HBV—DNA基因型与肝癌的预测性相关研究

HBV-DNA 基因型与肝癌发生的预测性相关研究为山东医科大学承担的山东省科委研究课题。本课题从分子水平上首次系统地对乙肝、肝癌患者的血清、淋巴细胞、肝癌/肝组织内 HBV-DNA 存在状态分布、定位及其复制活性等特点进行了系统分析研究,现报道如下:     

启东地区鸭群及鸭肝癌中人HBV变异体DNA存在的频率

以前曾报道从启东鸭肝癌中证实存在人HBV样DNA,并进行了分子克隆。经过全部DNA序列分析,证明它与鸭HBV(DHBV)无关,与人adw_2HBV有高度同源(98.6％),并在PreSr及C基因分别缺失1个和两个核苷酸,从而说明它是人HBV的突变体。本文报告了从新收集的上海地区32份、南京21份及启东19份鸭肝以及11例启东鸭肝癌标本,对DHBV及HBV DNA的存在状态作了分析。结果表明DHBVDNA在上述鸭肝或肝癌中普遍存在,阳性率在半数以上。但人的HBV DNA则完全不存在于上海和南京地区的鸭肝中。然而,在启东的19份鸭肝中,有3份人HBV DNA阳性。值得注意的是,在11例启东鸭肝癌样品中全部存在人HBV DNA序列。这结果提示,启东鸭肝癌中人HBV DNA的存在并非属偶然,而是带有普遍性和特征性。它进一步提示启东鸭肝癌病发与人的HBV DNA存在的病因学上的相关性。     

DNA甲基化与食管癌的关系

肿瘤细胞普遍存在DNA甲基化模式的改变,DNA甲基化异常包括原癌基因低甲基化和抑癌基因高甲基化。近年来研究结果表明,在食管癌发生过程中同样存在相关抑癌基因启动子区甲基化导致的基因表达的紊乱。其中由DNA甲基转移酶（DNA methyltransferase,DNMT）和去甲基化酶的活性改变导致的抑癌基因CpG岛超甲基化的研究,已成为食管癌发病机制研究中的热点之一。综述DNA甲基化的特点及其抑制基因转录和表达的分子生物学机制;DNA异常甲基化与食管癌发生发展的关系;食管癌相关肿瘤抑制基因的甲基化谱构成了食管癌独特的表遗传学标志;目前常用的甲基化检测手段及各方法的优缺点;DNA甲基化和去甲基化研究的展望及所需要解决的的问题等。     

重度饮酒与HBV相关肝癌危险性的关系

目的了解饮酒与乙型肝炎病毒(hepatitisBvirus,HBV)感染在肝细胞癌(hepatocellularcarcinogenesis,HCC)发生中的相互作用。方法采用病例对照研究方法,在江苏省淮安市以219例HCC患者为病例组,219例非HCC的上消化道恶性肿瘤患者,如胃癌、胆囊癌及胰腺癌等为对照组。通过问卷调查采集HCC危险因素信息,用ELISA法测定血标本中乙型肝炎病毒表面抗原(hepatitisBsurfaceantigen,HBsAg),并应用非条件Logistic回归模型计算比数比(oddsratio,OR)和95%可信区间(confidenceinterval,CI)。结果HBsAg阳性、轻度饮酒和重度饮酒(≥80mL/d乙醇)的多变量OR及其95%CI分别为28.2(10.6～75.5)、2.2(0.9～4.9)和1.2(0.5～3.1),轻度饮酒HBsAg阳性的多变量OR及其95%CI为28.6(11.7～70.2),而重度饮酒和HBsAg阳性的协同OR为50.3(19.2～131.6)。结论HBV感染是淮安市HCC的重要危险因素。饮酒虽然不是HCC的独立危险因素,但重度饮酒可增加罹患HBV相关HCC的危险性。     

HBV感染对原发性肝癌患者血清AFP值的影响

本文报道测定１０８例ＰＨＣ者的血清ＡＦＰ值和ＨＢＶ感染标记。７１例（６８．５％）ＡＦＰ值〉２０μｇ／Ｌ，其中６５例ＡＦＰ值〉１００μｇ／Ｌ；ＨＢｓＡｇ阳性者９６例（８８．９％），ＨＢｓＡｇ阴性者１２例（１１．１％），ＨＢｓＡｇ阳性者ＡＦＰ值的中位数为５０５μｇ／Ｌ（２０～２２３４μｇ／Ｌ）显著高于ＨＢｓＡｇ阴性者的２０μｇ／Ｌ（２０～３８３μｇ／Ｌ），ＨＢｓＡｇ阳性者ＡＦＰ值升高的峰值年龄组与ＰＨ     

胃癌组织RASAL1基因启动子甲基化及临床意义研究

目的:研究胃癌组织中RASAL1基因启动子的甲基化状态并分析其与临床病理资料的关系,初步探讨RASAL1基因启动子甲基化在胃癌发生发展中的意义。方法:取40例胃癌患者手术标本,使用甲基化特异性PCR(MSP)技术分别检测胃癌组织及相应癌旁组织中RASAL1基因启动子的甲基化状态,并分析其与胃癌临床病理特征的关系。结果:胃癌组织和癌旁组织中RASAL1基因启动子甲基化率分别为70%(28/40)和30%(12/40),胃癌组织中RASAL1基因启动子的甲基化发生率明显高于癌旁组织,P<0.01。胃癌组织中RASAL1基因启动子的甲基化率与性别(P=0.622)、年龄(P=0.168)无明显相关;但与分化程度(P=0.006)、肿瘤大小(P=0.043)、侵袭深度(P=0.015)和淋巴结转移相关(P=0.010);在肿瘤体积大、分化程度差、侵袭深度较深和有淋巴结转移的胃癌组织中,RASAL1基因启动子甲基化率明显增高。结论:胃癌组织中RASAL1基因启动子区域甲基化发生率高,且与分化程度及进展阶段相关,提示RASAL1基因启动子的异常甲基化可能参与了胃癌的发生发展过程。     

广州地区肝细胞癌与HCV,HBV感染关系的病例对照研究

在肝细胞癌（ＨＣＣ）中度流行区广州地区，对６４例ＨＣＣ患者抗－ＨＣＶ和ＨＢｓＡｇ状况按性别、年龄配对和１：２数量比设对照组进行了病例对照研究。结果表明：ＨＣＣ组抗－ＨＣＶ和ＨＢｓＡｇ阳性率分别为１８．７５％（１２／６４）和８４．３８％（５４／６４），分别显著高于对照组的２．３４％（３／１２８，Ｐ＜０．００１）和１４．０６％（１８／１２８，Ｐ＜０．００１）。与抗－ＨＣＶ、ＨＢｓＡｇ双阴性组比较，仅抗－ＨＣＶ阳性，仅ＨＢｓＡｇ阳性时发生ＨＣＣ的相对危险度分别为４６．７１和４３．７９，二者双阳性时上升至７０．０７。显示ＨＣＶ、ＨＢＶ均与ＨＣＣ显著相关，两者重叠感染具有协同致癌作用。作者提出预防和控制ＨＢＶ、ＨＣＶ感染对降低ＨＣＣ发病率意义重大。     

CpG岛甲基化表型及OPCML基因甲基化与肝细胞癌发生的关系

背景与目的：CpG岛甲基化表型（CpG island methylator phenotype,CIMP）涉及到多个基因启动子同时甲基化,具有肿瘤特异性,与多种肿瘤的发生或预后相关。但有关肝癌CPG岛甲基化表型的研究罕见报道。OPCML（opioid-binding protein／cell adhesion molecule—like）基因目前多为针对上皮性卵巢癌的研究．被认为是卵巢癌的候选抑癌基因。本研究旨在探讨CIMP及OPCML基因与肝癌的发生是否有关。方法：运用甲基化特异性PCR方法检测50例肝细胞癌组织及48例癌旁组织中OPCML、p15、SOCS-1、GST-P、RAR．b、p16、p73、p14、MGMT和hMLHl基因的甲基化状况。结果：肝癌组织甲基化率普遍比相应癌旁组织甲基化率高：OPCML（70．0％VS．64．6％）、p15（58．0％VS．50．0％）、SOCS-1（78．0％VS．50．0％）、GST—P（56．0％VS．27．1％）、RAR-b（30．0％vs．6．3％）、p16（26．0％vs．14．6％）、p73（16．0％vs．0％）、p14（36．0％vs．27．1％）、MGMT（16．0％vs．10．4％）和hMLH1（18．O％VS．4．2％）。SOCS-1,GST-p,RAR-b,p16和p73基因甲基化率在肝癌组与癌旁组差异有显著性（P〈0．05）,其它基因两组之间的甲基化率差异无显著性。CIMP阳性组（同时具有≥3个位点甲基化）复发时间较早,1年无瘤生存率为18．2％,而CIMP阴性组（具有〈3个位点甲基化）复发时间较晚,1年无瘤生存率为75．0％（P〈0．05）。结论：肝癌中存在着CpG岛甲基化表型（CIMP）。CIMP可作为肝癌患者预后判断的指标之一：OPCML基因甲基化可能在肝癌的发生中发挥重要的作用。     

乙型肝炎病毒感染与原发性肝癌相关的危险度分析

目的:分析原发性肝癌(PHC)患者血清中乙型肝炎病毒(HBV)标志情况,探讨HBV感染与PHC危险性的关系。方法:收集在中国医科大学附属第一医院介入科住院的PHC患者224例作为病例组,同时期在此科住院的其他非肝病患者280例作为对照组,记录其血清HBV标志,与既往乙型肝炎和肝硬化等情况进行病例对照研究。结果:PHC组HBV总感染率为68.22%(146/214),以HBsAg阳性(65.42%,140/214)和抗-HBe阳性(30.84%,66/214)为主,对照组总感染率为7.63%(20/262),其中以HBsAg(3.44%,9/262)和抗-HBe(7.63%,20/262)为主。病例组感染率明显高于对照组(χ2=200.1,P<0.000 1),优势比OR=27.4(95%CI为16.0~46.9)。结论:HBV慢性感染是PHC的一个危险因素,在HBV慢性感染者中进行PHC筛查,对早期诊断PHC提高其临床疗效有重要意义。     

Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma

High rate of chronic hepatitis B virus (HBV) infection and p16 promoter methylation were found in the majority of hepatocellular carcinoma (HCC). To investigate the potential linkage between high rate of p16 methylation and HBV infection, p16 methylation was detected with methylation-specific polymerase chain reaction (PCR), and HBV markers were examined with real-time PCR and immunologic method. p16 methylation was detected in 5.5% of patients with hepatitis B, 9.1% of noncancerous liver, 36.6% of cirrhotic liver tissue, and 70.5% of cancerous tissue of HCC, primarily in cirrhotic (46.7%) and cancerous tissue (90.6%) with HBV infection. In noncancerous tissue, p16 methylation could only be detected in samples with HBV infection, although no significant difference, the frequency of p16 methylation in noncancerous tissue with HBV infection was higher than those without it. The results showed that, in cancerous, cirrhotic, or noncancerous tissues, the frequency of p16 methylation in samples with HBV infection was higher than those without it, suggesting possible association between HBV infection and p16 methylation. The result of HBV-DNA analysis showed that 96.1% (49/51) samples with p16 methylation also showed detectable HBV-DNA; it signifies that replication and/or integration of HBV may contribute to high rate of p16 methylation in hepatocarcinogenesis. Generally, these results indicate that persistent HBV infection may be associated with high rate of p16 methylation, and involved in development of HCC through this way.     

A meta-analysis of case-control studies on the combined effect of hepatitis B and C virus infections in causing hepatocellular carcinoma in China

We investigated whether concurrent infection by hepatitis B virus (HBV) and hepatitis C virus (HCV) in China, a hyperepidemic area for these infections, was associated with a higher risk of causing hepatocellular carcinoma (HCC) than each infection alone in a meta-analysis in China, 32 case-control studies involving 3201 cases and 4005 controls, identified from a computer-based literature search from 1966 to 2004. The pooled odds ratio and 95% confidence interval (CI) for HBsAg positivity was 14.1 (95% CI: 10.6-18.8); for anti-HCV/HCV RNA positivity was 4.6 (95% CI: 3.6-5.9); for HBsAg positivity and anti-HCV/HCV RNA negativity were 15.6 (95% CI: 11.5-21.3); for HBsAg negativity and anti-HCV/HCV RNA positivity were 8.1 (95% CI: 5.0-13.0); and positivity for both HBsAg and anti-HCV/HCV RNA was 35.7 (95% CI: 26.2-48.5). We conclude that HBV and HCV infections are important independent risk factors for HCC in China, and that dual infection by HBV and HCV is associated with a higher risk of causing HCC than each infection alone, suggesting a synergism between HBV and HCV.     

Hepatitis B virus reactivation in hepatocellular carcinoma patients undergoing transcatheter arterial chemoembolization therapy

Aim: The effect of transcatheter arterial chemoembolization (TACE) therapy on hepatitis B virus (HBV) reactivation in hepatocellular carcinoma (HCC) patients with prior resolved hepatitis B is not fully understood. Methods: From January 2006 to December 2010, 43 hepatitis B surface antigen (HBsAg)‐negative/anti‐hepatitis B core antigen (HBc) positive patients with newly diagnosed unresectable HCC were enrolled in the study. All underwent TACE therapy. Results: Four patients (9.3%) developed HBV reactivation with mild/moderate hepatitis. The median number of TACE cycles received was 3.5 (range 3–4 cycles). The median time interval between the occurrence of HBV reactivation and the completion of TACE therapy was 3 months (range 1–5 months) and their median HBV DNA level was 1.58 × 10⁴ IU/mL (range, 1.65 × 10³–6.42 × 10⁴ IU/mL). After the introduction of lamivudine at the occurrence of HBV reactivation, all had resolution of hepatitis. An exploratory analysis indicated that significant predictors of HBV reactivation included increased serum total bilirubin coexisting with cirrhosis and the total number of cycles of TACE received. Conclusion: The administration of TACE therapy may increase the risk of HBV reactivation in HBsAg‐negative/anti‐HBc‐positive patients diagnosed with unresectable HCC. Further studies are warranted to explore the optimal management of HBV reactivation in patients with prior resolved hepatitis B.     

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.     

Smad4 expression in hepatocellular carcinoma differs by hepatitis status

Aims: Primary hepatocellular carcinoma (HCC) is a common malignancy often related to hepatitis viralinfection. Smad4 is known to mediate the TGF-β pathway to suppress tumorigenesis. However, the function ofSmad4 in HCC is still controversial. In this study we compared levels of Smad4 in HCC tissues with or withouthepatitis virus infection and adjacent normal-appearing liver. Methods: Samples from HCC patients wereanalyzed for Smad4 protein and mRNA expression by immunohistochemistry (IHC), RT-PCR and Westernblotting. Results: We found that tumor tissues expressed less Smad4 mRNA and protein than the adjacent tissues.Most HCC tumor tissues were negative for Smad4 in IHC staining, while the majority of adjacent tissues werepositively stained. Interestingly, protein levels were higher in HCC tissues with viral hepatitis than those withoutvirus infection. Suppression of expression appeared closely related to HCC, so that Smad4 appears to functionas a tumor suppressor gene (TSG). Conclusion: Patients with hepatitis viral infection, at higher risk for HCC,exhibited increased Smad4 protein expression suggesting hepatitis virus may modulate Smad4 expression, whichis functionally distinct from its putative role as a TSG. Smad4 expression may thus be an applicable marker fordiagnosis and/or a target to develop therapeutic agents for HCC.     

Hepatitis B and C viruses in the etiology of hepatocellular carcinoma; a study in Greece using third-generation assays

Objectives: The purpose of this study was to describe the role of hepatitis B virus (HBV) and hepatitis C virus (HCV) in the etiology of hepatocellular carcinoma (HCC). Methods: During a 4-year period from January 1995 to December 1998, blood samples and questionnaire data were obtained from 333 incident cases of HCC from Athens, Greece, as well as from patients in two control groups, also from Athens. Controls were 272 metastatic liver cancer (MLC) patients and 360 patients hospitalized for injuries or eye, ear, nose or throat conditions. Coded sera were tested for hepatitis B surface antigen (HBsAg) and antibodies to hepatitis C virus (anti-HCV) by third-generation enzyme immunoassays. Results: The odds ratios (with 95% confidence intervals) in logistic regression modeling comparing the HCC cases to the combined control series were 48.8 (30.5–78.3) for the presence of HBsAg and 23.2 (11.4–47.3) for the presence of anti-HCV. The odds ratio for concurrent infection with HBV and HCV was 46.2 (9.9–216.6) compared to infection with neither virus. Conclusions: Although HBV and HCV are both important causes of HCC in this study population the data do not suggest, neither do they conclusively refute, a super-additive interaction between the two infections in the development of this malignancy. In this population, 58% of HCC cases can be attributed to HBV, 12% to HCV, and 3% to dual infection with these viruses.