CDKN2/p16甲基化的研究与结直肠癌

抑癌基因CDKN2／p16在结直肠癌等多种肿瘤中缺失、点突变及甲基化，导致肿瘤的发生发展。甲基化是其失活的主要机制。应用甲基化特异PCR。检测恶性肿瘤患者外周血中的甲基化改变，在早期诊断、监测术后复发和判定预后方面，具有重要的临床意义。     

GATA5甲基化在结直肠癌血浆和粪便中的检测及临床诊断价值

背景与目的：结直肠癌(colorectal cancer,CRC)是全球发病率第三,死亡率第四的恶性肿瘤。遗传学及表观遗传学的改变引发抑癌基因甲基化表达沉默是CRC发生的重要原因,本研究旨在探讨血浆、粪便GATA5启动子甲基化检测在CRC临床诊断中的应用价值。方法：收集34例健康体检者和43例CRC患者的血浆和配对的粪便标本。采用甲基化特异性PCR法(methylation specific PCR method,MSP)检测其血浆、粪便中GATA5甲基化水平,并分析其与临床病理特征相关性。结果：MSP结果显示CRC患者血浆、粪便中GATA5启动子甲基化率(60.74%、76.60%)高于健康者发生率(26.47%、32.35%),差异均有统计学意义(P值分别为0.006 7,0.000 2)。CRC患者血浆甲基化发生率与临床分期(P=0.000 5)、淋巴结转移(P=0.020)密切相关,而粪便GATA5甲基化水平与临床病理特征无统计学意义。结论：检测粪便GATA5甲基化水平并辅以血浆GATA5甲基化水平可作为一种简单、非侵入、敏感及特异的方法应用于CRC患者的临床诊断。     

DNA甲基化在结直肠癌发生发展中的研究进展

结直肠癌（ｃｏｌｏｒｅｃｔａｌｃａｎｃｅｒ,ＣＲＣ）是研究得最深入的肿瘤之一 ,其发生发展的各阶段都有很明显的遗传改变。首先是正常上皮细胞的增生 ,然后是异常腺管和腺瘤的形成 ,最后转变成浸润性癌。各阶段遗传改变的研究阐明了结直肠癌发生发展的分子机理。尽管研究已有很大的进展 ,对结直肠癌的发生发展还有许多问题有待研究 ,如结直肠癌发生率为什么随年龄增加 ,遗传改变为什么不能完整地解释许多散发性肿瘤？５ 甲基胞嘧啶（５ｍＣ）存在于５′ ＣｐＧ ３′这样的二核苷酸回文结构中 ,ＣｐＧ二核苷酸在人类基因组中只占１０% ,…     

基因启动子区甲基化与结直肠癌研究进展

近年来研究表明,表观遗传修饰的ＤＮＡ甲基化在结直肠癌的发生发展中具有重要的作用。在结直肠癌中普遍存在ＤＮＡ甲基化,对于肿瘤的早期诊断具有重要的指导意义。因为ＤＮＡ甲基化的可逆性,可能为肿瘤的治疗提供靶点。从ＤＮＡ甲基化方面寻找预测结直肠癌药物疗效的分子标志物,有可能成为推动结直肠癌个体化治疗的新方向。     

Kiss-1基因启动子甲基化与结直肠癌的关系

目的探讨结直肠癌组织中Kiss-1基因启动子甲基化状态与临床病理学特征的关系及临床意义。方法采用甲基化特异性PCR检测142例正常结直肠组织、23例结直肠腺瘤组织、73例结直肠癌组织和相应癌旁组织中Kiss-1基因启动子甲基化状态。结果结直肠癌组织中Kiss-1基因启动子甲基化阳性率为82.2%,癌旁组织为30.1%,结直肠腺瘤组织为21.7%,正常结直肠组织为6.3%,总体比较差异有统计学意义(P<0.05)。Kiss-1基因启动子甲基化阳性率与结直肠癌的分化程度、浸润深度、淋巴结转移及远处转移有关(P<0.05)。结论 Kiss-1基因启动子甲基化可能促进结直肠癌的发生、发展与转移,Kiss-1基因启动子甲基化水平有可能成为评估结直肠癌转移风险及预后的指标之一。     

DNA甲基化在结直肠癌发生发展中的研究进展

结直肠癌(colorectal cancer,CRC)是研究得最深入的肿瘤之一,其发生发展的各阶段都有很明显的遗传改变.首先是正常上皮细胞的增生,然后是异常腺管和腺瘤的形成,最后转变成浸润性癌.各阶段遗传改变的研究阐明了结直肠癌发生发展的分子机理.尽管研究已有很大的进展,对结直肠癌的发生发展还有许多问题有待研究,如结直肠癌发生率为什么随年龄增加,遗传改变为什么不能完整地解释许多散发性肿瘤?     

食管鳞癌中p16基因启动子区甲基化及其表达

 目的探讨食管鳞癌（ESCC）p16基因甲基化的状况及其表达与食管鳞癌临床病理特征之间的关系。方法采用甲基化特异性PCR方法（MSP）分别检测75例食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用Envision免疫组化法检测食管癌组织及癌旁组织的p16蛋白的表达。结果75例标本中，食管癌组织、癌旁组织和切缘细织p16基因甲基化率分别为41．3％（31／75）、13．3％（10／75）和6．67％（5／75）。癌组织和癌旁组织P16蛋白的阳性表达率分别为29．3％（22／75）和56．7％0（17／30）。31例癌组织p16基因甲基化阳性标本中有2例（6．4％）检测到P16蛋白的表达，而44例癌组织p16基因甲基化阴性标本中有20例（45．5％）检测到P16蛋白的表达。食管癌组织p16基因甲基化率显著高于癌旁组织和切缘组织（P〈0.01），P16蛋白表达与p16基因甲基化呈负相关。p16基因启动子区甲基化与食管癌的组织学分级、肿瘤部位无明显相关，与临床分期、淋巴转移密切相关。结论p16基因甲基化在食管癌发生发展中起着重要作用，食管鳞癌的分期和淋巴结转移与p16基因甲基化之间有密切关系。     

粪便DNA甲基化检测在结直肠癌早期诊断中的研究进展

粪便基因甲基化作为一种新的、敏感度较高、特异性中等的无创性结直肠肿瘤标志物,能根据其阳性结果对疑似病例进行进一步检测,可作为筛查诊断结直肠肿瘤的一种重要的辅助手段。目前已有较多研究报道检测粪便中基因异常甲基化对于早期结直肠癌（colorectal cancer,CRC）筛查有较高的敏感性和特异性,但尚无任何一种甲基化基因真正地应用于临床实践。本文对目前结直肠癌早期粪便DNA甲基化检测的国内外相关研究成果进行综述,总结粪便甲基化检测的潜在临床价值。     

Promoter hypermethylation of the ADAM23 gene in colorectal cancer cell lines and cancer tissues

Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell‐to‐cell and cell‐to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation‐specific PCR (MSP) and bisulfate‐modified DNA sequencing analysis. Methylated cells were treated with 5‐aza‐2′‐deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70–90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5‐aza‐2′‐deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down‐regulated by aberrant promoter hypermethylation during the progression of colorectal cancer. © 2008 Wiley‐Liss, Inc.     

Cancer stem cell marker expression and methylation status in patients with colorectal cancer

The number of individuals diagnosed with colorectal cancer (CRC) has been on an alarming upward trajectory over the past decade. In some countries, this cancer represents one of the most frequently diagnosed types of neoplasia. Therefore, it is an important demand to study the pathology underlying this disease to gain insights into the mechanism of resistance to treatment. Resistance of tumors to chemotherapy and tumor aggressiveness have been associated with a minor population of neoplastic cells, which are considered to be responsible for tumor recurrence. These types of neoplastic cells are known as cancer stem cells, which have been previously reported to serve an important role in pathogenesis of this malignant disease. Slovakia has one of the highest incidence rates of CRC worldwide. In the present study, the aim was to classify the abundance of selected stem cell markers (CD133, CD166 and Lgr5) in CRC tumors using flow cytometry. In addition, the methylation status of selected genomic regions of CRC biomarkers (ADAMTS16, MGMT, PROM1 (CD133), LGR5 and ALCAM) was investigated by pyrosequencing in a cohort of patients from Martin University Hospital, Martin, Slovakia. Samples from both primary tumors and metastatic tumors were tested. Analysis of DNA methylation in the genomic regions of indicated five CRC biomarkers was also performed, which revealed the highest levels of methylation in the A disintegrin and metalloproteinase with thrombospondin motifs 16 and O6-methyguanine-DNA methyl transferase genes, whereas the lowest levels of methylation were found in genes expressing prominin-1, leucine-rich repeat-containing G-protein-coupled receptor 5 and activated leukocyte cell adhesion molecule. Furthermore, tumor tissues from metastases showed significantly higher levels of CD133⁺ cells compared with that in primary tumors. Higher levels of CD133⁺ cells correlated with TNM stage and the invasiveness of CRC into the lymphatic system. Although relatively small number of samples was processed, CD133 marker was consider to be important marker in pathology of CRC.     

胃癌患者外周血与胃癌组织中BRCA1基因甲基化的关系及其意义

目的:检测胃癌患者外周血及胃癌组织中BRCA1基因启动子甲基化状态,并探讨两者的关系及意义。方法:采用甲基化特异性PCR(methylation-specific polymerase chain reaction,MSP)技术检测37例胃癌患者外周血及胃癌组织中BRCA1基因启动子甲基化状态。结果:胃癌患者外周血和胃癌组织中BRCA1基因启动子甲基化率分别为40.5%(15/37)和48.6%(18/37),二者相关系数r=0.848(P=0.0004)。结论:胃癌患者外周血与胃癌组织中BRCA1基因启动子存在高比例的甲基化,而且两者甲基化率有良好的一致性。胃癌患者外周血BRCA1基因启动子甲基化的检测具有简便、快捷、可靠的特点,外周血BRCA1基因启动子甲基化有可能成为治疗胃癌的靶点。     

Detection and clinical significance of CEACAM5 methylation in colorectal cancer patients

Colorectal cancer (CRC) is a globally common cancer, and the serum carcinoembryonic antigen (sCEA) is widely applied as a diagnostic and prognostic tumor marker in CRC. This study aimed to elucidate the mechanism of CEA expression and corresponding clinical features to improve prognostic assessments. In CRC cells, hypomethylation of the CEACAM5 promoter enhanced CEA expression in HCT116 and HT29 cells with 5-aza-2′-deoxycytidine (5-Aza-dC) treatment. Our clinical data indicated that 64.7% (101/156) of CRC patients had an sCEA level above the normal range, and 76.2% (77/101) of those patients showed a lower average CpG methylation level of the CEACAM5 promoter. The methylation analysis showed that both CRC cell lines and patient samples shared the same critical methylation CpG regions at −200 to −500 and −1000 to −1400 bp of the CEACAM5 promoter. Patients with hypermethylation of the CEACAM5 promoter showed features of a BRAF mutation, TGFB2 mutation, microsatellite instability-high, and preference for right-sided colorectal cancer and peritoneal seeding presentation that had a similar clinical character to the consensus molecular subtype 1 (CMS1) of colorectal cancer. Additionally, hypermethylation of the CEACAM5 promoter combined with evaluated sCEA demonstrated the worst survival among the patients. Therefore, the methylation status of the CEACAM5 promoter also served as an effective biomarker for assessing disease prognosis. Results indicated that DNA methylation is a major regulatory mechanism for CEA expression in colorectal cancer. Moreover, our data also highlighted that patients in a subgroup who escaped from inactivation by DNA methylation had distinct clinical and pathological features and the worst survival.     

TMEM196基因在肺癌中的甲基化修饰与表达调控

目的:分析TMEM196基因在肺癌组织中的甲基化情况以及甲基化发生对TMEM196基因表达的影响。方法:采用MSP方法检测肺癌组织、癌旁正常对照组织及肺癌细胞株TMEM196基因甲基化率;用去甲基化药物5-氮-2'-脱氧胞苷(5-aza-dC)处理肺癌细胞后,采用RT-PCR法检测TMEM196 mRNA表达水平。 结果:肺癌组织中TMEM196基因甲基化发生率为57%(28/49),而正常对照组织中该基因甲基化发生率为0(0/20)。TMEM196基因甲基化与肿瘤的分化程度(P=0.012)和临床分期显著相关(P=0.007),而与患者的年龄、性别、吸烟以及肿瘤的病理分类无显著相关(P>0.05)。TMEM196基因在肺癌细胞株H1975和H1650中高甲基化且表达缺失,去甲基化药物(5-aza-dC)处理细胞后TMEM196 mRNA表达明显升高,说明TMEM196基因表达可能受甲基化调控。结论:TMEM196基因甲基化与肺癌的发生发展密切相关,推测该基因通过DNA甲基化失活参与了肿瘤的发生。     

Epidermal growth factor receptor (EGFR) gene promoter methylation and cetuximab treatment in colorectal cancer patients

Background:

Epidermal growth factor receptor (EGFR) promoter methylation may be responsible for the loss of EGFR expression in neoplastic cells. The primary aim of our study was to verify a possible correlation between EGFR gene promoter methylation and clinical outcome in metastatic colorectal cancer patients receiving chemotherapy with irinotecan and cetuximab.

Methods:

Colorectal samples from patients treated with irinotecan–cetuximab were analysed for EGFR promoter methylation and EGFR immunohistochemistry.

Results:

Fifty-two patients were analysed. Thirty patients (58%) showed EGFR promoter hypermethylation. In EGFR promoter methylated and EGFR promoter unmethylated patients, we observed a partial response in 3 (10%) and 13 (59%) patients, respectively (P=0.03), progressive disease was obtained in 19 (63%) and 2 (9%) patients, respectively, with EGFR promoter methylated and EGFR promoter unmethylated tumours (P=0.0001). Median progression-free survival was 2.4 months in patients showing EGFR promoter methylated tumours and 7.4 months for those who had EGFR promoter unmethylated tumours (P<0.0001; Figure 1). Median overall survival was 6.1 months in patients showing EGFR promoter methylated tumours and 17.8 months for those who had EGFR promoter unmethylated tumours (P<0.0001; Figure 2). EGFR promoter hypermethylation, after confirmation in larger data set, may represent a valuable asset in further studies investigating EGFR as a therapeutic target in colorectal cancer.Open in a separate windowFigure 1Kaplan–Meier curves for median progression-free survival (PFS) of colorectal cancer patients treated with irinotecan and cetuximab with EGFR promoter methylated and without EGFR promoter methylated tumours (2.4 vs 7.4 months, P<0.0001).Open in a separate windowFigure 2Kaplan–Meier curves for median overall survival (OS) of colorectal cancer patients treated with irinotecan and cetuximab with EGFR promoter methylated and without EGFR promoter methylated tumours (6.1 vs 17.8 months, P<0.0001).     

SOX2在结直肠癌表达的临床观察

目的：观察SOX2在结直肠癌组织的表达情况,探讨SOX2在结直肠癌中表达的临床意义。方法收集103例有60～125个月（中位随访时间84个月）随访资料的结直肠癌及其相应的正常结直肠黏膜标本,应用免疫组化方法检测SOX2的表达,分析SOX2表达与临床特征的关系。结果结直肠癌组织中SOX2蛋白的表达率显著高于癌旁正常肠黏膜（P＜0.001）;SOX2表达与结直肠癌分化程度显著负相关（P＜0.01）,与结直肠癌TNM分期、淋巴结转移个数及远处转移程度正相关（P＜0.05）。SOX2表达与结直肠癌患者术后5年生存率和3年无瘤生存率也具有相关性（P＜0.05）。结论 SOX2蛋白异常表达可作为提示结直肠癌预后判断的重要参考指标。