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 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
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
|Figure 2. Click for enlargement
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
|Figure 4. Click for enlargement
DD3 was initially described in the 1993 abstract
3). Further development of PCA3 was performed in the laboratories
of Jack A. Schalken,
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
|Figure 5. Click for
enlargement and legend
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
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
Evolution to Present-Day Test
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
PCA3 molecular assay employing the technologies of Target Capture,
Transcription Mediated Amplification (TMA), and Hybridization Protection
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,
|Figure 7. Click for enlargement
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
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
|Figure 9. Click for enlargement and legend
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
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
|Figure 10. Click for enlargement
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
PCA3 Score vs PSA Testing (Figure
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
|Figure 11. Click for enlargement
, 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.
|Figure 12. Click for enlargement and legend
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.
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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.
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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.
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