The need for a lower total PSA cut-off value with PSA assays ...

The need for a lower total PSA cut-off value with
PSA assays calibrated to the new WHO standard
The recalibration of PSA assays to the new World Health Organisation WHO 96/670 standard
has certainly led to improved harmonisation of testing, but it has not yet resulted in the complete
conformity expected by many clinicians and laboratory managers. There is also an urgent need
for manufacturers to redefine a valid clinical decision limit after PSA assay recalibration to WHO
standard, in order to maintain a similar clinical performance of the measurement. The consequence
of not providing a new cut-off value is that a higher number of PSA results may be interpreted as
negative in the presence of prostate cancer.
by Dr V. Jarrige
Prostate cancer is the second leading cause of cancer
death in men, exceeded only by lung cancer. It is the
most common form of malignancy in Europeans,
with its incidence increasing more than with any
other cancer over the last 20 years. According to WHO,
about 190,000 new cases occur each year. Prostatespecific
antigen (PSA) is the serum biomarker most
widely used for early detection of prostate cancer and
monitoring of patients with the disease. As men age,
PSA levels generally rise. The most frequent reasons
for elevated serum PSA values are benign prostatic
hyperplasia (BPH) or prostate cancer. Measurements
of total PSA allow detection of cancer as early as five
years before symptoms appear [1].
Clinicians have always been aware that PSA assays
may differ significantly from one manufacturer to
another; the drive towards recalibration was designed
to address this issue. However, recalibration alone has
no impact on the improvement in the clinical interpretation
of results and may have led to the erroneous
assumption that all assays are now the same.
More European countries now request laboratories to
use PSA assays that are calibrated to the WHO 96/670
standard. However, this standard is not the one that
was used to establish the clinically relevant PSA cut-off
value of 4.0 ng/mL. (This was defined using the original
Hybritech Tandem-R PSA calibration system).
Despite this, the cut-off value of 4.0 ng/mL is still
used with the WHO-standardised assay from many
manufacturers. Different results can still be produced
by different PSA assays even if they are standardised
to the same WHO standard; this is because variations
in assay architecture and antibody choice prevent full
achievement of standardisation. The use of the WHO
standard thus only improves harmonisation between
assays [2]. The use of the 4.0 ng/mL cut-off with results
Figure 1. Impact of different PSA standardisation
interpretation of results and cut-off definitions.
Q u A l i T y c O n T r O l
generated by WHO calibrated assays can affect the
clinical sensitivity and specificity of the assay, meaning
that the results need to be interpreted differently
[Figure 1].
Despite the still-occurring differences in results from
different WHO-calibrated assays for either PSA or
free PSA, it is nevertheless highly desirable for these
assays to be traceable to WHO reference preparations.
More and more companies are developing solutions
to that end. As an example, Beckman Coulter has
decided to develop a second calibration protocol for
its Access Hybritech PSA and Access Hybritech free
PSA assays that allows traceability to WHO reference
preparations while retaining the calibration to the
original Hybritech Tandem-R PSA and Hybritech
Tandem-R free PSA assays. Beckman Coulter have
redefined the cut-off value for their new WHO
96/670 recalibrated Access Hybritech PSA assay to 3.1
ng/mL, but remind users that this is not automatically
applicable to other methods.
Hybritech - the first gold standard for PSA
testing
In 1986, the Hybritech Tandem-R assay became the first
PSA assay to be approved for prostate cancer monitoring
by the Food and Drug Administration (FDA) in
the US. This set the benchmark for all PSA assays that
followed. In 1994, 4.0 ng/mL was identified as the most
appropriate clinical decision point for the detection of
prostate cancer for the Hybritech PSA assay [3]. The
original Hybritech Tandem-R calibration was based on
an internal reference preparation of purified human
PSA, and the clinical PSA cut-off was established as
4.0 ng/mL on the basis of results of samples from
over 6,600 men who were tested by the assay
calibrated with the Hybritech calibration system [4].
The original Hybritech Tandem-R assay is now available
on Beckman Coulter Access immunoassay systems.
The assay is used in conjunction with digital rectal
examination (DRE) to aid in the detection of prostate
cancer in men aged 50 years or older, to assess their
prognosis and to monitor the effectiveness of any
treatment. When used in conjunction with Hybritech
free PSA assay, it also helps to differentiate between
prostate cancer and benign conditions. The efficacy
of early prostate cancer detection using PSA testing
is currently the purpose of the 10-year European
Randomised Study for Screening of Prostate Cancer
Figure 2. Equimolar recognition of complexed and free PSA
forms in serum samples is essential for accurate PSA testing:
a non-equimolar assay may increase both false positive
and false negative results.
(ERSPC), which is expected to report its findings
between 2008 and 2010. Hybritech PSA is the method
of choice for the ERSPC study.
The need for equimolarity in PSA testing
There were significant variations in PSA results among
the early non-equimolar PSA assays. Equimolar
recognition of free and complexed PSA forms is
essential for accurate PSA testing. Inaccurate quantification
of PSA around the 4.0 ng/mL cut-off can yield
a false positive or a false negative result, depending on
the direction of the distortion, and may thus lead to
inappropriate management of the patient [Figure 2].
In order to compensate for the non-equimolar
response of some PSA assays, Thomas Stanley, a
clinical urologist at Stanford University, proposed in
the mid-1990s a PSA standard containing both complexed
and free PSA in a ratio of 90:10 respectively [5].
This became the basis for the new standard adopted
by WHO in 1999, which has its mass assigned using a
method based on the use of a particular molar extinction
coefficient, different from the original Hybritech
standard. Although the original intention was to
establish an ‘equimolarity standard’, this has actually
led to the creation of a new WHO ‘mass standard’
for PSA.
However, many clinicians are still unaware that
restandardising a PSA assay from an original
Hybritech calibration to a WHO calibration may
result in potential under-recovery of PSA values. This
is because assays calibrated to the new WHO standard
show a negative bias in mass units compared
with the Hybritech calibrated assays [6]. The original
Hybritech Tandem-R calibration was based on an
internal reference preparation of purified human
PSA and yields PSA results about 20% higher than
the new WHO standard [6]. The European Group

on Tumour Markers (EGTM) clearly
recommends that "every laboratory
report should contain the name of the
assay used and a valid reference range,
specifically generated for this assay."
In addition, some European countries
require laboratories to report PSA and
free PSA values standardised to WHO
96/670 and 96/668, respectively.
recalibrating for WHO
standards
In order to verify the Hybritech standard
against the WHO standard, Beckman
Coulter Immunodiagnostics (BCI)
developed WHO primary calibrators to
determine the ‘offset’ when compared
directly against the original Hybritech
calibration. This indicated how much
adjustment was needed for both total and
free PSA across the range of the assay.
When Beckman Coulter carried this out
it was found an adjustment of 20% was
needed for both assays.
The validation process involved a statistical
analysis of how the WHO standardisation
impacts on the original Hybritech
data used to establish the 4.0 ng/mL
cut-off [2]. During this process it became
apparent that the different calibrations
yielded different results, indicating that
the 4.0 ng/mL cut-off would not provide
optimal clinical sensitivity and specificity
for the WHO calibration. The 3.1
ng/mL cut-off for the WHO calibrated
method was compared against the original
Hybritech data set that was used to
determine the cut of 4.0 ng/mL. From
this, the new cut-off of 3.1 ng/mL was
verified as being appropriate [Table 1].
A sensitivity and specificity of 81.6% and
48.0% respectively was maintained at this
cut-off [4].
Use of the new cut-off of 3.1 ng/mL
does not impact the clinical sensitivity
of tests involving
the ratio of free
PSA to total PSA
(percentage of
free PSA) [Table
2] because the
WHO calibration
values for both
total and free PSA
exhibit similar
differences (approximately 20%) from
the Hybritech calibration values.
Table 1. WHO calibration 3.1 ng/mL total PSA cut-off is clinically
equivalent to Hybritech calibration 4.0 ng/mL cut-off.
Why is a lower PSA cut-off important
after WHO
recalibration?
When samples taken from men with
prostate cancer were evaluated using
a 3.1 or a 4.0 ng/mL cut-off for the
WHO calibration, use of the higher
cut-off missed 15% of prostate cancers
which were
detected by using
the cutoff of 3.1
ng/mL [Table 3].
Table 3a shows
PSA results for
cancer patients
using a 4.0 ng/
mL cut-off with
the Hybritech
calibration and
a 3.1 ng/mL
Table 2. Equivalent percentage free PSA results for the Hybritech and
WHO calibrations.
cut-off with the
WHO calibration.
Table 3b
shows the PSA results for the same cancer
patients when their samples were
analysed using a 4.0 ng/mL cut-off with
both the Hybritech and the WHO calibrations.
It is apparent that using
the 4.0 ng/mL cut-off with the
WHO calibration fails to pick up
38 of the 255 patients (15%) whose
prostate cancer may be missed as a
result. Laboratories need to make two
key points clear to their clinicians.
First, there is a new cut-off value of 3.1
ng/mL for the WHO calibrated Access
Hybritech PSA assay. Secondly, this
new cutoff cannot be automatically
applied to other methods, as cutoff
validation must be assay specific
according to EGTM. It is important
that other manufacturers invest in the
individual validation and verification
of their WHO calibrated assays.
Further information
Technical information for Beckman Coulter assays
on the Hybritech calibration and WHO calibration
options, the lower total PSA cut-off required when
calibrating to the WHO standard and QC values
for Hybritech and WHO calibrations are available
from mjeremaes@beckmancoulter.com. Please note
that the WHO ca calibrated assay is not currently
available in the US.
references
1. H. Ballentine Carter et al.
Longitudinal Evaluation of Prostate-
Specific Antigen Levels in Men With
and Without Prostate Disease. JAMA
1992; 267: No 16.
2. Blijenberg BG, Storm BN, Kruger
As published in April 2007
AE and Schroder
FH. On the standardisation
of total
prostate-specific
antigen: an exercise
with two referencepreparations.
Clin Chem
Lab Med 1999;
37: 545-552.
3. Catalona WJ
et al. Selection
of optimal PSA
cutoff for early
detection of prostate cancer, ROC
curves. J Urol 1994; 152:2037–2042.
4. Catalona WJ et al. Comparison of
digital rectal examination and serum
prostate specific antigen in the early
detection of prostate cancer: Results
of a multicenter clinical trial of 6,630
men. J Urol 1994; 151: 1283–1290.
5. Stamey TA, Teplow DB, Graves
HC. Identity of PSA purified from
seminal fluid by different methods:
comparison by amino acid analysis
and assigned extinction coefficients.
Prostate 1995; 27: 198–203.
6. Link RE et al. Variation in prostate
specific antigen results from 2
different assay platforms: Clinical
impact on 2,304 patients undergoing
prostate cancer screening. J Urol
2004; 171: 2234–2238.
7. Semjonow A, Albrecht W, Bialk P,
Gerl A, Lamerz R, Schmid HP and van
Poppel H: Tumour markers in prostate
cancer: EGTM recommendations.
Anticancer Res 1999; 19: 2799-2801.
Table 3. Distribution, by PSA cut-off, of subjects tested for prostate
cancer by PSA test.
The author
Veronique Jarrige, Ph.D.
European Scientific Manager,
Immunodiagnostics,
Beckman Coulter Europe,
c/o 22 rue Juste-Olivier,
P.O. Box 1044,
1260 Nyon 1, Switzerland
Tel +33 490 50 64 72
e-mail: vjarrige@beckman.com
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