BJUI. Methylated genes as potential biomarkers in prostate cancer

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1 2009 THE AUTHORS. JOURNAL COMPILATION 2009 BJU INTERNATIONAL Mini Reviews PHÉ ET AL. BJUI BJU INTERNATIONAL Methylated genes as potential biomarkers in prostate cancer Veronique Phé, Olivier Cussenot* and Morgan Rouprêt* Academic Department of Urology of la Pitiè-Salpétriére Hospital and of Tenon Hospital, GHU Est, Assistance Publique-Hopitaux de Paris, Facultè de Mèdecine Pierre et Marie Curie, University Paris VI, Paris, and *CeRePP, Centre d Etudes et de Recherche sur les Pathologies Prostatiques, Paris, France Accepted for publication 6 November 2009 Prostate cancer is the most common malignancy of the urogenital tract. Although controversial, prostate-specific antigen (PSA) testing is widely used for screening and follow-up of prostate cancer, but because of its limited specificity and sensitivity, PSA is not an ideal test. We currently lack the necessary tools to differentiate between latent disease with little likelihood of clinical manifestation and aggressive tumours that are likely to metastasize and lead to potentially lethal disease. DNA methylation is an important epigenetic mechanism of gene regulation and plays essential roles in tumour initiation and progression. Currently, aberrant promoter hypermethylation has been investigated in specific genes from the following groups: tumour-suppressor genes, proto-oncogenes, genes involved in cell adhesion, and genes involved in cell-cycle regulation. Glutathione S-transferase P1 (GSTP1) has been shown to be a biomarker for prostate cancer. Other genes, e.g. CD44, PTGS2, E-cadherin, CDH13, and cyclin D2 have been found to be prognostic markers for prostate cancer. In cell samples derived from the urine, the presence of the hypermethylation of either GSTP1 or RASS1a has been shown to be both sensitive and specific for detecting prostate cancer. Several studies have found that analysis of hypermethylation using a panel of tumoursuppressor genes yielded better results for detecting prostate cancer than the analysis of single-gene methylation. Hence, these different panels (e.g. GSTP1, APC, PTGS2, T1G1 and EDNRB) are of interest for detecting prostate cancer. Also, the methylation profile of multiple regulatory genes might be altered at the time of cancer relapse. Thus, preliminary results on the use of the methylation status of specific genes as potential tumour biomarkers for the early diagnosis and the risk stratification of patients with prostate cancer are promising. KEYWORDS prostate cancer, biomarkers, gene promoter, prognosis, recurrence, DNA methylation, epigenetic INTRODUCTION Prostate cancer is the most commonly detected male cancer and the second leading cause of male cancer deaths in the western world [1]. However, only about half of patients diagnosed will develop significant symptoms, and <20% will die from their disease [2]. The care of patients after their initial diagnosis and treatment is difficult due the lack of specific biomarkers [3]. While PSA can be used for screening and to detect recurrences before the development of clinical disease, its baseline values do not allow prognostic prediction of disease behaviour [3]. Also, although changes in PSA values over time (i.e. PSA velocity) can be used to derive information on tumour phenotype, there is an urgent clinical need for an accurate prognostic marker that could be used to identify men at high risk of relapse and metastasis [4,5]. A promising biomarker recently investigated centres on aberrant DNA promoter hypermethylation [4,6,7]. This epigenetic change occurs widely in various types of cancerous cells and always affects the same promoter regions [6,8,9]. In this review, we present the potential impact of aberrant promoter methylation analysis in the diagnosis and prognosis of prostate cancer. METHYLATION ANALYSIS Most of the techniques used to detect DNA methylation are based on PCR methods, and are therefore extremely sensitive [10 12]. The bisulphite modification-pcr amplification approaches, e.g. quantitative methylationspecific PCR, Methyl-Light, MSP, etc., rely on a methylation change at the PCR primer binding site (and or Taqman probe site for quantitative PCR). As such, these tests are prone to false-negative results (the gene promoter might have been methylated, but not at the primer binding site). New platforms featuring the capture of methylated DNA might be less prone to this problem, but these platforms have not yet replaced the bisulphite-based approaches [13]. Compared with other molecular structures, such as mrna and certain proteins, the use of DNA as the substrate to detect tumour markers has several advantages. First, DNA molecules are very stable and, in contrast to mrna and many proteins, can survive under harsh conditions for long periods. Most importantly, relatively intact DNA can be isolated from formalin-fixed, paraffinembedded tissue. Second, unlike proteins, nucleic acids can be amplified by PCR and related techniques, thus allowing measurements on very small test samples [10 12]. The genes undergoing methylation during the early phases of tumorigenesis could potentially be used as markers for identifying 1364 JOURNAL COMPILATION 2010 BJU INTERNATIONAL 105, doi: /j x x

2 TABLE 1 Genes that are frequently methylated in prostate cancer Genes Name Sensitivity, % Specificity, % Significance References GSTP-1 Glutathione S-transferase P [6,10,15 17,20,23] CDKN2A Cyclin-dependent kinase inhibitor 2 A (p16) [6,17,20,23] CCND2 Cyclin D2 32 [21] p14b 9.5 [15] MGMT O-6-methylguanine DNA methyltransferase [6,17,20,23] ASC Apoptosis-associated Speck-like protein 37 Recurrence [6] containing a CARD AR Androgen receptor 8 28 All stages, IFWDP [19,22] ESR1 Oestrogen receptor [22] ESR2 Oestrogen receptor [22] RARβ Retinoic acid receptor β [10,15,16] EDNRB Endothelin receptor type B [6,16,23] RASSF1A Ras association domain family protein [15,17,20,23] isoform A MDR1 Multidrug resistance receptor All stages, IFWDP [14 16,43] CDH13 Cadherin Recurrence [6,15,20] APC Familial adenomatous polyposis [6,10,15,17,18,20,23] TIMP3 TIMP metallopeptidase inhibitor 3 4 [6,17,23] CDH1 E-cadherin All stages [15,18,20,23] IFWDP (>50% of metastatic tumours) CD All stages [6,23] IFWDP [16,23,24] T1G All stages observed in IFWDP LAMA 3 α-3 laminin 44 [9] LAM B3 β-3 laminin 18 [9] LAM C2 γ-3 laminin 41 [9] SCAV 1 Caveolin 1 90 [47] PTGS2 Prostaglandin endoperoxide synthase [10,16] RUNX3 Runt-related transcription factor 3 44 [6,18] WIF1 WNT inhibitory factor 1 28 [6] COX2 Cyclo-oxygenase 2 78 [18,23] IFWDP, increasing frequency with disease progression. individuals at increased risk of developing malignancy, or for aiding in the diagnosis of early malignancy, while those genes undergoing methylation during the progression of malignancy could potentially be used as prognostic markers [4,5]. GENES OF INTEREST IN PROSTATE CANCER Several genes of interest are shown in Table 1 [6,9,10,14 24]. There are differences in the pattern of gene methylation in tumours derived from different organs. Genes involved in the following cellular processes are frequently disrupted by CpG island hypermethylation: (i) DNA damage repair, glutathione S-transferase P1 (GSTP1) and MGMT; (ii) tumour-suppressor gene activation, CDKN2A and CDH1; (iii) hormonal response, androgen receptor, oestrogen receptor and retinoid acid receptor β (RARβ); (iv) cell cycle control, CDKN2A and RASSF1A; (v) tumour cell invasion/metastasis, CDH, CD44, and APC. For many of these genes, promoter hypermethylation is often the main or only mechanism underlying their loss of function in prostate cancer. Inappropriate silencing of these genes can contribute to tumour initiation, progression and metastasis. Some of the hypermethylation occurs during the early stages of tumour progression in the multi-step process of prostate carcinogenesis. Hypermethylation can also correlate with pathological grade or clinical stage, and contribute to the invasiveness, metastasis, and/or androgen independence of prostate tumours. GSTP1 GSTP1 is involved in the metabolism, detoxification and elimination of potentially genotoxic foreign compounds, and thus acts to protect cells from DNA damage and cancer initiation. Suppression of GSTP1 activity can result in enhanced susceptibility to DNA damage and increased cancer incidence. Much work has been reported on the JOURNAL COMPILATION 2010 BJU INTERNATIONAL 1365

3 PHÉ ET AL. TABLE 2 The most frequently used panels of hypermethylated genes to detect prostate cancer, from current reports Panels Sample type Sensitivity, % Specificity, % PPV, % NPV, % AUC References GSTP1, RASSF1a, RARβ, and APC Urine 86 [5] GSTP1 and APC Urine [12] GSTP1, APC and RARβ Urine [29] GSTP1, APC and RARβ Urine [25] GSTP, APC, and PTGS2 Prostate tissue [10] GSTP or APC Prostate tissue [10] APC or RARβ Prostate tissue [10] APC or PTGS2 or RAR_ Prostate tissue [10] APC or RARβ or GSTP1 Prostate tissue [16] T1G1 or GSTP1 Prostate tissue [16] T1G1 and APC Prostate tissue [16] T1G1 and EDNRB Prostate tissue [16] T1G1 and GSTP1 Prostate tissue [16] T1G1 and RARβ Prostate tissue [16] GSTP1 and RARβ Prostate tissue [16] CDH13 and or ASC Prostate tissue [6] PPV, positive predictive value; NPV, negative predictive value; AUC, area under the receiver operating curve. development of GSTP1 methylation as a biomarker that could be used to detect prostate cancer. GSTP1 promoter hypermethylation constitutes the best DNAbased biomarker for the disease currently available because it is present in up to 90% of prostate cancer tissues (and therefore has a specificity of %) and is only rarely present in benign prostate tissue [6,10,15 17,20,23]. Moreover, GSTP1 methylation has been frequently found in samples from patients with prostatic intraepithelial neoplasia (PIN) (70%). Other genes undergoing hypermethylation in PIN at a considerable frequency include RARβ2 (20%), MGMT (92%), and RASSF1A (64%) [18]. Proliferative inflammatory atrophy (PIA), especially focal PIA, has also been considered to be a precursor lesion for the development of prostate cancer and/or high-grade PIN. Normally, PIA lesions express elevated levels of GSTP1 in response to increased oxidative stress. GSTP1 methylation also occurs in PIA lesions (6%), but to a much lesser extent than occurs in PIN [18]. The potential role that GSTP1 hypermethylation and subsequent inactivation plays in prostate carcinogenesis in premalignant prostate lesions has not been well established. INTEREST IN THE USE OF GENE PANELS The use of a single gene locus to discriminate malignant from benign cells has several drawbacks. First, the maximum sensitivity of a test using a single gene can only be as high as the frequency of hypermethylation at a specific CpG locus. Second, noncancerous tissues can, in some cases, harbour CpG island hypermethylation at the same gene locus as cancerous tissue. Furthermore, methylation of a single gene locus can occur in cancers other than prostate or even in benign disease. Investigating the methylation status of several genes might have more discriminatory power for detection prostate cancer. Recently, several groups have attempted to simultaneously analyse the methylation status of many genes to determine the fingerprints of prostate cancer methylation. For example, the panel including GSTP, APC and PTGS2 has a sensitivity of 89% for detecting prostate cancer [10]. Panels of genes can also be used for urine assays, e.g. the panel of GSTP, RASSF1a, RARβ and APC has a sensitivity of 86% for detecting prostate cancer [5] and the panel of GSTP1, RARβ and APC has a negative predictive value of 87% [25]. However, most studies of gene panels show that the gain in sensitivity is accompanied by a loss of specificity. The reality is that screening for prostate cancer might not be the most significant public health challenge [26]; rather, distinguishing prostate cancer that poses a threat of morbidity and mortality might be the real unmet need. As such, perhaps the specificity could be the most important attribute for any new prostate cancer test. In addition, it could be more useful to obtain prognostic information from the use of gene panels. For instance, hypermethylation at PTGS2, RARβ and EDNRB has been shown to be inversely correlated with PSA recurrence-free survival, according to a nomogram, and has potential prognostic value [10]. CpG island hypermethylation at several gene loci was previously detected in men with advanced disease, but no single gene was consistently hypermethylated in men with hormone-refractory disease [14]. These results suggest that the CpG island hypermethylation status of a defined panel of genes might be a useful biomarker in men with hormone-refractory prostate cancer. Table 2 lists the most frequently described hypermethylated gene panels in current reports. TESTING FOR HYPERMETHYLATION USING BODILY FLUIDS It is highly desirable for methylated genes present in the blood or other biological fluids to have a high specificity for cancer if these markers are to be used in screening for early malignancy. Efforts are also underway to develop noninvasive methods of quantifying the methylation of genes in bodily fluids. A summary of the genes that have been found to be frequently methylated in prostate cancer is listed in Table 3 [5,11,12,27 34] JOURNAL COMPILATION 2010 BJU INTERNATIONAL

4 TABLE 3 Methylation biomarkers for prostate cancer detection in bodily fluids samples Genes Sample type Sensitivity, % Specificity, %e References GSTP1 Biopsy [5,11,12,27,29 32] Urine [28,33,34] Plasma/serum [7,28,34] Sperm [28] RARβ Urine [5,12] APC Urine [5,12] RASSF1A Urine 77.9 [5] CDH1 Urine 30.5 [5] MGMT Urine 14.7 [5] P14 Urine 6.3 [5] TIMP3 Urine 43.2 [5] high incidence of promoter methylation observed in non-neoplastic prostate tissue [37]. The relationship between diet and methylation is also important to study, because diet is an easily modifiable risk factor. PROGNOSIS AND RELAPSE Several clinicopathological scoring systems have been developed to identify patients at greatest risk of recurrence after initial treatment [38,39]. However, many patients defined as low-risk still develop a recurrence. There is an urgent clinical need for accurate prognostic markers that could be used to identify men at high risk of relapse and metastasis [2 4]. GSTP1 protein is detectable in the urine of patients with prostate cancer, especially after prostate biopsy and/or prostatic massage [5,27]. After prostatic massage, methylation has been detected in 2% of patients diagnosed with BPH, 29% diagnosed with PIN, 68% diagnosed with early prostate cancer, and 78% diagnosed with locally advanced or metastatic disease [28]. However, there were discrepancies in the results of other studies that might reflect sample or assay differences. It has already been reported that patients with cancer have higher levels of cell-free DNA than healthy individuals, as well as patients with different nonmalignant diseases [4]. Thus, multiple gene-methylation analysis in circulating cell DNA could be a good biomarker for the early detection of prostate cancer recurrence. As circulating cells contribute most of the DNA in whole blood, researchers have hypothesized that most of the detected methylation of DNA was from circulating tumour cells. However, it remains still possible that there might be some contamination with free serum DNA [4]. Furthermore, GSTP1 promoter hypermethylation was detectable in 75% of plasma samples obtained from patients with newly diagnosed prostate cancer and in 36.8% of patients being treated for prostate cancer [35]. The detection of prostate cancer methylation signatures in bodily fluids samples has implications for the identification of high-risk patients and for monitoring residual disease after curative surgery. Detection of GSTP1 methylation in the serum of men with localized disease before treatment has been associated with a four times greater risk of biochemical recurrence after surgery [7]. This finding suggests that the detection of preoperative serum GSTP1 methylation might indicate the presence of aggressive disease. However, this report was the first to study this topic and larger studies with longer follow-up periods are needed to evaluate its significance in patients whose tumours develop into androgen-independent tumours. DNA METHYLATION AND RISK FACTORS FOR PROSTATE CANCER Although the molecular causes underlying racial differences in the incidence and clinical behaviour of prostate cancer have not been well characterized, preliminary findings suggest that differences in gene methylation could play a role. The frequency of CD44 hypermethylation was 1.7 times higher in African-American men with prostate cancer than in their Caucasian-American counterparts. Hypermethylation of CD44 and GSTP1 have independently been correlated with poor pathological findings in Asian, but not American, men [36]. Many genes that are aberrantly hypermethylated in cancer could also be aberrantly hypermethylated due to ageing. Because many hypermethylated genes encode proteins with tumour-suppressor activity, it is possible that age-related gene methylation/ inactivation increases a cell s susceptibility to neoplastic transformation. Thus, agedependent gene methylation might be an important early event in leading to cancer initiation. Such a mechanism could link the age dependence of prostate cancer to the Several studies have now shown that the methylation index, defined as the ratio of methylated genes to the total number of genes analysed, correlates with clinicopathological indicators of poor prognosis [14,18,20]. Correlations between methylation status and indicators of poor prognosis have also been made for several individual genes, including LAMA3, CDH13, cyclin D2 and TIG1, but these need to be confirmed in more robust studies because results for several genes (APC, CD44, E- cadherin, MDR1, RARβ2 and RASSF1A) are inconsistent and come from isolated reports [9,20,21,24,40]. However, the methylation frequencies of E-cadherin, PTGS2, and RUNX3 have been correlated with an increased risk of PSA recurrence independent of Gleason score or pathological stage [18,20,23]. In addition, quantitative studies have shown a relationship between increasing methylation levels of certain genes (GSTP1, APC, RARβ2 and EDNRB) and tumours with an advanced pathological stage and/or a high Gleason score [17,23]. Woodson et al. [41] found that methylation of GSTP1 and RARβ2 was not associated with recurrence; however, they found that CD44 and PTGS2 methylation significantly predicted recurrence. In a multivariate model, the combined methylation profile of CD44 and PTGS2 was found to be an independent predictor of biochemical recurrence (associated with a nine times greater risk of recurrence) after adjusting for Gleason grade. In addition, survival analysis showed the combined CD44 and PTGS2 methylation status was associated with shorter time to recurrence [41]. CD44 and PTGS2 methylation might therefore predict biochemical JOURNAL COMPILATION 2010 BJU INTERNATIONAL 1367

5 PHÉ ET AL. recurrence in patients undergoing radical prostatectomy and, if validated in larger studies, could be used to identify patients with aggressive cancer. However, the methylation profile of multiple regulatory genes might also be altered at the time of cancer relapse. In a previous study, we found that more hypermethylation of GSTP1, RASSF1a, APC and RARβ was detected in the diagnostic sample from the patients with cancer than in controls [4]. Also, patients with progressive disease had significantly greater methylation levels of these four genes than other patients. Patients at risk of disease progression have higher detectable concentrations of promoter hypermethylation in circulating cells than those without progression. The extent of this hypermethylation increases during disease progression and can be used to identify the extent and duration of treatment response in prostate cancer. DNA HYPOMETHYLATION AND PROSTATE CANCER DNA hypomethylation leads to elevated mutation rates by destabilizing the genome and promoting loss of heterozygosity in regions containing tumour-suppressor genes. It might also lead to chromosomal instability through its decondensing effect on chromatin structure. Significant levels of hypomethylation have been reported in 97% of primary prostate cancers, implying that hypomethylation plays a major role at all stages of tumorigenesis [8]. Hypomethylation might also target CpG islands in single-copy gene promoters, leading to proto-oncogene activation. Hypomethylation and abnormal expression of bone morphogenetic protein-6 in metastatic prostate cancer is thought to play a role in osteoblastic skeletal metastases [42]. The interrelationship between DNA hypo- and hypermethylation in malignancy is interesting. The balance between hyper- and hypomethylation might be important in predicting clinical outcomes such as disease recurrence [8]. Recent findings have provided evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes, and occur heterogeneously during prostate cancer progression and metastatic dissemination [43]. INTEREST IN DAILY PRACTICE It is clear from the information presented that methylated genes are promising biomarkers for prostate cancer [13,40,44 46]. However, all of the studies reported to date have mostly been retrospective and of small scale and/or with limited samples. To be used in routine clinical practice, these preliminary findings must be confirmed in studies with a high level of evidence. Moreover, the additional information that these assays provide must be clinically relevant, i.e. they must provide information that improves patient outcomes, enhances patient quality of life, or leads to reduced healthcare costs. A major unresolved analytical issue is the optimum system to determine gene methylation in the clinical setting. Currently, many approaches are available for measuring gene methylation in research laboratories. Quantitative methylation-specific PCR examining the methylation status of targeted genes is commercially available for use on tissue derived from prostate biopsy. However, it probably has inadequate sensitivity for clinical utility when used to predict the risk of prostate cancer on repeat biopsy after an initial negative biopsy. Urine testing has shown good sensitivity and much higher specificity than PSA for predicting prostate cancer on biopsy. However, ongoing validation in appropriate patient subsets is needed. THE FUTURE Research into cancer-dependent epigenetic DNA methylation is rapidly developing and might lead to advances in the understanding of the molecular basis of cancer development and progression [4,6,12,14]. The development of novel methods that involve genome-wide screening promises to lead to the identification of many new genes that are hypermethylated in cancer. These genes could be used as components of biomarker panels that can be used to better detect human cancers and assess an individual s risk of recurrence and/or progression after diagnosis. In addition, future research on gene methylation could provide novel insights into age-related, race-related, and hereditary forms of cancer. Finally, because DNA methylation represents a reversible DNA lesion, future research on the development of therapeutic agents that act on this epigenetic system could lead to advances in the treatment of specific cancers. CONCLUSION Early results from the use of the methylation status of specific genes as potential tumour biomarkers for the early diagnosis and the risk stratification of patients with prostate cancer are promising. 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7 PHÉ ET AL. 42 Tamada H, Kitazawa R, Gohji K, Kitazawa S. Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer. J Bone Miner Res 2001; 16: Yegnasubramanian S, Haffner MC, Zhang Y et al. DNA hypomethylation arises later in prostate cancer progression than CpG island hypermethylation and contributes to metastatic tumor heterogeneity. Cancer Res 2008; 68: Troyer DA, Lucia MS, de Bruine AP et al. Prostate cancer detected by methylated gene markers in histopathologically cancer-negative tissues from men with subsequent positive biopsies. Cancer Epidemiol Biomarkers Prev 2009; 18: Ellinger J, Muller SC, Stadler TC, Jung A, von Ruecker A, Bastian PJ. The role of cell-free circulating DNA in the diagnosis and prognosis of prostate cancer. Urol Oncol 2009; doi: /j.urolonc Sep 16 [Epub ahead of print] 46 Watson JA, Watson CJ, McCrohan AM et al. Generation of an epigenetic signature by chronic hypoxia in prostate cells. Hum Mol Genet 2009; 18: Woodson K, Hanson J, Tangrea J. A survey of gene-specific methylation in human prostate cancer among black and white men. Cancer Lett 2004; 205: Correspondence: Morgan Rouprêt, Hôpital Pitié-Salpétrière, Boulevard de l hôpital, Paris, France. morgan.roupret@psl.aphp.fr Abbreviations: GSTP1, glutathione S-transferase P1; RARβ, retinoid acid receptor β; PIN, prostatic intraepithelial neoplasia; PIA, proliferative inflammatory atrophy JOURNAL COMPILATION 2010 BJU INTERNATIONAL