【作者】 孙健；

【导师】 钦伦秀； 叶青海； 任宁； 贾户亮； 周海军；

【作者基本信息】 复旦大学 ， 外科学， 2009， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma,HCC)(以下简称肝癌)是诊治困难,预后极差的一种肿瘤,是全球第六位最常见的恶性肿瘤和第三位最主要的癌症死因。转移复发是影响肝癌术后生存的最大障碍。肝癌转移复发是多因素参与的多环节病理过程,是肝癌自身基因改变和宿主微环境相互作用的结果。迄今,尚无一个基因或基因组合能够用于临床预测肝癌预后。研究肝癌本身生物学特性和宿主免疫相关因素可能更利于理解肝癌转移复发的机制以及临床预测。本课题在我们既往研究的基础上选择与肝癌侵袭转移相关的骨桥蛋白(osteopontin,OPN)、CK19、MMP-2、uPA、APC,和宿主炎症免疫微环境有关的IFNG、CRP、CSF1、IL-2以及CD8等,研究其在早期肝癌中的变化,并观察其变化趋势对肝癌预后的影响,以期从一个新的角度预测肝癌转移复发。第一部分传统肝癌临床病理分期系统预测术后生存复发能力的评价目的比较肝癌常用分期系统AJCC/UICC TNM分期、中国分期、日本TNM分期、UNOS TNM分期、CLIP评分、JIS分期、BCLC分期预测肝癌切除术后生存和复发能力。方法选取2002年3月至2003年3月间515例行根治性切除术的肝细胞癌作为研究对象。用单因素和多因素回归方法分析各分期系统对总生存率和无瘤生存率的影响。结果515例肝癌患者的4年总生存率为58%,无瘤生存率为49%。单因素分析显示肿瘤数目、肿瘤大小、血管侵犯、Edmondson-Steiner分级、甲胎蛋白水平和AJCC/UICC TNM分期、CLIP分期、BCLC分期、LCSGJ分期、JIS分期、UNOS TNM分期、中国分期是影响肝癌切除术后生存的危险因子。而肿瘤数目、大小、血管侵犯、甲胎蛋白及上述各分期系统也显著影响肝癌术后的无瘤生存。多因素Cox回归分析显示Edmondson-Steiner分级、JIS分期和UNOS TNM分期是影响肝癌生存的独立预测因素,而多因素分析发现AJCC/UICC TNM分期和BCLC分期是肝癌复发的独立危险因素。小结Edmondson-Steiner分级、JIS分期和UNOS TNM分期是影响肝癌生存的独立预测因素。而AJCC TNM分期和BCLC分期是影响肝癌术后复发的独立危险因素。第二部分血浆骨桥蛋白和甲胎蛋白水平可预测早期肝细胞癌术后复发目的检测OPN在HBV阳性早期HCC病人外周血中的表达,分析OPN表达水平和术后复发及生存间的关系,评价其在早期肝癌中预测复发及预后的作用。方法用酶联免疫吸附法(ELISA)检测68例HBV阳性早期HCC外周血OPN的表达水平,分析血浆OPN表达水平和临床病理特征、术后复发和生存间的关系。结果68例早期HCC患者血浆OPN中位水平为82.51 ng/ml(63.15ng/ml-110.45 ng/ml)。直径＜5 cm的早期肝癌血浆OPN水平明显低于直径≥5 cm的肝癌(75.16 ng/ml vs 104.76 ng/ml,P=0.003)。以100 ng/ml作为阈值将病例分为2组:低OPN组(＜100 ng/ml,n=45)和高OPN组(≥100 ng/ml,n=23)。高OPN组的HCC术后复发率明显高于低OPN组(52.2%vs 24.4%,P=0.022)。而高AFP组复发率也明显高于低AFP组,分别为45.5%和12.5%(P=0.006)。低OPN组和高OPN组的总生存率分别为95.4%和76.7%(P=0.032,log-rank),无瘤生存率分别为72.5%和25.9%(P=0.037,log-rank)。低AFP组和高AFP组的总生存率分别为100%和82.8%(P=0.045,log-rank),无瘤生存率分别为87.1%和35.6%(P=0.010,log-rank)。Cox单因素和多因素分析表明血浆OPN和组织学分级是总生存率和无瘤生存率的独立危险因素。小结血浆OPN表达水平和血清AFP水平与早期肝癌的复发及预后相关,有望成为早期肝癌术后复发预测的有用指标。第三部分CD8+肿瘤浸润淋巴细胞预测早期肝细胞癌术后复发的研究目的评价CD8+淋巴细胞计数,CK19和CD34标记的微血管密度在早期肝癌中预测术后复发及生存的作用。方法用免疫组织化学染色法对141例早期肝癌进行分析。研究CD8+淋巴细胞计数、CK19和CD34标记的微血管密度和早期肝癌患者术后复发及生存的相关性。结果CK19和CD34标记的微血管密度与早期肝癌术后生存及复发无明显相关性。CD8+淋巴细胞在癌组织内和癌旁组织内均有分布,癌旁组织内CD8+淋巴细胞浸润和临床病理特征无相关性,癌组织内CD8+淋巴细胞数和肿瘤大谐矢合喙?r=-0.227)。以18个/200×视野为阈值,癌组织内CD8+淋巴细胞浸润和术后复发明显相关。癌旁组织CD8+淋巴细胞计数和术后生存及复发无关。癌组织内CD8+淋巴细胞数和患者无瘤生存率及总生存率均相关,癌组织内CD8淋巴细胞阳性患者的4年总生存率和无瘤生存率均为100%,阴性者分别为71%和74%。血清AFP水平和术后复发明显相关,而且CD8淋巴细胞阳性伴低AFP的患者无瘤生存率优于CD8淋巴细胞阴性伴高AFP者。用Cox回归进行多因素分析显示癌组织内CD8淋巴细胞阳性是无瘤生存率的独立危险因子。小结癌组织内CD8+淋巴细胞和术前血清AFP水平与早期肝癌术后复发相关,有可能成为预测早期肝癌术后复发的标记物。第四部分癌组织中APC和IFNG mRNA表达预测肝细胞癌预后的研究目的本研究的目的是评价和肝癌转移及炎症免疫相关的APC、OPN、uPA、MMP2、CRP、CSF1、IFNG和IL-2基因预测肝癌预后的能力。方法应用实时荧光定量RT-PCR方法检测112例HCC组织中APC、OPN、uPA、MMP2、CRP、CSF1、IFNG、IL-2的mRNA水平的表达,进而分析上述基因在肝癌组织内mRNA水平的表达和术后复发及生存的相关性。结果IL-2、CSF1、CRP、MMP2、uPA和OPN对早期肝癌生存及复发缺乏鉴别能力。在对IFNG和APC的表达与预后关系的分析显示:IFNG和APC的高表达组和低表达组之间总生存率有显著性差异,IFNG高表达组和低表达组间的2年总生存率分别为71.2%和34.4%,P=0.011;APC高表达组和低表达组间的2年总生存率分别为77.9%和31.8%,P=0.004。多因素Cox回归分析则显示APC表达水平是肝癌总生存率的独立预测因素。小结APC和IFNG在肝癌组织内表达和总生存率有关,APC mRNA水平的表达是肝癌总生存率的独立预后因素。结论1.AJCC TNM分期和BCLC分期可将肝癌根据生存率分类,并且是术后复发的独立危险因素,但是因为传统分期系统不能反映肝癌全部生物学特性,因而尚不能准确预测肝癌预后,尤其是早期肝癌。2.术前血浆OPN表达以及癌组织内CD8+淋巴细胞和APC mRNA表达可作为标记物将早期肝癌按不同预后进一步分类。3.早期肝癌中CD8+淋巴细胞浸润伴AFP表达下降的患者无瘤生存率明显改善,癌组织中APC表达增高伴IFNG表达增高的患者预后明显好于两者同时降低的患者,提示肝癌预后受到肝癌转移相关基因和炎症免疫相关基因共同的影响。潜在应用价值1.AJCC/UICC分期和BCLC分期是肝癌术后复发的独立危险因素,这两种分期系统对于肝癌复发预测和治疗决策有重要意义。2.术前血浆OPN表达以及肝癌组织CD8+淋巴细胞浸润和APC mRNA表达可将早期肝癌进一步分期,鉴别复发高危人群,提高治疗效果。3.肝癌预后同时受肝癌转移相关基因和宿主炎症免疫相关基因的影响,这一观点对于今后治疗模式的改进和预测模型的研究有重要的启示。创新点1.发现在早期肝癌中术前血浆OPN表达水平、肿瘤浸润性CD8+淋巴细胞和APC mRNA表达与肝癌预后相关,有可能成为预测预后的标记物,对早期肝癌进一步分期。2.发现早期肝癌预后受肝癌转移相关基因和宿主炎症免疫相关基因的影响,为今后早期肝癌治疗模式的改进和新的复发预测模型的研究提供了线索。更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is a common malignancy worldwide.Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients.Like other cancers,the progression and metastasis of HCC is a multifactorial and multistage pathogenesis.The metastasis of HCC also depends on multiple interactions of metastatic cells with favorable host microenviroment.Although many molecular markers have identified and shown potential prognostic values for HCC patients,so far,no any biomarker has been used to clinical routine practice. Investigating the tumor itself as well as the favorable host microenviroment maybe useful for understanding the mechanisms of HCC metastasis and prognosis prediction. In this study,based on the previous findings,we select the invsion-related molecules including osteopontin(OPN) and CK19,and inflammatory/immune-related cytokines such as IFNG and IL-2,and also tumor-infiltrated lymphocytes to validate their predictive value in early-stage HCC.Meanwhile,we study the correlation of their balance to HCC prognosis in order to understand metastasis of HCC in a new way.PartⅠEvaluation of the prognostic capabilities of traditional staging systems of hepatocellular carcinomaObjective The aim of this study was to compare the ability of different staging systems(American Joint Committee on Cancer/International Union Against Cancer [AJCC/UICC],China classification system[CS],Japanese TNM,United Network for Organ Sharing[UNOS],Cancer of the Liver Italian Program[CLIP],Japan Integrated Staging[JIS],and Barcelona Clinic Liver Cancer[BCLC]) to predict survival and recurrence after liver resection for hepatocellular carcinoma(HCC).Methods Five hundred and fifteen consecutive patients who underwent liver resection for HCC were identified between March 2002 and March 2003.End points were overall survival(OS) and recurrence-free survival(RFS).Sequential stage-wise discrimination of each system was evaluated using Cox regression. Results Four-year overall survival rates were 58%;recurrence-free survival rates were 49%.In the univariate analyses of various clinical variables in the cohort,tumor number,tumor size,vascular invasion,Edmondson-Steiner grade,alpha-fetoprotein (AFP),and several staging systems(TNM,CLIP,BCLC,LCSGJ,JIS,UNOS-TNM and CS) were revealed to be significant predictors for survival;tumor number,tumor size,vascular invasion,AFP and several staging systems(TNM,CLIP,BCLC, LCSGJ,JIS,UNOS-TNM,and CS) were significant predictors for tumor recurrence after surgery.The multivariate Cox regression model for survival showed that Edmondson-Steiner grade,JIS stage and UNOS-TNM were main prognostic factors in HCC.However,the multivariate Cox regression model for recurrence revealed that AJCC/UICC and BCLC were independent predictors of recurrence.Conclusions The Edmondson-Steiner grade,JIS and UNOS TNM staging system can provides the discrimination of survival in patients undergoing liver resection for HCC. The AJCC/UICC and BCLC were independent predictors of recurrence for HCC.PartⅡThe prognostic significance of preoperative plasma levels of osteopontin in patients with early-stage hepatocellular carcinomaObjective to evaluate the prognostic value of preoperative plasma osteopontin(OPN) levels in patients with HBV-positive early-stage hepatocellular carcinoma(HCC).Methods Plasma OPN levels were detected by ELISA in 68 patients with HBV-positive early-stage HCC.Results The median plasma OPN level of patients was 82.51 ng/ml(range 63.15-110.45 ng/ml).Plasma OPN levels in patients with tumor size≥5 cm in diameter were significantly higher than that of patients with tumor size＜5 cm in diameter(104.76 ng/ml versus 75.16 ng/ml,P=0.003).100 ng/ml as cutoff can divide the patients into two groups:the higher plasma OPN group and the lower plasma OPN group.The higher plasma OPN group recurrence rate was significantly higher than the lower plasma OPN group(52.2%versus 24.4%,P=0.022).Meanwhile 20 ng/ml as cutoff can divide the patients into two groups:the higher serum AFP group and the lower serum AFP group.The two groups recurrence rate was 45.5%and 12.5% respectively,P=0.006.A higher plasma OPN level was a leading independent prognostic factor for both overall survival(OS) and recurrence-free survival(RFS) in univariate and multivariate Cox models.Conclusions The preoperative plasma OPN level and serum AFP level in patients with HBV-positive early stage hepatocellular carcinoma can be used as a predictive marker for early stage HCC recurrence and prognosis.PartⅢThe prognostic significance of CD8+ lymphocytic infiltration in patients with early-stage hepatocellular carcinomaObjective To evaluate the prognostic value of CD8+ lymphocytic infiltration、CK19 and MVD in patients with HBV-positive early-stage hepatocellular carcinoma(HCC).Methods The clinical data of 141 patients with HBV-positive early-stage hepatocellular carcinoma were collected.Immunohistochemistry was used to detect the CD8+ lymphocytic infiltration、CK19 and MVD in intratumoral and peritumoral tissues.Results CK19 and CD34 labelled MVD failed to show correlation with prognosis. CD8+ lymphocytes in peritumoral tissues failed to show correlation with the clinicopathological factors but CD8+ lymphocytes in intratumoral tissues showed adverse correlation with size of tumor(r=-0.227).Eighteen per 200×field was defined as the cutoff,CD8+ lymphocytes in intratumoral areas other than in peritumoral areas was significantly associated with HCC recurrence rates.CD8+ lymphocytes in intratumoral areas was significantly associated with 4-year overall survival(OS) and relapse-free survival(RFS).The OS and RFS of patients with CD8+ lymphocytic infiltration both were 100%,that without CD8+ lymphocytic infiltration were 71 and 74%respectively.300 ng/ml was defined as the cutoff,the level of serum alpha fetoprotein(AFP) was significantly associated HCC recurrence rates.The RFS of patients with CD8+ lymphocytic infiltration and negative AFP was superior to patients without CD8+ lymphocytic infiltration and positive AFP.In multivariate Cox models,CD8+ lymphocytic infiltration in intratumoral areas was independent prognosis factor for RFS.Conclusions CD8+ lymphocytic infiltration in intratumoral areas and serum AFP level in patients with early stage of HCC can be used as a predictive marker for early stage of HCC. PartⅣThe Prognostic Significance of APC、IFNG mRNA Levels in Patients With Early- Stage Hepatocellular CarcinomaObjective The aim of this study was to evaluate clinical significance of several biomarkers associated with metastasis and microenvironment in patients with hepatocellular carcinoma who underwent liver resection.Methods One hundred and twelve patients with HCC were evaluated for the expression of adenomatous polyposis coli(APC),osteopontin(OPN),urokinase-type plasminogen activator(uPA),matrix metalloproteinases(MMP2),C-reactive protein (CRP),macrophage colony stimulating factors(CSF1),γ-interferon(IFNG), interleukin-2(IL-2) by real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR).The association of intratumoral expression of these genes of patients with tumor recurrence and survival was analyzed.Results The clinical outcome did not significantly differ in high expressive group and low expressive group of MMP2,IL-2,CSF1,CRP,uPA and OPN.Patients with high levels of IFN-gamma and APC expression had significantly longer overall survival. The 2-year overall survival was 34.4%and 71.2%for patients with low and high IFN-gamma expression,respectively(P=0.011),and the 2-year overall survival was 31.8%and 77.9%for patients with low and high APC expression,respectively(P =0.004).Application of multivariate Cox regression analysis showed APC expression to be an independent prognostic factor for overall survival.Conclusions Elevated APC and IFN-gamma expression correlates with improved clinical outcome in patients with hepatocellular carcinoma.Conclusions1.AJCC/UICCTNM and BCLC staging systems can divide HCC into subsets that behave differently from each other,are independent risk factors for recurrence of HCC.Traditional staging system can only predict approximate outcome as they merely reflect the likely biological behavior of the HCC based on clinicopathological factors.2.Preoperative plasma osteopontin(OPN) level,intratumoral CD8+ lymphocytes and APC mRNA level can act as biomarkers to subdivide early-stage HCC into subgroups that have different outcomes. 3.The patients with CD8+ lymphocytic infiltration obviously in intratumoral areas and lower serum AFP level have better recurrence-free survival.Similarly,patients with high level of APC and high level of IFNG in tumor have better overall survival.The potential application of this work1.AJCC TNM and BCLC staging system are independent risk factors for recurrence of HCC.And they are important for prognosis prediction and therapy decision-making.2.Preoperative plasma OPN levels,intratumoral CD8+ lymphocytes and APC mRNA levels can act as biomarkers to subdivide early-stage HCC into subgroups that may improve the selection of patients for adjuvant therapy.3.Prognosis of patients with HCC depends on characteristics of tumor cells and host microenvironment.This viewpoint has important value for improvement of new treatment strategies and investigation of prognosis prediction for HCC.Originalities of this work1.This is the first report investigating the association between the preoperative plasma osteopontin(OPN) levels,intratumoral CD8+ lymphocytes and APC mRNA levels and early-stage HCC.2.For the first time,this study suggested that prognosis of patients with early-stage HCC depends on characteristics of tumor cells and host microenvironment.It should offer additional clues for treatment of early-stage HCC and prognosis prediction..更多还原