Elsevier

European Urology

Volume 46, Issue 2, August 2004, Pages 182-187
European Urology

DD3PCA3 RNA Analysis in Urine – A New Perspective for Detecting Prostate Cancer

https://doi.org/10.1016/j.eururo.2004.06.004Get rights and content

Abstract

Objectives: Serum tPSA lacks specificity. The DD3PCA3 gene is highly specific for prostate cancer and is detectable in prostate cancer cells shedded into urine after rectal palpation. A newly developed nucleic acid sequence based amplification assay (uPM3™) for detecting DD3PCA3 RNA in urine samples was evaluated prospectively in patients referred for prostate cancer detection.

Methods: The uPM3™ assay simultaneously detects the relative expression of DD3PCA3 RNA and PSAmRNA as a marker for prostate cells in urine. Urine samples were collected after attentive digital rectal palpation prior to transrectal guided prostate biopsy. Samples were provided as a single void specimen (20–30 ml), stabilized in phosphate buffer and centrifuged. Lysis was performed on cell pellets DD3PCA3 RNA and PSAmRNA were extracted and amplification was performed using isothermic nucleic acid based amplification (NASBA). The two targets were detected in real-time using specific beacons as probes in a thermostated spectrofluorimeter. Parameters of the amplification curve were defined after a logistic curve fitting routine and a classification tree model was constructed to predict the outcome of patients (i.e. cancer and non-cancer).

Results: 201 patients were included in this prospective study. 158/201 analyzed urine samples contained enough prostate cells sufficient for DD3PCA3 analysis (79% adequacy rate). Prostate cancer was found in 62 (39%) of the evaluable patients. Overall sensitivity, specificity, positive predictive value and negative predictive value for the uPM3™ assay at a cut-off 0.5 probability were 82%, 76%, 67% and 87% respectively as compared to 98%, 5%, 40% and 83% respectively for tPSA (at a cutoff of 2.5 ng/ml). In the tPSA categories <4, 4–10 and >10 ng/ml sensitivity was 73%, 84% and 84% and specificity was 61%, 80% and 70%, respectively. The AUC (area under the curve) was 0.87 (CI 0.81–0.92).

Conclusion: The uPM3™ assay showed excellent clinical performances and a specificity far superior to tPSA.

Introduction

Early detection of prostate cancer is today mainly based on measurement of serum prostate specific antigen (tPSA) and, to a lesser extent, on digital rectal examination, with an aggressive biopsy policy with any sign of abnormality [1]. By lowering the threshold cut-off where biopsies are performed the sensitivity of tPSA as a biochemical marker may exceed to 90%, but the positive predictive value in the “grey zone “of 2.5–10 ng/ml drops to <25% [2]. As a result 4 out of 5 patients biopsied because of tPSA values in this range will show no cancer at biopsy. The use of tPSA derivatives, tPSA correlations to prostate volume and changes over time and sophisticated statistical models improve specificity somewhat but still do not solve the specificity problem [3]. More prostate cancer specific biomarkers are needed.

Better understanding of the molecular mechanisms of prostate cancer oncogenesis and improved analytical techniques like differential display analysis have identified prostate cancer specific genes such as PSMA, PCGEM-1, PSCA, PDEF, Prostase, NKX3.1 and DD3PCA3 [4]. The DD3PCA3 gene (“differential display code 3”) is localized at chromosome 9q21–22 and was found to be 10–100 fold overexpressed in 53 of 56 human prostate cancer samples in a Northern blot analysis whereas it was not expressed in adjacent non-malignant prostatic tissues [5]. Using the more sensitive reverse transcription polymerase chain reaction no DD3PCA3 transcripts were demonstrated in a wide range of normal human tissues and other human malignant tumors, rendering it the most prostate cancer specific gene identified so far [6]. The current understanding is that the DD3PCA3 gene expresses a non coding messenger RNA in epithelial prostate cells and functions as a polyadenylated RNA transcript, but no cytoplasmic protein results from its transcription [7].

With quantitative RT-PCR using dual time-resolved fluorescence from mRNA of DD3PCA3 prostate cancer samples cells have been shown to have a median 66 fold up-regulation as compared to normal prostate tissues [5]. Transcriptional activity showed no correlation to grade and significant overexpression was already demonstrable in prostate tissues containing <10% prostate cancer cells in a background of predominately non-malignant cells [4]. This permits the detection of prostate cancer cells shedded into urine. Utilizing the DD3PCA3 quantitative RT-PCR TRF hybridization assay Hessels et al. [4] evaluated urines of 108 men with a serum tPSA levels 3–10 ng/ml after digital rectal massage. With the DD3PC3/PSA mRNA ratio and transrectal ultrasound guided biopsy as a reference parameter an area under the curve of the Receiver Operating Characteristic Curve (ROC Curve) of 71% was obtained. With a cut-off value of 200 × 10−3, this reflects a sensitivity of 67% and specificity of 83%—a dramatic improvement as compared to serum tPSA [4].

Quantitative RT-PCR techniques require highly specialized laboratories and can not reasonably be utilized for routine clinical use. Recently a nucleic acid sequence based amplification assay (NASBA) for qualitative simultaneous detection of DD3PCA3 mRNA and PSAmRNA as a housekeeping gene in urine was introduced (uPM3™ DiagnoCure, Quebec, Canada) [8], [9]. We studied its usefulness prospectively in patients coming to prostate biopsy because of an abnormal tPSA and/or suspicious findings at DRE.

Section snippets

Materials and methods

201 patients with an elevated serum tPSA level and/or abnormal digital rectal examination referred to our department for prostate biopsy were included in this prospective study between June 2002 and December 2003. Approval was obtained from the Institutional Ethics Committee and written informed consent from each patient. Patients who had had any transurethral manipulation, radiotherapy, were on hormonal therapy, had an indwelling catheter or acute urinary infection before biopsy were excluded

Results

Urine samples from 201 patients were analyzed for DD3PCA3 RNA expression in the study period. 74/201 (37%) of patients were positive for prostate cancer on biopsy. 158/201 (79%) collected urine samples were found positive for PSAmRNA expression and thus were identified as evaluable samples which contain sufficient prostate cells to perform DD3PCA3 analysis (79% adequacy rate). There was no apparent difference in clinical parameters and the type of cancers detected among the overall group and

Discussion

Novel approaches in molecular technology seem to overcome hurdles in detecting prostate cancer cells in urinary samples and therefore prostate cancer diagnosis from urine is coming into the realm of clinical practice [13], [14]. Analysis of urinary sediments to detect prostate cancer cells after prostate massage was performed for GSTP1 hypermethylation and showed a specificity of 98% and a sensitivity of 73% in 92 patients [15], [16]. Telomerase activity is known to be expressed in at least 90%

Conclusions

The uPM3™ assay offers a new minimally invasive early detection tool for prostate cancer. In this prospective clinical study, it outperformed standard serum PSA at 4 and 2.5 ng/ml cut-offs in terms of specificity and PPV and had an overall accuracy of 78%.
Editorial Comment

Jack Schalken (Nijmegen, Netherlands)

[email protected]

Summary: Test for the more accurate diagnosis of prostate cancer, using state-of-the-art molecular technology is now validated in independent study.

With the rapidly

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