【作者】 吴李鸣；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2008， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma,HCC)是最常见的肝脏恶性肿瘤,占全世界肿瘤发生率的第五位,全球每年约有37万多患者被新诊断为HCC。由于中国人群中的乙型肝炎病毒携带率高达10%,从而使中国成为肝癌最高发的地区之一。尽管采用了以手术切除为基础的各种综合治疗(包括放射、化学、靶向、肝移植等治疗手段),但是目前HCC的预后依然不佳。肝移植已经成为治疗HCC,尤其是小肝癌的根治性手段,但是其5年总体生存率仍然在47-61%之间。究其原因,和其他所有的治疗手段一样,肝移植也不可避免的存在着治疗后转移复发这个瓶颈问题,从而严重制约了肝癌的治疗效果。加之供体紧张的因素,国内外始终对肝癌肝移植的入选标准争论不一。为了让更多的肝癌患者能受惠于肝移植,移植学界围绕肝癌肝移植后肿瘤复发这一核心问题,始终致力于寻找能早期、有效预测移植后肝癌复发的标记物,从而为建立科学合理的肝癌肝移植受者准入标准提供更多的有效信息。目前大多数移植中心从肿瘤大小、数量、组织学分级、血管侵犯等临床组织学指标方面评价肝癌肝移植的预后及预测复发,特别是血管侵犯被认为是影响术后肝癌复发及预后的唯一独立危险因素,但对移植后肝癌复发的预测,尤其是术前的早期预测,仍然缺乏特异性的生物学标记。近年来,随着对癌转移认识的不断深入和人类基因组学的巨大发展,人们逐步意识到肝癌的发生发展是一个涉及到多基因改变、调控的复杂过程,基于恶性肿瘤基因改变、调控的相关研究成为热点。在基因改变的多种形式中,基因的单核苷酸多态性(single nucleotide polymorphism,SNP)无疑是最受研究者关注的一种基因改变形式。另一方面,作为新兴的表观遗传学和表观基因组学最重要的研究内容,关于CpG岛的甲基化所致抑癌基因转录失活,进而影响肿瘤发生、发展这一问题也成为研究热点。然而,通过文献回顾发现,有关SNP和DNA甲基化在肝移植后肝癌复发预测中的价值国内外均尚无报道。因此在本课题研究的两部分中,针对当前肝癌肝移植面临的生物学标记缺乏这一热点问题,分别取HCC肝移植病人的外周血和肿瘤组织标本检测多种肿瘤相关基因的SNP和DNA甲基化,探讨二者对肝移植后肝癌复发的预测价值,为进一步提高肝癌肝移植的早期复发预测能力,建立适合中国国情的肝癌肝移植受者准入标准奠定个体基因方面的实验基础。第一部分、基因的单核苷酸多态性(SNP)对肝移植后肝癌复发预测的价值研究。目的:探讨多种基因的SNPs在肝移植后肝癌复发预测中的价值。方法:选取MDR1(C1236T,G2677T/A,C3435T)、MMP-2(-1306 C/T)、MMP-9(-1562 C/T)、IL-1α(-889 C/T)、IL-1β(-31 T/C,-511 C/T)、IL-6(-572G/C)、IL-8(-251T/A)、IL-10(-1082A/G,-819T/C,-592A/C)、TNF-α(-308G/A)和TGF-β1(-509T/C,+869C/T)共10个基因上的16个SNP位点(上述位点已知可能与人类不同恶性肿瘤的发生、侵袭、转移乃至预后相关),应用多聚酶链反应-限制性片断长度多态性分析法(PCR-RFLP)和DNA测序方法对99例HCC肝移植病人的外周血DNA样本进行上述位点的SNP检测,分析SNP与临床病理学指标及移植术后的3年无复发生存的关系。结果:本研究将MDR1 2677位点为AA、AT、GA的患者统称为2677A携带型,TT、GT、GG的患者称为2677A非携带型。结果发现2677A携带型与术后不复发显著相关(P=0.019)。在2677A携带型与2677A非携带型两组之间,复发的比例分别为34.5%(10/29)和55.7%(39/70),两组的3年无复发生存率分别为46.2%和22.0%,前者的中位无复发生存时间显著长于后者(44.2月vs.10.5月,P=0.015)。关于MMP-2基因-1306位点,则发现CC基因型与术后肿瘤复发显著相关(P=0.029)。73例CC型受者中,有42例(57.5%)复发,而20例CT型受者中的复发数仅为6例(30%)。MMP-2-1306 CT杂合子与CC纯合子两组之间的3年无复发生存率分别为64.4%和35.5%,前者的平均无复发生存时间显著长于后者(30.4 vs.19.3月,P=0.019)。此外,尽管IL-10-1082 GA型的频率不高,但9例GA型受者中有8例复发(88.9%),而81例AA型受体中复发数为40(49.4%)。结果表明:GA基因型与术后肿瘤复发显著相关(P=0.024)。IL-10 -1082 AA纯合子与GA杂合子两组的3年无复发生存率分别为37.1%和0,前者的平均无复发生存时间显著长于后者(23.5 vs.5.7月,P=0.010)。本实验还发现:上述三个位点的SNPs与病人的其它临床病理学指标如性别、年龄、是否合并门静脉癌栓(PVTT)、术前AFP水平、组织学分级、肿瘤大小、肿瘤数目均无关(P＞0.05)。除了上述三个SNPs外,其它基因的SNPs与移植后肝癌复发均无关。Cox单因素分析结果显示:除了PVTT、术前AFP水平、组织学分级、肿瘤大小、肿瘤数目以外,MDR1 2677A携带型、MMP-2-1306CT型、IL-10-1082AA型均与无复发显著相关。多因素分析结果表明:除术前AFP水平和肿瘤大小外,MDR1 2677A非携带型(危险系数:2.447;95%可信区间:1.123-5.329;P=0.024)是影响复发的独立危险因素。结论:MDR1 2677A携带型、MMP-2-1306CT型、IL-10-1082AA型的HCC病人移植后复发风险较低,尤其MDR1 2677A携带型是影响复发的独立危险因素,对移植后肝癌复发的预测具有潜在的重要价值。第二部分、DNA甲基化对肝移植后肝癌复发预测的价值研究。目的:探讨抑癌基因XAF1的DNA甲基化及其蛋白表达在肝移植后肝癌复发预测中的潜在价值。方法:采用逆转录-聚合酶链反应(RT-PCR)检测反甲基化药物5-aza-dC作用于人肝癌SMMC-7721、HepG2、Hep3B细胞系前后XAF1基因转录水平的变化。免疫组化(IHC)检测HCC患者病肝石蜡标本中的XAF1蛋白表达水平。甲基化特异性PCR(MSP)检测上述细胞在5-aza-dC作用前后甲基化的变化情况及HCC患者肝癌组织中的甲基化频率。进而分析XAF1基因启动子的甲基化及其蛋白的表达与肝移植后肝癌复发之间的关系。同时,也对XAF1基因的表达与其启动子甲基化之间的关系进行了探讨。结果:体外实验发现XAF1基因在SMMC-7721细胞发生了完全甲基化,mRNA表达完全缺失,HepG2和Hep3B细胞发生了部分甲基化,mRNA表达极低。去甲基化药物5-aza-dC分别作用于上述三种细胞后均能上调其mRNA表达。表明DNA的甲基化与XAF1在肝脏肿瘤细胞中的表达缺失或低表达密切相关。MSP结果显示:65例HCC组织标本中XAF1的甲基化频率为66.1%(43/65)。IHC结果显示:与之匹配的49例HCC组织标本中,有30例XAF1蛋白表达降低,且其中的27例(90.0%)检测到甲基化,而XAF1表达未改变的19例病人中仅4例(21.1%)检测到甲基化,统计学分析发现XAF1启动子的甲基化与XAF1在HCC组织中蛋白的表达降低显著相关(P＜0.001)。分析了XAF1基因的甲基化及其蛋白表达与临床病理指标的关系后发现:XAF1的甲基化与术前血清中AFP高表达成正相关,即AFP＞400μg/L的病人的甲基化频率明显高于AFP≤400μg/L的病人(P=0.099)。而XAF1蛋白的低表达与门静脉癌栓(P=0.018)、AFP高表达(P=0.003)和肿瘤大小(P=0.017)有关。行MSP的65例HCC中,复发者中XAF1甲基化频率为71.1%(27/38),而无复发者中甲基化频率为59.3%(16/27),XAF1甲基化与术后肿瘤复发无关(P=0.426)。而行IHC的49例HCC中,复发者中XAF1蛋白表达未改变的病例占21.4%(6/28),而无复发者中蛋白表达未改变的病例则占61.9%(13/21),显著高于前者,表明XAF1蛋白的低表达与肝癌复发显著相关(P=0.007)。在19例XAF1表达未改变者中仅有6例(31.6%)复发,30例XAF1低表达者中则有22例(73.3%)复发。两者的3年无复发生存率分别为61.8%和18.5%,前者的平均无复发生存时间显著长于后者(27.9 vs.12.6月,P=0.001)。多因素分析结果显示XAF1在HCC组织中的低表达是影响肝移植术后肝癌复发的独立影响因素(危险系数:4.219;95%可信区间:1.689-10.526;P=0.002)。结论:DNA甲基化是影响XAF1基因表达的关键因素但不是唯一的因素;XAF1的蛋白表达而非XAF1基因启动子的甲基化是预测肝移植后肝癌复发的潜在的生物学标记物。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is the most common primary malignant tumor in liver,which ranks fifth in overall frequency,and around 372,000 new cases of HCC are diagnosed annually.China has one of the highest prevalent areas of HCC,mainly because of chronic hepatitis B carders accounting for more than 10%of its population.The prognosis of patients with HCC is generally poor,even treated with combined therapy based on surgery,including resection,radiotherapy,chemotherapy, targeted therapy and liver transplantation(LT).LT has been accepted as a potentially curative option for patients with small HCC.However,long-term survival(at 5 years) remains 47-61%due to the high incidence of recurrence.Similar to other therapy option,recurrence of HCC after transplantation remains one of the most prevalent causes of poor long-term survival.Given the growing organs shortage and the risk of aggressive recurrence,selection of candidates for LT is a crucial factor and still a debate.Efforts are focused on identifying tumor characteristics to improve predictive power of recurrence and to establish optimal recipient selection criteria that can define a subset of HCC patients standing to benefit from LT.Conventional prognostic markers for tumor recurrence are clinicopathological characteristics such as tumor size,tumor number,histopathologic grading,vascular invasion etc. Especially,vascular invasion is regarded as a sole independent risk factor for poor prognosis.However,there are still no specific biomarkers,especially pre-transplant molecular markers,to reliably predict recurrence after LT.With the improvement of recognition for tumor recurrence and metastasis,as well as great advancement on genomics,it is gradually accepted that carcinogenesis and progress of HCC is a course involving multiple genetic alteration and modulation. Recently,numerous studies have focused on the genetic alteration or modulation in a wide range of human cancers.As an important type of genetic alteration,single nucleotide polymorphisms(SNPs)of genes have been investigated in many diseases, such as cancer,inflammation and immunologic disease.On the other hand,CpG island hypermethylation at the promoter region of many tumor suppressor genes have been shown to be associated with regeneration,carcinogenesis,and tumor progression.However,no study has examined the predictive value of SNPs and DNA methylation in tumor recurrence of HCCs after transplantation.In this study, we investigated 16 SNPs of 10 genes and hypermethylation of XAF1 gene in association with the risk of HCC recurrence after LT,and determined whether some polymorphisms or hypermethylation of XAF1 could provide further discriminative genetic power over current recipient selective systems for HCC patients undergoing LT. PartⅠ.Predictive value of single nucleotide polymorphism(SNP)in tumor recurrence of patients with hepatocellular carcinoma after liver transplantationAim:To evaluate the association between SNPs of genes and the risk of HCC recurrence after LT.Methods:Genomic DNA of 99 HCC patients undergoing LT was extracted from peripheral blood lymphocytes.A total of 16 SNPs of 10 genes,including MDR1 (C1236T,G2677T/A,C3435T)、MMP-2(-1306 C/T)、MMP-9(-1562 C/T)、IL-1α(-889 C/T),IL-1β(-31 T/C,-511 C/T)、IL-6(-572G/C)、IL-8(-251T/A)、IL-10(-1082A/G,-819T/C,-592A/C)、TNF-α(-308G/A)and TGF-β1(-509T/C, +869C/T),were enrolled in this study.Genotyping was performed rising polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP).The association between polymorphisms and clinicalpathological parameters,as well as 3-years recurrence-free survival after LT was assessed.Results:To make our description laconic,we labeled patients carrying at least one variant A allele(AA,GA,or AT)as 2677A carrier group,and TT,GT,or GG as 2677A non-carrier one in this study.The association between recurrence-free and 2677A carrier was significant(P=0.019).In 2677A carrier group and 2677A non-carrier group,the patients with recurrence were 34.5%(10/29)and 55.7% (39/70),respectively.The 3-year recurrence-free survival rate was 46.2%in 2677A carrier group compared with 22%in 2677A non-carrier group.The median recurrence-free survival for 2677A carrier group was significantly longer than that for 2677A non-carrier group(44.2 vs.10.5 months,P=0.015).In addition,the association between MMP-2 -1306 C/T polymorphisms and recurrence was significant(P=0.029).Recurrence of HCC was identified in 42 of 73(57.5%) patients with CC homozygous,and 6 of 20(30%)patients with CT heterozygous. The cumulative 3-year recurrence-free survival in patients with CC homozygous was 35.5%,compared to that of 64.4%in patients with CT heterozygous.The mean recurrence-free survival for heterozygous CT patients was significantly longer than that for homozygous CC patients by Kaplan-Meier estimates(30.4 versus 19.3 months,P=0.019).In spite of the low frequency of IL-10-1082 GA genotype,we observed that GA genotype was related to the increased risk of tumor recurrence(P =0.024).In IL-10 -1082 GA genotype and AA genotype,the patients with recurrence were 88.9%(8/9)and 49.4%(40/81),respectively.The cumulative 3-year recurrence-free survival in patients with AA homozygous was 37.1%, compared to that of 0%in patients with GA heterozygous.The mean recurrence-free survival for heterozygous AA patients was significantly longer than that for homozygous GA patients(23.5versus 5.7months,P=0.010).In the relationship between above three polymorphisms and clinicopathological data,there were no statistical significance in the distribution of selected clinicopathological parameters as to age,gender,portal vein tumor thrombi(PVTT),preoperative alpha-fetoprotein (AFP)level,or histopathologic grading,tumor size and tumor number after LT between different genotypes(P＞0.05).Meanwhile,there were no significant association between other polymorphisms of genes and tumor recurrence after LT. Univariate analysis revealed that the clinicopathological parameters could provide significant predictive values for recurrence including PVTT,preoperative AFP level, histopathologic grading,tumor size,tumor number,as well as above three SNPs including MDR1 2677A carrier,MMP-2 C-1306T and IL-10 A-1082G.The multivariate analysis revealed that preoperative AFP level,tumor size,and MDR1 2677A carrier status were independent factors for predicting recurrence-free survival (HR:2.447;95%CI:1.123-5.329;P=0.024). Conclusions:Our results illustrated that the MDR1 2677A carrier,MMP-2 C-1306 T and IL-10 A-1082G polymorphisms were associated with the reduced risk of tumor recurrence after LT.Especially,MDR1 2677A carrier status may serve as a potential biomarker for tumor recurrence after LT.PartⅡ.Predictive value of DNA methylation in tumor recurrence of patients with hepatocellular carcinoma after liver transplantationAim:To investigate the methylation status and expression level of XAF1 in HCCs treated with LT,and to evaluate potential predictive value for tumor recurrence.Methods:The XAF1 mRNA expression in three liver cancer cell lines (SMMC-7721,HepG2 and Hep3B)was detected by RT-PCR before and after a DNA methyltransferase(DNMT)inhibitor,5-aza-2’-deoxycytidine(5-aza-dC) treatment.Meanwhile,methylation status of XAF1 in three cell lines and HCC tissue samples from 65 cases treated with LT was measured by methylation-specific polymerase chain reaction(MSP).Immunohistochemistry was also used to assess the XAF1 protein expression in matched 49 HCC tissue samples.We then analyzed the predictive value of expression and methylation of XAF1 in tumor recurrence of HCC after LT.Furthermore,the relationship between methylation status and expression patterns of XAF1 in HCC was analyzed.Results:RT-PCR showed that the mRNA level of XAF1 was very low in HepG2 and Hep3B,while undetectable in SMMC-7721.MSP indicated that XAF1 gene was completed or partially methylated.However,5-aza-dc reactivated XAF1 mRNA expression in SMMC-7721 and upregulated the mRNA level of XAF1 in HepG2 and Hep3B,suggesting that DNA methylation plays a causal role in the transcriptional regulation of XAF1 in HCC.In 65 HCC tissue samples,the methylation frequency of XAF1 was 43 of 65(66.1%).We proved the relationship between methylation status and expression patterns of XAF1 in HCC tissues.In the 30 cases of XAF1 low expression group,27(90.0%)cases displayed hypermethylation in the XAF1 promoter region,whereas 4 of 19 cases(21.1%)in the unchanged expression group (P＜0.001).In the relationship between methylation and the clinicopathological data, we found that XAF1 methylation was more frequent in HCC,with AFP＞400μg/L than those with AFP≤400μg/L(P=0.009).Concerning the correlation between XAF1 expression and the clinicopathological data,our results showed that XAF1 expression positively correlated with PVTT(P=0.018),preoperative AFP level(P= 0.003)and tumor size(P=0.017).The methylated rates of XAF1 in patients with recurrence and without recurrence were 71.1%(27/38)and 59.3%(16/27), respectively.And there were no statistically significant differences between the methylation status and the risk of postoperative recurrence(P=0.426).However,the unchanged expression rates of XAF1 in patients with recurrence of HCC and without recurrence of HCC was 21.4%(6/28)and 61.9%(13/21),respectively.The association between XAF1 unchanged expression and recurrence-free was significant(P=0.007).Recurrence of HCC was identified in 6 of 19(31.6%) patients with XAF1 unchanged expression,and 22 of 30(73.3%)patients with XAF1 low expression.The cumulative 3-year recurrence-free survival in patients with unchanged expression is 61.8%,compared to that of 18.5%in patients with low expression.The mean recurrence-free survival for XAF1 unchanged expression patients was significantly longer than that for XAF1 low expression patients(27.9 versus 12.6 months,P=0.001).Multivariate analysis revealed that XAF1 expression level might be a novel independent factor for predicting recurrence-free survival (HR:4.219;95%CI:1.689-10.526;P=0.002).Conclusions:Promoter hypermethylation is one critical,but not sole mechanism for the gene silencing of XAF1 in HCC.The protein level of XAF1 rather than methylation status of XAF1 gene may be a potential biomarker for tumor recurrence after LT.更多还原