Nijmegen, Netherlands
Nijmegen, Netherlands

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Leyten G.H.J.M.,Radboud University Nijmegen | Hessels D.,Noviogendix | Jannink S.A.,Noviogendix | Smit F.P.,Noviogendix | And 10 more authors.
European Urology | Year: 2014

Background Prostate cancer antigen 3 (PCA3) and v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2-ERG) gene fusions are promising prostate cancer (PCa) specific biomarkers that can be measured in urine. Objective To evaluate the diagnostic and prognostic value of Progensa PCA3 and TMPRSS2-ERG gene fusions (as individual biomarkers and as a panel) for PCa in a prospective multicentre setting. Design, setting, and participants At six centres, post-digital rectal examination first-catch urine specimens prior to prostate biopsies were prospectively collected from 497 men. We assessed the predictive value of Progensa PCA3 and TMPRSS2-ERG (quantitative nucleic acid amplification assay to detect TMPRSS2-ERG messenger RNA [mRNA]) for PCa, Gleason score, clinical tumour stage, and PCa significance (individually and as a marker panel). This was compared with serum prostate-specific antigen and the European Randomised Study of Screening for Prostate Cancer (ERSPC) risk calculator. In a subgroup (n = 61) we evaluated biomarker association with prostatectomy outcome. Outcome measurements and statistical analysis Univariate and multivariate logistic regression analysis and receiver operating curves were used. Results and limitations Urine samples of 443 men contained sufficient mRNA for marker analysis. PCa was diagnosed in 196 of 443 men. Both PCA3 and TMPRSS2-ERG had significant additional predictive value to the ERSPC risk calculator parameters in multivariate analysis (p < 0.001 and resp. p = 0.002). The area under the curve (AUC) increased from 0.799 (ERSPC risk calculator), to 0.833 (ERSPC risk calculator plus PCA3), to 0.842 (ERSPC risk calculator plus PCA3 plus TMPRSS2-ERG) to predict PCa. Sensitivity of PCA3 increased from 68% to 76% when combined with TMPRSS2-ERG. TMPRSS2-ERG added significant predictive value to the ERSPC risk calculator to predict biopsy Gleason score (p < 0.001) and clinical tumour stage (p = 0.023), whereas PCA3 did not. Conclusions TMPRSS2-ERG had independent additional predictive value to PCA3 and the ERSPC risk calculator parameters for predicting PCa. TMPRSS2-ERG had prognostic value, whereas PCA3 did not. Implementing the novel urinary biomarker panel PCA3 and TMPRSS2-ERG into clinical practice would lead to a considerable reduction of the number of prostate biopsies. © 2012 European Association of Urology.


Leyten G.H.J.M.,Radboud University Nijmegen | Hessels D.,Noviogendix | Smit F.P.,Noviogendix | Jannink S.A.,Noviogendix | And 11 more authors.
Clinical Cancer Research | Year: 2015

Purpose: Serum PSA (sPSA) testing has led to the identification of patients with indolent prostate cancer, and inevitably over-treatment has become a concern. Progensa PCA3 urine testing was shown to improve the diagnosis of prostate cancer, but its diagnostic value for aggressive prostate cancer is limited. Therefore, urinary biomarkers that can be used for prediction of Gleason score > 7 prostate cancer in biopsies are urgently needed. Experimental Design: Using gene expression profiling data, 39 prostate cancer biomarkers were identified. After quantitative PCR analysis on tissue specimens and urinary sediments, eight promising biomarkers for the urinary detection of prostate cancer were selected (ONECUT2, HOXC4, HOXC6, DLX1, TDRD1, NKAIN1, MS4A8B, PPFIA2). The hypothesis that biomarker combinations improve the diagnostic value for aggressive prostate cancer was tested on 358 urinary sediments of an intention-to-treat cohort. Results: A urinary three-gene panel (HOXC6, TDRD1, and DLX1) had higher accuracy [area under the curve (AUC), 0.77; 95% confidence interval (CI), 0.71-0.83] to predict Gleason score >7 prostate cancer in biopsies compared with Progensa PCA3 (AUC, 0.68; 95% CI, 0.62-0.75) or sPSA (AUC, 0.72; 95% CI, 0.65-0.78). Combining the three-gene panel with sPSA further improved the predictive accuracy (AUC, 0.81; 95% CI, 0.75-0.86). The accuracy of the three-gene predictive model was maintained in subgroups with low sPSA concentrations. Conclusions: The urinary three-gene panel (HOXC6, TDRD1, and DLX1 ) represents a promising tool to identify patients with aggressive prostate cancer, also in those with low sPSA values. The combination of the urinary three-gene panel with sPSA bears great potential for the early diagnosis of patients with clinically significant prostate cancer. © 2014 American Association for Cancer Research.


PubMed | University of Amsterdam, Noviogendix, St Elisabeth Hospital, CWZ Hospital and 3 more.
Type: Journal Article | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2015

Serum PSA (sPSA) testing has led to the identification of patients with indolent prostate cancer, and inevitably overtreatment has become a concern. Progensa PCA3 urine testing was shown to improve the diagnosis of prostate cancer, but its diagnostic value for aggressive prostate cancer is limited. Therefore, urinary biomarkers that can be used for prediction of Gleason score 7 prostate cancer in biopsies are urgently needed.Using gene expression profiling data, 39 prostate cancer biomarkers were identified. After quantitative PCR analysis on tissue specimens and urinary sediments, eight promising biomarkers for the urinary detection of prostate cancer were selected (ONECUT2, HOXC4, HOXC6, DLX1, TDRD1, NKAIN1, MS4A8B, PPFIA2). The hypothesis that biomarker combinations improve the diagnostic value for aggressive prostate cancer was tested on 358 urinary sediments of an intention-to-treat cohort.A urinary three-gene panel (HOXC6, TDRD1, and DLX1) had higher accuracy [area under the curve (AUC), 0.77; 95% confidence interval (CI), 0.71-0.83] to predict Gleason score 7 prostate cancer in biopsies compared with Progensa PCA3 (AUC, 0.68; 95% CI, 0.62-0.75) or sPSA (AUC, 0.72; 95% CI, 0.65-0.78). Combining the three-gene panel with sPSA further improved the predictive accuracy (AUC, 0.81; 95% CI, 0.75-0.86). The accuracy of the three-gene predictive model was maintained in subgroups with low sPSA concentrations.The urinary three-gene panel (HOXC6, TDRD1, and DLX1) represents a promising tool to identify patients with aggressive prostate cancer, also in those with low sPSA values. The combination of the urinary three-gene panel with sPSA bears great potential for the early diagnosis of patients with clinically significant prostate cancer.


Hendriks R.J.,Radboud University Nijmegen | Dijkstra S.,Radboud University Nijmegen | Jannink S.A.,NovioGendix | Steffens M.G.,Isala Hospital | And 3 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2016

Background: PCA3 and ERG are mRNA-based prostate cancer (PCa) specific biomarkers that can be detected in urine. However, urine is a complex substrate that can be separated in several fractions. In this study we compared the levels of PCa-specific biomarkers (PCA3 and ERG) and KLK3 as prostate-specific reference gene in three urine substrates-whole urine, urinary sediment (cell pellet) and exosomes-and evaluated the influence of performing a digital rectal examination (DRE) prior to urine sampling. Methods: First-voided urine samples were prospectively obtained before and after DRE from 29 men undergoing prostate biopsies. The urine was separated in whole urine, cell pellet and exosomes and the biomarker levels were measured with RT-qPCR. Results: PCa was identified in 52% (15/29) of men. In several samples the mRNA levels were below the analytical limit of detection (BDL). The biomarker levels were highest in whole urine and significantly higher after DRE in all substrates. In PCa patients higher levels of PCA3 and ERG were found in all urine substrates after DRE compared to non-PCa patients. Conclusions: This is the first study in which urinary PCa-specific biomarker levels were compared directly in three separate urine fractions. These results suggest that whole urine could be the urine substrate of choice for PCa-diagnostics based on analytical sensitivity, which is reflected directly in the high informative rate. Moreover, the significant positive effect of performing a DRE prior to urine sampling is confirmed. These findings could be of influence in the development of PCa-diagnostic urine tests. © 2016 by De Gruyter 2016.


Pfeiffer M.J.,Radboud University Nijmegen | Smit F.P.,NovioGendix | Sedelaar J.P.M.,Radboud University Nijmegen | Schalken J.A.,Radboud University Nijmegen
Molecular Medicine | Year: 2011

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth. © 2011 The Feinstein Institute for Medical Research.


Salagierski M.,Radboud University Nijmegen | Salagierski M.,Medical University of Lódz | Verhaegh G.W.,Radboud University Nijmegen | Verhaegh G.W.,Nijmegen Center for Molecular Life science | And 5 more authors.
Prostate | Year: 2010

BACKGROUND. PCA3 is one of the most prostate cancer (PrCa)-specific markers described so far. Recently, a new genomic structure of PCA3 as well as new flanking and overlapping gene transcripts has been identified. Furthermore, a co-regulation of PCA3 and its overlapping gene PRUNE2(BMCC1) has been suggested. Our aim was to assess the diagnostic performance of a new PCA3 isoform (PCA3-TS4) and to study the interactions between PCA3 and BMCC1in PrCa. METHODS. Weused SYBR Green quantitative (q)PCR with specific primers to compare PCA3 and BMCC1 expression of normal versus tumor tissue of human prostate. PCA3-TS4 plasmid was created to calculate the absolute amounts of PCA3 transcripts. The androgen regulation of PCA3 and BMCC1 expression was studied in LNCaP and 22Rv1 cells stimulated with 5α-dihydrotestosterone. RESULTS. We have not found any relevant diagnostic advantage of the PCA3-TS4 isoform over the "classical" PCA3 isoform in our group of PrCa patients. Additionally, PCA3-TS4 appears to be only a minor PCA3 transcript. We were also unable to confirm the hypothesis that BMCC1 isoforms are androgen-induced in vitro. CONCLUSIONS. Despite the presence of the recently identified marginal PCA3 transcripts in human PrCa, the previously described major PCA3 isoform still constitutes the best target for diagnostic purposes. PCA3 and BMCC1 are overlapping genes in reverse orientation that do not appear to be co-regulated. © 2009 Wiley-Liss, Inc.


Dijkstra S.,Radboud University Nijmegen | Birker I.L.,University of Amsterdam | Smit F.P.,NovioGendix | Leyten G.H.J.M.,Radboud University Nijmegen | And 5 more authors.
Journal of Urology | Year: 2014

Purpose Urinary biomarker tests for diagnosing prostate cancer have gained considerable interest. Urine is a complex mixture that can be subfractionated. We evaluated 2 urinary fractions that contain nucleic acids, ie cell pellets and exosomes. The influence of digital rectal examination before urine collection was also studied and the prostate cancer specific biomarkers PCA3 and TMPRSS2-ERG were assayed. Materials and Methods Urine samples were prospectively obtained before and after digital rectal examination from 30 men scheduled for prostate biopsy. Cell pellet and exosomes were isolated and used for biomarker analysis. Analytical and diagnostic performance was tested using the Student t-test and ROC curves. Results Unlike the exosome fraction, urinary sediment gene expression analysis was compromised by amorphous precipitation in 10% of all specimens. Digital rectal examination resulted in increased mRNA levels in each fraction. This was particularly relevant for the exosomal fraction since after digital rectal examination the number of samples decreased in which cancer specific markers were below the analytical detection limit. Biomarker diagnostic performance was comparable to that in large clinical studies. In exosomes the biomarkers had to be normalized for prostate specific antigen mRNA while cell pellet absolute PCA3 levels had diagnostic value. Conclusions Exosomes have characteristics that enable them to serve as a stable substrate for biomarker analysis. Thus, digital rectal examination enhances the analytical performance of biomarker analysis in exosomes and cell pellets. The diagnostic performance of biomarkers in exosomes differs from that of cell pellets. Clinical usefulness must be prospectively assessed in larger clinical cohorts. © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.


Dijkstra S.,Radboud University Nijmegen | Leyten G.H.J.M.,Radboud University Nijmegen | Jannink S.A.,NovioGendix | De Jong H.,NovioGendix | And 3 more authors.
Prostate | Year: 2014

BACKGROUND. To monitor systemic disease activity, the potential of circulating tumor cells (CTCs) bears great promise. As surrogate for CTCs we measured KLK3, PCA3, and TMPRSS2-ERG messenger RNA (mRNA) in the peripheral blood mononuclear cell (PBMC) fraction from a castration-resistant prostate cancer (CRPC) patient cohort and three control groups. Moreover, biomarker response to docetaxel treatment was evaluated in the patient group. METHODS. Blood samples from 20 CRPC patients were analyzed at four different time points (prior to docetaxel treatment, at 9 weeks, 27 weeks, and 2 months after treatment). Blood was drawn once from three control groups (10 age-matched men, 10 men under 35 years of age, 12 women). All samples were analyzed for KLK3, PCA3, and TMPRSS2-ERG mRNA by using a quantitative nucleic acid amplification assay with gene-specific primers in the complementary DNA synthesis. RESULTS. At baseline, mRNA for KLK3 was detected in 17 (89%, 95% CI 76-100%), PCA3 in 10 (53%, 95% CI 30-75%), and TMPRSS2-ERG in seven of 19 evaluable patients (37%, 95% CI 15-59%). In contrast, the blood samples from all 32 healthy volunteers were reproducible negative for all markers. In response to docetaxel treatment, KLK3 levels decreased in 80% (95% CI 60-100%), PCA3 in 89% (95% CI 68-100%), and TMPRSS2-ERG in 86% (95% CI 60-100%) of patients. CONCLUSIONS. The feasibility of a highly sensitive modified nucleic acid amplification assay to assess KLK3, PCA3, and TMPRSS2-ERG mRNA in the PBMC fraction from CRPC patients was demonstrated. Moreover, response of these markers to systemic treatment was shown. © 2014 Wiley Periodicals, Inc.


Robert G.,Radboud University Nijmegen | Robert G.,Bordeaux Pellegrin University Hospital | Jannink S.,NovioGendix | Smit F.,NovioGendix | And 5 more authors.
Prostate | Year: 2013

BACKGROUND The prostate cancer gene 3 (PCA3) and TMPRSS2:ERG gene fusion are promising prostate cancer (PCa) specific biomarkers. Our aim was to simultaneously quantify the expression levels of PCA3 and TMPRSS2:ERG in a panel of benign prostatic hyperplasia (BPH), normal prostate adjacent to PCa (NP) and PCa tissue samples, to provide a rational basis for the understanding of the false-positive and false-negative results of the urine assays. METHODS The tissue samples were carefully histopathologically characterized to obtain homogeneous groups. The mRNA was isolated, transcribed into cDNA and the relative expressions of PCA3 and TMPRSS2:ERG were measured using a quantitative real-time polymerase chain reaction. The expression levels of PCA3 and TMPRSS2:ERG were compared between the different groups. RESULTS We included 48 BPH, 32 NP, and 48 PCa. The PCA3 expression levels progressively increased from BPH to NP (3 times) and finally to PCa (30 times). There were one false-positive sample and seven false-negative samples. The TMPRSS2:ERG gene fusion was found in 8.3% of the BPH, 15.6% of the NP, and 50% of the PCa samples. The use of TMPRSS2:ERG in the PCA3 negative cases allowed diagnosis of four of the seven false-negative samples and added one false-positive, but we had to define a cut-off value to avoid eight false-positive results. CONCLUSIONS Considering tissue expression of the markers, most of the false-negative results of the PCA3 test were corrected by TMPRSS2:ERG (57%) and the combination of both had a higher sensitivity for PCa diagnosis. Some of the control samples did express TMPRSS2:ERG and a cut-off value had to be defined to avoid false-positive results. Prostate 73: 113-120, 2013. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.


PubMed | NovioGendix, Radboud University Nijmegen and University of Indonesia
Type: Journal Article | Journal: The Journal of urology | Year: 2015

Dihydrotestosterone is the main active androgen in the prostate and it has a role in prostate cancer progression. After androgen deprivation therapy androgen receptor signaling is still active in tumor cells. Persistent intratumor steroidogenesis and androgen receptor changes are responsible for this continued activity, which influences the efficacy of prostate cancer treatment. We hypothesized that combining a 5-reductase inhibitor and an antiandrogen would block intratumor androgen synthesis and androgen receptor protein activity. Thus, it would act synergistically to reduce tumor cell proliferation.The expression level of 5-reductase and androgen receptor in endocrine therapy nave prostate cancer and castration resistant prostate cancer tissues, and cell line models was determined by microarray and quantitative polymerase chain reaction analysis. Intracellular androgen was measured with radioimmunoassay. Tumor cell proliferation was determined using coloric MTT assay. The synergistic effects of combination treatments on tumor cell proliferation were calculated using the Chou-Talalay equation.In all prostate cancer cases 5-reductase-1 and 3 were up-regulated. Androgen receptor was up-regulated in metastatic prostate cancer and castration resistant prostate cancer cases. The 5-reductase inhibitor dutasteride effectively decreased dihydrotestosterone production in prostate cancer and castration resistant prostate cancer cell lines. Furthermore, dutasteride combined with the novel antiandrogen enzalutamide synergistically suppressed endocrine therapy nave prostate cancer and castration resistant prostate cancer cell proliferation.In this study the combination of a 5-reductase inhibitor and (novel) antiandrogens synergistically inhibited tumor cell proliferation. These findings support clinical studies of combinations of a 5-reductase inhibitor and (novel) antiandrogens as first line treatment of prostate cancer and castration resistant prostate cancer.

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