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Int. J. Cancer: 70, 52–56 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
INCREASED ANALYTICAL SENSITIVITY OF RT-PCR OF PSA mRNA DECREASES
DIAGNOSTIC SPECIFICITY OF DETECTION
OF PROSTATIC CELLS IN BLOOD
Wolfgang HENKE1*, Monika JUNG1, Klaus JUNG1, Michael LEIN1, Horst SCHLECHTE1, Christoph BERNDT2, Birgit RUDOLPH3,
Dietmar SCHNORR1 and Stefan A. LOENING
1Klinik und Poliklinik für Urologie, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt Universität zu Berlin,
D-10098 Berlin, Germany
2Institut für Pathologische und Klinische Biochemie, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt Universität zu
Berlin, D-10098 Berlin, Germany
3Institut für Pathologie, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt Universität zu Berlin, D-10098 Berlin,
Germany
The diagnostic specificity of the detection of disseminated
prostatic cells by reverse-transcriptase polymerase chain
reaction (RT-PCR) of PSA mRNA was investigated. A sensitive nested PCR was developed. In blood samples from 10
healthy female and 10 healthy male persons examined by
RT-PCR, mRNA of PSA was detected 3 times in each group.
In the groups of patients suffering from benign prostate
hyperplasia and prostate cancer, 6 of 11 and 5 of 12, respectively, gave positive RT-PCR results. With increasing analytical sensitivity of the RT-PCR of PSA mRNA, the diagnostic
specificity of the assay is decreased. Further development of
this diagnostic method requires the introduction of the
quantitative PCR which may make possible discrimination
between prostatic and non-prostatic source of PSA mRNA by
quantification. Int. J. Cancer, 70:52–56, 1997.
r 1997 Wiley-Liss, Inc.
Early detection of metastases is of great relevance for the therapy
of prostate cancer. Recently, the detection of mRNA of prostatespecific antigen (PSA) by reverse-transcriptase polymerase chain
reaction (RT-PCR) has been introduced to identify prostatic cells in
blood (Moreno et al., 1992), lymph nodes (Deguchi et al., 1993)
and bone marrow (Wood et al., 1994). Provided that the amplified
mRNA is expressed exclusively in the cell of interest, its detection
outside the original organ identifies disseminated cells. PSA is
thought to be expressed only in the prostatic cell (Oesterling, 1991)
and thus fulfills the requirement of this method. The expression of
PSA is not tumor-specific, so the malignancy or metastatic power
of the disseminated cell cannot be evidenced.
Authors using PSA mRNA as an indicator of prostatic cells
disseminated in blood obtained positive RT-PCR results in the
range from 25% (Israeli et al., 1994a) to 80% (Cama et al., 1995)
of patients with metastatic disease. The diversity of published
results indicates that either the blood of some patients with
metastatic disease does not contain PSA-expressing cells or the
sensitivity of the applied method is limited. Nevertheless, RT-PCR
allows the study of such interesting questions as the intra-operative
spillage and hematogenous dissemination of prostate-cancer cells
during radical prostatectomy (Eschwège et al., 1995; Oefelein et
al., 1996) or the prediction of treatment failure following radical
prostatectomy (Olsson et al., 1996).
The diagnostic power of RT-PCR of PSA mRNA depends on the
reliability of the applied method. Therefore, we are interested in
increasing the sensitivity of this approach. The present study demonstrates that increased analytical sensitivity of RT-PCR leads to the
detection of PSA mRNA in the blood of healthy females and males.
MATERIAL AND METHODS
Subjects
We studied 43 subjects, 10 of whom were healthy females and 10
healthy males; 11 were patients with benign prostatic hyperplasia
and 12 were patients with prostate cancer without additional
disease. The average ages (median) of the blood donors, the
patients with hyperplasia and the cancer patients were 43, 67 and
64 respectively. The diagnosis of benign prostate hyperplasia was
based in 6 cases on histological analysis of tissue obtained by
transurethral resection of the gland and in 5 cases on conventional
clinical data (digital rectal examination, transrectal sonogram). The
cancer diagnosis was verified by histology of surgically removed
specimens (6 cases were stages T1 to T3N0M0, and 6 were
T2N1M0 to T4N2M0).
Isolation of mononucleated cells from blood and RNA extraction
Blood samples were collected in 10 ml EDTA-K monovettes
(Sarstedt, Nümbrecht, Germany) at 1 week pre-operatively and at
least 3 weeks after digital rectal examination or prostate needle
biopsy. Mononucleated cells were harvested from blood by FicollPaque (Pharmacia, Uppsala, Sweden) gradient centrifugation. Total
RNA was isolated by the guanidinium-thiocyanate/phenol/
chloroform extraction method (Chomczynski and Sacchi, 1987)
using the TRIzol reagent (GIBCO BRL, Eggenstein, Germany).
The RNA pellet obtained after precipitation with isopropanol and
washing with ethanol was allowed to air-dry and then re-dissolved
in 0.1% diethylpyrocarbonate-treated water. The RNA content was
measured spectrophotometrically at 260 nm.
Reverse transcription
Total RNA (1 µg) was used for cDNA synthesis, applying the
SuperScript II reverse-transcriptase assay (GIBCO BRL) according to the manufacturer’s instructions. RNA and 0.5 µg oligo(dT)12–18 primers were first denaturated for 10 min at 70°C,
chilled on ice for 1 min, and then incubated for 50 min at 42°C in
20 µl of a reaction mixture containing 20 mM Tris-HCl buffer, pH
8.4, 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM
deoxynucleoside triphosphate mix (dNTP), and 200 U of SuperScript II reverse transcriptase. The reaction was terminated by
heating for 15 min at 70°C. The assay was completed by incubation
with RNase H for 20 min at 37°C.
PCR
The synthesized cDNA (10 µl) were used in PCR. The 50-µl PCR
assay contained the following components: 16.7 mM (NH4)2SO4,
67 mM Tris-HCl buffer, pH 8.8, 0.02% (w/v) gelatine, 10 mM
mercaptoethanol, 100 µM dNTP mix and 2.5 U Taq DNA
polymerase (Promega, Heidelberg, Germany). The mixture was
overlaid with mineral oil (Sigma, Deisenhofen, Germany), heated
in the UNO thermal cycler (Biometra, Göttingen, Germany) at
94°C for 4 min and exposed to 40 cycles of heating at 48°C for 1
min, at 72°C for 1 min and at 94°C for 1 min. The last cycle was
*Correspondence to: Universitätsklinikum Charité, Medizinische Fakultät
der Humboldt-Universität zu Berlin, Forschungsabteilung der Klinik für
Urologie, Schumannstrasse 20–21, D-10098 Berlin, Germany. Fax: (049)
30/28021402. E-mail: Henke@rz.charite.hu-berlin.de.
Received 6 June 1996; revised 5 September 1996.
DETECTION OF PROSTATIC CELLS IN BLOOD BY RT-PCR
53
FIGURE 1 – Scheme of primer localization on PSA mRNA. Symbols:
M, I, K and W, primers published by Moreno et al. 1992), Israeli et al.
1994a), Katz et al. (1994) and Wood et al. 1994); subscripts s and a,
sense and anti-sense primer.
finished after prolonged annealing at 72°C for 3 min by cooling to
4°C. Then 3 µl of the first PCR were employed for the second PCR,
which followed the protocol of the first round.
Primers
The primer sets published by Moreno et al. (1992) and Wood et
al. (1994) as well as the outer primer sets of Israeli et al. (1994a)
and Katz et al. (1994) were included in our investigations. Figure 1
illustrates the binding sites of these primers on exons 3, 4, and 5 of
PSA mRNA. Finally, we tested different primer combinations with
nested PCR. PSA displays a high degree of homology with human
glandular kallikrein. The published primers are selected to avoid
co-amplification of this prostatic mRNA. b-globin primers were
used to verify the integrity of RNA as described (Jaakkola et al.,
1995).
Agarose-gel electrophoresis, digestion by SacI, and sequencing
The PCR products were separated by agarose-gel electrophoresis
using 2% agarose (Nusieve, FMC Bioproducts, Rockland, ME) and
visualized with ethidium bromide. The PCR product was digested
with the restriction enzyme SacI (Promega) according to the
manufacturer’s instructions.
PCR products were sequenced by the dideoxynucleotide-chaintermination method on the automated DNA sequencer A.L.F.
(Pharmacia, Freiburg, Germany). The second PCR round was
performed with a biotinylated primer, used to separate the NaOHtreated double DNA strand by streptavidin-coated magnetic beads
(Dynal, Hamburg, Germany), and a fluorescein-labeled sequencing
primer nested in a 3-base-pair distance to the original primer. The
sequencing reaction was done by the sequenase system.
Cell culture
LNCaP cells were grown in RPMI-1640 medium supplemented
with 10% FCS, 125 kU penicillin, and 125 mg/l streptomycin.
Confluent monolayers were washed twice with 2 ml of PBS at
room temperature and detached using trypsin/EDTA solution
(0.05%, 0.02%) at 37°C. Serial dilutions of LNCaP cells in PBS
were added to 5 ml blood of female subjects, mixed gently and
treated as described above.
RESULTS
We investigated detection of the mRNA of PSA with a nested
PCR consisting of 40 cycles per each round. To optimize the
sensitivity of this assay, several combinations of published primers
were tested. Finally, the primer combination 1, 2, and 3 consisting
of Is/Ia and Ms/Ma, Is/Ka and Ks/Ia, and Ks/Ka and Ws/Wa were
investigated in the first and second rounds of PCR (Fig. 1). The
assay sensitivity was determinated by dilution series of PSAgenerating LNCaP cells using blood of healthy females. Whereas
FIGURE 2 – Sensitivity of the detection of prostatic cells by RT-PCR
of PSA mRNA tested by dilution series of LNCAP cells in whole blood.
Lanes 3–5, 6–8 and 9–11, primer combinations 1, 2 and 3, respectively,
using 50, 5 and 1 LNCaP cells/106 leukocytes (ascending lane number);
lane 1, water control; lanes 2 and 12, DNA marker IX (Boehringer,
Mannheim, Germany).
TABLE I – DETECTION OF PSA mRNA BY NESTED RT-PCR IN BLOOD
OF HEALTHY FEMALE AND MALE SUBJECTS AND IN PATIENTS SUFFERING
FROM BENIGN PROSTATE HYPERPLASIA
AND PROSTATE CANCER
Source
Number
Positive RT-PCR
results (%)
Healthy females
Healthy males
Benign prostate hyperplasia patients
Prostate-cancer patients
10
10
11
12
3 (33)
3 (33)
6 (54)
5 (42)
an average of 1 LNCaP cell per 106 leukocytes is detected by using
primer combination 1, primer combinations 2 and 3 give lower
sensitivity, about 5 and 50 LNCaP cells per 106 leukocytes
respectively (Fig. 2). Further investigations were performed with
primer combination 1.
Analyzing blood from 10 healthy female and 10 healthy male
subjects by nested RT-PCR, we detected PSA mRNA 3 times in
each group (Table I). In the groups of patients suffering from
benign prostate hyperplasia and prostate cancer, 6 of 11 and 5 of 12
gave positive RT-PCR results (Fig. 3). Controls performed without
added RNA or reverse transcriptase were always negative, so it is
unlikely that the positive results are caused by cross-contamination.
Since the primers of the second PCR bind in exon 3 and exon 4, the
amplicon of PSA cDNA can be clearly distinguished from that of
the intron-containing genomic DNA (Moreno et al., 1992), which
might contaminate the RNA.
The amplicon of PSA has a cleavage site for the restriction
enzyme SacI. Digestion of the amplicon with SacI produced 2
restriction fragments of the expected size (Fig. 4). Human glandular kallikrein cDNA, the sequence of which has a high degree of
homology with that of PSA, has no SacI restriction site. Thus, the
amplification of human glandular kallikrein mRNA is ruled out.
The identity of the desired PCR products was verified by
sequencing the amplicon by the dideoxynucleotide-chain-termination method with a fluorescein-labeled primer using the sequenase
reaction. The determined sequences of the amplicons from one
healthy female and one healthy male, as well as from one
HENKE ET AL.
54
TABLE II – SUMMARY OF PUBLISHED RESULTS OBTAINED WITH RT-PCR OF
PSA AND PSM IN BLOOD OF PATIENTS WITH METASTATIC PROSTATE CANCER
Author
mRNA
Moreno et al. (1992)
Seiden et al. (1994)
Katz et al. (1994)
Jaakkola et al. (1995)
Ghossein et al. (1995)
Cama et al. (1995)
PSA
PSA
PSA
PSA
PSA
PSA
PSM
PSA
PSM
PSA
PSM
Israeli et al. (1994a)
Loric et al. (1995)
FIGURE 3 – Detection of PSA mRNA in blood of persons without
prostate cancer. Lanes 2 to 6, PSA primers; lanes 6 and 11, water
control; lanes 7 to 11, b-globin primers; lanes 1 and 12, DNA marker
IX; lane 2, male subject; lanes 3 and 4, female subject; lane 5, patient
with benign prostate hyperplasia.
FIGURE 4 – Digestion of PCR product by the restriction enzyme
SacI. PCR product of a healthy female before and after digestion with
SacI (lanes 1 and 2). DNA marker IX (lane 3) and water control (lane
4).
benign-prostate-hyperplasia patient, agree with those published for
PSA by Lundwall (1989).
DISCUSSION
Our data show that mRNA of PSA can be detected in the blood of
healthy female and male subjects with a sensitive RT-PCR applying
nested primers. Data from controls make it unlikely that the
positive results are caused by cross-contamination, human glandularkallikrein cDNA or genomic DNA. Our results are in contrast to
data of several other studies (Moreno et al., 1992; Katz et al., 1994;
Seiden et al., 1994; Ghossein et al., 1995; Israeli et al., 1995;
Jaakkola et al., 1995; Oefelein et al., 1996). However, Smith et al.
(1995) and O’Hara et al. (1996) also detected PSA mRNA by a
Patients with
metastatic
prostate
cancer
12
35
18
18
76
20
24
33
Positive
RT-PCR
results (%)
4 (33)
11 (31)
14 (78)
9 (50)
26 (34)
16 (80)
10 (50)
6 (25)
16 (67)
17 (51)
28 (85)
sensitive nested PCR in non-prostatic cell lines and in blood from
healthy men and women. Zarghami and Diamandis (1996) found
PSA mRNA in breast tumors of female patients.
From the published data, we conclude that the analytical
sensitivity of the RT-PCR is one determinant for the diagnostic
significance of this assay. The sensitivity of our assay was
optimized by an increased number of cycles per PCR and selection
of one from 3 primer combinations investigated. Primer combination 1, incorporating primer sets published by Israeli et al. (1994a)
and Moreno et al. (1992), gave the highest sensitivity in our
experimental conditions. The finding that primer selection is
decisive for the sensitivity of PCR is in agreement with the
experience of He et al. (1994).
We evaluated the assay sensitivity by dilution series of LNCaP
cells with blood and detected an average of 1 LNCaP cell per 106
leukocytes. With whole blood used as diluting medium, the same
sensitivity has been also found by other authors (Seiden et al.,
1994; Jaakkola et al., 1995; Loric et al., 1995). In several reports,
the assay sensitivity was measured by diluting LNCaP cells with
isolated non-prostatic cells (Deguchi et al., 1993; Israeli et al.,
1994a,b; Katz et al., 1994; Wood et al., 1994; Ghossein et al.,
1995). The sensitivity determined in this way can hardly be
compared with that based on whole blood, since recovery of
LNCaP cells in the mononuclear fraction was not evaluated. The
dilution of cDNA (Israeli et al., 1994a) with water or of RNA
isolated from LNCaP cells with non-prostatic RNA (Seiden et al.,
1994; Oefelein et al., 1996) has no practical relevance for the
characterization of the assay sensitivity.
Table II summarizes results obtained with RT-PCR of PSA and
prostate-specific membrane antigen (PSM), a further protein assumed to be specifically expressed in prostatic cells (Israeli et al.,
1994b), from blood of patients with prostate cancer. Israeli et al.
(1994a) or Loric et al. (1995), targeting on PSA, detected prostate
cells in only 25 or 51%, respectively, of patients with metastatic
cancer. However, prostate cells are present in blood, as can be
concluded from the data obtained with RT-PCR of mRNA of PSM
(Israeli et al., 1994a; Loric et al., 1995), of at least 67 or 85% of the
same patients. Obviously, the RT-PCR of PSA mRNA performed
by these authors does not detect all cases of disseminating prostate
cells. The RT-PCR applied by Cama et al. (1995) seems to be
characterized by an inverse relationship of the sensitivity for
mRNA of PSA and PSM, since their methods detected prostate
cells in 80 and 50% of the investigated patients respectively. The
authors, investigating mRNA of both PSA and PSM, checked the
sensitivity of the respective method by dilution series of LNCaP
cells. No sensitivity differences are described for the 2 targets
(Israeli et al., 1994a; Cama et al., 1995; Loric et al., 1995). Thus,
the method currently used to evaluate the sensitivity gives only a
rough estimation of the analytical sensitivity.
Eschwège et al. (1995) investigated the hematogenous dissemination of prostatic cells during radical prostatectomy by RT-PCR of
DETECTION OF PROSTATIC CELLS IN BLOOD BY RT-PCR
PSM mRNA and found that anti-androgen therapy reduces the
hematogenous spread. Hamdy and Neal (1996) pointed out that
androgen ablation depressed the number of tumor cells by apoptosis. Therefore, the drop in numbers of disseminating prostate cells
below the assay sensitivity should be considered as one possible
reason for the finding of Eschwège et al. (1995).
Olsson et al. (1996) evaluated the role of RT-PCR of PSA mRNA
in predicting disease recurrence. Comparing the predictive power
of pre-operative serum PSA, Gleason score and PSA mRNA, the
RT-PCR assay has the lowest univariate relative risk. The applied
RT-PCR method gave positive results in 85% of patients with
confirmed metastatic prostate cancer.
In the light of published data, the sensitivity of the RT-PCR
should be increased, to reduce the influence of methodical uncertainty on the result of studies. Diamandis and Yu (1995) pointed to
several methodological facts that could explain the requirement to
increase the analytical sensitivity of this approach. Such an
increase of analytical sensitivity by applying a nested PCR implies,
in agreement with Smith et al. (1995) and O’Hara et al. (1996),
with a decrease in diagnostic specificity. The assumption that PSA
is specifically expressed in prostate cells is now being revised.
Thus, immunoreactive PSA is ultrasensitively-assayed in normal
endometrial tissue and amniotic fluid, also in breast, ovarian, liver,
kidney, adrenal, colon, parotid and lung tumors (Diamandis, 1995).
More than one thousand female sera were analyzed with an
ultrasensitive PSA assay. In 17% of persons in this group, PSA was
measurable (Yu and Diamandis, 1995). Randell et al. (1996) found
detectable concentrations of PSA in many cord sera and in sera
55
from male and female neonates. Although most children at ages 1
to 10 years had undectable PSA, it was found in a few. The lack of
any apparent sex-related difference again suggests that nonprostatic sources may contribute to serum PSA (Randell et al.,
1996). Knowledge of the biological functions of PSA, originally
attributed solely to the liquefaction of seminal fluid, is increasing.
Published results suggest that PSA is either a growth factor or a
growth-factor regulator (Diamandis, 1995).
Besides growing evidence of PSA expression in non-prostatic
tissues, some data show that increased analytical sensitivity of PCR
may be connected with decreased diagnostic specificity. Schoenfeld et al. (1994) tested RT-PCR of cytokeratin 19 mRNA for
detecting breast-cancer micrometastases in axillary lymph nodes.
With a sensitivity increased by nested PCR, this mRNA was found
in lymph nodes without cancer. Biernaux et al. (1995) found
mRNA of the major bcr-abl fusion gene, presently seen as the
hallmark of chronic myeloid leukemia, in the blood of healthy
individuals, by means of a very sensitive nested RT-PCR.
Using RT-PCR of PSM mRNA positive results with healthy
female and male subjects are not described in the literature. Further
development of this assay might therefore be concentrated on PSM
mRNA as an indicator of disseminating prostate cells. Nevertheless, the introduction of competitive RT-PCR should improve the
reliability of this method, since the assay sensitivity can be
controlled and the signal intensity can be quantified, e.g., by a
microtiter-plate-based assay (Galvan et al., 1995). Quantification
might make possible discrimination between non-prostatic and
prostate-derived mRNA.
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