual medication was evaluated according to substance classes. Tissue microarrays from corresponding tumor specimen were immunohistochemically (IHC) stained for Cyclin D1 and Ki67 and semi-quantitatively evaluated. Uni- and multivariate analyses were used to compare survival outcomes. For the comparison of IHC expression according to medication subgroups, Kruskal-Wallis analysis was performed.

Results: Median follow-up was 57.93 months (95% CI 53.27-69.43) and median OS accounted for 181.12 months (129.72-237.17). Univariate analysis identified pathological standard variables (T-stage > T2, Grading > G2, L1, N1, M1, sarcomatoid subtype, necrosis) as significant determinants of OS. Moreover, statin use (p = 0.009) and sartan use (p = 0.032) were significantly associated with improved OS. Multivariate analysis identified M1-stage (p < 0.001), statin and sartan use (p = 0.003 and p = 0.033, respectively) as independent prognosticators of survival. Expression of Ki67 was significantly reduced in patients with statin use (p = 0.013), while Cyclin D1 expression showed no correlation with comedication.

Conclusions: Concomitant intake of statins and sartans identifies as an independent predictor of OS in RCC, and reduced Ki67 expression was significantly associated with statin use. Further evaluation of drug repurposing approaches with these substances in RCC appear warranted.
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http://dx.doi.org/10.1007/s00432-019-02914-2DOI Listing
July 2019

Determination of Free-PSA (fPSA) and fPSA/PSA-Ratio Using A Point-of-Care Device.

Clin Lab 2019 Jan;65(1)

Background: Prostate specific antigen (PSA) and free PSA (fPSA) are important tools for diagnosing prostate cancer (PC). Efforts are continuously undertaken to provide more patient-centered healthcare. The application of point-of-care (POC) systems for laboratory analyses represents a step in this direction. Previous investigations on total PSA measurements using a POC system (concile® Ω100 POC reader) showed good concordance with standard laboratory measurements. For the same POC reader a novel system for fPSA was developed. In the current study, we prospectively evaluated the quality of the POC system for fPSA.

Methods: Sixty-four patients undergoing PSA measurements in our outpatient clinic between 06/2015 and 09/2015 were enrolled in the study. We measured total PSA (tPSA) and fPSA with a POC reader system (concile® Ω100) and a standard laboratory system (Siemens Immulite 2000®) and compared the respective results using linear regression analyses for PSA, fPSA, and fPSA/tPSA ratio (%fPSA).

Results: The coefficients of determination (r²) for fPSA and %fPSA were 0.85 (p < 0.001) and 0.82 (p < 0.001) in the subgroup with total PSA between 4 and 10 ng/mL. In the subgroup with tPSA ≤ 4 ng/mL, r² for fPSA concile® was 0.55 (p < 0.001) and 0.10 (p = 0.088) for %fPSA. In the subgroup of tPSA > 10 ng/mL the r² for fPSA and %fPSA was 0.50 (p = 0.022) and 0.50 (p = 0.022), respectively.

Conclusions: The POC fPSA values correlated well with the laboratory analyses, specifically in the clinically relevant diagnostic range of tPSA 4 - 10 ng/mL. These results complement the tPSA data obtained previously and indicate the reliability of the fPSA method and the resulting %fPSA score.
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http://dx.doi.org/10.7754/Clin.Lab.2018.180710DOI Listing
January 2019

DNA methylation of neural EGFL like 1 (NELL1) is associated with advanced disease and the metastatic state of renal cell cancer patients.

Oncol Rep 2018 Dec 24;40(6):3861-3868. Epub 2018 Sep 24.

Department of Urology, Hannover Medical School, D‑30625 Hannover, Germany.

Recent studies have shown that NELL1 expression is silenced epigenetically in human renal cell cancer (RCC) tissues and in RCC cell lines. However, it remains unknown whether NELL1 promoter methylation observed in clinical specimens might be associated with the clinicopathology or survival of patients with RCC. We analyzed NELL1 DNA methylation in tissues from patients with RCC and in adjacent normal renal tissues. In addition, we evaluated NELL1 methylation in cell lines derived from different urogenital tumors (prostate cancer, urothelial cancer and RCC). We performed regression analyses to determine whether NELL1 methylation is associated with clinicopathological parameters and recurrence‑free survival (RFS). This cross‑sectional study included 98 patients with RCC and 63 paired tumor and adjacent normal tissue samples. We analyzed a locus in the intron 1 region of NELL1 with pyrosequencing. We performed in silico analysis of NELL1 methylation in the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) data set (n=284 patients), which served as a validation study. Statistical analyses were performed with the two‑sided paired t‑test for paired tumor and adjacent normal samples. We used logistic regression for subgroup comparisons and Cox regression for RFS comparisons. The mean methylation level was 6.8% higher in RCC tissues compared to paired adjacent normal tissues (paired t‑test, P<0.001). Methylation levels in RCC were associated with advanced disease (P=0.002), the presence of distant metastases (P=0.004), and shorter RFS (P=0.035, HR: 4.15). In silico validation with TCGA KIRC data for adjacent loci also demonstrated that high relative methylation levels were associated with adverse clinicopathology and shortened RFS. Our results suggest that NELL1 methylation contributes to RCC disease progression. This finding could provide a clinical marker to complement recent functional analyses in tumor models.
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http://dx.doi.org/10.3892/or.2018.6732DOI Listing
December 2018

Selective Inhibition of the Lactate Transporter MCT4 Reduces Growth of Invasive Bladder Cancer.

Mol Cancer Ther 2018 12 27;17(12):2746-2755. Epub 2018 Sep 27.

Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada.

The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma remains mostly unknown. The aim of this study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of urothelial carcinoma and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with urothelial carcinoma. Silencing of MCT4 was performed using siRNAs in urothelial carcinoma cell lines. Effects of MCT4 inhibition on cell growth, apoptosis, and production of reactive oxygen species (ROS) were assessed. Moreover, effects on lactate efflux were determined. The effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation and increased RNA and protein expression were associated with worse overall survival (OS). Inhibition of MCT4 led to a reduction in cell growth, induction of apoptosis, and an increased synthesis of ROS. MCT4 inhibition resulted in intracellular accumulation of lactate. , stable knockdown of MCT4 reduced tumor growth. The expression of MCT4 in urothelial carcinoma is associated with features of aggressive tumor biology and portends a poor prognosis. Inhibition of MCT4 results in decreased tumor growth and Targeting lactate metabolism via MCT4 therefore provides a promising therapeutic approach for invasive urothelial carcinoma, especially in the basal subtype.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0107DOI Listing
December 2018

Calgranulin A (S100A8) Immunostaining: A Future Candidate for Risk Assessment in Patients with Non-Muscle-Invasive Bladder Cancer (NMIBC).

Adv Ther 2018 11 19;35(11):2054-2068. Epub 2018 Sep 19.

Department of Urology and Urologic Oncology, Hannover University Medical School, Hannover, Germany.

Introduction: There is an urgent need to identify patients with bladder cancer (BC) who are at high risk of recurrence or progression. Calgranulin A is a strong marker for muscle-invasive or advanced BC and recent studies have shown its potential for identifying patients at risk even in non-muscle-invasive bladder cancer (NMIBC). The present study examines risks of recurrence and progression dependent on immunostaining with calgranulin A in NMIBC.

Methods: Calgranulin A protein expression was evaluated through the immunohistochemistry of 158 randomly selected, transurethrally resected BC specimens of separate patients (pTa 89, pT1 69) using tissue microarrays. Kaplan-Meier survival analysis and Cox regression were performed to determine whether calgranulin A expression is associated with recurrence-free survival (RFS), progression-free survival (PFS), or cancer-specific survival (CSS).

Results: Calgranulin A expression is significantly different between pTa and pT1 tumors (p = 0.000, Mann-Whitney U test) and between tumor grades (p = 0.015, Kruskal-Wallis test). Kaplan-Meier estimates produced significant results for low and high calgranulin A expression concerning RFS [5y-RFS 70.4 ± 4.0% vs. 35.9 ± 12.5%, median RFS not reached (NR) vs. 12.0 ± 4.4 month, p = 0.029, log-rank test], PFS (5y-PFS 90.3 ± 2.7% vs. 51.5 ± 14.0%, median PFS NR in both groups, p = 0.000, log-rank test), and CSS (5y-CSS 92.9 ± 2.6% vs. 70.7 ± 12.4%, median CSS NR in both groups, p = 0.005, log-rank test). Calgranulin A remained an independent factor for RFS (p = 0.024, HR 2.43) and PFS (p = 0.002, HR 5.92) according to the multivariate Cox regression model.

Conclusions: Calgranulin A expression in NMIBC, detected through immunohistochemistry, is a promising marker for the identification of NMIBC patients at high risk of recurrence and progression.
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http://dx.doi.org/10.1007/s12325-018-0789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224005PMC
November 2018

Transketolase like 1 (TKTL1) expression alterations in prostate cancer tumorigenesis.

Urol Oncol 2018 10 16;36(10):472.e21-472.e27. Epub 2018 Aug 16.

Department of Urology, Eberhard Karls University Tübingen, 72076 Tübingen, Germany. Electronic address:

Background: Prostate cancer (CaP) is the most common nonepidermal cancer in elderly males. Due to its heterogeneity and high variability in regards to clinical outcome and therapeutic response, urologists' handling of this disease remains a challenge. The objective of this study was to assess Transketolase like 1 (TKTL1) expression in benign prostatic tissue, peritumoral tissue and in CaP (in different stages of disease), and its correlation with clinicopathological findings, in order to detect if TKTL1 expression is associated with CaP tumorigenesis.

Methods: In total, 100 tissue samples were included: (i) 22 benign specimens, (ii) 46 specimens with nonmetastatic CaP, and (iii) 32 specimens from patients with metastatic CaP. From the tissue microarray slides, we evaluated immunohistochemically the expression of the TKTL1 protein, using the H-score.

Results: The TKTL1 protein expression pattern ranges from a low level in benign prostatic tissue (100 [57.5-105]), moderately low in peritumoral tissue (135.42 [100-195.16]), moderate expression in nonmetastatic CaP (200 [172.19-254.38]) to high in metastatic CaP (300 [222.50-300]). A significant rise of TKTL1 mean expression was seen throughout disease progression. A significant difference was also found in TKTL1 expression between peritumoral tissue and benign tissue.

Conclusion: The results obtained in this study suggest that pentose phosphate pathway and its key enzyme TKTL1 is altered throughout the CaP tumorigenesis, and this pathway merits further investigation.
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http://dx.doi.org/10.1016/j.urolonc.2018.06.010DOI Listing
October 2018

Systemic Alterations of Wnt Inhibitors in Patients with Prostate Cancer and Bone Metastases.

Dis Markers 2018 18;2018:1874598. Epub 2018 Jul 18.

Department of Urology, Eberhard Karls University of Tübingen, Tübingen, Germany.

Purpose: Dickkopf-1 (DKK-1) and sclerostin seem to inhibit osteoblast activity by blocking the Wnt pathway, which leads to progression of metastatic prostate cancer (PC). However, it is unknown whether serum levels of these proteins are altered in PC patients with or without metastasis. The aim of this study was to assess DKK-1 and sclerostin serum levels in PC patients, including patients with bone metastases.

Methods: The study cohort ( = 143) consisted of 53 controls with benign prostatic hyperplasia (BPH), 43 with localized PC (PC cM0), and 47 had PC with metastasis (PC cM1). Serum levels of DKK-1 and sclerostin were measured by enzyme-linked immunosorbent assay. Results were compared using the Kruskal-Wallis tests; post hoc analysis was performed using the Tukey-Kramer test.

Results: Mean DKK-1 levels in patients with BPH (2809.4 pg/ml) ( < 0.001) as well as PC cM1 (2575.5 pg/ml) ( = 0.001) were significantly higher than in patients with PC cN0 cM0 (1551.8 pg/ml). Among PC cM1 patients, median DKK-1 levels were significantly lower in patients with castration-resistant disease compared to those with hormone-sensitive PC ( = 0.02); in contrast, sclerostin concentrations were elevated ( = 0.04). DKK-1 correlated with PSA in the cM1 group ( = 0.03) and sclerostin correlated with PSA in the PC group (0.01).

Conclusions: DKK-1 is involved in the progression of PC. DKK-1-mediated inhibition of osteoblasts, which contributes to tumor progression and osteolytic metastases, may also play a role in the development of metastases with osteoblastic features. The use of DKK-1 antibodies should be considered for studies including metastatic PC patients.
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http://dx.doi.org/10.1155/2018/1874598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079590PMC
December 2018

Extended periprostatic nerve distributions on the prostate surface confirmed using diffusion tensor imaging.

BJU Int 2019 06 29;123(6):995-1004. Epub 2019 Jan 29.

Department of Urology, Eberhard-Karls University Tuebingen, Tuebingen, Germany.

Objective: To perform a descriptive microscopic study of prostatectomy specimens from 19 patients which anatomically characterizes the distributions of periprostatic nerve qualities, and to visualize these using diffusion tensor imaging (DTI).

Materials And Methods: Serial whole-mounted sections were stained for cholinergic (neuronal nitric oxide synthase), adrenergic (tyrosine hydroxylase) and sensory (calcitonin gene-related peptide) nerves. Extracapsular stained nerves were counted by prostate surface sector, and classified by diameter. Stain-related relative density was calculated, and distribution patterns were evaluated. To better visualize the reported neuronal structures and independently confirm our findings, nerve concordance in five male volunteers was investigated using a 3-Tesla magnetic resonance imaging-DTI system.

Results: At the base, cholinergic nerves were distributed from the anterolateral to posterior sectors, continuing posterolaterally (mid-section) into the posterolateral-posterior sector toward the apex. Adrenergic nerves were distributed across the anterolateral-posterior sectors at the base, with the course narrowing to the posterolateral-posterior sectors at the mid- and apical levels. Sensory fibres were found posterolaterally posteriorly at the base, continuing posterolaterally over the mid- and apical levels. Although it was not possible to determine the different nerve qualities, DTI confirmed histological findings from the base to the apex.

Conclusions: Different types of nerve fibres were found to vary in distribution. When linked to possible functional aspects of the different nerve types, this morphological evidence may be of importance to further protect function after radical prostatectomy (RP). To our knowledge, this is the first time that DTI has confirmed reported histological findings in nerve-sparing RPs. DTI could be an important tool with which to correlate nerves to tumour for better preoperative planning and to incorporate imaging into treatment.
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http://dx.doi.org/10.1111/bju.14508DOI Listing
June 2019

Simultaneous Extraction of RNA and Metabolites from Single Kidney Tissue Specimens for Combined Transcriptomic and Metabolomic Profiling.

J Proteome Res 2018 09 23;17(9):3039-3049. Epub 2018 Aug 23.

Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology , Stuttgart , Germany and University of Tübingen, Tübingen, Germany.

Tissue analysis represents a powerful tool for the investigation of disease pathophysiology. However, the heterogeneous nature of tissue samples, in particular of neoplastic, may affect the outcome of such analysis and hence obscure interpretation of results. Thus, comprehensive isolation and extraction of transcripts and metabolites from an identical tissue specimen would minimize variations and enable the economic use of biopsy material which is usually available in limited amounts. Here we demonstrate a fast and simple protocol for combined transcriptomics and metabolomics analysis in homogenates prepared from one single tissue sample. Metabolites were recovered by protein precipitation from lysates originally prepared for RNA isolation and were analyzed by LC-QTOF-MS after HILIC and RPLC separation, respectively. Strikingly, although ion suppression was observed, over 80% of the 2885 detected metabolic features could be extracted and analyzed with high reproducibility (CV ≤ 20%). Moreover fold changes of different tumor and nontumor kidney tissues were correlated to an established metabolomics protocol and revealed a strong correlation ( r ≥ 0.75). In order to demonstrate the feasibility of the combined analysis of RNA and metabolites, the protocol was applied to kidney tissue of metformin treated mice to investigate drug induced alterations.
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http://dx.doi.org/10.1021/acs.jproteome.8b00199DOI Listing
September 2018

Characterization of the breast cancer resistance protein (BCRP/ABCG2) in clear cell renal cell carcinoma.

Int J Cancer 2018 12 25;143(12):3181-3193. Epub 2018 Sep 25.

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.

The efflux transporter breast cancer resistance protein BCRP/ABCG2 is well-known for its contribution to multi-drug resistance in cancer. Its relevance in cancer biology independent from drug efflux remains largely elusive. Our study aimed at elucidating the biological relevance and regulatory mechanisms of BCRP/ABCG2 in clear cell renal cell carcinoma (ccRCC) and disease progression. Two independent ccRCC-cohorts [Cohort 1 (KIRC/TCGA): n = 453, Cohort 2: n = 64] were investigated to elucidate BCRP/ABCG2 mRNA and protein expression and their association with survival. The impact of BCRP/ABCG2 on response to sunitinib treatment was investigated in two independent sunitinib-treated ccRCC-cohorts based on mRNA levels. Moreover, underlying regulatory mechanisms for interindividual variability of BCRP/ABCG2 expression were systematically assessed. Owing to redundant functional properties, mRNA and protein expression of the multidrug resistance protein MDR1/ABCB1 were additionally evaluated in these cohorts. In independent ccRCC-cohorts, low BCRP/ABCG2 and MDR1/ABCB1 mRNA and protein expression were associated with severity (e.g., tumor stage) of ccRCC and poor cancer-specific survival. BCRP/ABCG2 and MDR1/ABCB1 mRNA expression were linked to decreased progression-free survival after sunitinib treatment. Germline and somatic variants influenced interindividual variability of BCRP/ABCG2 expression only moderately. miR-212-3p and miR-132-3p were identified to regulate BCRP/ABCG2 posttranscriptionally by interaction with the ABCG2 3'UTR as confirmed through reporter gene assays in RCC cell lines. In summary, BCRP/ABCG2 expression in ccRCC underlies considerable interindividual variability with impact on patient survival and response to sunitinib treatment. While germline or somatic genetic variants and DNA methylation cannot explain aberrant BCRP/ABCG2 expression, miR-212-3p and miR-132-3p were identified to contribute to posttranscriptional regulation of BCRP/ABCG2.
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http://dx.doi.org/10.1002/ijc.31741DOI Listing
December 2018

Clinical utility of the S3-score for molecular prediction of outcome in non-metastatic and metastatic clear cell renal cell carcinoma.

BMC Med 2018 07 5;16(1):108. Epub 2018 Jul 5.

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, 70376, Stuttgart, Germany.

Background: Stratification of cancer patients to identify those with worse prognosis is increasingly important. Through in silico analyses, we recently developed a gene expression-based prognostic score (S3-score) for clear cell renal cell carcinoma (ccRCC), using the cell type-specific expression of 97 genes within the human nephron. Herein, we verified the score using whole-transcriptome data of independent cohorts and extend its application for patients with metastatic disease receiving tyrosine kinase inhibitor treatment. Finally, we sought to improve the signature for clinical application using qRT-PCR.

Methods: A 97 gene-based S3-score (S3) was evaluated in a set of 52 primary non-metastatic and metastatic ccRCC patients as well as in 53 primary metastatic tumors of sunitinib-treated patients. Gene expression data of The Cancer Genome Atlas (n = 463) was used for platform transfer and development of a simplified qRT-PCR-based 15-gene S3-score (S3). This S3-score was validated in 108 metastatic and non-metastatic ccRCC patients and ccRCC-derived metastases including in part several regions from one metastasis. Univariate and multivariate Cox regression stratified by T, N, M, and G were performed with cancer-specific and progression-free survival as primary endpoints.

Results: The S3-score was significantly associated with cancer-specific survival (CSS) in 52 ccRCC patients (HR 2.9, 95% Cl 1.0-8.0, P = 3.3 × 10) as well as progression-free survival in sunitinib-treated patients (2.1, 1.1-4.2, P = 2.2 × 10). The qRT-PCR based S3-score performed similarly to the S3-score, and was significantly associated with CSS in our extended cohort of 108 patients (5.0, 2.1-11.7, P = 5.1 × 10) including metastatic (9.3, 1.8-50.0, P = 2.3 × 10) and non-metastatic patients (4.4, 1.2-16.3, P = 1.6 × 10), even in multivariate Cox regression, including clinicopathological parameters (7.3, 2.5-21.5, P = 3.3 × 10). Matched primary tumors and metastases revealed similar S3-scores, thus allowing prediction of outcome from metastatic tissue. The molecular-based qRT-PCR S3-score significantly improved prediction of CSS by the established clinicopathological-based SSIGN score (P = 1.6 × 10).

Conclusion: The S3-score offers a new clinical avenue for ccRCC risk stratification in the non-metastatic, metastatic, and sunitinib-treated setting.
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http://dx.doi.org/10.1186/s12916-018-1088-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033218PMC
July 2018

Transurethral Resection of Bladder Tumors: Next-generation Virtual Reality Training for Surgeons.

Eur Urol Focus 2019 Sep 22;5(5):906-911. Epub 2018 May 22.

University Tuebingen, Dept. of Urology, Tuebingen, Germany. Electronic address:

Background: The number of virtual reality (VR) simulators is increasing. The aim of this prospective trial was to determine the benefit of VR cystoscopy (UC) and transurethral bladder tumor resection (TURBT) training in students.

Design, Setting, And Participants: Medical students without endoscopic experience (n=51, median age=25 yr, median 4th academic year) were prospectively randomized into groups A and B. After an initial VR-UC and VR-TURBT task, group A (n=25) underwent a video-based tutorial by a skilled expert. Group B (n=26) was trained using a VR training program (Uro-Trainer). Following the training, every participant performed a final VR-UC and VR-TURBT task. Performance indicators were recorded via the simulator. Data was analyzed by Mann-Whitney U test.

Intervention: VR cystoscopy and TURBT.

Results And Limitations: No baseline and post-training differences were found for VR-UC between groups. During baseline, VR-TURBT group A showed higher inspected bladder surface than group B (56% vs 73%, p=0.03). Subgroup analysis detected differences related to sex before training (male: 31.2% decreased procedure time; 38.1% decreased resectoscope movement; p=0.02). After training, significant differences in procedure time (3.9min vs 2.7min, p=0.007), resectoscope movement (857mm vs 529mm, p=0.005), and accidental bladder injury (n=3.0 vs n=0.88, p=0.003) were found. Male participants showed reduced blood loss (males: 3.92ml vs females: 10.12ml; p=0.03) after training.

Conclusions: Measuring endoscopic skills within a virtual environment can be done easily. Short training improved efficacy and safety of VR-TURBT. Nevertheless, transfer of improved VR performance into real world surgery needs further clarification.

Patient Summary: We investigated how students without endoscopic experience profit from simulation-based training. The safe environment and repeated simulations can improve the surgical training. It may be possible to enhance patient's safety and the training of surgeons in long term.
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http://dx.doi.org/10.1016/j.euf.2018.04.011DOI Listing
September 2019
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