Protein profiling of post-prostatic massage urine specimens by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to discriminate between prostate cancer and benign lesions

Abstract

Post-prostatic massage urine specimens (PMUS) are expected to be rich in proteins originating from the prostatic acini. In this study, we created a PMUS bank consisting of 57 samples obtained from patients with biopsy-proven prostate cancer (PC) and 56 samples from subjects with biopsy-proven benign lesions to analyze protein profiles of PMUS by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Strong anion-exchange (Q10), weak cation-exchange (CM10) and immobilized metal affinity capture (IMAC30) ProteinChip Arrays were used for protein profiling. In PC samples, single-marker analysis detected 49 mass peaks that were significantly up-regulated and 23 peaks that were significantly down-regulated, compared with peaks obtained from benign lesion samples. To confirm reproducibility we performed additional three rounds of assay using CM10 chip with pH 4.0 binding buffer. Among these significant peaks, a peak of m/z 10788 was significant throughout all 4 rounds of assays. For hierarchical clustering analysis (HCA), we used the 72 peaks which revealed significant differences in single-marker analysis. The heat map discriminated PC from benign lesions with a sensitivity of 91.7% and a specificity of 83.3%. Therefore, SELDI-TOF MS profiling of PMUS can be applied to differentiate patients with PC from cancer-free subjects. However, further investigation is required to verify the usefulness of this method in clinical practice.

Full text

Abstract. Post-prostatic massage urine specimens (PMUS) are
expected to be rich in proteins originating from the prostatic
acini. In this study, we created a PMUS bank consisting of
57 samples obtained from patients with biopsy-proven prostate
cancer (PC) and 56 samples from subjects with biopsy-proven
benign lesions to analyze protein profiles of PMUS by
surface-enhanced laser desorption/ionization time-of-flight
mass spectrometry (SELDI-TOF MS). Strong anion-exchange
(Q10), weak cation-exchange (CM10) and immobilized metal
affinity capture (IMAC30) ProteinChip Arrays were used for
protein profiling. In PC samples, single-marker analysis
detected 49 mass peaks that were significantly up-regulated
and 23 peaks that were significantly down-regulated, compared
with peaks obtained from benign lesion samples. To confirm
reproducibility we performed additional three rounds of assay
using CM10 chip with pH 4.0 binding buffer. Among these
significant peaks, a peak of m/z 10788 was significant
throughout all 4 rounds of assays. For hierarchical clustering
analysis (HCA), we used the 72 peaks which revealed
significant differences in single-marker analysis. The heat map
discriminated PC from benign lesions with a sensitivity of
91.7% and a specificity of 83.3%. Therefore, SELDI-TOF MS
profiling of PMUS can be applied to differentiate patients with
PC from cancer-free subjects. However, further investigation
is required to verify the usefulness of this method in clinical
practice.
Introduction
Prostate cancer (PC) is the most common type of cancer in
men and the second highest cause of cancer death in the
United States (1). Recently, mortality rates for PC have been
increasing dramatically in Japan (2). Early detection of PC has
become easier by measuring prostate-specific antigen (PSA);
however, an urgent need exists for novel biomarkers to
improve the specificity of PC detection.
A number of innovations have been made to improve the
specificity of PSA testing. The most successful of these,
measurement of alternative molecular forms of PSA expressed
as the percentage of free PSA, improves the diagnostic
specificity of PSA testing (3,4) and can decrease the number
of false-negative prostate biopsies by ~20-25% (5). Moreover,
PSA velocity, age-specific PSA, PSA density and proPSA
have been postulated to improve the specificity of PSA testing
(6). However, the incidence of PC is shown as high as 22%
among men with a normal PSA range, 2.6-4.0 ng/ml (7).
Furthermore, PSA testing is almost organ-specific, but not
cancer-specific, because elevated serum concentrations are
also found in benign diseases, such as benign prostatic
hypertrophy and prostatitis. Therefore, great emphasis has
been placed on the need to discover novel biomarkers for use
in PC diagnosis.
Proteomic techniques applied to serum or plasma may
provide valuable information regarding protein markers or
patterns of markers that could possibly be used to improve
cancer detection (8). Serum protein profiling with surface
enhanced laser desorption/ionization time-of-flight mass
spectrometry (SELDI-TOF MS) has been shown to detect
cancers, including PC (9). In addition, several case-control
studies have reported excellent validity for PC detection
(10-13).
Serum proteomic approaches have not provided useful
biomarkers for PC in a clinical setting. In order to address this
problem, we conducted a proteomic study on protein
originating from prostate acini obtained by non-invasive
sampling. Post-prostatic massage urine specimens (PMUS),
ONCOLOGY REPORTS 21: 73-79, 2009 73
Protein profiling of post-prostatic massage urine
specimens by surface-enhanced laser desorption/ionization
time-of-flight mass spectrometry to discriminate
between prostate cancer and benign lesions
AKIKO OKAMOTO1, HAYATO YAMAMOTO1, ATSUSHI IMAI1, SHINGO HATAKEYAMA1,
IKUYA IWABUCHI1, TAKAHIRO YONEYAMA1, YASUHIRO HASHIMOTO1, TAKUYA KOIE1,
NORITAKA KAMIMURA1, KAZUYUKI MORI1, KANEMITSU YAMAYA2and CHIKARA OHYAMA1
1Department of Urology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562;
2Oyokyo Kidney Research Institute, 90 Kozawa Yamazaki, Hirosaki 036-8243, Japan
Received August 5, 2008; Accepted September 29, 2008
DOI: 10.3892/or_00000191
_________________________________________
Correspondence to: Dr Chikara Ohyama, Department of Urology,
Hirosaki University Graduate School of Medicine, 5 Zaifu-cho,
Hirosaki 036-8562, Japan
E-mail: coyama@cc.hirosaki-u.ac.jp
Key words: protein profile, prostate cancer, prostate massage
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·73

which have been established as diagnostic samples for
prostatitis (14), are expected to be rich in proteins originating
from prostatic acini. Moreover, to our knowledge, this is the
first detailed study describing protein profiling of PMUS by
SELDI-TOF MS.
Materials and methods
Post-prostatic massage urine specimen (PMUS). A flowchart
of this study is illustrated in Fig. 1. PMUS was collected
after digital rectal examination (5 strokes per lobe). Urine
was voided into urine collection cups, briefly centrifuged
(10 min at 2,000 x g), aliquotted, frozen immediately and
stored at -80˚C until protein profile analysis. The PMUS
bank consisted of 57 samples from patients with biopsy-
proven PC and 56 samples from subjects with biopsy-proven
benign lesions. The study was approved by the Institutional
Ethics Committee and a written consent was obtained from
all subjects who participated in the study.
Protein concentration measurement and prostate-specific
antigen assay. Protein concentration of PMUS was
measured by Immage 800 (Beckman Coulter Inc., Brea, CA,
USA) and prostate-specific antigen (PSA) in serum was
measured by Immulite 1000 (Siemens, Deerfield, IL, USA).
Prostate biopsy and pathological diagnosis. After collection
of PMUS, 10 or 12 prostate needle biopsy samples were
transrectally obtained by ultrasound guidance, using an 18 G
needle. The 2002 TNM staging system (15) was used to assign
the stage and the up-dated Gleason grading system from the
International Society of Urological Pathology (ISUP) (16) was
used for tumor grading.
Analysis of protein profiles. PMUS samples were briefly
centrifuged (10 min at 20,000 x g) and the supernatants
were subjected to protein profiling. Protein profiles of the
PMUS samples were obtained by using weak cation-
exchange (CM10), strong anion-exchange (Q10) and
immobilized metal affinity capture (IMAC30) ProteinChip
Arrays (Bio-Rad, Fremont, CA, USA). The ProteinChip
Arrays were assembled into a deep-well type Bioprocessor
assembly (Bio-Rad). Prior to sample loading, Q10 and
CM10 arrays were equilibrated with 150 μl of binding
buffer (for Q10, 50 mM Tris-HCl, pH 8.0; for CM10, 100 mM
sodium acetate, pH 4.0 and 50 mM HEPES, pH 7.0).
Before the samples were loaded, IMAC30 arrays were
charged with Cu2+ by adding 50 μl of 100 mM CuSO4.
After incubation for 5 min, the arrays were quickly rinsed
with water to remove the unbound metal and the surface
was further washed with 50 μl of 100 mM sodium acetate,
pH 4.0. The arrays were then equilibrated with 150 μl of
binding buffer (100 mM sodium phosphate with 0.5 M NaCl,
pH 7.0).
A 10 μl-portion of PMUS was mixed with 30 μl of 2%
CHAPS/9 M urea/50 mM Tris-HCl, pH 9.0 and further diluted
with 60 μl of binding/washing buffer. All arrays were then
incubated with 100 μl of diluted sample for 60 min on a shaker
and washed 3 times with 150 μl of binding buffer. After rinsing
with water, the arrays were removed from the Bioprocessor
assembly and air-dried. After air-drying, a 1.0 μl aliquot of
50% saturated sinapinic acid solution (dissolved in 50%
acetonitrile containing 0.5% trifluoroacetic acid) was added
twice and allowed to dry.
The ProteinChip Arrays were then transferred to the
ProteinChip System Series 4000 (Bio-Rad) which generates
nanosecond laser pulses from a UV-emitting pulsed nitrogen
laser (373 nm). External mass calibration was performed with
protein standards: porcine dynorphin (2148 Da), human
adrenocorticotropic hormone (2934 Da), bovine insulin ß-chain
(3496 Da), human insulin (5808 Da), recombinant hirudin
(6964 Da), bovine cytochrome C (12230 Da), equine
myoglobin (16951 Da), bovine carbonic anhydrase (29023 Da)
and enolase from Saccharomyces cerevisiae (46671 Da). All
assays were repeated twice.
The protein expression patterns were analyzed using
CiphergenExpress Data Manager software, version 3.0
(Bio-Rad), which generates consistent mass peak sets
(clusters) across multiple spectra and enables automatic
comparison. Each cluster was treated as a single protein or
peptide fragment. All data were normalized by the software's
total ion current normalization function, following the
manufacturer's instructions. Spectra between 2500 and
150000 mass-to-charge ratios (m/z) were selected for analysis.
Automatic peak detection was carried out for peaks with
signal/noise ratio >2.5. The Mann-Whitney U test was used to
compare intensities of clustered peaks between the 2 sample
groups.
Single-marker analysis. To identify a candidate peak, we used
CM10, Q10 and IMAC 30 chips. To confirm reproducibility,
we carried out additional 3 rounds of analysis by CM10
chip with pH 4.0 binding buffer. PMUS were randomly
selected from each group for each round. For the first round
analysis, we randomly selected 12 samples each from the
PC-PMUS pool and the benign lesion-PMUS pool. For the
second, third and fourth round analyses, we randomly
selected 10, 8 and 29 samples from each group, respectively
(Fig. 1). Finally, 37 PC samples and 39 benign lesion
samples were examined for single-marker analysis.
Demographic data on the subjects are shown in Table I.
OKAMOTO et al: PROTEIN PROFILE OF URINE AFTER PROSTATE MASSAGE
74
Figure 1. The study design. The PMUS sample bank consisted of 57 samples
from patients with biopsy-proven PC and 56 samples from subjects with
biopsy-proven benign lesions. PMUS samples were randomly selected for
protein profiling. Overall, 39 samples from benign lesions and 37 samples
from PC were subjected to single-marker analysis. Twelve samples from
each pool were subjected to hierarchical clustering analysis (HCA).
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·74

Hierarchical clustering analysis (HCA). HCA was performed
to create a heat map using CiphergenExpress Data Manager
software, version 3.0 (Bio-Rad). For HCA analysis, we
used the 72 peaks, which revealed significant differences in
single-marker analysis. Clinicopathological data on the
subjects whose PMUS were subjected to HCA are shown in
Table II.
Results
In normal urine samples from healthy subjects, protein cannot
be detected. However, protein concentration of PMUS was
successfully measurable in all specimens as shown in Table I.
Mean protein concentration of PMUS from patients with PC
was 102.3 μg/ml and that from benign lesion was 113.5 μg/ml.
ONCOLOGY REPORTS 21: 73-79, 2009 75
Table I. Demographic data on subjects for single-marker analysis.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
B (n=39) PC (n=37)
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Age (years) (mean; range) 68.0 (53-81) 70.3 (53-79)
Serum PSA (ng/ml) (mean; range) 6.9 (2.1-12.5) 15.7 (4.4-111.2)
PMUS protein conc. (μg/ml) (mean; range) 102.3 (27.2-353.5) 113.5 (25.1-338.1)
Gleason score (mean; range) - 7.3 (5-9)
Clinical stage - T1cN0M0-T4N1M1
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
PMUS, post-prostate massage urine specimen; B, benign lesion and PC, prostate cancer.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Table II. Clinicopathological data on subjects used for hierarchical clustering analysis (HCA).
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Case Age Serum PSA Pathology Gleason Clinical PMUS
(year) (ng/ml) score stage concentration (μg/ml)
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
B1 74 6.8 B - - 140.4
B2 70 7.2 B - - 27.2
B3 72 7.3 B - - 123.6
B4 75 8.8 B - - 41.3
B5 61 2.5 B - - 278.1
B6 68 6.6 B - - 57.1
B7 57 6.6 B - - 76.0
B8 60 12.3 B - - 57.1
B9 66 4.1 B - - 38.1
B10 71 4.9 B - - 53.9
B11 70 8.3 B - - 34.9
B12 68 5.2 B - - 70.7
PC1 74 5.2 PC 7 T2aN0M0 45.7
PC2 76 12.2 PC 7 T1cN0M0 291.9
PC3 74 11 PC 7 T2aN0M0 271.3
PC4 67 15.2 PC 7 T3N0M1 25.1
PC5 72 5.8 PC 9 T3N0M0 46.5
PC6 71 76.8 PC 9 T3N0M0 40.7
PC7 76 9.2 PC 7 T2aN0M0 144.8
PC8 68 7.8 PC 9 T2aN0M0 102.3
PC9 75 15.5 PC 7 T2aN0M0 44.4
PC10 77 34.5 PC 7 T3N0M0 86.5
PC11 63 8.6 PC 7 T2aN0M0 86.5
PC12 75 8.8 PC 7 T2bN0M0 138.1
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
PMUS, post-prostate massage urine specimen; B, benign lesion and PC, prostate cancer.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·75

PMUS protein concentration ranged from 25.1 μg/ml to
353.5 μg/ml. Therefore, we diluted PMUS sample with 2%
CHAPS/9 M urea/50 mM Tris-HCl to obtain final protein
concentration of 25.0 μg/ml.
For single-marker analysis, peak intensities detected by
three kinds of chips were compared between the 2 groups.
As a demonstrable example, protein profiles of the first
assay round are presented in Fig. 2. The peak intensity of m/z
4761 in the PC group was significantly higher than peaks in
the benign lesion group (P=0.0010; Fig. 3a). The receiver
OKAMOTO et al: PROTEIN PROFILE OF URINE AFTER PROSTATE MASSAGE
76
Figure 2. Protein profiling using CM10 chip with pH 4.0 buffer. The peak
intensity of m/z 4761, surrounded by line, was markedly higher in the PC
group than in the benign lesion group.
Figure 4. Heat map based on the results of protein profiling, using hierarchical clustering analysis. Horizontal line below the heat map represents case number.
Vertical line represents the 72 significant peaks and chip conditions, which correspond to peak information presented in Table IV. According to the heat map,
we were able to discriminate PC from benign lesions with 91.7% sensitivity and 83.3% specificity. B, benign lesion and PC, prostate cancer.
Figure 3. Difference in peak intensity of m/z 4761 between PC and benign
lesion groups. (a) The peak intensity of m/z 4761 was significantly higher in
the PC group (P=0.0010). (b) The receiver operating characteristic curve
(ROC) was plotted for m/z 4761. The area under the curve (AUC) on the ROC
plot was 0.917.
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·76

operating characteristic curve (ROC) of m/z 4761 is shown in
Fig. 3b. The area under the curve (AUC) on the ROC was
0.917.
In PC samples, single-marker analysis detected 49 mass
peaks that were significantly up-regulated and 23 peaks that
were significantly down-regulated, compared with peaks
obtained from benign lesion samples. Statistical data and
chip conditions of these peaks are shown in Table III.
To confirm reproducibility, we repeated the assay four
times in total using by CM10 chip with pH 4.0 binding buffer.
Random selection from PMUS bank for repeated single-
marker analysis caused some overlaps of samples. So, finally
we analyzed 37 PC samples and 39 benign lesion samples.
Results with repeated single-marker analysis are summarized
in Table IV. Although the significantly increased or decreased
peaks varied in each assay round, peaks of m/z 10788 showed
significantly lower intensity in the PC group throughout all
assay rounds. The peak of m/z 5384, which showed
significantly lower intensity in 3 assay rounds, is deduced to
be a double charge of the peak of m/z 10788.
ONCOLOGY REPORTS 21: 73-79, 2009 77
Table III. Statistical data and chip conditions of significant
peaks detected in single-marker analysis.
–––––––––––––––––––––––––––––––––––––––––––––––––
M/Z P-value ROC area Chip condition
–––––––––––––––––––––––––––––––––––––––––––––––––
Up-regulated in PC
2670 0.0027 0.861 CM10 pH 4.0
2776 0.0282 0.750 CM10 pH 4.0
2797 0.0243 0.722 CM10 pH 4.0
2978 0.0056 0.806 CM10 pH 4.0
3003 0.0010 0.861 CM10 pH 4.0
3023 0.0005 0.889 CM10 pH 4.0
3174 0.0282 0.778 Q10 pH 8.0
3204 0.0496 0.722 IMAC30
3272 0.0111 0.778 IMAC30
3375 0.0079 0.806 Q10 pH 8.0
3461 0.0012 0.889 CM10 pH 4.0
3496 0.0022 0.889 CM10 pH 4.0
3721 0.0018 0.861 CM10 pH 4.0
3773 0.0027 0.833 CM10 pH 4.0
3786 0.0496 0.722 Q10 pH 8.0
3851 0.0022 0.861 CM10 pH 4.0
3897 0.0377 0.750 CM10 pH 4.0
3938 0.0209 0.750 CM10 pH 4.0
3997 0.0243 0.778 Q10 pH 8.0
4026 0.0079 0.806 CM10 pH 7.0
4028 0.0002 0.917 CM10 pH 4.0
4056 0.0243 0.778 Q10 pH 8.0
4478 0.0377 0.750 CM10 pH 4.0
4544 0.0002 0.944 CM10 pH 4.0
4582 0.0243 0.778 CM10 pH 4.0
4761 0.0010 0.917 CM10 pH 4.0
4763 0.0282 0.778 CM10 pH 7.0
4781 0.0022 0.889 CM10 pH 4.0
4828 0.0153 0.778 Q10 pH 8.0
4862 0.0209 0.778 Q10 pH 8.0
4968 0.0001 0.944 CM10 pH 4.0
5017 0.0047 0.833 Q10 pH 8.0
5817 0.0209 0.778 CM10 pH 4.0
6200 0.0377 0.750 Q10 pH 8.0
6481 0.0027 0.833 CM10 pH 4.0
8030 0.0039 0.833 IMAC30
8037 0.0007 0.889 CM10 pH 4.0
8202 0.0179 0.778 CM10 pH 4.0
8309 0.0179 0.806 CM10 pH 4.0
8871 0.0056 0.806 CM10 pH 4.0
9098 0.0056 0.806 CM10 pH 4.0
9102 0.0327 0.722 IMAC30
9207 0.0433 0.750 CM10 pH 4.0
9281 0.0047 0.833 CM10 pH 4.0
9780 0.0111 0.833 CM10 pH4.0
Table III. Continued.
–––––––––––––––––––––––––––––––––––––––––––––––––
M/Z P-value ROC area Chip condition
–––––––––––––––––––––––––––––––––––––––––––––––––
Up-regulated in PC
9905 0.0056 0.806 CM10 pH 4.0
9990 0.0209 0.750 CM10 pH 4.0
-------------------------------------------------------------------------
Down-regulated in PC
4702 0.0496 0.778 Q10 pH 8.0
4827 0.0496 0.722 IMAC30
5333 0.0496 0.722 CM10 pH 4.0
5339 0.0433 0.750 Q10 pH 8.0
5384 00039 0.833 CM10 pH 4.0
5395 0.0067 0.806 Q10 pH 8.0
7281 0.0153 0.778 Q10 pH 8.0
7589 0.0094 0.778 Q10 pH 8.0
7764 0.0027 0.861 Q10 pH 8.0
10668 0.0111 0.806 Q10 pH 8.0
10677 0.0153 0.778 IMAC30
10678 0.0153 0.806 CM10 pH 4.0
10778 0.0094 0.778 IMAC30
10782 0.0079 0.806 Q10 pH 8.0
10788 0.0067 0.806 CM10 pH 4.0
10888 0.0179 0.806 CM10 pH 4.0
10985 0.0067 0.833 Q10 pH 8.0
10995 0.0111 0.806 CM10 pH 4.0
11201 0.0153 0.778 CM10 pH 4.0
11397 0.0079 0.833 CM10 pH 4.0
13909 0.0111 0.833 IMAC30
28094 0.0433 0.750 IMAC30
38025 0.0111 0.778 IMAC30
–––––––––––––––––––––––––––––––––––––––––––––––––
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·77

To create a heat map 72 significant peaks which identified
in the first round single-marker analysis were used. According
to the heat map based on the data from these 72 significant
peaks (Fig. 4), we were able to discriminate PC from benign
lesions with a sensitivity of 91.7% and a specificity of
83.3%.
Discussion
SELDI-TOF and matrix-assisted laser desorption/
ionization-TOF MS have been recognized as the most
common techniques for protein profiling (17). These
techniques have been applied to discover a novel biomarker
for PC (11-13,18). However, recent studies emphasize on
the limited usefulness of proteomic approach for identifying
candidates for serum proteins (19). To overcome these
problems, we conducted a proteomic study using PMUS. Most
proteins synthesized in prostatic epithelium are secreted
into prostatic acini and drained into the prostatic duct.
Thus, PMUS is expected to be rich in proteins originating
from prostatic epithelium. As demonstrated in the present
study, protein concentration in PMUS was much higher than
in urine. Moreover, this finding suggests that an abundant
source of proteins in PMUS originates from the prostatic
acini.
Lack of reproducibility in SELDI-TOF MS whole-serum
proteomic profiling has also been noted (20). To overcome the
weak point in reproducibility, we repeated our assays. For
single-marker analysis we performed 4 rounds of SELDI-TOF
MS analysis.
In the first single-marker analysis, we found 49 peaks that
were significantly up-regulated in the patients with PC and
23 peaks that were significantly down-regulated. During the
4 rounds of assays, significant peaks varied from round to
round. Among the significant peaks, a peak of m/z 10788
remained significant throughout all the rounds. Furthermore,
the peak of m/z 5384, which showed significantly lower
intensity in 3 assay rounds, may be a double charge of the peak
of m/z 10788. Therefore, we believe that the peak of m/z
10788 could be a promising single-marker for early detection
of PC. Further study is required focusing on the structural
analysis for this peak.
For HCA analysis, we used 72 peaks that proved significant
in the first-round assay. In spite of the small sample size, we
were able to discriminate biopsy-proven PC from benign
lesions with high sensitivity and specificity by using the
heat map. Especially, its high specificity of 83.3% is
remarkably higher than that of PSA test for the detection of
PC (6,7). However, this method along with single-marker
analysis, requires further investigation with a large number of
samples.
In this preliminary study, we postulated that promising
urine markers, originating from prostatic acini, can be obtained
by prostatic massage. Our assays identified a potential marker
to differentiate patients with PC from cancer-free subjects.
However, as stated previously (21), all candidate markers must
be strictly evaluated through multi-step checkpoints including
accurate methods for detecting markers, single institutional
pilot studies and rigorous validation in retrospective and
prospective studies.
Acknowledgements
This study was supported by a grant from the CREST (Core
Research for Evolutional Science and Technology) project of
the Japan Science and Technology Agency.
OKAMOTO et al: PROTEIN PROFILE OF URINE AFTER PROSTATE MASSAGE
78
Table IV. Significant peaks detected by repeated single-marker analysis using by CM10 with pH 4.0 binding buffer.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Assay round
1st 2nd 3rd 4th
–––––––––––––––– –––––––––––––––– –––––––––––––––– ––––––––––––––––
P-value AUC P-value AUC P-value AUC P-value AUC
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Up-regulated
(m/z)
4761 0.0012 0.917 0.0112 0.861 NS - NS -
5817 0.0209 0.778 0.0413 0.742 NS - 0.0338 0.643
8037 0.0007 0.889 NS - NS - 0.0351 0.609
8871 0.0056 0.806 NS - NS - 0.017 0.679
9098 0.0218 0.806 NS - NS - 0.0218 0.648
9780 0.0111 0.833 NS - NS - NS -
Down-regulated
(m/z)
5384 0.0039 0.833 NS - 0.0157 0.844 0.0393 0.617
10788 0.0067 0.806 0.0162 0.783 0.0274 0.797 0.0474 0.644
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
NS, not significant and AUC, area under the curve.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
73-79 3/12/2008 10:18 Ì ™ÂÏ›‰·78

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