Download Molecular forms of prostate specific antigen (PSA) in serum: clinical

– February/March 2011
14
Tumour markers
Molecular forms of prostate specific
antigen (PSA) in serum: clinical and
analytical implications
Prostate specific Antigen (PSA) is widely used as a disease biomarker
for diagnosis and monitoring of prostate cancer (PCa). Numerous
different immunoassays are available for the measurement of
PSA and its subforms in serum. The assays can be referenced to
different laboratory standards and are not interchangeable. Patients
and physicians should be aware of which assay was used, and
longitudinal monitoring should be performed with the same test.
by Dr Katharina Braun, Dr David Ulmert and Dr Hans Lilja
Prostate-specific antigen (PSA) is a kallikrein-related peptidase encoded by a five
exon gene 7.1 kb (KLK3), one of fifteen
genes clustered in a 280 kb locus on the long
arm of chromosome 19 in the cytogenic
region q13.3-4 [1]. KLK3 (encoding PSA)
and KLK2 (encoding kallikrein-related
peptidase 2 or hK2) share approximately
80% amino acid sequence identity and the
two proteins are produced and secreted at
highly abundant levels by prostate epithelium although some expression can also
be detected in certain other extra-prostatic
tissues [2].
PSA is synthesised as a 261-amino-acid
(aa) pre-pro precursor that is processed to a
non-catalytic zymogen through removal of
a ≈17-aa signal peptide upon transfer to the
endoplasmic reticulum, whereas the short
activation peptide must be released, e.g. by
hK2, to convert the non-catalytic ≈244-aa
zymogen to the mature 237-aa catalytic
single-chain PSA [2].
Originally called gamma-seminoprotein,
a seminal fluid protein was identified in
1966 and characterised in 1971 by Hara et
al [3]. The authors anticipated that the protein would be a potential marker for seminal fluid applicable in the field of forensic
medicine. In 1979, PSA was purified from
prostatic tissue, and was later found to be
identical to gamma-seminoprotein [4]. Subsequently, several studies recognised PSA
as a potential marker for PCa [5]. The first
assay for PSA in serum was developed by
Kuriyama et al [6] shortly after Papsidero
and coworkers [5] identified PSA in blood.
PSA is synthesised in normal prostate epithelium, benign prostate hyperplasia (BPH)
and all stages of prostate adenocarcinoma.
The concentration of PSA in seminal fluid
is up to 10⁶ fold higher than in blood [7].
The median concentration of tPSA in blood
is ≈0.7 ng/mL in healthy men at early middle age [8], whereas in advanced cancer the
amount of PSA in the blood can increase up
to 10,000 fold [7].
Although recent data from the large population-based randomised trials in Europe
and the US have demonstrated that PSAbased prostate cancer screening can reduce
mortality from prostate cancer by about half
after fourteen years, these important benefits
are tempered by considerable overdetection
and consequential risks for overtreatment
associated with current screening modalities [9]. Risk of prostate cancer diagnosis,
metastasis and death from prostate cancer
are very strongly associated with concentration of PSA in blood [8]. This strong rationale explains the widespread use of PSA as a
key biomarker to assess disease risk, monitor therapeutic intervention and disease
recurrence and as a key component in
various prognostic models.
Molecular forms of PSA in serum
PSA added to blood in vitro exists in three
forms: one fraction will occur complexed with
inactivating protease inhibitors, one portion
as non-complexed non-catalytic PSA, and a
third as active PSA entrapped by macroglobulins [10]. However, the “total PSA” (tPSA)
detected in clinical samples comprises the
sum of the concentration of both free PSA
and PSA complexed to protease inhibitor
ACT [11]. Data from the original discovery
and characterisation of the proportion of free
PSA versus PSA-ACT complexes suggested a
mean free-to-total PSA ratio of 22% (range
7-50%) in patient’s serum samples [11]. Based
on PSA-measurements at early middle age
in a large, highly representative populationbased cohort of men, the median proportion
of free-to-total PSA in blood has later been
shown to be ≈33% (IQR 28%; 38%) [12].
Complexed PSA
In the blood circulation, the majority of noncatalytic PSA is covalently complexed with
the protease inihibitor α1-antichymotrypsin
(ACT or SERPINA5). Active PSA can also
be enveloped by α-macroglobulins such as
α2-macroglobulin (A2M) and pregnancy
zone protein (PZP) [10]. Unlike the interactions with ACT, the complex-formation
with A2M or PZP does not inactivate PSA
although it blocks catalytic PSA from access
to protein substrates [10]. It is noteworthy
that such macromolecules mask epitopes recognised by commercially available assays and
thus stay undetected by these methods [1,11].
Since the original discovery in the early 1990s
it has been carefully documented that the proportion of PSA-ACT is higher in men with
PCa compared to men with BPH [6,10], that
the free-to-total PSA ratio is an independent
predictor of prostate cancer risk [9], and that
the free-to-total PSA ratio enhances discrimination of men with BPH from those with evidence of PCa beyond that of total PSA alone
[13]. A systematic review and meta-analysis of
66 subsequent studies found that the free-tototal PSA ratio (“%fPSA”) enhanced the accuracy in predicting the diagnostic outcome of
a prostate biopsy compared to that based on
tPSA alone [14].
Free PSA and subforms
The non-complexed, free PSA in blood
is a mixture of different inactive forms
circulating unattached to any plasma
proteins. These inactive forms can be
separated into two main fractions: single chain “intact” forms with or without
truncated remainders of the short activation peptide, and forms that are inactive due to internal cleavages. The most
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studied forms of the latter subgroup are
PSA with internal cleavages at Lys145Lys146 (“nicked PSA”) or cleavages at
Lys182-Ser183 (“BPSA”) [15].
While free PSA in men with BPH is correlated with a higher ratio of internally cleaved
PSA, increased concentration of intact noncomplexed forms and truncated precursor
forms of PSA are found in patients with
presence of prostate cancer [16].
Serum PSA measurement
More than 80 antibodies against PSA have
been very carefully characterised based on
their binding regions on the protein [17].
Because of the high degree of amino acid
sequence identity between PSA and hK2
(80% identity), many monoclonal antibodies
(MAbs) against PSA cross-react with hK2 –
also with identical binding affinity to each of
these two highly similar proteins.
Three distinct antigenic regions of PSA can
be identified in reference to the ability to recognise free PSA, both free and complexed
PSA, and the cross reactivity with hK2 [17].
Non-linear antigenic domains that are in
close proximity to amino acids 86-91 are
highly specific for free PSA. Epitopes specific
for PSA without cross reactivity with hK2 are
located at or close to amino acids 158-163.
The shared epitopes between PSA and hK2
are located close to amino acids 3-11, which
are close to the identical amino-terminal end
of both proteins. Knowledge of antibody specificity is important for selecting appropriate
antibody pairs when designing immunoassay
[17]. Numerous commercial immunoassays
are available for the measurement of PSA
and its subforms in serum. Specific assays
for fPSA as well as dual assays for fPSA and
tPSA, PSA-ACT assays and hK2 assays have
been developed [18 - 21]. Additionally assays
detecting different proPSA forms have been
made available for use in research.
There appears to be no access to any of the
antigenic PSA epitopes subsequent to a stable complex that formed between PSA and
α2 Macroglobulin (A2M), which makes
measurement of PSA-A2M technically complicated and not clinically informative [22].
Alternatively, denaturation with sodium
dodecyl sulphate at high pH can be used to
disrupt the PSA-A2M complex and release
PSA from this complex, which then can be
detected with a conventional ELISA [23].
During the past two decades, multiple studies
compared PSA values measured by commercially available immunoassays with - at least
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in part - inconsistent and conflicting results.
Graves et al compared the polyclonal assay
from Yang laboratories with the Hybritech
two site monoclonal assay in 1990 using
samples from a group of 27 patients, and
found a two-fold difference in PSA-levels
between the two assays [24]. Semjonow et al
reported a correction factor of 0.94 to 2.35
when comparing Beckman Coulter Access
and Hybritech Tandem E assays in 1996 [25].
In contrast to these findings, Roehrborn et al,
comparing three monoclonal based assays in
a group of 86 patients (Hybritech Tandem E,
Abbott ImX and Tosoh AIA 600) found no
differences for total PSA [26]. Leewansangtong et al also reported a high correlation
of PSA level ranges between the Hybritech
Tandem E and Abbott AxSYM assay [27].
Figure 1 illustrates one of the novel duallabel monoclonal antibody tests designed
to selectively measure both fPSA as well as
simultaneously enabling detection of total
PSA with equimolar detection of free PSA
and PSA-ACT complex [12].
In 1994, the Second Stanford Conference
on International Standardisation of International Standards proposed the use of a standard produced by Stamey et al. This standard
calibrator is composed of 90% PSA-ACT
www.cli-online.com & search 25049
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and 10% fPSA, similar to the distribution
found in the circulation of PCa patients [28].
This 90:10 PSA preparation was established
as the World Health Organisation standard
(WHO 96/670) [29]. PSA assays using the
WHO 96/670 standard yield 20-25% lower
PSA values than those using the Hybritech
standards [30].
In 2004 Link et al compared the Beckman
Coulter Access and Bayer Centaur system
as well as the third generation DCP Immulite System, and found higher PSA values
measured with Access than Centaur and
similar results with the Centaur and Immulite systems [31]. Blijenberg et al compared
the Hybritech Tandem E, Beckman Coulter
Access, DCP Immulite, Roche Diagnositcs
Elecsys and Defia Prostatus systems and
showed similar measurements for total
PSA but not for fPSA [32].
These findings were confirmed by two
recent studies comparing equimolar assays
calibrated to WHO standards. Kort et al
compared tPSa, fPSa and cPSA in 70 samples in 6 different assays (Beckman Coulter
Access, Abbott ARCHITECTS and Abbott
AxSYM, Bayer Centaur, DPC Immulite
2000, Roche Modular Analytics E170).
Results showed variation in values for tPSA
from 0.5 to 1.0µg/L and for fPSA from 0.12
to 0.40µg/L. Overall results showed less
diversity for tPSA than fPSA, but tPSA
assays were still not interchangeable [33].
Stephan et al investigated the interchangeability of tPSA, fPSA and %fPSA between
Beckman Coulter Access, DPC Immulite
2000, Abbott AxSYM, Bayer Centaur and
Roche Diagnositcs Elecsys assays and still
found significant interassay variability.
This may be due to the different epitope
specificity of the antibodies used [34].
Figure 1. Design of immunoassay for simultaneous
measurement of free, uncomplexed forms of PSA
and total PSA. Monoclonal antibodies coated on
plate as capture antibody for free and complexed
forms in equimolar fashion (Mab1). Monoclonal
antibodies to detect PSA-ACT and free PSA (Mab2)
and monoclonal antibodies accessible for fPSA
epitope only (Mab3), both measureable
with fluorescence (27).
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Tumour markers
Conclusion
Since the introduction of WHO 96/670
Standards and development of tPSA-assays
designed to detect free PSA and PSA-ACT
on an equimolar basis, inter-assay variability has decreased – in particular regarding
tPSA values. Nevertheless results of commercially available tPSA assays are not yet
interchangeable, not uniformly standardised, and with no widely accepted conversion factor to correct the accuracy. Large
discrepancies in fPSA values may result in
clinical misinterpretation as the decision to
consider a prostate biopsy may be based on
the ratio of fPSA to tPSA.
Persisting discrepancies between assays
result from a combination of the overall design, epitope specificity and affinity
of capture and detector antibodies, use
of monoclonal or polyclonal antibodies,
cross-reactivity and non-specific interferences, as well as standardisation. Physicians
should therefore be aware of which assay
and standards have been used and note
whether the same test is also being used for
longitudinal monitoring of their patients.
Acknowledgements
Grant support: Swedish Cancer Society, Swedish
Research Council (Medicine), The Tegger Foundation, Lund University Medical Faculty ALF grants,
the National Cancer Institute [P50-CA92629], the
Sidney Kimmel Center for Prostate and Urologic
Cancers, David H. Koch through the Prostate
Cancer Foundation, Fundación Federico SA, and
German Association of Urology (DGU), Ferdinand Eisenberger research grant Competing interest declaration: Dr Hans Lilja holds patents for free
PSA and hK2 assays.
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The authors
Katharina Braun1,5, David Ulmert 1,3,4 and
Hans Lilja 1,2,3
Departments of 1Surgery (Urology), 2Clinical
Laboratories, and Medicine, Memorial SloanKettering Cancer Center, New York, USA
Departments of 3Laboratory Medicine, and
4
Urology, Lund University, Skåne University
Hospital, Malmö, Sweden
5
Department of Urology, Marienhospital
Herne, University Bochum, Herne, Germany
Corresponding author:
Hans Lilja, MD, PhD.
Memorial Sloan-Kettering Cancer Center
Department of Clinical Laboratories, Urology,
1275 York Avenue, Box 213, New York, NY
10065, USA
e-mail: [email protected]