【作者】 徐瑞凤；

【导师】 廖旺军； 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2011， 硕士

【摘要】 研究背景和目的表皮生长因子受体(epidermal growth factor receptor, EGFR)是一个具有酪氨酸激酶活性的细胞膜受体,属于细胞表面erbB受体家族中的一员。当EGFR与其配体结合后,受体发生磷酸化而引起细胞内一些适配器分子与之结合,从而引起下游一系列信号通路的活化。在众多EGFR细胞内信号转导通路中,RAS/RAF/MEK/ERK以及PI3K/AKT通路是其中最重要的两条通路。其主要参与细胞的增殖、生长、浸润、迁移、血管生成与存活等。鉴于EGFR及其下游信号传导通路和肿瘤发生、发展、侵袭、转移之间的密切关系,因而EGFR成为靶向治疗的重要靶点。当前以EGFR为靶点的分子靶向药物主要有2类：一类为针对EGFR胞外域的单克隆抗体,代表药物有西妥昔单抗、帕尼单抗；另一种是针对EGFR胞内部分的小分子酪氨酸激酶抑制剂,代表药物为吉非替尼和厄洛替尼。两类药物均能有效的阻止下游受体依赖的信号转导途径,包括ERK/MAPK和P13K/AKT信号转导通路。目前在胃癌中取得一定疗效的EGFR抑制剂主要是西妥昔单抗,多个Ⅱ期临床研究分别观察了西妥昔单抗联合不同的化疗方案一线治疗晚期胃癌的有效性和安全性。但治疗总的有效率并不高,并且其价格昂贵,因而限制了其临床合理应用。如何准确预测西妥昔单抗的疗效,做到“有的放矢”,从而实现个体化治疗显得尤为重要。但西妥昔单抗在胃癌中疗效的预测因子相关研究较少。目前已证实K-ras突变型的结直肠癌患者不能从西妥昔单抗的治疗中获益,后续的研究报道EGFR信号传导通路上重要的调节因子B-raf、PIK3CA的突变及PTEN表达缺失的结直肠癌患者亦不能从西妥昔单抗的治疗中获益。是否像结直肠癌一样,K-ras、B-raf、PIK3CA基因突变及PTEN表达缺失是西妥昔单抗治疗获益的负性预测因子呢?因此,有必要了解中国人胃癌中K-ras、B-raf、PIK3CA基因的突变频率。基因的突变频率不但受地域,种族的影响,也与DNA质量、样本中肿瘤细胞的相对含量及检测方法有很大关系。目前临床上常用基因突变的检测方法为直接测序法,但传统的测序方法敏感度低,要求标本中至少20%的肿瘤含量。并且临床检测所用标本大多数为石蜡包埋组织,其DNA完整性较差。且由于肿瘤遗传异质性,肿瘤细胞与正常细胞混杂。因此应用直接测序法检测基因突变成功率相对较低,且检测流程复杂漫长,数据分析要求高、不易普及。本研究采用ARMS法检测K-ras、B-raf、PIK3CA基因突变,以了解K-ras、B-raf及PIK3CA基因在胃癌中的突变频率。ARMS (amplification refractory mutation system)即突变特异性扩增系统,是等位基因特异性PCR与荧光探针技术的综合。该方法灵敏度高,只需1%的肿瘤含量,且检测流程简单快速,数据分析要求低。胃癌是消化道最常见的恶性肿瘤,在世界范围内因肿瘤死亡的病例中,胃癌占第二位,严重地影响了人类的健康。和其它恶性肿瘤一样,胃癌的发病机制非常复杂,是多基因、多因素作用的结果。据报道,胃癌的发生与一百多种基因及其蛋白产物相关,包括癌基因、抑癌基因、细胞周期相关基因等。多种致癌因素在不同阶段作用于不同的基因,导致基因结构及表达水平的改变,其中包括癌基因的异常激活和抑癌基因的失活。RAS是一种癌基因,在细胞信号传导通路中起关键作用。RAS基因家族包括3个结构相似的基因即K-ras, H-ras和N- ras,编码相对分子量为21 kDa的膜相关蛋白,即p21蛋白。p21蛋白是细胞信号传递的枢纽,它的激活可导致细胞无限制增殖和永生化,致使肿瘤发生。关于H- ras的研究甚少。研究报道,H- ras基因mRNA在正常腮腺组织内处于不表达或微弱表达状态,而涎腺样囊性癌组织(SACC)中H- ras呈高水平异常表达,且与SACC的恶性程度、浸润性和转移性等密切相关。PTEN是近年来发现的一种肿瘤抑制基因,研究表明PTEN是唯一一种可以使脂类去磷酸化的抑癌基因,它可以使PIP3去磷酸化为PIP2,使其失去第二信使的作用,从而下调PI3K/AKT信号通路。然而在人类多种恶性肿瘤组织中,都存在PTEN表达和功能的异常。基于上述的研究背景和存在的问题,本研究目的如下：(1)本研究采用ARMS法检测K-ras、B-raf、PIK3CA基因突变,以了解K-ras、B-raf及PIK3CA基因在胃癌中的突变频率,进一步了解K-ras、B-raf、PIK3CA基因突变状态对患者生存的影响；(2)采用免疫组化的方法检测H-ras、PTEN在胃癌中的阳性表达率,以了解H-ras、PTEN的表达对患者生存的影响,及H-ras、PTEN的表达和患者性别、年龄、肿瘤的分化程度、转移灶数目之间的关系。研究方法1.K-ras、B-raf、PIK3CA基因突变频率的检测从1986位胃癌患者中筛选出143例符合入选标准的随访资料完整的Ⅳ期胃癌患者,提取样本DNA。应用ARMS法对K-ras常见突变类型G12D、G12A、G12V、G12S、G12R、G12C、G13D进行检测；对B-raf所报道的常见突变类型V600E进行检测；对PIK3CA所报道的外显子9常见突变类型E542K/E545D、E545K,外显子20常见突变类型H1047R、H1047L进行检测。分析K-ras、B-raf、PIK3CA基因突变状态与患者生存期之间的关系。2.H-ras、PTEN在胃癌中的表达和临床意义从1986位胃癌患者中筛选出143例符合入选标准的随访资料完整的Ⅳ期胃癌患者。应用免疫组化的方法分别检测H-ras、PTEN蛋白在胃癌中的表达情况；分析H-ras、PTEN蛋白的表达与患者各临床病理参数(性别、年龄、肿瘤分化程度、肿瘤转移灶数目)之间的关系；分析H-ras、PTEN蛋白的表达与胃癌患者生存期之间的关系。3.统计学方法应用SPSS 13.0软件进行统计学处理。对K-ras、B-raf、PIK3CA基因突变状态与患者生存期之间的关系进行Kaplan-Meier法生存分析；用χ2检验和Fisher’s精确概率法分别对H-ras、PTEN蛋白的表达与患者各临床病理参数(性别、年龄、肿瘤分化程度、肿瘤转移灶数目)之间的关系进行统计学分析；对H-ras、PTEN蛋白的表达与胃癌患者生存期之间的关系进行Kaplan-Meier法生存分析。研究结果1.K-ras、B-raf、PIK3CA基因的突变频率所检测的样本中K-ras、B-raf、PIK3CA基因的突变例数分别为2、1、4例,突变频率分别为1.4%(2/143)、0.7%(1/143)、2.8%(4/143)。2例K-ras突变,突变类型均为G12D；1例B-raf V600E突变；4例PIK3CA突变,突变类型为H1047L、E542K/E545D各1例,E545K 2例。所有样本中未检测到2种或2种以上突变类型。2. K-ras、B-raf, PIK3CA基因突变状态与患者生存期之间的关系Kaplan-Meier法生存分析结果显示：K-ras突变型胃癌患者的生存期显著低于K-ras野生型患者(χ2=8.128,P=0.004),而B-raf、PIK3CA基因不同突变状态患者生存期之间无显著差异。3.H-ras蛋白的表达及其与患者各临床病理参数之间的关系排除脱片和无法判定结果的样本,123例样本中,H-ras蛋白表达阳性者78例,阴性者45例,阳性表达率63.4%(78/123)。统计结果显示H-ras蛋白表达和患者的性别、年龄、肿瘤的分化程度及肿瘤的转移灶数目无显著相关性,并且H-ras蛋白的表达和患者的生存期之间亦无显著相关性。4.PTEN蛋白的表达及其与患者各临床病理参数之间的关系排除脱片和无法判定的样本,100例样本中,PTEN蛋白表达阳性者89例,阴性者11例,阳性表达率89.0%(89/100)。PTEN蛋白表达阳性和阴性患者中,位生存时间分别为10.7个月、5.6个月。PTEN蛋白表达阳性和阴性患者生存期之间差异有统计学意义(χ2=5.198,P=0.023)。统计结果示PTEN蛋白的表达和患者的性别、年龄、肿瘤的分化程度无关,和肿瘤的转移灶数目密切相关,转移灶数目大于3个的患者,PTEN蛋白表达阳性率较低,具有统计学意义(P=0.024)。研究结论1.在所检测的Ⅳ期初治胃癌患者中,K-ras、B-raf、PIK3CA突变频率较低。2.K-ras不同突变状态患者之间的生存期有显著差异,而B-raf和PIK3CA不同突变状态患者之间的生存无显著差异,提示K-ras基因状态可以在某种程度上预测患者的预后。3. H-ras蛋白在胃癌中的阳性表达率63.4%,但统计分析结果显示H-ras蛋白的表达和患者的性别、年龄、肿瘤的分化程度、肿瘤的转移灶数目及患者生存期之间无显著相关性,但H-ras蛋白在胃癌发生、发展中的作用,值得进一步探讨。4.PTEN蛋白在胃癌中的阳性表达率89.0%,统计分析结果显示PTEN蛋白表达和患者生存期之间显著相关,其表达阳性的患者生存期较长。PTEN蛋白的表达和患者的性别、年龄、肿瘤的分化程度无关,和肿瘤的转移灶数目密切相关。提示PTEN蛋白表达下调或缺失在胃癌的侵袭转移及患者不良预后中发挥重要作用。更多还原

【Abstract】 Background and objectiveEpidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that belongs to erbB family. Ligand binding to the extracellular domain leads to EGFR activation,which then homodimerizes, resulting in the phosphorylation of the intra-cellular tyrosine kinase initiating a series of intracellular signals. The Ras/Raf/MEK /ERK and PI3K/PTEN/AKT signaling cascades are two main pathways which play critical roles in the transmission of signals from EGFR to regulate cell proliferation, growth, invasion, migration, angiogenesis, survival and so on.Because of EGFR and its downstream signaling pathways playing critical roles in tumorgenesis,it has been an important target for molecular targeted therapy.Two main anti-EGFR strategies are currently in clinical development:monoclonal anti-bodies that are directed at the ligand-binding extracellular domain. Cetuximab and panitum-umab are the typical drugs;Another is low-molecular-weight tyrosine kinase inhibit-ors such as Gefitinib and Erlotinib which compete with ATP for binding to the tyro-sine kinase portion of the receptor.Both the two main durgs could effectively prevent the downstream receptor-dependent signal transduction pathways, including ERK/ MAPK pathway and P13K/AKT pathway. At present, Cetuximab has been used to treat gastric cancer.The efficacy and safety of cetuximab combined with different chemotherapy regimens in first-line treatment of advanced gastric cancer were obs-erved in several phase II clinical studies. However, the low treatment efficiency and it’s expensive price limited its clinical reasonable applications. How to predict the efficacy of cetuximab in treatment gastric cancer is especially important.Neverthel-ess, the research about predictors of cetuximab in treatment gastric cancer is very less. It has been confirmed that K-ras gene mutation could lead to failure of anti-EGFR monoclonal antibody therapy.The follow-up studys reported that B-raf, PIK3CA mutation and PTEN loss in patients with colorectal cancer could not get benefits from the treatment of cetuximab. Whether it is the same as colorectal cancer, K-ras、B-raf, PIK3CA mutation and PTEN loss is the negative indicator to predict the efficacy of anti-EGFR therapy. Therefore, it is necessary to understand K-ras, B-raf, PIK3CA mutant frequency in Chinese gastric cancer.Gene mutant frequency is not only influenced by geographical area, racial,but also by the DNA quality, content of tumor cells and detection methods. Currently, DNA direct sequencing is one of the most widely used methods for analysing gene mutation. But sequencing is not exquisitely sensitive which requires at least 20% tumor content in the samples. Nevertheless the samples for clinical testing are most of paraffin-embedded tissue in which DNA integrity is poor. In addition, tumour specimens are heterogeneous. They can contain surrounding and infiltrating normal cells, and not all tumour cells are identical.Thus,the success rate of DNA direct seq-uencing is relatively low in detecting the gene mutations. In addition, this assay is routinely a long testing process, resulting in difficult to spread. In this study, ARMS assays were used to detect K-ras、B-raf and PIK3CA mutations in order to under-stand the mutant frequency in gastric cancer. Amplification refractory mutation system(ARMS) is the combine of allele-specific PCR and fluorescent probe. This assay is sensitive, routinely being able to detect at least 1% mutant in a normal DNA background, and are quick and easy to use.Gastric cancer is the most common gastrointestinal malignancy and the second most common cause of cancer death worldwide with approximately one million cases diagnosed annually. Like other malignancies, the pathogenesis of gastric cancer is very complex, which is the results of multiple genes and factors. It has reported that the incidence of gastric cancer was related with over one hundred kinds of genes and their protein products including oncogenes, tumor suppressor genes, cell cycle-related genes. Carcinogenesis is a multistep process featuring the accumulation of several ge-netic alterations, including the abnormal activation of oncogenes and the inactivation of tumour suppressor genes. Ras constitutes a family of proto-oncogenes which plays a key role in the cell signaling pathways encoding small G-proteins with a molecular weight of 21 kDa (p21). Three different members contribute to the RAS gene family known as K-ras, H-ras, and N -ras. P21 protein plays a key role in the cell signal-ing and its activation can lead to unlimitedproliferation and immortalization of cells, resulting in tumorigenesis. It has reported that H -ras mRNA in normal parotid gland tissue is weak expression or absence of expression. But in salivary adenoid cystic car-cinoma (SACC), the H -ras shows abnormal expression of a high level which is clo-sely correlated with the differentiation, infiltration and metastasis of SACC.PTEN has been identified as the the only tumor suppressor gene that can make PIP3 dephospho-rylate into PIP2 resulting in losting the role of second messenger.Thus, it can down deregulate PI3K/AKT signaling pathways. However, there is a PTEN expression and function abnormalities in a variety of human malignant tumors.Based on the above background and problems, the purpose of this study are as follows:(1) Apply ARMS assay to detect K-ras,B-raf, PIK3CA gene mutations in order to understand the mutant frequency in gastric cancer, and further understand the influences of K-ras,B-raf, PIK3CA mutant status on the survival of patients with gastric cancer; (2) Apply immunohistochemical method to detect H-ras, PTEN exp-ression in gastric cancer in order to understand the relationship between H-ras,PTEN expression and patients’ survival,gender, age, tumor differentiation and the number of metastasis.Research Methods1.The detection of K-ras,B-raf, PIK3CA mutant frequency143 cases were screened which met the inclusion criteria from 1986 patients with gastric cancer of stage IV.DNA was extracted from paraffin-embedded tissue of gast-ric cancer. The commonly reported mutant types G12D、G12A、G12V、G12S、G12R、G12C. G13D of K-ras, V600E of B-raf, mutations in exon 9 (E542K/E545D and E545K) and mutations in exon 20 (H1047R and H1047L) of PIK3CA were dete-cted respectively by ARMS assay in real-time PCR reactions.Analyze the relationship between K-ras, B-raf, PIK3CA mutant status and the survival of patients.2.The expression H-ras, PTEN protein in gastric cancer and its clinical significance143 cases were screened which met the inclusion criteria from 1986 patients with gastric cancer of stageⅣ. H-ras, PTEN protein expression in gastric cancer were detected by immunohistochemical methods. Analyze the relationship between H-ras, PTEN protein expression with clinicopathological parameters including patients’sur-vival,gender, age, tumor differentiation and numbers of metastasis.3.Statistical MethodsSPSS 13.0 software was used for statistical analysis. Kaplan-Meier survival analysis was used to analyze the relationship between K-ras, B-raf, PIK3CA mutant status and patients’ survival. And the relationships between the expression of H-ras, PTEN protein and their clinical-pathologic features including patients’ survival,gender, age, tumor differentiation and numbers of metastasis were analyzed using Chi-square test and Fisher’s exact test.Meanwhile,the relationships between the expression of H-ras, PTEN protein and survival of patients with gastric cancer were analyzed by Kaplan-Meier survival analysis.Results1.Frequencies of K-ras, B-raf and PIK3CA mutationsIn the samples collected from 143 GC patients, the mutant frequency of K-ras, B-raf and PIK3CA was 1.4%(2/143),0.7%(1/143),2.8%(4/143) respectively. No samples bear 2 or more mutations. All the two mutatd cases of K-ras carried G12 D mutation. One cases of B-raf carried V600E mutation.There were one cases carri-ed E542K/E545D mutation,while two cases E545K mutations of PIK3CA. 2.The relationship between K-ras,B-raf, PIK3CA mutant status and patients’surv-ivalThe results of Kaplan-Meier survival analysis showed that survival of patients with K-RAS mutant-type was significantly lower than that of K-ras wild-type (χ2= 8.128, P=0.004). But, there were no significant differences in cumulated survival between patients with mutations of B-raf, PIK3CA gene and those with wild-type ones.3.The relationships between the expression of H-ras protein and their clinical-patho logic featuresEliminating the off-chip and undeterminable samples, there were 123 samples left. Positive expression of H-ras protein was detected in 78 cases,while negative expression in 45 cases. The positive rate of H-ras protein was 63.4%(78/123). There were no significant associations between the expression of H-ras protein and patient’ gender, age, tumor differentiation and numbers of metastasis. 4.The relationships between the expression of PTEN protein and their clinical-pathol-ogic featuresPositive expression of PTEN protein was detected in 89 cases,while negative expression in 11 cases. The positive rate of PTEN protein was 89.0%(89/100). The median survival time of patients who detected positive expression and negative expr-ession of PTEN protein was 10.7months and 5.6 months respectively. There were significant differences in cumulated survival between patients with positive expressi-on of PTEN protein and those with negative ones (χ2=5.198, P=0.023).The statistic results show that there were no significant associations between the expression of PTEN protein and patient’gender,age,tumor differentiation.But there was significant association between the expression of PTEN protein and numbers of metastasis. The positive rate of PTEN protein in patients whose metastatic lesions numbers more than 3 was significant lower than that of less than 3 (P=0.024)Conclusions1.The mutant frequency of K-ras,B-raf, PIK3CA detected in the initial treatment of gastric cancer patients with stageⅣwas low.2.There was significant differences in cumulated survival between patients with mut-ations of K-ras gene and those with wild-type ones,but not for B-raf,PIK3CA genes genes.This results suggest that the status of K-RAS may predict the prognosis of patients in a certain way.3.The positive rate of H-ras protein was 63.4%.The statistic analysis showed that th-ere were no significant associations between the expression of H-ras protein and patient’ gender, age, tumor differentiation and numbers of metastasis. However, it is worth further studying the role of H-ras protein playing in pathogenesis and develo-pment of gastric cancer.4.The positive rate of PTEN protein was 89.0%. There were significant differences in cumulated survival between patients with positive expression of PTEN protein and those with negative ones.The survival of patients with positive expression of PTEN protein was longer than that of negative ones.The statistic analysis showed that there were no significant associations between the expression of PTEN protein and patient’ gender, age, tumor differentiation. While, the expression of PTEN protein correlated closely with numbers of metastasis. The results suggest that PTEN loss or its’low level expression play an important role in metastasis and poor prognosis of patients with gastric cancer.更多还原