【作者】 朱磊；

【导师】 卢焕元；

【作者基本信息】 中南大学 ， 外科学， 2009， 硕士

【摘要】 表皮生长因子(Epidermal Growth Factor,EFG)是生长因子家族的重要成员之一,EGF通过与表皮生长因子受体(Epidermal GrowthFactor Receptor,EGFR)结合后启动其下游的信号传导通路而发挥一系列生理及病理作用。EGFR是由1186个氨基酸残基构成,分子量为170kD的一种跨膜糖蛋白。EGFR主要位于细胞质膜上,属于Ⅰ型酪氨酸激酶受体亚族,具有酪氨酸激酶的活性。EGFR稳定的表达于多种上皮组织,以及间质和神经源性组织。不同器官发生的实体瘤也高表达EGFR,如头颈部癌、卵巢癌、宫颈癌、膀胱癌和食管癌等。EGFR受到EGF等配体激活后通过二聚化引发胞内域形成酪氨酸激酶活性,由此启动一系列信号转导通路调节正常细胞的生长和分化,增加肿瘤细胞的侵袭力、促进血管生成、抑制肿瘤细胞的凋亡。EGFR在肿瘤中的高度表达及其在肿瘤细胞生长、分化中起着重要作用的这些特点,使EGFR成为具有良好前景的肿瘤诊断和治疗的靶点。由于EGFR在肿瘤细胞的生长、修复和存活等方面具有极其重要的作用,它的过度表达通常预示患者预后差、转移快、对化疗药抗拒、激素耐药、生存期较短等,因此以EGFR为靶点是一种比较合理的抗癌策略。目前肿瘤分子靶向治疗中研究较多的是以gefitinib(Iressa)和erlotinib(Tarceva)为代表的EGFR酪氨酸激酶抑制剂(EGFR-tyrosinekinase inhibitor,EGFR-TKI)。早期临床试验表明EGFR酪氨酸激酶抑制剂(EGFR-TKI)在部分化疗耐药的非小细胞肺癌(NSCLC)中仍然有较好的疗效。统计显示EGFR酪氨酸激酶抑制剂(EGFR-TKI)在女性、腺癌、不吸烟患者和东亚人群中疗效更佳。有研究表明EGFR酪氨酸激酶区突变与EGFR酪氨酸激酶抑制剂(EGFR-TKI)疗效密切相关。EGFR酪氨酸激酶抑制剂(EGFR-TKI)已在美国、日本及澳大利亚获准临床用于单药治疗晚期非小细胞肺癌(NSCLC),对治疗前列腺癌、乳腺癌、结肠癌等的大规模临床研究尚在进行中。EGFR酪氨酸激酶抑制剂(EGFR-TKI)仅对存在EGFR酪氨酸激酶区域外显子(Exon)19(e19)位点、外显子21的L858R位点有突变的非小细胞肺癌有确切疗效,此突变目前发现外显子19(e19)位点基因缺失(Deletion)(以下简称EGFR mutations(del)),和外显子21的L858R位点点突变(Point mutation)(以下简称EGFR mutations(p)),EGFR酪氨酸激酶抑制剂(EGFR-TKI)对外显子19(e19)位点突变的患者治疗效果优于外显子21的L858R位点有突变的非小细胞肺癌患者,且证实上述基因突变与多种因素有关,近年来的一些研究发现EGF、EGFR在肝癌中也存在过度表达,且与肝癌的形成、发生、发展有密切关系。本实验拟在肝癌组织中检测EGFR、EGFR mutations(EGFR mutations(del)和EGFR mutations(p))的表达及其与临床病理关系,为肝癌的分子靶向治疗提供理论依据和临床指导。目的在肝癌组织中检测EGFR、EGFR mutations(EGFRmutations(del)和EGFR mutations(p))的表达及分析其与临床病理关系,为肝癌的分子靶向治疗提供理论依据和临床指导。方法选取78例肝细胞癌(HCC)手术切除的新鲜标本和6例正常肝组织,10%福尔马林固定,常规石蜡包埋,每个标本行连续切片,厚4um。应用SP免疫组化法,以EGFR、EGFR mutations(del)和EGFRmutations(p)的单克隆抗体对组织切片进行染色。用X~2检验、Spearman’s非参数分析对EGFR、EGFR mutations(del)和EGFRmutations(p)在肝癌组织中的表达与临床病理关系进行分析,对患者术后生存期与各项参数进行Cox多因素回归分析和Kaplan-Meier生存分析。结果1.EGFR在正常肝组织中阳性表达率为66.7%(4/6),EGFRmutations(del)阳性表达率为0(0/6),EGFR mutations(p)表达率为0(0/6);EGFR在肝癌组织中阳性表达率为69.2%(54/78),EGFRmutations(del)阳性表达率为21.8%(17/78),EGFR mutations(p)表达率为9.0%(7/78)。肝癌组织表达EGFR与正常肝组织无差异性(X~2=0.171,P=0.680)、正常肝组织未见EGFR mutations表达,肝癌组织表达EGFRmutations(del)高于EGFR mutations(p)(X~2=4.924,P＜0.05)。2.肝癌组织EGFR mutations的表达情况与女性(r=0.743,p＜0.05)呈正相关,与术前AFP值(r=-0.519,p＜0.05)、乙肝(r=-0.567,p＜0.05)、肝硬化(r=-0.245,p＜0.05)、病理分级(r=-0.273,p＜0.05)呈负相关;肝癌组织表达EGFR mutations(del)与女性(r=0.453,p＜0.05)呈正相关,与术前AFP值(r=-0.243,p＜0.05)、乙肝(r=-0.693,p＜0.05)、肝硬化(r=-0.328,p＜0.05)、病理分级(r=-0.343,p＜0.05)呈负相关;肝癌组织表达EGFR mutations(p)与女性(r=0.390,p＜0.05)呈正相关,与乙肝(r=-0.657,p＜0.05)、病理分级(r=-0.347,p＜0.05)呈负相关,其余参数与EGFR、EGFR mutations的表达无关。上述所有参数与EGFR的表达无关。3.肿瘤病理分级(P＜0.001)和术前AFP值(P＜0.001)是该组肝癌患者术后生存期的主要影响因素。该组肝癌组织EGFR及EGFRmutations表达阴性的患者和表达阳性的患者术后生存期无明显差别(P＞0.05)。结论1.正常肝组织中存在EGFR的表达,未发现EGFR mutations(EGFR mutations(del)和EGFR mutations(p))的表达;肝癌组织中存在EGFR、EGFR mutations(EGFR mutations(del)和EGFR mutations(p))的表达。2.肝癌组织EGFR mutations、EGFR mutations(del)的表达与女性呈正相关,与术前AFP值、乙肝、肝硬化、病理分级呈负相关;肝癌组织表达EGFR mutations(p)与女性呈正相关,与乙肝、病理分级呈负相关,其余参数与EGFR、EGFR mutations的表达无关。上述所有参数与EGFR的表达无关。3.肝癌组织EGFR、EGFR mutations(EGFR mutations(del)和EGFR mutations(p))的表达不是肝癌患者的独立预后因素。更多还原

【Abstract】 Objective:To detect the expression of EGFR and EGFR mutations (EGFR mutations(del) and EGFR mutations(p)) in hepatocellular carcinoma tissue,explore their relationship with clinical pathology and provide theoretical basis and clinical guideline for the molecular target treatment of hepatocellular carcinoma.Methods:78 cases of fresh specimens were resected in hepatocellular carcinoma(HCC) operation and 6 cases of normal hepatic tissue were chosen as control,all fixed with 10%formalin and conventionally embedded with paraffin.Each specimen was consecutively cut into slices of 4um thickness.Slices were stained by the monoclonal antibody of EGFR,EGFR mutations(del) and EGFR mutations(p) with SP immunohistochemical method.The expression of EGFR and EGFR mutations(EGFR mutations(del) and EGFR mutations(p)) in hepatocellular carcinoma tissue and their relationship with clinical pathology were analyzed with X2 test and Spearman’s non-parametric analysis.Cox multi-factor regression analysis and Kaplan-Meier survival analysis were used to analyze the survival time of patients after operation and various parametersRESULTS:1.The positive rate of EGFR,EGFR mutations(del) and EGFR mutations(p) was 66.7%(4/6),0(0/6)and 0(0/6) respectively in normal hepatic tissue,and the positive rate of EGFR,EGFR mutations(del) and EGFR mutations(p) was 69.2%(54/78),21.8%(17/78) and 9.0%(7/78) respectively in hepatoma tissue.The expression of EGFR had no significance between normal hepatic tissue and hepatocellular carcinoma tissue(X~2=0.171,P=0.680),no expression of EGFR mutations was observed in the normal hepatic tissue.The positive rate of EGFR mutations(del) was higher than that of EGFR mutations(p)(X~2=4.924, P＜0.05).2.The expression of EGFR mutations was positively correlated with women(r=0.743,p＜0.05),and had a negative correlation with preoperative AFP value(r=-0.519,p＜0.05),hepatitis B(r=-0.567,p＜0.05), hepatic cirrhosis(r=-0.245,p＜0.05) and pathological grading (r=-0.273,p＜0.05);The expression of EGFR mutations(del) was positively correlated with women(r=0.453,p＜0.05),and had a negative correlation with preoperative AFP value(r=-0.243,p＜0.05),hepatitis B(r=-0.693,p＜0.05),hepatic cirrhosis(r=-0.328,p＜0.05) and pathological grading(r=-0.343,p＜0.05);The expression of EGFR mutations(p) in liver tissue was positively correlated with women(r=0.390,p＜0.05),and had a negative correlation with hepatitis B(r=-0.657,p＜0.05)and pathological grading(r=-0.347,p＜0.05).The remaining not-mentioned parameters were unrelated to the expression of EGFR and EGFR mutations.All of the above parameters had nothing to do with the expression of EGFR.3.Tumor pathological grading(P＜0.001) and preoperative AFP value(P＜0.001) were main influencing factors on postoperative survival time of patients with liver cancer in the group.Postoperative survival time had no significant difference between the patients with positive expression of EGFR and EGFR mutations and others with negative expression(P＞0.05).CONCLUSION:1.The expression of EGFR existed in normal hepatic tissue,the expression of EGFR mutations(del) and EGFR mutations(p) was not found in normal hepatic tissue,while EGFR、EGFR mutations(del) and EGFR mutations(p) all expressed in hepatocellular carcinoma.2.The expression of EGFR mutations and EGFR mutations(del) was positively correlated with women,and had a negative correlation with preoperative AFP value,hepatitis B,hepatic cirrhosis and pathological grading;The expression of FGFR mutations(p)in liver tissue was positively correlated with women,and had a negative correlation with hepatitis B and pathological grading.The remaining not-mentioned parameters were unrelated to the expression of EGFR and EGFR mutations.All of the above parameters had nothing to do with the expression of EGFR.3.The expression of EGFR,EGFR mutations(del) and EGFR mutations(p) in hepatocellular carcinoma was not an independent prognostic factor for patients with liver cancer.更多还原