MINIMALLY INVASIVE PROSTATE CANCER DIAGNOSIS BY GLUTHATIONE S-TRANSFERASE P 1 ) GENE METHYLATION ANALYSIS IN SERUM SPECIMENS

ORIGINAL ARTICLES MINIMALLY INVASIVE PROSTATE CANCER DIAGNOSIS BY GLUTHATIONE S-TRANSFERASE P 1 ) GENE METHYLATION ANALYSIS IN SERUM SPECIMENS Raluca Dumache 1a, Victor Dumitrascu 2, Radu Minciu 3, Dana David 1, Anca Tudor 4, Bogdan Bumbacila 5a, Maria Puiu 6 REZUMAT Introducere: Cancerul de prostată (CaP) reprezintă a doua cauză de mortalitate în rândul bărbaţilor pe plan mondial. Hipermetilarea genei glutation S-transferaza P 1 ) apare cel mai frecvent la debutul procesului de carcinogeneză prostatică. Obiective: Scopul acestui studiu a fost de a analiza valoarea diagnostică a hipermetilării genei din probele de ser în diferenţierea pacienţilor cu diagnostic de CaP, respectiv hiperplazie benignă de prostată (HBP) prin metode minim invazive. Material şi metode: Hipermetilarea genei a fost investigată la nivelul ADN-ului genomic extras din probele de ser provenite de la 91 bărbaţi cu diagnosticul de CaP şi 94 cu HBP. ADN-ul genomic extras a fost supus tratării bisulfitice şi analizat prin metoda metilării specifice reacţiei de polimerizare în lanţ, fiind apoi corelat cu parametrii clinico-patologici ai pacienţilor. Rezultate: Nivele ale hipermetilării genei au fost prezente în cazul a 89 din 91 (92.86%) bărbaţí cu CaP şi în cazul a 10 din 94 (11%) bărbaţi din lotul control cu HBP. Concluzii: În acest studiu am evaluat abilitatea genei GSTP1 de a diferenţia pacienţii cu CaP şi HBP din probele de ser prin metode minim invazive. Cuvinte cheie: cancer de prostată, hiperplazie benignă de prostată, glutation S-transferaza P 1, metilare specifică reacţiei de polimerizare în lanţ ABSTRACT Introduction: Prostate cancer (PCa) represents the most commonly diagnosed type of malignancy among men in Western countries, and the second cause of cancer-related deaths among men worldwide. Alterations in the methylation patterns of promoter CpG islands have been associated with the transcriptional inhibition of genes in many human cancers, including PCa. Objective: The aim of our study was to analyse the diagnostic value of aberrant promoter hypermethylation of gene glutathione S-transferase P 1 ) in serum DNA to discriminate between prostate cancer (PCa) and benign prostatic hyperplasia (BPH) patients by minimally invasive methods. Material and methods: Aberrant promoter hypermethylation was investigated in DNA isolated from serum samples of 91 patients with diagnostic of PCa and 94 with BPH (control subjects). Extracted genomic DNA was bisulfite treated and analyzed using methylation-specific polymerase chain reaction (MS-PCR). Results: Promoter hypermethylation of gene was detected in serum samples from 89 of 91 (92.86%) patients with PCa. Serum samples from the 94 controls without genitourinary cancer, revealed promoter hypermethylation of gene in 10 (10.6%) of the 94 patients. Receiver operating curve (ROC) included clinico-pathological parameters such as: serum PSA levels, pathological stage, Gleason score, hypermethylation status of gene, and gave a predictive accuracy of 96% with a sensitivity and specificity of 98% and 89%, respectively. Conclusions: In our study we have evaluated the ability of gene to discriminate between PCa and BPH patients in serum samples,by minimally invasive methods. Key Words: prostate cancer, benign prostatic hyperplasia (BPH), glutathione S-transferase P 1, methylation-specific polymerase chain reaction INTRODUCTION 1 Department of Biochemistry, Victor Babes University of Medicine and Pharmacy, Timisoara, 2 Department of Laboratory Medicine, Clinical Emergency County Hospital Timisoara, 3 Department of Urology, 4 Department of Medical Informatics and Biostatistics, 5 Department of Clinical Pharmacy, 6 Department of Medical Genetics, Victor Babes University of Medicine and Pharmacy, Timisoara a Because of the equal contribution to this work, both authors should be regarded as first author. Correspondence to: Raluca Dumache, MD, PhD, Department of Biochemistry, Victor Babes University of Medicine and Pharmacy, 2 E. Murgu Sq., 300041 Timisoara Email: raluca.dumache@umft.ro Received for publication: Oct. 13, 2011. Revised: Dec. 14, 2011. Prostate cancer (PCa) represents the most prevalent malignancy among men and the second cause of cancer related deaths worldwide. 1 If diagnosed in its early stages, when the tumor is confined to the prostatic capsule, PCa can be treated. Diagnosis and clinical management of PCa are often confounded because of the lack of symptoms and the absence of minimally invasive diagnostic techniques that could be used to detect the early stages of the disease. The only molecular biomarker used for early detection and recurrence monitoring after radical prostatectomy is prostate-specific antigen (PSA). 2 Measurement of the serum PSA levels alone is neither sensitive, nor specific for a definite diagnosis of prostate adenocarcinoma. 158 TMJ 2011, Vol. 61, No. 3-4

It has been demonstrated that most of the patients who had either an abnormal finding on digital rectal examination (DRE) or elevated serum PSA levels, require transrectal biopsies with ultrasound guidance (TRUS). Serum PSA levels are increased in benign prostatic hyperplasia (BPH), prostatitis, or prostatic ischemia. 3 Approximatelly one third from these men are found to have a negative biopsy result. Using these approaches, about 25% of men with false-negative prostate biopsy, are prone to develop PCa. It is known that the process of carcinogenesis develops in time, because of multiple molecular events, which include changes in gene expression,through epigenetic mechanisms. 4 The epigenetic alterations have been detected first in genomic DNA isolated from tissue samples from different types of tumors, especially DNA hypermethylation in the promoter region of tumor suppressor genes. Cytosine-phosphate guanine islands (CpG) are susceptible to hypermethylation in unknown growth conditions, and may develop some pathways leading to the development of certain types of tumors. 5 Using molecular biotechnology, some types of cancers have been detected from several bodily fluids, including urine in urological cancers, saliva in head and neck cancer, sputum and bronchoalveolar lavage in lung cancer. 6 Unlike RNA and proteins alterations, DNA methylation biomarkers are stable in bodily fluids and occur in definite regions, unlike DNA mutations. 7 Therefore, DNA methylation biomarkers might be used as noninvasive biomarkers in early detection of cancer and in monitoring the disease outcome. The earliest and commonest alteration which occurs during prostate carcinogenesis is represented by the hypermethylation of the glutathione S-transferase P 1 ) gene. hypermethylation has been detected by methylation-specific polymerase chain reaction (MS-PCR) method, in various bodily fluids such as urine, ejaculates or blood serum or plasma. hypermethylation has been reported to be present in up to 100% in PCa tissue, in up to 2/3 of high grade prostatic intraepithelial neoplasia (HGPIN), rarerly in benign prostatic hyperplasia(bph) and is absent in normal prostatic tissues. 8,9 AIM OF THE STUDY The main objective of our study was to determine the clinical utility of this new biomarker in serum samples to distinguish between PCa patients and BPH patients by minimally invasive methods. MATERIALS AND METHODS Patients and samples collection In our study we included 91 cases with PCa histological confirmed and 94 BPH cases (cancerfree controls), hospitalized between January 2008 to February 2010 in the Department of Urology, Clinical County Emergency Hospital Timisoara. The study was conducted in accordance with The World Medical Association Declaration of Helsinki from 2008 statements and written informed consent was obtained from each patient. The eligibility criteria for the PCa patients selection were: 1. Clinical tumor stage I or II; 2. No clinical evidence of lymph nodes or distant metastases; 3. No treatment with hormone or radiation therapy before blood samples collection The average patient age ± SD in the BPH group was 61 ± 8 years and 63 ± 6 years in the PCa group. At the time of enrollment, the 91 patients presented tumors that were clinical stage I (n=54 [59%]) and stage II (n=37 [41%]). After the pathologic examination of radical prostatectomy specimens, enrolled patients were grouped according to tumor stage, as follows: pt 2, n=47 (52%), and pt 3, n= 44(48%). Patients were grouped according to Gleason score (GS) intervals as follows: 3 through 4, n= 17 (19%); 5 through 6, n=39 (43%), 7 through 10, n=35 (38%). Preoperative serum PSA levels were in range of 4.0 to 34 ng/ml. 1. Blood collection and DNA isolation Five milliliters of blood were drawn and collected in a serum separator tube containing clot activator and gel (Vacutainer, Becton Dickinson, USA). Tubes were inverted 8 times and centrifuged within 2 hours of collection for 10 minutes at 1500 X g. Using ZR Serum DNA (Zymo Research, U.S.A) we extracted DNA from 1 ml serum following the manufacturer s protocol, and stored it at 80 ºC until further analysis. 2. Bisulfite treatment and methylation-specific PCR (MS-PCR) analysis Using EZ DNA Methylation Kit (Zymo Research, Orange, CA) protocol, 2 μg of genomic DNA from each patient sample was treated with denaturation buffer, sodium bisulfite (converting unmethylated cytosine residues to uracil), and desulfonation buffer, with elution of the bisulfite-modified DNA into 10 μl of buffer. Two sets of primers were used to amplify each region of interest: one pair recognized a sequence in which CpG sites are unmethylated (bisulfite modification to UpG) and the other recognized a sequence in which CpG sites are methylated (modified Raluca Dumache et al 159

by bisulfite treatment).the reaction volume was of 50 μl containing: 10 μl of distillated water in which we added 25 μl Taq Polymerase mix, 2.5 μl from each forward and reverse primer (Eurogentec, Belgium). To amplify the regulatory region of GSTP1 we used 2 μl of the bisulfite-modified DNA as a template for the MS-PCR reaction. The PCR conditions were as follows: Hot start at 95ºC for 5 minutes (to fully denaturate the bisulfite modified genomic DNA), 35 amplification cycles (94 ºC for 30 seconds for denaturation, 58ºC for 30 seconds for primer annealing and 72ºC for 60 seconds for extension), and a final full extension at 75ºC for 4 minutes. The primers used for the amplification reaction were as follows: Forward primers: 5 -TTCGGGGTGTAGCGGTCGTC-3 (methylated); 5 -GATGTTTGGGGTGTAGTGGTTGTT-3 (unmethylated); Reverse primers: 5 -GCCCCAATACTAAATCACGACG-3 (methylated); 5 -CCACCCCAATACTAAATCACAACA-3 (unmethylated) Sodium bisulfite-modified DNA from healthy donors lymphocites served as unmethylated negative control. To detect the methylation levels of gene GSTP1 in patients with PCa and BPH we separated electrophoretically the MS-PCR products on a 2% Seakem agarose gel (Lonza, Switzerland) and visualized them under an ultraviolet (U.V) transluminator (Vilbert Lourmat, France). Figure 1. Serum molecular detection of hypermethylation by MS- PCR analysis. Statistical analysis Data were analyzed by SPSS statistical package version 12 (SPSS Inc, Chicago,USA), with χ 2 test, Spearmann rank correlations and Mann-Whitney tests to comparison between groups and finding the correlations. A P-value < 0.05 was considered significant. RESULTS Correlations between the clinicopathological features and GSTP1 hypermethylation: 1. Hypermethylation status of gene in serum samples We observed that PCa patients undergoing disease progression had significantly increased methylation levels of gene when compared with BPH patients (Chi square test, p<0.001). In PCa patients, 89 (97.8%) of 91 presented hypermethylated levels of gene, whereas 10 (10.6%) of the 94 BPH patients had hypermethylated GSTP1 gene. The sensitivity and specificity of hypermethylation levels in discriminating PCa patients from BPH were determined by receiver operating curve (ROC) analysis. gene had a sensitivity of 98%, a specificity of 89% and yielded an area under the curve (AUC) of 0.936 (95 % CI; 0.895 to 0.977; p< 0.001), as presented in Figure 2. Figure 2. Receiver operating curve (ROC) analysis of gene hypermethylation levels in prostate cancer patients. 2. Hypermethylated levels of gene correlates with serum PSA levels According to Spearman rank-correlation, a significantly correlation has been found between serum PSA levels and GSTP1 hypermethylation in PCa patients (r=0.831; p<0.001). 3. Correlation of pathologic tumor stage with hypermethylation levels In PCa patients with pt 3 stage we observed significantly increased methylation levels of gene GSTP1, when compared with pt 2 stage (p<0.001; Mann-Whitney test). 4. Correlation of Gleason score (GS) with pathologic tumor stage According to the Spearman rank-correlation test, a significant correlation between GS and the pathological stage exists (r=0.749; p<0.001). Increased GS score were significantly associated with pt 3 stage. 160 TMJ 2011, Vol. 61, No. 3-4

DISCUSSION The evaluation of serum samples obtained from PCa patients, presents some advantages because, unlike tissue biopsy or imagistics, blood sampling is a minimally invasive method which does not present the risk of morbidity, and can be repeated to monitor the changes which occur during disease progression or to detect the recurrence of the disease. 10,11 The main goal of our study was to determine whether serum detection of methylation levels can discriminate PCa men from those with BPH by minimally invasive methods, and the second aim of the study was to investigate possible correlations between methylation levels and different clinico-pathological parameters. Sensitivity of 98% and specificity of 89% in distinguishing malignant cells, were determined by receiver operator curve (ROC) and the discriminatory power of the test was given by the area under the curve (AUC) which was 0.936 (95% CI;0.895 to 0.977; p<0.001). 12 The results obtained by us, can be considered good for a minimally invasive serum diagnostic test. The different levels of hypermethylation between neoplastic and nonneoplastic prostatic tissue,suggests the fact that measurement of hypermethylation levels could be more useful in distinguishing men at low risk for PCa from those with a clinically silent PCa,in comparison with the measurement of serum PSA levels. 13 De Marzo et al. reported in their studies that methylation levels of gene are present in men with proliferative inflamatory atrophy (PIA) and highgrade prostatic intraepithelial neoplasia (HGPIN), indicating that methylation occurs at the beginning of prostate carcinogenesis. 14 Detection of serum hypermethylation in patients with negative biopsy should be sufficient evidence to warrant the concern of the presence of an occult disease. The presence of methylation levels of gene in 10 (10.6%) men with previously BPH, indicates that they could harbor an occult microscopic foci of PCa in the context of BPH, which was omitted by prostatic biopsy. During the study, repeat biopsies have been performed to the 10 patients with BPH found with hypermethylated levels in serum samples. All of them (100%) have been found to have cancer on repeat biopsy. Abnormal methylation levels found in serum samples may help in identification of men who are at risk for harboring prostate cancer despite negative prostate biopsy. Henrique et al. found in serum samples from PCa patients hypermethylation levels ranging between 15 % and 70%. 15 In our study we found the hypermethylation levels up to 90%. These results could be explained by the use of quantitative MS-PCR rather than a conventional MS-PCR method. MS-PCR seems to be more sensitive in serum and currently, no evidence showed that qms-pcr could be more specific. Also, Maruyama et al. reported in one study the correlations between hypermethylation levels of gene from serum samples in PCa patients and Gleason score, serum PSA levels and pathologic stage. 16 Patients with elevated serum PSA levels can have either PCa or BPH, and repeat biopsies can provide up to 20% detection rate after an initial negative biopsy. Because only 30-40% of patients with serum PSA levels between 4 and 10 ng/ml have PCa, there are a lot of patients with negative prostate biopsies who would benefit from improved ability to discriminate between cancer and benign lesions, by noninvasive diagnosis methods, as described in our study. 17 The limitations of our study include the small number of patients and the lack of long-term followup. Further studies in the area of noninvasive detection of PCa include : the detection of PCa in its early stages from voided urine samples using a panel of tumoral biomarkers by qms-pcr method, and the detection of disease recurrence in men following radical prostatectomy from preoperative serum samples. CONCLUSIONS In our study we have demonstrated the feasibility of a novel clinical strategy, which is based on minimally invasive molecular test that can be used to aid to current investigation methods for prostate cancer detection. The efficacy of this minimally invasive test for early molecular detection of PCa is important in developing future clinical management algorithms and in establishing indications regarding the surveillance or repeat biopsy. REFERENCES 1. Garcia M, Jemal A, Ward EM, et al. Global Cancer Facts & Figures 2007. Atlanta (GA): American Cancer Society, 2007:1-50. 2. Ahmed H. Promoter methylation in prostate cancer and its application for the early detection of prostate cancer using serum and urine samples. Biomark Cancer 2010:17-33. 3. Phe V, Cussenot O, Roupret M. Methylated genes as potential biomarkers in prostate cancer. BJU Int 2010;105:1364-70. 4. Schulz WA, Hoffmann MJ. Epigenetic mechanisms in the biology of prostate cancer. Semin Cancer Biol 2009;19(3):172-80. 5. Hoque MO, Lee J, Begum, et al. High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density Raluca Dumache et al 161

single-nucleotide polymorphism array. Cancer Res 2003;63:5723-26. 6. Ellinger J, Muller SC, Stadler TC, et al. The role of cell-free circulating DNA in the diagnosis and prognosis of prostate cancer. Urol Oncol 2011;29(2):124-9. 7. Lee WH, Morton RA, Epstein JI, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA 1994;91:11733-7. 8. Ellinger J, Bastian PJ, Haan KI,et al. Noncancerous PTGS2 DNA fragments of apoptotic origin in sera of prostate cancer patients qualify as diagnostic and prognostic indicators. Int J Cancer 2008;122:138-43. 9. Boddy JL, Gal S, Malone PR, et al. The role of cell-free DNA size distribution in the management of prostate cancer. Oncol Res 2006;16:35-41. 10. Reibenwein J, Pils D, Horak P, et al. Promoter hypermethylation of GSTP1, AR, and 14-3 sigma in serum of prostate cancer patients and its clinical relevance. Prostate 2007;67:427-32. 11. Brooks JD, Weinstein M, Lin X, et al. CG island methylation changes near the GSTP1 gene in prostatic intraepithelial neoplasia. Cancer Epidemiol Biomark Prev 1998;7:531-6. 12. Thompson IM, Ankerst DP, Chi C, et al. Operating characteristics of prostate-specific antigen in men with an initial PSA level of 3.0 ng/ ml or lower. JAMA 2005;294:66-70. 13. Dumache R, Miclea F, David D, et al. Aspects of molecular genetics in prostate cancer. Romanian Journal of Rare Diseases, 2010;Suppl.1:71-2. 14. De Marzo AM, Marchi VL, Epstein JI, et al. Proliferative inflammatory atrophy of the prostate. Implications for prostatic carcinogenesis. Am J Pathol 1999;155:1985-92. 15. Jeronimo C, Usadel H, Henrique R, et al. Quantitative GSTP1 hypermethylation in bodily fluids of patients with prostate cancer. Urology 2002;60:1131-5. 16. Maruyama R, Toyooka S, Gazdar AF, et al. Aberrant promoter methylation profile of prostate cancer and its relationship to clinicopathological features. Clin Cancer Res 2002;8:514-9. 17. Nelson WG, De Marzo AM, Isaacs WB. Prostate cancer. N Engl J Med 2003;349:366-81. 162 TMJ 2011, Vol. 61, No. 3-4