【作者】 黄华；

【导师】 钦伦秀；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 肝细胞癌(Hepatocellular carcinoma, HCC)是世界上最常见、恶性程度最高的肿瘤之一,其病死率在全球所有癌症中占第3位。手术切除仍是目前肝癌获得长期生存的主要手段,但术后5年复发率高达60%以上(小肝癌也达40%-50%)。转移复发已成为进一步延长肝癌病人生存期、提高远期疗效的瓶颈问题,也是最终攻克肝癌的关键。在肝癌术后病人中区分高、低复发风险人群有相当的困难,特别是那些没有明显血管侵犯、局部淋巴结转移或远处转移的早期病人。鉴定相关的分子标签可以为肝癌患者预测临床结果提供额外的有益信息,并为术后甄别需要接受辅助治疗的病人提供帮助。以往大量研究指出,Thl和Th2类细胞因子在多种人类恶性肿瘤的进展中起重要作用。此前我们与美国国立卫生研究院(NIH)的合作研究构建了包含17个免疫细胞因子的预测模型预测肿瘤复发。本课题组在此基础上采用微流体低密度表达芯片技术(qRT-PCR),应用生物信息学机器学习方法对以上预测模型优化后发现癌旁IL-2、IL-15基因表达可以作为早期肝癌术后预后的独立预测因子。以上是基于基因转录水平的研究,然而Th1/Th2类细胞因子蛋白是否可以作为肝癌的临床预测指标尚不十分肯定。本课题组前期基于较小样本量的初步研究发现在蛋白水平IL-2和IL-15也与肝癌的复发相关。基于其他恶性肿瘤的研究也提示Th1型细胞因子可以减少肿瘤的转移复发,而Th2细胞因子表达增高则可增加术后复发率。特别是IL-10在许多恶性肿瘤中被认为是预后不良的分子标签。基于肿瘤微环境的研究成果令人振奋,但是肝癌的转移复发除与微环境免疫状态密切相关外,更与肝癌细胞自身的转移特性密切相关。我们的前期研究发现调控肝癌转移潜能的基因改变在其原发瘤阶段即已存在,此为转移理论的重要进展。研究还发现骨桥蛋白Osteopontin, OPN)表达升高与肝癌转移密切相关。骨桥蛋白是一种分泌型的非胶原化富含唾液酸的磷酸化糖蛋白,OPN正常情况下参与体内许多生理过程。许多文献报道OPN表达增高与恶性肿瘤演进、侵袭转移密切相关。为此,本研究中我们在前期研究的基础上首先通过ELISA方法在一组包含192例样本的队列中检测12种Th1/Th2相关细胞因子,发现IL-2,IL-15和IFN-γ与肝癌复发相关,IL-2是最佳的预测预后的因子,并进一步扩大样本量通过高通量的组织芯片技术加以验证。同时利用组织芯片检测肝癌组织中OPN的水平,分析其与免疫相关因子联合应用在预测肝癌预后中的作用。本研究拟从蛋白水平检测癌旁非癌肝组织中免疫相关因子的表达,研究其与肝癌复发的关系。我们利用酶联免疫吸附反应(enzyme-linked immunosorbent assays ELISAs)定量检测192例(cohort A训练组n=128,测试组n=64)肝癌患者癌旁肝组织中12个Th1/Th2相关细胞因子。同时用组织芯片免疫组化技术在包含261例患者的独立阵列中(cohort B)进行验证,评价癌旁微环境中免疫相关细胞因子的表达,在组织中的分布情况及其与术后肿瘤复发之间的关系。结果发现,无复发组肝癌患者中,癌旁肝组织中IL-2,IL-15和IFN-γ蛋白表达显著高于复发组。Cox逐步回归分析发现,仅有IL-2是效果最佳的独立预测因子(HR for recurrence=0.4, P=0.001; HR for death=0.5, P=0.019)。这一结果在队列A的测试组及队列B(HR for recurrence=0.4; P<0.0001; HR for survival=0.6; P=0.005)中都得到了验证。本研究中我们还发现,在癌旁肝组织中IL-2主要存在于肝细胞内,而非淋巴细胞。癌旁肝组织中的IL-2,IL-15和IFN-γ蛋白水平与肝癌的复发有关,但IL-2是预测复发及总体生存的最佳的预测因子,可以基于癌旁肝组织中的IL-2水平将肝癌患者分为不同预后的亚群。本研究的另一结论是在癌旁肝组织中IL-2主要存在于肝细胞中,而非淋巴细胞中。本研究拟从转录水平及蛋白水平研究外周血单个核细胞及血清中Th1/Th2平衡状况,探索其对肝癌预后的预测作用。我们首先收集肝癌患者、乙型肝炎肝硬化无肝癌患者及健康对照组的血清,通过先进的流式微球捕获技术(Cytometric Bead Array, CBA)检测血清Th1/Th2类细胞因子。同时收集与之配对的外周血单个核细胞(PBMC),行qRT-PCR检测,了解机体全身(血清细胞因子蛋白水平及外周血单个核细胞基因转录水平)免疫状态改变及其与肝癌复发及预后的关系。我们的研究发现肝癌患者、乙型肝炎肝硬化无肝癌组及正常人,血清IL-2(P=0.6907),IFN-γ(P=0.459)、IL-4(P=0.2044)和IL-10(P=0.0837)表达无显著差异。外周单个核细胞(PBMC),行qRT-PCR检测IL-2(P=0.2959),IFN-γ(P=0.8225)、IL-4(P=0.2968)和IL-10(P=0.3239)mRNA表达组间亦无明显差异。本研究中检测的4种细胞因子其表达水平与肝癌患者的总生存时间及至复发时间无显著相关性。本研究尚未在血清或外周单个核细胞(PBMC)中发现可用于预测肝癌预后的免疫炎症相关因子。我们前面的研究发现IL-2是预测复发及总体生存的最佳的预测因子。Osteopontin (OPN)在恶性肿瘤的发生发展中起重要作用,与多种恶性肿瘤的预后关系密切。有研究认为OPN可以起类似促炎因子作用可增强Th1类细胞因子的表达,我们通过western blot及细胞免疫荧光染色,分析不同转移潜能肝癌细胞株OPN表达情况,流式微球捕获技术分析细胞培养上清中的IL-2,IFNγ,IL-4和IL-10的表达量,了解OPN是否与Th1或Th2类细胞因子的表达存在关联。并检测肝癌癌旁正常肝组织中IL-2以及癌组织中OPN的表达,研究两者的相关性及其与肝癌复发的关系。我们采用配对标本组织芯片免疫组化技术在含有261例患者的队列(cohort B)进行研究,评价癌旁正常肝组织IL-2以及癌组织中OPN的表达,在组织中的分布情况以及两者的相关性及其与术后肿瘤复发及肿瘤侵袭能力指标之间的关系。分析OPN联合IL-2在肝癌复发中的预测作用。结果,未发现OPN和炎症相关细胞因子表达有明显相关性,癌旁IL-2和癌内OPN是互不干扰的相对独立的预测肝癌预后的指标。癌旁IL-2与肝癌术后复发关系密切,IL-2高表达有较好的总体生存率及更长的至复发时间(TTR：P<0.0001,0S：P=0.004)。癌组织内OPN高表达的肝癌患者预后显著差于OPN低表达的患者(TTR：P=0.0007,0S：P=0.0214)。OPN联合IL-2表达可以充当肝癌预后的有效的预测因子(TTR：P<0.0001,0S：P=0.0039)。其预测效能优于两个指标单一使用时的预测效能,亦优于传统的临床病理指标。1.癌旁微环境炎症免疫相关因子与肝癌术后复发及总体生存密切相关。癌旁肝组织中的IL-2表达水平与肝癌的复发预后有关,IL-2是预测复发及总体生存的最佳的预测因子。2.在癌旁肝组织中IL-2主要存在于肝细胞中,而癌旁T淋巴细胞中IL-2很少存在。3.本研究尚未在血清或外周单个核细胞(PBMC)中发现免疫炎症相关因子可用于预测肝癌的预后。但不能就此认为肝癌的预后与机体全身免疫状态无关。4.癌旁IL-2和癌内OPN表达水平是预测肝癌术后生存及复发的独立的预测因子,癌旁肝组织中IL-2联合癌内OPN可提高肝癌术后复发的预测效能。5.肝癌的转移复发是一个复杂的过程。无论“土壤(微环境)”还是“种子(癌细胞本身)”在肝癌的复发中都起着重要的作用,预防肝癌术后复发要从多靶点着手,多途径干预,既针对“土壤”又针对“种子”才有可能取得理想的效果。1.首先报道癌旁肝组织中的IL-2和IL-15蛋白表达水平与肝癌的复发预后有关,IL-2是预测复发及总体生存的最佳的预测因子。2.我们首次发现在癌旁肝组织中IL-2主要存在于肝细胞中,而非淋巴细胞中。并用连续切片定位染色方法加以验证。3.本研究同时从癌旁微环境和肿瘤细胞本身着手,证实癌旁IL-2和癌内OPN水平是肝癌预后的独立预测指标,既针对“土壤(微环境)”又针对“种子(肿瘤细胞本身)”,为早期肝癌的复发寻找分子标签。1.癌旁IL-2和癌内OPN水平能够提供预测肝癌病人生存和复发的有益信息,尤其针对早期病人效果更显著。这一发现有助于我们界定和选择高复发风险人群采取个体化措施预防复发。2.本研究中的方法实施方便,便于在临床应用,一旦成熟临床极易推广。“基于炎症因子的肝癌患者术后转移复发多分子预测试剂盒”已经申请国家专利,申请号：200810208150.6。3.调控癌旁微环境,调变肿瘤细胞侵袭特性,多方出击方能战胜肿瘤。本研究为临床上控制肿瘤复发,提高肝癌的远期疗效提供新的思路。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. It has an extremely poor prognosis, mainly attributed to the high frequency of intrahepatic metastatic recurrence. It is a challenge to identify patients who are at a greater risk for tumor recurrence after curative treatment for HCC, particularly in those with early stage disease who do not have significant vascular invasion, or regional lymph node or distant metastasis. Identification of molecular markers could provide supplemental and useful information for predicting clinical outcome in patients with a given stage of disease and improve the selection of patients for adjuvant therapies after resection. Studies of many kinds of human malignancies have shown that Th1-and Th2-like cytokines may play a prominent role in tumor progression and metastasis.In a recent study of gene expression profiling, we found that the liver tissues in patients with metastatic HCC had lower levels of pro-inflammatory Thl-like cytokines and remarkably higher levels of anti-inflammatory Th2-like cytokines in comparison to those without metastatic HCC. However, whether the protein expression levels of these Thl/Th2 cytokines can be used to predict the clinical outcome of HCC is not known. In our previous research, we found that IL-2 or IL-15 of immunity-associated genes signature should allow a sensitive and specific monitoring of survival in HCC. The accumulated evidence has clearly demonstrated that a dominant Th2-like cytokine profile and a decrease in Thl-like cytokines are associated with the metastatic phenotype. High IL-10 levels had a worse postresection outcome, and IL-10 may be a predictor marker of the of HCC patients after surgery.The research based on the micro-environment has achieved exciting results, but, as we know the metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. In our previous work, we collaborated with the National Cancer Insititute (NCI) of USA, Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. An OPN-neutralizing antibody efficiently blocked in vitro invasion and in vivo pulmonary metastasis of HCC cells. The current study was based on our previous findings and the concept that proteins affect cancer progression, thus serving as markers of disease and therapeutic targets. We examined the protein levels of 12 Thl/Th2 cytokines in noncancerous liver tissue samples from 192 patients (cohort A) with HCC using ELISA methods. Of the 12 tested cytokines, three (IL-2, IL-15, and IFN-y) were found to correlate with clinical outcome. IL-2 had the best prognostic performance in the training set, which was then further validated in the test set (n=64). The prognostic value of IL-2 was further confirmed in another independent cohort (cohort B) of 261 patients using in situ immunohistochemical staining. At the same time, we examined OPN expression in tumor tissue microarrays (TMA) containing 261cases (cohort B) in order to investigate the predictive value of peritumoral IL-2 and intratumoral OPN for the prognosis of HCC patients.Thl/Th2-like cytokine mRNA levels in noncancerous hepatic tissues from hepatocellular carcinoma (HCC) patients are associated with metastases and recurrence. The present study evaluated the prognostic values of Thl/Th2 cytokine in HCC patients.Two independent cohorts of 453 HCC patients were enrolled. Twelve Thl/Th2 cytokines in noncancerous hepatic tissues from cohort A (n=192) were quantified with enzyme-linked immunosorbent assays. This cohort was split into training and test sets. Prognostic cytokines were identified from the training set, validated with the testing set, and confirmed in cohort B (n=261) using in situ immunohistochemical staining.IL-2, IL-15 and IFN-γprotein levels in noncancerous liver tissues were significantly greater in HCC patients without than in those with tumor recurrence. On Cox regression stepwise variable selection analyses, IL-2 was the optimal independent predictor of outcome (HR for recurrence=0.4, P=0.001; HR for death=0.5, P=0.019), rather than IL-15. This was confirmed in the testing set and validated in cohort B (HR for recurrence=0.4; P<0.0001; HR for survival=0.6; P=0.005). Another finding was that IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient。The aim of this study was to assess the value of Thl/Th2-like cytokine for predicting tumor recurrence after curative resection in serum and peripheral blood mononuclear cell of hepatocellular carcinoma patients.Blood samples from HCC patients and liver cirrhosis patients were collected for cytokine quantitation. Because Thl/Th2-like cytokine concentrations were too low, we performed the cytometric bead array (CBA) assay, which utilizes the sensitivity of amplified fluorescence detection. The expression levels of cytokine mRNA in peripheral blood mononuclear cell was assessed by qRT-PCR.No significant difference in serum IL-2 (P=0.6907),IFN-γ(P=0.459)、IL-4 (P=0.2044) and IL-10 (P=0.0837) level was found between the HCC patients (n=71), CG (n=9)and NG(n=13). Moreover, the serum IL-2, IFN-γ、IL-4 and IL-10level had no significant associations with OS or TTR in HCC patients using the median levels as cutoff. Similar results were also found in PBMC. The finding was that, unlike in liver tissues, serum IL-2 levels were not significantly correlated with tumor recurrence or survival of HCC patients.Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. It is generally classified as a pro-inflammatory cytokine because it modulates cell-mediated immunity by promoting the Thl response. In our present study, we found that peritumoral IL-2 was the optimal predictor for both tumor recurrence and overall survival time. The aim of this study was to explore the relationship of OPN and Thl/Th2 cytokines(IL-2, IFN-γ, IL-4 and IL-10)expression, then assess the value of OPN and IL-2 for predicting tumor recurrence after curative resection in hepatocellular carcinoma patients. Tissue microarrays of 261 (cohort B) HCC patients were used to detect the expressions of OPN and IL-2. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rankt tests.No significant association was found between OPN and Thl/Th2 cytokines expression in cell lines. In a tissue microarray-immunohistochemical analysis, we found that patients with higher levels of intratumoral OPN and lower levels of peritumoral IL-2 had a significantly poorer prognosis than patients with lower OPN and higher IL-2 levels. The combination of OPN and IL-2 expression thus served as an effective prognosticator.These findings suggest that IL-2 alone or in combination with OPN may act as an independent indicator for HCC patients after curative resection.1. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.2. IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.3. The third important finding was that, unlike in liver tissues, serum and PBMC immune/inflammatory levels were not significantly correlated with tumor recurrence or survival of HCC patients.4. Peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.5. The metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. To prevent recurrence of HCC we must focus on them all. 1. We firstly demonstrated that HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 and IL-15 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.2. For the first time, we found IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.3. We firstly confirmed that peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.Measuring the IL-2 protein level in noncancerous hepatic tissues could provide useful information for predicting tumor recurrence and prognosis in patients, even those with early stage HCC. This finding may potentially enable us to identify and select high-risk patients for effective adjuvant therapy. Regulation of the immune/inflammatory response may be a helpful strategy to control tumor relapse and further improve the treatment outcome of HCC patients. It has been applied for the patent (" Multiple inflammatory cytokines prediction kit for postoperative recurrence and metastasis of hepatocellular carcinoma"; Chinese patent applying number is 200810208150.6)更多还原