Molecular Biomarker in Prostate Cancer: The Role of CpG Island Hypermethylation

Abstract

CpG island hypermethylation may be one of the earliest somatic genome alterations to occur during the development of multiple cancers. Recently, aberrant methylation patterns for different tumors have been reported. We present a comprehensive review of the literature describing the role of CpG island hypermethylation of DNA from prostatic tissue and bodily fluids from men with prostate cancer. We reviewed the literature to evaluate CpG island hypermethylation in tissue and bodily fluids of men with primary and metastatic prostate cancer. Additionally, we reviewed the literature with respect to CpG island hypermethylation patterns in other urological malignancies. Using modern analytic methods, CpG island hypermethylation detection can be achieved. In men with prostate cancer, correlations between specific gene regulatory region hypermethylation analyses and Gleason score, pathologic stage and tumor recurrence have been demonstrated. CpG island hypermethylation may serve as a useful molecular biomarker for the detection and diagnosis of patients with prostate cancer.

Full text

Review
Molecular Biomarker in Prostate Cancer: The Role
of CpG Island Hypermethylation
Patrick J. Bastian
a,b,1
, Srinivasan Yegnasubramanian
c
, Ganesh S. Palapattu
a
,
Craig G. Rogers
a
, Xiaohui Lin
b,d
, Angelo M. De Marzo
a,b,d,e
, William G. Nelson
a,b,c,d,e,
*
a
The James Buchanan Brady Urological Institute, Department of Urology, The Johns Hopkins University School of Medicine,
Baltimore, MD 21231-1000, USA
b
Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
c
Department of Pharmacology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
d
The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
e
Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Accepted 29 July 2004
Available online 1 September 2004
Abstract
CpG island hypermethylation may be one of the earliest somatic genome alterations to occur during the
development of multiple cancers. Recently, aberrant methylation patterns for different tumors have been
reported. We present a comprehensive review of the literature describing the role of CpG island hypermethlytion
of DNA from prostatic tissue and bodily fluids from men with prostate cancer. We reviewed the literature to
evaluate CpG island hypermethylation in tissue and bodily fluids of men with primary and metastatic prostate
cancer. Additionally, we reviewed the literature with respect to CpG island hypermethylation patterns in other
urological malignancies.Using modern analytic methods, CpG island hypermethylation detection can be
achieved. In men with prostate cancer, correlations between specific gene regulatory region hypermethylation
analyses and Gleason score, pathologic stage and tumor recurrence have been demonstrated. CpG island
hypermethylation may serve as a useful molecular biomarker for the detection and diagnosis of patients with
prostate cancer.
#2004 Published by Elsevier B.V.
Keywords: Prostate cancer; DNA methylation; Hypermethylation; CpG island; GSTP1;
Gluthatione S-transferase; Epigenetics
1. I n t r o d u c t i o n
Prostate cancer is the most common serious cancer
in men and the second leading cause of cancer related
deaths in the United States and Western Europe. Since
the beginning of the prostate specific antigen (PSA)
testing era in the early 1990s, the number of men with
non-palpable prostate cancer has significantly
increased [1,2]. Autopsy studies and the recent Prostate
Cancer Prevention Trial (PCPT) have revealed a higher
prevalence of prostatic cancer than anticipated by PSA
screening alone [3–5]. The lifetime risk of developing
prostate cancer is 1 in 6, whereas the lifetime risk of
death due to metastatic prostate cancer is 1 in 30 [6].It
is estimated that 230,110 new cases will be diagnosed
in 2004 and 29,900 men will die of the disease in the
US in 2004 [6].
Because the prevalence of diagnosed prostate cancer
has increased dramatically during the PSA era, many
European
Urology European Urology 46 (2004) 698–708
* Corresponding author. Tel. +1 410 614 1661; Fax: +1 410 502 9817.
E-mail address: patrick.bastian@ukb.uni-bonn.de (P.J. Bastian),
bnelson@jhmi.edu (W.G. Nelson).
1
Co-corresponding author. Klinik und Poliklinik fur Urologie, Univer-
sitatsklinikum Bonn, Rheinische Friedrich-Wilhelms Universitat Bonn,
Sigmund-Freud-Str. 25, 53129 Bonn, Germany. Tel. +49 228 287 5109;
Fax: +49 228 287 4285.
0302-2838/$ – see front matter #2004 Published by Elsevier B.V.
doi:10.1016/j.eururo.2004.07.022

clinicians feared that widespread use of PSA testing for
prostate cancer screening might detect a large number
of clinically insignificant tumors, thus resulting in
potentially unnecessary treatment [7,8]. PSA screen-
ing, regardless of the threshold value, has certain well
documented limitations with regards to sensitivity and
specificity for the detection of prostate cancer [9].
Ultrasound-guided prostate biopsy is the gold standard
method for the diagnosis of prostate cancer. The pre-
cise biopsy strategy in terms of number of samples and
sampled area, however, remains controversial [10,11].
Moreover, non-diagnostic, yet clinically-suspicious,
lesions, such as a small focus of atypical glands (also
referred to as ASAP or atypical, small acinar prolif-
eration) or high grade prostatic intra-epithelial neopla-
sia, necessitate further evaluation in the absence of
obvious cancer in the specimen [12]. Molecular studies
have revealed important information about prostate
cancer development and progression. Multiple immu-
nohistochemistry tools to aid the diagnosis of prostate
cancer have been developed, but as of yet, none of these
alone or in conjunction have been able to definitively
diagnose prostate cancer [13–17]. Clearly, more sen-
sitive and specific biomarkers for prostate cancer diag-
nosis would be useful.
Epigenetic alterations, i.e., alterations in gene
expression without changes in the DNA sequence, in
human cancer were first described in 1983 [18].
Somatic epigenetic genome changes include global
genomic hypomethylation, promotor hypermethyla-
tion of CpG islands and loss of imprinting. The most
common somatic genome alteration during prostate
cancer development appears to be the hypermethyla-
tion in the regulatory region of certain genes, most
commonly in the promoter of the p-class glutathione-
S-transferase (GSTP1) gene [19,20]. Genomic imprint-
ing is an epigenetic alteration in the zygote or gamete
that causes expression of a specific parental allele of a
gene in somatic cells of the offspring. Loss of imprint-
ing can involve activation of the normally silent allele
of a growth promoting gene, or silencing of the nor-
mally expressed allele of a growth inhibitory gene. In
this review we describe the potential use of CpG island
hypermethylation as a molecular marker for prostate
cancer screening, detection and diagnosis.
2. DNA hypermethylation
The dinucleotide sequence CpG often carries the
modified base 5-methyl-cytosine (
5-m
C).
5-m
C can be
maintained in the genome through DNA replication via
the activity of DNA methyltransferases. CpG islands
are clusters (400 to 2000 bp) of CpG dinucleotides
without
5-m
C modifications that are present at the
transcriptional regulatory regions of many genes
[21]. Aberrant methylation of CpG islands are now
recognized to be one of the most common soma-
tic alterations in human cancers [22–24]. These
potentially reversible epigenetic changes inhibit trans-
cription and lead to gene silencing. DNA methyltrans-
ferases (DNMTs),
5-m
C-binding proteins (MBD) and
histone deactylases couple DNA methylation and tran-
scriptional repression [22–24]. In general, only methy-
lation of CpG islands within or surrounding the
transcriptional promotor region is associated with gene
silencing in cancer. That is to say, methylation in other
regions of genes, even within CpG island like
sequences, does not inhibit transcription [24]. Typi-
cally, transcriptional repression occurs as a consec-
quence of the assembly of a repressive chromation
structure containing MBDs, histone deacetylaces,
and other proteins, preventing DNA polymerase at
hypermethylated transcriptional promoter regions
[25].
3. Detection of hypermethylation
As DNA markers of cancer, CpG island hyper-
methylation changes have the advantage of being more
stable than RNA or proteins. Moreover, hypermethyla-
tion changes are common in many types of cancer.
Furthermore, specific CpG island hypermethylation
profiles can be observed in several different types of
cancer [26]. Because the position of CpG islands
within regulatory regions is similar across most
patients, detection of hypermethylation at CpG islands
is simpler than that of mutations in cancer. Also,
because of this commonality, a single assay can be
used for detection in all patients [22].
Currently, two major strategies for detection of CpG
island hypermethylation are employed, although new
technologies are constantly emerging. The first detec-
tion method uses
5-m
C-sensitive restriction enzymes
(Fig. 1), while the second approach uses sodium
bisulfite conversion (Fig. 2) to detect CpG island
hypermethylation.
5-m
C-sensitive restriction enzyme
detection assays include Southern blot (SB) analysis
and polymerase chain reaction amplification of DNA
(RE-PCR). Bisulfite conversion based assays include
bisulfite genomic sequencing (BGS) [27] and bisulfite
modification of DNA followed by either selective
polymerase chain reaction amplification (MSP) [28]
or quantative, real-time methylation specific polymer-
ase chain reaction (Q-MSP) [29]. Currently, the SB
P.J. Bastian et al./ European Urology 46 (2004) 698–708 699

analysis has been replaced with more sensitive tests.
The RE-PCR is a very sensitive technique in detecting
single hypermethylated alleles [30], employing treat-
ment with a restriction endonuclease that selectively
cuts its recognition site only if it does not contain
5-m
C
prior to PCR amplification. If the CpG island at the
restriction enzyme recognition site does not contain
a
5-m
C, the DNA is cut and no product can be detected
after PCR amplification. False positive detection of
CpG island hypermethylation with this method may be
the result of incomplete digestion of unmethylated
CpG island targets by restriction enzymes. Currently,
the most common approach is MS-PCR [31]. In this
technique, genomic DNA is first treated with bisulfite,
which deaminates unmethylated C bases to produce U
bases while
5-m
C in CpG islands remains unchanged.
This initial steps converts methylation patterns into
DNA sequence alterations that can be detected with
specifically designed PCR primers. One drawback of
this technique is limited sensitivity, as bisulfite treat-
ment can damage DNA and yield to inefficient PCR
amplification. BGS is a very labor intensive test and
due to limited sensitivity in detecting CpG island
hypermethylation it is not under development as a
clinical test [32].
4. Hypermethylation in prostate cancer
4.1. CpG island hypermethylation at GSTP1
Multiple studies assessing the CpG island hyper-
methylation status in prostate cancer and other human
cancers have been recently reported. The first study of
CpG island hypermethylation in prostate cancer found
significant CpG island hypermethylation at the GSTP1
promotor region [19]. Since then, multiple studies have
reported on the hypermethylation of the GSTP1 reg-
ulatory region in prostate cancer (Table 1 and Fig. 4).
The GSTP1 hypermethyltion status has since been
investigated in 1071 cases from a total of 24 studies
and has been shown to be hypermethylated in over 81%
of the cases examined. Although most studies used the
same primers, there are differences in the sensitivity
(Fig. 2). This may be due to differences in the tissue
processing and assay conditions. GSTP1 encodes for
the p-class Glutathione S-transferase (GST-p). GSTs
P.J. Bastian et al. / European Urology 46 (2004) 698–708700
Fig. 1. Detection of GSTP1 hypermethylation in prostate cancer with
restriction enzyme based assays [19,30,49,52,64]. On top, the regulatory
region of the GSTP1 gene is shown in red from base pair 851 to +239
relative to the transcriptional start site (represented by a green arrow).
Vertical black bars indicate CpG dinucleotides. For Southern blot assay the
restriction enzyme cutting site is shown as a pink vertical bar (base pair
562 and 564) within the southern blot probe. For restriction enzymes
PCR assays, the PCR amplicon is shown along with all restriction enzyme
cutting sites (vertical pink lines) within the amplicon. Fig. 2. Detection of GSTP1 hypermethylation in prostate cancer with
bisulfite treatment based assays [37–41,45,48,57,61–64,66–69,71,
72–86,88–102]. On top, the regulatory region of the GSTP1 gene is shown
in red from base pair 851 to +239 relative to the transcriptional start site
(represented by a green arrow). Vertical black bars indicate CpG dinucleo-
tides. For bisulfite genomic sequencing, the amplicons are indicated as
horizontal black lines. For methylation specific PCR arrows indicate the
primer annealing sites. For real time methylation specific PCR, the taqman
probe hybridization site is shown between the specific primers.

are an enzyme family that can detoxify reactive che-
mical species by catalyzing their conjugation to
reduced glutathione [33]. Thus, GSTP1 likely serves
as a ‘‘caretaker’’ gene [34], defending prostate cells
against genomic damage mediated by carcinogens or
various oxidants [20,35,36]. Loss of GSTP1 function
may render prostatic cells sensitive to carcinogenesis
driven by inflammation and diet.
4.2. CpG island hypermethylation at various loci
Using all of the currently available techniques to
detect DNA hypermethylation, 45 genes have been
examined in prostate cancer tissue (Table 1). Most
reports have analyzed one gene at a time, but more
recent studies have assayed several genes simulta-
neously and have identified prostate cancer specific
gene hypermethylation profiles [37–41]. Combining
the literature on CpG island hypermethlyation in pros-
tate cancer with that from data on similar studies from
bladder and renal cancer, a profile that distinguishes
prostate cancer from these other genito-urinary cancers
can be formulated (Fig. 3).
A recent comphrehensive study by Yegansubrama-
nian et al. assesses the extent of hypermethylation in 16
different genes in prostate cancer. Using the quantita-
tive, real-time MSP the authors noted that using various
combinations of GSTP1,APC,RASSF1a,PTGS2 and
MDR1 CpG island hypermethylation can distinguish
primary prostate cancer from benign prostate tissue
with sensitivities of 97.3%–100% and a specificity of
92%–100% [40]. In contrast to most other studies,
benign prostate tissues were obtained from brain-dead
transplant tissue donors. Although not age-matched to
the study cohort, these tissue samples resembled
healthy prostate upon histopathological analysis. Most
studies use normal prostate tissue adjacent to prostate
cancer as controls, leading to the yet unsolved question
whether normal tissue adjacent to cancer is, although
histopathologically benign, the same in terms of certain
molecular changes.
4.3. CpG island hypermethylation in non-cancerous
tissue
In addition to being methylated in prostatic cancer
tissue, some genes are also methylated in benign
prostatic tissue or benign prostatic hyperplasia
(BPH) (Fig. 4). In non-cancerous prostate tissues,
various groups have reported methylation of CpG
islands at EDNRB (up to 91%) [42],HIC (up to
100%) [38,40],ER (up to 60%–80%) [43,44] and
RASSF1a. However, other groups have reported that
methylation at the CpG islands of these genes is highly
P.J. Bastian et al. / European Urology 46 (2004) 698–708 701
Fig. 3. A comparison of the overall frequency of hypermethylation at various CpG islands in prostate cancer [19,30,37–41,45,49,52,57,61,63–71,72–86,
88–102], renal cell carcinoma [69,103–118] and urothelial cancer of the urinary bladder [26,119–131]. The overall frequency of methylation at each CpG island
was calculated by dividing the total number of cases studied in the literature by the total number of cases that were methylated at each CpG island. This
comparison suggests that a hypermethylation profile can uniquely identify each urological malignancy.

specific for cancerous tissue and did not find any
methylation at these genes in benign tissues. These
observed differences in the frequency of CpG island
methylation in benign tissues is probably due to the
differences in the number of CpG dinucleotides inter-
rogated as well as their genomic position. For instance,
Jeronimo et al., using an MSP assay with primers
containing 6 CpG dinucleotides that amplified a region
that was 139 to 9 relative to the translational start
site, found that 91% of benign tissues were methylated
at the EDNRB CpG island [42]. In contrast, Yegnasu-
bramanian et al., using a real time MSP assay with
primers and probe containing 9 CpG dinucleotides that
amplified a region that was 271 to 122 relative to
P.J. Bastian et al. / European Urology 46 (2004) 698–708702
Ta b l e 1
A review of hypermethylated genes in prostate cancer tissue
Gene Number
of studies
Number
of samples
a
Number of
hypermethylated samples
b
Overall
frequency (%)
c
Range
(%)
d
References
GSTP1 22 1071 874 81.61 36–100 [19,30,37–41,45,49,52,57,61,63–71]
CDH1 6 367 61 16.62 0–100 [37,40,41,63,72,73]
MGMT 5 352 39 11.08 0–88 [37–41]
DAPK 4 320 44 13.75 0–36 [37,38,40,41]
p16/CDKN2a 8 313 50 15.97 0–66 [37,39–41,74–77]
RASSF1a 9 274 199 72.63 53–96 [37,40,41,78,79]
RARbeta 3 234 159 67.95 53–79 [37,38,80]
APC 3 211 114 54.03 27–90 [37,40,41]
CD44 4 191 94 49.21 32–77.5 [62,81–83]
HIC1 2 182 181 99.45 99–100 [38,40]
TIMP3 2 182 6 3.3 0–6[38,40]
AR 5 181 25 13.81 0–15 [38,40,84–86]
p14 4 158 6 3.8 0–10.81 [39–41,74]
EDNRB 4 147 96 65.31 49–100 [40,42,87,88]
PTGS2 2 110 72 65.45 21.62–88 [40,41]
Cyclin D2 1 101 32 31.68 n/a [89]
CDH13 1 101 31 30.69 n/a [37]
FHIT 1 101 15 14.85 n/a [37]
MDR1 1 73 64 88 n/a [40]
ESR1 1 73 14 19.18 n/a [40]
p15/CDKN2b 2730 0 0[40]
hMLH1 1 73 0 0 n/a [40]
ER alpha A 2 70 65 92.86 90–95 [85,90]
ER alpha B 2 70 64 91.43 90–92 [85,90]
PR-B 2700 0 0[85,90]
PR-A 2700 0 0[85,90]
ER alpha C 2700 0 0[85,90]
VEGFR1 1 63 24 38.1 n/a [91]
ER beta 2 61 53 86.89 79–100 [44,85]
P27 3 61 10 16.39 6–38 [39,74,92]
TIG1 1 50 26 52 n/a [93]
RB1 2 48 4 8.33 6–12.5 [39,74]
P21 2 48 4 8.33 6–12.5 [39,74]
RUNX3 1 37 1 40.54 n/a [41]
THBS1 1 37 10 27.03 n/a [41]
TNFRSF6 1 32 4 12.5 n/a [94]
ER 1 31 28 90.32 n/a [43]
17p 1 26 25 96 n/a [95]
ZNF185 1 25 11 44 n/a [96]
Inhibin alpha subunit 1 24 7 29.17 n/a [97]
Caveolin-1 1 22 20 90.91 n/a [98]
TSLC1 1 22 7 31.82 n/a [99]
NEP 1 2 3 14.29 n/a [100]
PTEN 1 16 0 0 n/a [74]
P73 1 16 0 0 n/a [74]
p16/p14 1 11 8 72.73 n/a [76]
a
Sum of number of cancerous samples across all studies.
b
Sum of number of cancerous samples that were hypermthylated at each CpG island across all studies.
c
Overall frequency calculated by diversion of number of hypermethylated samples from the toal number of samples.
d
The minimum to maximum frequency reported by all studoes at each CpG island; n/a: not available.

the translational start site, reported that none of the
benign tissues were methylated at the EDNRB CpG
island [40]. Therefore, it appears that the number and
genomic position of CpG dinucleotides interrogated in
the MSP assay may significantly affect the frequency
of methylation in various prostate tissues.
4.4. Correlation with clinical parameters
In addition, aberrant methylation at EDNRB has
been correlated with Gleason grade and pathological
stage of prostate cancer [40]. The gene encoding for
COX2, PTGS2, has been found to be methylated in
88% of cases and has been correlated with an increased
risk of PSA recurrence independently of Gleason score
or pathological stage [40]. Recently, Kang et al. [41]
described a correlation between hypermethylation at
APC,RASSF1a and RUNX3 with characteristics (PSA
value and Gleason score) associated with a poor prog-
nosis. These studies suggest that assessment of methy-
lation status may be useful as a prognostic marker for
prostate cancer.
4.5. CpG island hypermethylation in precursor
lesions
One explanation for why CpG island hypermethyla-
tion has been observed in non-neoplastic tissue may be
that the examined tissues also contained prostate cancer
or prostatic intraepithelial neoplasia (PIN) cells. To
address this problem, Nakayama et al. used laser cap-
ture microdissection (LCM) to selectively recover cells
from normal prostatic epithelium, PIN, prostate cancer
and proliferative inflammatory atrophy (PIA) lesions
[45]. PIA has been implicated to play a role in prostate
carcinogenesis as a potential precursor to PIN and
prostate cancer [46]. Nakayama et al. found GSTP1
hypermethylation in 6% of the PIA lesions, 69% of the
PIN lesions and 91% of the prostate cancer specimens.
Importantly, no hypermethylation was reported in nor-
mal prostate epithelium or benign prostatic hyperplasia
despite the fact these tissue were microdissected from
cancer containing prostates [45] (Fig. 4).
4.6. CpG island hypermethylation in biopsy tissue
The ultimate proof of a diagnosis of prostate cancer
is the needle core biopsy. Although the biopsy is widely
used and a standard urological procedure, no standard
has been set in terms of sampling number of cores and
the regions of the prostate to be biopsied [47]. False
negative results, possibly from sampling error, can
further cloud patient management. In addition, the
diagnosis of prostate cancer from biopsy material
can be challenging for pathologists, as several entities
can imitate the histological appearance of prostate
cancer histologically [32]. To circumvent these issues,
Harden et al. (Q-MS-PCR) and Chu et al. (RE-PCR)
have examined the use of CpG island hypermethylation
at GSTP1 in small tissue samples obtained by prostate
biopsy [48,49]. They found that by using PCR
based detection techniques they were able to clearly
distinguish neoplastic prostate from non-neoplastic
prostate tissue. In another report, Goessl et al. used
MSP to detect GSTP1 CpG island hypermethylation
from biopsy needle washes and was able to detect it in
70% of the prostate cancer cases surveyed [50]. Gon-
P.J. Bastian et al. / European Urology 46 (2004) 698–708 703
Fig. 4. A comparison of the overall frequence of CpG island hpermethylation in normal prostate, benign prostatic hyperplasia (BPH), prostatic intraepithelial
neoplasia (PIN)and prostate cancer (PCA) [19,30,37–41,45,49,52,57,61,63–86,88–102]. The overall frequency was determined as described in the legendof Fig. 3.

zalgo et al. examined urine collected after prostate
biopsy and detected GSTP1 hypermethylation in 50%
of the patients with histologically proven prostate
cancer [51]. In this study 33% of patients without
prostate cancer or PIN also exhibited GSPT1 hyper-
methylation in post-biopsy urine DNA [51]. Interest-
ingly, two patients with a negative initial biopsy and
positive post-biopsy urine analysis for GSTP1 hyper-
methylation had a subsequent biopsy that was positive
for prostate cancer.
4.7. CpG island hypermethylation in metastatic
disease
Relatively few studies have examined the extent of
aberrant hypermethylation in metastatic sites of pros-
tate cancer [40,52,53]. In one such report, Yegnasubra-
manian et al. examined prostate cancer specimens that
included metastatic disease to multiple regions such as
lymph nodes, lung, liver and bone and found that the
hypermethylation pattern in prostate cancer metastases
mimicked that of the primary tumor and tended to show
greater differences within patients with multiple sites of
metastases than within sites across methylated patients
[40].Ko
¨llermann et al. studied lymph nodes obtained
during standard radical prostatectomy and pelvic lym-
phadenectomy for organ-confined prostate cancer in
patients with subsequent PSA relapse [53]. Although all
of these men had histologically benign lymph nodes,
GSTP1 hypermethylation was detected in 90% of the
lymph nodes at the time of surgery.
5. Hypermethylation in bodily fluids of
prostate cancer patients
For any markerto become clinically useful, it must be
present and detetctable in easily accessible sites such as
peripheral blood,urine, ejaculate or prostatic secretions.
To date, only CpG island hypermethylation of the
GSTP1 promotor has been examined in a small number
of studies in bodily fluids from patients with prostate
cancer (Table 2). Peripheral blood specimens are easy
available and are a part of current prostate cancer
screening and detection modalities. Although cell-free
circulating DNA has been detected in the plasma and
serum as early as 1948 [54], circulating DNA in the
plasma and serum of patients with urological malig-
nancies was only recently described [55]. Aside from
free circulating prostate DNA, prostate cancer DNA
may be present in the circulation as a result of intravas-
cular cell death of prostate cancer cells or circulating
phagocytic cells that have ingested prostate cancer cells
P.J. Bastian et al. / European Urology 46 (2004) 698–708704
Ta b l e 2
Studies of GSTP1 hypermethylation in bodily fluids of prostate cancer patients
Study Detection method
a
Frequency (%) Reference
Normal BPH
b
PIN
c
PCA
d
Urine
Carins et al. MS-PCR 27 [61]
Goessl et al. MS-PCR 2 29 73 [62]
Goessl et al. MS-PCR 3 76 [58]
Jeronimo et al. MS-PCR 3.2 30 [56]
Gonzalgo et al. MS-PCR (after biopsy) 33 67 58 [51]
Ejaculate
Goessl et al. MS-PCR 0 50 [58]
Suh et al. RE-PCR 44 [60]
Plasma
Goessl et al. MS-PCR 0 72 [57]
Goessl et al. MS-PCR 0 56 (T2-3) [58]
Goessl et al. MS-PCR 0 93 (T4 or mets) [58]
Jeronimo et al. MS-PCR 0 36 [56]
Prostatic secretion
Gonzalgo et al. MS-PCR
Primer Set A 76 [59]
Primer Set B 54 [59]
Biopsy washings
Goessl et al. MS-PCR 0 67 100 [50]
a
MS-PCR: methylation specific PCR; RE-PCR:
5-m
CpG-sensitive restriction enzyme PCR.
b
Benign prostatic hyperplasia.
c
Prostatic intraepithilial neoplasia.
d
Prostate cancer.

[32]. Jeronimo et al. detected GSTP1 hypermethylation
in the plasma of 36% of men with organ confined
prostate cancer [56]. Goessl et al. found GSTP1 hyper-
methylation in plasma of 56% of pathological stage T2–
3N0M0 and in 93% of pathological stage T4N+ or M+
prostate cancer patients [57,58]. The clinical use of these
studies remains unclear, but appears promising.
Other bodily fluids that can be collected and tested
for CpG island hypermethylation include urine, ejacu-
late and prostatic secretions. DNA can appear in fluids
of the urinary tract by cell shedding into the prostatic
ducts. Gonzalgo et al. recently reported that GSTP1
hypermethylation was detectable in 86% of prostatic
secretion specimens from men undergoing radical
prostatectomy [59]. Suh et al. was the first to demon-
strate the usefulness of GSTP1 hypermethylation for
detecting prostate cancer in the ejaculate [60]. They
found GSTP1 hypermethylation in the ejaculate of
approximately 50% men with prostate cancer [60].
Cairns et al. demonstrated GSTP1 hypermethylation
in urine of 27% of men with early stage prostate cancer
[61]. After one minute of prostatic massage, Goessl et
al. were able to detect GSTP1 hypermethylation in 73%
of men with prostate cancer [62]. By presumably
enriching the urine with prostate cells, prostatic mas-
sage appears to increase the detection rate of hyper-
methylated GSTP1 in men with prostate cancer in
comparison to a routine urine sample. This would also
seem to explain the high detection rates observed by
Gonzalgo et al. in post-biopsy urine specimens [51].A
prospective evaluation of GSTP1 hypermethylation in
bodily fluids for prostate cancer has yet to be published.
Additional studies using prostate specific marker
panels are also warranted to distinguish prostate cancer
from other cancers such as kidney, urinary bladder or
gastrointestinal cancers.
6. Conclusion
The hypermethylation of CpG islands represents a
somatic, epigenetic event that almost uniformly arises
during prostate caricnogenesis. Using modern detection
assays, CpG island hypermethylation of multiple pros-
tate cancer specific genes has become a promising
molecular marker for prostate cancer diagnosis and
detection. By applying these techniques to readily avail-
able clinical specimens such as urine or blood the current
ability to diagnosis prostate cancer may be improved.
Acknowledgements
This work was supported by NIH/NCI grant R01
CA70196 and NIH/NCI SPORE grant P50 CA58236.
W.G. Nelson has a patent (United States patent
5.552.277) titled ‘‘Genetic Diagnosis of Prostate
Cancer’’.
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