Articles

Collaborative Review – Prostate Cancer

Contemporary Role of Prostate Cancer Antigen 3 in the Management of Prostate Cancer

By: Marco Aupricha lowast , Anders Bjartellb, Felix K.-H. Chunc, Alexandre de la Tailled, Stephen J. Freedlande, Alexander Haesef, Jack Schalkeng, Arnulf Stenzlh, Bertrand Tombali and Henk van der Poelj

European Urology, Volume 60 Issue 1, November 2011, Pages 1045-1054

Published online: 01 November 2011

Keywords: PCA3, Prostate biopsy, Prostate cancer detection, Staging, Targeted therapy

Abstract Full Text Full Text PDF (712 KB)

Abstract

Context

Newly discovered biomarkers ideally should prove clinical usefulness, provide additional detection, staging, and prognosis information to improve individual risk assessment, and potentially permit targeted cancer therapy.

Objective

To review, display, and evaluate the current evidence regarding the biologic and analytic approach of urinary prostate cancer gene 3 (PCA3) in prostate cancer (PCa) detection, staging, and prognosis, and its therapeutic potential.

Evidence acquisition

A systematic and comprehensive Medline search was performed using the Medical Subject Headings search terms PCA3, DD3, UPM3, prostate cancer, cell-lines, prostate tissue, prostate biopsy, detection, diagnosis, radical prostatectomy, staging, grading, progression, and gene therapy. Results were restricted to English-language papers published within the period 1999–2011.

Evidence synthesis

The PCA3 gene is highly overexpressed in specific PCa cell lines and prostatic tumours. In 2006, a simple and robust urine test (Progensa) became commercially available. Despite its costs, prostate cancer antigen 3 (PCA3) is superior to prostate-specific antigen (PSA) and percent free PSA in the early detection of PCa. PCA3 improves the diagnostic accuracy of externally validated nomograms among men with an elevated PSA undergoing biopsy. PCA3 independently predicts low-volume disease and pathologically insignificant PCa but is not associated with locally advanced disease and is limited in the prediction of aggressive cancer. Preliminary data demonstrate that combining PCA3 with other new biomarkers further improves diagnostic and prognostic accuracy. Finally, findings of the first PCA3-Gene-ViroTherapy study suggest therapeutic potential by exploiting PCA3 overexpression.

Conclusions

PCA3, integrated in novel biopsy nomograms or risk stratification tools, can be used to counsel or confirm biopsy indications. If confirmed in further studies, using PCA3 together with established staging risk factors could assist clinicians in specific pretreatment decision making. So far no evidence for the usefulness of PCA3 in active surveillance programs has been presented.

Take Home Message

Prostate cancer antigen 3 (PCA3), a novel prostate cancer biomarker, can be used to interpret biopsy indications and may assist clinicians in pretreatment decision making. Its combination with other new biomarkers and with PCA3-based gene therapy concepts holds great promise for the near future.

Keywords: PCA3, Prostate biopsy, Prostate cancer detection, Staging, Targeted therapy.

1. Introduction

In 1999, through a joint effort of researchers at Radboud University in Nijmegen in the Netherlands and Johns Hopkins University in the United States, Bussemakers et al. identified the DD3 gene (later called prostate cancer antigen 3 [PCA3]), which is highly overexpressed in prostatic tumours [1]. Several studies confirmed this, reporting significantly higher PCA3 messenger RNA (mRNA) expression in malignant versus nonmalignant prostatic tissue [2], [3], [4], [5], [6], and [7]. However, most of these analyses did not find significant correlations between PCA3 tissue levels and tumour stage and aggressiveness [5], [6], [7], and [8].

Three different assays measuring urinary PCA3 mRNA after digital rectal examination (DRE) were then developed [3], [9], and [10]. Out of prostate cells shed into urine after prostate massage, these assays measure PCA3 mRNA, which is highly upregulated in neoplastic prostate tissue, simultaneously with prostate-specific antigen (PSA) mRNA, which is not upregulated in prostate cancer (PCa) and used to normalise for the amount of prostate-specific RNA in the molecular test sample.

After the third-generation PCA3 assay (Progensa) [10] attained Conformité européenne (CE) approval in 2006, several clinical studies were conducted to evaluate PCA3 as a novel diagnostic marker, to counsel or confirm biopsy indications, and to rule out aggressive cancer at biopsy. The clinical staging significance of preoperative urinary PCA3 was assessed to identify pathologic favourable and/or unfavourable features, such as small-volume/insignificant PCa, locally advanced disease, and aggressive disease. Based on promising findings from previous studies, the novel marker was further evaluated in its ability to predict biopsy progression in men undergoing active surveillance (AS) and as a first-line diagnostic test in prescreened men. (Table 1)

Table 1 Selected prostate cancer antigen 3 studies stratified according to their clinical scenario

Clinical scenario/end points Reference Patients, n Risk factors Results
Early detection
Initial, repeat Bx

Prediction of PCa at Bx
Deras et al. [23] 570 PSA, DRE, age, PV

PCA3
AUC-PSA: 0.55

AUC-PCA3: 0.69

AUC-PSA+PV+DRE+PCA3=0.75
Initial, repeat Bx

Prediction of PCa at Bx
Chun et al. [28] 809 PSA, DRE, age

PV, Bx-history

PCA3
AUC-PSA: 0.53

AUC-PCA3: 0.68

AUC-PSA+DRE+age+PV+Bx-history+PCA3-17=0.73
Repeat Bx

Prediction of PCa at Bx
Marks et al. [15] 226 PSA

PCA3
AUC-PSA: 0.52

AUC-PCA3: 0.68
Repeat Bx

Prediction of PCa at Bx
Haese et al. [14] 463 PSA, DRE, age, %fPSA, PV

PCA3
AUC-PSA: 0.60

AUC-%fPSA: 0.58

AUC-PCA3: 0.66

AUC-age+PSA+%fPSA+PV+DRE+PCA3=0.71
Screening

Prediction of PCa at Bx
Roobol et al. [31] 721 PSA, DRE, age, %fPSA, PV

PCA3
AUC-PSA: 0.58

AUC-PCA3: 0.64
Active surveillance

Prediction of Bx progression
Tosoian et al. [17] 294 PSA, age, %fPSA, PV

PCA3
Progression at Bx (yes vs no):

PSA: 5.0 vs 4.0; p=0.051

%fPSA: 18 vs 21; p=0.013

PCA3: 47 vs 33; p=0.131
Preoperative staging
Prediction of pathologic features Nakanishi et al. [34] 83 PSA, DRE, age, race, PV, PPC

PCA3
Prediction of tumour volume <0.5 ml

AUC-PSA: 0.63

AUC-PPC: 0.73

AUC-PCA3: 0.76
Prediction of pathologic features Whitman et al. [35] 72 PSA, DRE, age, race, PV, Bx-GS, PPC, PCA3 Prediction of extracapsular extension:

AUC-PSA: 0.73

AUC-PCA3: 0.73

AUC-PSA+PCA3+Bx-GS=0.90
Correlation to pathologic features Hessels et al. [36] 70 PSA, DRE, age, PV, Bx-GS

PCA3
Correlation of PCA3 to pathologic features:

Tumour volume <0.5 ml: p=0.680

Insignificant PCa: p=0.496

Extracapsular extension: p=0.765

Prostatectomy GS ≥7: p=0.199
Prediction of pathologic features Auprich et al. [38] 305 PSA, DRE, age, race, PV, Bx-GS, PPC, PCA3 Prediction of tumour volume <0.5 ml:

AUC-PSA+PPC+Bx-GS+PCA3=0.84

Prediction of insignificant PCa:

AUC-PPC+Bx-GS+PCA3=0.92

PCa=prostate cancer; Bx=biopsy; PSA=prostate-specific antigen; DRE=digital rectal examination; PV=prostate volume; PCA3=prostate cancer antigen 3; AUC=area under the curve; Bx-hist.=biopsy history; %fPSA=percent free PSA; PPC=percent positive cores at biopsy; GS=Gleason score.

Beyond these clinical implications, further research was also directed at evaluating its potential use in combination with other new biomarkers and as a novel target for PCa therapy, which all reflects a highly promising future for PCA3.

This review considers the current clinical evidence investigating PCA3's clinical role in screening, early detection, AS, and preoperative staging. Several significant aspects such as test applicability and robustness, combinations with other new biomarkers, and potential further applications of PCA3 are also discussed.

2. Evidence acquisition

A systematic review of the literature was performed in February 2011 using the National Library of Medicine's PubMed database (1999–2011). This search included both Medical Subjects Heading (MeSH) search terms and free-text protocols. Specifically, the MeSH search was conducted by combining the following terms retrieved from the MeSH browser provided by Medline: PCA3, DD3, UPM3, prostate cancer, cell lines, prostate tissue, prostate biopsy, detection, diagnosis, radical prostatectomy, staging, grading, progression, and gene therapy. The search results were subsequently restricted to the English language. This approach resulted in 47 PCA3 studies, selected by the 10 authors to discuss in this collaborative review, including 9 basic papers, 19 and 7 reports on diagnostic and staging, respectively, and 12 studies on new diagnostic and therapeutic concepts of PCA3.

3. Evidence synthesis

3.1. History of PCA3: from characterisation to development of a urine test

Bussemakers et al. were the first to identify and characterise the DD3PCA3 or PCA3DD3 gene (Fig. 1) comparing PCa tissue with nonmalignant prostatic tissue. Using a reverse transcriptase polymerase chain reaction (RT-PCR) method, they detected PCA3 overexpressed in cancerous tissue with low expression in benign prostatic tissue and not measurable in the normal tissue of numerous organs such as the prostate, testis, bladder, kidney seminal vesicles, brain, and lung. Specifically, the expression of PCA3, a noncoding RNA located on chromosome 9q21–22 whose function is unknown, is highly prostate specific, and it was overexpressed in 94.6% of tumour lesions, in only 1 of 7 human PCa cell lines (lymph node carcinoma of the prostate), and in none of 18 nonmalignant prostate samples [1].

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Fig. 1 The prostate cancer antigen 3 (PCA3) gene, located at chromosome 9q21-22, consists of four exons, and exon 2 is often skipped by alternative splicing. Three alternative polyadenylations can occur in exon 4 (4a, 4b, and 4c). Most frequently, the transcript contains exons 1, 3, 4a, and 4b. Reprinted with permission from Nature Publishing Group: Hessels et al., Nature Reviews Urology, copyright 2009 [56].

Using a quantitative real-time RT-PCR technique, comparing PCA3 with human telomerase reverse transcriptase, de Kok et al. demonstrated a 34-fold versus 6-fold higher mRNA expression in malignant compared with nonmalignant prostatic tissue, even if the samples contained <10% tumour (Fig. 2) [2].

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Fig. 2 Prostate cancer antigen 3 (PCA3) messenger RNA (mRNA) expression in prostatic tissue. Box plots representing the expression of PCA3 mRNA comparing (A) benign prostatic tissue (median: 2.4×105; range: 0.2×105 to 10.1×105); (B) prostate tumour containing ≤10% prostate cancer (PCa) cells (median: 25.3×105; range: 66.0×105 to 166.0×105), and (C) prostate tumour containing>10% PCa cells (median: 158.4×105; range: 7.0×105 to 994.0×105). Reprinted with permission from Elsevier: Hessels et al., European Urology, copyright 2003 [3].

These fundamental findings promoted the development of a PCA3 diagnostic test. Based on the idea of prostate cells shedding into the urine after prostate massage, Hessels et al. reported first on PCA3 mRNA measurement in sedimented urine. In detail, the first voided proportion of urine was collected after a thorough rectal examination of the prostate, consisting of exactly three strokes, with firm pressure to depress the prostate surface for several millimetres, applied from the base to the apex and from the lateral to the median line for each lobe. PSA mRNA was used to normalise the test for the number of prostate cells in the urine sediment. Although PSA expression is constant in normal cells and weakly (1.5-fold) downregulated in PCa cells, the so-called PCA3 score, which is the ratio between PCA3 mRNA over PSA mRNA multiplied by 1000, was presented as a new diagnostic tool. Using a determined PCA3-PSA cut-off of 200×103, test sensitivity and specificity of 67% and 83%, respectively, were achieved in 108 patients undergoing prostate biopsy and suspected of harbouring PCa based on a PSA level >3 ng/ml [3].

Using the second-generation PCA3 test (uPM3 assay), Tinzl et al. at first compared the new urinary marker to PSA among men undergoing initial and repeat biopsy for an elevated PSA. Although the informative rate (79%) in this study was inferior to current third-generation assays, PCA3 outperformed PSA, achieving a sensitivity and specificity of 82% and 76% versus 87% and 16%, respectively [9]. Despite this remarkable diagnostic improvement, 21% of all samples would not have provided any information about patients’ risk of PCa. Using the same assay, Fradet et al. confirmed this diagnostic superiority (PCA3 vs PSA: area under the curve [AUC] 81% vs 40%) in the first multicentre cohort of 443 men undergoing 6- to 10-core random biopsy, although again among men preselected for biopsy based on an elevated PSA [11].

In 2006, Groskopf et al. presented a prototype of a quantitative, validated PCA3-based urine test using post-DRE whole-urine specimens further processed in a single-tube format, which is thus fundamentally different from earlier developed assays [3] and [9]. Assay stability was evaluated in archived urine specimens stored at either 4°C or 30°C. The PCA3-to-PSA ratio at 4°C remained within a 20% range of the initial values after 2 wk. However, at 30°C, a significant degradation of PCA3 reflected the test's instability at room temperature. Median PCA3 to PSA mRNA ratio values showed a statistically significant difference comparing 52 healthy (<45 yr of age), 52 biopsy-negative, and 16 biopsy-positive men (4.5 vs 27.0 vs 81.8; p<0.01) [10].

The analytic performance of the newly developed PCA3 assay subsequently was intensively tested in a multicentre evaluation (n=179) conducted by Sokoll and coworkers. They confirmed the need of an attentive DRE, whether performed with three or eight strokes (p=0.85), to provide high informative test rates up to 95.5% with<18%, 15%, and 10% total, intra-assay, and interassay variations, respectively. PCA3 scores of biopsy-positive men demonstrated high correlation (97%; p<0.0001) when two different research sites were compared [12]. Overall, the informative rate of the third-generation PCA3 assay (Gen-Probe, Progensa) was significantly improved over the previously reported 79% by Tinzl et al. [9] and is reported to range from 94% to 100% [10], [12], [13], [14], [15], [16], [17], [18], [19], and [20].

Although the US Food and Drug Administration approval process is currently ongoing, the CE approved the PCA3 test in November 2006 to assist clinicians in counselling and confirming initial and repeat biopsy indications.

3.2. Clinical applicability of prostate cancer antigen 3 for early detection

Despite substantial improvements in early detection due to PSA [21] and [22], a main limitation remains the high proportion of men detected with nonmalignant findings at first or subsequent biopsy. Therefore, one of the most important clinical rationales of a meaningful PCA3 application aims at reducing the number of potentially unnecessary biopsies.

Marks and coworkers first tested this in 226 consecutive men undergoing repeat biopsy. They demonstrated PCA3's superiority over PSA (AUC: 0.68 vs 0.52; p=0.008). Using 35 as the most balanced PCA3 score cut-off resulted in a sensitivity, specificity, and odds ratio of 58%, 72%, and 3.6, respectively. Identification of the optimal threshold value was obtained by calculating the highest summary index of the probability of a true positive (equals sensitivity) and a true negative (equals specificity) test result. Unfortunately, comparable with earlier studies [2] and [3], median PCA3 scores in aggressive PCa (Gleason score [GS] <7 vs GS ≥7) were not significantly different [15].

Subsequently, US and European prospective multicentre trials were conducted in patients undergoing initial or repeat biopsy by Deras et al. [23] and first or second repeat biopsy by Haese et al. (Fig. 3) [14]. Within these studies comparable diagnostic accuracies of US and European men at first repeat biopsy (AUC: 0.68 vs 0.65) were reported. Interestingly, PCA3 predicted biopsy outcome in European men on second repeat biopsy with slightly increased accuracy compared with those on first repeat biopsy (AUC: 0.67 vs 0.65). This aspect, which seems counterintuitive, stands in contrast to the findings of Deras et al. [23] and needs to be validated in further PCA3 biopsy protocols. Both studies revealed conflicting results in PCA3's association with cancer aggressiveness, which is discussed in the next section of this review. However, both studies demonstrated that combining PCA3 with established biopsy risk factors such as age, PSA, DRE, prostate volume, and percent free PSA (%fPSA) improved the diagnostic accuracy in multivariable regression models. A subgroup analysis (n=301) of the European multicentre study performed by Ploussard et al. confirmed PCA3's univariable superiority over %fPSA as a predictor of repeat biopsy outcome (AUC: 0.69 vs 0.57) [24].

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Fig. 3 The correlation of prostate cancer antigen 3 (PCA3) to repeat biopsy outcome. Bars represent the probability of a positive repeat biopsy expressed in percentages according to different PCA3 score ranges. Reprinted with permission from Elsevier: Haese et al., European Urology, copyright 2008 [14].

In line with previous studies, incorporating PCA3 in the Prostate Cancer Prevention Trial risk calculator (PCPT-RC) improved the diagnostic accuracy compared with the established biopsy risk factors (AUC: 0.65 vs 0.70) [25]. Applying the most stringent statistical criteria following Kattan [26] and [27], Chun et al. demonstrated in a large mixed biopsy patient cohort from Europe and North America (n=809) that PCA3 independently predicted PCa, and its addition to established PCa risk factors (age, PSA, DRE, prostate volume, and biopsy history; Fig. 4) significantly improved predictive AUC of the base model between 2% and 5% [28]. In fact, these PCA3-based biopsy nomograms were recently externally validated and thus represent novel tools that may assist clinicians in counselling and confirming biopsy indications [29].

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Fig. 4 The prostate cancer antigen 3 (PCA3) biopsy nomogram. This recently externally validated nomogram combines established biopsy risk factors such as age, digital rectal examination (DRE), total serum prostate-specific antigen (PSA), prostate volume, and history of previous biopsy together with PCA3 score to predict cancer on prostate initial and repeat biopsy. Instructions for physicians: To obtain nomogram-predicted probability of prostate cancer, locate patient values at each axis. Draw a vertical line to the “Point” axis to determine how many points are attributed for each variable value. Sum the points for all variables. Locate the sum on the “Total Points” line to be able to assess the individual probability of cancer on prostate biopsy on the “Probability of prostate cancer at biopsy” line. Reprinted with permission from Elsevier: Chun et al., European Urology, copyright 2009 [28].

In this context, Perdona and coworkers directly compared the updated PCPT-RC, including PCA3, and Chun's PCA3-based nomogram. They demonstrated significantly better discriminative power of the updated PCPT-RC (AUC: 0.80 vs 0.72; p=0.04) yet superior calibration for Chun's nomogram. Finally, decision curve analysis revealed a higher net benefit for Chun's nomogram, resulting in up to 21% of avoided unnecessary repeat biopsies at the expense of missing up to 6.8% of cancers [30].

Regarding health care expenses, it must be acknowledged that different European countries have different reimbursement systems. In general, urinary PCA3s are more expensive than PSA measurements. For example, at an Austrian tertiary referral centre, expenses for a total serum PSA versus urinary PCA3 measurement are €32.4 versus €243.1. However, because PCA3 use is related to avoiding up to 67% of repeat biopsies procedures compared with PSA [14], the avoided expenses for a prostate biopsy (approximately €266.8) and its potential further follow-up diagnostic interventions (eg, follow-up PSA, %fPSA) should also be acknowledged. Moreover, biopsy-related anxiety, discomfort, and complications may be spared in a substantial number of patients.

Taken together, using mainly data from two multicentre trials, PCA3 was confirmed as a reliable predictor of PCa at biopsy, demonstrating superiority over PSA and %fPSA. Its combination with established risk factors demonstrated improved accuracy and applicability of newly developed diagnostic tools to assist clinicians in biopsy decision making among men who already met an established criteria for biopsy (ie, elevated PSA or abnormal DRE). However, whether PCA3 should be used as a continuous coded variable or whether different cut-offs (eg, PCA3 score 35) should be used at each specific biopsy scenario (eg, initial, first repeat, second repeat) must be addressed in future trials.

3.3. Potential use of prostate cancer antigen 3 in screening and active surveillance

Most recently, PCA3 was assessed as a first-line screening test within the European Randomised Study of Screening for Prostate Cancer trial. A PCA3 score ≥10 demonstrated a positive predictive value of 17.1 compared with 18.8 for a PSA value ≥3.0 ng/ml. Interestingly, PCA3 versus PSA missed substantially fewer cancers (32.0% vs 64.7%) and serious cancers (26.3 vs 57.9%). Because this unique study evaluated a PSA-prescreened cohort (third round or more; 33% had a negative first biopsy), a consecutive study in unscreened patients, avoiding attribution bias, should be conducted to further assess PCA3 as a potential screening marker [31].

Recently, Tosoian et al. assessed PCA3's ability to rule out clinically significant PCa in men undergoing AS as defined by the biopsy criteria of Epstein et al. [32]. A trend towards higher median PCA3 scores in patients with GS upgrading at follow-up biopsy (72 vs 50.8; p=0.08) was recorded. However, at adjusted multivariable Cox regression analysis, PCA3 did not represent an independent risk factor of biopsy progression (p=0.15) [17]. Based on the limitations that the number of events was small (n=38) and that PCA3 was assessed only once at the time of first diagnosis but not repeatedly during the follow-up biopsies, so far no evidence for the usefulness of PCA3 in AS programs has been presented. Because PCA3 does not represent a useful marker to monitor PCa aggressiveness at biopsies [2], [3], [15], and [23], its role in risk assessment during AS needs to be tested in larger studies with repeated PCA3 score measures.

3.4. Prostate cancer antigen 3: a prognostic marker to predict tumour volume, stage, and grade?

Given that PCA3 is highly overexpressed in PCa tissue and improves the prediction of biopsy outcome, several studies have focused on its potential ability to predict PCa stage and aggressiveness before definitive therapy.

Bostwick et al. at first reported on 24 patients undergoing radical prostatectomy (RP) after being diagnosed with PCa based on a suspicious urinary UPM3 test. The assessed RP specimens demonstrated no difference in cancer volume, location, stage, and GS compared with RP specimens of men diagnosed with PCa based on PSA or suspicious DRE findings [33].

Using the Progensa PCA3 assay, Nakanishi and coworkers analysing 83 RP samples reported that the urinary PCA3 score significantly correlated with tumour volume (TV), GS, and independently predicted small-volume diseases (TV<0.5ml), demonstrating the highest univariable AUC of 0.76. Using 25 as a PCA3 score cut-off to predict small-volume tumours in combination with low grade (GS<7) resulted in a sensitivity and specificity of 70% and 73.3%, respectively. However, it is important to note that the number of events was limited (n=10) [34]. Similarly, Whitman et al. confirmed PCA3's correlation to TV and identified it as an independent predictor (p<0.01) of extracapsular extension (ECE) resulting in a multivariable AUC of 0.90 when combined with PSA and biopsy GS. In contrast to Nakanishi, Whitman et al. could not find a significant association of PCA3 with pathologic GS [35]. In contrast, Hessels et al. and van Gils et al. demonstrated neither a significant correlation of PCA3 to pathologic grading nor to TV and pathologic stage in a cohort combining 132 patients [36] and [37].

The largest published series so far on urinary PCA3's correlation to clinicopathologic features (n=305) demonstrated that the multivariable AUC of low-volume disease (+2.4% to +5.5%) and insignificant PCa models (+3% to +3.9%) improved when PCA3 was added to standard clinical risk factors (Fig. 5). On the other end of the spectrum, there was no significant correlation between PCA3 and adverse features such as ECE and seminal vesicle invasion, and its significance on aggressive PCa models (RP GS ≥7) was reported to be limited [38]. Similar results were reported on 106 consecutive men undergoing RP due to clinically low-risk disease (PSA<10 ng/ml, T1c–T2a, and biopsy GS<7). Low urinary PCA3 scores and favourable biopsy criteria (<33% or 3-mm tumour; <3 positive cores) independently predicted small TV (<0.5ml) and insignificant PCa. Again, the urinary PCA3 score, combined with established risk factors in multivariable logistic regression models, was not significantly associated with high-grade and locally advanced disease [39].

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Fig. 5 Prediction of small-volume and insignificant prostate cancer with preoperative prostate cancer antigen 3 (PCA3). Receiver operating characteristic curve analyses and area under the curve (AUC) for predicting (A) tumour volume <0.5ml and (B) pathologically confirmed insignificant prostate cancer.BxGS=biopsy Gleason score; PPC=percentage of positive cores; PSA=prostate-specific antigen. Reprinted by permission from Elsevier: Auprich et al., European Urology, copyright 2011 [38].

The assumption that higher PCA3 scores are associated with more aggressive cancer is based on the hypothesis that with increasing dedifferentiation, PCa cells become more invasive and could therefore more easily be shed into the ductal system of the prostatic gland after DRE or that larger tumours simply have more surface area over which to shed PCA3 [37] and [39]. Although most studies, especially in RP cohorts, failed to confirm this hypothesis [35], [36], [37], [38], and [39], some authors suggest that, following Gleason's scoring system [40], tumours with pattern 4 and 5 increasingly lose their glandular differentiation and luminas, disabling cells to be shed into urine after DRE in correlation with their TV. Therefore, potentially higher PCA3 mRNA tissue levels, resulting from larger tumour masses, might not be adequately measured by the urinary test [38].

Taken together, evaluations on PCA3's potential prognostic ability, which are currently based on a relatively small number of patients (n=722), revealed that the novel diagnostic marker independently predicts small-volume and insignificant PCa. However, PCA3 is not significantly associated with locally advanced disease and has limited value in the prediction of aggressive tumours.

3.5. Potential prostate cancer antigen 3 score alterations over time and following bioptic or medical intervention

Within the placebo arm of the Reduction by Dutasteride of Prostate Cancer Events trial, urinary PCA3, PSA, and %fPSA were available at the year 2 and year 4 follow-up biopsy in 1072 men (age: 50–75 yr; PSA: 2.5–10 ng/ml; one previous negative 6- to 12-core biopsy). On univariable analyses for the prediction of year 4 biopsy outcome based on year 2 biomarker values, PCA3 score was exclusively found as a significant predictor for a positive follow-up biopsy at year 4. Interestingly, PCA3 scores in biopsy-positive men only slightly increased (+15.7%) within the study period [20].

Larre et al, evaluating urinary PCA3 scores before and 2h after prostate biopsy, recently reported no significant difference of measured PCA3 scores, neither in all men (18%; p>0.05) nor in PCa-positive men (1.5%; p>0.05) [41]. Despite the small number of patients (n=15) and increased rate of PCA3 score alterations compared with the reported intra- and interassay variations (14% and 10%) by Sokoll et al. [12], this study suggests a certain robustness of PCA3 even towards interventional effects on the prostatic tissue.

In this context, the influence of dutasteride (5α-reductase inhibitor [5-ARI]) on prostatic markers was assessed by Gils et al. [42]. In 16 men with benign prostatic hyperplasia (BPH) and 9 men with clinically localised PCa (all treated with 5-ARI), PSA, testosterone, dihydrotestosterone (DHT), and urinary PCA3 were measured at baseline and 1, 2, and 3 mo thereafter. As expected, dutasteride reduced DHT (>90%), halved PSA levels, decreased prostate volume (10–16%), and increased testosterone (20–30%). In contrast, 5-ARI treatment had a widely variable effect on PCA3 scores, which increased (75–284%) and decreased (14–77%) over time, irrespective of whether patients with or without PCa were observed [42]. This needs to be taken into account when counselling patients on dutasteride who are designated for a PCA3 test.

3.6. New perspectives

3.6.1. Combination of prostate cancer antigen 3 with new biomarkers

Since PCA3 was introduced as highly PCa specific and as a clinically useful marker to predict biopsy outcome, its combined use with other new tumour markers may further improve its diagnostic accuracy. Therefore, transcripts of a fusion between the transmembrane-serine protease gene (TMPRSS2) and the v-ets erythroblastosis virus E26 oncogene (ERG) were evaluated in combination with PCA3 in the post-DRE urine of 108 patients undergoing prostate biopsy. It is of note that TMPRSS2-ERG fusion transcripts were only found in 59% of the primary PCa tissue specimens, and the included patients did not represent a typical biopsy cohort because PCa detection rate was quite high with 72% due to PSA levels ranging from 1.1 to 1619 ng/ml. Urine sediments of men diagnosed with PCa were positive for TMPRSS2-ERG fusion transcripts and PCA3 (cut-off: 48) in 37% and 62%, respectively. Combining both markers improved the sensitivity to 73%, yet a considerable decreased specificity of 63%, compared with 93% of TMPRSS2-ERG fusion alone [43].

In addition, Laxman and coworkers evaluated golgi membrane protein 1 (GOLM1), serine peptidase inhibitor, Kazal type 1 (SPINK1), PCA3, and TMPRSS2-ERG fusion in sedimented urine of men before biopsy (n=216) or RP (n=60). A multivariable regression model for the detection of PCa including these four biomarkers improved the diagnostic AUC from 0.662 (for PCA3 alone) to 0.758, respectively [44]. When α-methylacyl-coenzyme racemase (AMACR) and PCA3 from post-DRE urine was assessed in patients undergoing prostate biopsy due to suspicion of PCa, both markers demonstrated an improved AUC over PSA (0.65 vs 0.67 vs 0.59). Using AMACR (cut-off: 10.7) and PCA3 (cut-off: 19.9) within a combined model resulted in a high sensitivity and specificity of 81% and 84% versus 70% and 71% versus 72% and 59% for AMACR versus PCA3 alone, respectively [45]. Comparable findings were demonstrated by Rigau et al. using PCA3 together with prostate-specific G-protein coupled receptor in urine sediments after prostatic massage from 215 patients presented for prostate biopsy. Rigau et al. reported an increased specificity of 44.4% at an assumed sensitivity of 90% for the combined test compared with each biomarker used as a stand-alone test (25.4% vs 23.9%) [46].

Despite the fact that the reported studies used PCA3 cut-off values (19.9, 48) different from the more established cut-off value of 35 reported in previous studies [14], [15], [23], [24], [28], and [29], a substantial improvement in predicting biopsy outcome was demonstrated by combining PCA3 and new biomarkers in a limited number of patients. If these promising results could be confirmed by further studies, combinations of new biomarkers including PCA3 may potentially offer an interesting new perspective on the early detection and staging of PCa. However, because to date most of the markers combined with PCA3 are still in their experimental phase, it remains to be assessed which marker panel has the greatest potential to improve predictive ability compared with established markers.

3.6.2. Detection of prostate cancer antigen 3 in circulating tumour cells

In PCa patients, circulating tumour cells (CTCs) are correlated with a poor prognosis [47]. For this reason detection of specific biomarkers found in prostatic CTCs could potentially indicate an advanced and aggressive stage of disease.

In 2008, Väänänen et al. described a quantitative RT-PCR assay for the detection of PCA3 mRNA in peripheral blood and evaluated 67 patients with locally advanced (n=23) and metastatic diseases (n=9), respectively. Interestingly, only two patients were found positive for PCA3 mRNA in peripheral blood samples [48]. In contrast, Marangoni et al, evaluating preoperative peripheral blood samples, detected PCA3 mRNA expression in 25 (62.5%) of 40 patients with PCa compared with 15 (37.5%) of 40 BPH patients [49]. Patients presenting with progressive castrate-resistant PCa demonstrated significantly overexpressed levels of PCA3 in CTCs from peripheral blood [50]. This is in line with findings reported by Jost et al, using an immunomagnetic CTC enrichment method to assess peripheral blood from 67 PCa patients. Although none of the androgen-dependent patients tested positive for PCA3, 5 (31%) of 16 androgen-independent patients were found positive for CTC-PCA3 [51].

In summary, the detection of PCA3 mRNA expression in CTCs from peripheral blood proved to be feasible, although what role this has in identifying patients with poor prognosis is unknown because the data to date are quite limited and further studies are needed.

3.6.3. Prostate cancer antigen 3 as a novel gene therapy target

Because Van der Poel et al. demonstrated the high PCa specificity of PCA3 and using a specific diphtheria toxin model highlighted its potential use as a precursor to suicide gene therapy [52], a combination of PCA3's promoter region driving expression of a suicide gene could be used to process novel PCa therapies. In theory, this combined therapeutic construct will bind, interact, and finally induce cell death in PCa tissue, and nonmalignant and nonprostatic cells will not be involved in this highly specific therapeutic cascade.

Based on this concept, Fan and coworkers developed an oncolytic adenovirus (Ad.DD3-E1A-IL-24), in which replication is driven by the PCA3DD3 promoter, carrying the therapeutic gene interleukin (IL)-24. Its in vitro and in vivo effects were investigated in DU-145 cell lines and in DU-145 xenograft tumours in nude mice. In five of six treated mice, tumours were completely eliminated within 50 d. Most remarkably, all mice stayed alive until the end of observation [53]. Despite nonnegligible discrepancies regarding the therapeutic effect of Ad.DD3-E1A-IL-24 in vitro and in vivo, this study demonstrated “Gene-ViroTherapy's” excellent antitumoural efficacy in an initial small single tumour model study in mice. Therefore further investigations on PCA3's potential role in PCa gene therapy should be intensively promoted in the future.

3.7. Actual role of prostate cancer antigen 3 in clinical routine

In the European Association of Urology Guidelines 2011 [54], the use of PCA3 in the detection setting is classified as experimental and therefore cannot be recommended. However PCA3's value in identifying PCa, especially in men with an initially negative biopsy, is supported by growing level 2a evidence [14], [15], [20], [24], [25], [28], [29], [30], and [55]. Therefore, PCA3 may be most clinically relevant in the repeat biopsy setting, using a cut-off of 35 to confirm repeat prostate biopsy indication.

4. Conclusions

This systematic review of PCA3 studies published within the last 12 yr reveals that the PCA3 gene is highly PCa specific, and its application on initial technically challenging research assays to today's standardised commercially available platforms offers simple applicability and reliable robustness. Based on PCA3's confirmed superiority over PSA and %fPSA in the further stratification of men selected for biopsy based on an elevated PSA and/or abnormal DRE, its combined use with established biopsy risk factors, particularly within nomograms or risk calculators, may assist clinicians in counselling and confirming biopsy indications. Based on relatively small patient numbers, PCA3 was identified to independently predict small-volume and insignificant PCa; however, PCA3 was not associated with advanced disease and was limited in the prediction of aggressive cancer in men undergoing RP. In several pilot studies, PCA3 together with other potential new markers improved the multivariable accuracy in predicting biopsy outcome, although by only a modest amount (2–5%). Finally, the implementation of the PCA3 promoter in developing new highly PCa-specific gene therapies represents a promising perspective in the near future.

Author contributions: Marco Auprich had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Auprich, Bjartell, Chun, Freedland, Haese, van der Poel, Schalken, Stenzl, de la Taille, Tombal.

Acquisition of data: None.

Analysis and interpretation of data: None.

Drafting of the manuscript: Auprich.

Critical revision of the manuscript for important intellectual content: Auprich, Bjartell, Chun, Freedland, Haese, van der Poel, Schalken, Stenzl, de la Taille, Tombal.

Statistical analysis: None.

Obtaining funding: None.

Administrative, technical, or material support: None.

Supervision: Chun.

Other (specify): None.

Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.

Funding/Support and role of the sponsor: None.

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Footnotes

a Department of Urology, Medical University of Graz, Graz, Austria

b Department of Urology, Skåne University Hospital Malmö, Malmö, Sweden

c Department of Urology, University Clinic Eppendorf, Hamburg, Germany

d Department of Urology, CHU Henri Mondor, Paris, France

e Department of Surgery, Durham VA Medical Center and the Duke Prostate Center, Division of Urological Surgery, Departments of Surgery and Pathology, Duke University School of Medicine, Durham, NC, USA

f Martini Clinic Prostate Cancer Center, University Clinic Eppendorf, Hamburg, Germany

g Department of Experimental Urology, Radboud University Nijmegan Medical Center, Nijmegen, Netherlands

h Department of Urology, University Hospital and Faculty of Medicine, Tübingen, Germany

i Department of Urology, Clinique universitaire Saint Luc, Université catholique de Louvain, Brussels, Belgium

j Department of Urology, The Netherlands Cancer Institute, Amsterdam, Netherlands

lowast Corresponding author. Department of Urology, Medical University of Graz, Auenbruggerplatz 5/6, 8036 Graz, Austria.

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