【作者】 杨艳丽；

【导师】 陈丽荣；

【作者基本信息】 浙江大学 ， 肿瘤学， 2010， 硕士

【摘要】 背景：食管癌是发生在食管上皮组织的恶性肿瘤,占所有恶性肿瘤的2%,鳞状细胞癌是主要组织学类型。我国是食管鳞状细胞癌的高发国家,又是食管鳞状细胞癌死亡率最高的国家。由于早期症状不明显,绝大多数病人就诊时已属中晚期,预后不佳。目前,以手术为主,结合放化疗的综合治疗方案渐已成熟,但五年生存率仍低于20%。近年来,随着肿瘤分子生物学的发展,分子靶向治疗让癌症病人看到了新曙光,渐成为抗肿瘤治疗的重要组成部分,并已在各类肿瘤的治疗中取得了重大进展,尤其针对表皮生长因子受体(epidermal growth factor receptor,EGFR)的临床研究成果不菲。EGFR广泛分布在人体上皮细胞膜表面,并在多种上皮性肿瘤中存在高表达,其介导的信号转导效应包括肿瘤细胞的增殖、迁移、凋亡抑制和血管生成等诸多方面,因此,EGFR是目前最有应用前景的特异性治疗靶点之一。然而在肿瘤发生发展中不同人群可能存在着遗传学差异,而且目前还没有关于中国人食管鳞状细胞癌EGFR蛋白表达及其与基因状态和临床病理参数相关性等方面研究的实验数据,因此阐明EGFR蛋白表达与基因状态的生物学意义对于临床筛选适宜EGFR靶向治疗的患者有着十分重要的意义；此外,Akt, B-raf分别是EGFR受体信号转导PI3K／Akt和Ras/Raf/MEK/ERK通路中位于枢纽位置的相关分子,而且越来越多的资料显示,AKT激酶及其介导PI3K／Akt通路和Raf激酶及其介导的Ras/Raf/MEK/ERK通路在肿瘤的发生发展中起着重要作用。另有证据表明,大多数肿瘤并非由单一信号转导通路所支配,因此,针对多靶点治疗可能会取得更大疗效,但目前国内外对其在食管鳞状细胞癌中的联合表达研究并不多。在食管鳞状细胞癌中Akt和B-raf的表达是否与EGFR表达存在相关性以及是否能提示患者的预后目前尚无定论。为了帮助临床选择适合EGFR靶向治疗及多靶点联合治疗的食管鳞状细胞癌患者,我们进行了以下检测：(1)食管鳞状细胞癌EGFR蛋白过表达及基因状态分析；(2)食管鳞状细胞癌Akt和B-raf蛋白表达；(3)EGFR蛋白表达与基因状态之间的相关性；(4)EGFR蛋白表达与其下游分子Akt和B-raf蛋白表达的相关性；(5)以上各参数与临床病理参数之间的联系；(6)生存分析。目的：探讨EGFR蛋白过表达和基因扩增在食管鳞状细胞癌发生发展中的作用及两者的相关性,以及EGFR下游信号分子Akt和B-raf蛋白表达水平及与临床病理参数的联系,为食管鳞状细胞癌患者的EGFR靶向治疗及多靶点联合治疗的提供依据。方法：采用IHC分析食管50例正常鳞状上皮,50例鳞状上皮反应性增生,50例低级别鳞状上皮内肿瘤,42例高级别鳞状上皮内肿瘤,和105例鳞状细胞癌EGFR蛋白表达情况；采用FISH分析食管84例鳞状细胞癌,18例鳞状上皮内肿瘤,和18例正常鳞状上皮中EGFR基因状态；此外,采用IHC分析食管11例正常鳞状上皮,16例鳞状上皮内肿瘤,105例鳞状细胞癌Akt,B-raf蛋白表达情况。统计学处理用SPSS15.0软件完成,计数资料采用卡方检验,生存分析采用Kaplan-Meier;分析和Log Rank检验,P<0.05为差异具有显著性。结果：在食管50例正常鳞状上皮,50例鳞状上皮反应性增生,50例低级别鳞状上皮内肿瘤,42例高级别鳞状上皮内肿瘤和105例鳞状细胞癌中EGFR蛋白过表达率分别为0(0／50),2%(1／50),4%(2／50),76%(32／42),86%(90／105)。EGFR在正常鳞状上皮,鳞状上皮内肿瘤和鳞状细胞癌中具有显著统计学意义(P<0.05)。在105例食管鳞状细胞癌中有90例(86%)EGFR过表达,其中3+者55例,2+者35例,EGFR的表达与性别,年龄,肿瘤分化程度和淋巴结转移无关(均P>0.05),与肿瘤浸润浓度及TNM分期有关(P<0.05)。在84例食管鳞状细胞癌,18例鳞状上皮异型增生组织,和18例正常食管组织中EGFR FISH阳性率分别为33.3%(28／84),0(0／18)和0(0／18)。在84例食管鳞状细胞癌中,EGFR FISH阳性共28例,其中基因扩增20例(24%),高多体性8例(9.5%)；EGFR FISH阴性共56例,其中二体性35例,低三体性17例,高三体性4例。在食管鳞状细胞癌中,EGFR FISH阳性与肿瘤浸润深度,淋巴结转移,TNM分期有关(P<0.05),而与其它临床病理参数无关。EGFR基因状态异常与蛋白过表达相关(P<0.05)。Kaplan-Meier生存分析显示累积生存率与EGFR FISH检测相关,FISH检测阳性者,累积生存率小于FISH阴性者,Log Rank检验P<0.05。在11例正常食管组织中,16例鳞状上皮内肿瘤,和105例食管鳞状细胞癌中,Akt蛋白阳性率分别为82%(9／11),88%(14／16)和67%(70／105)；B-raf蛋白阳性率分别为27%(3／11),44%(7／16)和73%(77／105)；B-raf在食管不同组织中表达差异具有显著统计学意义(P<0.05),Akt在不同食管组织中表达差异无显著统计学意义(P>0.05)。Akt的表达与性别、淋巴结转移和TNM分期无关(P>0.05),而与年龄,肿瘤分化程度及浸润深度有关(P<0.05)。B-raf的表达与性别,年龄及肿瘤分化程度无关(P>0.05),而与淋巴结转移和TNM分期有关(P<0.05)。食管鳞状细胞癌中EGFR与B-raf的表达具有显著相关性(P<0.05),而与Akt表达不具有相关性(P>0.05)。结论：EGFR蛋白表达与基因异常参与食管鳞状细胞癌的发生发展,EGFR及其下游分子Akt和B-raf表达水平亦与食管鳞状细胞癌的生物学行为相关,为食管鳞状细胞癌的抗EGFR靶向药物治疗提供了重要的科学依据。更多还原

【Abstract】 Background:Esophageal carcinoma is one of the most frequent malignancies in China, and squamous cell carcinoma (SCC) is the main histological type. It generally has a poor prognosis because it is usually in an advanced stage at the time of diagnosis. Despite recent progress in chemotherapeutic, radiotherapeutic and surgical treatment, the five-year survival rate is still less than 20%. In recent years, with the development of tumor molecular biology, molecular target therapy has become an important treatment and has made significant progress, especially the epidermal growth factor receptor (EGFR) target therapy. EGFR is widely distributed in human epithelial cell membrane, and the majority of epithelial tumors have high expression of EGFR. Signal transduction effect mediated by EGFR includes tumor cell proliferation, migration, apoptosis inhibition, and angiogenesis and so on. Therefore, EGFR is one of the most promising molecular targets, but the eligibility criteria for EGFR target therapy in esophageal carcinomas are not fully established. There is little research to evaluate ESCC EGFR overexpression and gene status in China. But it is of importance for selecting patients who may respond to EGFR target therapy. In addition, Akt and Raf are located in the important position in the PI3K/Akt and Ras/Raf/MEK/ERK pathway respectively, which mediated by EGFR. More and more information demonstrate:AKT kinase and its mediated PI3K/Akt pathway, and the Raf kinase and its mediated Ras/Raf/MEK/ERK pathway play an important role in tumor occurrence and development. In addition, there are evidences that most of the tumors are not dominated by a single signal transduction pathway, so multi-target therapy may achieve greater efficacy. However, there is little research for their co-expression in esophageal carcinoma at home and abroad. Whether Akt or B-raf expression is well correlated with EGFR expression and whether they can prompt the prognosis of patients is currently uncertain. In order to help to identify those patients who may benefit from anti-EGFR therapy and/or multi-target therapy, we sought to clarify:(1) the ESCC EGFR expression and gene status; (2) the ESCC Akt and B-raf expression; (3) the correlation between EGFR expression and gene status; (4) the correlation of EGFR, Akt, and B-raf in ESCC; (5) the correlation between the above parameters and clinicalpathological parameters; (6) survival analysis.Objectives:To investigate the protein overexpression and gene status of EGFR and expression of EGFR downstream signal molecular Akt, B-raf in ESCC, and help to identify patients who may benefit from EGFR target therapy and mutil-target therapy.Methods:IHC was performed to analyze the expression of EGFR in 50 cases of normal esophageal tissue,50 cases of squamous epithelial reactive hyperplasis,50 cases of low grade intraepithelial neoplasia,42 cases of high grade intraepithelial neoplasia, and 105 cases of ESCC. FISH was performed to analyze the gene status of EGFR in 84 cases of ESCC,18 cases of squamous epithelial dysplasia, and 18 cases of normal esophageal tissue. In addition, IHC was performed to analyze the expression of Akt, B-raf in 11 cases of normal esophageal tissue,16 cases of squamous epithelial dysplasia and 105 cases of ESCC.Statistical analysis was performed using SPSS 15.0. x2 test and Kaplan-Meier survival analysis was performed, and P< 0.05 was considered statistically significant. Results:The IHC-positive rates of EGFR in 50 cases of normal esophageal tissue,50 cases of squamous epithelial reactive hyperplasis,50 cases of low grade intraepithelial neoplasia,42 cases of high grade intraepithelial neoplasia, and 105 cases of ESCC were 0(0/50),2%(1/50),4%(2/50),76%(32/42), and 86%(90/105) respectively. The difference of expression of EGFR among different esophageal groups had statistically significance (p<0.05). Among the 105 cases of ESCC, overexpression of EGFR was found in 90 cases (86%), of which 55 cases scored 3+for EGFR staining and 35 cases scored 2+staining. In ESCC, the expression of EGFR was significantly correlated with depth of invision and TNM stage (p<0.05), but not with other parameters. The FISH-positive rates of EGFR in 84 cases of ESCC,18 cases of squamous epithelial dysplasia, and 18 cases of normal esophageal tissue were 33.3%(28/84),0(0/18) and 0(0/18) respectively. In ESCC, EGFR gene amplification was found in 20(24%) cases, high polysomy in 8(9.5%) cases, disomy in 35 cases, low trisomy in 17 cases, high trisomy in 4 cases; EGFR FISH-positive was significantly correlated with depth of invasivion, lymph node metastasis and TNM staging(p<0.05). EGFR FISH-positive was significantly associated with overexpression of EGFR. The IHC-positive rates of Akt in 11 cases of normal esophageal tissue,16 cases of squamous epithelial dysplasia, and 105 cases of ESCC were 82%(9/11),88%(14/16), and 67%(70/105); B-raf 27%(3/11),44%(7/16), and 73%(77/105). The difference of expression of B-raf among different esophageal groups had statistically significance(p < 0.05), while that of the expression of Akt had no statistically significance. In ESCC, the expression of Akt was significantly correlated with age, differentiation and depth of invision(p< 0.05), the expression of B-raf was significantly correlated with lymph node metastasis and TNM stage(p< 0.05). The relationship of EGFR expression to B-raf in ESCC was significant(p< 0.05). Conclusions:EGFR overexpression and gene abnormality are involved in the tumorigenesis and development of esophageal squamous cell carcinomas. Expression of EGFR and its downstream molecules Akt and B-raf are related to biological behaviors of esophageal squamous cell carcinomas. Our data provided an important basis for anti-EGFR target therapy of esophageal squamous cell carcinomas.更多还原