PCA3: A Genetic Marker of
Prostate Cancer

Autumn, 2006-- The advent of molecular diagnostics has brought the promise of a specific test for prostate cancer (CaP), the urinary PCA3 gene test. Widespread testing with prostate-specific antigen (PSA) has increased the numbers of prostate biopsies to perhaps one million annually in the U.S. However, serum PSA levels are not specific for CaP. Thus, approximately four men with elevated PSA levels undergo prostate biopsies to find one with cancer, and some cancerous men with "normal" PSA levels escape detection with this measurement. Early studies of the urinary PCA3 gene test indicate this new marker has a much greater degree of CaP specificity than PSA testing.

Limitations of PSA Testing for Prostate Cancer
Within the prostate gland, benign prostatic hyperplasia (BPH) cells contain a concentration of PSA several fold higher than adjacent cancer cells1, which seriously undermines the theoretical basis of CaP testing with PSA. Thompson, in data from the Prostate Cancer Prevention Trial, where biopsies were obtained irrespective of PSA levels, has shown

"There is no cutpoint of PSA with simultaneous high sensitivity and high specificity for monitoring healthy men for prostate cancer, but rather a continuum of prostate cancer risk at all values of PSA ( Table 1) 2."

Figure1. Click for enlargement and legend

And Stamey, an early advocate of PSA testing, has declared, " Serum PSA levels are no longer related to prostate cancer, but only to the volume of BPH present3." Why? Because the disease has changed! Nowadays, instead of finding large primary cancers in the prostate as seen 20 years ago, the usual findings are multiple small lesions, where the serum PSA coming from the cancer is overwhelmed by the BPH contribution (Figure 1). Despite these changes, nearly 30,000 men will still die of CaP this year, and an accurate test for the disease is an urgent priority. The major foibles of PSA testing for CaP were recently detailed in a USRF website posting (http://usrf.org/news/10Foibles_of_PSA/index.htm).

Discovery of the PCA3 Gene
At about the same time that PSA testing was starting to gain widespread adoption, a young molecular biologist from Holland began post-doctoral work at The Brady Urological

Figure 2. Click for enlargement and legend

Institute of Johns Hopkins University. There, in the early 1990s, in the laboratory of. William B. Isaacs, Marion Bussemakers performed studies on human prostate tissue using the technique of differential display, a then newly-described method to identify gene expression in different tissues. During this series of experiments, an mRNA was discovered that appeared highly specific for prostate cancer (Figure 2) 4.

Figure 4. Click for enlargement and legend

The gene could not be found in any of the existing gene databases. Bussemakers and Isaacs called their new gene DD3, referring to its appearance in the display, and they concluded that it "..might be useful in prostate cancer detection (Figure 3)." The gene was ultimately found to be over-expressed in 53 of 56 prostate cancers and absent from 18 other normal human tissues. Further study revealed the new gene to be a noncoding RNA, which could be mapped to chromosome 9q21-22 (Figure 4).

DD3 was initially described in the 1993 abstract shown (Figure 3). Further development of PCA3 was performed in the laboratories of Jack A. Schalken,

Figure 5. Click for enlargement and legend

Bussemakers' supervising professor at University Hospital, Nijmegen, The Netherlands. Among the important contributions from Nijmegen were the first clinical demonstration of the specificity of PCA3, its measurability in urine, and the importance of denoting PCA3 expression vis-á-vis a background of normal prostate epithelial genetic material5,6. Interest in urinary prostate cells, which had been generally abandoned years before, was then resurrected, and urinary PCA3 nresearch studies were soon instituted by Yves Fradet in Laval University in Canada. A prototype urine assay known as uPM3 was developed at Diagnocure. During this time the nomenclature for DD3 was formally changed to PCA3. The PCA3 pioneers are shown in Figure 5.

Why Molecular Markers?
Molecular biology may be defined as the branch of biology focused on the formation, structure, and function of DNA, RNA and proteins, and their roles in the transmission of genetic information. The central theme of molecular biology is as follows: Information encoded in a sequence of the DNA strand, passes to molecules of RNA through a process called transcription. RNA acts as a messenger (mRNA) to pass the information to proteins through a process called translation. The message transcribed from the gene is therefore translated into a protein product that is specialized for a particular function based on the instruction stored in the gene. With the sequencing the human genome, molecular biologists became faced with another hurdle, determining the function of individual genes and their protein products. Knowing the function of each gene is essential to biotechnology, a branch of engineering that focuses on using such knowledge for the development of molecular markers and treatments for diseases in man, such as prostate cancer. Thus a gene is the fundamental unit of storage and transmission of cell biology and to know the genetic make-up is to know the potential direction of development of that biological unit. How genetic information is passed and how cancer may develop when this process goes awry is shown in online videos from the National Cancer Institute (http://www.cancer.gov/newscenter/benchmarks-vol1-issue1/Video ).

Initial Clinical Experience with PCA3
While the PCA3 gene was clearly discovered in Isaacs' lab at Hopkins, it was at Schalken's institution in Nijmegen , The Netherlands where the gene was initially translated from lab to clinic5,7. The earlier work of Bussemakers and Isaacs was confirmed and expanded. A method to accurately quantify the gene in urine was developed, using RTqPCR. Receiver operating characteristics for PCA3 (tumor vs benign cells) were shown to be remarkable, with area under the curve (AUC) of 0.985, ie accuracy of the test at the cellular level was nearly perfect. The median upregulation of PCA3 from normal to tumor tissue was found to be 34 fold5, increasing to 66 fold in tumor tissue containing more than 10% cancer cells7. This upregulation in cancer tissues provided a theoretical basis for detecting presence of the gene in tissues containing only a small number of cancer cells, against a background of low expression by many normal or BPH prostate cells, i.e., "..in tissue biopsies and bodily fluids5." Thus, the importance of denoting PCA3 as a ratio with PSA mRNA, a surrogate for background prostate epithelial cell nuclear material, was established. Importantly, a practical application was confirmed: the PCA3 ratio determined in voided urine, especially after light prostatic massage, or 'attentive' digital rectal exam, was shown to be a sensitive and specific test for CaP in the host7.

In clinical trials from Canada8 and Austria9, the potential diagnostic value of the PCA3 urine test was soon established. In these two trials, more than 700 men undergoing prostate biopsy donated urine after attentive digital rectal exam. When the urinary sediment contained enough prostate epithelial nuclear material to be evaluated, the PCA3-to-PSA mRNA urinary levels exhibited a 66-82% sensitivity and 76-89% specificity for cancer. Both values compare quite favorably with accuracy of PSA. However, using the early assay method, approximately 15%-20% of PCA3 samples were deemed "non-evaluable" because the urine did not contain a sufficient quantity of PSA mRNA to allow detection of background genetic material.

Evolution to Present-Day Test

Figure 6. Click for enlargement and legend

In the clinical trials cited above, gene testing was performed at DiagnoCure, a Canadian biotech company founded by Dr. Yves Fradet. Fradet had obtained the PCA3 patent from the group at Nijmegen. The gene was then known as uPM3, and the test was a qualitative assay. In November 2003, Gen-Probe, Inc (San Diego, CA) acquired from DiagnoCure exclusive worldwide diagnostic rights to this new prostate cancer gene, known now, according to standard nomenclature, as PCA3. Gen-Probe soon developed a quantitative

Figure 7. Click for enlargement and legend

PCA3 molecular assay employing the technologies of Target Capture, Transcription Mediated Amplification (TMA), and Hybridization Protection (HPA). (Figure 6)10. In collaboration with Urological Sciences Research Foundation (USRF) of Culver City, CA, clinical testing of the Gen-Probe assay began in early 2004, and the first presentation of data from that work was made at the Gordon Research Conference on Biomarkers in January, 2005 (Figure 7).

The Target Capture process allows the PCA3 RNA target to be "captured" with magnetic beads, and eliminates inhibitors in urine specimens. (Figure 6). This maximizes the molecular PCA3 assay sensitivity and specificity. Transcription Mediated Amplification (TMA) achieves a 10 billion-fold amplification of target RNA in less than 1 hour. The Hybridization Protection Assay (HPA) uses specific DNA probe labeled with an acridinium ester detector molecule that can emit a chemiluminescent signal. The acridinium ester on the hybridized probes is protected within the double helix, and allows chemilumininescence when exposed to detection reagents. Because both TMA and HPA occur in a single test tube, there is no transfer of reagents out of the assay reaction tube, thus eliminating the risk of carryover contamination.

Figure 8. Click for enlargement and legend

The PCA3 test is actually a dual assay in which both PCA3 and PSA mRNA are separately quantified and the ratio of the two, the PCA3 Score, is determined. The ratio is used because the denominator, PSA mRNA, establishes the amount of prostate-specific nuclear material in the specimen. A low level of PCA3 is expressed by normal prostate cells, and if absolute concentration of PCA3 were used, a high Score might be obtained from a

Figure 9. Click for enlargement and legend

specimen rich only in normal prostate cells. Thus, the PCA3 Score tells the expression of PCA3 corrected for the background of normal or BPH epithelial cells present in the specimen. (Figure 8). In early clinical testing it was soon determined that the higher the urinary PCA3 Score, the greater the likelihood of prostate cancer. (Figure 9).

In addition to normalizing PCA3 signal, measurement of PSA mRNA also serves to confirm that the yield of prostate specific RNA is sufficient to generate a valid or "informative" test. Without a certain minimum amount of prostate-specific genetic material in the sample, the test is deemed "non-informative." An attentive digital rectal exam (3 sweeps on each side of the prostate), performed just prior to urine specimen collection, improves the informative rate from approximately 80% to greater than 95%. The informative rate now being obtained with the new assay is likely attributable to both the attentive DRE and the increased sensitivity of the new assay technologies explained above.

Current Use and Availability of PCA3 Testing
In presentations at the 2006 American Urological Association meeting (J.Urol., 175: 174-6 (S), 2006), in recent data gathered on approximately 1000 men, the Gen-Probe PCA3 test was shown to exhibit a high degree of sensitivity and specificity for prostate cancer. For cancer vs non-cancer, a specificity of 76% at 50% sensitivity (PCA3 cutoff = 35 copies/copy of PSA mRNA), with an area under the ROC curve (AUC) of 0.680, was reported by Fradet's group. By comparison, serum tPSA specificity was only 22% for the same men. In addition, the quantitative PCA3 Score correlated with the probability of positive biopsy in this population: at low PCA3 Scores (< 5) the biopsy positive rate was only 20%, while at PCA3 Scores > 100 the risk of positive biopsy was 67%. A suggestion was presented in Schalken's recent data that some correlation with Gleason grade and cancer volume may also be present. In data from USRF, almost no overlap was seen in PCA3 Scores from men with cancer and men with only BPH, confirming the specificity of the test. PCA3 RNA is uniformly undetectable in urine from post-radical prostatectomy patients, even following attentive DRE.

A particularly important role of the new marker appears to be in men with persistently elevated serum PSA levels, but a negative initial biopsy. In such men, who constitute a large problematic group, the odds ratio for the PCA3 test to predict cancer upon re-biopsy is 3.6, compared to only 1.2 for serum PSA testing11 (Table 2).

Figure 10. Click for enlargement and legend

PCA3 testing is highly dependent on the cutoff Score used to determine a "positive" or "negative" test, because sensitivity and specificity vary reciprocally with the Score. The higher the cutoff, the greater the specificity and the lower the sensitivity; the lower the cutoff, the greater the sensitivity and the lower the specificity. Thus, although the test is now available commercially, physicians must be cautious in interpreting the lab report and should know the performance characteristics of the assay, before decisions are based on a "positive" or "negative" test result. U.S. laboratories currently offering the PCA3 test commercially include Bostwick Laboratories, Richmond, VA (http://www.bostwicklaboratories.com/about/PCA3.html) and AmeriPath Laboratories, Palm Beach Gardens, FL (http://www.ameripathgu.com/PCA3/). The test is not currently approved by the US FDA. The method of specimen collection is shown in Figure 10.

PCA3 Score vs PSA Testing  (Figure 11)

Figure 11. Click for enlargement and legend

In comparison with serum levels of PSA, the urinary PCA3 Score appears to be highly specific for prostate cancer. While serum PSA levels are known to be influenced by volume of BPH tissue, age, inflammation, trauma, and use of 5 alpha-reductase inhibitors (finasteride, dutasteride), preliminary data indicate that these factors do not appear to influence PCA3 Scores. For example, standard teaching is to draw blood for PSA levels before rectal exam, for fear the exam might cause spurious elevations in serum PSA. However, an attentive DRE actually increases the "informative" rate of PCA3 determinations and is, in fact, recommended. In Figure 12

Figure 12. Click for enlargement and legend

, the effect of prostate volume is shown on both PSA and PCA3 in the same group of adult men. Clearly, PSA is directly related to prostate volume, while PCA3 is not. Unpublished data from USRF indicate that the same is likely to be true for age and use of 5ARI drugs. Thus, with the caveat that data are limited, the urinary PCA3 Score appears to offer a great specificity advantage over serum PSA levels in the early diagnosis of prostate cancer.

Conclusion and Future Directions
The PCA3 gene, a noncoding segment of mRNA located on chromosome 9, is over-expressed by prostate cancer cells in comparison with all other cells studied. The differential expression is great, permitting detection of the gene in nuclear material from cancer cells shed into urine after attentive digital rectal exam. Thus, urinary PCA3 appears useful as a highly specific marker for prostate cancer. While the early data look promising, the PCA3 test must still be regarded as a 'work in progress," from several perspectives. PCA3 expression is denoted against a background of prostate-specific genetic material, a PCA3 Score, ie, a ratio of PCA3 to PSA mRNA, and normative values have only been defined in a preliminary fashion. Factors regulating PCA3 gene expression are not yet clearly defined, but the great confounds of serum PSA levels (prostate volume, age, trauma) appear to affect PCA3 to a much lesser degree than PSA. Additional clinical research trials, now in an organizational phase, should provide further guidelines for widespread application of the urinary PCA3 Score.

1. Magklara A, Scorilas A, Stephan C, Kristiansen GO, Hauptmann S, Jung K and Diamandis EP: Decreased concentrations of prostate-specific antigen and human glandular kallikrein 2 in malignant versus nonmalignant prostatic tissue. Urology. 56: 527-32, 2000.

2. Thompson IM, Ankerst DP, Chi C, Lucia MS, Goodman PJ, Crowley JJ, Parnes HL and Coltman CA, Jr.: Operating characteristics of prostate-specific antigen in men with an initial PSA level of 3.0 ng/ml or lower. Jama. 294: 66-70, 2005.

3. Stamey TA, Caldwell M, McNeal JE, Nolley R, Hemenez M and Downs J: The prostate specific antigen era in the United States is over for prostate cancer: what happened in the last 20 years? J Urol. 172: 1297-301, 2004.

4. Bussemakers MJ, van Bokhoven A, Verhaegh GW, Smit FP, Karthaus HF, Schalken JA, Debruyne FM, Ru N and Isaacs WB: DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res. 59: 5975-9, 1999.

5. de Kok JB, Verhaegh GW, Roelofs RW, Hessels D, Kiemeney LA, Aalders TW, Swinkels DW and Schalken JA: DD3(PCA3), a very sensitive and specific marker to detect prostate tumors. Cancer Res. 62: 2695-8, 2002.

6. Schalken JA, Hessels D and Verhaegh G: New targets for therapy in prostate cancer: differential display code 3 (DD3(PCA3)), a highly prostate cancer-specific gene. Urology. 62: 34-43, 2003.

7. Hessels D, Klein Gunnewiek JM, van Oort I, Karthaus HF, van Leenders GJ, van Balken B, Kiemeney LA, Witjes JA and Schalken JA: DD3(PCA3)-based molecular urine analysis for the diagnosis of prostate cancer. Eur Urol. 44: 8-15; discussion 15-6, 2003.

8. Fradet Y, Saad F, Aprikian A, Dessureault J, Elhilali M, Trudel C, Masse B, Piche L and Chypre C: uPM3, a new molecular urine test for the detection of prostate cancer. Urology. 64: 311-5; discussion 315-6, 2004.

9. Tinzl M, Marberger M, Horvath S and Chypre C: DD3PCA3 RNA analysis in urine--a new perspective for detecting prostate cancer. Eur Urol. 46: 182-6; discussion 187, 2004.

10. Groskopf J, Aubin SM, Deras IL, Blase A, Bodrug S, Clark C, Brentano S, Mathis J, Pham J, Meyer T et al.: APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer. Clin Chem. 52: 1089-95, 2006.

11. Marks LS, Fradet Y, Deras IL, Blase A, Mathis J, Aubin SMJ, Cancio AT, Desaulniers M, Ellis WJ, Rittenhouse HG, Groskopf J: Prostate cancer specificity of PCA3 Urinary Gene Test. In Press, Urology, 2006.