人肺癌细胞中TSLC1基因表达缺失与DNA甲基化的相关性研究

背景与目的TSLC1在多种肿瘤中表达下调或失活,其表达下调与DNA高甲基化有很大关系。本研究旨在探索TSLC1在肺癌细胞中的表达缺失与其启动子区DNA甲基化的关系。方法采用RT-PCR和Real-time PCR方法检测TSLC1在正常肺组织和3种肺癌细胞系(A549、NCI-H446和Calu-3)中的表达谱;运用亚硫酸氢盐修饰后测序(bisulfite sequencing)方法检测上述正常肺组织和肺癌细胞中TSLC1启动子区的甲基化状态;应用甲基化转移酶抑制剂5-氮-2-脱氧胞苷(5-Aza-dC)处理上述细胞后,采用Real-time PCR方法检测处理前后TSLC1的表达变化。结果TSLC1在正常肺组织和A549细胞中表达,其启动子区DNA未发生甲基化;而在NCI-H446和Calu-3细胞中表达缺失,其启动子区DNA发生高甲基化,并且5-Aza-dC处理NCI-H446和Calu-3细胞后可促进TSLC1的表达。结论TSLC1在肺癌细胞中的表达缺失是由其启动子区的DNA高甲基化引起。     

散发性Peutz-Jeghers综合征患者LKB1基因突变情况

目的研究LKB1基因突变和甲基化在散发性Peutz-Jeghers综合征(P-J综合征)患者中的作用。方法提取5例散发性Peutz-Jeghers综合征患者外周血和其中3例的大肠息肉组织DNA,采用PCR法分析其序列的突变情况,MSP法检测基因启动子区域甲基化情况。结果在所有患者外周血和大肠息肉组织DNA中均未发现有病理意义的突变位点,在1例发生癌变的息肉组织DNA中检测到LKB1基因的甲基化。结论并非所有P-J综合征的患者都出现LKB1基因的序列突变,P-J综合征的发病可能存在其他的分子机制,但甲基化状态的改变可能是其息肉发生癌变的机制。     

急性髓系白血病RASSF1A基因启动子区异常甲基化临床意义研究

目的 RASSF1A基因异常甲基化可能参与血液肿瘤的发生,并为微小残留疾病(minimal residual de-sease,MRD)监测、分层、预后评估及靶向治疗提供依据.本研究旨在分析RASSF1A基因启动子区异常甲基化在急性髓系白血病(acutemyeloid leukemia,AML)中的临床意义.方法 选取2005-01-01-2013-03-01解放军总医院(113例)以及第一附属医院(39例)住院患者和门诊体检患者,共152例AML患者骨髓标本以及15例健康供者骨髓标本纳入本项研究.提取基因组DNA,并进行DNA硫化修饰;设计重亚硫酸盐测序PCR(bisulfite sequencing PCR,BS-PCR)引物以及甲基化特异性PCR(methylation specific PCR,MS-PCR)引物,进行PCR扩增,进而电泳分析以及DNA序列分析.同时对RASSF1A高甲基化组以及低甲基化组的血液学特点、骨髓原始细胞比例、细胞遗传学异常、基因异常、完全缓解率和总生存期进行统计学分析.结果 MS-PCR分析结果显示,RASSF1A基因在15例健康人中呈完全非甲基化状态,在152例AML患者中有38例出现启动子区高甲基化状态,其甲基化阳性率为25％.4例MS-PCR阳性AML患者经BS-PCR测序分析后,显示RASSF1A甲基化率分别为88.2％、85.5％、78.6％和92.7％,而在4例MS-PCR阴性患者RASSF1A基因启动子区甲基化率分别为10％、11.8％、12.7％和6.8％,4例健康供者RASSF1A基因启动子区甲基化率分别为5.0％、9.1％、8.2％和7.3％.进而通过统计学分析发现携带RASSF1A基因高甲基化的AML患者易合并存在ASXL1基因突变或DNMT3A基因突变.携带RASSF1A基因高甲基化的AML患者,其无进展生存期以及总生存期较短.结论 RASSF1A基因启动子区异常甲基化可能参与AML的发生,同时可能为AML分层诊治以及预后评估提供分子理论依据.     

CDH1基因启动子甲基化与宫颈癌临床病理特征的关系

目的:检测Cadlherin 1(CDH1)基因启动子区CpG岛甲基化状态,并分析CDH1基因甲基化率与临床病理特征的关系.方法:运用甲基化特异性PCR(MSP)检测95例宫颈癌标本及对照组40例正常宫颈组织的CDH1基因启动子甲基化状态,荧光定量PCR检测高危型HPV DNA.分析CDH1基因甲基化状态与不同临床病理特征之间的联系.结果:宫颈癌组CDH1基因甲基化率明显高于正常对照组(分别为49.5%,12.5%,P<0.01).宫颈癌CDH1基因甲基化检测和高危型HPV DNA检测结果有较好的一致性(Kappa=0.332,P<0.001).鳞癌组甲基化率较腺癌组高(分别为66.2%,7.4%,P<0.01).CDH1甲基化阳性率随肿块增大及FIGO分期升高而增高(P<0.05).不同年龄、肿瘤浸润深度及有无淋巴结转移和CDH1基因甲基化未见明显关系(P>0.05).结论:宫颈癌CDH1基因启动子甲基化与高危型HPV具有良好的一致性.CDH1基因启动子高甲基化为频发事件,并与不同宫颈癌病理类型相关,可作为宫颈癌诊断及分型的生物学标志物.     

降落PCR及克隆测序法检测DNA甲基化的实验研究

目的 探索优化PCR及测序法检测DNA甲基化的实验条件,检测肺癌hsa-mir-34b(mir-34b)基因启动子DNA甲基化谱。方法 肺癌细胞株A549 DNA进行亚硫酸盐处理后,设计引物扩增mir-34b基因启动子序列,使用普通PCR、巢式PCR和降落(Touchdown)PCR方法以及不同DNA聚合酶进行扩增,甲基化谱检测采用PCR直接测序和克隆后测序两种方法。结果 降落 PCR与巢式PCR方法相结合优于普通PCR及单独的巢式PCR或降落 PCR。HotStart酶优于普通Taq酶及Ex Taq酶。直接PCR测序法的测序图普遍出现套峰难以判读,PCR产物克隆后测序可见清晰的测序峰,甲基化位点可明确判读,并可以结合测序克隆数目提高甲基化检测敏感度。结论 对于重亚硫酸盐处理的DNA甲基化状态检测,推荐使用HotStart酶、降落 PCR与巢式PCR相结合、克隆测序法检测基因甲基化谱,序列图明确且敏感度高。     

血浆Ras相关区域家族蛋白1A基因甲基化在肝细胞癌分子诊断中的价值

目的:建立一种可检测微量DNA标本中DNA甲基化的甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-qPCR)方法,并运用该技术探讨血浆Ras相关区域家族蛋白1A(Ras association domain family1A,RASSF1A)基因甲基化检测在肝细胞癌(hepatocellutar carcinoma,HCC)非侵入性诊断中的价值。方法:用MSRE HhaⅠ消化DNA样品,再用qPCR技术分析酶切结果,建立检测RASSF1A基因甲基化的MSRE-qPCR方法。以45例肝组织(20对HCC患者手术切除肿瘤标本及匹配非癌组织和5例正常肝)为材料,测试该方法的应用价值;运用亚硫酸氢盐测序PCR(bisulf ite sequencing PCR,BSP)技术进行进一步验证,并与甲基化特异性PCR(methylation specif ic PCR,MSP)方法相比较。再运用该技术检测150例血浆标本(包括72例HCC患者、37例肝硬化或慢性肝炎等良性病变患者和41例健康对照)的RASSF1A基因甲基化状态,并分析其与HCC患者临床病理参数的关系。结果:MSRE-qPCR法可定量检测低至1%以下的RASSF1A甲基化片段。20例HCC组织中有14例(70%)发生RASSF1A高甲基化,对应非癌组织中RASSF1A甲基化阳性率为25%,而5例正常肝组织均为阴性。MSRE-qPCR结果经BSP验证无误,且与MSP检测结果具有较好的一致性。HCC患者血浆RASSF1A甲基化阳性率(47/72,65.3%)显著高于健康对照(1/41,2.4%)和肝良性病变组(3/37,8.1%),差异均有统计学意义(P<0.0001)。联合检测血浆RASSF1A甲基化与血清AFP可显著提高HCC诊断效率。结论:建立的MSRE-qPCR方法要求样本少、操作简便、成本低廉,可定量检测RASSF1A基因甲基化水平。血浆RASSF1A甲基化分析对于HCC的非侵入性诊断具有重要价值。     

卵巢肿瘤组织和血清中RASSF1A基因甲基化检测及临床意义的研究

目的：检测卵巢肿瘤组织和血清中RASSF1A基因启动子区异常甲基化，探讨卵巢肿瘤组织和血清中RASSF1A基因甲基化与卵巢癌的关系。方法：收集新鲜的卵巢肿瘤组织及其对应的血清标本65例，10名健康志愿者血清标本。采用半巢式甲基化特异性PCR技术分别检测肿瘤组织DNA和血清游离DNA中RASSF1A基因启动子异常甲基化情况。结果：卵巢癌患者血清和癌组织中，RASSF1A基因甲基化的检出率分别为20．0％和31．4％。卵巢良性肿瘤组织和血清以及健康志愿者血清中，均未发生RASSF1A基因异常甲基化。肿瘤组织中，RASSF1A基因的甲基化状况与血清中出现RASSF1A基因甲基化关系密切，P〈0．01；卵巢癌患者血清和癌组织中，RASSF1A基因的甲基化异常改变与肿瘤的临床分期、细胞分化程度以及组织学类型无明显相关性，P〉0．05；与淋巴结转移密切相关，P〈0．05。结论：分析血清DNA的RASSF1A基因异常甲基化有可能成为辅助卵巢癌诊断的有效方法之一。     

BRCA1基因甲基化及甲硫氨酸合成酶与乳腺癌发病的关系

背景与目的:启动子异常甲基化是肿瘤发生的早期事件,肿瘤组织中存在的DNA甲基化异常可以概括为广泛低甲基化伴局部高甲基化.甲硫氨酸合成酶(methionine synthase,MS)是参与甲基供体生成的关键酶,为DNA的甲基化提供甲基.本研究探讨抑癌基因BRCA1 mRNA在乳腺癌组织中的表达及启动子区甲基化状态,及MS基因mRNA表达与BRCA1基因甲基化的关系.方法:应用RT-PCR及甲基化特异性PCR(methylationspecific PCR,MSP)技术检测乳腺癌组织、相应癌旁组织(距癌>3 cm)和乳腺良性病变组织中BRCA1 mRNA的表达及其启动子甲基化状态,并检测MS mRNA的表达水平.结果:乳腺癌组织、癌旁组织及良性病变组织BRCA1mRNA表达差异有统计学意义(P<0.05);乳腺癌组织中BRCA1基因启动子区甲基化检出率明显高于癌旁组织和良性病变组织(χ2=7.631,P<0.05),BRCA1基因启动子区甲基化与散发性乳腺癌组织学分级、雌激素受体(estrogen receptor,ER)相关(P<0.05).乳腺癌组织MS基因表达量明显低于乳腺良性病变及乳腺癌旁组织(P<0.05).MS mRNA的表达与BRCA1基因甲基化的发生有相关性(r=0.419,P<0.05).结论:BRCA1甲基化能够增加乳腺癌的患病风险,MS基因可能通过影响部分肿瘤相关基因发生甲基化而调控其表达.     

非小细胞肺癌中hMLH1启动子的甲基化

目的 探讨在非小细胞肺癌（NSCLC）中DNA修复基因hMLH1启动子甲基化与基因转录失活的关系,观察5-Aza-CAR的干预作用。方法 甲基化特异的PCR（methyl-specific PCR,MSP）和RT-PCR法分别测定基因的甲基化率和转录水平。结果 NSCLC癌／癌旁组织中的甲基化率分别是hMLH1为55％和14％;〉65岁组hMLH1甲基化率明显高于≤65岁组（P〈0．01）,hMLH1甲基化率随吸烟指数增高而升高（P〈0．05或P〈0．01）;hMLH1的甲基化率随TNM临床分期进展而逐渐增加（P〈0．05或P〈0．01）。甲基化的NSCLC标本hMLH1基因mRNA转录下降或失活,在细胞株水平5-Aza-CdR处理后hMLH1恢复转录活性。结论 启动子甲基化是调节hMLH1转录活性的重要机制,5-Aza-CDR具有逆转甲基化而恢复转录的作用。     

三氯乙烯对B6C3F1雄性小鼠肝脏和肾脏中细胞增殖相关基因表达和DNA甲基化的影响

目的:三氯乙烯(TCE)是环境中广泛存在的工业污染物,可引起小鼠肝癌,但对肾癌发生未见显著影响。本研究通过检测TCE对小鼠肝脏和肾脏细胞增殖和DNA甲基化调控相关基因表达以及对DNA甲基化的影响,探讨TCE引起小鼠肝癌的分子机制。方法:将6周龄B6C3F1雄性小鼠随机分成3组,每组4只,分别以0、500和1 000 mg/kg剂量的TCE连续灌胃5 d。以荧光定量PCR方法检测TCE染毒小鼠肝脏、肾脏中与细胞增殖以及DNA甲基化调控相关基因的mRNA表达水平,以结合重亚硫酸盐的限制性内切酶方法检测Cdkn1a启动子区和重复序列的DNA甲基化水平。结果:与对照组相比,TCE可引起小鼠肝脏中细胞增殖相关基因Cdkn1a、Jun和Mki67的mRNA水平显著升高(P均<0.05),且呈剂量反应关系。同时1 000 mg/kg TCE染毒小鼠肝脏中主要DNA甲基化调控基因Dnmt3a、Dnmt3b和Tet2的mRNA水平降低(P均<0.05),Uhrf1 mRNA的表达升高(P均<0.05)。TCE染毒还导致肝脏内Cdkn1a启动子区的DNA甲基化水平降低,但对肾脏中相关基因及DNA甲基化水平无显著影响。结论:TCE引起的细胞增殖相关基因表达升高及DNA甲基化异常可能在其促进小鼠肝癌发生中起重要作用。     

Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients.

We prospectively analyzed p15 methylation patterns in 25 surgically resected tumors and 130 plasma, serum, and buffy coat samples from hepatocellular carcinoma (HCC) patients, controls with chronic hepatitis/cirrhosis, and healthy subjects. Using methylation-specific PCR, we demonstrated for the first time p15 promoter methylation in 64% of tumors and 25% (4 of 16) of patients' plasma and serum samples. Concurrent p15 and p16 methylation was shown in 48% of tumors, and p15/p16 methylation was detected in the plasma/serum of 92% (11 of 12) of patients. Of note, 75% of 12 patients with concurrent tumor methylation developed clinical metastasis/recurrence (P = 0.027). In buffy coat samples, p15 methylation was detected in all eight patients with tumor p15 methylation, suggesting the presence of circulating tumor cells. None of the control samples were methylation positive. Our data underscore the important role(s) of p15 and p16 methylation in hepatocarcinogenesis and tumor progression. Among 92% (23 of 25) of patients with tumor p15/p16 methylation, circulating tumor DNA and HCC cells were detected in the peripheral blood of 87% (20 of 23) of patients. The combination of these epigenetic markers may prove valuable for noninvasive HCC diagnosis and disease monitoring.     

肝细胞癌血浆LINE-1启动子区甲基化水平的研究

目的 探讨肝细胞癌（HCC）患者血浆中长散布核元件-1（LINE-1）基因启动子区CpG位点甲基化水平变化的临床意义。方法 应用亚硫酸氢盐测序 PCR（BSP）检测33例HCC患者（HCC组）、18例肝硬化患者（肝硬化组）及16例健康志愿者（正常组）的外周血血浆中LINE 1启动子区的CpG位点甲基化程度。结果 HCC组的LINE-1启动子区多个CpG位点甲基化水平及整体甲基化水平下降;进一步通过ROC曲线分析发现CpG位点1、位点7、位点8的甲基化水平对诊断HCC的能力较优,其敏感度和特异度分别为80.0％和78.8％、81.8％和71.4％、82.9％和75.8％。结论 LINE-1基因启动子区甲基化水平在HCC中明显下降,其中CpG位点1、位点7、位点8的甲基化水平可能对HCC诊断有意义。     

Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients.

PURPOSE AND EXPERIMENTAL DESIGN: Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC). RESULTS: RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive. CONCLUSIONS: Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.     

Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA.

PURPOSE: Most hepatocellular carcinomas (HCC) are diagnosed at an advanced stage. Hypermethylation of CpG islands in promoter regions is now recognized as an important early event in carcinogenesis and detection of methylated DNA has been suggested as a potential biomarker for early detection of cancer. There are no studies on epigenetic changes in samples from HCC patients before diagnosis. We explored the possible diagnostic value of aberrant promoter hypermethylation of three tumor suppressor genes in serum DNA for early detection of HCC. EXPERIMENTAL DESIGN: Aberrant promoter hypermethylation was investigated in DNA isolated from the serum of 50 HCC patients who provided repeated blood samples before diagnosis and 50 controls enrolled in a cancer screen program in Taiwan. Methylation-specific PCR was used to determine the methylation status of p16, p15, and ras association domain family 1A (RASSF1A). RESULTS: Among cases, aberrant methylation was found in serum DNA 1 to 9 years before clinical HCC diagnosis. RASSF1A had the highest frequency of hypermethylation with 35 (70%) cases having at least one positive sample compared with 22 (44%) for p16 and 12 (22%) for p15. Six subjects were hypermethylation negative for all three genes. For the 50 controls, promoter hypermethylation was found in three and two subjects for RASSF1A and p16, respectively; none had methylation of p15. A receiver operating characteristic curve that included clinical risk factors (age, HBsAg status, anti-hepatitis C virus status, smoking, and alcohol status) and hypermethylation biomarkers gave an overall predictive accuracy of 89% with sensitivity and specificity 84% and 94%, respectively. CONCLUSIONS: The analysis of epigenetic changes on RASSF1A, p16, and p15 tumor suppressor genes in serum DNA may be a valuable biomarkers for early detection in populations at high risk of HCC.     

肝细胞癌多基因甲基化异常及其临床意义

目的 探讨肝细胞癌中多基因甲基化异常的发生率,研究肝细胞癌中多基因甲基化异常的临床意义.方法 收集60例肝细胞癌及相应的癌旁组织、16例肝炎后肝硬化组织、5例慢性肝炎和5例正常肝组织,筛选消化道肿瘤中APC、RASSF1A、p16、GSTP1、MGMT、DAPK、SOCS-1和RIZ18个甲基化异常频率高的肿瘤抑制基因,应用甲基化特异件聚合酶链反应(MSP)检测8个肿瘤抑制基因在所有标本中的甲基化状态.比较不同基因甲基化与非甲基化肝细胞癌患者的临床病理特征和生存情况.结果 肝细胞癌组织中,RASSF1A、APC、GSTP1、p16、RIZ1和MGMT基因的甲基化率分别为95.0%、90.0%、73.3%、65.0%、61.6%和60.0%,均高于相应的癌旁组织(均P＜0.05);癌旁组织中,MGMT、GSTP1和RIZ1基因的甲基化率分别为41.6%、40.0%和25.0%,均高于肝硬化组织(均P＜0.05).p16基因甲基化的肝细胞癌患者的平均年龄大于非甲基化者;巨块性肝细胞癌中,MGMT基因甲基化者的比例高于非甲基化者;MGMT基基甲基化者的无瘤生存期短于非甲基化者.结论 不同基因在肝细胞癌、癌旁和肝硬化组织中的甲皋化率差异显示了肝细胞癌发生中渐进的表观遗传学改变;GSTP1、RIZ1和MGMT基因的甲基化异常具有肿瘤风险评估和早期诊断价值,而MGMT基因的甲基化异常同时具有预后评估价值.     

Transforming growth factor-beta1 as a useful serologic marker of small hepatocellular carcinoma

BACKGROUND: Although alpha-fetoprotein (AFP) is a useful serologic marker of hepatocellular carcinoma (HCC), it has been reported insufficiently sensitive in detecting small HCCs. Plasma transforming growth factor-beta1 (TGFbeta1) has been reported to be elevated in HCC patients compared with liver cirrhosis patients. It has been reported that TGFbeta1 mRNA was overexpressed in HCC, especially in patients with small HCC and well-differentiated HCC compared with patients with liver cirrhosis. The current study investigated the usefulness of TGFbeta1 compared with AFP in the diagnosis of small HCCs. METHODS: Thirty-eight patients with small HCC (< or = 3 cm), 31 patients with liver cirrhosis only, and 23 normal volunteers were studied. Using plasma TGFbeta1 and serum AFP levels measured at the time of diagnosis, the sensitivities and specificities were calculated in the diagnosis of small HCCs. RESULTS: Plasma TGFbeta1 and serum AFP levels were significantly higher in patients with small HCC than in those with liver cirrhosis. In diagnosing small HCCs, the cut-off values of plasma TGFbeta1 and serum AFP were 800 pg/mL and 200 ng/mL, respectively, where the specificities were over 95%. At the cut-off level of plasma TGFbeta1 and serum AFP, the sensitivities were 68% and 24%, respectively. CONCLUSIONS: The current results suggest that TGFbeta1 may be a useful serologic marker in detecting HCCs earlier because it shows higher sensitivity than, with specificity as, AFP in the diagnosis of small HCCs.     

Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients

Elevated aflatoxin B(1)-albumin adducts (AFB(1)-Alb) have been associated with an increased risk for HCC development. However, there are no studies in humans, correlating albumin adducts in blood with liver DNA adducts. Forty frozen tumor tissues and 39 paired plasma samples from HCC patients were collected in Taiwan, to determine the relationship between albumin adducts in blood and DNA adducts in liver tissue as well as mutations in p53 and methylation of p16. AFB(1)- and polycyclic aromatic hydrocarbon (PAH)-DNA adducts in tissue and albumin adducts in plasma were determined by immunohistochemistry and competitive ELISA, respectively. Plasma AFB(1)-Alb adducts in subjects with low, medium and high levels of AFB(1)-DNA adducts in tumor tissues were 51.0 +/- 36.5, 70.5 +/- 48.1 and 84.9 +/- 48.2 fmol/mg, respectively (p(trend) = 0.05). No significant correlation was found for PAH. Fourteen of 40 (36%) tissues were positive for mutant p53 protein by immunohistochemistry; 11 of 40 tissue DNA samples (28%) were positive for p53 mutations, but not their corresponding plasma DNAs. p16 was methylated in 24 of 40 (62%) tissues and 12 of 39 (32%) plasma DNAs. Significant correlations were observed between AFB(1)-Alb adducts and p53 mutations and p16 methylation. These data suggest that genetic, epigenetic and environmental exposure biomarkers in plasma may help in estimating the risk for the development of HCC.     

Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer.

PURPOSE: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation. EXPERIMENTAL DESIGN: TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution. RESULTS: TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett's metaplasia alone, 57.5% in dysplastic Barrett's esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2'-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression. CONCLUSIONS: TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.