摘要
肝细胞肝癌是我国常见的恶性肿瘤,好发于40岁到60岁的男性,恶性程度高,发展迅速,虽然手术是当前肝癌的主要治疗手段,使得肝癌由不治之症变成部分可治,但手术后的高复发率尤其是肝内复发转移仍然是影响肝癌预后的首要因素。任何事物的发生发展都是内因和外因共同作用的结果,肝癌的肝内复发转移同样取于肝癌组织本身和癌旁正常肝脏组织。
肿瘤组织中可以概括为肿瘤细胞和肿瘤细胞所处的微环境两个主要部分。既往的研究主要集中在肿瘤细胞本身,包括肿瘤细胞癌基因和抑癌基因的改变,肿瘤细胞分化程度,表型改变等等。然而,现代研究发现,肿瘤所处的微环境在肿瘤的发生发展中同样有非常重要的意义。肿瘤微环境是肿瘤细胞赖以生长、增殖和进展的生存环境,包括与肿瘤细胞密切联系的细胞因子,生长因子、间质细胞、炎症免疫因子和细胞、电解质、pH值以及其他众多因素等等。
有学者将肿瘤的转移比作“种子和土壤”。在肝癌研究领域,传统的研究都是研究原发肝癌本身。然而,肝癌以外的肝组织却常常被视作正常的组织而忽略了对其进行研究。事实上,这些肝脏组织尤其是癌旁的肝组织,不仅仅是肝内复发的最常见区域,而且在原发肝癌生长时就有能够为肝癌细胞的转移扩散提供趋化的方向和适应生存的微环境以支持扩散的肿瘤细胞生长增殖形成微转移灶。癌旁微环境这些特点即使在原发肿瘤很小的时候就已经具备了。
在肿瘤微环境的研究中,人们发现缺氧时人体实体肿瘤的普遍特征。实体肿瘤内部缺氧主要和两大因素有关：氧的利用增加,氧的供给减少。例如肿瘤细胞的异常的分裂增殖,细胞高代谢率以及细胞的大量坏死,凋亡、自噬等生命反应均导致肿瘤组织快速完全的消耗氧；然而氧的供给不足却可能是肿瘤缺氧的主要原因：肿瘤细胞的排列不规则会影响氧从血管向细胞中的扩散,更重要的是肿瘤内血管紊乱,如形成盲端,癌栓、血栓导致微血管的阻塞等等,还有肿瘤病人本身的贫血以及红细胞运送氧的能力下降,这些因素都能引起氧的供给不足,从而造成肿瘤组织内缺氧微环境的存在。
缺氧对肿瘤细胞的特性有重要的意义,研究证实,缺氧与肿瘤细胞的发生、增殖、凋亡、白噬和代谢等均有密切的联系。
肿瘤细胞和正常细胞最明显的区别之一就是肿瘤细胞长期处于缺氧的生存环境下。肿瘤细胞不但本身能够适应缺氧,而且缺氧已经成为肿瘤细胞赖以生长增殖和保持其特性的生存环境。这种情况的出现,也许和缺氧对肿瘤细胞的自然选择有关。缺氧不但控制着细胞的增殖而且还控制着细胞的凋亡。从细胞开始恶变到形成成恶性程度高的肿瘤的过程中,那些能够适应缺氧微环境的恶变细胞变存活下来并继续增殖继而形成高度恶性的肿瘤组织,而不适环境的细胞则通过凋亡等途径消失在历史的长河中。研究还发现,除了影响肿瘤细胞本身的特性,缺氧还和肿瘤组织中的炎症,血管新生等病理特征有密切联系。存在即合理,既然缺氧是人类实体肿瘤中普遍的特征,肿瘤组织的大部分生命活动都可能与缺氧微环境存在或多或少的联系。
虽然人们很早就开始肿瘤与缺氧的关系,但一直以来都缺少很好的工具,直到人们发现缺氧诱导因子(HIFs)为止。与缺氧引起细胞外源性标记的改变不同,HIFs是缺氧状态下产生最重要的一类内源性转录因子,是引起众多缺氧反应的上游因子。其中,HIF-1是是分布最广,重要性最大,研究得最为透彻的一个,HIF-1α是其发挥功能的单体。HIF-1α的表达和缺氧的严重程度呈正相关,同类组织,缺氧越严重的区域HIF-1α表达越高。而且,在缺氧发生后的短时间内,细胞内HIF-1α就迅速升高到一定水平。
当前的研究已证实,HIF-1α可直接调控60多个目的基因,而且不断有新的目的基因不断被发现,是缺氧影响组织代谢和微环境改变的主要纽带。在肿瘤组织内,HIF-1α与肿瘤的进展有密切联系。HIF-1α可以调控细胞凋亡与增殖,使得肿瘤选择性地增殖那些更适合在缺氧微环境中生存的肿瘤克隆。HIF-1α调控与糖酵解的关键酶,使肿瘤细胞具有在缺氧下也能生存的特性。更重要的,HIF-1α可以促进血管新生,上调多种血管生长因子及其受体。HIF-1α还能促进基质金属蛋白酶(MMPs)分泌增加,降解基质,也可调控与细胞粘附和运动相关的受体和配体,如CXCR4, c-Met, SDF-1等,进而促进肿瘤细胞的扩散和侵袭。HIF-1α还可以促进COX-2, NF-κB等炎症相关因子的转录,与微环境中的炎症网络联系,从而改变微环境中细胞因子,炎症因子和生长因子,在影响肿瘤细胞生长增殖和血管新生等方面加速肿瘤进展。此外,HIF-1α作为细胞内的转录因子,与肿瘤细胞内的癌基因有密切联系,如缺氧条件下肿瘤细胞MYC转录增加,而MYC和HIF-1α又有错综复杂的联系,从而影响HIF-1α如在代谢和血管新生等方面的调控通路。
本研究中,通过研究肝癌内HIF-1α转录水平和蛋白水平的的表达情况,结合临床资料,揭示了HIF-1α与肝癌临床病理特征的关系以及对肝癌术后生存和复发的预后意义。同时研究肝癌内COX-2、MMP7和MMP9等炎症相关因子,VEGF、PDGFRA等血管生成因子和原癌基因MYC,从而研究HIF-1α对肝癌预后的影响以及对肝癌组织内炎症,血管新生和MYC的关系以及它们的临床意义。另一方面,我们也研究了癌旁HIF-1α的表达情况,同时研究癌旁趋化因子SDF-1α的表达,进而研究癌旁HIF-1α和SDF-1α的关系以及它们对肝癌复发转移的影响。
第一部分
肝癌组织内HIF-1α对肝癌复发转移的临床意义
肿瘤缺氧在各种基础研究中已经证实对肿瘤的侵袭转移有重要影响,而且在临床方面,很多肿瘤如胃癌,肾癌,乳腺癌等均发现HIF-1α的高表达与这些肿瘤患者的预后和转移复发有关。然而在肝癌中,却很少有研究同时从转录水平和蛋白水平来验证HIF-1α对肝癌术后生存和复发的预后意义以及与肝癌临床病理特征的关系。
本研究中,我们从2002年到2005年在复旦大学附属中山医院进行肝癌根治性切除的病例中随机取了110例肝细胞肝癌病例,用Real time RT-PCR技术检测了这些肝癌组织标本中HIF-1αmRNA的表达水平,用统计软件X-tile计算出最佳的cutoff值,将这些病例分成HIF-1αmRNA高表达组和低表达组,再用Fisher精确值法分析两组病例肝癌临床病理特征的区别以及COX风险比例模型分析HIF-1αmRNA对肝癌术后生存和复发的预后意义。然后,我们用免疫组化以及组织芯片技术检测了该110个病例肝癌组织内HIF-1α蛋白情况,参照既往的文献对免疫组化结果进行评分,将这些病例分HIF-1α蛋白阳性组和阴性组,再进步分析两组之间肝癌临床病理特征的区别以及HIF-1α蛋白对肝癌术后生存和复发的影响。
结果发现：1、肝癌组织内HIF-1αmRNA的表达水平与肝癌术后的生存和复发相关,是肝癌复发预后的独立影响因子(P=0.012 for OS; P=0.004 for DFS), HIF-1αmRNA高表达组的总体生存率和无瘤生存率明显低于低表达组。2、HIF-1a mRNA和肝癌有无血管侵犯和BCLC分期有关,HIF-1αmRNA高表达组血管侵犯率明显增高(P=0.033), BCLC分期晚期比例相对低表达组较大(P=0.041)。3、肝癌组织内HIF-1α蛋白的表达水平也是肝癌术后生存和复发的独立预后因子(P=0.021 for OS; P=0.007 for DFS) 4、HIF-1α蛋白也可肝癌有无血管侵犯和BCLC分期有关,HIF-1α蛋白阳性组血管侵犯率明显增高(P=0.027), BCLC分期晚期比例相对阴性组较大(P=0.039)。
以上结果表明,肝癌组织内HIF-1α不管是在转录水平还是在蛋白水平,都是肝癌术后生存和复发的独立预后因素。肝癌组织内HIF-1α表达高则预示着术后生存下降,复发的可能性增加。肝癌组织内HIF-1α的表达,不管是在转录水平还是在蛋白水平,均和血管侵犯率和BCLC分期相关。HIF-1α高表达预示着血管侵犯的可能性增加同时患者处于BCLC分期的晚期可能性较大。深入研究HIF-1α可以在将来为肝癌提供良好的诊断和治疗靶点。
第二部分
肝癌组织内HIF-1α与肝癌内炎症,血管新生以及MYC的关系
缺氧是实体肿瘤中重要的特征之一,是肿瘤的进展的关键因素之一。然而,肿瘤仍然中存在其它很多病理反应,如炎症、免疫、血管新生等等,这些病理反应与缺氧微环境存在着错综复杂的联系,而且相关因子在肿瘤的侵袭转移中也发挥着重要作用。癌基因MYC在很多基础研究中也正是和HIF-1α存在错综复杂的联系。然而,在肝癌微环境的研究中,很少有研究能从临床角度来揭示缺氧和炎症,血管新生以及MYC的关系。
在第一部分中,我们从2002年到2005年在复旦大学附属中山医院进行肝癌根治性切除的病例中随机取了110例肝细胞肝癌病例,用RT-PCR技术检肝癌组织中HIF-1αmRNA表达水平。在此同时,我们还检测了该组病例中一些炎症相关指标(COX-2, MMP7, MMP9),血管新生相关指标(VEGF, PDGFRA)以及原癌基因MYC作为第二部分的研究。我们用Spearman相关分析了HIF-1α与其他指标的关系。然后我们用X-tile计算出这些指标的最佳cutoff值,将其分成高表达组和低表达组,用COX风险比例模型分析这些指标对肝癌术后生存和复发的预测意义。
结果发现,1、HIF-1α和COX-2, MMP7,MMP9, PDGFRA以及MYC的表达水平呈正性相关。其中,COX-2 (P＜0.001, r=0.708), MMP7 (P＜0.001, r=0.593), MMP9 (P＜0.001, r=0.384), VEGF (P=0.183, r=0.128), PDGFRA (P＜0.001, r=0.493), MYC (P=0.016,r=0.230),然而VEGF与HIF-1α的相关性(P=0.183,r=0.128)并无统计学意义。2、在多因素分析中,COX-2 (P=0.004)、MMP7(P=0.010)和PDGFRA (P=0.010)是OS的独立预后因子,而COX-2 (P=0.010)和PDGFRA (P=0.038)是DFS的独立预后因子,而COX-2, MMP7和PDGFRA是与HIF-1α相关性最大的三个指标。
以上结果表明,肝癌组织内HIF-1α和肝癌组织的炎症反应,血管新生有密切联系,和原癌基因MYC也有一定关系。HIF-1α可能是肝癌组织内形成炎症,血管新生等病理特征的关键因素。而这些炎症和血管新生相关因素也促进了肿瘤的进展和复发。HIF-1α不仅本身对肝癌术后复发有最佳的预测价值(P值最小,风险比例值最大),而且与HIF-1α相关性越大的因子对肝癌的预后效果也越大,如COX-2, PDGFRA和MMP7。所以,HIF-1α是影响肝癌的预后的关键因素,可能通过影响肝癌内炎症、血管新生等因素而实现部分效应。与HIF-1α相关的炎症和血管新生等因素可成为诊断肝癌病人预后的重要指标,也为肝癌的治疗提供新的靶点。
第三部分
肝癌癌旁HIF-1α与趋化因子SDF-1α的关系以及对肝癌复发转移的影响
肝内转移复发时肝癌最常见的特征,过去被认为是正常肝组织的术后余肝经癌旁常被学者所忽视。事实上,肝脏组织不仅是肝癌术后复发的主要场所,也被越来越多的研究证实其组织特征和肝癌的肝内复发存在密切联系。间质细胞衍生因子(SDF-1α)是一种趋化因子,在很多体外研究中被证实能趋化CXCR4+阳性的肿瘤细胞,也包括肝癌细胞。很多基础研究证实SDF-1α是HIF-1α的目的基因之一。肝癌病人的非癌肝脏组织中,也存在缺氧的状况,可能与肿瘤引起的炎症免疫反应和肝癌细胞侵犯血管形成癌栓等因素有关。我们首次从临床角度来研究癌旁HIF-1α和SDF-1α的关系以及两者对肝癌术后复发转移到临床意义。
本研究中,我们从2002年到2006年在复旦大学附属中山医院进行肝癌根治性切除的病例中随机取了240例肝细胞肝癌癌旁组织标本制作成组织芯片,用免疫组化技术检测癌旁HIF-1α和SDF-1α的蛋白表达。我们使用平均光密度的方法对免疫组化结果进行了评估,再按中位值将每个指标分成阳性组和阴性组。用Spearman相关分析和Fisher精确值法检测两个指标的相关性。用COX风险比例模型分析了两个指标对肝癌术后生存和复发的影响。由于癌旁的肝脏组织中,肝细胞和成纤维细胞在细胞总数中占大部分,我们选择了人肝细胞株L02和人成纤维细胞株LX2,用氯化钴模拟缺氧条件,检测缺氧状况下两种细胞HIF-1α和SDF-1α的表达情况。
结果我们发现,1、癌旁SDF-1α是肝癌术后生存和复发的独立预后因素(多因素：P=0.021 for OS, P=0.012 for DFS)。而且,癌旁SDF-1α对肝癌术后复发的影响主要与术后两年内的早期复发有关,而与晚期复发无关。2、在免疫组化结果显示癌旁HIF-1α和SDF-1α呈正性相关(P＜0.001, r=0.305), Western-blot结果中,肝细胞和成纤维细胞中HIF-1α和SDF-1α在缺氧条件下也都同时增高。3、癌旁HIF-1α对肝癌生存和复发的影响与SDF-1α相异。癌旁高表达HIF-1α反而有降低复发率提高总体生存率的趋势(单因素分析：P=0.062 for OS;P=0.064 for DFS).而且,癌旁SDF-1α和HIF-1α两者均阳性时并不是预测肝癌预后的良好指标(单因素：P=0.418 for OS; P=0.394 for DFS),而癌旁SDF-1α阳性加HIF-1α阴性才是肝癌预后的独立因素(多因素：P=0.022 for OS; P=0.009 forDFS)。
以上结果表明,癌旁趋化因子SDF-1α的表达增高,有促进肝癌的肝内复发的可能,降低肝癌病人术后生生存率。而SDF-1α引起的复发主要和原发肿瘤扩散引起的早期复发关系较大。癌旁HIF-1α虽然对SDF-1α具有正性调控作用,但癌旁HIF-1α的高表达并不增加肝癌的肝内复发。癌旁组织中SDF-1α可以作为肝癌术后复发的诊断指标,并在防治肝癌肝内复发转移方面提供新的治疗靶点。癌旁HIF-1α的复杂影响对肿瘤缺氧相关诊疗技术提出更高的要求。
结论
1,不管在转录水平还是蛋白水平,肝癌组织内HIF-1α与肝癌的临床病理特征密切相关,而且是预测肝癌术后生存与复发的独立因素。
2,肝癌组织内HIF-1α和多种炎症,新生血管相关因子以及MYC密切相关,HIF-1α对调控肝癌组织内的病理反应有重要意义。
3,与HIF-1α相关性较大的某些炎症,血管新生相关因子：如COX-2、PDGFRA等也是肝癌生存和复发的独立预后因素。
4,癌旁趋化因子SDF-1α可以促进肝癌术后的肝内复发,尤其是早期复发。
5,癌旁HIF-1α虽然可以正性调控SDF-1α的表达,但并不增加肝癌术后的复发转移。
创新点
1,首次同时从基因转录和蛋白水平同时研究肝癌组织内HIF-1α的重要临床意义,为HIF-1α相关诊断和治疗技术提供依据。
2,首次用临床统计方法研究和报道了肝癌组织内HIF-1α和炎症、血管新生等病理特征的关系,为缺氧微环境在肿瘤研究领域中的重要性提供了依据。
3,首次发现和报道了肝癌癌旁趋化因子SDF-1α与肝癌术后肝内复发转移的关系,为研究癌旁趋化因子网络在肝癌肝内转移中作用提供了新的视野。
4,首次报道了癌旁HIF-1α对肝癌术后生存和复发的临床意义,为缺氧相关治疗技术在重视癌旁组织的作用方面提出了新的要求。
5,首次用临床统计的方法通过大量病例研究HIF-1α与SDF-1α的关系,为以后的相关研究和诊疗技术的发展提供了依据。
潜在的应用价值
1,揭示了HIF-1α在肝癌组织内炎症、血管新生等各种病理反应中起着关键作用,对未来肿瘤研究方向有一定参考价值。
2,肝癌组织内HIF-1α本身及相关指标可以作为肝癌预后的诊断指标,并提供新的治疗靶点
3,癌旁组织中SDF-1α可以作为肝癌术后复发的诊断指标,并在防治肝癌肝内复发转移方面提供新的治疗靶点。
4,癌旁HIF-1α的复杂影响提示了在肿瘤缺氧相关研究和诊疗技术需要重视癌旁组织。
Hepatic cell carcinoma (HCC) is one of the most common cancer in China and often occurred in the male between 40 and 60 years old, charactered with fast relapse and high mortality. Liver resection gave HCC patients the chance to escaped from death and some patients might obtain a complete cure. However, the frequent recurrence especially the recurrence in residual liver after resection was the main problem and seriously reduced the HCC patients survival rate and made the outcome dismal. All things happened for its intrinsic factor and extrinsic factor. The event of intrahepatic recurrence after resection also determined by the feature of original caner and the residual liver tissue especially the peritumoral liver tissue.
HCC as a human solid tumor, consist of the tumor cells and microenvironment where the tumor cells survive and proliferation. The previous study mainly focused on tumor cell itself, such as oncogene and antioncogene, differentiation and phenotype of tumor cells and so on. However, modern research revealed that the tumor microenvironment also crucial to the tumor progression. Tumor environment contained a lot of cells and molecular correlated with tumor cell, such as kinds of cytokines, growth factors, inflammation cells and factors, stromal cells, nutrition, pH and electrolytes and so on.
Tumor progression or metastasis was described as "seed and soil". In HCC research area, traditional research mainly focused on the features of the primary tumor itself. However, the residual liver tissue, which used to be thought as normal tissue, was often neglected. In fact, the residual liver tissue especially the peritumor liver tissue not only serves as the most common field where a visible intrahepatic recurrence occurs after resection, but also provides the microenvironment of the chemoattracting direction and nourishment for original cancer cells dissemination and micrometastasis formation. The feature of peritumoral liver tissue microenvironment had existed even if the primary HCC was very small.
In the field of tumor environment, hypoxia was confirmed to be a common character in human solid tumor including HCC. Hypoxia in solid tumor can be caused by two reasons:the increased oxygen consumption and the decreased oxygen supply. The character of tumor cells determined the high oxygen consumption rate in tumor tissue, such as the abnormal mitotic division, rapid proliferation, high metabolism rate, as well as extensive apoptosis, autophagy and necrosis which also consume a lot of oxygen. However, the most important reason for tumor hypoxia was the low supply of oxygen. Aberrant vasculogenesis, including caecum of vessels, thrombus, embolus from cancer cells and so on, make the ischemia aera in tumor tissue. The irregular arrangement of intratumor cells made the oxygen more differcultly diffuse to intracellar organelles of the target cells. Besides, the anemia or dysfunction of erythrocytes in tumor patients also reduced the oxygen supply. All these caused a hypoxia microenvironment in tumor.
Tumor hypoxia was very important in the feature of tumor cell. A lot of research have found that tumor hypoxia is closely correlated to tumor cells proliferation, apoptosis, autophagy, metabolism and so on. It is the most obvious difference between tumor cells and normal cells that tumor cells survived in the hypoxia microenvironment all the time but the normal cells not. Tumor hypoxia was not only suitabled for the survival of tumor cells but also a necessary condition for keeping and developing the feature of the tumor cell itself. This may be correlated to the natural selection for tumor cells by hypoxia. Hypoxia can regulate the cell apoptosis. From the beginning of the tumor progression, only the specific malignant cell which can survive in the hypoxia condition was selected to continue to proliferation and formed the macroscopic tumor. While the others that can not be suitable for the hypoxia condition in tumor were disappeared in the process of tumor formation by apoptosis and other ways. Hypoxia not only affect the character of the tumor cell itself, but also have close correlation to the feature of intratumor pathologic reaction such as inflammation, immunity, angiogenesis and so on. "Existence in possible" tells us that hypoxia as a common "existence" in cancer may correlate to most of the feature and action of the malignant neoplasm.
Although the research of tumor hypoxia had a long history, there was few good tools in this field until hypoxia induced factors (HIFs) were discovered. HIFs was a sort of transcription factors upregulated in hypoxia. They are endogenous factors regulating a lot of target genes which were different with those exogenous markers induced by hypoxia condition. In the family, HIF-1 was the one that had most importance and widely distribution with HIF-1αas the functional unit. HIF-1αexpression level is positively correlated to oxygen concentration. In the same kind of tissues, HIF-1αexpression level is higher in those cells with more severe hypoxia. And the intracellular HIF-1αprotein could reach a high level as soon as the cell was sensitive to the hypoxia.
There were more than sixty HIF-1αdirect target genes were confirmed by previous research. And more possible target genes need to be confirmed. The proteins generated by HIF-1αtarget genes often play the crucial roles in cell actions and microenvironment variation that were responding to hypoxia. Intratumoral HIF-la had close correlations to the tumor progression. HIF-la could regulate the apoptosis and proliferation of tumor cells and provide the natural selection of those suitable for hypoxia to form the macroscopic tumor. HIF-1αcould regulate the key enzymes in anaerobic glycolysis which make the tumor cells survive in hypoxia microenvironment. More importantly, HIF-1αplay a important role in the metastasis or invasion of tumor. HIF-1αcould regulate the angiogenesis in the progression of tumor by upregulating kinds of related factors and receptors. HIF-1αalso increased the expression of matrix metalloproteinases (MMPs) which can degrade the matrix. Besides, HIF-1αcan regulate the receptors in the tumor cell surface related the cell adhesion and mobility or their ligands secretion, such as CXCR4, c-Met, SDF-1 and so on. HIF-1αas a transcription factor also correlated to the variation of oncogene in tumor cells such as MYC. In some research, hypoxia feature increased MYC expression which also affecting the HIF-1-depended regulating pathway in metabolism, angiogenesis and so on.
In our research, we detected the expression of HIF-1αmRNA as well as protein. Combining with the complete clinical date, we analyzed the correlations between HIF-la and the clinicopahologic features, as well as the prognosis value in HCC after resection. We also detected the factors related to inflammation such as COX-2 MMP7和MMP9, those related to angiogenesis such as VEGF、PDGFRA and MYC oncogene. Then we analyzed the correlation between HIF-1αand these factors. The prognostic value of all factors in HCC after resection was assessed. On the other hand, we detected HIF-1αexpression in peritumor tissue as well as the chemokine SDF-1α. The correlation between peritumoral SDF-1αand HIF-1αwas analysed. Their prognostic value in recurrence and survival of HCC after resection was also discussed.
Part One The clinical significance of intratumoral HIF-1αin survival and recurrence of HCC after resection
Tumor hypoxia has been confirmed to be a crucial role in tumor invasion or metastasis in a lot of basic research. And in clinical study, HIF-la high expression was correlated to recurrence and survival in some kinds of tumor, such as gastric cancer, renal cancer, breast cancer and so on. However, few reports studied the clinical significance of both the HIF-1αmRNA and protein in HCC.
In our study, we randomly chose 110 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2005. The HIF-la mRNA in the tumor was detected by Real time RT-PCR. The cutoff value was determined by X-tile and the specimens were divided to high and low expression groups. Fisher s exact test was carried out to analyse the correlation with clinicopathologic features. Cox proportional hazards regression model was carried out to assese the prognostic value of HIF-la mRNA in the survival and recurrence of HCC after curative resection. We also detected the HIF-la protein expression in the tumor of these 110 patients by immnohistochemisty and tissue microarray. The degree of the expression was evaluated and classified by the criterion in previous researches. Then the specimens were separated into HIF-1αprotein staining positive and negative groups. The correlations between HIF-1αprotein and clinic pathologic features was analyzed in the same methods as HIF-1αmRNA, as well as the prognostic value of HIF-1αprotein for recurrence and survival in HCC patients after resection.
The results showed that:(a) In multivariable analysis, intratumoral HIF-1αmRNA was an independent prognostic factor for survival (P=0.012) and recurrence (P=0.004) of HCC after resection. (b) HIF-1αmRNA was correlated to vascular invasion (P=0.033)and BCLC stage (P=0.041). Intratumoral HIF-1αmRNA high expression had more proportion of tumor cell vascular invasion and advanced BCLC stages. (c) Intratumoral HIF-1αprotein level was also an independent prognostic factor for OS (P=0.021) and DFS (P=0.007) in multivariable analysis. (d) HIF-1αprotein also correlated to vascular invasion and BCLC stages. HIF-1αpositive staining group was more likely to suffer vascular invasion and advanced BCLC stages.
These results suggested that both HIF-1αmRNA and protein were independent prognostic factors for survival and recurrence of HCC after curative resection. Increased HIF-1αin tumor correlated to high recurrence rate and low survival. Intratumoral HIF-1αalso increased the probability of vascular invasion and high HIF-1αoften accompanied with advanced BCLC stages. A further investigation to HIF-1αmay bring us new and good diagnostic and therapeutic tools in HCC.
Part Two
The correlations between intratumoral HIF-la and inflammation, angiogenesis as well as MYC
Tumor hypoxia was an important feature and one of the crucial ingredient in the procession of malignant tumor. However, there were also other pathologic actions in the tumor tissue, such as inflammation, immunity, angiogenesis and so on. There were complicated relations between hypoxia and other pathologic actions in tumor and related factors also play an important role in tumor invasion and metastasis. The MYC oncogene was also showed complicated relation to HIF-la in some investigations. However, few research showed the correlations between hypoxia and inflammation, angiogenesis as well as MYC in the clinical view.
In this part, we analyzed these problems. In part one, we randomly chose 110 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2005. We carried out Real time RT-PCR to detected intratumral HIF-la mRNA. And in the same procedure, we detected the mRNA of other factors for this part analysis. This factors included inflammation related factors (COX-2, MMP7, MMP9), angiogenesis related factors (VEGF, PDGFRA) and oncogene MYC. Spearman's correlation test was carried out to assess the correlations between HIF-1αmRNA and these factors. The cutoff value of these factors also determined by X-tile. And all the specimens were divided into high and low expression group in each factor. Multivariable and univariable Cox proportional hazards regression model was carried out to assess the prognostic value in HCC survival and recurrence after curative resection.
The results showed that:(a) Significant positive correlations were observed between HIF-1αmRNA and the putative markers of inflammation, angiogenesis and MYC:COX-2 (P＜0.001, r=0.708), MMP7 (P<0.001, r=0.593), MMP9 (P＜0.001, r=0.384), VEGF (P=0.183,r=0.128), PDGFRA (P＜0.001, r=0.493), MYC (P=0.016, r=0.230). However, there were no significant correlation between mRNA expression level of HIF-1αand VEGF (P< 0.183, r= 0.128). (b) In multivariable analysis, COX-2 (P=0.004)、MMP7 (P=0.010) and PDGFRA (P=0.010) was an independent prognostic factor for OS. While COX-2 (P=0.010) and PDGFRA (P=0.038) was an independent prognostic factors for DFS. And of all the six parameters, COX-2, MMP7 and PDGFRA were the top three factors with the largest correlation coefficients to HIF-1α.
These results suggested that intratumoral HIF-1αwas closed correlated to inflammation, angiogenesis and oncogene MYC. HIF-1αmay be a crucial role in the pathologic actions in tumor. The inflammation, angiogenesis related factors also advanced the tumor progress and increased the recurrence. In all the factors, HIF-1αwas an independent prognostic factor for both survival and recurrence in HCC with the smallest P value as well as the largest hazard ratio for recurrence. And other factors correlated to HIF-1αclosely also were the independent prognostic factor for OS or DFS, such as COX-2, PDGFRA and MMP7. We thought that HIF-1αwas the crucial factor affecting the prognosis of HCC, which may be in association with inflammation and angiogenesis. HIF-1αrelated factors including those functioned in inflammation, angiogenesis and so on may serve us new diagnostic markers and therapeutic targets in HCC.
Part Three
The correlation between peritumoral HIF-1αand SDF-1αas well as their clinical significance
Intrahepatic recurrence and metastasis was the most common feature in HCC after resection. The residual liver tissue which used to be thought as normal tissue was often neglected. In fact, the peritumoral liver tissue not only serves as the field where a visible intrahepatic recurrence occurs after resection, but also affected intrahepatic recurrence and metastasis in more and more investigation. SDF-1α, as a chemokine ligand, played an important role in direct migration of HCC cells with CXCR4 positive. Some basic research also confirmed that SDF-1αwas a target gene of HIF-1αin hypoxia condition. Peritumoral live tissue also suffered hypoxia due to some reactions to tumor, such as direct presssion, inflammation, immunity and vascular invasion. In this study, we firstly confirmed the correlation between SDF-la and HIF-1αin the clinical view and firstly detected the clinical significance of peritumoral SDF-1αand HIF-1αin HCC after resection.
In our study, we randomly chose 240 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2006. The tissue microarray and immunohistochemical technology were carried out to detected the HIF-1αand SDF-1αprotein. The density of SDF-1αand HIF-1αpositive staining was assessed by mean optical density. Cut-point value of experiment results was determined by the median and the specimens were divided into HIF-1αor SDF-1αprotein staining positive and negative groups. The correlations between SDF-la and HIF-1αwas analyzed by Spearman's correlation test and Fisher s exact test. Cox Proportional Hazards Regression model was carried out in univariate and multivariate survival analyses.
The results showed that:(a) Peritumoral SDF-1αwas an independent prognostic factor for survival and recurrence in HCC after resection (P=0.021 for OS, P=0.012 for DFS). In addition, peritumoral SDF-1αhad prognostic value for early recurrence of HCC (P=0.017) in multivariate analysis, but not for late recurrence (P=0.111 in univariate analysis). (b) Significant positive correlation was observed between HIF-1αand SDF-1αexpression in peritumoral tissue (P＜0.001, r=0.305). (c) However, peritumoral HIF-1αhad a propensity of reduced recurrence and prolonged survival with boardline significance in univariate analysis (P=0.062 for OS and P=0.064 for DFS in univariate analysis) which was different to peritumoral SDF-1α. Furthermore, the combination of negative HIF-1αand positive SDF-1αwas an independent prognostic factor for increased recurrence (P=0.022) and reduced survival (P=0.009) in multivariate analysis. While both positive had not significance just in unvariate analysis (P=0.418 for OS and P=0.394 for DFS).
These results suggested that peritumoral SDF-1αcould increase the recurrence and reduce the survival, especially the early intrahepatic recurrence of HCC after resection which was mainly from the metastasis and proliferation of residual tumor cells derived from the original tumor. Peritumoral HIF-1αmight partly upregulate SDF-1α, but did not increase the recurrence. Attach importance to peritumoral SDF-1αmay develop new anti-metastatic strategies in HCC. The role of peritumoral HIF-1αshould not be neglected when we thought hypoxia was a therapeutic target for HCC.
Conclusions
1. Intratumroal HIF-1αmRNA and protein were correlated to the clinicopathologic features of HCC and were all independent prognostic factors in survival and recurrence of HCC after resection.
2. Intratumoral HIF-1αmay influence HCC biological behaviors and affect the tumor inflammation, angiogenesis and act in concert with the oncogene MYC.
3. The inflammation and angiogenesis related factors such as COX-2 and PDGFRA, which were closely correlated to HIF-1α, also has prognostic value in survival and recurrence of HCC after resection.
4. Peritumoral SDF-1αincreased the recurrence, especially the early recurrence of HCC after resection.
5. Peritumoral HIF-1αmight upregulate SDF-1αexpression, but did not increase the recurrence of HCC after resection.
Novelty
1. For the first time, we detected clinical significance of the intratumoral HIF-1αin both mRNA and protein levels. It provided the basis for HIF-1αrelated diagnostic and therapeutic technology in the future.
2. For the first time, we detected the correlations between intratumoral HIF-1αand the tumor pathologic actions such as inflammation and angiogenesis. It suggested that hypoxia was crucial in tumor microenvironment.
3. For the first time, we reported the peritumoral SDF-1αcorrelated to the intrahepatic recurrence of HCC after resection. It confirmed again that peritumoral chemokines played an important role in the intrahepatic recurrence and metastasis of HCC.
4. For the first time, we detected the clinical significance of peritumoral HIF-1αin survival and recurrence of HCC after resection. In provide a new viewpoint in tumor hypoxia related therapy.
5. For the first time, we detected the correlations between peritumoral HIF-1αand SDF-1αin the clinical view by large quantity of patients, which provided the proof for the further related investigations.
Potential application
1. Intratumoral HIF-1αplay the crucial role in the pathologic actions in HCC such as inflammation and angiogenesis, which was valuable for the further related research.
2. The clinical significance of intratumoral HIF-1αsuggested that related markers will be valuable in the diagnostic and therapeutic practice for HCC.
3. The clinical significance of peritumoral SDF-la suggested that it could be provided as the good diagnostic marker and therapeutic target for intrahepatic recurrence of HCC after resection.
4. The complex affections of peritumoral HIF-la suggested that peritumor tissue should not be neglected when the tumor hypoxia was thought as a therapeutic target.
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