【作者】 史炯；

【导师】 钦伦秀；

【作者基本信息】 复旦大学 ， 疾病蛋白质组学， 2009， 博士

【摘要】 原发性肝细胞癌在全球癌症病死率中已上升至第3位,在国内居第2位;我国肝癌病人占全球每年因肝癌死亡人数的53%。虽然肝癌研究与治疗已取得很大进展,根治切除术后的十年生存率已提高到30%左右,但即使是小肝癌根治性切除,5年内仍有60-70%的病人出现转移复发,因此探索肝癌转移复发的机制,寻找有效的抑制途径,探索肝癌术后预测因子,准确区分患者,特别是发现早期肝癌的预后因子已成为进一步提高肝癌生存率的关键。基于传统的肿瘤预测因子只能粗略区分不同分期的HCC,对于外科临床实践中常见的早期HCC预测效果较差,本研究拟在原有工作基础上,分析癌周免疫细胞因子表达构成、优化肝癌术后预后预测指标,从基因和蛋白水平进一步验证和优化癌周免疫相关因子的预测标签、探讨其对预后的影响,为向临床应用转化奠定基础。第一部分基于癌周免疫炎症因子的肝癌转移预测标签的验证与优化及其对早期肝癌预后预测价值的评价:基因表达水平研究目的:早期肝癌患者预后预测仍然困难,我所前期发现癌旁组织17个免疫炎症相关细胞因子表达谱可以较准确预测肝癌术后早期复发。本研究拟进一步在早期肝癌患者中验证及优化此标签,检验其对早期肝癌术后预后预测的效果。方法:微流体低密度表达芯片技术(384孔qRT-PCR array)检测HCC癌旁组织的20个免疫细胞因子(包括17个预测标签因子和文献报告的3个复发转移相关因子)mRNA表达水平,应用生物信息学的机器学习方法构建有效的细胞因子预测模型,分析其中BCLC极早期和早期病人细胞因子表达水平与肝癌复发及预后的关系,检验其预测效果。结果:121例患者中复发和无复发组临床病理特征无显著差别,Cox多因素分析仅有肿瘤大小(P=0.03)对术后复发有显著影响。Cox风险比例回归模型分析20个细胞因子分别对复发的影响,影响大小排序后IL-10(P=0.004),IL-15(P=0.025),IL-2(P=0.013)为前三位,即这三个细胞因子表达情况对术后复发影响较大。IL-2、IL-15等在无复发患者中表达明显升高,患者平均生存期51.2个月,有镜下癌栓组IL-2 mRNA表达量平均值低于无镜下癌栓组(P=0.001)。基于20个细胞因子mRNA表达谱的预测标签在多因素分析中独立于其他临床病理指标,对复发影响风险比OR=5.2(P=2.71E-6);对生存多因素分析风险比为OR=2.5(P=0.012)。进一步筛选发现基于C4.5的决定树模型发现包含IL-2单细胞因子的预测模型可以较准确地预测该组病人复发情况(P=4.28E-5,AUC=0.81,准确率76%),IL-2预测效能高于单包含IL-15的模型。Cox风险回归分析IL-2 mRNA表达水平是肝癌复发的独立影响因子,并且可作为肝癌术后总体生存期预后指标。IL-15对于肝癌术后远期复发具有预测作用,IL-2与IL-15表达显著相关,但是IL-2预测效能高于IL-15。108例病人应用生物信息学方法分为训练组和测试组。ROC曲线确定IL-2或IL-15细胞因子表达临界值,训练组根据阈值分为高风险组和低风险组,Kaplan-Meier分析显示高风险组无瘤生存期显著短于低风险组(P=0.0003),高风险组总生存期也短于低风险组(P=0.016)。测试组分为高复发风险组(21例)和低复发风险组(15例)。高复发风险组无瘤生存期显著低于低分显著(P=0.002),总生存期在两组之间无显著差别(P=0.456)。单独应用IL-2和IL-15细胞因子mRNA作为预测因子的复发预测效能通过ROC曲线评价,曲线下面积AUC=0.73;准确率=70%(P=0.020),与训练组准确性相当。ROC曲线分析IL-2作为独立预测因子准确率可达80%。结论:20个细胞因子的预测模型预测早期肝癌术后复发的准确率70%,优化后单独IL-2和IL-15细胞因子构建早期肝癌术后预测模型,IL-2mRNA水平预测术后复发率准确率可达80%,癌旁IL-2、IL-15基因表达可以作为TNMI期肝癌术后预后的独立预测因子。第二部分基于癌周免疫炎症因子的肝癌转移预测标签的验证与优化及其对早期肝癌预后预测价值的评价:蛋白水平研究目的:前一部分研究从mRNA水平验证了已建立的包含有17个免疫细胞因子肝癌转移预测标签可以准确预测早期肝癌的术后复发,并筛选出IL-2和IL-15基因水平可作为独立预后因子。本研究中,我们从蛋白水平进一步在另一组具有更长随访期的早期病人中验证及优化癌周免疫因子蛋白表达对早期肝癌术后复发的预测作用。方法:选取187例早期肝癌切除病人癌旁肝组织,采用优化后裂解液总蛋白抽提法,提取癌旁组织总蛋白,ELISA法检测经筛选的第一部分20个免疫炎症细胞因子蛋白表达水平。ROC曲线法分析细胞因子对预后的预测作用,确定细胞因子蛋白水平阈值,根据阈值将病人分为高、低表达组。单因素和多因素Cox回归分析蛋白表达谱与肝癌术后存活、复发的关系。应用生物信息学方法,进一步优化蛋白预测因子。结果:187例患者临床病理特征单因素分析仅有HBsAg(OR=2.3,95%CI:1.1-4.5)对术后复发有显著影响,多因素分析均无显著意义。Cox风险比例回归模型分析20个细胞因子蛋白水平对复发的影响,按影响大小排序后IL-2(P=0.013)、IL-15(P=0.025),为前两位。在蛋白水平,IL-2(OR=2.9,95%CI:1.7-5.2;P=2.68E-5)和IL-15(OR=1.9,95%CI:1.2-3.2;P=0.005)与肿瘤复发密切相关。生物信息学分析,训练组IL-2(P=0.002)和IL-15对术后无瘤生存期影响显著,高IL-2和(或)IL-15表达水平的HCC病人术后复发率较低,无瘤生存期显著长于低IL-2和IL-15表达组,其中显著性分别为IL-2(P=1.02E-5) IL-15(P=0.004)。癌旁肝组织的IL-2和IL-15蛋白表达相关性较强(P＜0.001)。结论:IL-2和IL-15蛋白表达是早期肝癌病人术后复发的可靠标签,可以区分不同预后的病人,预测肿瘤术后复发风险。第三部分肝癌癌周组织浸润淋巴细胞表型及表面标志物表达目的:癌旁组织免疫炎症相关细胞因子与肿瘤进展及预后关系密切,可以作为预后预测因子,但是癌旁免疫微环境中免疫细胞的状态变化,及其对细胞因子的影响还不清楚,本部分初步分析肝癌患者组织浸润淋巴细胞组织分布与表型及外周血淋巴细胞表型变化与预后的关系。方法:免疫组织化学染色分析肝癌组织及癌旁组织浸润淋巴细胞表型,检测T细胞或B细胞表面标志物:CD3、CD8、CD4、CD20、CD19、Foxp3,分析浸润淋巴细胞表型及数目和肿瘤临床特征及预后的关系;检测癌旁组织石蜡切片HLA-Ⅰ、HLA-Ⅱ、CD80、CD86表达情况。流式细胞仪检测肝癌病人外周血T细胞CD3、CD8、CD4分析其比例变化。结果:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织,癌周淋巴细胞与患者肝炎病史及肝硬化相关,主要分布于间质、汇管区。浸润细胞以CD3+T细胞为主,其中以CD8+T细胞为主;CD4染色在多数病例为阴性,Foxp3仅在个别病例13.8%(15/109)存在。肿瘤浸润淋巴细胞B细胞表面标志CD20、CD19均为阴性。统计分析发现,肿瘤组织内CD8+T细胞浸润数量与预后正相关,而癌周浸润淋巴细胞数目与患者转移及复发无显著关系。癌旁组织HLA-Ⅰ、HLA-Ⅱ、CD80、CD86表达均与肝癌复发及预后无明显关系,但CD86高表达患者有预后好的趋势;HLA-Ⅱ低表达患者预后略差。结论:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织(P＜0.01),癌周淋巴细胞与患者肝炎病史及肝硬化相关,肿瘤及癌周浸润细胞以CD3+CD8+T细胞为主;CD4染色在多数病例为阴性,Foxp3仅在个别病例存在。肿瘤组织内CD8+T细胞浸润数量与预后相关,而癌周浸润淋巴细胞表面标志物表达增加,可能影响肝癌术后预后。全文总结1.肝细胞癌癌旁组织炎症/免疫微环境细胞因子蛋白和基因表达有其特异性,相关细胞因子与早期肝癌术后预后有关。2.细胞因子IL-2或IL15与肿瘤血管侵袭负相关,IL-2和IL-15癌旁组织的蛋白和基因水平均可以预测早期肝癌术后复发,IL-2蛋白水平对预后复发预测准确性可达76%,具有潜在的临床应用价值。3.癌旁组织CD8+T细胞功能性缺失可能造成微环境细胞因子分泌改变,为今后癌周免疫微环境细胞因子来源提供线索。更多还原

【Abstract】 Hepatocellular carcinoma （HCC） is the fifth most common cancer worldwide and also the third most common cause of cancer-related death, in China, mortality rate had rised second. It has an extremely poor prognosis, attributed to the high frequency of intrahepatic metastatic recurrence. To date, the only effective approaches for patients with HCC are resection or liver transplantation. But even to small HCC, the recurrence rate still was up to 60-70% in 5 years. It is most important to find effective mechanism of metastasis and recurrence aimed to improving the prognosis of HCC. Base on traditional staging system, the patients were hard to predict who had samillar clinical character. How to identify stratify subgroups with different outcome still were great difficulties.Immunological/ inflammatory mechanisms are important in the surveillance of malignancy and control of tumor progression. In this study, we focused on HCC noncancerous tissue immune microenvironment to further validate and refine a gene or protein signature of cytokine which can predict prognosis of HCC after curative treatment.Part APurpose: Hepatocellular carcinoma （HCC） is a common malignancy, but the prognosis remains poor due to metastasis and recurrence after curative hepatectomy. A 17-cytokine gene expression signature in non-cancerous hepatic tissue from patients with active metastatic HCC was recently found to predict HCC venous metastases. We examined whether a more refined cytokine gene expression profile of noncancerous liver tissue could predict survival of early stage HCC after curative hepatectomy.Methods: We analyzed a 20-cytokine gene expression profile in noncancerous liver tissue from 121 Chinese patients with TNM stage I～II of HCC using TaqMan^@ low density arrays. We then used supervised hierarchical clustering and log-rank test to analyze the prognostic ability of the cytokine gene profile. Using methods of bioinformatics, build a predictical model, then refined it to explore optimal factor.Results: Clinicopathologic characteristics Cox analysed hadn’t find signifinant effection on recurrence except tumor size （HR = 1.793, 95%CI: 1.056-3.045 ;P = 0.03）. Cox analyzed effection on recurrence IL-10, IL-2, IL-15 were ranked in the first three sites. The expression of other Th2 cytokines, including IL-4, IL-6 and IL-8, were decreased in most samples tested compared to normal pool tissuse, but no remarkable relativity was observed with the survival and HCC recurrence. The mRNA-based predictor with logistic regression method, which employed all 20 cytokine gene expression, was excellent recurrence predictor in gene dataset(P= 4.28E-5, the area under the curve=0. 81, accuracy=0.76 on tranining dataset. After refined the model, the expression levels of IL-2 （P = 0. 001） and IL-15 （P=0. 0002） correlated inversely and significantly with intrahepatic microvascular invasion. In the univariate analysis, only the mRNA levels of IL-15 and IL-2, preferable than the traditional clinical characteristics, were significantly associated with HCC recurrence. In the multivariate analysis, the mRNA levels of both IL-2 （HR=2. 0; P = 0.015） and IL-15 （HR=2.0; P = 0.011） were independent predictors of HCC recurrence. Higher mRNA levels of IL-2 （P = 0. 016） or IL-15 （P = 0. 001） were associated with a significantly better relapse-free survival of HCC patients compared to those with lower levels of these cytokines. IL-2 and IL-15 had significant correlation in the noncancerous liver tissues using the Spearman test (Spearman’sρ= . 487; P = 1.88×10^-7).Conclusions: IL-2 or IL-15 of immunity-associated genes signature should allow a sensitive and specific monitoring of survival in HCC.Part BPurpose: By analyzing the gene expression profiles of noncancerous hepatic tissue from patients with hepatocellular carcinoma （HCC）, we recently identified a 17-cytokine signature that can predict HCC metastasis. In this study, we further validated and refined this signature in protein level for tumor recurrence over a long follow-up period in a much larger and specific cohort of patients with early HCC.Methods: The protein expression levels of 20 cytokines in the noncancerous hepatic tissues from 187 patients with early stage of HCC were determined using ELISA assays. Univariate and multivariable Cox proportional hazards model and Kaplan-Meier method were used for analyzing the association of their expression profiles with tumor recurrence and survival of patients with HCC. To determinate the threshold values by ROC analyze.Results: Ranked 20 cytokines by a univariate Cox model for recurrence （Table 1）. Of all 20 proteins, IL-2 （P = 0.013）, IL-15 （P = 0.049） were the top two significant cytokines for recurrent classification. The protein expression levels of IL-2 (P = 2. 68×10^-5) and IL-15 （P = 0. 005） correlated strongly with tumor recurrence, and these correlations had been validated in mRNA levels. The patients with higher level （s） of IL-2 and/or IL-15 had a significantly better relapse-free survival compared with those with lower level （s） of IL-2 (P = 1.02×10^-5) and/or IL-15 （P = 0.004）. The expression levels of IL-2 and IL-15 correlated with each other. Furthermore, the expression levels of IL-2 and IL-15 correlated inversely and significantly with intrahepatic microvascular invasion.Conclusion: The IL-2 and IL-15 are useful markers for stratifying the patients with early stage of HCC into subgroups with different probabilities of tumor recurrence, and predicting the patients’ prognosis after surgical treatment.Part CPurpose: By analyzing the CD molecular expression profiles ofnoncancerous hepatic tissue from patients with hepatocellular carcinoma,we want to identify signature of tumor infiltrating lymphocytes whichmaybe affect the cytokines expression between different outcome HCCpatients. In this study, we identified TIL subtype by some lymphocyticdifferentiation antigen and lymphocyte surface mark.Methods: The lymphocyte surface marks of tumor infiltrating lymphocytesin the noncancerous hepatic tissues from 146 HCC patients were determinedusing IHC. Univariate and multivariable Cox proportional hazards model and Kaplan-Meier method were used for analyzing the association of their expression levels with tumor recurrence and survival of patients with HCC. Results: The phenotype and cell numbers of tumor-infiltrating lymphocytes （TILs） in noncancerous hepatic tissues of HCC were analyzed via immunohistochemistry of sections from patients undergoing surgery for HCC and via flow cytometry of peripheral blood mononuclear cells. Tumor-infiltrating T-cell function and activation status were assessed via CD80, CD86. More than 86. 4% patients of TILs were quiescent as measured via CD4+ or Foxp3 expression, while more than 90% of CD3（+） T cells expressed CD8+. The proportion of Th cell was quite low compared with the ratio of CD8+ T cell. The proportion in distant nontumor tissue CD19 and CD20 was zero. The proportion T cells subgroup isolated from HCC circulating whole blood didn’t show significant shift comepared with normal control, CD4+ T:CD8+ T=1.167±1.04、CD+8 T:CD3+T=0. 288±0.116; CD4+T: CD3+T0.429±0.178. The proportion of CD8+ cells in noncancerous hepatic tissues was higher than in blood （P = 0.000）. The expression of HLA-I、HLA-II、CD80、CD86 in noncancerous tissues didn’t show significant different between different prognostic group, only showed some tendency for outcome. There had no definite proof about the increasing IL-2 or IL-15 only form activation of Th1, maybe they can be secreted by hepatic cell. Conclusion: TILs in HCC noncancerous hepatic tissues are increased and contain a subpopulation of CD3+CD8+ T cells. Activation functional deletion of tumor-infiltrating T cells could damage tumor-specific immunotherapy.更多还原