巢式甲基化特异性PCR 检测食管癌病人血清中p16 基因启动子区过甲基化

 目的 检测食管鳞状细胞癌（squamous cell carcinoma，SCC）患者外周血血清中p16基因启动子区的甲基化状态，探讨p16基因启动子的过甲基化在食管鳞状细胞癌筛查及早期诊断中的意义。方法 利用巢式甲基化特异性PCR（nMSP）法检测食管鳞状细胞癌患者外周血血清与正常人血清中p16基因启动子的甲基化状态，并与普通甲基化特异性PCR（MSP）法进行了比较。结果 56例SCC血清样品中nMSP法发现34例p16基因启动子的过甲基化，MSP法只检出15例，而22例正常人血清中都未检测到p16基因启动子的过甲基化。测序结果进一步验证了方法的可靠性。结论 利用巢式MSP（nMSP）法检测外周血血清中p16基因启动子的甲基化，可为食管癌的筛查、早期诊断及预后判断提供有价值的信息。     

中国非小细胞肺癌患者血清DAPK基因、p16基因启动子甲基化的分析

Objective： To evaluate the clinical significance of the aberrant methylation of DAPK gene and p16 gene in sera from 65 NSCLC patients from Nanjing General Hospital of Nanjing Command, China. Methods： A methylation-specific PCR （MSP） was performed for the detection of promoter hypermethylation of DAPK gene and p16 gene in blood DNA from 65 cases of NSCLC, and to analyze the relation of the aberrant methylation of DAPK gene and p16 gene and the clinicopathological data. Results： 30.8% （20/65） of the sera from 65 cases of NSCLC showed hypermethylation for DAPK promoter and 43.1% （28/65） the same for p16 promoter, whereas no methylated DAPK gene promoter and p16 gene promoter were found in sera from the patients with lung benign diseases and normal controls. Methylated DAPK gene promoter and p16 gene promoter in sera were not closely correlated with the pathological classification, stage, metastasis and differentiation in NSCLC. Conclusion： Detection of the aberrant methylation of DAPK gene and p16 gene in blood DNA from NSCLC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.     

Hypermethylation of p16INK4a in Chinese lung cancer patients: biological and clinical implications

Promoter hypermethylation of the p16INK4a gene was investigated in 111 cases of tumor tissue, as well as in 136 circulating plasma and 95 sputum samples from Chinese patients with primary lung cancer, using a modified protocol of semi-nested methylation-specific-PCR (MSP). The results showed hypermethylated p16 sequence in 80.2% of tumor tissues and frequencies of 75.7 and 74.7% in plasma and sputum specimens, respectively. Among the patients, 50 cases of matched plasma, sputum and tumor tissue from the same individual were analyzed. Of these, hypermethylation of the p16 promoter was detected in 84.0% of the tumor tissues, with frequencies of 72.0 and 76.0% in the corresponding plasma and sputum, respectively. Notably, only patients whose tumor tissue showed hypermethylation of p16 exhibited the same aberrant methylation in their sputum and/or plasma. Hypermethylation of p16 in sputum and plasma samples may provide a more sensitive approach to molecular diagnosis of lung cancer than relying on conventional cytological analysis. Our data show that a combination of cytological analysis of sputum and examination of p16 hypermethylation in sputum and plasma identified 92.0% (46/50) of the lung cancer patients studied, offering an effective means of early detection of lung cancer.     

Aberrant promoter methylation in Chinese patients with non-small cell lung cancer: patterns in primary tumors and potential diagnostic application in bronchoalevolar lavage.

This study was aimed at defining patterns of aberrant gene methylation in non-small cell lung cancer (NSCLC) in Chinese patients and its use in detecting cancer cells in bronchoalveolar lavage (BAL). The methylation-specific PCR (MSP) was used to study methylation of the p16, retinoic acid receptor-beta (RARbeta), death-associated protein (DAP) kinase, and O(6)-methylguanine-DNA-methyltransferase (MGMT) genes in 75 NSCLCs [44 adenocarcinomas and 31 squamous cell carcinomas (SCCs)] and 68 BALs from suspected lung cancers. More females had adenocarcinoma than SCC (11 of 44 versus 2 of 31, P = 0.04). Aberrant methylation in at least one gene was found in 63 of 75 (84%) NSCLCs. p16, RARbeta, DAP kinase, and MGMT methylation was similar in adenocarcinoma and SCC. However, females with NSCLC showed more frequent p16 methylation than males (12 of 13 versus 36 of 62, P = 0.02), because of more frequent p16 methylation in female adenocarcinomas (10 of 11 versus 17 of 33, P = 0.02). This sexual difference was not observed in RARbeta, DAP kinase, and MGMT. At 92%, the frequency of p16 methylation in Chinese female NSCLC is one of the highest known. For BAL, MSP and cytological analysis showed concordant and discordant results in 25 of 68 and 43 of 68 samples. Of 41 MSP+/cytology- cases, 35 were eventually shown to have malignant lung lesions, 4 were at high risk but had no evidence of lung cancer, and 2 were lost to follow-up. There were two MSP-/cytology+ cases. Frequent gene methylations were seen in Chinese NSCLC patients. More frequent p16 methylation was seen in female patients. MSP is a useful molecular adjunct for cancer cell detection in BAL samples.     

肺癌患者外周血血浆中p16基因异常甲基化的检测

目的 分析肺癌患者外周血血浆中p16基因启动子区异常甲基化发生情况,探讨血浆中p16基因异常改变作为肺癌临床辅助诊断分子生物学标志物的可能性。方法 利用半巢式甲基化特异性PCR技术,检测了137例肺癌患者血浆和112例相对应肿瘤组织DNA p16基因的异常甲基化情况。结果 在血浆和肿瘤组织中的p16基因甲基化检出率分别为75．2％和80．4％;其中鳞癌、腺癌、腺鳞癌和小细胞肺癌患者血浆标本的阳性率分别为77．9％,65．1％,75．1％和91．7％。血浆DNAp16基因甲基化仅在肿瘤组织存在同样甲基化的病例中被检出。血浆和肿瘤组织中,p16基因的甲基化异常改变与肿瘤的分期、分型无明显相关性。结论 分析血浆DNA的p16基因异常甲基化有可能成为辅助肺癌诊断的有效方法之一。     

Detection of p16 promoter hypermethylation in serum of gastric cancer patients

For the early detection of tumor-related aberrant DNA in the serum of patients, we examined promoter hypermethylation of the p16 gene using methylation-specific PCR (MSP) in paired tumor and serum samples of 60 gastric cancer patients. Aberrant p16 methylation was found in 23 of 60 (38%) primary gastric cancers, but in none of the corresponding gastric mucosae. Of these 23 patients, 6 (26%) exhibited the same alteration in their serum DNA. As a control, we screened for aberrant methylation in the serum DNA of 37 patients with gastric cancers whose corresponding tumor DNA had no methylation in the p16 promoter. We also screened for methylation in the serum DNA of 16 non-cancer individuals. No methylation was found in serum DNA of these control groups. Our results suggest that p16 methylation would be a good marker for the detection of tumor DNA in the serum of primary gastric cancer patients. (Cancer Sci 2003; 94: 418–420)     

中国非小细胞肺癌患者血清DAPK基因、p16基因启动子甲基化的分析(英文)

目的分析65名中国南京军区南京总医院的非小细胞肺癌(NSCLC)住院患者血清中DAPK、p16启动子区域甲基化的改变状况及其临床意义。方法运用甲基化特异性PCR技术,检测65例NSCLC患者血清DAPK、p16基因启动子区域甲基化的改变情况,并分析与临床病理资料的关系。结果 NSCLC患者血清DAPK基因甲基化检出率为30．8％(20/65),p16基因甲基化检出率为43．1％(28/65),而正常对照组和良性肺部疾病组血清未检出DAPK基因、p16基因甲基化,DAPK基因、p16基因甲基化检出率与NSCLC病理类型、分期及转移状态无明显关系。结论 DAPK基因、p16基因检出启动子区域异常甲基化是NSCLC早期辅助诊断的分子标志物之一。     

Hypermethylation of p16(INK4a) and p15(INK4b) genes in non-small cell lung cancer.

Hypermethylation of CpG island is a common mechanism by which tumor suppressor genes are inactivated. The tumor suppressor genes p16(INK4a) and p15(INK4b) are important components of the cell cycles. We have studied the feasibility of detecting tumor-associated aberrant p16(INK4a) and p15(INK4b) methylation in non-small cell lung cancer (NSCLC) using methylation-specific PCR. We found a high frequency of hypermethylation of the p16(INK4a) gene in 17 of 45 cases of NSCLC. In this study, there was no difference between the clinicopathological features or overall survival of patients with and without p16(INK4a) methylation. On the other hand, p15(INK4b) promoter hypermethylation is rare (5/45) in lung cancer and occurs in association with p16(INK4a) methylation. The overall survival of patients with p15(INK4b) methylation was markedly shortened in this series. We also analyzed cells in bronchial washings, and p16(INK4a) methylation was detected in 4 of 17 cases of NSCLC. Moreover, 1 of 10 plasma samples from patients with NSCLC was positive for p16(INK4a) methylation. Our results suggest a possible prognostic role of p15(INK4b) methylation in NSCLC, and that the detection of aberrant p16(INK4a) methylation in both bronchial washings and plasma may be useful for cancer diagnosis.     

p16(INK4A) Hypermethylation detected by fluorescent methylation-specific PCR in plasmas from non-small cell lung cancer.

PURPOSE: The p16(INK4A) tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16(INK4A) hypermethylation in precursor lesions of NSCLC and in body fluids of individuals at risk makes it a potential candidate for early disease detection. However, the current low sensitivity of p16(INK4A) hypermethylation detection in plasma limits its consideration in a diagnostic grid. EXPERIMENTAL DESIGN: A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16(INK4A) promoter hypermethylation in 35 NSCLC and paired plasma samples and in 15 plasmas from healthy donors. F-MSP sensitivity was investigated in combination with microsatellite alterations on 3p (evaluated by fluorescent PCR), K-ras mutations (determined by a mutant-enriched PCR), and quantification of circulating DNA. Assay results were analyzed by two-sided chi(2) or Fisher's exact tests. RESULTS: p16(INK4A) promoter hypermethylation, detectable by F-MSP in 22 of 35 NSLCs (63%) and in 12 of 22 (55%) plasmas from patients with methylated tumors, was independent of microsatellite alterations (detectable in 57% of tumors and 50% of paired plasmas), K-ras mutations (detectable in 31% of tumors but in no paired plasma), or amount of circulating DNA. p16(INK4A) methylation in association with microsatellite alterations identified 62% (18 of 29) of plasma samples from patients presenting the same alteration in their tumors, and its sensitivity increased to 80% when combined with the amount of circulating DNA. CONCLUSIONS: The establishment of F-MSP remarkably improved p16(INK4A) promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16(INK4A) hypermethylation might contribute to a diagnostic grid for NSCLC.     

Aberrant Promoter Methylation Profile in Pleural Fluid DNA and Clinicopathological Factors in Patients with Non-small Cell Lung Cancer

The aim of this study was to investigate the prognostic value of hypermethylation of tumor suppressor genesin patients with non-small cell lung cancer (NSCLC). In samples from 34 lung patients with malignant pleuraleffusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of thepromoters of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, rasassociation domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK),and retinoic acid receptor ß (RARß).There is no association between methylation status of five tumor suppressorgenes including MGMT, p16INK4a, RASSF1A, DAPK and RARß in pleural fluid DNA and clinicopathologicalparameters including clinical outcome. Aberrant promoter methylation of tumor suppressor genes in pleuralfluid DNA could not be a valuable prognostic marker of NSCLC patients with malignant pleural effusion.     

肺癌患者组织样品中p16基因的异常甲基化

目的 分析肺癌患者组织样品中p16基因启动子区域异常甲基化的改变情况,评价该指标作为辅助临床诊断的分子标记物的价值。方法 运用甲基化特异性PCR技术,检测51例原发性肺癌患者的肿瘤组织、外周血血浆和痰标本中p16基因启动子区域的异常甲基化改变。结果 在43例肿瘤组织、36例血浆和39例痰标本中检测到了p16基因异常甲基化。凡在肿瘤组织检出p16基因甲基化阳性的病例,其血浆和(或)痰标本也为阳性;而肿瘤组织p16基因甲基化阴性的病例,其血浆和痰标本也为阴性。将血浆和痰标本的p16甲基化分析与痰细胞学检查相结合,可以发现92．2％的肺癌病例。结论 利用半巢式甲基化特异性PCR进行的血浆和痰标本p16基因甲基化分析,有可能成为辅助肺癌诊断的分子生物学指标。     

Fluorescent methylation-specific polymerase chain reaction for DNA-based detection of prostate cancer in bodily fluids

Promoter hypermethylation of the glutathione S-transferase P1 gene (GSTP1) is the most frequent DNA alteration in prostatic carcinoma. Because this epigenetic DNA alteration can be reliably detected by methylation-specific PCR (MSP), we applied this new technique for molecular detection of prostate cancer in various human bodily fluids. We investigated GSTP1 promoter hypermethylation in DNA isolated from plasma, serum, ejaculate, and urine after prostate massage and from prostate carcinoma tissues from 33 patients with prostate cancer and 26 control patients with benign prostatic hyperplasia (BPH). Fluorescently labeled MSP products were analyzed on an automated gene sequencer. Whereas GSTP1 promoter hypermethylation was not detectable by MSP in prostate tissue and bodily fluids from patients with BPH, we found it in 94% of tumors (16 of 17), 72% of plasma or serum samples (23 of 32), 50% of ejaculate (4 of 8) and 36% of urine (4 of 11) from patients with prostate cancer. Additionally, MSP identified circulating tumor cells in 30% (10 of 33) of prostate cancer patients. Analysis of GSTP1 promoter hypermethylation by MSP thus provides a specific tool for molecular diagnosis of prostate cancer in bodily fluids.     

PROMOTER HYPERMETHYLATION OF p16 GENE IN PRE- AND POST-OPERATIVE PLASMA OF PATIENTS WITH GASTRIC ADENOCARCINOMA

Objective: To detect promoter hypermethylation of p16 gene in matched pre- and post-operative plasma of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. Methods: Tissue samples, pre- and post-operative plasma of 84 patients were collected. Plasma of 15 healthy people was collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). Results: Among 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) tumor-adjacent tissues and 12 (14.3%) pre-operative plasma, while negative in plasma of healthy people. For positive plasma cases, the paired tumor tissues were confirmed to be methylated.Within available 30 pairs of matched pre- and post-operative plasma, 6 pre-operative plasma was positive, and only 1 of 6 plasma remained hypermethylated after surgery. The results detected by HPLC exactly matched those by gel-electrophoresis. Conclusion: The alteration of status of p16 hypermethylation in post-operative plasma is considered the consequences of surgical intervention. Although p16 hypermethylation has no role in pre-operative staging of gastric cancer, detecting hypermethylated p16 in plasma could be utilized in monitoring patients after surgery.     

非小细胞肺癌pl6/CDKN2基因失活的研究

目的：分析中国人非小细胞肺癌中ｐ１６／ＣＤＫＮ２基因失活的情况,探讨该基因在肺癌发生中的作用。方法：选取与ｐ１６基因紧密连锁的Ｄ９Ｓ１７４８位点,对１７例临床切除的原发性肺癌标本进行微卫星不稳定性分析,用甲基化特异性ＰＣＲ（ｍｅｔｈｙｌａｔｉｏｎ－ｓｐｅｃｉｆｉｃ ＰＣＲ, ＭＳＰ）检测 ｐｌ６基因启动子区 ＣｐＧ岛甲基化状况,用免疫组织化学法检测Ｐ１６蛋白表达情况。结果：微卫星不稳定性分析结果表明,在１３例Ｄ９Ｓ１７４８位点存在多态性的肿瘤ＤＮＡ中有 ９例（６９ ．２％）发生了杂合性缺失（ｌｏｓｓ ｏｆ ｈｅｔｅｒｏｚｙｇｏｓｉｔｙ, ＬＯＨ）。 ＭＳＰ的结果显示,有 ７０． ６％（１２／１７）的肿瘤组织存在ｐ１６启动子区的异常高甲基化。在本研究中,８２．４％（１４／１７）的肿瘤组织可以检测出一种或两种ｐ１６基因的异常改变。对此１７例肿瘤标本进行的免疫组织化学分析显示,有 １３例 Ｐ１６蛋白表达阴性,其中 ９２ ３％（１２／１３）存在一种或两种ｐ１６基因的异常改变。免疫组化结果与ｐ１６基因分子遗传学改变情况基本吻合。结论：作为一种抑癌基因,ｐ１６在多种肿瘤组织中都有异常改变。我们的研究表明,ｐ１６基因失表达是非小细胞肺癌     

痰标本p16和MGMT基因甲基化对肺癌的诊断价值

目的：分析肺癌患者痰标本中p16和MGMT基因启动子区甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标志物的价值。方法：运用甲基化特异性PCR技术,检测77例原发性肺癌患者痰标本和部分对应肿瘤组织(53例),以及30例正常对照者痰标本中p16和MGMT基因启动子区域的甲基化改变。结果：49例(63．6%)肺癌患者痰标本中检测到了pi6基因异常甲基化,34例(44．2%)检测到了MGMT基因异常甲基化,77例患者痰标本中2个基因中至少有1个基因出现甲基化为64例(83．1%),对肿瘤的检出灵敏度较高。长期吸烟史是影响肺鳞状细胞癌痰标本p16(P=0．001)基因启动子区甲基化的因素。随TNM分期增高,肺鳞状细胞癌患者痰标本中p16基因甲基化比例增高(P=0．021)；随TNM分期增高,肺腺癌患者痰标本MGMT基因甲基化比例增高(P=0．023)。对照组正常人痰液标本未发现p16和MGMT基因启动子区甲基化。结论：痰标本中p16和MGMT基因甲基化是临床肺癌辅助诊断的有效生物标志物之一。     

非小细胞肺癌INK4a和ARF基因甲基化与其蛋白共表达关系的探讨

目的:分析INK4a和ARF基因启动子甲基化与其蛋白共表达之间的关系.方法:选择前期实验中p14ARF和p16INK4a蛋白共表达阴性的非小细胞肺癌(NSCLC)患者35例,共表达阳性的NSCLC患者20例作为研究对象,分别称为(p14 p16)阴性组和(p14 p16)阳性组.运用甲基化特异性PCR(MSP)方法对两组患者癌组织INK4a和ARF基因启动子的甲基化状态进行检测.结果:(p14 p16)阴性组有18例发生INK4a基因甲基化,(p14 p16)阳性组有2例发生INK4a基因甲基化,两组差异有显著意义(P＜0.01).(p14 p16)阴性组有8例发生ARF基因启动子甲基化,(p14 p16)阳性组有2例发生ARF基因启动子甲基化,两组差异无显著意义(P＞0.05).INK4a和ARF基因异常申基化相互之间无显著相关性(P＞0.05).结论:NSCLC患者肺癌组织INK4a基因甲基化是导致p16INK4a蛋白表达阴性的重要机制;INK4a和ARF基因甲基化可能是相对独立的事件.     

Tumor-derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications

Progressive p16 methylation has been associated with metastasis and invasive phenotypes in many cancers. Loss of E-cadherin (CDH1) function contributes to breast cancer progression by promoting cell proliferation, invasion and metastasis. Using methylation-specific PCR, aberrant hypermethylation of p16 and CDH1 in tumor and plasma was analyzed and correlated with levels of serum protein markers, carcinoembryonic antigen (CEA) and carcinoma antigen 15-3 (CA15.3), in 36 patients with invasive ductal breast cancer. Aberrant p16 methylation was found in 11% (4/36) of primary tumors and 8% (3/36) of plasma samples. Aberrant CDH1 methylation was detected in 25% (9/36) of primary tumors and 20% (7/36) of plasma samples. p16 and/or CDH1 hypermethylation was found in 31% (11/36) of primary breast carcinomas and 82% (9/11) of breast cancer patients with tumoral methylation showing identical epigenetic changes in plasma. The 25 patients without tumoral methylation did not show epigenetic changes in the plasma. Tumoral p16 methylation was significantly associated with advanced tumor stage (p=0.028; Fisher's exact test), tumor size (p=0.017) and nodal metastasis (p=0.002). However, p16 methylation in plasma was only associated with nodal metastasis (p=0.012). Altogether, aberrant p16 methylation in plasma and elevated serum CEA level were associated with advanced tumor stage (p=0.033), tumor size (p=0.022) and extensive nodal metastasis (p=0.003). With clinical implications, p16 hypermethylation in plasma and/or raised serum CEA levels may prove useful as diagnostic and prognostic markers for breast cancer.     

Detection of aberrant methylation as a tumor marker in serum of patients with gastric cancer

BACKGROUND: This study aimed to detect hypermethylation in serum DNA from patients with gastric cancer at various stages and to assess the assay for early detection of primary and recurrent disease. MATERIALS AND METHODS: Preoperative serum samples were obtained from 109 patients with gastric cancer. Blood samples were subjected to methylation-specific polymerase chain reaction analysis to determine the methylation status of the promoter region of the p16 and E-cadherin genes. RESULTS: Forty patients (37%) showed hypermethylation of the promoter region in one or both genes (p16, 20 patients; E-cadherin, 26 patients). Of 36 patients with early-stage gastric cancers, 10 (28%) showed aberrant methylated signals. No aberrant methylation was detected in the serum DNA from 10 healthy volunteers. CONCLUSION: Aberrant promoter hypermethylation may prove useful as a new serum marker for gastric cancer.     

Gene promoter hypermethylation in oral rinses of leukoplakia patients--a diagnostic and/or prognostic tool?

Leukoplakia is the most frequent oral precancerous lesion and shows a variable rate of malignant transformation. We hypothesised that the detection of molecular alterations, like the promoter hypermethylation of DNA, in oral cytological samples from patients at risk of developing primary or recurrent tumours could be a valuable diagnostic and prognostic tool in the management of these lesions. Two groups of patients with differing risks of developing oral squamous cell carcinoma (OSCC) were analysed. DNA was extracted from the oral rinse of each patient. The methylation status of the p16, p14 and MGMT gene promoters was determined using a methylation-specific polymerase chain reaction (MSP). Methylation of p16 and MGMT was observed in 44 and 56% of the oral samples, respectively. Only 12% of the cases showed p14 methylation. DNA hypermethylation was more frequent in patients with previous OSCC. DNA promoter hypermethylation is frequent during early oral carcinogenesis and even more so in the later stages. MSP using oral rinses is a non-invasive and highly sensitive technique which could be used to monitor patients with precancerous and cancerous oral lesions.