Molecular Biomarker in Prostate Cancer: The ... - European Urology

European Urology

European Urology 46 (2004) 698–708

Review

Molecular Biomarker in Prostate Cancer: The Role of CpG Island Hypermethylation Patrick J. Bastiana,b,1, Srinivasan Yegnasubramanianc, Ganesh S. Palapattua, Craig G. Rogersa, Xiaohui Linb,d, Angelo M. De Marzoa,b,d,e, William G. Nelsona,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. Introduction 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) * Corresponding author. Tel. +1 410 614 1661; Fax: +1 410 502 9817. E-mail address: [email protected] (P.J. Bastian), [email protected] (W.G. Nelson). 1 Co-corresponding author. Klinik und Poliklinik fur Urologie, Universitatsklinikum 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

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

P.J. Bastian et al. / European Urology 46 (2004) 698–708

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 screening, 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 precise 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 proliferation) or high grade prostatic intra-epithelial neoplasia, 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 immunohistochemistry 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 sensitive and specific biomarkers for prostate cancer diagnosis 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 hypermethylation of CpG islands and loss of imprinting. The most common somatic genome alteration during prostate cancer development appears to be the hypermethylation in the regulatory region of certain genes, most commonly in the promoter of the p-class glutathioneS-transferase (GSTP1) gene [19,20]. Genomic imprinting 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 imprinting can involve activation of the normally silent allele of a growth promoting gene, or silencing of the normally 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-mC). 5-mC can be maintained in the genome through DNA replication via the activity of DNA methyltransferases. CpG islands

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are clusters (400 to 2000 bp) of CpG dinucleotides without 5-mC 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 somatic alterations in human cancers [22–24]. These potentially reversible epigenetic changes inhibit transcription and lead to gene silencing. DNA methyltransferases (DNMTs), 5-mC-binding proteins (MBD) and histone deactylases couple DNA methylation and transcriptional repression [22–24]. In general, only methylation 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]. Typically, transcriptional repression occurs as a consecquence 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 hypermethylation changes have the advantage of being more stable than RNA or proteins. Moreover, hypermethylation 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 detection method uses 5-mC-sensitive restriction enzymes (Fig. 1), while the second approach uses sodium bisulfite conversion (Fig. 2) to detect CpG island hypermethylation. 5-mC-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 polymerase chain reaction (Q-MSP) [29]. Currently, the SB

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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.

analysis has been replaced with more sensitive tests. The RE-PCR is a very sensitive technique in detecting single hypermethylated alleles [30], employing treatment with a restriction endonuclease that selectively cuts its recognition site only if it does not contain 5-mC prior to PCR amplification. If the CpG island at the restriction enzyme recognition site does not contain a 5-mC, 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-mC 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 treatment 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].

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 dinucleotides. 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.

4. Hypermethylation in prostate cancer 4.1. CpG island hypermethylation at GSTP1 Multiple studies assessing the CpG island hypermethylation 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 regulatory 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


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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.

are an enzyme family that can detoxify reactive chemical 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 simultaneously and have identified prostate cancer specific gene hypermethylation profiles [37–41]. Combining the literature on CpG island hypermethlyation in prostate 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 Yegansubramanian et al. assesses the extent of hypermethylation in 16 different genes in prostate cancer. Using the quantitative, 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

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Table 1 A review of hypermethylated genes in prostate cancer tissue Gene

Number of studies

Number of samplesa

Number of hypermethylated samplesb

Overall frequency (%)c

Range (%)d

References

GSTP1 CDH1 MGMT DAPK p16/CDKN2a RASSF1a RARbeta APC CD44 HIC1 TIMP3 AR p14 EDNRB PTGS2 Cyclin D2 CDH13 FHIT MDR1 ESR1 p15/CDKN2b hMLH1 ER alpha A ER alpha B PR-B PR-A ER alpha C VEGFR1 ER beta P27 TIG1 RB1 P21 RUNX3 THBS1 TNFRSF6 ER 17p ZNF185 Inhibin alpha subunit Caveolin-1 TSLC1 NEP PTEN P73 p16/p14

22 6 5 4 8 9 3 3 4 2 2 5 4 4 2 1 1 1 1 1 2 1 2 2 2 2 2 1 2 3 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1

1071 367 352 320 313 274 234 211 191 182 182 181 158 147 110 101 101 101 73 73 73 73 70 70 70 70 70 63 61 61 50 48 48 37 37 32 31 26 25 24 22 22 2 16 16 11

874 61 39 44 50 199 159 114 94 181 6 25 6 96 72 32 31 15 64 14 0 0 65 64 0 0 0 24 53 10 26 4 4 1 10 4 28 25 11 7 20 7 3 0 0 8

81.61 16.62 11.08 13.75 15.97 72.63 67.95 54.03 49.21 99.45 3.3 13.81 3.8 65.31 65.45 31.68 30.69 14.85 88 19.18 0 0 92.86 91.43 0 0 0 38.1 86.89 16.39 52 8.33 8.33 40.54 27.03 12.5 90.32 96 44 29.17 90.91 31.82 14.29 0 0 72.73

36–100 0–100 0–88 0–36 0–66 53–96 53–79 27–90 32–77.5 99–100 0–6 0–15 0–10.81 49–100 21.62–88 n/a n/a n/a n/a n/a 0 n/a 90–95 90–92 0 0 0 n/a 79–100 6–38 n/a 6–12.5 6–12.5 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a

[19,30,37–41,45,49,52,57,61,63–71] [37,40,41,63,72,73] [37–41] [37,38,40,41] [37,39–41,74–77] [37,40,41,78,79] [37,38,80] [37,40,41] [62,81–83] [38,40] [38,40] [38,40,84–86] [39–41,74] [40,42,87,88] [40,41] [89] [37] [37] [40] [40] [40] [40] [85,90] [85,90] [85,90] [85,90] [85,90] [91] [44,85] [39,74,92] [93] [39,74] [39,74] [41] [41] [94] [43] [95] [96] [97] [98] [99] [100] [74] [74] [76]

a b c d

Sum of number of cancerous samples across all studies. Sum of number of cancerous samples that were hypermthylated at each CpG island across all studies. Overall frequency calculated by diversion of number of hypermethylated samples from the toal number of samples. The minimum to maximum frequency reported by all studoes at each CpG island; n/a: not available.

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 interrogated 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, Yegnasubramanian 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


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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 legend of Fig. 3.

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 prognosis. These studies suggest that assessment of methylation 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 hypermethylation 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 capture 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 normal 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-

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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 hypermethylation in post-biopsy urine DNA [51]. Interestingly, two patients with a negative initial biopsy and positive post-biopsy urine analysis for GSTP1 hypermethylation 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 prostate cancer [40,52,53]. In one such report, Yegnasubramanian 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 lymphadenectomy 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 marker to 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 malignancies was only recently described [55]. Aside from free circulating prostate DNA, prostate cancer DNA may be present in the circulation as a result of intravascular cell death of prostate cancer cells or circulating phagocytic cells that have ingested prostate cancer cells

Table 2 Studies of GSTP1 hypermethylation in bodily fluids of prostate cancer patients Study

Detection methoda

Frequency (%) Normal

Urine Carins et al. Goessl et al. Goessl et al. Jeronimo et al. Gonzalgo et al.

MS-PCR MS-PCR MS-PCR MS-PCR MS-PCR (after biopsy)

Ejaculate Goessl et al. Suh et al.

MS-PCR RE-PCR

Plasma Goessl et al. Goessl et al. Goessl et al. Jeronimo et al.

MS-PCR MS-PCR MS-PCR MS-PCR

Prostatic secretion Gonzalgo et al.

Biopsy washings Goessl et al. a b c d

Reference b

c

BPH

PIN

2 3 3.2

29

MS-PCR

[61] [62] [58] [56] [51]

0

50 44

[58] [60]

0 0 0 0

72 56 (T2-3) 93 (T4 or mets) 36

[57] [58] [58] [56]

76 54

[59] [59]

100

[50]

33

67

0 5-m

PCA 27 73 76 30 58

MS-PCR Primer Set A Primer Set B

MS-PCR: methylation specific PCR; RE-PCR: Benign prostatic hyperplasia. Prostatic intraepithilial neoplasia. Prostate cancer.

d

CpG-sensitive restriction enzyme PCR.

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[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 hypermethylation 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, ejaculate 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 demonstrate 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 massage appears to increase the detection rate of hypermethylated 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

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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 prostate cancer specific genes has become a promising molecular marker for prostate cancer diagnosis and detection. By applying these techniques to readily available 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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