【作者】 黄凌；

【导师】 吴一龙；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2011， 博士

【摘要】 肺癌是最常见的恶性肿瘤之一,近年来肺癌的发病率与死亡率已经位居恶性肿瘤之首。随着分子生物学研究的深入,肺癌的治疗模式正在发生转变,分子靶向治疗已经成为除手术、化疗、放疗等传统手段以外的重要治疗方法。靶向治疗的优势是特异性杀伤肿瘤细胞而相对少地损伤正常组织。表皮生长因子受体(epidermal growth factor receptor, EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitors, TKIs),包括吉非替尼和厄洛替尼,和EGFR的单克隆抗体西妥昔单抗等在肺癌治疗上获得的显著疗效是转化医学的成功范例。如何更加有效和更加完全地抑制这些通道以进一步改善患者生存,另外一些作用机制完全不同的靶向治疗的研究正在进行中。肺癌的主动免疫治疗方法将会延长患者寿命并提高其生命质量,但是合适的靶位点的选择仍是一个难题。MAGE-A3是CT (cancer/testis)基因家族中的一员,在除外睾丸组织的人类正常组织中该基因沉默,虽然睾丸细胞表达MAGE-A3基因,却由于缺乏Ⅰ类递呈分子使得睾丸细胞无MAGE-A3抗原表达。目前的研究表明肿瘤细胞表达的MAGE抗原肽与特定的HLA-Ⅰ类分子结合形成复合体能被机体特异性CTL细胞识别和杀伤,所以MAGE-A3蛋白被认为是真正的肿瘤特异性抗原,已经发现它在许多恶性肿瘤上表达,例如恶性黑色素瘤、NSCLC、膀胱癌、头颈部肿瘤、食道鳞状细胞癌和肝细胞癌等。治疗NSCLC的MAGE-A3蛋白质疫苗在早期的临床研究中显示出较好的疗效,2002年GSK生物制药公司启动的MAGE-A3重组蛋白联合AS02B免疫佐剂系统治疗完全切除后的ⅠB期或者Ⅱ期NSCLC的双盲、随机、安慰剂对照Ⅱ期临床研究发现,MAGE-A3 ASCI治疗组比安慰剂对照组的无疾病生存时间延长33%。据此,MAGE-A3 ASCI国际多中心双盲、随机、安慰剂对照Ⅲ期临床研究入组病灶完全手术切除后,无论是否接受辅助化疗的ⅠB、Ⅱ期或者ⅢA期NSCLC患者,其主要终点是无疾病生存时间,预计入组2270例患者,是目前评估NSCLC的候选治疗方法的最大规模的临床研究。该研究正在进行中,有望证实MAGE-A3 ASCI能够带给患者临床获益。目前发现,MAGE-A3在NSCLC患者的阳性表达率随人种的不同而有差异,已有报道全球总的阳性率26%,欧洲35.7%,台湾13%,新加坡11%,香港20%,韩国和日本均约30%。造成阳性率差异的原因很多,其一是研究方法的不同,不同的方法敏感度不同,目前采用的检测方法主要为免疫组化方法和RT-PCR方法,是造成MAGE-A3表达率不同的原因之一。另一方面,目前的研究结果认为,MAGE--A3的表达与各项临床因素相关。Josip Grah等人发现NSCLC患者中鳞状细胞癌(65.1%) MAGE-A3/4的阳性表达率明显高于腺癌(18.9%)。Sang Hyun Kim等学者发现韩国NSCLC的MAGE-A3/6的阳性表达率为50%,其中鳞癌64%高于腺癌的31.6%,男性56.0%高于女性的21.7%,与年龄、TNM分期和淋巴结转移情况无关。也有研究认为MAGE-A3的表达与临床分期相关,Ⅱ期(49.5%) NSCLC患者明显高于Ⅰ期(29.5%)。在GSK的Ⅱ期临床研究人群中发现各临床分期的MAGE-A3阳性表达率,ⅠA期16%,ⅠB期34%,Ⅱ期36%,Ⅲ期48%。据此,目前报道的MAGE-A3在肺癌的表达率不一的原因可为检测方法、人群种族、疾病分期等临床因素不同所致。MAGE-A3的表达与肿瘤患者生存的关系也有不同的观点。Sang Hyun Kim等认为MAGE-A3/6表达与NSCLC患者的生存无关。Gure AO等则发现NSCLC患者MAGE-A3的表达是疾病预后的独立相关因素。Perez D等学者发现MAGE-A3等CT抗原阳性表达的GIST患者的DFS明显比表达阴性患者短。巴西一项对喉癌的研究发现MAGE抗原的表达与总生存时间无关,而另一项关于头颈鳞癌的报道则认为,MAGE-A4表达阳性是与其他临床参数无关的独立预后指标。MAGE-A基因亚家族位于染色体Xq28上,其家族成员具有高度的相似度,其中MAGE-A3和MAGE-A6基因具有95%同源性。MAGE-A3有44个已知的SNP位点,其中12个与MAGE-A6共享。目前对于MAGE-A3基因SNP与MAGE-A3的表达水平以及对肿瘤病人的生存影响方面的研究尚未见到文献报道。最常见的EGFR突变是19外显子缺失(E19del[LREA缺失],发生率为45%)和21外显子突变(L858R,发生率40%)。关于EGFR作为NSCLC患者预测因子和预后因子的研究中,目前由于大多数报道集中于EGFR突变的药物治疗预测作用上,这些药物敏感性突变的预后作用还不清楚。关于EGFR基因突变和表达与MAGE-A3基因表达水平的研究尚无文献报道。本课题拟研究1. NSCLC患者MAGE-A3基因表达水平,2.已知5个MAGE-A3基因SNP位点人群频率,3.该人群EGFR基因表达水平与EGFR基因突变情况,通过分析MAGE-A3基因表达水平及SNP位点基因频率与年龄、性别、吸烟状况、组织学类型及肿瘤临床分期等的关系,进一步明确MAGE-A3在中国NSCLC患者的表达情况及SNP人群频率；分析MAGE-A3基因表达水平及SNP频率与生存时间的关系,寻找NSCLC潜在的预后因子；分析EGFR基因表达水平与基因突变及它们与MAGE-A3mRNA表达水平及SNP基因频率的关系,探讨两者之间可能存在的关联,为NSCLC患者的靶向治疗和免疫治疗提供实验室依据。本课题由三部分组成：第一部分NSCLC患者MAGE-A3 mRNA表达水平与生存和临床因素的关系目的MAGE-A3抗原在除了睾丸、胎盘以外的正常组织中不表达,而表达于NSCLC在内的多种肿瘤细胞,MAGE-A3抗原蛋白能够与HLA-Ⅰ分子结合被递呈后激活CTL细胞,激发人体主动免疫机制以杀灭肿瘤细胞。作为免疫治疗的靶点,MAGE-A3基因表达水平是选择合适治疗人群的基础。已有的研究对MAGE-A3表达水平及其与肿瘤临床分期、性别、年龄、及组织学类型等的关系报道不完全一致。本研究拟采用RT-PCR方法检测NSCLC患者肿瘤组织的MAGE-A3mRNA表达水平,分析MAGE-A3表达水平与临床参数的关系,以及MAGE-A3表达水平与生存时间的关系,探讨NSCLC患者潜在的预后因子,同时为MAGE-A3 ASCI治疗提供实验室依据。方法应用反转录和实时定量PCR方法检测了212例冰冻组织标本的MAGE-A3基因mRNA表达水平(包括112例腺癌和100例鳞癌)。MAGE-A3 mRNA表达阳性与临床参数的关系采用卡方检验,应用Kaplan-Meier法和log-rank检验进行生存资料分析。应用Cox回归分析对临床参数及MAGE-A3基因mRNA表达水平进行多因素生存分析。临床参数对MAGE-A3 mRNA表达水平的多因素分析应用二项分类Logistic回归分析。结果单因素分析表明MAGE-A3表达水平与年龄、性别、吸烟状态、组织学类型相关(P<0.05),与临床分期无关(P=0.622)。多因素分析表明,年龄、性别和组织学类型是肿瘤组织MAGE-A3 mRNA表达水平的3个重要的影响因素,P值分别为0.034,0.048,和0.016, OR(95%CI)值分别为1.984(1.052-3.742),0.440(0.195-0.993),2.216 (1.158-4.242)。MAGE-A3基因mRNA表达阳性患者的生存时间较阴性表达患者长(P=0.047)。分层分析表明,临床Ⅱ期患者(χ2=4.389,P=0.036)、非吸烟患者(χ2=7.890,P=0.005)、腺癌患者(χ2=7,066,P=0.008)MAGE-A3阳性表达与生存呈正相关。多因素分析表明,肿瘤临床分期是影响生存的独立预后因素(P<0.001, OR=2.013,95%C1:1.547-2.620)。结论年龄、性别和组织学类型是NSCLC患者肿瘤组织表达MAGE-A3的重要影响因素,老年、男性、鳞癌NSCLC患者肿瘤MAGE-A3表达水平较高。MAGE-A3基因mRNA阳性表达与生存时间呈正相关,各项临床参数中只有肿瘤临床分期是影响生存的独立预后因素。第二部分NSCLC患者MAGE-A3基因多态性/突变与生存和临床因素的关系目的生物标志物能够作为NSCLC的预后和预测因子,尤其是EGFR基因突变作为TKI药物治疗的敏感性预测因子的应用,使得基因多态性/突变状态与生存及临床参数的关系的研究越来越深入。MAGE-A3基因SNP位点的人群频率及基因突变情况与NSCLC患者生存时间及年龄、性别、组织学类型及临床分期等临床参数的关系未见文献报道。本研究拟通过PCR产物测序法,分析5个已知MAGE-A3基因SNP位点的人群频率及其与患者生存、临床参数的关系,发现肿瘤潜在的预后和预测因素。方法对212例(112例腺癌,100例鳞癌)NSCLC患者的冰冻组织标本的全基因组DNA标本进行分段PCR,应用PCR产物纯化测序方法,对MAGE-A3基因进行目标片段基因测序,用Bioedit软件读图并分析SNP位点的碱基频率,继而分析SNP基因型与生存时间及各临床参数的关系。采用卡方检验及Kaplan-Meier和log-rank检验生存分析进行统计学分析。结果201标本完成了相应DNA片段的测序工作。本研究入组的NSCLC人群中,SNP位点rs5970360基因型CC (139/201,0.692), CT (62/201,0.308); rs5925210基因型分别为CC (135/201,0.672), CG (66/201,0.328); rs5970361位点基因型的人群频率为CC (140/201,0.697), CA (61/201,0.303); rs5925211SNP位点除了两例为AT基因型且均为MAGE-A3表达阴性外,其余199例均为AA基因型,人群频率为AT (2/201,0.010), AA(199/201,0.990); rs35123853的SNP位点全部为GG基因型,后两个SNP基因型及人群频率与NCBI基因库中的资料不同。MAGE-A3基因420位点cattccc碱基插入突变及MAGE-A3基因rs5970360、rs5925210、rs5970361、rs5925211、rs35123853共5个已知SNP位点的基因型与MAGE-A3mRNA表达水平进行卡方检验,结果表明rs5970360、rs5925210和rs5970361位点基因型与MAGE-A3mRNA表达水平无关,P值分别为0.035、0.134及0.153。MAGE-A3基因表达水平与MAGE-A3基因420位点7碱基插入突变有关,该位点的野生型组、杂合突变组和纯合突变组这三组MAGE-A3基因mRNA表达阳性率分别为27.6%、5.9%、37.3%,三组之间的差别有统计学意义(P=0.035),420位点纯合子组(30.3%)阳性表达率高于杂合子组(5.9%)(P=0.045)。420位点基因型与性别(χ2=31.297,P<0.001)和吸烟(χ2=9.266,P=0.002)有关,多因素分析表明性别是420位点基因型的唯一影响因素(χ2=16.948,P<0.001)。本研究中MAGE-A3基因SNP位点rs5970360[CC/CT]、rs5925210[CG/CC]、rs5970361 [CC/CA]、rs5925211[AT/AA]的基因型及420位点的cattccc插入突变与患者生存时间的log-rank检验生存分析发现,各组基因型及420位点碱基插入突变的中位生存时间无统计学差异(P=0.434,0.370,0.895,0.212,0.940)。结论MAGE-A3基因mRNA表达水平与基因420位点的7碱基序列cattccc插入突变有关(p=0.035), MAGE-A3基因420位点基因型纯合子的基因阳性表达率高于杂合子,其纯合状态与性别有关,MAGE-A3基因420位点7碱基插入突变可能是MAGE-A3表达水平相关的功能性突变。MAGE-A3基因mRNA表达水平与SNP位点rs5970360[CC/CT]、rs5925210[CG/CC]、rs5970361 [CC/CA]、rs5925211 [AT/AA]、rs35123853 [GG]的基因型无关。SNP位点rs5970360[CC/CT]、rs5925210[CG/CC]、rs5970361 [CC/CA]、rs5925211 [AT/AA]、rs35123853[GG]基因型及420位点的7碱基序列插入与NSCLC患者的生存时间无关。第三部分NSCLC患者MAGE-A3基因表达水平与EGFR基因突变及其表达之间的关系目的EGFR基因突变已经被作为TKI小分子靶向药物治疗NSCLC的预测因子。最常见的NSCLC患者EGFR突变是19外显子缺失(E19del[LREA缺失],发生率为45%)和21外显子突变(L858R,发生率40%)。目前大多数报道集中于E19del(LREA)和L858R的药物治疗预测作用上,这些药物敏感性突变的预后作用还有争议。EGFR突变状态和基因表达水平的关系尚不明确。已有的资料表明EGFR突变优势人群为腺癌,而MAGE-A3基因表达的优势人群为鳞癌,EGFR基因突变和表达水平与MAGE-A3mRNA表达水平的关系及对生存的影响未见报道。本研究分析NSCLC患者EGFR基因突变和基因mRNA表达水平与MAGE-A3mRNA表达水平的关系,分析它们对生存时间的影响,以期明确EGFR和MAGE-A3基因可能存在的联系,为NSCLC患者联合应用TKI小分子靶向治疗和肿瘤主动免疫治疗提供理论依据。结果206例标本完成了EGFR基因突变检测,212例标本完成了EGFRmRNA表达水平qPCR检测,并对以上检测结果进行统计学分析。本研究人群EGFR基因总突变率为26.2%,外显子18突变率1.0%,外显子19突变率10.9%,外显子20突变率4.0%,外显子21突变率11.4%。单因素分析EGFR基因突变率与临床参数的关系,结果显示EGFR基因突变率女性患者高于男性(43.3%vs 19.9%,P=0.001),不吸烟患者高于吸烟患者(38.5%vs13.4%,P<0.001),腺癌高于鳞癌(46.2%vs6.0%,P<0.001)。多因素分析表明,吸烟状况和组织学类型是肿瘤组织EGFR基因突变的2个重要的影响因素,非吸烟患者的EGFR基因突变率高于吸烟患者(P= 0.016, OR=0.390,95%CI:0.181-0.840);腺癌EGFR基因突变率高于鳞癌(P<0.001, OR=0.092,95%CI:0.036-0.231)。EGFR突变患者的mRNA表达水平较EGFR基因野生型患者增高(4.02±0.40 vs 3.79±0.49,P=0.002)。多因素分析表明,EGFR基因外显子19突变和外显子21突变是影响肿瘤组织EGFR mRNA表达水平的2个重要的影响因素,χ2值分别为3.681、6.805,P值分别为0.055和0.009。MAGE-A3基因表达水平与EGFR基因突变(P=0.857)及基因表达水平(P=0.988)之间均无相关性。EGFR基因突变与MAGE--A3基因420位点突变及SNP位点rs5970360、rs5925210、rs5970361基因型无关。,EGFR基因mRNA表达水平与MAGE-A3基因纯合型插入突变及SNP位点rs5970360、rs5925210、rs5970361的优势基因型CC有关。EGFR基因突变状态与NSCLC患者生存时间无关(P=0.505)。EGFR基因mRNA表达水平以对数转换后取中位数为界值分为EGFR高表达组(lgEGFR≥median)和低表达组(lgEGFR<median,106例),生存分析表明两组的中位生存期无差异(56.9±8.5月(95%CI：40.3-73.6月)和76.0±13.75月(95%CI：49.0-102.9月,P=0.743)。分层分析发现55例EGFR突变患者(P=0.096)和106例EGFRmRNA (?)(?)表达患者(P=0.093) MAGE-A3阳性表达组生存时间有延长趋势。结论单因素分析表明女性、不吸烟、腺癌患者的EGFR基因突变率明显高于男性、吸烟、鳞癌患者,多因素分析表明吸烟状况和组织学分型是EGFR基因突变的2个重要影响因素。EGFR基因突变患者基因mRNA表达水平较野生型患者明显增高,其中19外显子和21外显子突变是影响EGFR表达的重要因素。MAGE-A3基因表达水平与EGFR基因突变及基因表达水平之间均无相关性。EGFR基因突变和基因表达水平均与生存时间无关。分层分析发现EGFR突变患者和EGFRmRNA低表达患者中MAGE-A3阳性表达组生存时间有延长趋势。EGFR基因突变与MAGE-A3基因420位点突变及SNP位点rs5970360、rs5925210、rs5970361基因型无关。EGFR基因mRNA表达水平与MAGE-A3基因420位点纯合型突变及SNP位点rs5970360、rs5925210、rs5970361优势基因型CC型相关。本研究通过研究212例本中心收集的NSCLC患者的组织标本的MAGE-A3和EGFR基因的mRNA表达水平和MAGE-A3基因多态性/突变及EGFR基因突变情况,分析这些数据与临床分期、性别、年龄、吸烟状况、组织学类型以及生存时间的关系,得到以下结论：1.年龄、性别和组织学类型是NSCLC患者肿瘤组织表达MAGE-A3的重要影响因素。2. MAGE-A3mRNA阳性表达与生存时间呈正相关,在临床Ⅱ期、非吸烟、腺癌患者更加明显。3. MAGE-A3基因420位点的7碱基序列cattccc插入突变与基因mRNA表达水平有关,与生存时间无关,考虑MAGE-A3基因420位点插入型突变可能是表达相关的功能性突变。性别和吸烟状态与该位点基因型(纯合子/杂合子)有关,多因素分析表明性别是重要的影响因素。4. MAGE-A3已知SNP位点rs5970360[CC/CT]、rs5925210[CG/CC]、rs5970361[CC/CA]、rs5925211[AT/AA]、rs35123853[GG]基因型与基因表达水平和患者的生存无关。5.吸烟状况和组织学分型是EGFR基因突变的2个重要影响因素。EGFR基因突变患者基因mRNA表达水平较野生型患者明显增高,多因素分析发现外显子19和21突变是影响EGFR基因mRNA表达水平的重要影响因子。6. EGFR基因]nRNA表达水平与MAGE-A3基因420位点纯合型突变及SNP位点rs5970360、rs5925210、rs5970361的优势基因型明显相关。7. MAGE-A3基因表达水平与EGFR基因突变及基因表达水平之间均无相关性。8. EGFR基因突变和基因表达水平均与生存时间无关。MAGE-A3抗原是NSCLC抗原特异性免疫治疗的靶点,该疫苗已经在Ⅱ期临床研究取得良好的疗效,并进入Ⅲ期临床研究阶段。我们的研究探讨MAGE-A3在NSCLC肿瘤组织表达的规律,发现MAGE-A3基因420位点7碱基插入型突变可能是影响MAGE-A3mRNA表达的功能性突变,为NSCLC的ASCI治疗分层选择优势人群提供实验室依据。研究表明作为NSCLC患者TKIs治疗的有效预测因子,EGFR基因突变状态和基因mRNA表达水平均不是NSCLC患者的预后因子。EGFR基因突变患者其mRNA表达水平增高。但无论是EGFR突变还是EGFRmRNA表达水平均与MAGE-A3表达无关。更多还原

【Abstract】 Lung cancer is among the most common malignancies, and remains, at the same time, among the most difficult to treat. Despite recent developments of early diagnosis and treatment, the statistics are sobering:lung cancer is by far the most common cancer worldwide in men and in women. Less than 15% of those diagnosed with lung cancer are alive 5 years following their diagnosis. Inability to predict which patients will benefit from which treatment results in significant morbidity and mortality. Increasing understanding of tumour biology, and in particular of the molecular pathways contributing to tumour growth, has led to the development of innovative medications for the treatment of NSCLC.MAGE-A3 is a member of the cancer/testis gene family. This human gene is silent in all normal tissues except the testis but with no antigen presentation because of the lack of classⅠpresenting molecules in the testis cells expressing the gene. TheMAGE-A3 protein is thus considered as a truly tumor-specific antigen with expression in a variety of tumors such as melanoma, NSCLC, bladder and head and neck cancer, but also squamous esophageal cancer and hepatocarcinoma. Recently, clinical investigation of cancer immunotherapy has been very active and several approaches have been evaluated in PhaseⅢtrials. In particular, the characterisation at the molecular level of tumour- specific antigens MAGE-A3 has led to develop Antigen-Specific Cancer Immunotherapeutic(ASCI) against NSCLC.MAGRIT is a PhaseⅢtrial has thus been designed to evaluate the efficacy of the MAGE-A3 immunotherapeutic in patients with MAGE-A3-positive, completely resected stageⅠB,ⅡorⅢA NSCLC, and who received or did not receive adjuvant chemotherapy. From interim premature data of MAGRIT trial, MAGEA3 expression levels were mainly different between histology (24% in AC vs 45% in SCC) as well as regional population (East Asian vs Europe and America). MAGEA3 expression is positive about 35.7% in Europe vs 26% in international groups, which further showed that MAGEA3 is positive in Taiwan (13%), Singapore (11%) and Hongkong (20%) that is lower than that in Korea and Japan groups (both～30%).Which reason underlying the differential MAGE A3 expression between regions is unknown. From point of gene expression regulation, potential differential polymorphisms in MAGE A3 gene between racial populations may contribute to differential gene expression regulation and the relevant mRNA expression level. For clinical patients selection, the relationship between MAGEA3 expression and EGFR mutation status and expression is unknown. As EGFR mutation has been greatly implicated in patient selection for specific EGFR inhibitors like Gefitinib or Erlotinib in clinical setting, one question to be asked is if MAGEA3 targeted patients are mutually exclusive or inclusive to EGFR expression/muation? Answer to this question will help to determine the extent of MAGEA3 used in combination with EGFR inhibitors.Chapter 1 Prognosis of MAGE-A3 mRNA expression levels and the realtionship with the clinical parameters of lung cancer patientsMethodsReverse transcriptase and real-time polymerase chain reaction (RT-PCR) were used to evaluate the MAGE-A3 mRNA expression levels in 212 frozen tissue samples (112 adenocarcinoma,100 squamous carcinoma) of stageⅠB,ⅡorⅢA NSCLC patients. A tumor sample was considered MAGE-3 positive when its MAGE-3 expression level after beta-actin normalization was equal or greater than the cut-off value determined by 1% Gerl (GSK company). The MAGE-A3 mRNA expression positive rate and clinical parameters were analyzed by Chi Square test. The Kaplan-Meier method and the log-rank test were used for survival analysis. ResultsOf these 212 patients,100 were squamous cell carcinoma,110 were adenocarcinoma; 102 wereⅠB stage,63 wereⅡB stage, and 47 wereⅢA stage; 151 were male,61 were female; The mean age of these patients was 60.91 years, range from 26-82 years. The MAGE-A3 positive rate is 27.4%(58/212) in all of these patients. Of them, the positive rate is higher in squamous cell carcinoma than in adenocarcinoma (37.0% vs 18.8%, X2=8.854, P=0.003), higher in male than in female (32.5% vs 14.8%, X2=6.846, P=0.009), and higher in smokers than in non-smokers (36.3% vs 19.1%, X2=7.863, P=0.005). Our results provided no evidence for a relationship between MAGEA3 expression and tumor stage, or age. However, with regard to certain subgroups, such as smokers, male, squamous cell carcinoma samples, MAGE-A3 gene expression positive rate was significantly higher than others (45.2% vs 20.0%, X2=13.974, P<0.005).The MAGE-A3 expression showed significant association with good survival(P=0.047). A multivariate analysis enabled us to conclude that the TNM staging is the only independent predictive factor.Conclusion The present study showed that the MAGE-A3 is highly positive expressed in male, smokers and squamous cell histology of patients with non-small cell lung cancer. We conclude that the MAGE-A3 expression is associated with good prognosis in NSCLC.Chapter 2 Study on the polymorphisms and mutations of MAGE-A3 gene in NSCLC patientsMethodsPotential MAGE-A3 gene polymorphisms analysis by PCR-base sequencing. Six pairs of primers were designed for amplifying fragments of MAGEA3 gene. PCR products will be purified and labeled with ABI BigDye 3.1 agents, and then for sequencing on the ABI 3130 genetic analyzer platform. Sequence results were collected and compared with a MGEA3 reference sequence in NCBI GenBank. The SNPs’genotype and clinical parameters were analyzed by Chi Square test. The Kaplan-Meier method and the log-rank test were used for survival analysis.ResultsThe sequences of SNPs’ sites (rs5970360、rs5925210、rs5970361、rs5925211、rs35123853) of MAGE-A3 gene in 201cases NSCLC patients were assayed by PCR and DNA sequencing methods. There are no relationship of sites’ genotype of rs5970360、rs5925210、rs5970361 with MAGE-A3 mRNA expression (P=0.035,0.134,0.153). MAGE-A3 mRNA expression level were associated with the 7-bases "cattccc" inserting at site420, the frequency of PCR products as MAGE-A3^ MAGE-A3 and -A6 and MAGE-A6 were 27.6%、5.9%、37.6% respectively(P=0.035). There were no relationship between these SNPs’genotype (rs5970360[CC/CT]、rs5925210[CG/CC]、rs5970361[CC/CA]、rs5925211[AT/AA]) and survival, the 7-bases inserting at site 420 as the same(P=0.434,0.370,0.895,0.212,0.940 respectively).ConclusionThere were no relationships between the polymorphisms and MAGE-A3 mRNA expression, and no relationship with survival.Chapter 3 Relationship between EGFR expression level and it’s mutation and MAGE-A3 mRNA expression in NSCLC patientsMethodsEGFR mutation status containing Exons 18,19,20 and 21 were amplified using 4 pairs of primers respectively, and then run PCR reactions, PCR products were subject to sequencing as mentioned above for MAGE A3 polymorphism analysis. EGFR expression level:The same cDNA samples used for MAGE A3 expression tests will be tested for EGFR expression as well. Specific primers and TaqMan probe for EGFR will be used for quantifying EGFR expression at mRNA level by real-time qRT-PCR reactions. Delta Ct will be acquired by subtraction of Ct values of EGFR and endogenous gene beta-actin.ResultsTotal 206 samples finished the detecting of EGFR mutation and EGFR RT-PCR. Of them, the positive rate is higher in adenocarcinoma than in squamous cell carcinoma (46.2% vs 6.0%, P<0.001), higher in female than in male (43.3% vs 19.9%, P=0.001), and higher in non-smokers than in smokers (38.5% vs 13.4%, P<0.001). Our results provided no evidence for a relationship between MAGEA3 expression and EGFR mutation along with EGFR mRNA expression, also no relationship between EGFR mutation/expression and survival. With regard to certain subgroups, such as 55 cases EGFR mutation patients(P=0.096) and 106 cases lower EGFR expression level patients(P=0.093), survival was prolonged in MAGE-A3 positive expression group. A multivariate analysis enabled us to conclude that the smoking status and histologic type is the independent predictive factor of EGFR mutation.ConclusionThe present study showed that the EGFR is highly positive expressed in female, non-smokers and adenocacinoma histology of patients with non-small cell lung cancer. A multivariate analysis was to conclude that the smoking status and histologic type is the independent predictive factor of EGFR mutation. MAGE-A3 expression got no relationship with EGFR mutation and mRNA expression.更多还原