Human Cancer Biology

Molecular Detection of Localized Cancer Using Quantitative Methylation-Specific PCR on Urinary Cells Obtained Following Prostate Morgan Roupre“ t,1Vincent Hupertan,2 David R. Yates,1James W.F. Catto,1Ishtiaq Rehman,1Mark Meuth,1 Sylvie Ricci,3 Roger Lacave,3 Ge¤ raldine Cancel-Tassin,2 Alexandre de laTaille,5 Franc¸ois Rozet,6 Xavier Cathelineau,6 Guy Vallancien,6 Freddie C. Hamdy,1and Olivier Cussenot2,4

Abstract Purpose: The diagnosis of localized is difficult due to a lack of cancer-specific biomarkers. Many patients require repeat prostate biopsies to diagnose the disease. We investi- gated whether aberrant promoter hypermethylation in prostatic fluid could reliably detect prostate cancer. Experimental Design: Urine samples were collected after prostate massage from 95 patients withlocalized prostate cancer undergoing radical (63 pT 1,31pT2,and1pT3)and from 38 control patients. Ten genes (GSTP1, RASSF1a, ECDH1, APC, DAPK, MGMT, p14, p16, RARb2,andTIMP3) were investigated using quantitative real-time methylation-specific PCR. Receiver operator curves were generated. Results:The frequency of gene methylation ranged from 6.3% (p14 )to83.2%(GSTP1)inpros- tate cancer patients. At least one gene was hypermethylated in 93% of cancer patients. The specificity of methylation was 0.74. Methylation was significantly more frequent (P < 0.05) in cancer than control patients for all genes except p14 and p16 . According to receiver operator curve analysis, the four-gene combination of GSTP1 (0.86), RASSF1a (0.85), RARb2 (0.80), and APC (0.74) best discriminated malignant from nonmalignant cases. The sensitivity and accuracy of this four-gene set were 86% and 89%, respectively. Conclusions: The presence of aberrant methylation in urinary cells obtained after prostate mas- sage is significantly associated withprostate cancer. A panel of four genes could stratify patients into low and high risk of having prostate cancer and optimize the need for repeat prostatic biopsies.

Prostate cancer is the most commonly detected male cancer for the disease are low,regardless of the threshold value used and the second leading cause of male cancer deaths in the (either 2.5 or 4.0 ng/mL; refs. 3–5). PSA may in fact be a better United States and in Europe (1). Its prognosis is directly related marker of benign prostate hypertrophy than of prostate cancer. to stage at diagnosis and treatment (2). However,the early and To confirm the diagnosis of cancer,the gold standard method is specific diagnosis of prostate cancer is difficult due to lack of ultrasound-guided (6). This is an uncomfort- cancer-specific biomarkers. The most common marker used is able procedure with frequent morbidity. Markers that distin- prostate-specific antigen (PSA),whose specificity and sensitivity guish benign from clinically silent malignant disease are urgently needed to improve the care of men with prostate cancer and reduce the number of unnecessary biopsies. Cancer arises through the accumulation of multiple molec- ular genetic events (7). These include changes in DNA sequence 1 Authors’ Affiliations: Institute for Cancer Studies and Academic Unit, or content (inherited and acquired) and changes in gene University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom and 2CeRePP group, EA3104, University Paris VII; 3Departments of Tumor Biology and expression through epigenetic mechanisms (8–10). The devel- 4Urology, Groupement Hospitalier et Universitaire-Est Assistance Publique- opment of diagnostic and screening tools that detect these Ho“ pitaux de Paris, University Paris VI; 5Department of Urology, Hospital Henri alterations (and the corresponding neoplastic cells) with a high Mondor, University Paris XII; and 6Department of Urology, Institut Mutualiste specificity and sensitivity would constitute a major step forward Montsouris, University ParisV, Paris, France in oncological care (7,8). One mechanism of tumor suppressor Received 10/11/06; revised 12/1/06; accepted 12/28/06. The costs of publication of this article were defrayed in part by the payment of page gene inactivation seems to be methylation of the cytosine- charges. This article must therefore be hereby markedadvertisement in accordance guanine dinucleotides (CpG islands) within gene promoter with18 U.S.C. Section 1734 solely to indicate this fact. regions. Hypermethylation at various gene loci has been Requests for reprints: Morgan Roupre“ t, Institute for Cancer Studies, Royal detected in several urologic malignancies,including prostate Hallamshire Hospital, G Floor, Glossop Road, S10 2JF Sheffield, United Kingdom. cancer. Most studies have been done on prostate tissue Phone: 44-114-271-1648; Fax: 44-114-271-2268; E-mail: mroupret@ club- internet.fr. (11–14) but some have used urine (15,16),sometimes F 2007 American Association for Cancer Research. obtained after prostate massage (17,18). To our knowledge, doi:10.1158/1078-0432.CCR-06-2467 there are,however,few studies that have investigated an

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All patients had undergone pre-radical prostatectomy diagnostic Table 1. Clinical characteristics of prostate cancer prostatic biopsies because of a digital abnormality or and control patients an increase in PSA. Exclusion criteria were clinical T3 prostate cancer at digital rectal examination and a history of urothelial tumors. Pathologic Cancer Control stage of prostate cancer is detailed in Table 1 according to the tumor- patients (%), patients (%), n =95 n =38 node-metastasis 2002 classification (22). Urine samples were obtained from 95 consecutive radical prostatectomy patients and from 38 age- Age at diagnosis (y) matched males (controls) with no history of genitourinary malignancy, Median 63 62 negative prostate biopsies,and with or without benign prostate Range 56-82 57-79 hypertrophy (Table 1). Median number of prostate biopsies was 10 Preoperative serum PSA (range,8-12) in both groups according to our local protocol. Radical Median (ng/mL) 7.28 4.9 prostatectomy patients underwent prostate massage by digital rectal Range 3.5-26.3 1.1-10.2 V4 ng/mL 5 (5.3) 7 (18.4) examination (at least 1 min) in the operating room to obtain prostate 4-8 ng/mL 47 (49.5) 22 (57.9) secretions at the external urethral meatus. The first urine stream was 8.1-12 ng/mL 35 (36.8) 9 (23.7) then collected by urethral catheterization of the bladder. Control >12 ng/mL 8 (8.4) — patients also underwent prostate massage to collect urine sediments Pathologic stage either before a set of biopsies or an endoscopy or a medical visit. pT2a 32 (33.7) — DNA extraction from urine. Urine samples (40-120 mL) were pT2b 14 (14.7) — immediately placed in Carbovax-PEG. They were centrifuged at 4jC pT2c 40 (42.1) — (800 g  10 min). The pelleted material,including exfoliated cells,was pT 9 (9.5) — 3a resuspended in 3 mL PBS and recentrifuged (800 g  10 min). DNA Gleason score — 4-5 8 (8.4) — was extracted from the wash urine suspension using Qiagen Tissue and 6 47 (49.5) — Blood kits (Qiagen,Valencia,CA). Its concentration was measured on 7 35 (36.8) — an aliquot with Hoechst 33258 as DNA-binding fluorochrome (DyNA 8 4 (4.2) — Quant 200,Hoefer Pharmacia Biotech,San Francisco,CA). The DNA 9-10 1 (1.1) — extracts were frozen at À20jC until use. Methylation analysis. For methylation analysis,1 to 2 Ag DNA was treated with sodium bisulphite using the CpGenome kit (Chemicon, extended panel of methylation markers on urine sediment for Hampshire,United Kingdom) according to the manufacturer’s the detection of prostate cancer (19,20). instructions as described previously (23,24). The bisulfite-treated In the present study,we examined 10 genes that are either DNA was then used as a template for the quantitative fluorescence- silenced or activated by aberrant methylation mechanisms and/ based real-time methylation-specific PCR (QMSP) as described previously (19,20). Ten gene promoter regions [ GSTP1, Ras association or that play an important role in human neoplasia (12,19,21) domain family 1 isoform A (RASSF1a), CDH1 (E-cadherin), adenomatosis to establish a small,reliable set of prostate cancer markers,which polyposis coli (APC), DAPK, MGMT, p14, p16, retinoic acid receptor b2 could be assessed from urine obtained after prostate massage. (RARb2),and TIMP3] were investigated for aberrant methylation by QMSP. These genes have been examined previously and shown to be Materials and Methods of interest in early prostate cancer detection (12,19,21). For methyl quantification,PCR was done using bisulphite-specific primers to h- actin (as the internal reference gene). Universally methylated DNA Patients and urine collection. We analyzed urine samples collected (Chemicon) was used as a positive control. prospectively (October 2005-February 2006) from patients with QMSP Accuprime Taq polymerase (Invitrogen,Paisley,United localized prostate cancer who underwent laparoscopic radical prosta- Kingdom) using the ABI Prism 7000 Sequences Detection System. tectomy at the Institut Mutualiste Montsouris (Paris,France). Urine Primers and fluorescence-labeled probes were obtained from MWG sample collection was approved by the Ethics Committee of the Biotech (London,United Kingdom). A 10 AL PCR volume was used, Assistance Publique-Hoˆpitaux de Paris. All study participants gave their comprising 1 AL Accuprime buffer,0.25 AL Accuprime Taq polymerase, written informed consent. 5 pmol/L forward and reverse primers,5 pmol/L probe,0.2 AL Rox reference dye,and water. Sixty cycles of denaturation (95 jC for 45 s), annealing (specific primer temperature for 2 min),and extension (72 jC for 1 min) were used (Fig. 1). Statistical analysis. For quantification,the relative methylation levels for each promoter were calculated from the ratio of fluorescence released by the QMSP reaction and that from the h-actin reaction of same sample (gene/h-actin ratio). For analysis with respect to the presence of methylation,we used a threshold of 0.2 DRn. We have found previously that this correlates well with the absolute presence of methylation within sample using bisulphite sequencing (data not shown; Fig. 1). Correlations between methylation levels and clinico- pathologic variables were evaluated by the m2 test and Fisher’s exact test. The odds ratio [95% confidence interval (95% CI)] for each of the 10 gene loci was calculated. The sensitivity and specificity of methylation in discriminating malignant cells were determined by receiver operator curve (ROC) analysis. The discriminatory power of the test was given by the area under the ROC modified for censored data (1 = a perfect test; 0.5 = a worthless test). ROC analysis was also used to determine the methylation cutoff levels yielding optimal sensitivity and specificity for Fig. 1. Representative QMSP image (RASSF1a gene promoter). the ‘‘best’’ set of gene loci. Continuous QMSP data were converted into

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Table 2. Frequency of aberrant methylation in urine DNA

Gene Methylation-positive urine* P c Odds ratio (95% CI) Cancer patients (%) Controls (%)

RASSF1a 74 (77.9) 3 (7.9) <0.0001 41.1 (11.5-147.1) CDH1 29 (30.5) 2 (5.3) 0.002 7.9 (1.8-35.1) APC 48 (50.5) 2 (5.3) <0.0001 18.4 (4.2-80.7) DAPK 27 (28.4) 2 (5.3) 0.003 7.1 (1.6-31.8) MGMT 14 (14.7) 1 (2.6) 0.046 6.4 (0.8-50.5) p16 11 (11.6) 2 (5.3) 0.268 2.3 (0.5-11.2) p14 6 (6.3) 1 (2.6) 0.39 2.5 (0.3-21.4) GSTP1 79 (83.2) 5 (13.2) <0.0001 32.6 (11-96.3) RARb2 59 (62.1) 1 (2.6) <0.0001 60.6 (7.9-461.3) TIMP3 41 (43.2) 0 (0) 0.001 2.1 (1.6-2.7)

*Methylation-positive: methylation level above the empirical cutoff level obtained by comparing patients and controls and maximizing the likelihood ratio for a positive test. Cancer patients (n = 95), controls (n = 38). cP value according to the m2 test. discrete binary data by considering a methylation level that was above cancer patients than in age-matched controls. The difference the calculated cutoff level as positive and by maximizing the likelihood reached statistical significance (P <0.05)foreightloci ratio for a positive test. Statistical analyses were done using SPSS version (RASSF1a, CDH1, APC, DAPK, MGMT, GSTP1, RARb2,and 12 (SPSS,Inc.,Chicago,IL). TIMP3; Table 2). There was no correlation between hyper- methylation and either tumor stage or grade in the current Results study. Hypermethylation of no single gene locus was signifi- cantly related to a clinicopathologic variable (preoperative In our patients,PSA (with a threshold of 4 ng/mL) had a serum PSA level,pathologic stage,or Gleason score; Table 3). sensitivity of 94.7% and a specificity of 18.4% for prostate We plotted the methylation levels for the four gene loci cancer. Methylation analysis of urine DNA samples successfully (GSTP1, APC, RASSF1a,and RARb2),which showed the diagnosed 88 of 95 cases of early prostate cancer (sensitivity, greatest statistical difference (P < 0.0001) in aberrant methyl- 0.93),and 74 of 95 (78%) of these samples were positive for at ation between prostate carcinoma and controls (Fig. 2). As least three genes. Of the 38 patients with no evidence of disease, shown,the relative amount of methylated promoter was much 28 showed no aberrant methylation at any promoter site higher in urine sediment from cancer patients than from (specificity,0.74). The frequency of aberrant methylation at controls. The estimated odds ratios for these four genes ranged CpG islands ranged from 6.3% (p14) to 79% (GSTP1; Table 2). from 18.4 to 60.6 (Table 2). All four ROC curves are plotted in Methylation at all 10 gene loci was more frequent in prostate Fig. 3. The area under the ROC was 0.86 (95% CI,0.79-0.93)

Table 3. Cross-table between positive promoter methylation in urine DNA and clinical variables of prostate cancer patients

n No. methylation-positive patients* RASSF1a CDH1 APC DAPK MGMT P16 P14 GSTP1 RARB2 TIMP3

Preoperative PSA (ng/mL) V45 4 031 0003 4 5 4-8 47 39 17 28 14 7 6 2 42 32 19 8.1-12 35 24 9 16 10 3 4 3 28 21 13 >12 8 7 3 1 2 4 1 1 6 2 4 Pathologic stage

T1c 63 53 9 25 11 6 2 2 55 40 24 T2a 22 15 12 18 12 4 4 2 17 12 9 T2b 6 4 522 1313 2 5 T2c 3 1 221 2103 2 2 T3a 1 1 111 1111 1 1 Gleason score 4-5 8 5 2 1 1 1 0 0 4 3 6 6 47 37 11 27 15 6 7 2 40 25 14 7352813178 422312819 8 4 3 223 2113 2 1 9-10 1 1 1 1 1 1 1 1 1 1 1

*Methylation-positive: methylation level above the empirical cutoff level obtained by comparing patients and controls and by maximizing the likelihood ratio for a positive test.

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Fig. 2. Methylation levels of GSTP1 (A), RASSF1a (B), RARb2 (C), and APC (D)in urine sediment DNA of prostate cancer patients (n = 95) and controls (n =38).

for GSTP1,0.85 (95% CI,0.77-0.92) for RASSF1a,0.8 (95% this study. No urine sediment DNA sample has yet given a CI,0.73-0.87) for RARb2,and 0.74 (95% CI,0.65-0.82) for positive GSTP1 methylation result in the absence of methyla- APC. Threshold values for distinguishing benign and malignant tion in the corresponding tumor (20,28). Although rare in cells with 95% specificity were calculated (1.0,1.0,0.34,and normal prostate tissue (29,30),GSTP1 methylation did occur 0.91 DRn for GSTP1, RASSF1a, RARb2,and APC,respectively). in 5 (13.2%) of our controls. It may be age related as promoter Combining these four genes cause the greatest discriminatory hypermethylation of certain genes in apparently normal tissues power. The theoretical sensitivity of the four-gene set was 86% seems to be associated with aging (29). Further follow-up is and its diagnostic accuracy was 89%. needed to determine whether our subjects with methylated markers but no evidence of cancer actually have disease. Discussion The use of a single gene locus to discriminate malignant from benign cells has drawbacks. First,the maximum sensitivity can Our study has confirmed that aberrant methylation can be only be as high as the frequency of hypermethylation at a specific detected in cells from post-prostate massage voided urine CpG locus. Second,noncancerous tissues can in some cases harbor specimens from radical prostatectomy patients with early CpG island hypermethylation at the same gene locus. Further- prostate cancer (8,10,17,18). The sensitivity of the 10 gene more,methylation of a single gene locus may occur in cancers loci in diagnosing cancer on these specimens was 93% (i.e., other than prostate cancer or even in benign disease. Investigating slightly higher than for preoperative voided or catheterized several genes may have more discriminatory power because the urine specimens; refs. 15–18). Prostate tissue and serum number of hypermethylated genes rises as prostate cancer samples have given similar results but they yield larger amounts progresses (8,19,21,25). A panel of carefully selected methylation of DNA for extraction (12,14,25,26). Prostate massage could markers for use on urine sediments might help both detect be a first step in increasing the shedding of prostate cells in and discriminate among a variety of urologic tumors. Cancers urine (10) and collecting enough material for QMSP. other than prostate cancer (e.g.,bladder and kidney cancer) can The most common somatic genome alteration during contribute cellular DNA to urine sediment (23,31–33). prostate cancer development seems to be hypermethylation in In our panel of 10 genes,3 genes besides GSTP1 proved to be the regulatory region of the promoter of the k-class glutathione of special interest: APC, RASSF1a,and RARb2. APC is a tumor S-transferase (GSTP1) gene (20,21,27). Its prevalence in body suppressor involved in down-regulating WNT/h-catenin signal- fluids (urine or serum) can reach 76% (15,17); it was 83% in ing but it also plays a role in cell-adhesion,stability of the

www.aacrjournals.org 172 3 Clin Cancer Res 2007;13(6) March 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology microtubular skeleton,and possibly apoptosis. APC was methylated in the urine sediment DNA of 50.5% of our patients. This is within the reported 27% to 95% range for APC hypermethylation in primary prostate tumors (11,34). RASSF1a is a tumor suppressor that interacts with Cdc20,an activator of the anaphase-promoting complex,to inhibit complex activity and prevent mitotic progression. RASSF1a was methylated in 77.9% of our patients. Epigenetic inactiva- tion of RASSF1a is observed in 53% to 71% of solid tumors and epithelial cancers,including prostate cancer (35,36). RAR h2 functions as a tumor suppressor in lung,breast,and gyneco- logic neoplasia. It is hypermethylated in 62.1% of prostate adenocarcinomas (37,38). In the present study,RAR h2 clearly discriminated between neoplastic and nonneoplastic cells. Three of these genes (APC, RARb2,and GSTP1) are the same as those found to be most discriminatory between cancer tissue and benign prostate hypertrophy in a recent study (39). For most genes,we observed higher frequencies of promoter methylation than reported previously (19,21,40),although our results seem similar to those found by Hoque et al. (20) for many genes. In particular,we found less methylation of p16 and MGMT and more of GSTP1 and RARh2 than Hoque et al. (20). The reasons for differences in reported methyl frequency are unclear and may reflect patient selection [European versus North American patients (different PSA screening prevalences)], different materials examined (primary tumour tissue versus urine),or laboratory methodology (QMSP versus MSP). Our data once again confirm the importance of GSTP1 in the biology of prostate cancer and that for most loci levels of methylation Fig. 3. ROC curves for RASSF1a, APC, GSTP1,andRARb2 used to determine the methylation cutoff level for optimal sensitivity and specificity for each gene locus. are reproducible in different laboratories. With regards to tissue examined,most previous reports focus on prostate tissue rather than bodily fluids (19,21,40) and authors have reported technique may limit general acceptability of the test in routine results in line with our findings (26,37,41). With respect clinical practice. These assays may in future reduce the need for to methodology,quantitative method (QMSP) uses PCR repeat biopsies and have the potential to distinguish men at conditions that differ from those of conventional MSP and high risk of having prostate cancer from those with benign seems more sensitive to the latter (15,20) and enables tissue disease following PSA screening. Further work is also required differentiation by quantitative rather than just qualitative to investigate aberrant methylation as a possible predictive methyl analysis (20,41). biomarker of disease aggressiveness in prostate cancer,in an There is currently no consensus for managing high-risk men attempt to address the growing issue of overtreatment of with an abnormal digital rectal examination or persistently indolent disease (43). elevated PSA levels and a negative initial biopsy (5,6). Surveillance includes measuring the percentage of free PSA or Acknowledgments monitoring PSA kinetics (42). QMSP assays of aberrant Morgan Roupre“ t was awarded a European Urological Scholarship Programme methylation of key prostate cancer genes in urine DNA warrant from the European Association of Urology for performing the laboratory aspect of further multicenter validation,on larger numbers of patients, this work at the Academic Urology Unit and Institute for Cancer Studies, University although the necessity and variability of the prostatic massage of Sheffield.

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