【作者】 张宝燕；

【导师】 李向红； 吕有勇；

【作者基本信息】 中国人民解放军军医进修学院 ， 病理学与病理生理学， 2010， 博士

【摘要】 胃癌是世界上第四位常见恶性肿瘤,癌症第二大死因。国内临床调查显示胃癌被发现时90%以上处于进展期,即使施行了胃癌根治术,5年生存率仍然低。传统的组织病理学诊断是肿瘤诊断和治疗的基础,但即使组织学类型相同的肿瘤如采取相同的治疗方案,预后并不一致。因此在组织学分型基础上寻找能够有效评估预后、警示转移复发的临床、病理指标和分子标志物及其相应的靶向治疗药物成为胃癌研究工作者不懈探索的目标。Ras/Raf/MAPK和PI3K/Akt信号通路与肿瘤关系密切,受体酪氨酸激酶(RTKs)活化后可以激活这两条通路。表皮生长因子受体(Her-2)为RTKs家族一员,活化后可激活Ras/Raf/MAPK和PI3K/Akt通路,引发一系列细胞生物学改变。乳腺癌中发现Her-2与复发和预后有关,Her-2阳性的乳腺癌易复发、预后差。针对Her-2的靶向治疗药物曲妥珠单抗对Her-2阳性的乳腺癌病例的治疗已取得明确疗效。有资料显示Her-2在胃癌中也发挥重要作用,而且曲妥珠单抗已经进入胃癌治疗Ⅲ期临床试验,取得疗效,但Her-2在胃癌的表达与预后报道不尽一致,亟需有大样本的临床病理和预后数据。另一方面,Ras/Raf/MAPK信号通路通过下游的信号分子进入细胞核内作用于上皮间质转化(Epithelial Mesenchymal Transition, EMT)诱导转录因子Snail家族,使其下游基因E-cd表达下调。Slug/Snai2是该家族成员之一。北京肿瘤防治研究所前期的基因表达谱信息分析显示Slug在低分化胃癌中高表达,在高分化胃癌中低表达,而其下游的E-钙黏蛋白(E-cadherin,E-cd)的表达刚好与之相反,提示Slug和E-cd可能与胃癌的分化有关,有可能是与胃癌分化、预后相关的分子标志物。基于此,我们首先对486例胃癌病例进行WHO和Lauren组织学分型与预后再评估,结果显示发病年龄、有无脉管浸润、淋巴结转移情况(包括pN分期和N ratio分级)及pTNM分期是胃癌的独立预后因素,WHO和Lauren分型并非是胃癌的独立预后因子。其次我们分别用免疫组织化学和荧光免疫原位杂交技术检测了486例胃癌Her-2的过表达和基因扩增情况,并分析Her-2状态与胃癌临床病理的联系及预后意义,结果显示Her-2阳性率为10.93%,其阳性与WHO组织学分型(P=0.011)、分级(P=0.000)和Lauren分型(P=0.024)有关,乳头状腺癌和管状腺癌、组织学分级低和肠型胃癌病例Her-2阳性率更高,但Her-2状态与胃癌总体生存率无关(P=0.709),但在印戒细胞癌或弥漫型胃癌,有迹象表明Her-2阳性组较阴性组预后更差,但因HER2阳性例数较少,两组差异均无统计学意义。进一步,我们探讨了Slug和E-cd的表达及其与预后的关系。检测了Slug和E-cd在胃癌细胞系中mRNA和蛋白水平的表达并对两者的相关性进行分析,对高致瘤性的胃癌细胞株SGC7901进行Slug的化学干扰,证实其与E-cd的关系。然后利用组织芯片和免疫组织化学技术,检测Slug和E-cd在486例胃癌组织中的表达,分析其与胃癌浸润、转移的关系,并结合临床病理学特征和预后进行综合评价。细胞系实验结果显示在mRNA和蛋白水平,Slug在致瘤性高的胃癌细胞系BGC823、MGC803和SGC7901中均高表达,在致瘤性弱的胃癌细胞系AGS和N87中基本不表达。E-cd的表达趋势则与之相反,在致瘤性高的3株胃癌细胞系低表达,而在致瘤性弱的细胞系AGS和N87高表达。对高表达Slug的胃癌细胞系SGC7901进行化学干扰后,E-cdmRNA表达上调1.9～2.9倍。胃癌组织的检测结果显示Slug与E-cd的表达呈正相关,Slug的蛋白表达与患者年龄、肿瘤发生部位、浸润深度、WHO组织学分型、分级、Lauren分型、淋巴结转移和pTNM分期有关。生存分析显示在胃周淋巴结总数≥15个的病例组,Slug与pN、pTNM分期都是胃癌的独立预后因素,提示Slug可能是与胃癌预后相关的标志物。E-cd的蛋白表达水平与性别、年龄、肿瘤部位、WHO组织学分型、分级以及Lauren分型有关,但与肿瘤大小、浸润深度、脉管癌栓、淋巴结转移和pTNM分期无关。单因素及多因素生存分析都显示E-cd与胃癌预后无关,提示E-cd可能为一分化标志物,与胃癌预后无关。以上结果表明,WHO和Lauren组织学分型仍然是深入研究胃癌的基础；Her-2状态没有明确的预后价值；Slug与pN、pTNM分期都是胃癌预后独立因素；E-cd则更倾向于分化标志物,而与胃癌预后无关。更多还原

【Abstract】 Gastric carcinoma(GC) still remains the fourth most common cancer worldwide and is the second cause of cancer related death. Clinically more than 90% of GC cases are in advanced stage. Even though radical gastrectomy is performed, the prognosis is still dismal with a low 5-year survival rate. Histopathological diagnosis and classification is the foundation of subsequent treatment. However, prognosis differs even in cases with identical histological type and uniform therapy. Great efforts were made to correlate the clinicopathological parameters with prognosis and molecular markers have been searching in recent years to predict the prognosis.Ras/Raf/MAPK and PI3K/Akt signaling pathways play important roles in GC. Receptor tyrosine kinases (RTKs) can activate these two pathways. Her-2, as a member of epidermal growth factor receptors, belongs to RTKs which regulate many basic cellular processes through Ras/Raf/MAPK and PI3K/Akt. In the case of breast carcinoma, overexpression of Her-2 protein or amplification of Her-2 gene was associated with recurrence and poor prognosis. A new therapeutic agent trastuzumab as a monoclonal antibody to Her-2 has been shown to be effective in Her-2 positive breast carcinoma patients. Data also showed that Her-2 played an important role in GC development. Recently, a global clinical study was conducted to determine response to trastuzumab therapy in Her-2 positive GC patients. However the frequency of Her-2 positivity varied from different studies and its clinical significance in gastric cancer is yet not clear. A large-sample clinicopathological observation and analysis concerning Her-2 status is still needed.On the other hand, Ras/Raf/MAPK pathway may finally affect the transcription factor-Snail family which induce epithelial mesenchymal transition(EMT) by downregulating E-cd. Slug/Snai2 is a member of Snail family. According to the gene expression profiling of Beijing Institute of Cancer Research, transcription factor Slug mRNA level was increased in poorly differentiated GC, but decreased in well differentiated one. E-cd mRNA expression level was the reverse. So we hypothesized that Slug and E-cd may be correlated with differentiation and prognosis in GC.Thus firstly we re-evaluated prognostic significance of WHO and Lauren classification by reviewing HE sections of 486 gastric cancer patients who underwent curative gastrectomy from January 2001 to December 2004 in PLA General Hospital. Statistical analysis demonstrated that age, lymphatic/vascular invasion, lymph node metastasis (including pN stage and N ratio grade) and pTNM stage were independent prognostic factors. Although WHO and Lauren classification had significant influence on overall survival in univariate analysis, they lost their independent value in multivariate analysis.Subsequently, to clarify the potential clinical relevance of Her-2 status in gastric cancer, we evaluated Her-2 status in 486 cases of GC by applying immunohistochemistry(IHC) and Fluorescence in situ hybridiza-tion(FISH), then correlated its positivity with clinicopathological parameters and follow-up data. Her-2 positivity was found in 10.93% of GC cases. Her-2 positive status were common in cases with papillary and glandular adenocarcinomas according to WHO classification (P=0.011), in cases with a well or moderately differentiated GC by WHO histological grade (P=0.000) and in cases of intestinal type by Lauren classification (P=0.024). Survival analysis revealed that the overall survival between Her-2 positive and negative cases showed no significant difference (P=0.709). Yet in signet ring cell carcinoma or diffuse GC, the 5-year survival rate of Her-2 positive cases was 0%, indicating worse prognosis. However, the difference was not statistically significant probably due to the fewer Her-2 positive cases.In the third part of the study, we explored the significance of Slug and E-cd in GC cell lines as well as in 486 GC cases. Initially we examined their expression in 6 GC cell lines at mRNA and protein level respectively, analyzed the role of Slug in relation with E-cd through silencing of Slug by short hairpin RNA. Then we investigated Slug and E-cd expression using tissue array and IHC method. Meanwhile, we analyzed the association of Slug and E-cd expression with clinicopathological parameters and prognosis. An increased Slug expression was observed in BGC823、MGC803 and SGC7901 GC cell lines which had greater capacity of tumorgenesis, while Slug expression level was low in AGS and N87 at both mRNA and protein level. Inversely, E-cd expression was low in BGC823、MGC803 and SGC7901 GC cell lines, while it was increased in AGS and N87 GC cell lines at both mRNA and protein level. In MKN45 GC cell line, a sequence of 12 bases was found missing by gene sequencing. Transient Slug silencing in SGC7901 cell line induced an increase in E-cd mRNA level up to 2.9-fold. Positive expression of Slug and E-cd was observed in 56.2% and 69.1% GC cases, respectively, which was positively correlated. The expression of Slug correlated with age, tumor location, depth of invasion, WHO classification, WHO histological grade, Lauren classification, lymph node metastasis status and pTNM stage. Though univariate analysis revealed Slug expression was unrelated to survival, multivariate analysis indicated that Slug expression was an independent prognostic factor in addition to pN, pTNM stage. The expression of E-cd correlated with gender, age, tumor location, WHO classification, WHO histological grade and Lauren classification, but unrelated with tumor size, depth of invasion, lymphatic/vascular invasion, lymph node metastasis or pTNM stage. Neither univariate nor multivariate analysis showed E-cd expression had a significant impact on patient’s overall survival.In conclusion, precise WHO and Lauren classification remains the baseline study of GC. Her-2 status is not related to the prognosis.Slug expression is an independent prognostic factor in addition to pN, pTNM stage. E-cd tends to be a differentiation marker rather than a prognostic one.更多还原