ANTICANCER RESEARCH 33: 191-198 (2013) Study of Genetic and Epigenetic Alterations in Urine Samples as Diagnostic Markers for Prostate Cancer EFTHYMIOS DIMITRIADIS1, THEODOROS KALOGEROPOULOS2, STAMATIA VELAETI1, SOTIRIOS SOTIRIOU4, EVANGELOS VASSILIOU5, LOUKAS FASOULIS2, VASILIOS KLAPSAS2, MICHALIS SYNESIOU2, AIKATERINI APOSTOLAKI3, THEONI TRANGAS4 and NIKOLAOS PANDIS1 1Department of Genetics 2Urology and 3Pathology, St. Savvas Anticancer Hospital, Athens, Greece; 4Department of Biological Applications and Technologies, University of Ioannina, Ioannina, Greece; 5Department of Financial and Management Engineering, School of Business, University of Aegean, Chios, Greece Abstract. Background: Early diagnosis of prostate cancer and Prostate cancer is one of the most frequently diagnosed types identification of new prognostic factors remain main issues in of non-skin cancer and a leading cause of cancer-related prostate cancer research. In this study, we sought to test a deaths for men worldwide (1). Diagnosis and management panel of cancer-specific markers in urine samples as an aid for are complicated by the lack of cancer-specific markers to early cancer diagnosis. Materials and Methods: Sedimented assist for diagnosis during the early stages of the disease, and urine samples of 66 candidates for needle biopsy were tested. to predict and monitor response to therapy. Real time-polymerase chain reaction (RT-PCR) was applied to The use of prostate-specific antigen (PSA) as a screening detect the expression of transmembrane protease serine-2 and and monitoring marker for prostate cancer is widespread (2). Ets-related gene fusion (TMPRSS2–ERG), Ets-related gene Although PSA monitoring has led to higher prostate cancer (ERG), prostate cancer antigen-3 (PCA3), and serine peptidase detection rates, it has also substantial drawbacks. PSA is inhibitor kazal type-1 (SPINK1) transcripts. For testing of the specific for tissues of prostatic origin but is not cancer-specific. methylation status of Glutahione S-tranferase P (GSTP1) and Serum PSA levels are often elevated in benign prostatic Ras association domain family member-1(RASSF1A) promoter hyperplasia and prostatitis. Thus, the PSA testing is associated region, methylation-specific PCR (MSP-PCR) was applied. with a significant false-positive rate, with a high proportion Results: Among the tested parameters, the presence of (>50%) of the resulting biopsies proving negative for cancer. TMPRSS2–ERG (OR=9.044, 95% CI=2.207-37.066, Furthermore, studies have also indicated that low levels of PSA p=0.002), as well as a positive test result for PCA3 do not preclude prostate cancer, and that 15% of men with PSA (OR=7.549, 95% CI=1,858-30,672, p=0.005) were associated 0-4 mg/ml developed prostate cancer (3). Moreover large with the subsequent diagnosis of prostate cancer. A randomized trials assigned modest effects upon mortality rates multivariable logistic regression including all the significantly of PSA screening during the first decade of follow-up (4, 5). associated variables [prostate-specific antigen (PSA), digital Thus the need for non-invasive methods that can accurately rectal examination (DRE), TMPRSS2-ERG and PCA3], yielded assist in the early detection of prostate cancer, still exists. To a model with area under the receiver-operating characteristic address this issue, additional markers have been investigated curve (AUC) =0.894 (95% CI=0.772-1.00). Conclusion: A including genetic and epigenetic alterations. Among those the multiplexed quantitative PCR analysis on sedimented urine, in most promising are: Genes that are specifically overexpressed conjunction with the results of serum PSA levels and DRE, has in prostate cancer cells, such as prostate cancer antigen-3 the potential to accurately foresee subsequent needle biopsy (PCA3), alpha methyl-CoA racemase (AMACR), serine outcomes. On the basis of the above, algorithms may be peptidase inhibitor kazal type1 (SPINK1) (6-8); prostate designed to guide decisions for needle biopsy. cancer-specific gene alterations, mainly the fusion genes involving transmembrane protease serine-2 (TMPRSS2) and E-twenty six (ETS) family members (9); and prostate cancer- specific methylation alterations of gene promoter regions Correspondence to: Efthymios Dimitriadis, Department of Genetics, Glutahione S-tranferase P (GSTP1), Ras association domain St. Savvas Anticancer Hospital, Alexandras Ave 171, 11522, Athens, Greece. E-mail: [email protected] family member 1(RASSF1A) (10, 11). Taking advantage of the fact that prostate cells can be Key Words: Prostatic neoplasms, carcinoma, gene expression, detected in blood and urine, prostate cancer-specific markers diagnostic markers, urine. can be tested through a urine or blood diagnostic test (12). 0250-7005/2013 $2.00+.40 191 ANTICANCER RESEARCH 33: 191-198 (2013) The goal of the present study was to assess the diagnostic Table I. Sequence of primers, probes and annealing temperatures (TM). efficacy of a number of markers associated with malignant transformation when used individually or combined, in order Primer, probe Sequence TM to achieve a pre-biopsy prediction of prostate cancer and TMRSS2–ERGa2F cgc ggc agg aag cct ta 60˚C contribute to early diagnosis in a non-invasive manner. TMRSS2–ERGa2R tcc gta ggc aca ctc aaa caa c TMRSS2–ERG probe cag ttg tga gtg agg acc Materials and Methods PSA Fw gtc tgc ggc ggt gtt ctg 60˚C PSA Rw tgc cga ccc agc aag atc PSA probe cac agc tgc cca ctg cat cag ga Urine collection and DNA and RNA isolation. This study was PSA_SGfw gca tca gga aca aaa gcg tga 60˚C approved by the St. Savvas Anticancer Hospital Scientific PSA_SGRw cct gag gaa tcg att ctt cag Committee and informed consent was obtained from all participants. PCA3_SGFw cat ggt ggg aag gac ctg atg ata c 60˚C Urine samples were collected from 66 men who were admitted for PCA3_SGRw gat gtg tgg cct cag atg gta aag tc transrectal ultrasound (TRUS)-guided prostate biopsy at the SPINK1_SGFw caa aaa tct ggg cct tgc tga gaa c 60˚C Department of Urology in St. Savvas Anticancer Hospital on the SPINK1_SGRw agg cct cgc ggt gac ctg at basis of PSA level and/or abnormal DRE. At least 30 ml of urine ERG5-6_SGFw cgc aga gtt atc gtg cca gca gat 60˚C sample were collected from each patient immediately following a ERG5-6_SGRw cca tat tct ttc acc gcc cac tcc DRE. All patients included in this study were referred for prostate RASSFIA_methF gtg tta acg cgt tgc gta tc 56˚C biopsy for the first time. RASSFIA_methR aac ccc gcg aac taa aaa cga Urine was voided into sterile collection cups containing 5 ml of RASSFIA_unmethF ttt ggt tgg agt gtg tta atg tg 0.5 M EDTA. A minimum of 30 ml of the collected urine was RASSFIA_unmethR caa acc cca caa act aaa aac aa centrifuged at 5000 ×g for 20 min at 4˚C and the resulting cell pellet GSTP1_methF ttc ggg gtg tag cgg tcg t 56˚C was processed for DNA and RNA extraction. GSTP1_methR gcc cca ata cta aat cac gac g GSTP1_unmethF gat gtt tgg ggt gta gtg gtt gtt Total RNA and DNA was isolated using an RNA, DNA GSTP1_unmethR cca ccc caa tac taa atc aca aca extraction kit (NucleoSpin RNAXS, NucleoSpin RNA/DNA buffer set; Macherey-Nagel, Duren, Germany), according to the TMPRSS2: Transmembrane protease serine-2; ERG: Ets-related gene; manufacturer’s instructions. PSA: prostate-specific antigen; PCA3: prostate cancer antigen 3, SPINK1: serine peptidase inhibitor kazal type1; RASSF1A: Ras Quantitative reverse transcription-polymerase chain reaction association domain family member 1; GSTP: Glutahione S-tranferase (qRT-PCR). Quantitative RT-PCR was used to assess the P; SG: Syber Green. expression of four biomarkers [transmembrane protease serine2 and Ets related gene fusion (TMPRSS2–ERG), Ets related gene (ERG), PCA3, SPINK1] and the PSA transcript as control for prostate-derived cells. Total RNA (8 μl) was reverse-transcribed in 20 μl reactions using the SuperScript III First-strand Synthesis PCR conditions, as previously described (13, 14) (Table I). PCR Super Mix (Invitrogen, Carlsbad, California, USA) and random products were electrophoresed on 2.5% agarose gels and visualised hexamers as primers. with ethidium bromide staining. The cDNA was subjected to TaqMan PCR amplification for both the TMPRSS2–ERG fusion gene and PSA transcripts using primers Statistical analysis. Pre-biopsy clinical parameters were compared and PCR conditions, as described in Table I. between men diagnosed with prostate cancer vs. men without All PCR amplifications were performed in 20 μl reaction prostate cancer using Mann-Whitney tests for continuous variables mixture, containing 1× Platinum Quantitative PCR Super Mix and Fisher’s exact tests for categorical variables. Exact univariate (Invitrogen), 5 pmol of each primer and 5 pmol of TaqMan probes. logistic regression was used to examine the association between Syber Green qRT-PCR was applied for PCA3, ERG, SPINK1 and PSA levels, DRE outcome, PCA3 expression levels and PSA control expression using primers and PCR conditions described TMPRSS2–ERG fusion with the presence or absence of prostate in Table I. cancer upon prostate needle biopsy (PNB). All PCR amplifications were performed in 20μl reactions, The performance of each biomarker as a screening test was containing 1× Platinum Syber Green Quantitative PCR Super mix evaluated, and sensitivity, specificity, and the area under the (Invitrogen), and 5 pmol of each primer. receiver-operating characteristic curve (AUC), with 95% confidence The qPCR results for all the genes were calculated using intervals (CI), were calculated (15). A multivariable logistic RelQuant software (Roche Molecular Biochemicals, Mannheim, regression model predicting the diagnosis of prostate cancer on Germany) and are expressed as the ratio of the target gene/PSA biopsy was then developed using backward selection. The initial ×1000. The results were considered valid only if the PSA Ct was model contained PSA, DRE, PCA3 and TMPRSS2–ERG fusion as <35 cycles. potential predictor variables.