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.     

胰腺癌微卫星不稳定性、hMLH1启动子甲基化及其蛋白表达研究

目的探讨胰腺癌中微卫星不稳定性（MSI）与hMLH1启动子甲基化及蛋白表达之间的联系，揭示胰腺癌发生的分子机制。方法从35例胰腺癌患者的正常胰腺组织、癌组织中提取DNA；SSCP法检测标本中微卫星不稳定性发生情况；免疫组织化学法检测错配修复基因hMLH1在胰腺癌中的表达情况；MSP法检测hMLH1基因启动子甲基化状态。结果35例胰腺癌中微卫星高度不稳定7例，低度不稳定14例，稳定11例，正常组织中没有出现微卫星不稳定，两者之间差异有统计学意义（P〈0．05）。hMLH1在微卫星不稳定胰腺癌组织中常为缺失表达。在微卫星稳定胰腺癌组织中呈正常表达。35例胰腺癌中hMLH1启动子CpG岛甲基化发生率为60％（21/35）。正常组织中未发现甲基化，两者之间差异有统计学意义（P〈0．05）。结论与胰腺癌有关的错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制，而hMLH1的表达失活则可能导致MSI的产生，促进胰腺癌的发生。     

2007年《中华胃肠外科杂志》作者索引

白月奎胃间质瘤临床病理及预后分析1:21本科编辑部《中华胃肠外科杂志》第三届编辑委员会第一次全体会议纪要2:105蔡国响散发性结直肠癌CpG岛甲基子表型和基因组遗传学不     

散发性结直肠癌微卫星不稳定与hMHL1和hMSH2基因突变的研究

目的:探讨散发结直肠癌组织微卫星不稳定性与错配修复基因hMHL1和hMSH2蛋白表达的关系。方法:结直肠癌患者病理标本和正常肠壁组织(距肿瘤边缘10cm取材各40例)。选取微卫星位点(D2S123、BAT-26、D17S261、D17S799)进行PCR,PCR产物行毛细管电泳法检测。用免疫组化染色方法分析错配修复基因hMHL1和hMSH2在肿瘤组织的蛋白表达情况。结果:1)4个位点(D2S123、BAT26、D17S261、D17S799)的微卫星不稳定性检出率分别为:12.5%、17.5%、10%、7.5%。总的微卫星不稳定率为9/40(22.5%)。MSI-H表达为7例,均表现BAT26位点不稳定。MSI-L表达2例。2)所有标本错配修复基因hMSH2检测均正常表达。错配修复基因hMHL1表达阴性11份,结肠比直肠hMLH1蛋白的阴性表达率高(P0.01)。3)hMLH1表达阴性,是出现MSI的重要分子因素,hMLH1不表达和MSI相关显著(P0.01)。结论:1)错配修复基因突变引起微卫星不稳定性是散发性结肠癌发生的重要机制;2)部分微卫星不稳定性是由错配修复基因hMLH1不表达引起,其余的微卫星不稳定性可能涉及到其它错配修复基因。     

微卫星不稳定结直肠癌hMLH1和hMSH2及hMSH6种系突变与hMLH1启动子甲基化检测

目的探究微卫星不稳定（MSI）结直肠癌患者的hMLH1、hMSH2和hMSH6种系突变特征和hMLH1启动子甲基化状态。方法对前瞻性收集的34例MSI结直肠癌患者检测其hMLH1、hMSH2和hMSH6种系突变，并研究其肿瘤的hMLH1启动子甲基化状态。结果34例MSI结直肠癌中。共检测到MLH1基因启动子的甲基化19例（55．9％）。19例MSI—H结直肠癌中检测到MLH1基因的甲基化14例（73．7％）；15例MSI—L结直肠癌检测到MLH1基因的甲基化5例（33．3％）；两组差异有统计学意义（P〈0．05）。全组共发现8个hMSH2和hMSH6基因的突变，其中hMSH6基因突变3个，hMSH2基因突变5个。结论中国人MSI结直肠癌错配修复基因突变（未测到MLH1基因突变）和MLH1基因启动子的甲基化检出率可能有别于国外MSI结直肠癌。     

BNIP3基因甲基化与早期结直肠癌患者临床病理特征的关系

目的分析早期结直肠癌组织中BNIP3基因启动子区CpG岛甲基化与患者临床病理特征的关系,评价其在结直肠癌发生发展中的作用。方法用甲基化特异性PCR技术分析107例早期结直肠癌患者肿瘤组织中BNIP3基因启动子区CpG岛甲基化状态。结果本组结直肠癌组织标本中BNIP3基因启动子区CpG岛甲基化阳性率为58.9%(63/107)。右半结肠组肿瘤组织中BNIP3基因甲基化明显高于左半结肠组(P<0.001);低分化组明显高于高中分化组(P=0.002)。BNIP3基因甲基化在患者性别、年龄、肿瘤大小和分期等分组间无明显差异。结论早期结直肠癌组织中存在BNIP3基因启动子区CpG岛甲基化。BNIP3基因高甲基化与早期结直肠癌部位和分化程度密切相关。     

应用荧光多重聚合酶链反应法检测结直肠癌微卫星不稳定

目的建立一种敏感、稳定、高通量的结直肠癌微卫星不稳定(MSI)检测技术。方法105例散发性结直肠癌患者取其新鲜癌组织及正常肠黏膜,DNA提取,荧光多重聚合酶链反应(PCR)扩增5个微卫星位点,应用GeneScan方法分析PCR产物。结果105例结直肠癌患者26例(24.7%)存在微卫星不稳定,其中MSI-H 14例(13.3%),MSI-L 12例(11.4%),MSS 79例(75.3%)。D5S346、BAT26、BAT25、D17S250、D2S123突变率分别为5.6%、8.6%、10.5%、8.6%、10.5%。结论应用荧光多重PCR可以高通量检测结直肠癌微卫星不稳定,敏感性高,结果可靠,适合临床应用。     

结直肠癌γ-synuclein基因启动子CpG岛的去甲基化及其临床意义

目的 探讨结直肠癌中γ-synuclein基因的表达与启动子区CpG岛甲基化修饰之间的关系,以及γ-synuclein基因去甲基化水平与结直肠癌临床病理特征的关系.方法 采用半定量RT-PCR法检测30例结直肠癌和相应癌旁组织中γ-synuclein基因的表达及去甲基化试剂5-杂氮-2＇-脱氧胞苷（5-Aza-C）干预对结直肠癌细胞COLO205、LoVo和SW480的γ-synuclein基因表达的影响.采用亚硫酸氢盐修饰测序（BSP）法检测5-Aza-C处理前后CpG岛的甲基化情况.采用巢式甲基化PCR（NMSP）及实时荧光定量MSP法检测67例结直肠癌组织和30例相应癌旁组织中γ-synuclein基因的甲基化状态,并分析其与临床病理特征的关系.结果结直肠癌组织中γ-synuclein mRNA的表达水平0.66±0.34,高于相应癌旁组织的0.45±0.26,差异有统计学意义（P=0.011）.5-Aza-C处理后,COLO205、LoVo和SW480细胞γ-synuclein mRNA表达水平明显上升,同时γ-synuclein基因启动子区CpG岛的甲基化水平明显降低.80.0%（24/30）的结直肠癌组织和50.0%（15/30）的相应癌旁组织的γ-synuclein基因被去甲基化,结直肠癌组织中γ-synuclein基因去甲基化的趋势明显强于相应癌旁组织（P=0.030）.γ-synuclein基因的去甲基化水平与结直肠癌的淋巴结转移、临床病理分期及远处转移有关.结论 结直肠癌中γ-synuclein基因的上调表达与启动子区CpG岛的去甲基化状态有一定关联,γ-synuclein基因的去甲基化水平有望成为结直肠癌患者判断预后的潜在标志物.     

错配修复基因启动子甲基化与胰腺癌发生的关系

目的通过对胰腺癌错配修复基因启动子甲基化及蛋白表达的检测与微卫星不稳定性的分析,探讨胰腺癌发病的分子机制。方法从35例胰腺癌病人的正常胰腺组织、癌组织中提取DNA;MSP法检测hMLH1及hMSH2基因启动子甲基化状态;SSCP法检测标本中微卫星不稳定性发生情况;免疫组化法检测错配修复基因hMLH1及hMSH2在胰腺癌中的表达情况。结果35例胰腺癌中hMLH1启动子甲基化发生率为60％（21／35）,正常组织中未发现甲基化,两者之间有统计学差异（P〈0.05）;而对于hMSH2仅有1例胰腺癌出现启动子甲基化;35例胰腺癌中微卫星高度不稳定7例,低度不稳定14例,稳定11例,正常组织中没有出现微卫星不稳定,两者之间有统计学差异（P〈0.05）;在微卫星不稳定的21例胰腺癌组织中有19例（90％）出现hMLH1启动子甲基化现象,微卫星稳定的ll例胰腺癌组织中仅有2例（6％）出现hMLH1启动子甲基化。hMLH1启动子甲基化在微卫星不稳定胰腺癌组织中常为缺失表达或低表达,在微卫星稳定胰腺癌组织中呈正常表达。hMSH2无论在MSI-H或MSI-L胰腺癌与正常胰腺组织中均无缺失表达,仅部分低表达。结论胰腺癌组织中错配修复基因的缺陷主要是hMLH1启动子甲基化,与胰腺癌微卫星不稳定性及蛋白表达缺失有关,在胰腺癌发生过程中起重要作用,是胰腺癌发生的重要机制之一。     

高度微卫星不稳定性对结直肠癌病人的预后作用

微卫星不稳定性 (MSI)在 10 %～ 15%的散发性结直肠癌病人中出现 ,根据 MSI的水平可分为三型 :高度 MSI(MSI- H)、低度 MSI(MSI- L )、微卫星稳定(MSS)。作者通过检测散发性结直肠癌病人 MSI- H,确立 MSI- H与病人预后的关系。方法　 2 38例散发性结直肠癌病人 ,入选条件为 :肿瘤分期符合改良 ACPS- C期 (相当于 TNM 期 ) ,单纯手术治疗 ,未行化疗 ,随访时间至少 5年。从上述病人的石蜡包埋组织中分别提取正常及肿瘤 DNA,通过放射标记的 PCR技术扩增。作者检测了 7个微卫星标志系列 (BAT2 5、BAT2 6、c- myb、D5S346…     

Prognostic value of hMLH1 methylation and microsatellite instability in pancreatic endocrine neoplasms

BACKGROUND: The aberrant promoter methylation of the mismatch repair gene, hMLH1, is associated with microsatellite instability (MSI) in cancer cells and often is associated with a favorable prognosis. METHODS: Pancreatic endocrine neoplasms (PENs) were obtained from 48 patients who underwent surgical resection. Methylation-specific polymerase chain reaction was used to detect methylation in the hMLH1 promoter. Tumor MSI at loci BAT26, BAT25, D2S123, D5S346, and D17S250 was determined with microsatellite polymerase chain reaction. RESULTS: Hypermethylation of the hMLH1 promoter was present in 11 of 48 PENs (23%). Five of the 11 hMLH1-methylated PENs were found to be microsatellite unstable, and MSI was restricted to PENs with hMLH1 hypermethylation. Tumor recurrence at 2 years after surgical resection was significantly less common among the hMLH1-methylated PENs (11%), compared with the unmethylated PENs (35%; P=.038). Patients with hMLH1-methylated PENs experienced improved 5-year survival (100%) compared with patients with unmethylated tumors (56%; P=.010). Likewise, MSI-positive PENs were associated with improved survival compared with MSI-negative tumors (100% vs 59%; P=.017) at 5 years. CONCLUSION: As in hereditary nonpolyposis colorectal cancer in which MSI is associated with improved survival, methylation of hMLH1 leads to MSI in PENs and affords a favorable prognosis.     

Stage IV early gastric cancer: two cases with microsatellite instability

Background We experienced two rare cases of stage IV early gastric carcinoma with extensive lymph node metastasis (pT1N3) and microsatellite instability caused by methylation of the MLH1 gene. Materials and methods The clinicopathological features of patients with stage IV EGC were examined. Microsatellite instability (MSI) was analyzed using five microsatellite markers and MLH1 gene promoter methylation patterns were determined by methylation specific polymerase chain reaction (PCR) from paraffin-embedded tissue samples. Loss of hMLH1 was identified by immunohistochemical study. Results In both cases, the carcinomas were located in the antrum of the stomach, confined within the submucosa (SM3) and accompanied by endolymphatic tumor emboli. Immunohistochemical analysis for hMLH1 showed negative nuclear staining in the carcinoma cells. The tumor demonstrated methylation of the MLH1 gene and MSI-H because they manifested instability in three and two of the five markers tested. One patient is alive without any clinical, radiological, or pathological evidence of recurrence or metastasis at 37 months, while the other patient died after a cerebrovascular accident 5 months after surgery. Conclusion Our rare cases support that MSI caused by MLH1 promoter methylation and the loss of hMLH1 protein play an important role in stage IV EGC development.     

Accuracy of the routine detection of mutation in mismatch repair genes in patients with susceptibility to hereditary upper urinary tract transitional cell carcinoma

OBJECTIVE: To establish the clinical benefits of systematic testing for hMSH6 and hMLH1 mutations in the very rare patients with upper urinary tract transitional cell carcinomas (UUT-TCCs), a clinical predisposition for hereditary tumour and no mutation detected in hMSH2 gene. PATIENTS AND METHODS: In all, 164 UUT-TCC specimen blocks were screened for microsatellite instability (MSI); 27 (16%) had high MSI levels. Eight patients (30%) had clinical criteria suspicious of hereditary tumour; in three a mutation in hMSH2 was detected. For the other patients, clinical data were collated, and DNA gene sequences analysed to detect mutations in hMLH1 and in hMSH6 genes. RESULTS: Five patients were assessed (mean age at the diagnosis of UUT-TCC 65.2 years, sd 8, range 54-71; two aged < 60 years). Three patients had a personal history of hereditary nonpolyposis colorectal related-cancer (three colorectal). There were only mutations in hMSH2 gene detected, with none in hMSH6 and hMLH1. CONCLUSION: For the rare patients with UUT-TCC who are suspected of carrying mismatch repair gene mutations if no hMSH2 mutation is found by genetic testing, complementary DNA sequencing for hMLH1 and hMSH6 mutation does not seem to contribute and should not be recommended in daily practice.     

Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points

The aim of this study was to compare BRAF and KRAS, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status in each of the histologic categories, including end-point carcinomas with residual adenoma, of the serrated polyp neoplasia pathway and the traditional (nonserrated) adenoma-carcinoma sequence. Deoxyribonucleic acid (DNA) was extracted from the selected samples and assayed for BRAF, KRAS2 codon12, 13, CIMP using markers hMLH1, MGMT, MINT1, MINT2, p16, and MSI using an assay for BAT25 and BAT26. A BRAF mutation was present in 82% of serrated carcinomas (SCas), 62% of serrated adenomas (SAs), 83% of serrated polyps with abnormal proliferation (SPAPs-syn. sessile serrated adenoma [SSA]), 76% of microvesicular serrated polyps (MVSPs), and was not found in any of the histologic categories of the traditional adenoma-carcinoma sequence. KRAS2 mutations were found in 43% of the goblet cell serrated polyp (GCSP) category, 13% of MVSPs, 7% of SPAPs, and 24% of SAs; in 26% of large traditional adenoma (lTAs) compared with small traditional adenomas (sTAs) (0/30; P<0.005) and in 37.3% of traditional carcinomas (TCa). CIMP-H (>1 marker positive) was significantly more frequent in SPAP, SA, and SCa compared with MVSP (P<0.05); CIMP-H was present in 10% of sTAs but was found more frequently in lTA (44.4%; OR 7.2; P=0.007) and TCa (38.9%; OR 5.8; P=0.007). Higher CIMP levels (4 or more markers positive) were significantly more frequent in advanced categories of the serrated pathway (SAs [31%] and SCas [30%]) compared with lTAs [0%] and TCAs [3.4%] (OR 12.2; P=0.02). MSI-H was identified only in the adenocarcinoma component of SCas (9/11) or in the contiguous SAs (3/7). The findings indicate that a BRAF mutation is a specific marker for a serrated polyp pathway that has its origin in a hyperplastic polyp (MVSP) and a potential end point as MSI carcinoma. CIMP-High (CIMP-H) develops early in this sequence and MSI-H develops late. The data provided a less complete picture of a second serrated pathway, identified by a KRAS2 mutation in SAs, but showed that the progressive stages of both iterations of the serrated neoplasia pathway are separate and distinct from those of the traditional adenoma-carcinoma sequence.     

Microsatellite instability and MLH1 hypermethylation – incidence and significance in colorectal polyps in young patients

OBJECTIVE: Microsatellite instability (MSI) is observed in most hereditary nonpolyposis colorectal cancer-related colorectal cancers (CRC). The original Bethesda criteria recommends MSI testing in patients 相似文献   

膀胱移行细胞癌微卫星不稳定性表达及机理探讨

目的 探讨微卫星不稳定性在膀胱移行细胞癌的表达及其作用机制。方法 用PCR方法分析35例膀胱移行细胞癌尿沉渣标本中微卫星不稳定性表达;用RT-PCR方法监测5种人类错配修复基因在膀胱癌细胞系mRNA转录水平的表达;用PCR方法检测膀胱癌细胞系BIU-87中错配修复基因hMLH1的启动子区域出现异常甲基化。结果 35例膀胱癌患者尿沉渣中,有31例（88.6%)可检出微卫星不稳定性表现;5种人的错配修复基因在BIU-87膀胱癌细胞系中有hMLH1和hMSH2表达缺失,而在正常近曲小管细胞系中都有表达;BIU-87细胞错配修复基因hMLH1的启动子区域出现异常甲基化,应用去甲基化剂处理后可检测到hMLH1的启动子区域的表达,再次去除去甲基化剂后叠不能检测hMLH1的启动子区域。结论 微卫星不稳定性与错配修复基因表达有关。甲基化对膀胱癌细胞系BIU-87错配修复基因hMLH1的表达具有调控作用。     

Promoter hypermethylation: an important epigenetic mechanism for hMLH1 gene inactivation in head and neck squamous cell carcinoma.

OBJECTIVE: The hMLH1 gene is one of the mismatch DNA repair genes. Inactivation of the hMLH1 gene has been implicated in the tumorigenesis of many types of human cancers. In most sporadic forms of human cancers, promoter hypermethylation is responsible for hMLH1 gene inactivation. Lack of hMLH1 protein expression has been found in a subset of head and neck squamous cell carcinomas (HNSCCs). The purpose of this study was to investigate whether promoter hypermethylation causes hMLH1 gene inactivation in HNSCCs. STUDY DESIGN: hMLH1 protein expression was determined by immunohistochemical staining in 62 cases, whereas hMLH1 gene promoter methylation was analyzed by methylation-sensitive restriction enzyme digestion, followed by polymerase chain reaction, in 35 cases of HNSCCs. RESULTS: Sixteen (26%) of 62 cases of HNSCCs showed near-complete loss of hMLH1 protein expression on immunohistochemical staining. Twelve (92%) of 13 cases that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 17 (77%) of 22 cases positive for the protein were free of promoter methylation. CONCLUSIONS: Promoter hypermethylation may be an important mechanism for hMLH1 gene inactivation in a subset of HNSCCs.