ER基因CpG岛甲基化的表达及其临床意义

目的:研究ER基因表达及CpG岛甲基化状况与胃癌生物学行为和预后的关系.方法:分别应用免疫组化SP法和限制性内切酶PCR法分析91例胃癌ER的表达和30例胃癌ER墓因CpG岛甲基化的状况.结果:胃癌ER阳性率为38%(35/91),其中高分化腺癌10%(3/30),分别与低分化腺癌43%(13/30)、未分化腺癌53%(8/15)、粘液癌63%(5/8)及印戒细胞癌75%(6/8)比较皆具有显著性差异(P＜0.05);伴淋巴结转移组为55%(11/20),未转移组22.5%(9/40),差异有显著性(P＜0.05).正常胃粘膜ER基因CpG岛未甲基化,而胃癌呈高度甲基化40%(12/30)(P＜0.05).结论:ER在胃癌中的表达与浸润、转移有关,ER阳性胃癌生物学行为差,ER基因CpG岛甲基化可能是胃癌中ER失表达的分子机制.     

p16基因甲基化在乳腺癌发展中的作用

目的 研究p16基因甲基化在乳腺癌中的分布情况,并探讨其与乳腺癌发生、发展的关系.方法 采用甲基化特异的PCR技术检测四川省肿瘤医院乳腺科2008年3～9月期间收治的38例原发性乳腺癌组织及距离肿瘤>5 cm以远的正常乳腺组织中p16基因启动子区CpG岛甲基化频率.结果 乳腺癌组织中p16基因启动子区CpG岛甲基化频率[31.6%(12/38)]明显高于其正常乳腺组织[5.3%(2/38)],差异有统计学意义(P=0.003).有淋巴结转移的乳腺癌患者中p16基因甲基化频率[45.5%(10/22)]高于无淋巴结转移患者[12.5%(2/16)],差异有统计学意义(P=0.044).结论 p16基因启动子区CpG岛甲基化可能在乳腺癌发生、发展过程中起着重要作用.     

肝细胞癌抑癌基因和癌基因甲基化水平的检测及意义

目的:探讨DNA甲基化异常在肝癌发展中的作用.方法:应用甲基敏感的限制性内切酶及Southern杂交技术对18例肝细胞癌抑癌基因Rb、17p13.3位点基因CpG岛和癌基因c-myc3′端散在CpG的甲基化状态进行了检测.结果:1例肝透明细胞癌和41.7%(5/12)肝细胞癌分别存在Rb基因和17p13.3位点基因CpG岛的高甲基化;而c-myc基因3′端散在形式的CpG岛的甲基在61%的肝癌发生不同程度的丢失,这种高和低甲基化状态可同时存在于同一病例.甲基化异常可发生在早期肝癌并随肿瘤进展而加重.结论:本实验结果表明:肝细胞癌存在严重的DNA甲基化紊乱,可能是抑癌基因失活和癌基因激活的重要因素.     

尿液蛋白激酶Y甲基化联合前列腺影像数据与报告系统评分在前列腺癌早期诊断中的价值

目的探讨尿液蛋白激酶Y(PRKY)基因甲基化联合前列腺影像数据与报告系统(PI-RADS)评分在前列腺癌(PCa)中的诊断价值。方法收集2018年10月至2019年10月在苏州大学附属第二医院住院的51例可疑PCa患者的尿液, 提取DNA后通过焦磷酸测序法检测PRKY基因启动子区甲基化水平, 同时根据前列腺穿刺活检病理结果将患者分为PCa组和良性前列腺增生(BPH)组。采用Mann-Whitney检验分析两组患者年龄、前列腺特异性抗原(PSA)等临床指标的差异, 运用χ2检验分析两组患者尿液中的PRKY基因启动子甲基化状态。建立PRKY甲基化和PI-RADS评分的受试者工作特征曲线(ROC), 计算曲线下面积(AUC), 分析PRKY甲基化在PCa中的诊断价值, 并联合磁共振PI-RADS评分进行联合诊断。结果 PCa和BPH患者尿液中PRKY甲基化阳性率分别为79.17%(19/24)和25.93%(7/27), 差异有统计学意义(χ2=3.516, P<0.01)。ROC分析得出PRKY甲基化对于诊断PCa的曲线下面积(AUC)为0.813, PRKY甲基化联合PI-RADS...     

胰液中K-ras基因突变和p16基因甲基化改变联合检测在胰腺癌诊断中的初步研究

目的 探讨胰液中K-ras基因突变和p16基因启动子区5′CpG岛甲基化联合检测在胰腺癌诊断中的价值.方法 采用ERCP下放置鼻胰管收集胰液,采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)法检测胰液中K-ras基因12密码子点突变,PCR-MSP法检测胰液中p16基因甲基化状态.结果 所有胰液标本均成功抽提出DNA,30例胰腺疾病病人胰液标本同时进行了K-ras基因突变和p16基因启动子区5′CpG岛甲基化检测,其中20例胰腺癌病人胰液中K-ras基因突变率为70%(14/20),p16基因甲基化率为35%(7/20),8例慢性胰腺炎中K-ras基因突变率为25%(2/8),2例胰腺囊腺瘤病人中K-ras基因突变率为50%(1/2),慢性胰腺炎和胰腺囊腺瘤病人胰液中无p16基因甲基化.单纯胰液中K-ras基因突变检测诊断胰腺癌的敏感性为70%(14/20),特异性为70%(7/10),阳性预测值为82.4%(14/17),阴性预测值为53.8%(7/13),诊断准确性70%(21/30).胰液中p16基因甲基化与K-ras基因联合诊断胰腺癌的敏感性为70%(14/20),特异性为100%(10/10),阳性预测值为100%(14/14),阴性预测值为62.5%(10/16),诊断准确性80.0%(24/30).结论 鼻胰管收集的胰液可用于分子生物学检测,联合检测胰液中K-ras基因突变和p16基因启动子区5′CpG岛甲基化更有助于胰腺癌的诊断.     

乳腺癌患者血浆中E钙粘素基因启动子甲基化的检测与临床意义

目的探讨乳腺癌组织及外周血浆中E 钙粘素基因启动子的甲基化改变在乳腺癌诊断、复发中的作用。方法采用甲基化特异性PCR方法 (methylation specificPCR ,MSP) ,分别检测4 2例乳腺癌肿瘤组织、癌旁组织和外周血浆中游离DNAE 钙粘素基因的甲基化改变。结果 4 2例乳腺癌肿瘤组织中 ,E 钙粘素基因甲基化改变阳性率为 5 2 4 % (2 2 / 4 2 ) ,相应外周血浆中游离DNAE 钙粘素基因甲基化检出率为 33 3% (14 / 4 2 ) ,血浆中甲基化改变与肿瘤组织甲基化状况显著相关(P <0 0 0 1) ,肿瘤组织及血浆中E 钙粘素甲基化改变与乳腺癌病理特征无相关性。肿瘤组织无甲基化患者及良性疾病患者血浆中未发现E 钙粘素甲基化畸变。结论乳腺癌血浆中游离DNAE钙粘素甲基化改变检测在其早期诊断 ,评估疗效 ,监控复发及普查中应用具有一定的可行性。     

HGPIN患者血清PSA特征及首次穿刺活检HGPIN阳性针数对再次活检PCa检出率的影响

目的 探讨高级别前列腺上皮内瘤(high grade prostatic intraepithelial neoplasia,HGPIN)患者血清前列腺特异性抗原(prostate specific antigen,PSA)特征及首次穿刺活检HGPIN阳性针数对再次活检前列腺癌(PCa)检测率的影响.方法 选取2013年2月至2015年12月在本院行前列腺穿刺的患者共320例,均行直肠超声引导下前列腺穿刺活检,分析各类患者血清PSA差异,对结果为非PCa患者于6个月后再次穿刺活检.结果 320例患者首次穿刺活检病理结果显示:其中HGPIN患者80例(孤立型51例,多灶型29例),低级别前列腺上皮内瘤(LGPIN)患者45例,前列腺增生(BPH)患者128例,PCa患者67例;其中PCa患者血清PSA为35.20(13.01,60.55) ng/mL,明显高于BPH、LGPIN、孤立型和多灶型HGPIN患者(P＜0.05);多灶型HGPIN血清PSA为12.15(6.82,16.43) ng/mL,明显高于BPH、LGPIN、孤立型HGPIN患者(P＜0.05);BPH、LGPIN、孤立型HGPIN患者血清PSA比较差异无统计学意义(P＞0.05);多灶性HGPIN患者再次穿刺为PCa的比例为38.46％ (10/26),明显高于BPH、LGPIN和孤立型HG-PIN患者(P＜0.05);BPH、LGPIN和孤立型HGPIN再次穿刺为PCa的比例比较差异无统计学意义(P＞0.05).结论 多灶型HGPIN血清PSA水平高于孤立型HGPIN、BPH以及LGPIN,但低于PCa;多灶型HGPIN患者再次活检PCa的检出率显著高于其他患者.     

结直肠癌环氧化酶-2、错配修复酶-1微卫星不稳定与基因调控的关系

目的 检测临床手术切除42例结直肠癌及相应正常组织环氧化酶-2(COX-2)、错配修复酶-1(hMLH1)微卫星不稳定状态(MSI)及二种基因启动子甲基化,探讨它们与结直肠癌发生的关系.方法 采用聚合酶链反应(PCR)技术检测5个位点的MSI状态;甲基化特异性PCR(MSP)方法检测结直肠癌及正常组织COX-2、hMLH1二种基因启动子CpG岛甲基化状态.结果 42例结直肠癌中MSI总检出率为42.86%(18/42),5个位点的MSI检出率差异无统计学意义(P＞0.05).COX-2和hMLH1基因启动子CpG岛甲基化在42例结直肠癌中分别有13例和15例.MSI-H组中COX-2基因启动子CpG岛甲基化8例,且有6例结直肠癌同时出现hMLH1和COX-2基因启动子CpG岛甲基化.结论 MSI结直肠癌hMLH1基因启动子CpG岛甲基化率高于MSS结直肠癌,hMLH1基因启动子CpG岛甲基化有助于判断肿瘤类型.     

乳腺癌WWOX基因表达缺陷的意义及其与CpG岛甲基化的关系

目的 研究WWOX蛋白在乳腺癌组织和细胞系中的表达变化,分析其与WWOX基因启动子和第一外显子CpG岛甲基化的关系.方法 应用免疫组织化学染色检测乳腺癌组织和细胞系中WWOX蛋白表达,采用甲基化特异性PCR(methylation-specific PCR,MSP)分析乳腺癌组织和细胞系中WWOX基因CpG岛甲基化状况.结果 32.2%的乳腺癌组织和5.4%正常乳腺组织中WWOX蛋白表达缺失,差异有统计学意义(P＜0.01).乳腺癌组织WWOX表达异常与绝经后状态(P＜0.01)关系密切.Ⅰ、Ⅱ、Ⅲ期患者中WWOX蛋白表达缺失的比例分别为23.1%、28.6%和46.2%.55%的乳腺癌组织WWOX启动子CpG岛甲基化扩增,45%的乳腺癌组织WWOX第一外显子CpG岛甲基化扩增,癌旁组织中未出现CpG岛甲基化扩增.MDA-MB-231细胞WWOX基因CpG岛完全甲基化,而MCF-7细胞无甲基化产物扩增.结论 乳腺癌中广泛存在着WWOX基因CpG岛甲基化,是WWOX表达缺陷的重要机制,并且可能通过性激素受体信号途径在乳腺癌的发生、发展中发挥作用.     

肝癌患者E-cadherin基因启动子区CpG岛甲基化状态的研究

目的探讨抑癌基因E-cadherin基因启动子区CpG岛甲基化与人原发性肝癌(简称肝癌)发生、发展的关系,及其在肝癌早期诊断中的意义。方法用巢式甲基化特异性PCR对34例肝癌患者的癌组织、距离癌灶边缘>3 cm的远癌组织及10例未发生肝癌的肝硬变患者的肝硬变组织中E-cadherin基因启动子区CpG岛的甲基化状态进行检测。结果 E-cadherin基因启动子区CpG岛甲基化阳性率,在34例肝癌组织中为61.76%(21/34),明显高于远癌组织中的29.41%(10/34),P<0.05;在10例无肝癌的肝硬变组织中为50.00%(5/10),与肝癌组织和远癌组织比较差异均无统计学意义(P>0.05);在4例正常肝组织中均为甲基化阴性。联合检测癌组织E-cadherin基因CpG岛甲基化与血清甲胎蛋白两种分子标志物,肝癌的检出率可达82.35%。结论抑癌基因E-cadherin启动子区CpG岛甲基化可能在肝癌的发生、发展中起到重要作用,并且可能是早期事件,其在肝癌的临床诊断及治疗中的意义值得进一步深入研究。     

Prognostic value of CpG island hypermethylation at PTGS2, RAR-beta, EDNRB, and other gene loci in patients undergoing radical prostatectomy

OBJECTIVES: To evaluate CpG island hypermethylation in a set of candidate genes in prostate cancer (pCA) and its relationship to clinicopathologic parameters and a nomogram predicting prostate-specific antigen (PSA) recurrence after radical prostatectomy. MATERIALS AND METHODS: Tissues of 78 prostate carcinomas, 32 benign prostate hyperplasias (BPHs), and prostate cell lines (LNCaP, DU145, PC3, BPH-1) were examined with MethyLight polymerase chain reaction at 13 gene loci (APC, CDC6, CTNNB1, E-Cadherin, EDNRB, FGFR2, GSTP1, NAB2, PKCmu, PTGS2, RAR-beta, RASL11A, WWOX). RESULTS: APC, RAR-beta, PTGS2, GSTP1, EDNRB, and CTNNB1 (83%, 71%, 65%, 33%, 14%, 9%, respectively) were methylated in pCA but rarely or not methylated in BPH. NAB2 and CDC6 were hypermethylated frequently in pCA (92%, 67%, respectively) and in BPH (91%, 59%, respectively). FGFR2, WWOX, E-Cadherin, PKCmu, and RASLL1A did not display noteworthy methylation in pCA (0-1%) or in BPH. CpG island hypermethylation at APC, retinoic acid receptor beta (RAR-beta), and PTGS2 discriminated with a sensitivity of 65-83% and a specificity of 97-100% between BPH and pCA. The combination of various genes increased the diagnostic expressiveness. PTGS2 hypermethylation correlated with seminal vesicle infiltration (p=0.047), capsular penetration (p=0.004), and pT stage (p=0.014). RAR-beta methylation was accompanied by a higher cumulative Gleason score (p=0.042). The probability of PSA-free-survival calculated with a Kattan nomogram correlated inversely with CpG island hypermethylation at EDNRB, RAR-beta, and PTGS2. All prostate cancer cell lines displayed a varying degree of demethylation after 5-aza-2'deoxycytidine treatment. CONCLUSIONS: CpG island hypermethylation at various gene loci is frequent in prostate cancer and can distinguish between neoplastic and noncancerous tissue. Furthermore, hypermethylation at PTGS2, RAR-beta, and EDNRB inversely correlated with PSA-free-survival according to a Kattan nomogram and has potential prognostic value.     

Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer

BACKGROUND: DNA methylation has emerged as a promising biomarker for prostate cancer detection. In this report, we screened 36 candidate genes generated by a bioinformatic analysis of the human genome, and found that the melanoma cell adhesion molecule (MCAM) was an excellent candidate for cancer-specific methylation in prostate cancer. METHODS: Direct sequencing of bisulfite-treated genomic DNA, conventional methylation-specific PCR (MSP), real-time quantitative methylation-specific PCR, immunohistochemistry, colony formation assay, and statistical analysis. RESULTS: We found that the melanoma cell adhesion molecule (MCAM) gene promoter was specifically methylated in prostate cancer cell lines and primary prostate cancer (PCa) but not in non-neoplastic prostate (BPH) tissues by direct sequencing of bisulfite-treated genomic DNA and conventional methylation-specific PCR (MSP). Further analysis with quantitative MSP showed greater hypermethylation of the MCAM promoter (80%, 70/88) in primary prostate cancer compared to 12.5% (3/24) in BPH. Prostatic intraepithelial neoplasias (PIN), potential precursors of prostate carcinoma, showed an intermediate methylation rate of 23% (7/30). We further observed that MCAM promoter methylation was directly correlated with tumor stage (pT3+pT4) (P = 0.001) and Gleason score (P = 0.018) in primary prostate carcinoma. CONCLUSIONS: Our results suggest that MCAM promoter hypermethylation deserves further attention as a potential diagnostic prostatic DNA marker in human prostate cancer.     

肿瘤标记物P504s在前列腺癌中的表达及应用价值

目的探讨肿瘤标记物P504s在不同前列腺组织中的表达情况,及在诊断前列腺癌中的应用价值。方法采用免疫组化二步法,观察P504s和基底细胞标记物p63在不同前列腺病变组织中的表达情况。结果①P504s表达在前列腺癌根治标本、高等级上皮内瘤标本、前列腺增生标本中的阳性率分别为91.4%（32/35）、87.0%（20/23）和0（0/30）,良恶性组织的差别有显著性,在前列腺癌穿刺标本中阳性率85.7%（72/84）。p63染色在全部前列腺癌中呈阴性,在高等级上皮内瘤中为不连续或连续的阳性染色,在前列腺增生中为连续的阳性染色。②P504s表达与前列腺癌的分化程度、激素治疗、转移有关。结论P504s是一个特异的前列腺癌标记物,结合p63在鉴别诊断前列腺癌中有重要的临床应用价值。     

DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis

PURPOSE: We elucidated the significance of aberrant DNA methylation on multiple CpG islands and its correlation with DNA methyltransferase DNMT1 protein expression during urothelial carcinogenesis. MATERIALS AND METHODS: We examined the DNA methylation status on multiple CpG islands by methylation specific polymerase chain reaction and combined bisulfite restriction enzyme analysis in 12 specimens of normal urothelium, 23 of noncancerous urothelium showing no remarkable histological changes obtained from patients with bladder cancer (NBC) and 70 of transitional cell carcinoma (TCC). RESULTS: DNA methylation on CpG islands of the p16 (0%, 17% and 21%) and death-associated protein kinase (13%, 33% and 29%) genes, and methylated in tumor-2 (56%, 60% and 76%), 12 (0%, 6% and 30%), 25 (25%, 27% and 35%) and 31 (45%, 56% and 79%) clones was detected in normal urothelium, NBCs and TCCs, respectively. The incidence of concurrent DNA hypermethylation on 3 or more CpG islands in NBCs (38%) was significantly higher than that in normal urothelium (0%, p = 0.0455) and even higher in TCCs (59%, p = 0.0043). The incidence of the CpG island methylator phenotype in nonpapillary carcinomas (nodular invasive carcinomas and their precursors, ie flat carcinoma in situ, 71%) was significantly higher than in papillary carcinomas (40%, p = 0.0143). In all specimens examined concurrent DNA hypermethylation on 3 or more CpG islands significantly correlated with immunohistochemically evaluated DNMT1 protein over expression (p = 0.0167). CONCLUSIONS: DNA hypermethylation on multiple CpG islands in association with DNMT1 protein over expression may participate in multistage urothelial carcinogenesis even at the precancerous stage and particularly in the development of nodular invasive carcinomas of the bladder.     

Methylation and mutational analysis of p27(kip1) in prostate carcinoma

BACKGROUND: We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation. METHODS: Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS: With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were identified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated promotor methylation in all sequenced clones and the number of methylated base pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS: Mutational inactivation of p27(kip1) is a rare event in metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.     

AMACR(P504S)、P63、34βE12联合检测在前列腺癌早期诊断中的应用

目的:探讨AMACR(P504S)、P63、34βE12联合检测在前列腺癌(PCa)早期诊断中的临床应用价值。方法:应用即用型组合式单克隆抗体和双酶标记的免疫组化MaxvisionTM一步法检测42例PCa、12例高级别前列腺上皮内瘤变(HGPIN)和30例良性前列腺增生(BPH)穿刺活检标本中AMACR、P63、34βE12的表达情况。比较Glea-son评分各组中AMACR阳性表达情况。结果:AMACR、P63、34βE12抗原在PCa和BPH穿刺标本中的表达差异均有极显著性(P<0.01),PCa组织中AMACR阳性表达率为100%,无P63和34βE12表达;BPH组织中均无AM-ACR表达,P63和34βE12均高表达。HGPIN中AMACR的阳性表达率(91.67%)与BPH差异有极显著性(P<0.01),与PCa差异无显著性(P>0.05);P63和34βE12阳性表达率HGPIN(100%)与PCa差异有极显著性(P<0.01),而与BPH差异无显著性(P>0.05)。AMACR表达强弱与PCa的Gleason评分无关(P>0.05)。结论:AMACR是PCa高度敏感和特异的标志物,P63和34βE12联合标记基底细胞的特异性高,3者联合检测能增加前列腺穿刺活检标本诊断的准确性,在PCa早期诊断中具有重要的临床应用价值。     

HRK inactivation associated with promoter methylation and LOH in prostate cancer

OBJECTIVES: Recent studies in selected human tumors have demonstrated reduced expression of HRK with hypermethylation. Because no similar study has been performed specifically in prostatic lesions, we examined whether the methylation status of HRK is altered in prostate cancers. METHODS: We chose to analyze the hypermethylation status of HRK, the expression of HRK protein and mRNA with 12q13.1 loss of heterozygosity (LOH) and with p53 mutation, and lesion apoptotic indices as determined by transferase-mediated digoxigenin-tagged 16-desoxy-uridine-triphosphate nick end-labeling (TUNEL) assays in 53 prostate cancers. RESULTS: Twenty of the 53 prostate cancers (38%) demonstrated hypermethylation in either the promoter or in exon 1 and, more significantly, the loss of HRK expression observed in 14 cancers by immunohistochemistry (IHC) was associated with promoter methylation. In addition, high apoptotic indices in tumors were related to positive HRK expression. Prostate cancers demonstrating HRK methylation also showed methylation of multiple other genes, such as p14(ARF), p16(INK4a), O(6)-MGMT, and GTS-P, but, with the exception of one case, p53 mutations were not detected. When compared to tumors having a Gleason score (GS) of 5-6, a significant difference in the apoptotic indices was found among prostate cancers of GS 7 (P < 0.001) or GS 8-9 (P = 0.007). We also detected a close correlation between the loss of HRK expression and decreased apoptosis in GS 5-6 and GS 7 tumors (P = 0.008, P < 0.001, respectively). CONCLUSIONS: HRK appears to be inactivated principally by promoter hypermethylation in prostate cancers. We further suggest that the decreased expression of HRK may play an important role in tumor progression by modulating apoptotic cell death.     

The use of real-time quantitative polymerase chain reaction to detect hypermethylation of the CpG islands in the promoter region flanking the GSTP1 gene to diagnose prostate carcinoma

PURPOSE: We developed a real-time, quantitative, methylation sensitive polymerase chain reaction (PCR) protocol to analyze hypermethylation of the CpG islands in the promoter region of the pi class glutathione-S-transferase gene GSTP1 in prostate cancer tissue. MATERIALS AND METHODS: A total of 21 prostate cancer and 72 benign prostate hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was digested with restriction enzyme, followed by real-time quantitative PCR amplification. Cycle threshold values were used to determine whether cancer genome was present in these tissues. A cutoff cycle threshold value of 35 was arbitrarily assigned. Samples with a cycle threshold of 35 or less were considered positive for prostate cancer. Conventional nested PCR was also performed for comparison. RESULTS: The mean cycle threshold values plus or minus standard deviation in prostate cancer and BPH cases were 30.12 +/- 2.88 and 37.77 +/- 2.72, respectively. All prostate cancer samples analyzed showed positive results, while 5 of the 72 BPH samples tested positive. Conventional nested PCR data indicated that 19 of 21 prostate cancer cases were positive for the methylation change, while 71 of 72 BPH cases tested negative. The test limitations of real-time PCR and the nested PCR protocols were determined to be 0.048 and 0.64 ng. DNA, respectively. CONCLUSIONS: We established a novel protocol for detecting the methylation change in the 5' regulatory sequence flanking the GSTP1 gene. The sensitivity of this protocol was superior to that of conventional nested PCR. The data also suggest that this novel protocol may accurately discriminate prostate carcinoma from BPH.