Fatty acid binding protein 7 may be a marker and therapeutic targets in clear cell renal cell carcinoma.

BACKGROUND: To identify potential therapeutic target in clear cell renal cell carcinoma (ccRCC), we performed a transcriptome analysis. Our analysis showed that fatty acid binding protein 7 (FABP7) has the highest mean differential overexpression in ccRCC compared to normal kidney. We aimed to investigate the significance of FABP7 in ccRCC. METHODS: Immunohistochemical staining for 40 advanced ccRCC cases was performed to investigate correlation between clinicopathological parameters and FABP7. They were composed of 40-83 years old cases with 33 male, 22 cases with pT ≥ 3, 19 cases with M1, and 16 cases with grade 3. The effect of gene knockdown was analysed by a cell viability assay and invasion assay in FABP7-overexpressing cell lines (SKRC7 and SKRC10). RESULTS: Our immunohistochemical analysis showed that higher FABP7 expression significantly correlated with distant metastasis and poor cancer-specific survival (CSS; both p < 0.05). Functional suppression of FABP7 significantly inhibited SKRC10 cell growth (p < 0.05) and resulted in a significant reduction of the invasive potential (p < 0.01), but did not cause growth inhibition of SKRC7 cells. We found that The Cancer Genome Atlas Research Network (TCGA) database shows FABP6 and 7 as equally overexpressed in the FABP family. Functional suppression of fatty acid binding protein 6 (FABP6) resulted in significant growth inhibition of SKRC7 cells (p < 0.005). CONCLUSIONS: Functional suppression of FABP7 significantly reduced cell viability and invasive potential in a ccRCC cell line. FABP7 may play a role in progression in some metastatic ccRCCs. The suppressed function may be compensated by another FABP family member.

Detection of High-grade Prostate Cancer Using a Urinary Molecular Biomarker-Based Risk Score.

BACKGROUND: To reduce overdiagnosis and overtreatment, a test is urgently needed to detect clinically significant prostate cancer (PCa). OBJECTIVE: To develop a multimodal model, incorporating previously identified messenger RNA (mRNA) biomarkers and traditional risk factors that could be used to identify patients with high-grade PCa (Gleason score ≥7) on prostate biopsy. DESIGN, SETTING, AND PARTICIPANTS: In two prospective multicenter studies, urine was collected for mRNA profiling after digital rectal examination (DRE) and prior to prostate biopsy. The multimodal risk score was developed on a first cohort (n=519) and subsequently validated clinically in an independent cohort (n=386). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The mRNA levels were measured using reverse transcription quantitative polymerase chain reaction. Logistic regression was used to model patient risk and combine risk factors. Models were compared using the area under the curve (AUC) of the receiver operating characteristic, and clinical utility was evaluated with a decision curve analysis (DCA). RESULTS AND LIMITATIONS: HOXC6 and DLX1 mRNA levels were shown to be good predictors for the detection of high-grade PCa. The multimodal approach reached an overall AUC of 0.90 (95% confidence interval [CI], 0.85-0.95) in the validation cohort (AUC 0.86 in the training cohort), with the mRNA signature, prostate-specific antigen (PSA) density, and previous cancer-negative prostate biopsies as the strongest, most significant components, in addition to nonsignificant model contributions of PSA, age, and family history. For another model, which included DRE as an additional risk factor, an AUC of 0.86 (95% CI, 0.80-0.92) was obtained (AUC 0.90 in the training cohort). Both models were successfully validated, with no significant change in AUC in the validation cohort, and DCA indicated a strong net benefit and the best reduction in unnecessary biopsies compared with other clinical decision-making tools, such as the Prostate Cancer Prevention Trial risk calculator and the PCA3 assay. CONCLUSIONS: The risk score based on the mRNA liquid biopsy assay combined with traditional clinical risk factors identified men at risk of harboring high-grade PCa and resulted in a better patient risk stratification compared with current methods in clinical practice. Therefore, the risk score could reduce the number of unnecessary prostate biopsies. PATIENT SUMMARY: This study evaluated a novel urine-based assay that could be used as a noninvasive diagnostic aid for high-grade prostate cancer (PCa). When results of this assay are combined with traditional clinical risk factors, risk stratification for high-grade PCa and biopsy decision making are improved.

Comparative analysis of prostate cancer specific biomarkers PCA3 and ERG in whole urine, urinary sediments and exosomes.

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.

The role of HOXC6 in prostate cancer development.

BACKGROUND: Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored. METHODS: We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes. RESULTS: HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients. CONCLUSIONS: HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy.

Identification of a Candidate Gene Panel for the Early Diagnosis of Prostate Cancer.

PURPOSE: 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. 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.

KLK3, PCA3, and TMPRSS2-ERG expression in the peripheral blood mononuclear cell fraction from castration-resistant prostate cancer patients and response to docetaxel treatment.

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.

Prostate cancer biomarker profiles in urinary sediments and exosomes.

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.

Prospective multicentre evaluation of PCA3 and TMPRSS2-ERG gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer.

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.

High-resolution ERG-expression profiling on GeneChip exon 1.0 ST arrays in primary and castration-resistant prostate cancer.

OBJECTIVE: To assess whether oestrogen-regulated gene (ERG) expression analysis using GeneChip arrays can predict transmembrane protease, serine 2 (TMPRSS2)-ERG fusion. The expression level of the TMPRSS2-ERG gene was studied in various histological grades of prostate cancer and castration-resistant prostate cancer (CPRC). PATIENTS AND METHODS: GeneChip Affymetrix exon 1.0 ST arrays were used for expression profiling of ERG, erythroblast transformation-specific (ETS) variant gene 1 (ETV1), ETV4 and ETV5 genes in 67 prostate cancer tissue specimens. Real-time quantitative polymerase chain reaction analysis and in some cases DNA sequencing was used to validate the presence and the expression levels of TMPRSS2-ERG gene fusions. RESULTS: In our series of patients with prostate cancer over expression of the ERG gene predicted the presence of TMPRSS2-ERG rearrangements in almost all cases. ETS expression by itself outmatched the diagnostic performance of the ERG exons ratioing allowing equal detection of the less frequent ETS gene fusion transcripts. The gene fusions were expressed at significantly lower levels in CPRC but occurred more frequently than in primary prostate cancer. CONCLUSIONS: ERG expression analysis using GeneChip arrays appears to be an excellent diagnostic tool for identifying gene rearrangements. In coming years, measuring expression of the ETS gene family by itself might become a clinically relevant surrogate test to identify patients with fusion-positive prostate cancer. The variation of gene fusion expression levels, particularly in CPRC, needs to be taken into account when using quantitative molecular diagnosis of prostate cancer.

Rational basis for the combination of PCA3 and TMPRSS2:ERG gene fusion for prostate cancer diagnosis.

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.

Biomarkers for the diagnosis of prostatic inflammation in benign prostatic hyperplasia.

BACKGROUND: Chronic prostatic inflammation could be a central mechanism in benign prostatic hyperplasia (BPH) progression. Currently, the histological examination of prostate biopsies remains the only way to diagnose prostatic inflammation. Our objective was to find new noninvasive biomarkers for the diagnosis of prostatic inflammation. METHODS: Ninety BPH samples were investigated in two steps. First, a hypothesis was generated using a profiling procedure with a panel of 96 genes on an initial set of 30 samples. Then, the candidate biomarkers were validated on a large number of samples (n = 90). Gene expression was compared with the histological prostatic inflammation score based on the density and the confluence of lymphoid nodules. Finally, protein transcripts of the candidate biomarkers were investigated in urine samples and compared with clinical data. RESULTS: Of the 96 genes, nine were significantly correlated with the inflammation score on the initial set of patients. Four of them were validated on the complete set of patients: CCR7, CD40LG, CTLA4, and ICOS. ICOS and CTLA4 protein levels were readily measured in urine samples using a conventional ELISA procedure. High-ICOS expression in urine was associated with a higher post-void residual and a lower maximum urinary flow rate. CONCLUSIONS: Four genes were significantly upregulated at the mRNA level in the prostate tissue of patients with severe inflammation score. Two proteins were measured in urine samples, and were associated with maximum uroflowmetry and post-void residual. A prospective clinical study is needed to confirm their clinical relevance.

Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in Dutch prostate cancer patients.

BACKGROUND: The occurrence of the retrovirus xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been reported in prostate tissue of patients with prostate cancer (PrCa). Considering the potential great medical and social relevance of this discovery, we investigated whether this finding could be confirmed in an independent group of Dutch sporadic PrCa cases. METHODS: We investigated the occurrence of XMRV in fresh-frozen PrCa specimens of 74 PrCa patients from The Netherlands. Total RNA and DNA were isolated, subjected to cDNA synthesis, and analyzed by real-time PCR targeting conserved XMRV sequences. RESULTS: Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences in 100,000 cells by real-time PCR, demonstrating high sensitivity of the assay. XMRV sequences were detected in 3 out of 74 (i.e., 4%) PrCa specimens. The number of XMRV containing cells was extremely low (1 in 600-7,000 cells). This was corroborated by the fact that XMRV could not be detected in consecutive tissue sections of the initial XMRV-positive cases. CONCLUSIONS: XMRV was rarely detected, and at extremely low levels, in sporadic PrCa samples from Dutch patients. When XMRV would play a causal role in prostate carcinogenesis, integration of the provirus could be expected to be present in, at least, a proportion of tumor cells. Therefore, our data do not support the claim that there is an association between XMRV infection and PrCa in Dutch PrCa patients.

Predictive value of PCA3 in urinary sediments in determining clinico-pathological characteristics of prostate cancer.

PURPOSE: PCA3 urine tests have shown to improve the specificity in prostate cancer (PCa) diagnosis, and have thus the potential to reduce the number of unnecessary prostate biopsies and to predict repeat biopsy outcomes. In this study, PCA3 was correlated with clinical stage, biopsy Gleason score (GS), radical prostatectomy GS, tumor volume, and pathological stage to assess its potential as predictor of PCa aggressiveness. METHODS: In this study, 351 men admitted for prostate biopsies based on serum PSA levels >3 ng/ml, an abnormal DRE, and/or a family history of PCa were included. Post-DRE urinary sediments from 336 men were tested using a transcription-mediated amplification-based PCA3 test, and assay results were correlated with clinical stage and biopsy GS. In a sub-cohort of 70 men who underwent radical prostatectomy, the PCA3 values were correlated to their radical prostatectomy GS, tumor volume, and pathological stage. RESULTS: In this patient cohort we could not find a correlation between clinical stage, biopsy GS, radical prostatectomy GS, tumor volume, and pathological stage. CONCLUSIONS: The predictive value of PCA3 for PCa aggressiveness features as reported in earlier studies cannot be confirmed in our study. Experimental differences (urine sediments vs. whole urine) and cohort may explain this. The exact place of PCA3 as prognostic test for PCa remains the subject of investigation.

Differential expression of PCA3 and its overlapping PRUNE2 transcript in prostate cancer.

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 BMCC1 in PrCa. METHODS: We used 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 5alpha-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.

The time-resolved fluorescence-based PCA3 test on urinary sediments after digital rectal examination; a Dutch multicenter validation of the diagnostic performance.

PURPOSE: To improve the specificity in prostate cancer diagnosis and to prevent unnecessary prostate biopsies, especially in the serum prostate-specific antigen (PSA) "gray zone" between 3 and 15 ng/mL, the implementation of prostate cancer-specific markers is urgently needed. The recently discovered prostate cancer antigen 3 (PCA3) is such a promising prostate cancer marker. In a previous single institution study, the PCA3 urine test clearly proved to be of diagnostic value. Therefore, the diagnostic performance of the PCA3 urine test was validated in a multicenter study. EXPERIMENTAL DESIGN: The first voided urine after digital rectal examination was collected from a total of 583 men with serum PSA levels between 3 and 15 ng/mL who were to undergo prostate biopsies. We determined the PCA3 score in these samples and correlated the results with the results of the prostate biopsies. RESULTS: A total of 534 men (92%) had an informative sample. The area under the receiver-operating characteristic curve, a measure of the diagnostic accuracy of a test, was 0.66 for the PCA3 urine test and 0.57 for serum PSA. The sensitivity for the PCA3 urine test was 65%, the specificity was 66% (versus 47% for serum PSA), and the negative predictive value was 80%. CONCLUSIONS: In this multicenter study, we validated the diagnostic performance of the PCA3 urine test in the largest group studied thus far using a PCA3 gene-based test. This study shows that the PCA3 urine test, when used as a reflex test, can improve the specificity in prostate cancer diagnosis and could prevent many unnecessary prostate biopsies.

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.