多基因甲基化的检测在肝癌早期诊断中的价值

目的检测肝癌患者癌组织中p14、p15、p16、RB基因启动子异常甲基化状况,探讨其在肝癌诊断中的价值。方法采用甲基化特异性PCR（MSP）方法检测44例肝癌组织及其相对应41例肝硬化组织、44例癌旁组织中p14、p15、p16、RB的甲基化状况。对所得结果进行卡方检验,Fisher精准概率分析。结果肝硬化组织中p14、p15、p16、RB 4种基因的甲基化检出率分别为17.1%（7/41）、28.9%（11/41）、36.6%（15/41）和7.3%（3/41）;肝癌组织中4种基因的甲基化检出率分别为34.1%（15/44）,56.8%（25/44）,70.5%（31/44）和27.3%（12/44）。癌旁组织中检测出p16甲基化阳性产物4例,p15阳性产物1例。癌组织和肝硬化组织中4种基因的甲基化率明显高于癌旁组织（P〈0.05）;不同年龄、性别、肿瘤分化程度和临床分期的肝癌患者各个基因甲基化率没有明显差异。结论 p14、p15、p16、RB基因甲基化在肝癌中频发;多基因甲基化的联合检测在肝癌的早期诊断中有一定的价值。     

中国北方人群p14~（ARF）启动子甲基化与原发性肝癌的临床相关性

目的通过对中国北方人群原发性肝癌p14ARF启动子甲基化的分析,探讨p14ARF基因启动子甲基化与原发性肝癌的临床相关性。方法通过甲基化特异性PCR（MSP）方法检测111例原发性肝癌组织和22例癌旁组织中p14ARF基因启动子甲基化状况,然后与肿瘤种类、病因、病理以及肿瘤分期等临床数据进行关联。结果肝细胞癌组织及癌旁组织中p14ARF基因启动子甲基化频率分别为33.7%（29/86）和5.0%（1/20）,二者差异有统计学意义（P=0.010）。肝细胞癌p14ARF基因的启动子甲基化频率在不同肿瘤分期显著相关（P=0.027）,而与肿瘤细胞分化程度以及是否感染HBV无关,肝内胆管细胞癌p14ARF基因启动子甲基化频率在不同肿瘤分期、有无HBV感染以及不同肿瘤细胞分化程度之间的差异均无统计学意义。结论肝细胞癌中p14ARF基因启动子甲基化可能是p14ARF基因失活并影响TP53功能的方式之一,p14ARF基因启动子甲基化可能与肝细胞癌发生发展相关。     

食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。     

HIV-1感染对T淋巴细胞p16INK4A基因甲基化及其表达的影响

目的；p16^INK4A基因在与AIDS相关的疾病中的表达缺陷，DNA甲基化可使基因转录下调。本研究观察人类免疫缺陷病毒I型（HIV－1）感染对T淋巴细胞的p16^INK4A基因的表达影响及其途径。方法：建立感染有野生型和突变体HIV－1病毒的T淋巴细胞Hut78细胞系，以RT－PCR、定量PCR和Western blotting检测细胞感染后不同时间的DNA甲基化酶（DNMT）1、3a和3b的mRNA（提高40％以上）和蛋白质水平；并由甲基化特异PCR（MSP）了解p16^INK4A基因甲基化改变，及通过RT－PCR研究该基因的mRNA表达情况。结果：无论野生型抑或突变体HIV－1感染，都能使Hut78细胞中DNMT1表达增强，DNMT3a和DNMT3b无明显变化。HIV－1感染可引起p16^INK4A基因启动子区甲基化水平提高和基因转录水平下调。结论；HIV－1感染可使p16^INK4A甲基化水平升高和表达降低，且与该病毒有无复制无关。     

p16基因在原发性肝癌中表达缺损的研究

目的：探讨p16基因的异常表达与原发性肝癌发生、发展的关系。方法：应用PCR、SSCP分析及DNA序列测定技术研究了30例原发性肝癌组织中的p16基因外显子2（E2）的缺损情况。结果：（1）p16基因缺损的检出率与肝癌的肿瘤组织类型无关，在肝细胞癌与胆管细胞癌中p16基因缺损的检出率分别为33．33％和20．00％，两组间无显著差异（P＞0．05）；（2）p16基因缺损率的高低与组织学分级密切相关（P＜0．05）。结论：结果提示p16基因的缺损与原发性肝癌的组织学分级相关，它可能参与原发性肝癌的发生、发展过程。     

DNA甲基化和胃液固有荧光光谱联合检测在胃癌诊断中的意义

目的探索在胃癌患者胃液、外周血清中检测基因甲基化的可行性，并结合胃液稀释固有荧光光谱评价二者在胃癌诊断中的作用。方法采用甲基化特异性PCR方法，检测50例胃癌患者的原发肿瘤组织、外周血清和胃液脱落细胞的死亡相关蛋白（DAP）激酶、p16基因启动子区域甲基化状态，并以胃良性溃疡和慢性浅表性胃炎各20例、慢性萎缩性胃炎30例作为对照，同时检测胃癌患者和对照者的胃液稀释固有荧光光谱。结果50例胃癌患者的肿瘤组织、外周血清和胃液脱落细胞中p16和DAP激酶基因甲基化阳性率分别为74．4％和68．1％、52．0％和58．0％、58．6％和76．0％，20例慢性浅表性胃炎患者中均未检测到基因异常甲基化；20例胃溃疡患者溃疡周边组织、胃液脱落细胞中p16和DAP激酶甲基化阳性率为10．0％和20．0％、5．0％和15．0％，在外周血清中未检测到异常甲基化；30例慢性萎缩性胃炎患者胃黏膜组织、外周血清、胃液脱落细胞p16和DAP激酶基因甲基化阳性率分别为10．0％和23．3％、3．3％和3．3％、3．3％和20．0％。胃癌患者胃液固有荧光光谱强度较对照者明显增强（P〈0．05）；以P1FI≥111．8为分界点分析胃液固有荧光光谱诊断胃癌的敏感性和特异性分别为76．1％和78．6％。p16和DAP激酶基因甲基化和胃液固有荧光光谱结合后诊断胃癌的敏感性提高到95．6％和97．8％。结论胃癌患者外周血清及胃液脱落细胞中可检测到与原发肿瘤组织一致的基因异常甲基化，胃液固有荧光光谱和DNA甲基化联合对检测胃癌有良好的临床应用价值。     

乳腺癌组织中p16基因甲基化与p16、ER蛋白表达的相关性及意义

目的探讨p16基因甲基化在乳腺癌发生、发展中的作用。方法应用甲基化特异性PCR（MSP）联合测序检测58份乳腺癌及其癌旁组织中p16基因甲基化状态,应用免疫组化sP法检测p16及ER蛋白表达情况,对各指标间关系行Spearman相关分析。结果p16基因在乳腺癌中甲基化率为29,3％（17／58）,有淋巴结及远处转移者显著高于无转移者（P〈0．05）;p16甲基化及非甲基化者p16蛋白表达阻性（失表达）率分别为82．4％（14／17）、43．9％（18／41）,ER蛋白失表达率分别为76．5％（13／17）、24．4％（10／41）,P均〈0．05。结论p16基因甲基化在乳腺癌发生、发展中具有重要作用,机制可能与调节p16和ER蛋白表达有关。     

乳腺癌组织p16基因甲基化与ER、PR、HER2及p53表达的关系

目的探讨乳腺癌组织中p16基因甲基化与相关受体表达的相关性,进一步提高乳腺癌的诊断水平。方法采用甲基化特异性PCR（MSP）法检测86份乳腺癌组织及40份乳腺癌患者血清中p16基因的甲基化状态;采用免疫组化sP法检测乳腺癌组织中雌激素受体（ER）和孕激素受体（PR）、人类表皮生长因子受体2（HER2）及p53基因表达,分析各指标之间及与乳腺癌之间的关系。结果乳腺癌组织及血清中p16基因甲基化率分别为29．1％、27．5％;15例ER、PR、HER2均为阴性表达者（三阴乳腺癌）,p16基因甲基化率为86．67％（13／16）,非三阴乳腺癌71例,p16基因甲基化率为16．9％（12／71）,P〈0．01。p16基因甲基化与ER、PR蛋白表达呈负相关（r=-0．425、-0．512,P均〈0．05）,与HER2表达呈正相关（r=0．443,P〈0．05）;与p53表达无明显相关性。结论p16基因甲基化是乳腺癌中常见的分子改变,其与ER、PR、HER2联合检测可作为乳腺癌早期诊治及预后判断的重要指标。     

非小细胞肺癌患者血清DAPK基因、p16基因甲基化的联合检测

目的 分析非小细胞肺癌（NSCLC）患者血清中死亡相关蛋白激酶（DAPK）、p16启动子区域甲基化的改变状况及其联合检测的意义.方法 运用甲基化特异性PCR技术,检测89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30.3%（27/89）,p16基因甲基化检出率为43.8%（39/89）,联合检测两基因甲基化检出率为76.4%（68/89）,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.联合检测两基因优于各单基因检测.     

原发性肝细胞癌中p16基因甲基化及其蛋白表达分析

目的探讨p16基因的甲基化改变和p16蛋白表达在肝癌的发生发展过程中的作用。材料和方法收集原发性肝细胞癌(HCC)手术切除的新鲜标本10例,石蜡包埋标本3 3例,Ⅰ级8例,Ⅱ级2 1例,Ⅲ级14例(Edmonson分级) ,p16蛋白检测用免疫组化法(ABC) ,p16基因甲基化检测用甲基化特异PCR(MS_PCR)法。结果HCCp16蛋白缺失率65 1% ( 2 8/4 3 ) ,而癌旁组织缺失率11 5 % ( 3 /2 6) ,两者相比有显著差异(P <0 0 1)。Ⅰ～Ⅱ级HCCp16蛋白的缺失率与Ⅲ级相比明显较低(P <0 0 5 )。p16蛋白阳性的15例HCC标本,均未检出基因甲基化,p16蛋白缺失的2 8例标本,有19例检出基因甲基化,甲基化率44 2 %。10例癌旁肝组织有1例基因甲基化,HCC和癌旁组织相比甲基化率有明显差异(P <0 0 5 ) ,基因甲基化与p16蛋白缺失明显相关(P <0 0 1)。各级HCCp16基因启动子区甲基化率有明显差异(P <0 0 5 )。结论p16蛋白缺失与HCC的发生有密切关系,低分化的HCC和p16蛋白缺失有关,p16蛋白在判断其恶性程度方面有一定的价值。基因的甲基化可能是p16基因在HCC中的主要失活方式,HCC分化程度和p16基因甲基化之间有密切关系。     

pl6 promoter hypermethylation: A useful serum marker for early detection of gastric cancer

AIM: To determine pl6 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on pl6-protein expression, and correlation with clinical and histological features.METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate methylation status of p16 promoter. pl6-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections.RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. pl6-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking.CONCLUSION: pl6 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of pl6 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.     

Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma

One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14(ARF), p15(INK4B), p16(INK4A), p18(INK4C), and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P =.01) only 1 methylated gene. With the exception of p18(INK4C) the genes under study showed promoter methylation with frequency ranging from 82% (p16(INK4A) in HCC) to 33% and 39% (p15(INK4B) and p16(INK4A) in cirrhoses). In cases with only 1 methylated gene, p15(INK4B) in cirrhosis (8 of 17, 47%) and p16(INK4A) in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (> or = 3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15(INK4B) and p16(INK4A) in cirrhosis and p16(INK4A) in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis-HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications.     

p16 promoter hypermethylation： A useful serum marker for early detection of gastric cancer

AIM： TO determine p15 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS： Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction （MSP） was used to evaluate methylation status of p16 promoter, p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS： Methylation was detected in 44.2% （23/52） of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients （14/52）. Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases （32/52）, and was significantly associated with promoter hypermethylation （P 〈 0.001）. Methylation was significantly more frequent in higher pathological grades （P 〈 0.05）. Methylation was not associated with other clinicopathological features and environmental factors including Hpylori infection and smoking. CONCLUSION： p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.     

人肝细胞癌c-myc基因扩增、MTS1/p16基因变异和HBV感染的研究

]目的探讨原癌基因c-myc扩增、抑癌基因MTS1/p16变异以及HBV感染在人肝细胞癌(HCC)发生发展中的作用.方法应用差异PCR结合非变性聚丙烯酰胺凝胶电泳银染及激光扫描(d-PCR-PAGE-Laser)技术检测c-myc基因扩增,应用PCR结合单链构象多态(SSCP)银染法分析MTS1/p16基因变异,PCR检测HBVDNA.结果(1)29例配对肝细胞癌、癌旁组织中c-myc基因扩增阳性率分别为44.83%(13/29)和51.72%(15/29),两者差异无显著性,P＞0.05;但两者均显著高于肝硬化组织c-myc基因扩增的阳性率8.33%(1/12),P＜0.05.(2)只有3例(10.34%,3/29)肝细胞癌中发现MTS1/p16基因纯合性缺失,未发现MTS1/p16基因突变.(3)正常肝脏、肝硬化和肝细胞癌组织中HBVDNA的阳性率分别为14.29%(2/14)、66.67%(8/12)和96.55%(28/29),三者间差异具有高度显著性,P＜0.001,并且HBVDNA的阳性率随肝脏病情的加重而增高(b＝0.3986,P＜0.001).(4)29例肝细胞癌中c-myc基因扩增和HBVDNA存在与否无关(P＜0.01).结论(1)c-myc基因扩增和HBV感染与HCC的发生、发展密切相关,HCC中c-myc基因扩增和HBV感染之间无内在相关性.(2)HCC中MTS1/p16基因纯合性缺失和突变的发生频率较低.[     

Profile of hepatocellular carcinoma in India: An insight into the possible etiologic associations

BACKGROUND: Several etiologic factors including hepatitis viruses, alcohol and aflatoxin have been implicated in the pathogenesis of hepatocellular carcinoma (HCC). There is, however, limited information from the Indian subcontinent. METHODS: Seventy-four consecutive cases of HCC were studied. A detailed history, tests for hepatitis B virus (HBV; HBsAg, HBeAg, anti-HBe, IgG anti-HBc, anti-HBs and HBV-DNA), hepatitis C virus (HCV; anti-HCV and HCV-RNA) infection, liver histopathology and HBV-DNA integration by using Southern blot hybridization were studied. A p53 gene mutation was also studied by using PCR and single-strand conformation polymorphism. RESULTS: Hepatocellular carcinoma patients were predominantly males (mean age 49.5 +/- 14.0 years). Portal hypertension and cirrhosis were seen in 56 (76%) patients, more often (P < 0.05) in viral marker positive cases. Forty-five percent of patients had features of hepatic decompensation at presentation. Evidence of HBV infection was present in 53 (71%) patients. Twenty-six (49%) of these patients had either HBeAg + ve, HBV-DNA + ve (n = 12), or HBsAg - ve, HBV-DNA + ve (n = 14) forms of HBV infection. Hepatitis B virus DNA integration in the liver tissue was seen in 10 of 17 (59%) patients. Infection with HCV alone was detected in three (4%) and dual HBV and HCV infection in six (8%) patients. A majority (78.5%) of the chronic alcoholics had associated viral infection. The etiology of HCC remained undetermined in 15 (20%) patients. The p53 gene mutations were detected only in three of 21 (14%) liver tissues. Aflatoxin toxicity, oral contraceptive use or metabolic disorder were not seen. CONCLUSIONS: In India: (i) HBV infection is the predominant factor for the development of HCC, often related to mutant forms of HBV; (ii) a majority of the HCC patients have overt cirrhosis of the liver; and (iii) HCV and alcohol per se are uncommonly associated.     

Epigenetic inactivation of INK4/CDK/RB cell cycle pathway in acute leukemias

Dysregulation of cell cycle is important in oncogenesis. We analyzed the inactivation of the INK4 family CKI/CDK/RB pathway by gene promoter hypermethylation in leukemogenesis. The methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles of the p15, p16, p18, and RB genes was used to study five leukemic cell lines, 50 acute myeloid leukemia (AML) and 25 acute lymphoblastic leukemia (ALL) samples. None of the leukemic cell lines showed p18 and RB methylation. p15 was methylated in Raji, while p16 was methylated in U937 and Raji. In NB4 and Jurkat, both alleles of p15 and p16 appeared to be deleted. At diagnosis, p15 methylation occurred in 29 (58%) AML patients, and 10 (40.0%) ALL patients. p16 methylation occurred in two (4%) AML and two (8%) ALL patients. Only one each of AML and ALL patients had concurrent p15 and p16 methylation. None of the patients had methylation of p18 or RB. In AML, p15 methylation was associated with M2 subtype (p=0.018). Patients with and without p15 methylation had similar complete remission (CR) rates and projected 5-year overall survival (OS) or disease-free survival (DFS). Therefore, methylation inactivation of the INK4/CDK/RB pathway in leukemia involved primarily p15 and occasionally p16, but not p18 or RB. In AML, p15 gene methylation was associated with the M2 subtype, but was not prognostic for CR, OS, or DFS.