食管癌预后和单核苷酸多态性的关系
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摘要
1.研究背景
食管癌是世界上最常见的六大恶性肿瘤之一,其发病率位于全世界恶性肿瘤发病率的第八位,死亡率居全球癌症死亡率的第六位。我国是世界上食管癌发病率和死亡率最高的国家,在中国最常见的十大恶性肿瘤死亡率中,食管癌占第四位。中国每年食管癌发病和死亡人数占全世界的一半以上
食管癌预后很差,中晚期食管癌的自然生存7-8个月,治疗后的生存情况也很不令人满意,5年生存率在20%左右,虽然治疗方式有了很大的改进,然而其5年生存率几十年来未见明显提高。
“三早”工作已引起普遍重视,但食管癌患者就诊时80-90%属中晚期是目前的现实,提高这一大组病人的生存自然也就成为了改善食管癌肿瘤防治现状的一个决定性环节。中晚期患者生存普遍较差,但其中经治疗干预后仍有20%能长期生存,这就提出了一个科学问题,是什么原因造成了相似人群,却有不同的预后?无论在发病机制、自然生存、临床预后等方面,食管癌都呈现出明显的个体化特征。是环境因素的作用,还是先天遗传的差异?比较一致的看法是遗传和环境相互影响共同作用的结果,但二者的作用,孰轻孰重,以及所占比重尚不完全清楚。
预后研究历来都是广大科研和医务工作者关注的焦点,寻找预后的内在机制、建立预后客观的评价体系,进而能够对预后风险及早进行干预,对改善患者的生存、延长病人的生命,具有现实而重要的意义。
既往的研究多集中在外界环境、表观症状、临床病理特征等相关因素和预后的关系,如TNM分期、生活习惯、生存环境、治疗方式等。已取得了一些共识和成果,但还存在很多的争议和疑问,离临床实际工作要求尚有很大距离,足可见该问题的复杂性。
“所有医学都是遗传学”,任何疾病的发生、发展、转归及临床表型的背后都需要一定的遗传物质基础来支配。随着分子生物学和遗传学的发展,分子检测、基因筛选等成为了现实,人们在注意多种临床表观现象研究的同时,也更加重视这些现象背后的分子机制、遗传背景。基因和疾病的关系成为目前研究的热点,并取得了可喜的成果,如癌基因、抑癌基因、药物敏感基因、预后基因等等。预后因素的研究也进入到了一个新的领域,深入到了细胞甚至蛋白、基因等水平。
食管癌是一种复杂疾病,又称多基因病或多因子病。目前,人类基因组计划的完成和单核苷酸多态的单体型图谱构建完成(HapMap计划),为肿瘤的遗传分子基础研究提供了大量的数据支持。同时,经济、高效大通量基因分型技术,在一个反应内可同时检测到几万、几十万个SNPs(单核苷酸多态性)位点成为可能,即全基因组关联分析技术(Genome-wide Associate Study, GWAS)。本项目组运用GWAS技术发现了18个和食管癌密切相关的SNPs位点,在此基础上,将18个SNPs位点及其相关的基因和食管癌预后联系起来,对食管癌预后的分子机制进行研究。
与以往有关食管癌预后分子机制方面的研究相比,本研究具有以下特点：
1.检测的SNPs位点多。18个SNPs位点是本项目组利用GWAS技术对中国汉族1077食管癌病例和1733正常对照的506,666个位点中筛选发现的。和既往只限于个别基因和预后关系的研究相比,其覆盖面广、代表性强,具有更明显的种族特色和针对性。
2.样本量大,临床病理资料全,进入观察项目多。首先从9703病例中随访出有明确生存期的病人共有2290例,然后从21个预后相关项目中选择了13个常见的临床病理因素进行分析。
3.临床和基础相结合。预后看似个临床问题,其背后隐藏着复杂的分子生物学机制,把预后及相关临床病理因素和单核苷酸多态性研究相结合,既注重临床表型,又注重分子机制探讨。
4.流调工作和实验室验证相结合。把经过流调和统计分析后得到的食管癌预后相关基因在癌组织中进行蛋白表达检测,在分子水平验证基因对预后的影响。初步进行了基因的功能研究。
5.具有良好的前期工作基础。易感基因对肿瘤发生的作用和对肿瘤的进展、预后作用具有一致性,二者具有相似的生物学基础和研究方法。本次研究是本项目组“食管癌易感基因GWAS研究”的一个延伸,GWAS前期工作结果已发表在了Nature Genetics杂志上。
综上观点,本论文将分为三大部分：第一部分：食管癌预后和临床表型的关系；第二部分：食管癌预后及相关表型和单核苷酸多态性的关系；第三部分：预后相关基因在食管癌组织中的不同表达。
2.材料及方法
2.1研究对象
2.1.1研究对象入选标准
本研究主要是对病人进行生存分析,入选标准如下：
1)全部研究对象均为中国汉族人群；
2)均经手术治疗,排除放化疗因素的干扰；
3)有相关的临床病理资料(TNM分期、大体类型、食管癌部位、分化程度、浸润程度、淋巴结转移、血型等)；
4)家庭住址或电话登记详细并能随访到患者本人或家属；
5)登记有详细的病案号和病理号,有完整的病史资料。
6)所有研究对象之间均无亲缘关系。
本研究共调查食管癌患者9703例,部分来自医院住院患者,部分来自新农合患者,经上述标准严格筛选后,通过电话和家访等形式共随访6793例,得到随访资料齐全并且是经过手术治疗的病例2290例,随访截止日期为2011年3月1同。所有患者未接受任何放疗和化疗。为了排除由于患者在不同地区或者医院就诊可能引起的重复调查,调查时保证每个家庭只有一位先证者。
2.1.2研究对象一般情况
本次研究系观察生存期及相关因素,所以所统计资料均来源于有明确生存期的2290例患者。其中男性1447例,年龄最小22岁,最大81岁,平均年龄54.69±8.746岁；女性843例,年龄最小29岁,最大86岁,平均年龄54.31±8.478。
2.1.3研究对象病理学诊断
由至少两名病理科医师确诊,全部符合食管鳞癌的标准。并采用到医院查阅病历的方式,追踪到有完整的临床TNM分期、淋巴结转移、浸润程度、分化程度、肿瘤类型、食管癌病变部位、血型、手术方式并有明确的手术日期等记录。
2.2流行病学调查
本研究主要采用问卷调查的方式,调查内容主要包括：一般情况(年龄、性别、籍贯等),食管癌肿瘤家族史,初次诊断时间及治疗方式等。回顾性调查病人既往住院病历档案,补充患者的食管癌临床病理资料(食管癌部位,大体类型、分化程度,浸润程度和淋巴结转移等)。然后80%采用电话追踪随访和20%的家访完成生存期随访,随访周期为3个月,随访截止时间为2011年3月。所有调查员均经过严格培训,问卷标准统一。
2.3 18个SNPs位点基因分型
采用Qiagen DNA试剂盒提取血样中DNA,测浓度,电泳,标化至15-20ng/μl,根据本研究团队在前期利用GWAS技术对内镜和病理确诊的1077食管癌病例和1733正常对照做了506,666个SNPs位点全扫,发现和食管癌密切相关的18个SNPs位点,然后进行引物设计,用Sequenom MassArray技术对472例有完整随访资料的患者在18个SNPs位点进行基因分型,经过人群吻合度检验,观察值和预期值符合Hardy-Weinberg平衡,具有人群代表性。然后通过官方HapMap网站或NCBI网站查询18个SNPs位点所在的染色体及所对应的基因,如rs12263737和rs2274223位点对应于PLCE1基因、rs6140125和rs13042395位点对应于C20orf54基因等。
2.4免疫组织化学
69例食管癌手术切除标本来自各个医院,其中浸润程度达到固有层、粘膜下层和浅肌层者25例、达到纤维膜或邻近器官者44例,所有患者术前未接受任何放、化疗,并均经组织学证实为食管鳞癌。其中,28例有完整的临床随访资料,并且69例组织均有对应患者的18个SNPs位点的分型。免疫组织化学实验方法采用卵白素-生物素-辣根过氧化氢酶复合物(ABC)法完成。采用本实验室已建立的ABC法,进行定性和定量分析。
2.5统计学分析
采用SPSS17.0统计软件分析,Cox-regression分析判断影响食管癌患者预后的年龄、性别、家族史、高低发区、食管癌部位、大体类型、病理分化程度、淋巴结转移、TNM分期等临床因素,用Hardy-Weinberg平衡检验18个SNPs位点的人群吻合度,Cox回归、Kaplan-Meier和Log-rank检验分析18个SNPs位点与生存的关系,卡方检验比较18个SNPs位点在不同的临床表型中的遗传差别和免疫组化结果,检验标准取α=0.05。
3结果
3.1食管癌患者临床表型和预后的关系
3.1.1年龄分布与生存时间总体之间无统计学差异(Log-rank P=0.061)。但Cox回归分析结果显示65岁以上年龄组患者较35岁(包括35岁)以下年龄组死亡风险降低了45.7%(HR=0.643),同时差异达到统计学意义(P=0.023)。男、女生存时间没有明显差异(Log-rank P=0.104),男性患者较女性患者死亡风险增加(HR=1.078),但是差异未达到统计学意义(P＝0.137)。
3.1.2三种不同的好发部位在总体生存时间上差异并无统计学意义(Log-rankP＝0.342)。食管中段癌较下段癌死亡风险增加(HR=2.691),但差异未达到统计学意义(P=0.098)；食管上段癌较下段癌死亡风险增加(HR=2.008),差异有统计学意义(P=0.045)。
3.1.3食管癌总体生存在四种大体形态之间无统计学差异(Log-rank P=0.114),蕈伞型、溃疡型、髓质型较缩窄型死亡风险均降低(HR=0.588；HR=0.65；HR=0.838),)但差异只有溃疡型有统计学意义(P=0.05)。
3.1.4各种分化程度的患者生存时间无显著性差异(Log-rank P=0.542)。中分化和低分化患者较高分化患者死亡风险均增加(HR=1.762；HR=1.413),但是差异均未达到统计学意义(P=0.277；P=0.204)。
3.1.5早、中、晚期患者之间生存上存在显著的差异(Log-rank P=0.000),中、晚期患者较早期患者死亡风险增加(HR=1.702；HR=2.154),晚期患者与早期之间死亡风险差异有显著性统计学意义(P=0.004)。
3.1.6浸润程度达到肌层和纤维膜层的患者较粘膜层的患者生存时间较短(Log-rank P=0.028)。并且浸润程度达到肌层和纤维膜层的患者较粘膜层得患者死亡风险增加(HR=1.676；HR=1.752),且纤维膜层与粘膜层之间死亡风险差异有统计学意义(P=0.016)。
3.1.7无淋巴结转移的患者较有淋巴结转移的患者生存时间长(Log-rank P=0.000),Cox回归分析结果显示有淋巴结转移的患者较无淋巴结转移的患者死亡风险增加(HR=2.159),差异有显著性统计学意义(P=0.000)。
3.1.8高发区患者生存时间优于中高发区,中高发区患者生存期优于低发区患者(Log-rank P=0.003), Cox回归结果显示,高发区患者死亡风险较低发区患者有所下降(HR=0.647),且差异有显著性统计学意义(P=0.001)。
3.1.9食管癌家族史阳性的患者生存时间优于食管癌家族史阴性的患者(Log-rankP=1.18E-06),Cox回归结果显示,食管癌家族史阳性的患者死亡风险较食管癌家族史阴性的患者低(HR=0.64),差异有统计学意义(P=2.53E-06)。
3.1.10手术年代分布与患者的生存时间之间存在统计学差异(Log-rankP=3.49E-52)。手术方式与患者的生存时间之间也存在统计学差异(Log-rankP=2.31E-21)。90年代、2000年及以后两组死亡风险较70年代手术的患者均降低(HR=0.666；HR=0.432),差异均有统计学意义(P=6.52E-1和P=2.85E-2),弓下吻合和颈部吻合较弓上吻合的死亡风险均降低(HR=0.943；HR=0.814),且颈部吻合与弓上吻合之间死亡风险差异有统计学意义(P=0.046)。
3.1.11不同血型分布与患者的生存时间之间不存在统计学差异(Log-rankP=0.329)；Cox回归分析发现：B型、O型两组死亡风险和A型的患者基本相似。(HR=0.988和HR=0.967),差异均无统计学意义(P=0.882和P=0.692)。
3.2食管癌预后和18个SNPs位点基因型的关系
3.2.1 AHDC1基因rs4908343位点A>G基因型分布中,与突变纯合基因型GG携带者相比,野生纯合基因型AA携带者死亡风险降低(HR=0.54,95%CI=0.30-0.99)。与携带AA/GA基因型者相比,GG基因型携带者生存时间较短(Log-rank P=0.048)
3.2.2 PPP1R14A基因rs8102476位点T>C基因型分布的三种基因型携带者,携带TT和TC基因型的患者生存时间较短(Log-rank P=0.043)
3.3食管癌临床表型和18个SNPs位点基因型的关系
3.3.1 C20orf54基因rs6140125位点C>A基因型分布在食管癌不同的浸润程度(粘膜层、肌层、纤维膜)之间有统计学差异(P=0.007)。
3.3.2 IRX1基因rs11134032位点T>C基因型分布与食管癌发生部位有统计学差异(P=0.014)。
3.3.3 PPP1R14A基因rs8102476位点T>C基因型分布与食管癌淋巴结转移有统计学差异(P=0.037)。
3.3.4 PES1基因rs5753220位点C>T基因型分布与临床分期有统计学差异(P=0.033),且与淋巴结转移也有差异(P=0.044)。
3·4食管癌预后和基因多态性的分层分析
为控制潜在混杂因素对基因多态性和预后的影响,我们分别以年龄、性别、食管癌发生部位、大体类型、临床分期及浸润程度和淋巴结转移等为分层变量进行SNPs和预后关系的分层分析,结果如下：
3.4.1 AHDC1基因rs4908343A>G多态性改变分层后发现在男性患者组、家族史阴性组、食管癌晚期组、淋巴结转移阳性组中,与携带AA/GA基因型者相比,携带GG基因型者的死亡风险增加(HR=1.90; HR=1.85; HR=2.43; HR=2.66)
3.4.2 PES1基因多态性rs5753220C>T多态性改变分层后发现在高分化组、淋巴结转移阴性组中,,与携带TC/CC基因型者相比,携带TT基因型者死亡风险增加(HR=131.96；HR=1.98)
3.5食管癌预后和基因多态性的联合分析
将三个和预后相关的基因AHDC1基因rs4908343A>G、PPP1R14A基因rs8102476T>C和PES1基因rs5753220C>T位点基因多态性联合分析,发现携带预后不良基因型个数的增加,其死亡风险也增大(Log-rank P=0.01),趋势性P=-0.012。
3.6免疫组织化学结果
C20orf54和食管癌易感有关。预后分析中发现C20orf54与食管癌的浸润深度有关联,所以进行了该基因在食管组织中表达的验证。
C20orf54蛋白阳性表达部位主要在胞浆,食管癌组织阳性表达率为87%,但是阳性表达率和组织浸润程度、基因型及预后无关。
4.结论
4.1食管癌预后和临床分期、浸润程度、淋巴结转移、高/低发区、食管癌家族史、手术年代及手术方式有统计学相关。
4.2 AHDC1基因rs4908343A>G、PPP1R14A基因rs8102476T>C位点的基因多态性和食管癌预后相关；食管癌分层后PES1基因rs5753220C>T位点的多态性在高分化或者淋巴结阴性组和食管癌预后风险相关。携带危险基因型数目越多,死亡风险也随之增大。
4.3 C20orf54基因rs6140125位点、IRX1基因rs11134032位点、PPP1R14A基因rs8102476位点和PES1基因rs5753220位点基因多态性与食管癌临床表型相关。
4.4 C20orf54基因蛋白表达在食管癌组织中较强,但是表达率和肿瘤组织浸润程度、基因型分布及预后无关。
1. Background and Objective
Esophageal cancer (EC) is one of the six most common malignancies worldwide. The incidence of this disease is the eighth in the all carcinomas worldwide while its mortality rate is the sixth in the all cancers worldwide. Our country is the highest area of incidence and mortality rate of EC in the world while EC is the fourth most frequent cause of cancer-related deaths in China.The total incidence and deaths of EC in China takes up the half number of the world's each year.
The prognosis of EC is very poor. The natural survival time of advanced EC is 7-8 months approximately, the survival rate after treatment is also very unsatisfactory, and 5-year survival rate is 20% approximately. Although the therapy of EC has been improved greatly, the five-year survival rate of EC has not been significantly improved in recent decades.
Three early" works has been attracted widespread attention. But in fact, the 80-90% of patients with EC is in the middle and late period when they get to hospital. To improve the survival of this large group of patients has become a decisive link of the actuality of prophylaxis and treatment in EC. The prognosis of patients with EC in middle and late period is very poor generally, but after the intervention by the treatment there are still 20% of patients who can survive in a long run. It presents a scientific problem. What causes a different prognosis in a similar population? In terms of pathogenesis, natural survival and clinical outcome, EC emerged significant individual characteristics. The effects of environmental factors or differences in congenital genetic? More consistent view is the result of genetic and environmental interaction, but the role of the two, the pros and cons as well as the proportion of both has not been understood clearly.
The research for prognosis of disease has always been attracted by the researchers and doctors constantly. To search for the internal mechanism of prognosis and establish the impersonal evaluation system, it will proceed early intervention direct prognosis. It has realistic and important significance in the field to improve patient survival rates.
Previous studies were mostly concentrated in the interrelated factors such as external environment, apparent symptoms, clinical pathological characteristics of prognosis. These factors include TNM stage, living habits, environment, treatment methods and so on. Some consensus has been achieved, however there are still have many controversies and doubts. There is still a great distance from the work requirements of clinical practice. So we can see the complexity of this problem obviously.
"All medicines are genetics." The occurrence, development, prognosis and clinical phenotype of diseases, all have the genetic material basis. With the development of molecular biology and genetics, molecular detection and genetic screening have become reality. People who pay attention to the studies for multiple phenomenon of clinical must pay more attention to the molecular mechanisms and genetic background underlying these phenomena. The relationships between genes and disease has become the focus of current research and have achieved encouraging results, such as oncogenes, tumor-suppressor genes, drug-sensitive genes, the prognosis genes, etc. The research for prognostic factors also entered into a new field of the cell and gone so far as to protein and genes levels.
EC is a complex disease, also known as multi-genes disease or multi-factors disease. At present, the completion of the Human Genome Project and single nucleotide polymorphism haplotype map (HapMap plan) provide a large amount of data for the genetic study of tumor. At the same time, it becomes possibility that economic and efficient large-throughput genotyping technology can detect the tens of thousands or hundreds of thousands of SNPs within one response. It is known as the genome-wide association study (GWAS). Taking advantage of GWAS technology, our team found the 18 EC associative SNPs loci. On the basis, our study intends to observe the relationship between 18 SNPs loci and correlative genes and prognosis of EC, and then study the molecular mechanism of prognosis.
Compared with the previous studies for prognosis related to molecular mechanisms of EC, this project has the following characteristics:
(1). The number of detective SNPs loci is large.18 SNPs loci were selected and found from 506,666 SNPs loci of 1077 cases of patients with EC and 1733 controls in China by using GWAS technology. Compare with the previous studies for the relationships between limited genes and prognosis, this study have extensive coverage, intense representativeness, and more obvious racial characteristics and pertinence.
(2). The sample volume is large and the clinicopathological data are completed. The numbers of observational items are large. First of all, selected 2290 cases patients which have clear survival time by followed up from 9703 cases, and then selected 13 common clinicopathological factors which to be analyzed from 21 prognosis relative items.
(3). Combined the clinical study and basic medicine. Prognosis appears to be clinical problems, which has the complex molecular mechanisms. This study wants to know the interactions among the prognosis, clinicopathological factors and the single nucleotide polymorphisms.
(4). Combined epidemiological survey work and laboratory validation. Detect the protein expression of EC prognosis related genes in cancer tissue which were found by epidemiological inquiry and statistical analysis, and then validate the influence of prognosis by gene at the molecular level. Conduct a preliminary study for gene function.
(5). With a good foundation of preliminary work. The susceptibility genes of the tumor have consistency on the role of the occurrence, progress and prognosis, they have similar biology basis and research methods. This study is an extension of "the GWAS work for esophageal cancer susceptibility genes" by our project team, as well as GWAS results of preliminary work has been published in the journal of Nature Genetics.
In summary, this paper will be set to three parts. The first part:the relationship between EC prognosis and clinical phenotype; the second part:the relationship between EC prognosis with correlative phenotype and single nucleotide polymorphism; the third part:the expression changes of prognosis related genes in the tissues of EC.
2. Materials and methods
2.1 The object of study
2.1.1 The inclusion criteria of study object
This study aims at the survival analysis of patients, inclusion criteria as follows:
1) All subjects were Chinese Han population;
2) All subjects were treated by surgery, excluded radiotherapy and chemotherapy disturbances;
3) Relevant clinicopathological data (TNM stage, gross type, esophageal cancer location, the degree of differentiation, the degree of invasion, lymph node metastasis, blood type,etc);
4) Home address or phone were registered clearly and can follow-up to the patients or their families;
5) Medical record number and pathological number were detailed, as well as medical history informations were completed;
6) All of the subjects had no genetic relationship each other.
The study surveyed total of 9703 individuals with EC. These patients partly were hospital patients, partly from the new rural cooperative medical care system. After rigorous screening by the above criteria, followed up 6793 cases by telephone and home visits, get 2290 cases which have completed follow-up data and received surgical treatment. The follow-up deadline is March 1,2011. All the patients did not receive any preoperative radiotherapy and chemotherapy. In order to exclude duplicated investigation which results from the patients going to the different hospitals and regions, each family only has one proband at investigation.
2.1.2 The general situation of study object
The study observed survival time and related factors, so the statistics data were all from the 2290 cases of patients which had straightforward survival time. Therein,1447 male cases that the youngest was 22 years old, maximum age was 81 years and the mean ages were 54.69±8.746; and 843 female cases that the youngest was 29 years old, maximum age was 86 years and the mean ages were 54.31±8.478 years.
2.1.3 The pathology diagnosis of study object
All these patients were confirmed by at least two pathologists, meanwhile accord with diagnostic criteria for esophageal squamous cell carcinoma. And access medical records by going to the hospital, to track complete data included clinical TNM stage, lymph node metastasis, the degree of invasion, the degree of differentiation, tumor type, lesions location of EC, blood type, the type of surgical operation and a clear date of surgical operation.
2.2 The investigation of epidemiology
This study meanly uses the way of questionnaire survey. The contents of survey include:general condition (age, sex, native place etc.), family history of EC, the date of initial diagnosis and treatment methods. We conducted retrospective study of patients'records, added clinical pathological data of patients with EC (including location of EC, general types, the degree of differentiation, the degree of invasion, lymph node metastasis, etc). And then follow up survival time by telephone manner and home visits manner in the patients of 80% and 20% respectively. The follow-up period was 3 months. The deadline of followed up is March 2011. All investigators were trained strictly while the standard of questionnaire is unified.
2.3 Genotyping of 18 SNPs loci
Genomic DNA was extracted by using Qiagen Gene DNA kits and the concentration was measured, then electrophoresis, and normalized to 15ng-20ng/μl. According to our research team in the early study that made full scan of 506,666 SNP loci for 1077 cases of EC patients of endoscopic and pathological diagnosis and 1733 controls by using GWAS technology, as well as detected 18 SNP loci related closely with EC, and then primer design. By using Sequenom MassArray technology conducted genotyping for 18 SNP loci on 472 cases of patients which had complete follow-up data. Consistent with Hardy-Weinberg equilibrium, observed and expected value had people representative through the crowd goodness of fit tests. Then inquire the chromosome and corresponding genes which 18 SNPs loci located in through the official HapMap website or NCBI website, such as rs12263737 and rs2274223 loci corresponding to the PLCE1 gene, rs6140125 and rs13042395 sites corresponding to the C20orf54 gene, etc.
2.4 Immunohistochemistry
69 specimens of operation excises of EC from several hospital that include 25 cases were infiltrated to the lamina propria, submucosa and superficial muscle layer and 44 cases were infiltrated to the fiber membrane or the adjacent organs. All patients did not receive any radiotherapy and chemotherapy before operation while all specimens were confirmed esophageal squamous cell carcinoma by histology. Among them,28 patients had complete follow-up data, and the tissues of 69 corresponded cases had genotyping of 18 SNPs loci. Immunohistochemistry used the method of avidin-biotin-horseradish peroxide enzyme complex (ABC) that our laboratory had been established to conduct qualitative and quantitative detection.
2.5 Statistical analysis
Using SPSS 17.0 statistical software, Cox-regression analysis determine clinical factors that influence the prognosis of patients with EC, which include age, sex, family history, high and low incidence areas, location of EC, general types, the differentiation degree of pathology, lymph node metastasis, TNM staging,etc. Use Hardy-Weinberg equilibrium to test the crowd coincide degrees of 18 SNPs loci. Use Cox-regression, Kaplan-Meier and Log-rank test to analyse the relationship between the 18 SNPs loci and survival time. Compare the genetic differences of the 18 different SNPs loci in different Clinical phenotypes and examine immunohistochemical results by chi-square test (a=0.05 was considered as the test standard).
3. Results
3.1 The relationship between clinical characters of patients with EC and prognosis
3.1.1 There were no significant difference between the distribution of age and survival time (Log-rank P=0.061), however, Cox-regression analysis shown that >65 years age group of patients lowered the risk of death by 45.7% than≤35 years age group (HR=0.643), and the difference reached statistical significance (P=0.023). Male and female survival time differ unsignificantly (Log-rank P=0.104), and male patients had increased risk of death than female patients (HR=1.078), but the difference did not reach statistical significance (P=0.137).
3.1.2 The difference of overall survival time among three frequently occurred location of EC did not reach statistically significance (Log-rank P=0.342). The middle part of EC had higher cancer risk of death than the lower part (HR=2.691), but the difference did not reach statistical significance (P=0.098); The upper part of EC have higher cancer risk of death compared with the lower part (HR=2.008), and the difference was statistically significant (P=0.045).
3.1.3 The difference of overall survival time among four sub-types was no statistically significant (Log-rank P=0.114), mushroom type, ulcerative type, and medulla type had decreased risk of death than the narrow type (HR=0.588; HR=0.65; HR=0.838), but only the difference of ulcer type was statistically significant (P=0.05).
3.1.4 The difference of survival time among patients with various degree of differentiation was no significant (Log-rank P=0.542). The patients with moderately differentiated and poorly differentiated had higher risk of death than well differentiated (HR=1.762; HR=1.413), but the difference did not reach statistical significance (P=0.277; P=0.204).
3.1.5 The survival time among patients with early stage, metaphase and later period had significant difference (Log-rank P=0.000), The patients of metaphase and later period had increased risk of death than patients with early stage (HR=1.702; HR=2.154), The difference of risk of death between later period patients and early stage had statistical significance (P=0.004).
3.1.6 The patients with invasion to muscle and fiber layer had shorter survival time than that of invasion to mucosa (log-rank P=0.028). At the same time the patients with invasion to muscle and fiber layer have an increased risk of death than that of invasion to mucous layer (HR=1.676; HR=1.752), and the difference of the risk of death between patients with invasion to fiber layer and mucosa layer had statistical significance (P=0.016).
3.1.7 Patients without lymph node metastasis had longer survival time than patients with lymph node metastasis (Log-rank P=0.000);Cox regression analysis showed that patients with lymph node metastasis increased risk of death than patients without lymph node metastasis (HR=2.159), the difference had statistical significance (P=0.000).
3.1.8 The patients in the high incidence areas had longer survival time than that of middle-high incidence, as well as patients in the middle-high incidence had longer survival time than that of low-incidence areas (Log-rank P=0.003); Cox regression showed that the patients in high incidence had lower risk of death than patients in low-incidence areas (HR=0.647), and the difference had noteworthy statistical significance (P=0.001).
3.1.9 The patients with positive family history of EC had longer survival time than that with negative family history (Log-rank P=0.18E-06); Cox regression analysis showed that patients with positive family history of EC had lower risk of death compared with patients with negative family history of EC (HR=0.647), the difference was statistically significant (P=2.53E-06).
3.1.10 There was significantly difference between the distribution of surgical age and the survival time of patients (Log-rank P=3.49E-52), as well as the distribution of surgical methods (Log-rank.P=2.31E-21). The Nineties, after 2000 have the lower risk of death compared with surgery patients of the Seventies (HR=0.666; HR=0.432), the differences were statistically significant (P= 6.52E-1; P=2.85E-2); The patients with under the arch anastomosis and neck anastomosis had decreased risk of death than that of over the arch anastomosis (HR=0.943; HR=0.814), and difference of the risk of death between patients with neck anastomosis and over the bow anastomosis was statistically significant (P=0.046)
3.1.11 The distribution of different blood type and survival time of patients had no significant differences (Log-rank P=0.329);Cox regression analysis showed that the risk of patients of B type, O type and A type is almost similar and the difference had no significances (P=0.882;P=0.692)
3.2 The relationship between the genotype of 18 SNPs loci and prognosis of EC
3.2.1 In the gene distribution of AHDC1 gene rs4908343A>G genotype, compared with carriers of mutation homozygous GG genotype, the carriers of wild type homozygous AA genotype have reduced risk of death (HR=0.54,95% CI=0.30-0.99). Compared with carriers of AA/GA genotype, carriers of GG genotype had shorter survival time (Log-rank P=0.048).
3.2.2 Compared with the three genotype carriers of gene distribution of PPP1R14A gene rs8102476T>C genotype, patients carrying TT and TC genotype had shorter survival time (Log-rank P=0.043).
3.3 The relationship between 18 SNPs loci polymorphism and clinical phenotypes of EC
3.3.1 Genotype distribution diversities of C20orf54 gene rs6140125C>A polymorphism among different infiltrating degree of EC (mucosa, muscle, fiber coating) had statistically significance (P=0.007).
3.3.2 There was significantly different between IRX1 gene locus rs11134032 T>C genotype and esophageal cancer sites (P=0.014).
3.3.3 Genotype distribution of PPP1R14A gene rs8102476T>C polymorphism and lymph node metastasis had significantly statistical difference (P=0.037).
3.3.4 Genotype distribution of PES1 gene rs5753220C>T polymorphism and clinical stage had significantly statistical difference (P=0.033), as well as lymph node metastases (P=0.044).
3.4 The stratified analysis of gene polymorphism and prognosis of EC
To control the influence of potential confounding factors on gene polymorphism and prognosis, we conducted stratified analysis between SNPs and prognosis by stratified variables such as age, sex, location of EC, macroscopic type, clinical stage, the degree of invasion and lymph node metastases etc, the results as follows:
3.4.1 The stratified analysis of AHDC1 gene rs4908343A>G polymorphisms had found that carrier of GG genotype had increased risk of death compared with carrier AA/GA genotype in male patients stratified group, family history negative group, the later period group of EC, positive lymph node metastases group (HR=1.90; HR=1.85; HR=2.43; HR=2.66).
3.4.2 The stratified analysis of PES1 gene rs5753220C>T polymorphism found that carrier of TT genotype had increased the risk of death compared with carrier TC/CC genotype in well differentiated group, negative lymph node metastases group (HR=131.96; HR=1.98).
3.5 The joint analysis of gene polymorphism and prognosis of EC
Use joint analysis for the polymorphism the three genes, AHDC1 genes rs4908343A>G, PPP1R14A genes rs8102476T>C and PES1 genes rs5753220C>T related to prognosis, and show that patients with more genotypes of poor prognosis, the risk of death also increased (log-rank P=0.01), trend P=0.012.
3.6 The results of immunohistochemistry
C20orf54 and esophageal cancer susceptibility were related. In our study, C20orf54 was found associated with the invasion, so this gene was verificated in the expression of esophageal tissues.The positive expressions of protein mainly took place in endochylema. The positive expression rate of C20orf54 in tissues of EC was 87%, but the positive expression rate had no correlation with the degree of tissues infiltration, genotype and prognosis.
4 Conclusions
4.1 The prognosis of EC were statistically correlated with clinical stage, depth of invasion, lymph node metastasis, high/low-incidence areas, family history of EC, age of surgery and surgical methods.
4.2 The polymorphism of AHDC1 gene rs4908343A>G, PPP1R14A gene rs8102476T>C sites were correlated with prognosis of EC.In the stratified analysis, PES1 gene rs5753220C>T polymorphism was related with the prognoses in well differentiated group, negative lymph node metastases group.Moreover, the more quantity of carried risk genotypes, the more increases the risk of death.
4.3 C20orf54 gene rs6140125 locus, IRX1 gene rs11134032 locus, PPP1R14A gene rs8102476 locus and PES1 gene rs5753220 locus are associated with the clinical phenotype of EC.
4.4 The protein expressions of C20orf54 gene in tissues of EC are intense. But its expression rates are not associated with the degree of tissues depth of invasion, the distribution of genotype and prognosis.
食管癌是世界上最常见的六大恶性肿瘤之一,其发病率位于全世界恶性肿瘤发病率的第八位,死亡率居全球癌症死亡率的第六位。我国是世界上食管癌发病率和死亡率最高的国家,在中国最常见的十大恶性肿瘤死亡率中,食管癌占第四位。中国每年食管癌发病和死亡人数占全世界的一半以上
食管癌预后很差,中晚期食管癌的自然生存7-8个月,治疗后的生存情况也很不令人满意,5年生存率在20%左右,虽然治疗方式有了很大的改进,然而其5年生存率几十年来未见明显提高。
“三早”工作已引起普遍重视,但食管癌患者就诊时80-90%属中晚期是目前的现实,提高这一大组病人的生存自然也就成为了改善食管癌肿瘤防治现状的一个决定性环节。中晚期患者生存普遍较差,但其中经治疗干预后仍有20%能长期生存,这就提出了一个科学问题,是什么原因造成了相似人群,却有不同的预后?无论在发病机制、自然生存、临床预后等方面,食管癌都呈现出明显的个体化特征。是环境因素的作用,还是先天遗传的差异?比较一致的看法是遗传和环境相互影响共同作用的结果,但二者的作用,孰轻孰重,以及所占比重尚不完全清楚。
预后研究历来都是广大科研和医务工作者关注的焦点,寻找预后的内在机制、建立预后客观的评价体系,进而能够对预后风险及早进行干预,对改善患者的生存、延长病人的生命,具有现实而重要的意义。
既往的研究多集中在外界环境、表观症状、临床病理特征等相关因素和预后的关系,如TNM分期、生活习惯、生存环境、治疗方式等。已取得了一些共识和成果,但还存在很多的争议和疑问,离临床实际工作要求尚有很大距离,足可见该问题的复杂性。
“所有医学都是遗传学”,任何疾病的发生、发展、转归及临床表型的背后都需要一定的遗传物质基础来支配。随着分子生物学和遗传学的发展,分子检测、基因筛选等成为了现实,人们在注意多种临床表观现象研究的同时,也更加重视这些现象背后的分子机制、遗传背景。基因和疾病的关系成为目前研究的热点,并取得了可喜的成果,如癌基因、抑癌基因、药物敏感基因、预后基因等等。预后因素的研究也进入到了一个新的领域,深入到了细胞甚至蛋白、基因等水平。
食管癌是一种复杂疾病,又称多基因病或多因子病。目前,人类基因组计划的完成和单核苷酸多态的单体型图谱构建完成(HapMap计划),为肿瘤的遗传分子基础研究提供了大量的数据支持。同时,经济、高效大通量基因分型技术,在一个反应内可同时检测到几万、几十万个SNPs(单核苷酸多态性)位点成为可能,即全基因组关联分析技术(Genome-wide Associate Study, GWAS)。本项目组运用GWAS技术发现了18个和食管癌密切相关的SNPs位点,在此基础上,将18个SNPs位点及其相关的基因和食管癌预后联系起来,对食管癌预后的分子机制进行研究。
与以往有关食管癌预后分子机制方面的研究相比,本研究具有以下特点：
1.检测的SNPs位点多。18个SNPs位点是本项目组利用GWAS技术对中国汉族1077食管癌病例和1733正常对照的506,666个位点中筛选发现的。和既往只限于个别基因和预后关系的研究相比,其覆盖面广、代表性强,具有更明显的种族特色和针对性。
2.样本量大,临床病理资料全,进入观察项目多。首先从9703病例中随访出有明确生存期的病人共有2290例,然后从21个预后相关项目中选择了13个常见的临床病理因素进行分析。
3.临床和基础相结合。预后看似个临床问题,其背后隐藏着复杂的分子生物学机制,把预后及相关临床病理因素和单核苷酸多态性研究相结合,既注重临床表型,又注重分子机制探讨。
4.流调工作和实验室验证相结合。把经过流调和统计分析后得到的食管癌预后相关基因在癌组织中进行蛋白表达检测,在分子水平验证基因对预后的影响。初步进行了基因的功能研究。
5.具有良好的前期工作基础。易感基因对肿瘤发生的作用和对肿瘤的进展、预后作用具有一致性,二者具有相似的生物学基础和研究方法。本次研究是本项目组“食管癌易感基因GWAS研究”的一个延伸,GWAS前期工作结果已发表在了Nature Genetics杂志上。
综上观点,本论文将分为三大部分：第一部分：食管癌预后和临床表型的关系；第二部分：食管癌预后及相关表型和单核苷酸多态性的关系；第三部分：预后相关基因在食管癌组织中的不同表达。
2.材料及方法
2.1研究对象
2.1.1研究对象入选标准
本研究主要是对病人进行生存分析,入选标准如下：
1)全部研究对象均为中国汉族人群；
2)均经手术治疗,排除放化疗因素的干扰；
3)有相关的临床病理资料(TNM分期、大体类型、食管癌部位、分化程度、浸润程度、淋巴结转移、血型等)；
4)家庭住址或电话登记详细并能随访到患者本人或家属；
5)登记有详细的病案号和病理号,有完整的病史资料。
6)所有研究对象之间均无亲缘关系。
本研究共调查食管癌患者9703例,部分来自医院住院患者,部分来自新农合患者,经上述标准严格筛选后,通过电话和家访等形式共随访6793例,得到随访资料齐全并且是经过手术治疗的病例2290例,随访截止日期为2011年3月1同。所有患者未接受任何放疗和化疗。为了排除由于患者在不同地区或者医院就诊可能引起的重复调查,调查时保证每个家庭只有一位先证者。
2.1.2研究对象一般情况
本次研究系观察生存期及相关因素,所以所统计资料均来源于有明确生存期的2290例患者。其中男性1447例,年龄最小22岁,最大81岁,平均年龄54.69±8.746岁；女性843例,年龄最小29岁,最大86岁,平均年龄54.31±8.478。
2.1.3研究对象病理学诊断
由至少两名病理科医师确诊,全部符合食管鳞癌的标准。并采用到医院查阅病历的方式,追踪到有完整的临床TNM分期、淋巴结转移、浸润程度、分化程度、肿瘤类型、食管癌病变部位、血型、手术方式并有明确的手术日期等记录。
2.2流行病学调查
本研究主要采用问卷调查的方式,调查内容主要包括：一般情况(年龄、性别、籍贯等),食管癌肿瘤家族史,初次诊断时间及治疗方式等。回顾性调查病人既往住院病历档案,补充患者的食管癌临床病理资料(食管癌部位,大体类型、分化程度,浸润程度和淋巴结转移等)。然后80%采用电话追踪随访和20%的家访完成生存期随访,随访周期为3个月,随访截止时间为2011年3月。所有调查员均经过严格培训,问卷标准统一。
2.3 18个SNPs位点基因分型
采用Qiagen DNA试剂盒提取血样中DNA,测浓度,电泳,标化至15-20ng/μl,根据本研究团队在前期利用GWAS技术对内镜和病理确诊的1077食管癌病例和1733正常对照做了506,666个SNPs位点全扫,发现和食管癌密切相关的18个SNPs位点,然后进行引物设计,用Sequenom MassArray技术对472例有完整随访资料的患者在18个SNPs位点进行基因分型,经过人群吻合度检验,观察值和预期值符合Hardy-Weinberg平衡,具有人群代表性。然后通过官方HapMap网站或NCBI网站查询18个SNPs位点所在的染色体及所对应的基因,如rs12263737和rs2274223位点对应于PLCE1基因、rs6140125和rs13042395位点对应于C20orf54基因等。
2.4免疫组织化学
69例食管癌手术切除标本来自各个医院,其中浸润程度达到固有层、粘膜下层和浅肌层者25例、达到纤维膜或邻近器官者44例,所有患者术前未接受任何放、化疗,并均经组织学证实为食管鳞癌。其中,28例有完整的临床随访资料,并且69例组织均有对应患者的18个SNPs位点的分型。免疫组织化学实验方法采用卵白素-生物素-辣根过氧化氢酶复合物(ABC)法完成。采用本实验室已建立的ABC法,进行定性和定量分析。
2.5统计学分析
采用SPSS17.0统计软件分析,Cox-regression分析判断影响食管癌患者预后的年龄、性别、家族史、高低发区、食管癌部位、大体类型、病理分化程度、淋巴结转移、TNM分期等临床因素,用Hardy-Weinberg平衡检验18个SNPs位点的人群吻合度,Cox回归、Kaplan-Meier和Log-rank检验分析18个SNPs位点与生存的关系,卡方检验比较18个SNPs位点在不同的临床表型中的遗传差别和免疫组化结果,检验标准取α=0.05。
3结果
3.1食管癌患者临床表型和预后的关系
3.1.1年龄分布与生存时间总体之间无统计学差异(Log-rank P=0.061)。但Cox回归分析结果显示65岁以上年龄组患者较35岁(包括35岁)以下年龄组死亡风险降低了45.7%(HR=0.643),同时差异达到统计学意义(P=0.023)。男、女生存时间没有明显差异(Log-rank P=0.104),男性患者较女性患者死亡风险增加(HR=1.078),但是差异未达到统计学意义(P＝0.137)。
3.1.2三种不同的好发部位在总体生存时间上差异并无统计学意义(Log-rankP＝0.342)。食管中段癌较下段癌死亡风险增加(HR=2.691),但差异未达到统计学意义(P=0.098)；食管上段癌较下段癌死亡风险增加(HR=2.008),差异有统计学意义(P=0.045)。
3.1.3食管癌总体生存在四种大体形态之间无统计学差异(Log-rank P=0.114),蕈伞型、溃疡型、髓质型较缩窄型死亡风险均降低(HR=0.588；HR=0.65；HR=0.838),)但差异只有溃疡型有统计学意义(P=0.05)。
3.1.4各种分化程度的患者生存时间无显著性差异(Log-rank P=0.542)。中分化和低分化患者较高分化患者死亡风险均增加(HR=1.762；HR=1.413),但是差异均未达到统计学意义(P=0.277；P=0.204)。
3.1.5早、中、晚期患者之间生存上存在显著的差异(Log-rank P=0.000),中、晚期患者较早期患者死亡风险增加(HR=1.702；HR=2.154),晚期患者与早期之间死亡风险差异有显著性统计学意义(P=0.004)。
3.1.6浸润程度达到肌层和纤维膜层的患者较粘膜层的患者生存时间较短(Log-rank P=0.028)。并且浸润程度达到肌层和纤维膜层的患者较粘膜层得患者死亡风险增加(HR=1.676；HR=1.752),且纤维膜层与粘膜层之间死亡风险差异有统计学意义(P=0.016)。
3.1.7无淋巴结转移的患者较有淋巴结转移的患者生存时间长(Log-rank P=0.000),Cox回归分析结果显示有淋巴结转移的患者较无淋巴结转移的患者死亡风险增加(HR=2.159),差异有显著性统计学意义(P=0.000)。
3.1.8高发区患者生存时间优于中高发区,中高发区患者生存期优于低发区患者(Log-rank P=0.003), Cox回归结果显示,高发区患者死亡风险较低发区患者有所下降(HR=0.647),且差异有显著性统计学意义(P=0.001)。
3.1.9食管癌家族史阳性的患者生存时间优于食管癌家族史阴性的患者(Log-rankP=1.18E-06),Cox回归结果显示,食管癌家族史阳性的患者死亡风险较食管癌家族史阴性的患者低(HR=0.64),差异有统计学意义(P=2.53E-06)。
3.1.10手术年代分布与患者的生存时间之间存在统计学差异(Log-rankP=3.49E-52)。手术方式与患者的生存时间之间也存在统计学差异(Log-rankP=2.31E-21)。90年代、2000年及以后两组死亡风险较70年代手术的患者均降低(HR=0.666；HR=0.432),差异均有统计学意义(P=6.52E-1和P=2.85E-2),弓下吻合和颈部吻合较弓上吻合的死亡风险均降低(HR=0.943；HR=0.814),且颈部吻合与弓上吻合之间死亡风险差异有统计学意义(P=0.046)。
3.1.11不同血型分布与患者的生存时间之间不存在统计学差异(Log-rankP=0.329)；Cox回归分析发现：B型、O型两组死亡风险和A型的患者基本相似。(HR=0.988和HR=0.967),差异均无统计学意义(P=0.882和P=0.692)。
3.2食管癌预后和18个SNPs位点基因型的关系
3.2.1 AHDC1基因rs4908343位点A>G基因型分布中,与突变纯合基因型GG携带者相比,野生纯合基因型AA携带者死亡风险降低(HR=0.54,95%CI=0.30-0.99)。与携带AA/GA基因型者相比,GG基因型携带者生存时间较短(Log-rank P=0.048)
3.2.2 PPP1R14A基因rs8102476位点T>C基因型分布的三种基因型携带者,携带TT和TC基因型的患者生存时间较短(Log-rank P=0.043)
3.3食管癌临床表型和18个SNPs位点基因型的关系
3.3.1 C20orf54基因rs6140125位点C>A基因型分布在食管癌不同的浸润程度(粘膜层、肌层、纤维膜)之间有统计学差异(P=0.007)。
3.3.2 IRX1基因rs11134032位点T>C基因型分布与食管癌发生部位有统计学差异(P=0.014)。
3.3.3 PPP1R14A基因rs8102476位点T>C基因型分布与食管癌淋巴结转移有统计学差异(P=0.037)。
3.3.4 PES1基因rs5753220位点C>T基因型分布与临床分期有统计学差异(P=0.033),且与淋巴结转移也有差异(P=0.044)。
3·4食管癌预后和基因多态性的分层分析
为控制潜在混杂因素对基因多态性和预后的影响,我们分别以年龄、性别、食管癌发生部位、大体类型、临床分期及浸润程度和淋巴结转移等为分层变量进行SNPs和预后关系的分层分析,结果如下：
3.4.1 AHDC1基因rs4908343A>G多态性改变分层后发现在男性患者组、家族史阴性组、食管癌晚期组、淋巴结转移阳性组中,与携带AA/GA基因型者相比,携带GG基因型者的死亡风险增加(HR=1.90; HR=1.85; HR=2.43; HR=2.66)
3.4.2 PES1基因多态性rs5753220C>T多态性改变分层后发现在高分化组、淋巴结转移阴性组中,,与携带TC/CC基因型者相比,携带TT基因型者死亡风险增加(HR=131.96；HR=1.98)
3.5食管癌预后和基因多态性的联合分析
将三个和预后相关的基因AHDC1基因rs4908343A>G、PPP1R14A基因rs8102476T>C和PES1基因rs5753220C>T位点基因多态性联合分析,发现携带预后不良基因型个数的增加,其死亡风险也增大(Log-rank P=0.01),趋势性P=-0.012。
3.6免疫组织化学结果
C20orf54和食管癌易感有关。预后分析中发现C20orf54与食管癌的浸润深度有关联,所以进行了该基因在食管组织中表达的验证。
C20orf54蛋白阳性表达部位主要在胞浆,食管癌组织阳性表达率为87%,但是阳性表达率和组织浸润程度、基因型及预后无关。
4.结论
4.1食管癌预后和临床分期、浸润程度、淋巴结转移、高/低发区、食管癌家族史、手术年代及手术方式有统计学相关。
4.2 AHDC1基因rs4908343A>G、PPP1R14A基因rs8102476T>C位点的基因多态性和食管癌预后相关；食管癌分层后PES1基因rs5753220C>T位点的多态性在高分化或者淋巴结阴性组和食管癌预后风险相关。携带危险基因型数目越多,死亡风险也随之增大。
4.3 C20orf54基因rs6140125位点、IRX1基因rs11134032位点、PPP1R14A基因rs8102476位点和PES1基因rs5753220位点基因多态性与食管癌临床表型相关。
4.4 C20orf54基因蛋白表达在食管癌组织中较强,但是表达率和肿瘤组织浸润程度、基因型分布及预后无关。
1. Background and Objective
Esophageal cancer (EC) is one of the six most common malignancies worldwide. The incidence of this disease is the eighth in the all carcinomas worldwide while its mortality rate is the sixth in the all cancers worldwide. Our country is the highest area of incidence and mortality rate of EC in the world while EC is the fourth most frequent cause of cancer-related deaths in China.The total incidence and deaths of EC in China takes up the half number of the world's each year.
The prognosis of EC is very poor. The natural survival time of advanced EC is 7-8 months approximately, the survival rate after treatment is also very unsatisfactory, and 5-year survival rate is 20% approximately. Although the therapy of EC has been improved greatly, the five-year survival rate of EC has not been significantly improved in recent decades.
Three early" works has been attracted widespread attention. But in fact, the 80-90% of patients with EC is in the middle and late period when they get to hospital. To improve the survival of this large group of patients has become a decisive link of the actuality of prophylaxis and treatment in EC. The prognosis of patients with EC in middle and late period is very poor generally, but after the intervention by the treatment there are still 20% of patients who can survive in a long run. It presents a scientific problem. What causes a different prognosis in a similar population? In terms of pathogenesis, natural survival and clinical outcome, EC emerged significant individual characteristics. The effects of environmental factors or differences in congenital genetic? More consistent view is the result of genetic and environmental interaction, but the role of the two, the pros and cons as well as the proportion of both has not been understood clearly.
The research for prognosis of disease has always been attracted by the researchers and doctors constantly. To search for the internal mechanism of prognosis and establish the impersonal evaluation system, it will proceed early intervention direct prognosis. It has realistic and important significance in the field to improve patient survival rates.
Previous studies were mostly concentrated in the interrelated factors such as external environment, apparent symptoms, clinical pathological characteristics of prognosis. These factors include TNM stage, living habits, environment, treatment methods and so on. Some consensus has been achieved, however there are still have many controversies and doubts. There is still a great distance from the work requirements of clinical practice. So we can see the complexity of this problem obviously.
"All medicines are genetics." The occurrence, development, prognosis and clinical phenotype of diseases, all have the genetic material basis. With the development of molecular biology and genetics, molecular detection and genetic screening have become reality. People who pay attention to the studies for multiple phenomenon of clinical must pay more attention to the molecular mechanisms and genetic background underlying these phenomena. The relationships between genes and disease has become the focus of current research and have achieved encouraging results, such as oncogenes, tumor-suppressor genes, drug-sensitive genes, the prognosis genes, etc. The research for prognostic factors also entered into a new field of the cell and gone so far as to protein and genes levels.
EC is a complex disease, also known as multi-genes disease or multi-factors disease. At present, the completion of the Human Genome Project and single nucleotide polymorphism haplotype map (HapMap plan) provide a large amount of data for the genetic study of tumor. At the same time, it becomes possibility that economic and efficient large-throughput genotyping technology can detect the tens of thousands or hundreds of thousands of SNPs within one response. It is known as the genome-wide association study (GWAS). Taking advantage of GWAS technology, our team found the 18 EC associative SNPs loci. On the basis, our study intends to observe the relationship between 18 SNPs loci and correlative genes and prognosis of EC, and then study the molecular mechanism of prognosis.
Compared with the previous studies for prognosis related to molecular mechanisms of EC, this project has the following characteristics:
(1). The number of detective SNPs loci is large.18 SNPs loci were selected and found from 506,666 SNPs loci of 1077 cases of patients with EC and 1733 controls in China by using GWAS technology. Compare with the previous studies for the relationships between limited genes and prognosis, this study have extensive coverage, intense representativeness, and more obvious racial characteristics and pertinence.
(2). The sample volume is large and the clinicopathological data are completed. The numbers of observational items are large. First of all, selected 2290 cases patients which have clear survival time by followed up from 9703 cases, and then selected 13 common clinicopathological factors which to be analyzed from 21 prognosis relative items.
(3). Combined the clinical study and basic medicine. Prognosis appears to be clinical problems, which has the complex molecular mechanisms. This study wants to know the interactions among the prognosis, clinicopathological factors and the single nucleotide polymorphisms.
(4). Combined epidemiological survey work and laboratory validation. Detect the protein expression of EC prognosis related genes in cancer tissue which were found by epidemiological inquiry and statistical analysis, and then validate the influence of prognosis by gene at the molecular level. Conduct a preliminary study for gene function.
(5). With a good foundation of preliminary work. The susceptibility genes of the tumor have consistency on the role of the occurrence, progress and prognosis, they have similar biology basis and research methods. This study is an extension of "the GWAS work for esophageal cancer susceptibility genes" by our project team, as well as GWAS results of preliminary work has been published in the journal of Nature Genetics.
In summary, this paper will be set to three parts. The first part:the relationship between EC prognosis and clinical phenotype; the second part:the relationship between EC prognosis with correlative phenotype and single nucleotide polymorphism; the third part:the expression changes of prognosis related genes in the tissues of EC.
2. Materials and methods
2.1 The object of study
2.1.1 The inclusion criteria of study object
This study aims at the survival analysis of patients, inclusion criteria as follows:
1) All subjects were Chinese Han population;
2) All subjects were treated by surgery, excluded radiotherapy and chemotherapy disturbances;
3) Relevant clinicopathological data (TNM stage, gross type, esophageal cancer location, the degree of differentiation, the degree of invasion, lymph node metastasis, blood type,etc);
4) Home address or phone were registered clearly and can follow-up to the patients or their families;
5) Medical record number and pathological number were detailed, as well as medical history informations were completed;
6) All of the subjects had no genetic relationship each other.
The study surveyed total of 9703 individuals with EC. These patients partly were hospital patients, partly from the new rural cooperative medical care system. After rigorous screening by the above criteria, followed up 6793 cases by telephone and home visits, get 2290 cases which have completed follow-up data and received surgical treatment. The follow-up deadline is March 1,2011. All the patients did not receive any preoperative radiotherapy and chemotherapy. In order to exclude duplicated investigation which results from the patients going to the different hospitals and regions, each family only has one proband at investigation.
2.1.2 The general situation of study object
The study observed survival time and related factors, so the statistics data were all from the 2290 cases of patients which had straightforward survival time. Therein,1447 male cases that the youngest was 22 years old, maximum age was 81 years and the mean ages were 54.69±8.746; and 843 female cases that the youngest was 29 years old, maximum age was 86 years and the mean ages were 54.31±8.478 years.
2.1.3 The pathology diagnosis of study object
All these patients were confirmed by at least two pathologists, meanwhile accord with diagnostic criteria for esophageal squamous cell carcinoma. And access medical records by going to the hospital, to track complete data included clinical TNM stage, lymph node metastasis, the degree of invasion, the degree of differentiation, tumor type, lesions location of EC, blood type, the type of surgical operation and a clear date of surgical operation.
2.2 The investigation of epidemiology
This study meanly uses the way of questionnaire survey. The contents of survey include:general condition (age, sex, native place etc.), family history of EC, the date of initial diagnosis and treatment methods. We conducted retrospective study of patients'records, added clinical pathological data of patients with EC (including location of EC, general types, the degree of differentiation, the degree of invasion, lymph node metastasis, etc). And then follow up survival time by telephone manner and home visits manner in the patients of 80% and 20% respectively. The follow-up period was 3 months. The deadline of followed up is March 2011. All investigators were trained strictly while the standard of questionnaire is unified.
2.3 Genotyping of 18 SNPs loci
Genomic DNA was extracted by using Qiagen Gene DNA kits and the concentration was measured, then electrophoresis, and normalized to 15ng-20ng/μl. According to our research team in the early study that made full scan of 506,666 SNP loci for 1077 cases of EC patients of endoscopic and pathological diagnosis and 1733 controls by using GWAS technology, as well as detected 18 SNP loci related closely with EC, and then primer design. By using Sequenom MassArray technology conducted genotyping for 18 SNP loci on 472 cases of patients which had complete follow-up data. Consistent with Hardy-Weinberg equilibrium, observed and expected value had people representative through the crowd goodness of fit tests. Then inquire the chromosome and corresponding genes which 18 SNPs loci located in through the official HapMap website or NCBI website, such as rs12263737 and rs2274223 loci corresponding to the PLCE1 gene, rs6140125 and rs13042395 sites corresponding to the C20orf54 gene, etc.
2.4 Immunohistochemistry
69 specimens of operation excises of EC from several hospital that include 25 cases were infiltrated to the lamina propria, submucosa and superficial muscle layer and 44 cases were infiltrated to the fiber membrane or the adjacent organs. All patients did not receive any radiotherapy and chemotherapy before operation while all specimens were confirmed esophageal squamous cell carcinoma by histology. Among them,28 patients had complete follow-up data, and the tissues of 69 corresponded cases had genotyping of 18 SNPs loci. Immunohistochemistry used the method of avidin-biotin-horseradish peroxide enzyme complex (ABC) that our laboratory had been established to conduct qualitative and quantitative detection.
2.5 Statistical analysis
Using SPSS 17.0 statistical software, Cox-regression analysis determine clinical factors that influence the prognosis of patients with EC, which include age, sex, family history, high and low incidence areas, location of EC, general types, the differentiation degree of pathology, lymph node metastasis, TNM staging,etc. Use Hardy-Weinberg equilibrium to test the crowd coincide degrees of 18 SNPs loci. Use Cox-regression, Kaplan-Meier and Log-rank test to analyse the relationship between the 18 SNPs loci and survival time. Compare the genetic differences of the 18 different SNPs loci in different Clinical phenotypes and examine immunohistochemical results by chi-square test (a=0.05 was considered as the test standard).
3. Results
3.1 The relationship between clinical characters of patients with EC and prognosis
3.1.1 There were no significant difference between the distribution of age and survival time (Log-rank P=0.061), however, Cox-regression analysis shown that >65 years age group of patients lowered the risk of death by 45.7% than≤35 years age group (HR=0.643), and the difference reached statistical significance (P=0.023). Male and female survival time differ unsignificantly (Log-rank P=0.104), and male patients had increased risk of death than female patients (HR=1.078), but the difference did not reach statistical significance (P=0.137).
3.1.2 The difference of overall survival time among three frequently occurred location of EC did not reach statistically significance (Log-rank P=0.342). The middle part of EC had higher cancer risk of death than the lower part (HR=2.691), but the difference did not reach statistical significance (P=0.098); The upper part of EC have higher cancer risk of death compared with the lower part (HR=2.008), and the difference was statistically significant (P=0.045).
3.1.3 The difference of overall survival time among four sub-types was no statistically significant (Log-rank P=0.114), mushroom type, ulcerative type, and medulla type had decreased risk of death than the narrow type (HR=0.588; HR=0.65; HR=0.838), but only the difference of ulcer type was statistically significant (P=0.05).
3.1.4 The difference of survival time among patients with various degree of differentiation was no significant (Log-rank P=0.542). The patients with moderately differentiated and poorly differentiated had higher risk of death than well differentiated (HR=1.762; HR=1.413), but the difference did not reach statistical significance (P=0.277; P=0.204).
3.1.5 The survival time among patients with early stage, metaphase and later period had significant difference (Log-rank P=0.000), The patients of metaphase and later period had increased risk of death than patients with early stage (HR=1.702; HR=2.154), The difference of risk of death between later period patients and early stage had statistical significance (P=0.004).
3.1.6 The patients with invasion to muscle and fiber layer had shorter survival time than that of invasion to mucosa (log-rank P=0.028). At the same time the patients with invasion to muscle and fiber layer have an increased risk of death than that of invasion to mucous layer (HR=1.676; HR=1.752), and the difference of the risk of death between patients with invasion to fiber layer and mucosa layer had statistical significance (P=0.016).
3.1.7 Patients without lymph node metastasis had longer survival time than patients with lymph node metastasis (Log-rank P=0.000);Cox regression analysis showed that patients with lymph node metastasis increased risk of death than patients without lymph node metastasis (HR=2.159), the difference had statistical significance (P=0.000).
3.1.8 The patients in the high incidence areas had longer survival time than that of middle-high incidence, as well as patients in the middle-high incidence had longer survival time than that of low-incidence areas (Log-rank P=0.003); Cox regression showed that the patients in high incidence had lower risk of death than patients in low-incidence areas (HR=0.647), and the difference had noteworthy statistical significance (P=0.001).
3.1.9 The patients with positive family history of EC had longer survival time than that with negative family history (Log-rank P=0.18E-06); Cox regression analysis showed that patients with positive family history of EC had lower risk of death compared with patients with negative family history of EC (HR=0.647), the difference was statistically significant (P=2.53E-06).
3.1.10 There was significantly difference between the distribution of surgical age and the survival time of patients (Log-rank P=3.49E-52), as well as the distribution of surgical methods (Log-rank.P=2.31E-21). The Nineties, after 2000 have the lower risk of death compared with surgery patients of the Seventies (HR=0.666; HR=0.432), the differences were statistically significant (P= 6.52E-1; P=2.85E-2); The patients with under the arch anastomosis and neck anastomosis had decreased risk of death than that of over the arch anastomosis (HR=0.943; HR=0.814), and difference of the risk of death between patients with neck anastomosis and over the bow anastomosis was statistically significant (P=0.046)
3.1.11 The distribution of different blood type and survival time of patients had no significant differences (Log-rank P=0.329);Cox regression analysis showed that the risk of patients of B type, O type and A type is almost similar and the difference had no significances (P=0.882;P=0.692)
3.2 The relationship between the genotype of 18 SNPs loci and prognosis of EC
3.2.1 In the gene distribution of AHDC1 gene rs4908343A>G genotype, compared with carriers of mutation homozygous GG genotype, the carriers of wild type homozygous AA genotype have reduced risk of death (HR=0.54,95% CI=0.30-0.99). Compared with carriers of AA/GA genotype, carriers of GG genotype had shorter survival time (Log-rank P=0.048).
3.2.2 Compared with the three genotype carriers of gene distribution of PPP1R14A gene rs8102476T>C genotype, patients carrying TT and TC genotype had shorter survival time (Log-rank P=0.043).
3.3 The relationship between 18 SNPs loci polymorphism and clinical phenotypes of EC
3.3.1 Genotype distribution diversities of C20orf54 gene rs6140125C>A polymorphism among different infiltrating degree of EC (mucosa, muscle, fiber coating) had statistically significance (P=0.007).
3.3.2 There was significantly different between IRX1 gene locus rs11134032 T>C genotype and esophageal cancer sites (P=0.014).
3.3.3 Genotype distribution of PPP1R14A gene rs8102476T>C polymorphism and lymph node metastasis had significantly statistical difference (P=0.037).
3.3.4 Genotype distribution of PES1 gene rs5753220C>T polymorphism and clinical stage had significantly statistical difference (P=0.033), as well as lymph node metastases (P=0.044).
3.4 The stratified analysis of gene polymorphism and prognosis of EC
To control the influence of potential confounding factors on gene polymorphism and prognosis, we conducted stratified analysis between SNPs and prognosis by stratified variables such as age, sex, location of EC, macroscopic type, clinical stage, the degree of invasion and lymph node metastases etc, the results as follows:
3.4.1 The stratified analysis of AHDC1 gene rs4908343A>G polymorphisms had found that carrier of GG genotype had increased risk of death compared with carrier AA/GA genotype in male patients stratified group, family history negative group, the later period group of EC, positive lymph node metastases group (HR=1.90; HR=1.85; HR=2.43; HR=2.66).
3.4.2 The stratified analysis of PES1 gene rs5753220C>T polymorphism found that carrier of TT genotype had increased the risk of death compared with carrier TC/CC genotype in well differentiated group, negative lymph node metastases group (HR=131.96; HR=1.98).
3.5 The joint analysis of gene polymorphism and prognosis of EC
Use joint analysis for the polymorphism the three genes, AHDC1 genes rs4908343A>G, PPP1R14A genes rs8102476T>C and PES1 genes rs5753220C>T related to prognosis, and show that patients with more genotypes of poor prognosis, the risk of death also increased (log-rank P=0.01), trend P=0.012.
3.6 The results of immunohistochemistry
C20orf54 and esophageal cancer susceptibility were related. In our study, C20orf54 was found associated with the invasion, so this gene was verificated in the expression of esophageal tissues.The positive expressions of protein mainly took place in endochylema. The positive expression rate of C20orf54 in tissues of EC was 87%, but the positive expression rate had no correlation with the degree of tissues infiltration, genotype and prognosis.
4 Conclusions
4.1 The prognosis of EC were statistically correlated with clinical stage, depth of invasion, lymph node metastasis, high/low-incidence areas, family history of EC, age of surgery and surgical methods.
4.2 The polymorphism of AHDC1 gene rs4908343A>G, PPP1R14A gene rs8102476T>C sites were correlated with prognosis of EC.In the stratified analysis, PES1 gene rs5753220C>T polymorphism was related with the prognoses in well differentiated group, negative lymph node metastases group.Moreover, the more quantity of carried risk genotypes, the more increases the risk of death.
4.3 C20orf54 gene rs6140125 locus, IRX1 gene rs11134032 locus, PPP1R14A gene rs8102476 locus and PES1 gene rs5753220 locus are associated with the clinical phenotype of EC.
4.4 The protein expressions of C20orf54 gene in tissues of EC are intense. But its expression rates are not associated with the degree of tissues depth of invasion, the distribution of genotype and prognosis.
引文
[1]陈龙奇.制订2009第7版食管癌TNM分期标准[J].中国胸心血管外科临床杂志,2008,15(1)：52-55.
[2]王永岗,汪良骏,张庆斌,等.影响食管癌手术治疗患者预后的病理因素分析[J].实用癌症杂志.2001,16(5)：516-520.
[3]Zhang DW, Chen GY, Huang GJ, et al. Opreable squamous esophageal cancer: current results from the East [J]. World J Surg,1994,18:347-354.
[4]戒铁华,林鹏.胸段食管癌淋巴结转移的临床研究[J].中华胸心血管外科杂志,1994,10：242.
[5]陈建华,卫功铨,陈明耀,等.食管癌淋巴结转移的预后评价[J].中华肿瘤杂志,1994,16(6)：441.
[6]曹富民,王其彰,李保庆,等.食管鳞状细胞癌患者预后危险因素的分析[J].中国胸心血管外科临床杂志,2005,12(4)：250-252.
[7]张合林,何明,张方林,等.淋巴结转移数对食管癌预后和国际TNM分期的影响[J].中国肿瘤临床,2008,35(5)：252-255.
[8]李进东.王文光,许金良,等.胸段食管癌淋巴结转移特点的研究[J].中华医学杂志,2006,86(45)：3197-3200.
[9]Nishimaki T, Suzuki T, Kuwabara S, et al. Outcomes of extended radical esophagectomy for thoracic EC [J]. J Am Coll Surg,1998,186(3):306-312.
[10]李琮宁,李苹娟,吴会芳,等.河南食管癌高发区食管癌调查[J].郑州大学学报：医学版,2006,41(1)：32-34.
[11]王立东,樊慧,焦新英,等.河南食管癌高发区95对单卵双胞胎疾病谱和2323例食管癌患者家族史调查[J].中华肿瘤防治杂志,2006,13(20)：1521-1524.
[12]李卫东,王秀琴,张春林,等.山西省阳泉市部分人口食管癌遗传流行病学调查[J].中华医学杂志,1998,78(13)：203-206.
[13]Hu N, Dawsey SM, Wu M, et al. Familial aggregation of oesophageal cancer in Yangcheng County, Shanxi Province, China [J]. Eur J Cancer,1991,27(12):1336-1339.
[14]胡楠,贺立绩,韩小友,等.山西阳城622个食管癌阳性家族十年后随访[J].中华医学杂志,1990,70(34)：679-681.
[15]Chang CJ, Becher H, Blettner M, et al. Familial aggregation of esophagus cancer in a higher incidence area in China [J]. Int J Epidemilol,1997,26(2):1159-1165.
[16]Hu N, Dawsey S M, Wu M, et al. Familial aggregation of ESCC in Yangcheng County, Shanxi Province, China [J]. Int J Epidemiol,1992,21(5):877-882.
[17]Su H, Hu N, Shih J, et al. Gene expression Analysis of esophageal squamous cell carcinoma Reveals consistent molecular profiles related to a family history of upper gastrointestinal cancer [J]. Cancer Res,2003,63(14):3872-3876.
[18]Hu N, Roth MJ, Emmert-Buck MR, et al. Allelic loss in esophageal squamous cell carcinoma patients with and without family history of upper gastrointestinal tract cancer [J]. Clin Cancer Res,1999,5(11):3476-3482.
[19]Hu YC, Lam KY, Law S, et al. Profiling of differentially expressed cancer-related genes in esophageal squamous cell carcinoma (ESCC) using human cancer cDNA arrays: overexpression of oncogene MET correlates with tumor differentiation in ESCC [J]. Clin Cancer Res,2001,7(11):3519-3525.
[20]Tsuneoka M, Fujita H, Arima N, et al. Mina53 as a potential prognostic factor for esophageal squamous cell carcinoma [J]. Clin Cancer Res,2004,10(21):7347-7356.
[21]温登瑰,王士杰.食管癌高发区上消化道癌整体高发对病因及预防的启示[J].中国肿瘤临床,2008,35(20)：1150-1153.
[22]Jennifer A, Meyerhardt, Donna N, et al. Association of family history with cancer recurrence and survival among patients with stag III colon cancer [J]. JAMA,2008, 299(21):2515-2523.
[23]黄志刚,李学民,范增林,等.食管癌切除术后患者预后的 Cox 回归分析[J].中国公共卫生,2005,21(7)：784-785.
[24]李秀敏,赵志敏,张渊智,等.食管癌高发区食管癌患者遗传易感性对生存期的影响[J].实用医学杂志,2010,26(4)：675-678.
[25]耿贯一.流行病学[M].第2版.北京：人民卫生出版社,1997.303.
[26]Wang HW, Kuo KT, Wu YC, et al. Surgical results of upper thoracic esophageal carcinoma [J]. J Chin Med Assoc,2004,67(9):447-457.
[27]卢珠明,张华,王铭辉,等.胸段食管鳞癌淋巴结转移强度和淋巴结清扫手术方式分析[J].癌症,2006,25(5)：604-608.
[28]Siewert JR, Stein HJ, Feith M, et al. Histologic tumor type is an independent prognostic parameter in EC:lessons from more than 1,000 consecutive resections at a single center in the Western world [J]. Ann Surg,2001,234(3):360-367.
[29]Nozoe T, Saeki H, Ohga T, et al. Clinicopat hologic characteristics of esophageal SCC in younger patients [J]. Ann Thorac Surg,2001,72(6):1914-1917.
[30]陈建华,卫功铨,刘先本,等.淋巴结阴性食管癌的预后因素[J].河南肿瘤学杂志,1995,8(2)：107-108.
[1]AJ Brookes. The essence of SNPs [J]. Gene,1999,234(2):177-186.
[2]Wang DG, Fan JB, Sian CJ, et al. Large-scale identifieation mapping, and genotyping of single-nueleotide polymorphism in the human genome [J]. Seience,1998,280(5366): 1077-1082.
[3]Marshall E. Snipping away at genome patenting [J]. Science,1997,277(19):1752-1753.
[4]包广宇.单核昔酸多态性的研究进展及其应用[J].国外医学遗传分册.2003,26(1)：7-10.
[5]Kruglyak L. Prospects for whole-genome linkage disequilibrium mapping of common disease genes [J]. Nat Genet,1999,22(9):139-144.
[6]Kaessmann H, Zollner S, Gustafsson AC, et al. Extensive linkage disequilibrium in small human populations in Eurasia[J]. Am J Hum Cent,2002,70(3):673-685.
[7]罗怀容,施鹏,张亚平.单核普酸多态性的研究技术[J].遗传,2001,23(5)：471-476.
[8]Erichsen HC, Chanock SJ. SNPs in cancer research and treatment [J]. Br J Cancer,2004, 90(4):747-751.
[9]Biros E, Kohut A, Biros I, et al. A link between the p53 germ line Polymorphisms and white blood cells apoptosis in lung cancer patients [J]. Lung Cancer,2002,35(3):231-235.
[10]Botstein D, Risch N. Discovering genotypes underlying human phenotypes:past successes for mendelian disease, future approaches for complex disease [J]. Nat Genet,2003,33 Suppl:228-237.
[11]Wallenstein S, Hodge SE, Weston A. Logistic regression model for analyzing extended haplotype data[J]. Genet Epidemiol,1998,15(2):173-181.
[12]Keavney B, McKenzie CA, Connell JM, et al. Measured haplotype analysis of the angiotensin-I converting enzyme gene [J]. Hum Mol Genet,1998,7(11):1745-1751.
[13]Hiyama T, Yoshihara M, Tanaka S, et al. Genetic polymorphisms and esophageal cancer risk[J]. Int J Cancer,2007,121(8):1643-1658.
[14]王立东,中芳芳,崔娟,等Meta分析：基因多态变异与食管癌发病风险预测[J].郑州大学报(医学版),2011,46(2)(发表中).
[15]Frazer KA, Ballinger DG, Cox DR, et al. A second generation human haplotype map of over 3.1 million SNPs [J]. Nature,2007,449(7164):851-861.
[16]Pennisi E. Breakthrough of the year. Human genetic variation [J]. Science,2007,318 (5858):1842-1843.
[17]Easton DF, Eeles RA. Genome-wide association studies in cancer [J]. Hum Mol Genet, 2008,17(R2):R109-115.
[18]Wang LD, Zhou FY, Li XM, et al. Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54[J]. Nat Genet,2010,42(9):759-763.
[19]Lim J, Hao T, Shaw C, et al. A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration [J]. Cell,2006,125(4):801-814.
[20]Eto M, Senba S, Morita F, et al. Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle[J]. FEBS Lett,1997,410 (2-3):356-360.
[21]Nohria A, Grunert ME, Rikitake Y, et al. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease [J]. Circ Res,2006,99(12): 1426-1432.
[22]Seko T, Ito M, Kureishi Y, et al. Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle [J]. Circ Res,2003, 92(4):411-418.
[23]Ohki S, Eto M, Kariya E, et al. Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation[J]. J Mol Biol,2001,314(4):839-849.
[24]Kitazawa T, Eto M, Woodsome TP, et al. Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle[J]. J Physiol,2003,546(Pt 3):879-889.
[25]席文锦,王艳,娄运伟,等.CPI-17调控血管平滑肌表型对动脉粥样硬化形成的影响[J].山东大学学报(医学版),2010,48(6)：13-16.
[26]徐竞,明佳,李涛,等CPI-17、ILK、 ZIPK与PKCα、β在低氧大鼠血管平滑肌中的相互作用[J].创伤外科杂志,2009,11(1)：66-69.
[27]Yu YY, Pan YS, Zhu ZG. Homeobox genes and their functions on development and neoplasm in gastrointestinal tract [J]. Eur J Surg Oncol,2007,33(2):129-132.
[28]Bosse A, Zulch A, Becker MB, et al. Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system[J]. Mech Dev,1997,69(1-2):169-181.
[29]Cheng CW, Hui C, Strahle U, et al. Identification and expression of zebrafish Iroquois homeobox gene irxl [J]. Dev Genes Evol,2001,211 (8-9):442-444.
[30]Goriely A, Diezdel CR, Storey KG. c-Irx2 expression reveals an early subdivision of the neural plate in the chick embryo[J]. Mech Dev,1999,87(1-2):203-206.
[31]Bao ZZ, Bruneau BG, Seidman JG, et al. Regulation of chamber-specific gene expression in the developing heart by Irx4[J]. Science,1999,283(5405):1161-1164.
[32]于颖彦,计骏,陆云,等.胃癌表型与5p15.33位点等位基因丢失相关性研究[J].中华实验外科杂志,2005,22(10)：1190-1192.
[33]潘玉中,丁颖彦,计骏,等.胃癌中IRX1的表达与启动子区甲基化的相关性[J].上海交通大学学报(医学版),2007,27(5)：524-527.
[34]ML Allende, A Amsterdam, T Becker, et al. Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development [J]. Genes Dev,1996,10(24):3141-3155.
[35]Haque, J. Boger, S, et al. The murine pesl gene eneodes a nuclear protein containing a BRCT domain [J].Genomies,2000,70(2):201-210.
[36]Kinoshita Y, Jarell AD, Flaman JM, et al. Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells [J]. J Biol Chem,2001,276(9):6656-6665.
[37]Gessert S, Maurus D, Rossner A, et al. Pescadillo is required for Xenopus laevis eye development and neural crest migration [J]. Deve Biol,2007,310(1):99-112.
[38]Kim B, Bang S, Lee S, et al. Expression profiling and subtype-specific expression of stomach cancer [J]. Cancer Res,2003,63(23):8248-8255.
[39]Li Y, Hong X, Hussain M, et al. Gene expression profiling revealed novel molecular targets of docetaxel and estramustine combination treatment in prostate cancer cells[J]. Mol Cancer Ther,2005,4(3):389-398.
[40]Li Y, Sarkar FH. Gene expression profiles of genistein-treated PC3 prostate cancer cells[J]. J Nutr,2002,132(12):3623-3631.
[41]Charpentier AH, Bednarek AK, Daniel RL, et al. Effects of estrogen on global gene expression: identification of novel targets of estrogen action [J]. Cancer Res,2000,60(21): 5977-5983.
[42]Zhang H, Li J, Wang X, et al. The antibody preparation and expression of human Pescadillo [J]. Sci China C Life Sci,2007,50(3):298-304.
[43]Green P, Wiseman M, Crow YJ, et al. Brown-Vialetto-Van Laere syndrome, a ponto-bulbar palsy with deafness, is caused by mutations in c20orf54[J]. Am J Hum Genet,2010,86(3): 485-489.
[44]Bosch AM, Abeling NG, Ijlst L, et al. Brown-Vialetto-Van Laere and Fazio Londe syndrome is associated with a riboflavin transporter defect mimicking mild MADD:a new inborn error of metabolism with potential treatment [J]. J Inherit Metab Dis,2011,34(1): 159-164.
[45]Wang LD, Bi XL. A sequence variant in the phospholipase cepsilon C2 domain is associated with esophageal carcinoma and esophagitis [J]. Proc Am Assoc Cancer Res, 2011,52(3):234.
[46]沈琼,王东煜,蔡祥生,等.复合核黄素阻断治疗食管癌前增生的效果评价[J].中国肿瘤临床,1997,24(5)：331-334.
[47]林培中,张金生,戎振鹏,等.食管癌前病变的药物阻断性治疗——抗癌乙片,维胺酯和核黄素三年和五年的阻断效果[J].中国医学科学院学报,1990,12(4)：235-245.
[48]崔剑锋,胥永忠,刘新伏,等.四川盐亭核黄素盐干预试验红血球谷胱甘肽还原酶活性系数的动态变化[J].肿瘤防治研究,2006,33(2)：117-120.
[49]He Y, Ye L, Shan B, et al. Effect of riboflavin-fortified salt nutrition intervention on esophageal squamous cell carcinoma in a high incidence area, China [J]. Asian Pac J Cancer Prev,2009,10(4):619-622.
[50]Jemal A, Bray F, Center MM, et al. Global cancer statistics [J]. CA Cancer J Clin,2011, 61(2):69-90.
[1]Eriehsenl H C, Chanoek S J. SNPs in cancer researeh and treatment [J]. British Journal of Caneer,2004,90(4):747-751.
[2]Green P, Wiseman M, Crow Y J, et al. Brown-Vialetto-Van Laere Syndrome, a Ponto-Bulbar Palsy with Deafness, is caused by mutations in C20orf54[J]. The American Journal of Human Genetics,2010,86(3):485-489.
[3]沈琼,蔡祥生.复合核黄素阻断治疗食管癌前增生的效果评价[J].中国肿瘤临床,1997,24(5)：331-334.
[4]林培中,张金生,戎振鹏,等.食管癌前病变的药物阻断性治疗——抗癌乙片,维胺酯和核黄素三年和五年的阻断效果[J].中国医学科学院学报,1990,12(4)：235-245.
[5]崔剑锋,胥永忠,刘新伏,等.四川盐亭核黄素盐干预试验红血球谷胱甘肽还原酶活性系数的动态变化[J].肿瘤防治研究,2006,33(2)：117-120.
[6]He Y, Ye L, Shan B, et al. Effect of riboflavin-fortified salt nutrition intervention on esophageal squamous cell carcinoma in a high incidence area, China [J]. Asian Pac J Cancer Prev,2009,10(4):619-622.
[7]Wang LD, Zhou FY, Li XM, et al. Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54[J]. Nat Genet,2010,42(9):759-763.
[8]Bosch AM, Abeling NQ Ijlst L, et al. Brown-Vialetto-Van Laere and Fazio Londe Syndrome is associated with a riboflavin transporter defect mimicking mild MADD:a new inborn error of metabolism with potential treatment [J]. J Inherit Metab Dis,2011,34(1): 159-164.
[1]王波,徐德忠,张艳,等.西安市男性吸烟、饮酒与食管癌关系的Meta分析[J].西安交通大学学报(医学版),2003,24(3)：280-281,284.
[2]彭波,别文华,马祖帅,等.食管肿瘤地域、生活习惯等因素相关性研究[J].临床消化病杂志,2010,22(4)：245-246.
[3]Freng A, Daae LN, Engeland A, et al. Malignant epithelial tumors in the upper digestive tract: a dietary and socio medical case control and survival study [J]. Eur J Clin Nutr.1998, 52(4):271-278.
[4]Mariette C, Maurel A, Fabre S, et al. Preoperative prognostic factors for squamous cell carcinomas of the thoracic esophagus [J]. Gastroenterol Clin Biol,2001,25(5):468-472.
[5]Navarrete Aleman J, ofiate Ocafia LF, Herrereta Goepfert R, et al. Survival prognostic factors in a cohort of patients with esophageal carcinoma [J]. Rev Gastroenterol Mex.2004, 69(4):209.
[6]刘爱娜,黄镜,蔡锐刚,等,138例晚期食管癌的化疗疗效和预后因素分析[J].癌症,2008,27(4)：400.
[7]Nozoe T, Saeki H, Ohga T, et al. Clinicopat hologic characteristics of esophageal SCC in younger patients [J]. Ann Thorac Surg,2001,72(6):1914-1917.
[8]陈建华,卫功铨,刘先本,等.淋巴结阴性食管癌的预后因素[J].河南肿瘤学杂志,1995,8(2)：107-108.
[9]安丰山,黄金球,陈少湖.217例胸段食管癌淋巴结转移及其对预后影响的分析[J].癌症,2003,22(9)：974-977.
[10]李秀敏,赵志敏,张渊智,等.食管癌高发区食管癌患者遗传易感性对生存期的影响[J].实用医学杂志,2010,26(4)：675-678.
[11]温登瑰.家族史阳性与阴性食管癌预后差别的研究[c].2008,第五届中国肿瘤学术大会.
[12]Ishigami S, Natsugoe S, Tokuda K, et al. Prognostic value of intratumoral natural killer cells in gastric carcinoma[J]. Cancer,2000,88(3):577-583.
[13]段惠军,冼美生.食管癌免疫形态学反应与预后的关系[J].临床与实践病理学杂志,1985,1(2)：4-7.
[14]曾小澜,麦羡霞,马克荣,等.食管癌放疗前后免疫功能的变化与预后[J].中华放射肿瘤学杂志,1993,2(2)：86-87.
[15]Kubo A, Corley DA. body mass index and adenocarcinomas of the esophagus or gastric cardia, A systematic review and meta-analysis [J]. Cancer Epidemiol Biomarkers Prev, 2006,15(5):872.
[16]Merry AH, Schouten LJ, Goldbohm RA et al. Body mass index, height and risk of adenocarcinomas of the esophagus and gastric cardia, A prospective cohort study [J]. Gut, 2007,56(11):1503.
[17]Smith M, Zhou M, Whitlock G, et al. Esophageal cancer and boby mass index: Results from a prospective study of 220,000 men in china and a meta-analysis of published studies [J]. Int J Cancer,2008,122(7):1604.
[18]Hofheinz RD, Raab B, Mai S, et al. Impact of chemoradio therapy-induced anemia on survival in uniformly staged patients with locally advanced squamous cell carcinoma of the esophagus [J]. Onkologie,2004,27(5):462.
[19]Wang HW, Kuo KT, Wu YC, et al. Surgical result s of upper thoracic esophageal carcinoma [J]. J Chin Med Assoc,2004,67(9):447-457.
[20]Griffiths EA, Brummell Z, Gort hi G, et al. Tumor length as a prognostic factor in esophageal malignancy:univariate and multivariate survival analyses [J]. J Surg Oncol, 2006,93(4):258-267.
[21]曹富民,王其彰,李保庆,等.食管鳞状细胞癌患者预后危险因素的分析[J].中国胸心血管外科临床杂志,2005,12(4)：250-252.
[22]Eloubeidi MA, Desmond R, Arguedas MR, et al. Prognostic factors for the survival of patients with esophageal carcinoma in the U.S. The importance of tumor length and lymph node status [J]. Cancer,2002,95(7):1434-1443.
[23]祝淑钗,李任,李娟,等.非手术治疗胸段食管癌临床分期与预后关系的初步探讨[J].中华放射肿瘤学杂志,2004,13(3)：189-192.
[24]黄志刚,范增林,李学民,等.971例食管癌切除术后患者颅后的Cox回归分析[J].实用肿瘤杂志,2005,20(3)：263-266.
[25]张合林,何明,孟宪利,等.1146例胸段食管癌淋巴结转移的相关因素[J].中国肿瘤临床,2007,34(11)：650-652.
[26]黄金球,安丰山.影响胸段食管癌切除术后的预后因素的Cox模型分析[J].实用诊断与治疗杂志,2004,18(3)：178-180.
[27]谢映涛,王树钦,黄金球,等.影响胸段食管鳞癌切除术预后的因素[J].中华肖肠外科杂志,2004,7(6)：453-455.
[28]王永岗,汪良骏,张庆斌,等.影响食管癌手术治疗患者预后的病理因素分析[J].实用癌症杂志,2001,16(5)：516-517.
[29]Nishimaki T, Suzuki T, Kuwabara S, et al. Outcomes of extended radical esophagectomy for thoracic EC [J]. J Am Coll Surg,1998,186(3):306-312.
[30]Rizk NP, Venkatraman E, Bains MS, et al. Ameriean Joint Committee on cancer staging system does not accurately predict survival in patients receiving multimodality therapy for esophageal adenocarcinoma [J]. J Clin oncol,2007,25(5):507.
[31]Rizk N, venkatraman E, Park B, et al.The prognostic Importance of the number Of involved lymph nodes in esophageal cancer:ImPlications for revisions of the American Joint Committee on Cancer staging system [J]. J Thorac Cardiovasc Surg,2006,132(6): 1374.
[32]Kimura H,Konishi K, ArakawaH,et al. Number of lymph node metastases infulences survival in patients with thoracic esophageal carcinoma: therapeutic value of radiation treatment for recurrence [J]. Dis Esophagus,1999,12(3):205-208.
[33]陈建华,卫功铨,陈明耀,等.食管癌淋巴结转移的预后评价[J].中华肿瘤杂志,1994,16(6)：441-443.
[34]许运龙,郭昭扬.胸部食管癌转移淋巴结数与预后的临床研究[J].中华肿瘤杂志,2000,22(3)：244-246.
[35]王永刚,汪良俊,张德超,等.胸段食管鳞癌淋巴结转移特点及临床意义[J].中华肿瘤杂志,2000,22(3)：241-243.
[36]孙克林,张汝刚,张大为,等.食管癌淋巴结转移对手术治疗预后的影响[J].中华外科杂志,1995,33(4)：222-224.
[37]张合林,何明,张方林.淋巴结转移数对食管癌预后和国际TNM分期的影响[J].中国肿瘤临床,2008,35(5)：252-255.
[38]Tachikawa D, Inada S, Kotoh T, et al. An evaluation of malig-nancy and prognostic factors based on mode of lymph node metastasis in esophageal cancinoma [J]. Surg Today,1999, 29(11):1131-1135.
[39]Adachi W, Koike S, Nimura Y, et al. Clinicopathologic charac-teristics and postoperative outcome in Japanese and Chinese patients with thoracic oesphageal cancer [J]. World J Surg,1996,20(3):332-336.
[40]Casson AG, van Lanschot JJ. Improving outcomes after esophagectomy: the impact of operative volume [J]. J SurgOncol,2005,92(3):262-266.
[41]Siewert JR, Stein HJ, Feith M, et al. Histologic tumor type is an independent prognostic parameter in EC:lessons from more than 1,000 consecutive resections at a single center in the Western world [J]. Ann Surg,2001,234 (3):360-367.
[42]Mitani M, Kuwabara Y, Shinoda N, et al. Long 2 term survivors after the resection of limited esophageal small cell carcinoma [J]. Dis Esophagus,2000,13(3):259-261.
[43]Tajima Y, Nakanishi Y, Ochiai A, et al. Histopat hologic findings predicting lymph node metastasis and prognosis of patients with superficial EC:analysis of 240 surgically resected tumors [J]. Cancer,2000,88(6):1285-1293.
[44]Tachibana M, Kinugasa S, Yoshimura H, et al. Clinical outcomes of extended esophagectomy with three-field lymph node dissection for esophageal squamous cell carcinoma [J]. Am J Surg,2005,189(1):98-109.
[45]Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methy2 lation in the plasma and serum of liver cancer patients [J]. Cancer Res,1999,59(1):71-73.
[46]Hibi K, Robinson CK, Booker S, et al, Molecular detection of genetic alteration in the serum of colorectal cancer patient [J]. Cancer Res,1998,58(7):1405-1407.
[47]Goessl C, Heicappell R, Munker R, et al. Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma [J]. Cancer Res,1998,58 (20):4728-4732.
[48]Koible k, Vicrich CM, Pidde H, et al. Microsatellite alterations in serum DNA of patients with colorectal cancer [J]. Lab Invest,1999,79(9):1145-1150.
[49]Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells [J]. Cancer Res, 2001,61 (4):1659-1665.
[50]于金明,杨丽华,国前,等.流式细胞计测定食管癌细胞DNA含量和临床及病理特性的关系[J].中国肿瘤临床,1990,17(3)：150.
[51]麻国新,周道安,平育敏,等.食管癌DNA含量及P53蛋白表达与术前放疗[J].中华肿瘤杂志,1999,21(3),19-23.
[52]Matsuura H, Kuwano H, Morita M, et al. Predicting recurrence time of esophageal carcinoma through assessment of histologic factors and DNA ploidy [J]. Cancer,1991, 67(5):1406-1411.
[53]Watanzbe M, Kuwano H, Tanaka S, et al. Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of the esophagus [J]. Cancer,1999,85(11):2322-2328.
[54]Mukaida H, Toi M, Hirai T, et al. Clinical significance of the expression of epidermal growth factor and its receptor in esophageal cancer [J]. Cancer,1991,68(1):1422-1481.
[55]卢兆桐,君格平,孙晓明,等.食管癌DNA和RNA含量测定的临床意义[J].临床肿瘤学杂志,1997,2(3)：20.
[56]Kitagawa Y, Ueda M, Andol V, et al, Significance of int-2/hst-1 coamplification as a prognostic factor in patients with the esophageal squamous carcinoma [J]. Cancer Res, 1991,51(5):1504-1508.
[57]王永康,陈景寒,孟龙,等.食管鳞癌预后因素的病理学和免疫组织化学研究[J].中华病理学杂志,2000,29(4)：25-29.
[58]寇仁业,李劲松,孙桂武,等.食管癌C-erbB-2原癌基因的表达及意义[J].中国胸心血管外科临床杂志,1997,4(3)：157.
[59]寇仁业,李劲松,孙桂武,等C-erbB-2原癌基因的表达与食管癌患者的预后[J].临床肿瘤学杂志,1996,1(34)：5.
[60]Nagasawa S, Onda M, Sasajima K, et al. Cyclin D1 over expression as a prognostic factor in patients with esophageal carcinoma [J]. J Surg Oncol,2001,78(3):208-214.
[61]祝叔钗,翟福山,殷蔚伯,等.周期素D1蛋白表达对食管癌预后的影响[J].中华肿瘤杂志,1998,20(2)：129.
[62]Saito H, T sujitani S, Oka S, et al. The expression of murine double minute 2 is afavorable prognostic marker in esophageal squamous cell carcinoma without p53 protein accumulation [J]. Ann Surg Oncol,2002,9(5):450-456.
[63]Hashimoto N, Tachibana M, Dhar DK, et al. Expression of p53 and RB proteins in squamous cell carcinoma of the esophagus:their relationship with clinicopathologic characteristics [J]. Ann Surg Oncol,1999,6(5):489-494.
[64]李华川,陆十新.食管癌组织中Rb基因突变与表达的研究[J].中华肿瘤杂志,1993,15(6)：412.
[65]陈克能,张力健,师晓天.食管癌基因分子水平的研究进展[J].中国胸心血管外科临床杂志,1999,6(1)：57.
[66]Nozoe T, Korenaga D, Itoh S, et al. Clinico pathological significance of pRb2/p130 expression in squamous cell carcinoma of the esophagus [J]. J Cancer Res Clin Oncol, 2002,128(12):691-696.
[67]Shimada H, Nabeya Y, Okazumi S, et al. Prognostic significance of serum p53 antibody in patients with esophageal squamous cell carcinoma[J]. Surgery,2002,132(1):41-47.
[68]陈慰浙,马童丽.P53基因蛋白在食管鳞癌中的表达[J].实用肿瘤杂志,1994,9(1)：32.
[69]闫庆娜,战忠利.食管癌P53基因蛋白检测的临床意义[J].中国肿瘤临床,1998,25(9)：683.
[70]吕桂泉,陈旭峰,曹巍,等.食管鳞状细胞癌中P16基因变异的临床意义及地域性差异[J].中华肿瘤杂志.1999,21(5)：359.
[71]Singh SP, Lipman J, Goldman H, et al. Loss or altered subcellular localization of P27 in Barrett's associated adenocarcinoma [J]. Cancer Res,1998,58(15):1730.
[72]Lin LI, Lee PH, Wu CM, et al. Significance of nm23 mRNA expression in human hepotocellular carcinoma [J]. Anticancer Res,1998,18(1B):541-546.
[73]Wang CS, Lin KH, Hsu YC, et al. Distant metastasis of gastric cancer is associated with elerated expression of anti2metastatic nm23 gene [J]. Cancer Lett,1998,128(1):23-29.
[74]Wang LS, Chow KC, Wu YC, et al. In Inverse expression of di2 hydrodiol dehydrogenase and glutathione2 transferase in patients with esophageal squamous cell carcinoma [J]. Int J Cancer,2004,111 (2):246-251.
[75]Lin WK, Chu YL, Zhang F, et al. The relationship between HPV16 and expression of CD44v6, nm23H1 in esophageal squamous cell carcinoma [J]. Arch Virol,2005,15(5): 991-1001.
[76]华平,黄洪铮,张华,等.食管癌中CD44S、CD44V6和nm23基因蛋白表达的研究[J].中国肿瘤临床,1999,26(9)：666.
[77]李桂芳.转移抑制基因nm23与肿瘤转移[J].中国肿瘤临床,1996,23(10)：741
[78]沈金辉,曾纠文,蔡广玲,等.200例食管癌nm23/NDPK表达与淋巴结转移的关系[J].中国肿瘤临床,1998,25(4)：305.
[79]吴灵飞,姚俊国,张锦,等.nm23-H1蛋白在食管鳞癌中的表达及意义[J].中国肿瘤临床,1997,24(10)：778.
[80]李宣海,主编.肿瘤标志物的检测和临床[M].第1版.北京：人民卫生出版社,1997,177.
[81]吴名耀,郑瑞明,沈健,等.食管癌增殖细胞核抗原的免疫组织化学研究[J].中华病理学杂志,1993,22(4)：239.
[82]卢兆桐,张宝仁,孔宪涛,等.鳞状细胞癌抗原在食管癌临床的应用[J].中华胸心外科杂志,1994,10(3)：239.
[83]Charoenrat P, Rhys-Evans PH, Archer DJ, et al. C-erbB receptors in squamous cell carcinomas of the head and neck:clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors [J]. Oral Oncol,2002,38(1): 73-80.
[84]刘以民,郭永军,张国强,等.表皮生长因子受体在人食管鳞状细胞癌中的高表达及其对预后的影响[J].中华病理学杂志,1993,22(5)：303.
[85]战忠利,常新忠.食管癌中P-糖蛋白表达及其临床病理意义[J].中国肿瘤临床,1998,25(8)：588.
[86]杨国涛,王善政,孙鸿铖,等.食管鳞状细胞癌核仁组成区嗜银蛋白的研究[J].中华胸心血管外科杂志,1995,1 1(5)：288.
[87]Fukai Y, Fukuchi M, Masuda N, et al. Reduced expression of transforming growth factor-beta receptors is an unfavorable prognostic factor in human esophageal squamous cell carcinoma [J]. Int J Cancer,2003,104(2):161-166.
[88]Natsugoe S, Xiangming C, Matsumoto M, et al. Smad4 and transforming growth factor betal expression in patients with squamous cell carcinoma of the esophagus [J]. Clin Cancer Res,2002,8(6):1838-1842.
[89]Kyriazanos ID, Tachibana M, Dhar DK, et al. Expression and prognostic significance of S100A2 protein in squamous cell carcinoma of the esophagus [J]. Oncol Rep,2002,9(3): 503-510.
[90]Shimada H, Takeda A, Nabeya Y, et al. Clinical significance of serum vascular endothelial growth factor in esophageal squamous cell carcinoma [J]. Cancer,2001,92(3):663-669.
[91]Wallner G, Ciechanski A, Dabrow ski A, et al. Vascular endothelial growth factor and basic fibroblast growth factor in patients with squamous cell oesophageal cancer [J]. Folia Histochem Cytobiol,2001,39(Supp12):122-123.
[92]Mobius C, Freire J, Beeker 1, et al. VEGF-C expression in squamous cell carcinoma and adenocarcinoma of the esophagus [J]. Worid J Surg,2007,31(9):1768.
[93]Kii T, Takiuchi H, Kawabe S, et al. Evaluation of prognostic factors of esophageal squamous cell carcinoma (stageⅡ-Ⅲ) after concurrent chemoradiotherapy using biopsy specimens [J]. Jpn J Clin oncol,2007,37(8):583.
[94]Matsumoto S. Prognostic significance of platelet-derived growth factor-BB expression in human esophageal squamous cell carcinomas [J]. Anticancer Res,2007,27(4B):2409.
[95]Hsia JY, Chen CY, Chen JT, et al. Prognostic significance of caspase-3 expression in primary resected esophageal squamous cell carcinoma [J]. Eur J Surg Oncol,2003,29(1): 44-48.
[96]高文信,刘洪静,王颖,等Caspase-3在口腔粘膜癌前病变及鳞状细胞癌中的表达研究.现代口腔医学杂志,2003,17(2)：109-111.
[97]Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma [J]. Br J Cancer,2002,87(8):883-887.
[98]Cornford PA, Dodson AR, Parsons KF, et al. Heat shock protein expression independently predicts clinical outcome in prostate cancer[J]. Cancer Res,2000,60(24):7099-7105.
[99]曹富氏,王小玲,张祥宏,等.术后不同生存期患者食管鳞状细胞癌热休克蛋白27与70表达及临床意义[J].中国肿瘤临床,2004,31(8)：462-465.
[100]Nakajima M, Kuwano H, Miyazaki T, et al. Significant correlation between expression of heat shock proteins 27,70 and lymphocyte infiltration in esophageal squamous cell Carcinoma [J]. Cancer Lett,2002,178(1):99-106.
[101]Sarbia M, Bit tinger F, Porschen R, et al. Bcl-2 expression and prognosis in squamous cell carcainoma of the esophageal [J]. Int J Cancer,1996,69(3):324.
[102]Raouf AA, Evoy DA, Carton E, et al. Loss of Bcl-2 expression in Barrett's dysplasia and adenocarcinoma is associated with tumor progression and worse survival but not with response to neoadjuvant chemoradiation [J]. Dis Esophagus,2003,16(1):17-23.
[103]杨志江,李桂红,蒲红伟,等.食管癌组织中Bcl-2、cmyc、iNOS表达及其意义[J].陕西医学杂志,2008,37(6)：686-688.
[104]Parenti AR, Rugge M, Shiao YH, et al. Bcl-2 and P53 immunophenotypes in pre-invasive, early and advanced esophageal squamous cancer [J]. Histopathology,1997,31(3):430.
[105]黄莺,王康敏,何长成,等.Bcl-2和P16蛋白在食管癌中的表达及意义[J].肿瘤防治研究,2003,30(4)：259-261.
[106]曾亮,高平.Bcl-2、P53在食管鳞癌组织中的表达的临床意义[J].宁夏医学院学报,2008，30(3)：298-299.
[107]Konishi S, Ishiguro H, Shibata Y, et al. Decreased expression of DFF45/ICAD is correlated with a poor prognosis in patients with esophageal carcinoma [J]. Cancer,2002,95(12): 2473-2478.
[108]Kase S, Osaki M, Adachi H, et al. Expression of Fas and Fas lig and in esophageal tissue mucosa and carcinomas [J]. Int J Oncol,2002,20(2):291-297.
[109]Nishioka K, Doki Y, Shiozaki H, et al. Clinical significance of CDC25A and CDC25B expression in SCCs of the oesophagus [J]. Br J Cancer,2001,85(3):412-421.
[110]Takeno S, Noguchi T, Kikuchi R, et al. Prognostic value of cyclin B1 in patients with esophageal SCC [J]. Cancer,2002,94(11):2874-2881.
[111]Fukuchi M, Fukai Y, Masuda N, et al. High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma [J]. Cancer Res,2002,62(24):7162-7165.
[112]Basire A, Sabarier F, Ravet S, et al. High urokinase expression contributes to the angiogenic properties of endothelial cells derived from circulating progenitors. Thromb Haemost,2006,95(4):678-688.
[113]Torzewski M, SarbiaM, Verreet P, et al. Prognostic significance of urokinase-type plaseminogen activator expression in squamons cell carcinoma of the esophagus [J]. Clin Cancer Res,1997,3(12):2263-2268.
[114]Buskens CJ, Van Rees BP, Sivula A, et al. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus [J]. Gastroenterology,2002,122(7):1800-1807.
[115]Shamma A, Yamamoto H, Doki Y, et al. Up-ragulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus [J]. Clin Cancer Rus,2000,6(4):1229-1238.
[116]王顺文,高青.环氧化酶-2与食管癌临床病理特征及预后关系的研究[J].肿瘤防治杂 志,2004,10(11)：1051-1053.
[117]Nakanishi Y, Ochiai A, Akimoto S, et al. Expression of E-cadherin, alpha-catenin, beta-catenin and plakoglobin in esophageal carcinomas and its prognostic signifience: immunohistochemical analysis of 96 lesions[J]. Oncology,1997,54(2):158-165.
[118]Wu H, Lotan R, Menter D, et al. Expression of E-cadher in is associated with squamous differentiation I squamous cell carcinomas [J]. Anticancer Res,2000,20(3):1385-1390.
[119]林英城,吴名耀,李德锐.食管鳞癌ｙ-连接蛋白和钙粘附蛋白表达及其预后意义[J].肿瘤防治杂志,2001,8(5)：467-469.
[120]陈捷,王兰兰,等.食管癌患者手术前后机体免疫调节与反应性蛋白表达的分析[J].免疫学杂志,2006,22(4)：419-420.
[121]Nozoe T, Saeki H, and Sugimachi K. Significance of preoperative elevation of serum C-reactive protein as an indicator of prognosis in EC[J]. Am J Surg,2001,182(2):197-201.
[122]Nakamura T, Ide H, Eguchi R, et al. Cyfra21-1 is a useful marker for esophageal squamous cell carcinoma [J]. Cancer,1997,79(9):1467-1475.
[123]Shimada H, Nabeya Y, Okazumi S, et al. Prognostic significance of CYFRA21-1 in patients with esophageal squamous cell carcinoma[J]. J Am Coll Surg,2003,196:573-578.
[124]Maeda K, Chung YS, OgawaS, et al. Thymidine phosphorylas eplatelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma [J]. Br J Cancer,1996,73:884-888.
[125]Takebayashi Y, Miyadera K, Akiyama S, et al. Expression of thymidine phosphorylase in human gastric carcinoma [J], Jpn J Cancer Res,1996,87:288-295.
[126]Igarashi M, Dhar DK, Kubota, et al. The prognostic significance of microvessel density and thymidine phosphorylase expression in squamous cell carcinoma of the esophagus [J]. Cancer,1998,82:1225-1232.
[127]Shimada H, Takeda A, Shiratori T, et al. Prognostic significance of serumthymidine phosphorylase concentration in esophageal SCC[J]. Cancer,2002,94(7):1947-1954.
[128]张晓光,药立波,苏成.酪氨酸激酶受体EPh亚族的研究进展[J].生物化学与生物物理进展,1999,26：138.
[129]张晓光,阎小君,苏成芝,等.酪氨酸蛋白激酶受体EPh亚族新基因HEK在儿株人肿瘤细胞系的表达[J].第四军医大学学报,1998,19：577.
[130]Miyazaki T, Kato H, Fukuchi M, et al. EphA2 Over expression correlates with poor prognosis in esophageal squamous cell carcinoma[J]. Int J Cancer,2003,103(5):657-663.
[131]Kinch MS, Moore MB, HarPole DH. Predietive value of the EPhA2 recePtor Tyrosine kinase in lung cancer recurrence and survival [J]. Clin Caneer Res,2003,9:613.
[132]Miyazaki T, Kato H, Fukuehi M, et al. EPhA2 overexPression correlates With Poor Prognosis in esophageal squamous cell carcinoma [J]. Int J Caneer,2002,103:657.
[133]Schwartz M A. Integrin oncogenes and ancorage independence [J]. J CellBio,1997,139(3): 575-581.
[134]Miyazaki T, Kato H, Nakajima M, et al. FAK Over expression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma [J]. Br J cancer,2003,89(1):140-145.
[135]Miyazaki T, Kato H, Kimura H, et al. Evaluation of tumor malignancy in esophageal squamous cell carcinoma using different characteristic factors [J]. Anticancer Res,2005, 25(6B):4005-11.
[136]黄海花,吴秀浅,等.粘着斑激酶在食管癌中表达的临床意义[J].现代肿瘤医学,2006,14(1)：23-25.
[137]Medical Research Coucil Oesophageal Cancer Work Groph. Surgical research with or without preoperative chemotherapy in oesphageal cancer: a randomised controlled trial [J]. Lancet,2002,359(9319):1727-1733.
[138]Thirion PG, Michiel S, Le Maitre A, et al. On behalf of the Meta Analysis of chemotherapy in esophageal cancer collaborative groph. Individual patient data-based meta-analysis assessing pre-operative chemotherapy in resectable oesophageal carcinoma [J]. J Clin oncol,2007,25(18-suppl):4512.
[139]Raja SG. Does neoadjuvant chemotherapy improve survival in patients with resectable thoracic oesophageal cancer [J]. Interact Cardiovasc Thorac Surg,2007,6(5):661.
[140]汪楣,谷铣之,黄国俊.食管癌术前放射治疗的前瞻性临床研究[J].中华放射肿瘤学杂志,2001,10(3)：168-172.
[141]Sydney JA, William Duncan, Marc Gignoux. Preoperative Radiotherapy in Esophageal Carcinoma:A meta-analysis using Individual Patient Data (Oesophageal Cancer Collaborative Group)[J]. Int J Radiation Oncology Biol Phys,1998,41(3):579-583.
[142]安丰山,黄金球,谢映涛.食管癌新辅助放化疗的前瞻性临床研究[J].中华肿瘤杂志,2003,25(4)：376-379.
[143]Burmeister BH, Smithers BM, Fitzgerald L, et al. A randomized phase III trial of preoperative chemoradiation followed by surgery (CR-S) versus alone (S) for localized respectable cancer of the esophageal [J]. Proc Am Soc Clin Oncol,2002,21:518.
[144]Ioannis GK, Gail RW, Kristian F. Neoad juvant Treatment for resectable Cancer of the Esophageal and the gastroesophageal Junction: A meta analysis of randomized clinical trials [J]. Annals of Surgical Oncology,2003,10(7):754-761.
[145]John DU, Hari VB, Chris JB. A meta-analysis of randomized controlled trials that compared neoadjuvant chemotherapy and surgery to surgery alone for respectable esophageal cancer [J]. The American of surgery,2002,188(3):274-279.
[146]Tepper J, Krasna MJ, Niedzwiecki D, et al. Phase Ⅲ trial of trimodality therapy with cisplatin, fluorouracil, radiotherapy, and surgery compared with surgery alone for esophageal cancer: CALGB 9781[J]. J Clin Oncol,2008,26(7):1086-1092.
[147]肖泽芬,杨宗贻,梁军.食管癌根治术后预防性放射治疗的临床价值[J].中华放射肿瘤学杂志,2002,24(6)：608-611.
[148]Robccca W, R malthaner. Combined chemotherapy and radiotherapy (without surgery) compared with radiotherapy alone in localized carcinoma of the esophagus [J]. The Cochrane Library,2006(1):CD002092.
[149]Cooper JS, Guo MD, Herskovic A, et al. Chemoradiotherapy of locally advanced esophageal cancer. Long-term Follow-up of a prospective randomized trial (RTOG85-01) [J]. JAMA,1999,281(17):1623-1627.
[150]Conroy T, Yataghene Y, Etienne PL, et al. Definitive chemoradiotherapy (CRT) with FOLFOX4 or 5-FU-cisplatin as first line treatment for patients (pts) with inoperable esophageal cancer (IEC):Final results of a randomized phase Ⅱ study. J Clin Oncol[J], 2007,25(18-suppl):4532.
[2]王永岗,汪良骏,张庆斌,等.影响食管癌手术治疗患者预后的病理因素分析[J].实用癌症杂志.2001,16(5)：516-520.
[3]Zhang DW, Chen GY, Huang GJ, et al. Opreable squamous esophageal cancer: current results from the East [J]. World J Surg,1994,18:347-354.
[4]戒铁华,林鹏.胸段食管癌淋巴结转移的临床研究[J].中华胸心血管外科杂志,1994,10：242.
[5]陈建华,卫功铨,陈明耀,等.食管癌淋巴结转移的预后评价[J].中华肿瘤杂志,1994,16(6)：441.
[6]曹富民,王其彰,李保庆,等.食管鳞状细胞癌患者预后危险因素的分析[J].中国胸心血管外科临床杂志,2005,12(4)：250-252.
[7]张合林,何明,张方林,等.淋巴结转移数对食管癌预后和国际TNM分期的影响[J].中国肿瘤临床,2008,35(5)：252-255.
[8]李进东.王文光,许金良,等.胸段食管癌淋巴结转移特点的研究[J].中华医学杂志,2006,86(45)：3197-3200.
[9]Nishimaki T, Suzuki T, Kuwabara S, et al. Outcomes of extended radical esophagectomy for thoracic EC [J]. J Am Coll Surg,1998,186(3):306-312.
[10]李琮宁,李苹娟,吴会芳,等.河南食管癌高发区食管癌调查[J].郑州大学学报：医学版,2006,41(1)：32-34.
[11]王立东,樊慧,焦新英,等.河南食管癌高发区95对单卵双胞胎疾病谱和2323例食管癌患者家族史调查[J].中华肿瘤防治杂志,2006,13(20)：1521-1524.
[12]李卫东,王秀琴,张春林,等.山西省阳泉市部分人口食管癌遗传流行病学调查[J].中华医学杂志,1998,78(13)：203-206.
[13]Hu N, Dawsey SM, Wu M, et al. Familial aggregation of oesophageal cancer in Yangcheng County, Shanxi Province, China [J]. Eur J Cancer,1991,27(12):1336-1339.
[14]胡楠,贺立绩,韩小友,等.山西阳城622个食管癌阳性家族十年后随访[J].中华医学杂志,1990,70(34)：679-681.
[15]Chang CJ, Becher H, Blettner M, et al. Familial aggregation of esophagus cancer in a higher incidence area in China [J]. Int J Epidemilol,1997,26(2):1159-1165.
[16]Hu N, Dawsey S M, Wu M, et al. Familial aggregation of ESCC in Yangcheng County, Shanxi Province, China [J]. Int J Epidemiol,1992,21(5):877-882.
[17]Su H, Hu N, Shih J, et al. Gene expression Analysis of esophageal squamous cell carcinoma Reveals consistent molecular profiles related to a family history of upper gastrointestinal cancer [J]. Cancer Res,2003,63(14):3872-3876.
[18]Hu N, Roth MJ, Emmert-Buck MR, et al. Allelic loss in esophageal squamous cell carcinoma patients with and without family history of upper gastrointestinal tract cancer [J]. Clin Cancer Res,1999,5(11):3476-3482.
[19]Hu YC, Lam KY, Law S, et al. Profiling of differentially expressed cancer-related genes in esophageal squamous cell carcinoma (ESCC) using human cancer cDNA arrays: overexpression of oncogene MET correlates with tumor differentiation in ESCC [J]. Clin Cancer Res,2001,7(11):3519-3525.
[20]Tsuneoka M, Fujita H, Arima N, et al. Mina53 as a potential prognostic factor for esophageal squamous cell carcinoma [J]. Clin Cancer Res,2004,10(21):7347-7356.
[21]温登瑰,王士杰.食管癌高发区上消化道癌整体高发对病因及预防的启示[J].中国肿瘤临床,2008,35(20)：1150-1153.
[22]Jennifer A, Meyerhardt, Donna N, et al. Association of family history with cancer recurrence and survival among patients with stag III colon cancer [J]. JAMA,2008, 299(21):2515-2523.
[23]黄志刚,李学民,范增林,等.食管癌切除术后患者预后的 Cox 回归分析[J].中国公共卫生,2005,21(7)：784-785.
[24]李秀敏,赵志敏,张渊智,等.食管癌高发区食管癌患者遗传易感性对生存期的影响[J].实用医学杂志,2010,26(4)：675-678.
[25]耿贯一.流行病学[M].第2版.北京：人民卫生出版社,1997.303.
[26]Wang HW, Kuo KT, Wu YC, et al. Surgical results of upper thoracic esophageal carcinoma [J]. J Chin Med Assoc,2004,67(9):447-457.
[27]卢珠明,张华,王铭辉,等.胸段食管鳞癌淋巴结转移强度和淋巴结清扫手术方式分析[J].癌症,2006,25(5)：604-608.
[28]Siewert JR, Stein HJ, Feith M, et al. Histologic tumor type is an independent prognostic parameter in EC:lessons from more than 1,000 consecutive resections at a single center in the Western world [J]. Ann Surg,2001,234(3):360-367.
[29]Nozoe T, Saeki H, Ohga T, et al. Clinicopat hologic characteristics of esophageal SCC in younger patients [J]. Ann Thorac Surg,2001,72(6):1914-1917.
[30]陈建华,卫功铨,刘先本,等.淋巴结阴性食管癌的预后因素[J].河南肿瘤学杂志,1995,8(2)：107-108.
[1]AJ Brookes. The essence of SNPs [J]. Gene,1999,234(2):177-186.
[2]Wang DG, Fan JB, Sian CJ, et al. Large-scale identifieation mapping, and genotyping of single-nueleotide polymorphism in the human genome [J]. Seience,1998,280(5366): 1077-1082.
[3]Marshall E. Snipping away at genome patenting [J]. Science,1997,277(19):1752-1753.
[4]包广宇.单核昔酸多态性的研究进展及其应用[J].国外医学遗传分册.2003,26(1)：7-10.
[5]Kruglyak L. Prospects for whole-genome linkage disequilibrium mapping of common disease genes [J]. Nat Genet,1999,22(9):139-144.
[6]Kaessmann H, Zollner S, Gustafsson AC, et al. Extensive linkage disequilibrium in small human populations in Eurasia[J]. Am J Hum Cent,2002,70(3):673-685.
[7]罗怀容,施鹏,张亚平.单核普酸多态性的研究技术[J].遗传,2001,23(5)：471-476.
[8]Erichsen HC, Chanock SJ. SNPs in cancer research and treatment [J]. Br J Cancer,2004, 90(4):747-751.
[9]Biros E, Kohut A, Biros I, et al. A link between the p53 germ line Polymorphisms and white blood cells apoptosis in lung cancer patients [J]. Lung Cancer,2002,35(3):231-235.
[10]Botstein D, Risch N. Discovering genotypes underlying human phenotypes:past successes for mendelian disease, future approaches for complex disease [J]. Nat Genet,2003,33 Suppl:228-237.
[11]Wallenstein S, Hodge SE, Weston A. Logistic regression model for analyzing extended haplotype data[J]. Genet Epidemiol,1998,15(2):173-181.
[12]Keavney B, McKenzie CA, Connell JM, et al. Measured haplotype analysis of the angiotensin-I converting enzyme gene [J]. Hum Mol Genet,1998,7(11):1745-1751.
[13]Hiyama T, Yoshihara M, Tanaka S, et al. Genetic polymorphisms and esophageal cancer risk[J]. Int J Cancer,2007,121(8):1643-1658.
[14]王立东,中芳芳,崔娟,等Meta分析：基因多态变异与食管癌发病风险预测[J].郑州大学报(医学版),2011,46(2)(发表中).
[15]Frazer KA, Ballinger DG, Cox DR, et al. A second generation human haplotype map of over 3.1 million SNPs [J]. Nature,2007,449(7164):851-861.
[16]Pennisi E. Breakthrough of the year. Human genetic variation [J]. Science,2007,318 (5858):1842-1843.
[17]Easton DF, Eeles RA. Genome-wide association studies in cancer [J]. Hum Mol Genet, 2008,17(R2):R109-115.
[18]Wang LD, Zhou FY, Li XM, et al. Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54[J]. Nat Genet,2010,42(9):759-763.
[19]Lim J, Hao T, Shaw C, et al. A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration [J]. Cell,2006,125(4):801-814.
[20]Eto M, Senba S, Morita F, et al. Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle[J]. FEBS Lett,1997,410 (2-3):356-360.
[21]Nohria A, Grunert ME, Rikitake Y, et al. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease [J]. Circ Res,2006,99(12): 1426-1432.
[22]Seko T, Ito M, Kureishi Y, et al. Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle [J]. Circ Res,2003, 92(4):411-418.
[23]Ohki S, Eto M, Kariya E, et al. Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation[J]. J Mol Biol,2001,314(4):839-849.
[24]Kitazawa T, Eto M, Woodsome TP, et al. Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle[J]. J Physiol,2003,546(Pt 3):879-889.
[25]席文锦,王艳,娄运伟,等.CPI-17调控血管平滑肌表型对动脉粥样硬化形成的影响[J].山东大学学报(医学版),2010,48(6)：13-16.
[26]徐竞,明佳,李涛,等CPI-17、ILK、 ZIPK与PKCα、β在低氧大鼠血管平滑肌中的相互作用[J].创伤外科杂志,2009,11(1)：66-69.
[27]Yu YY, Pan YS, Zhu ZG. Homeobox genes and their functions on development and neoplasm in gastrointestinal tract [J]. Eur J Surg Oncol,2007,33(2):129-132.
[28]Bosse A, Zulch A, Becker MB, et al. Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system[J]. Mech Dev,1997,69(1-2):169-181.
[29]Cheng CW, Hui C, Strahle U, et al. Identification and expression of zebrafish Iroquois homeobox gene irxl [J]. Dev Genes Evol,2001,211 (8-9):442-444.
[30]Goriely A, Diezdel CR, Storey KG. c-Irx2 expression reveals an early subdivision of the neural plate in the chick embryo[J]. Mech Dev,1999,87(1-2):203-206.
[31]Bao ZZ, Bruneau BG, Seidman JG, et al. Regulation of chamber-specific gene expression in the developing heart by Irx4[J]. Science,1999,283(5405):1161-1164.
[32]于颖彦,计骏,陆云,等.胃癌表型与5p15.33位点等位基因丢失相关性研究[J].中华实验外科杂志,2005,22(10)：1190-1192.
[33]潘玉中,丁颖彦,计骏,等.胃癌中IRX1的表达与启动子区甲基化的相关性[J].上海交通大学学报(医学版),2007,27(5)：524-527.
[34]ML Allende, A Amsterdam, T Becker, et al. Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development [J]. Genes Dev,1996,10(24):3141-3155.
[35]Haque, J. Boger, S, et al. The murine pesl gene eneodes a nuclear protein containing a BRCT domain [J].Genomies,2000,70(2):201-210.
[36]Kinoshita Y, Jarell AD, Flaman JM, et al. Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells [J]. J Biol Chem,2001,276(9):6656-6665.
[37]Gessert S, Maurus D, Rossner A, et al. Pescadillo is required for Xenopus laevis eye development and neural crest migration [J]. Deve Biol,2007,310(1):99-112.
[38]Kim B, Bang S, Lee S, et al. Expression profiling and subtype-specific expression of stomach cancer [J]. Cancer Res,2003,63(23):8248-8255.
[39]Li Y, Hong X, Hussain M, et al. Gene expression profiling revealed novel molecular targets of docetaxel and estramustine combination treatment in prostate cancer cells[J]. Mol Cancer Ther,2005,4(3):389-398.
[40]Li Y, Sarkar FH. Gene expression profiles of genistein-treated PC3 prostate cancer cells[J]. J Nutr,2002,132(12):3623-3631.
[41]Charpentier AH, Bednarek AK, Daniel RL, et al. Effects of estrogen on global gene expression: identification of novel targets of estrogen action [J]. Cancer Res,2000,60(21): 5977-5983.
[42]Zhang H, Li J, Wang X, et al. The antibody preparation and expression of human Pescadillo [J]. Sci China C Life Sci,2007,50(3):298-304.
[43]Green P, Wiseman M, Crow YJ, et al. Brown-Vialetto-Van Laere syndrome, a ponto-bulbar palsy with deafness, is caused by mutations in c20orf54[J]. Am J Hum Genet,2010,86(3): 485-489.
[44]Bosch AM, Abeling NG, Ijlst L, et al. Brown-Vialetto-Van Laere and Fazio Londe syndrome is associated with a riboflavin transporter defect mimicking mild MADD:a new inborn error of metabolism with potential treatment [J]. J Inherit Metab Dis,2011,34(1): 159-164.
[45]Wang LD, Bi XL. A sequence variant in the phospholipase cepsilon C2 domain is associated with esophageal carcinoma and esophagitis [J]. Proc Am Assoc Cancer Res, 2011,52(3):234.
[46]沈琼,王东煜,蔡祥生,等.复合核黄素阻断治疗食管癌前增生的效果评价[J].中国肿瘤临床,1997,24(5)：331-334.
[47]林培中,张金生,戎振鹏,等.食管癌前病变的药物阻断性治疗——抗癌乙片,维胺酯和核黄素三年和五年的阻断效果[J].中国医学科学院学报,1990,12(4)：235-245.
[48]崔剑锋,胥永忠,刘新伏,等.四川盐亭核黄素盐干预试验红血球谷胱甘肽还原酶活性系数的动态变化[J].肿瘤防治研究,2006,33(2)：117-120.
[49]He Y, Ye L, Shan B, et al. Effect of riboflavin-fortified salt nutrition intervention on esophageal squamous cell carcinoma in a high incidence area, China [J]. Asian Pac J Cancer Prev,2009,10(4):619-622.
[50]Jemal A, Bray F, Center MM, et al. Global cancer statistics [J]. CA Cancer J Clin,2011, 61(2):69-90.
[1]Eriehsenl H C, Chanoek S J. SNPs in cancer researeh and treatment [J]. British Journal of Caneer,2004,90(4):747-751.
[2]Green P, Wiseman M, Crow Y J, et al. Brown-Vialetto-Van Laere Syndrome, a Ponto-Bulbar Palsy with Deafness, is caused by mutations in C20orf54[J]. The American Journal of Human Genetics,2010,86(3):485-489.
[3]沈琼,蔡祥生.复合核黄素阻断治疗食管癌前增生的效果评价[J].中国肿瘤临床,1997,24(5)：331-334.
[4]林培中,张金生,戎振鹏,等.食管癌前病变的药物阻断性治疗——抗癌乙片,维胺酯和核黄素三年和五年的阻断效果[J].中国医学科学院学报,1990,12(4)：235-245.
[5]崔剑锋,胥永忠,刘新伏,等.四川盐亭核黄素盐干预试验红血球谷胱甘肽还原酶活性系数的动态变化[J].肿瘤防治研究,2006,33(2)：117-120.
[6]He Y, Ye L, Shan B, et al. Effect of riboflavin-fortified salt nutrition intervention on esophageal squamous cell carcinoma in a high incidence area, China [J]. Asian Pac J Cancer Prev,2009,10(4):619-622.
[7]Wang LD, Zhou FY, Li XM, et al. Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54[J]. Nat Genet,2010,42(9):759-763.
[8]Bosch AM, Abeling NQ Ijlst L, et al. Brown-Vialetto-Van Laere and Fazio Londe Syndrome is associated with a riboflavin transporter defect mimicking mild MADD:a new inborn error of metabolism with potential treatment [J]. J Inherit Metab Dis,2011,34(1): 159-164.
[1]王波,徐德忠,张艳,等.西安市男性吸烟、饮酒与食管癌关系的Meta分析[J].西安交通大学学报(医学版),2003,24(3)：280-281,284.
[2]彭波,别文华,马祖帅,等.食管肿瘤地域、生活习惯等因素相关性研究[J].临床消化病杂志,2010,22(4)：245-246.
[3]Freng A, Daae LN, Engeland A, et al. Malignant epithelial tumors in the upper digestive tract: a dietary and socio medical case control and survival study [J]. Eur J Clin Nutr.1998, 52(4):271-278.
[4]Mariette C, Maurel A, Fabre S, et al. Preoperative prognostic factors for squamous cell carcinomas of the thoracic esophagus [J]. Gastroenterol Clin Biol,2001,25(5):468-472.
[5]Navarrete Aleman J, ofiate Ocafia LF, Herrereta Goepfert R, et al. Survival prognostic factors in a cohort of patients with esophageal carcinoma [J]. Rev Gastroenterol Mex.2004, 69(4):209.
[6]刘爱娜,黄镜,蔡锐刚,等,138例晚期食管癌的化疗疗效和预后因素分析[J].癌症,2008,27(4)：400.
[7]Nozoe T, Saeki H, Ohga T, et al. Clinicopat hologic characteristics of esophageal SCC in younger patients [J]. Ann Thorac Surg,2001,72(6):1914-1917.
[8]陈建华,卫功铨,刘先本,等.淋巴结阴性食管癌的预后因素[J].河南肿瘤学杂志,1995,8(2)：107-108.
[9]安丰山,黄金球,陈少湖.217例胸段食管癌淋巴结转移及其对预后影响的分析[J].癌症,2003,22(9)：974-977.
[10]李秀敏,赵志敏,张渊智,等.食管癌高发区食管癌患者遗传易感性对生存期的影响[J].实用医学杂志,2010,26(4)：675-678.
[11]温登瑰.家族史阳性与阴性食管癌预后差别的研究[c].2008,第五届中国肿瘤学术大会.
[12]Ishigami S, Natsugoe S, Tokuda K, et al. Prognostic value of intratumoral natural killer cells in gastric carcinoma[J]. Cancer,2000,88(3):577-583.
[13]段惠军,冼美生.食管癌免疫形态学反应与预后的关系[J].临床与实践病理学杂志,1985,1(2)：4-7.
[14]曾小澜,麦羡霞,马克荣,等.食管癌放疗前后免疫功能的变化与预后[J].中华放射肿瘤学杂志,1993,2(2)：86-87.
[15]Kubo A, Corley DA. body mass index and adenocarcinomas of the esophagus or gastric cardia, A systematic review and meta-analysis [J]. Cancer Epidemiol Biomarkers Prev, 2006,15(5):872.
[16]Merry AH, Schouten LJ, Goldbohm RA et al. Body mass index, height and risk of adenocarcinomas of the esophagus and gastric cardia, A prospective cohort study [J]. Gut, 2007,56(11):1503.
[17]Smith M, Zhou M, Whitlock G, et al. Esophageal cancer and boby mass index: Results from a prospective study of 220,000 men in china and a meta-analysis of published studies [J]. Int J Cancer,2008,122(7):1604.
[18]Hofheinz RD, Raab B, Mai S, et al. Impact of chemoradio therapy-induced anemia on survival in uniformly staged patients with locally advanced squamous cell carcinoma of the esophagus [J]. Onkologie,2004,27(5):462.
[19]Wang HW, Kuo KT, Wu YC, et al. Surgical result s of upper thoracic esophageal carcinoma [J]. J Chin Med Assoc,2004,67(9):447-457.
[20]Griffiths EA, Brummell Z, Gort hi G, et al. Tumor length as a prognostic factor in esophageal malignancy:univariate and multivariate survival analyses [J]. J Surg Oncol, 2006,93(4):258-267.
[21]曹富民,王其彰,李保庆,等.食管鳞状细胞癌患者预后危险因素的分析[J].中国胸心血管外科临床杂志,2005,12(4)：250-252.
[22]Eloubeidi MA, Desmond R, Arguedas MR, et al. Prognostic factors for the survival of patients with esophageal carcinoma in the U.S. The importance of tumor length and lymph node status [J]. Cancer,2002,95(7):1434-1443.
[23]祝淑钗,李任,李娟,等.非手术治疗胸段食管癌临床分期与预后关系的初步探讨[J].中华放射肿瘤学杂志,2004,13(3)：189-192.
[24]黄志刚,范增林,李学民,等.971例食管癌切除术后患者颅后的Cox回归分析[J].实用肿瘤杂志,2005,20(3)：263-266.
[25]张合林,何明,孟宪利,等.1146例胸段食管癌淋巴结转移的相关因素[J].中国肿瘤临床,2007,34(11)：650-652.
[26]黄金球,安丰山.影响胸段食管癌切除术后的预后因素的Cox模型分析[J].实用诊断与治疗杂志,2004,18(3)：178-180.
[27]谢映涛,王树钦,黄金球,等.影响胸段食管鳞癌切除术预后的因素[J].中华肖肠外科杂志,2004,7(6)：453-455.
[28]王永岗,汪良骏,张庆斌,等.影响食管癌手术治疗患者预后的病理因素分析[J].实用癌症杂志,2001,16(5)：516-517.
[29]Nishimaki T, Suzuki T, Kuwabara S, et al. Outcomes of extended radical esophagectomy for thoracic EC [J]. J Am Coll Surg,1998,186(3):306-312.
[30]Rizk NP, Venkatraman E, Bains MS, et al. Ameriean Joint Committee on cancer staging system does not accurately predict survival in patients receiving multimodality therapy for esophageal adenocarcinoma [J]. J Clin oncol,2007,25(5):507.
[31]Rizk N, venkatraman E, Park B, et al.The prognostic Importance of the number Of involved lymph nodes in esophageal cancer:ImPlications for revisions of the American Joint Committee on Cancer staging system [J]. J Thorac Cardiovasc Surg,2006,132(6): 1374.
[32]Kimura H,Konishi K, ArakawaH,et al. Number of lymph node metastases infulences survival in patients with thoracic esophageal carcinoma: therapeutic value of radiation treatment for recurrence [J]. Dis Esophagus,1999,12(3):205-208.
[33]陈建华,卫功铨,陈明耀,等.食管癌淋巴结转移的预后评价[J].中华肿瘤杂志,1994,16(6)：441-443.
[34]许运龙,郭昭扬.胸部食管癌转移淋巴结数与预后的临床研究[J].中华肿瘤杂志,2000,22(3)：244-246.
[35]王永刚,汪良俊,张德超,等.胸段食管鳞癌淋巴结转移特点及临床意义[J].中华肿瘤杂志,2000,22(3)：241-243.
[36]孙克林,张汝刚,张大为,等.食管癌淋巴结转移对手术治疗预后的影响[J].中华外科杂志,1995,33(4)：222-224.
[37]张合林,何明,张方林.淋巴结转移数对食管癌预后和国际TNM分期的影响[J].中国肿瘤临床,2008,35(5)：252-255.
[38]Tachikawa D, Inada S, Kotoh T, et al. An evaluation of malig-nancy and prognostic factors based on mode of lymph node metastasis in esophageal cancinoma [J]. Surg Today,1999, 29(11):1131-1135.
[39]Adachi W, Koike S, Nimura Y, et al. Clinicopathologic charac-teristics and postoperative outcome in Japanese and Chinese patients with thoracic oesphageal cancer [J]. World J Surg,1996,20(3):332-336.
[40]Casson AG, van Lanschot JJ. Improving outcomes after esophagectomy: the impact of operative volume [J]. J SurgOncol,2005,92(3):262-266.
[41]Siewert JR, Stein HJ, Feith M, et al. Histologic tumor type is an independent prognostic parameter in EC:lessons from more than 1,000 consecutive resections at a single center in the Western world [J]. Ann Surg,2001,234 (3):360-367.
[42]Mitani M, Kuwabara Y, Shinoda N, et al. Long 2 term survivors after the resection of limited esophageal small cell carcinoma [J]. Dis Esophagus,2000,13(3):259-261.
[43]Tajima Y, Nakanishi Y, Ochiai A, et al. Histopat hologic findings predicting lymph node metastasis and prognosis of patients with superficial EC:analysis of 240 surgically resected tumors [J]. Cancer,2000,88(6):1285-1293.
[44]Tachibana M, Kinugasa S, Yoshimura H, et al. Clinical outcomes of extended esophagectomy with three-field lymph node dissection for esophageal squamous cell carcinoma [J]. Am J Surg,2005,189(1):98-109.
[45]Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methy2 lation in the plasma and serum of liver cancer patients [J]. Cancer Res,1999,59(1):71-73.
[46]Hibi K, Robinson CK, Booker S, et al, Molecular detection of genetic alteration in the serum of colorectal cancer patient [J]. Cancer Res,1998,58(7):1405-1407.
[47]Goessl C, Heicappell R, Munker R, et al. Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma [J]. Cancer Res,1998,58 (20):4728-4732.
[48]Koible k, Vicrich CM, Pidde H, et al. Microsatellite alterations in serum DNA of patients with colorectal cancer [J]. Lab Invest,1999,79(9):1145-1150.
[49]Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells [J]. Cancer Res, 2001,61 (4):1659-1665.
[50]于金明,杨丽华,国前,等.流式细胞计测定食管癌细胞DNA含量和临床及病理特性的关系[J].中国肿瘤临床,1990,17(3)：150.
[51]麻国新,周道安,平育敏,等.食管癌DNA含量及P53蛋白表达与术前放疗[J].中华肿瘤杂志,1999,21(3),19-23.
[52]Matsuura H, Kuwano H, Morita M, et al. Predicting recurrence time of esophageal carcinoma through assessment of histologic factors and DNA ploidy [J]. Cancer,1991, 67(5):1406-1411.
[53]Watanzbe M, Kuwano H, Tanaka S, et al. Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of the esophagus [J]. Cancer,1999,85(11):2322-2328.
[54]Mukaida H, Toi M, Hirai T, et al. Clinical significance of the expression of epidermal growth factor and its receptor in esophageal cancer [J]. Cancer,1991,68(1):1422-1481.
[55]卢兆桐,君格平,孙晓明,等.食管癌DNA和RNA含量测定的临床意义[J].临床肿瘤学杂志,1997,2(3)：20.
[56]Kitagawa Y, Ueda M, Andol V, et al, Significance of int-2/hst-1 coamplification as a prognostic factor in patients with the esophageal squamous carcinoma [J]. Cancer Res, 1991,51(5):1504-1508.
[57]王永康,陈景寒,孟龙,等.食管鳞癌预后因素的病理学和免疫组织化学研究[J].中华病理学杂志,2000,29(4)：25-29.
[58]寇仁业,李劲松,孙桂武,等.食管癌C-erbB-2原癌基因的表达及意义[J].中国胸心血管外科临床杂志,1997,4(3)：157.
[59]寇仁业,李劲松,孙桂武,等C-erbB-2原癌基因的表达与食管癌患者的预后[J].临床肿瘤学杂志,1996,1(34)：5.
[60]Nagasawa S, Onda M, Sasajima K, et al. Cyclin D1 over expression as a prognostic factor in patients with esophageal carcinoma [J]. J Surg Oncol,2001,78(3):208-214.
[61]祝叔钗,翟福山,殷蔚伯,等.周期素D1蛋白表达对食管癌预后的影响[J].中华肿瘤杂志,1998,20(2)：129.
[62]Saito H, T sujitani S, Oka S, et al. The expression of murine double minute 2 is afavorable prognostic marker in esophageal squamous cell carcinoma without p53 protein accumulation [J]. Ann Surg Oncol,2002,9(5):450-456.
[63]Hashimoto N, Tachibana M, Dhar DK, et al. Expression of p53 and RB proteins in squamous cell carcinoma of the esophagus:their relationship with clinicopathologic characteristics [J]. Ann Surg Oncol,1999,6(5):489-494.
[64]李华川,陆十新.食管癌组织中Rb基因突变与表达的研究[J].中华肿瘤杂志,1993,15(6)：412.
[65]陈克能,张力健,师晓天.食管癌基因分子水平的研究进展[J].中国胸心血管外科临床杂志,1999,6(1)：57.
[66]Nozoe T, Korenaga D, Itoh S, et al. Clinico pathological significance of pRb2/p130 expression in squamous cell carcinoma of the esophagus [J]. J Cancer Res Clin Oncol, 2002,128(12):691-696.
[67]Shimada H, Nabeya Y, Okazumi S, et al. Prognostic significance of serum p53 antibody in patients with esophageal squamous cell carcinoma[J]. Surgery,2002,132(1):41-47.
[68]陈慰浙,马童丽.P53基因蛋白在食管鳞癌中的表达[J].实用肿瘤杂志,1994,9(1)：32.
[69]闫庆娜,战忠利.食管癌P53基因蛋白检测的临床意义[J].中国肿瘤临床,1998,25(9)：683.
[70]吕桂泉,陈旭峰,曹巍,等.食管鳞状细胞癌中P16基因变异的临床意义及地域性差异[J].中华肿瘤杂志.1999,21(5)：359.
[71]Singh SP, Lipman J, Goldman H, et al. Loss or altered subcellular localization of P27 in Barrett's associated adenocarcinoma [J]. Cancer Res,1998,58(15):1730.
[72]Lin LI, Lee PH, Wu CM, et al. Significance of nm23 mRNA expression in human hepotocellular carcinoma [J]. Anticancer Res,1998,18(1B):541-546.
[73]Wang CS, Lin KH, Hsu YC, et al. Distant metastasis of gastric cancer is associated with elerated expression of anti2metastatic nm23 gene [J]. Cancer Lett,1998,128(1):23-29.
[74]Wang LS, Chow KC, Wu YC, et al. In Inverse expression of di2 hydrodiol dehydrogenase and glutathione2 transferase in patients with esophageal squamous cell carcinoma [J]. Int J Cancer,2004,111 (2):246-251.
[75]Lin WK, Chu YL, Zhang F, et al. The relationship between HPV16 and expression of CD44v6, nm23H1 in esophageal squamous cell carcinoma [J]. Arch Virol,2005,15(5): 991-1001.
[76]华平,黄洪铮,张华,等.食管癌中CD44S、CD44V6和nm23基因蛋白表达的研究[J].中国肿瘤临床,1999,26(9)：666.
[77]李桂芳.转移抑制基因nm23与肿瘤转移[J].中国肿瘤临床,1996,23(10)：741
[78]沈金辉,曾纠文,蔡广玲,等.200例食管癌nm23/NDPK表达与淋巴结转移的关系[J].中国肿瘤临床,1998,25(4)：305.
[79]吴灵飞,姚俊国,张锦,等.nm23-H1蛋白在食管鳞癌中的表达及意义[J].中国肿瘤临床,1997,24(10)：778.
[80]李宣海,主编.肿瘤标志物的检测和临床[M].第1版.北京：人民卫生出版社,1997,177.
[81]吴名耀,郑瑞明,沈健,等.食管癌增殖细胞核抗原的免疫组织化学研究[J].中华病理学杂志,1993,22(4)：239.
[82]卢兆桐,张宝仁,孔宪涛,等.鳞状细胞癌抗原在食管癌临床的应用[J].中华胸心外科杂志,1994,10(3)：239.
[83]Charoenrat P, Rhys-Evans PH, Archer DJ, et al. C-erbB receptors in squamous cell carcinomas of the head and neck:clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors [J]. Oral Oncol,2002,38(1): 73-80.
[84]刘以民,郭永军,张国强,等.表皮生长因子受体在人食管鳞状细胞癌中的高表达及其对预后的影响[J].中华病理学杂志,1993,22(5)：303.
[85]战忠利,常新忠.食管癌中P-糖蛋白表达及其临床病理意义[J].中国肿瘤临床,1998,25(8)：588.
[86]杨国涛,王善政,孙鸿铖,等.食管鳞状细胞癌核仁组成区嗜银蛋白的研究[J].中华胸心血管外科杂志,1995,1 1(5)：288.
[87]Fukai Y, Fukuchi M, Masuda N, et al. Reduced expression of transforming growth factor-beta receptors is an unfavorable prognostic factor in human esophageal squamous cell carcinoma [J]. Int J Cancer,2003,104(2):161-166.
[88]Natsugoe S, Xiangming C, Matsumoto M, et al. Smad4 and transforming growth factor betal expression in patients with squamous cell carcinoma of the esophagus [J]. Clin Cancer Res,2002,8(6):1838-1842.
[89]Kyriazanos ID, Tachibana M, Dhar DK, et al. Expression and prognostic significance of S100A2 protein in squamous cell carcinoma of the esophagus [J]. Oncol Rep,2002,9(3): 503-510.
[90]Shimada H, Takeda A, Nabeya Y, et al. Clinical significance of serum vascular endothelial growth factor in esophageal squamous cell carcinoma [J]. Cancer,2001,92(3):663-669.
[91]Wallner G, Ciechanski A, Dabrow ski A, et al. Vascular endothelial growth factor and basic fibroblast growth factor in patients with squamous cell oesophageal cancer [J]. Folia Histochem Cytobiol,2001,39(Supp12):122-123.
[92]Mobius C, Freire J, Beeker 1, et al. VEGF-C expression in squamous cell carcinoma and adenocarcinoma of the esophagus [J]. Worid J Surg,2007,31(9):1768.
[93]Kii T, Takiuchi H, Kawabe S, et al. Evaluation of prognostic factors of esophageal squamous cell carcinoma (stageⅡ-Ⅲ) after concurrent chemoradiotherapy using biopsy specimens [J]. Jpn J Clin oncol,2007,37(8):583.
[94]Matsumoto S. Prognostic significance of platelet-derived growth factor-BB expression in human esophageal squamous cell carcinomas [J]. Anticancer Res,2007,27(4B):2409.
[95]Hsia JY, Chen CY, Chen JT, et al. Prognostic significance of caspase-3 expression in primary resected esophageal squamous cell carcinoma [J]. Eur J Surg Oncol,2003,29(1): 44-48.
[96]高文信,刘洪静,王颖,等Caspase-3在口腔粘膜癌前病变及鳞状细胞癌中的表达研究.现代口腔医学杂志,2003,17(2)：109-111.
[97]Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma [J]. Br J Cancer,2002,87(8):883-887.
[98]Cornford PA, Dodson AR, Parsons KF, et al. Heat shock protein expression independently predicts clinical outcome in prostate cancer[J]. Cancer Res,2000,60(24):7099-7105.
[99]曹富氏,王小玲,张祥宏,等.术后不同生存期患者食管鳞状细胞癌热休克蛋白27与70表达及临床意义[J].中国肿瘤临床,2004,31(8)：462-465.
[100]Nakajima M, Kuwano H, Miyazaki T, et al. Significant correlation between expression of heat shock proteins 27,70 and lymphocyte infiltration in esophageal squamous cell Carcinoma [J]. Cancer Lett,2002,178(1):99-106.
[101]Sarbia M, Bit tinger F, Porschen R, et al. Bcl-2 expression and prognosis in squamous cell carcainoma of the esophageal [J]. Int J Cancer,1996,69(3):324.
[102]Raouf AA, Evoy DA, Carton E, et al. Loss of Bcl-2 expression in Barrett's dysplasia and adenocarcinoma is associated with tumor progression and worse survival but not with response to neoadjuvant chemoradiation [J]. Dis Esophagus,2003,16(1):17-23.
[103]杨志江,李桂红,蒲红伟,等.食管癌组织中Bcl-2、cmyc、iNOS表达及其意义[J].陕西医学杂志,2008,37(6)：686-688.
[104]Parenti AR, Rugge M, Shiao YH, et al. Bcl-2 and P53 immunophenotypes in pre-invasive, early and advanced esophageal squamous cancer [J]. Histopathology,1997,31(3):430.
[105]黄莺,王康敏,何长成,等.Bcl-2和P16蛋白在食管癌中的表达及意义[J].肿瘤防治研究,2003,30(4)：259-261.
[106]曾亮,高平.Bcl-2、P53在食管鳞癌组织中的表达的临床意义[J].宁夏医学院学报,2008，30(3)：298-299.
[107]Konishi S, Ishiguro H, Shibata Y, et al. Decreased expression of DFF45/ICAD is correlated with a poor prognosis in patients with esophageal carcinoma [J]. Cancer,2002,95(12): 2473-2478.
[108]Kase S, Osaki M, Adachi H, et al. Expression of Fas and Fas lig and in esophageal tissue mucosa and carcinomas [J]. Int J Oncol,2002,20(2):291-297.
[109]Nishioka K, Doki Y, Shiozaki H, et al. Clinical significance of CDC25A and CDC25B expression in SCCs of the oesophagus [J]. Br J Cancer,2001,85(3):412-421.
[110]Takeno S, Noguchi T, Kikuchi R, et al. Prognostic value of cyclin B1 in patients with esophageal SCC [J]. Cancer,2002,94(11):2874-2881.
[111]Fukuchi M, Fukai Y, Masuda N, et al. High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma [J]. Cancer Res,2002,62(24):7162-7165.
[112]Basire A, Sabarier F, Ravet S, et al. High urokinase expression contributes to the angiogenic properties of endothelial cells derived from circulating progenitors. Thromb Haemost,2006,95(4):678-688.
[113]Torzewski M, SarbiaM, Verreet P, et al. Prognostic significance of urokinase-type plaseminogen activator expression in squamons cell carcinoma of the esophagus [J]. Clin Cancer Res,1997,3(12):2263-2268.
[114]Buskens CJ, Van Rees BP, Sivula A, et al. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus [J]. Gastroenterology,2002,122(7):1800-1807.
[115]Shamma A, Yamamoto H, Doki Y, et al. Up-ragulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus [J]. Clin Cancer Rus,2000,6(4):1229-1238.
[116]王顺文,高青.环氧化酶-2与食管癌临床病理特征及预后关系的研究[J].肿瘤防治杂 志,2004,10(11)：1051-1053.
[117]Nakanishi Y, Ochiai A, Akimoto S, et al. Expression of E-cadherin, alpha-catenin, beta-catenin and plakoglobin in esophageal carcinomas and its prognostic signifience: immunohistochemical analysis of 96 lesions[J]. Oncology,1997,54(2):158-165.
[118]Wu H, Lotan R, Menter D, et al. Expression of E-cadher in is associated with squamous differentiation I squamous cell carcinomas [J]. Anticancer Res,2000,20(3):1385-1390.
[119]林英城,吴名耀,李德锐.食管鳞癌ｙ-连接蛋白和钙粘附蛋白表达及其预后意义[J].肿瘤防治杂志,2001,8(5)：467-469.
[120]陈捷,王兰兰,等.食管癌患者手术前后机体免疫调节与反应性蛋白表达的分析[J].免疫学杂志,2006,22(4)：419-420.
[121]Nozoe T, Saeki H, and Sugimachi K. Significance of preoperative elevation of serum C-reactive protein as an indicator of prognosis in EC[J]. Am J Surg,2001,182(2):197-201.
[122]Nakamura T, Ide H, Eguchi R, et al. Cyfra21-1 is a useful marker for esophageal squamous cell carcinoma [J]. Cancer,1997,79(9):1467-1475.
[123]Shimada H, Nabeya Y, Okazumi S, et al. Prognostic significance of CYFRA21-1 in patients with esophageal squamous cell carcinoma[J]. J Am Coll Surg,2003,196:573-578.
[124]Maeda K, Chung YS, OgawaS, et al. Thymidine phosphorylas eplatelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma [J]. Br J Cancer,1996,73:884-888.
[125]Takebayashi Y, Miyadera K, Akiyama S, et al. Expression of thymidine phosphorylase in human gastric carcinoma [J], Jpn J Cancer Res,1996,87:288-295.
[126]Igarashi M, Dhar DK, Kubota, et al. The prognostic significance of microvessel density and thymidine phosphorylase expression in squamous cell carcinoma of the esophagus [J]. Cancer,1998,82:1225-1232.
[127]Shimada H, Takeda A, Shiratori T, et al. Prognostic significance of serumthymidine phosphorylase concentration in esophageal SCC[J]. Cancer,2002,94(7):1947-1954.
[128]张晓光,药立波,苏成.酪氨酸激酶受体EPh亚族的研究进展[J].生物化学与生物物理进展,1999,26：138.
[129]张晓光,阎小君,苏成芝,等.酪氨酸蛋白激酶受体EPh亚族新基因HEK在儿株人肿瘤细胞系的表达[J].第四军医大学学报,1998,19：577.
[130]Miyazaki T, Kato H, Fukuchi M, et al. EphA2 Over expression correlates with poor prognosis in esophageal squamous cell carcinoma[J]. Int J Cancer,2003,103(5):657-663.
[131]Kinch MS, Moore MB, HarPole DH. Predietive value of the EPhA2 recePtor Tyrosine kinase in lung cancer recurrence and survival [J]. Clin Caneer Res,2003,9:613.
[132]Miyazaki T, Kato H, Fukuehi M, et al. EPhA2 overexPression correlates With Poor Prognosis in esophageal squamous cell carcinoma [J]. Int J Caneer,2002,103:657.
[133]Schwartz M A. Integrin oncogenes and ancorage independence [J]. J CellBio,1997,139(3): 575-581.
[134]Miyazaki T, Kato H, Nakajima M, et al. FAK Over expression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma [J]. Br J cancer,2003,89(1):140-145.
[135]Miyazaki T, Kato H, Kimura H, et al. Evaluation of tumor malignancy in esophageal squamous cell carcinoma using different characteristic factors [J]. Anticancer Res,2005, 25(6B):4005-11.
[136]黄海花,吴秀浅,等.粘着斑激酶在食管癌中表达的临床意义[J].现代肿瘤医学,2006,14(1)：23-25.
[137]Medical Research Coucil Oesophageal Cancer Work Groph. Surgical research with or without preoperative chemotherapy in oesphageal cancer: a randomised controlled trial [J]. Lancet,2002,359(9319):1727-1733.
[138]Thirion PG, Michiel S, Le Maitre A, et al. On behalf of the Meta Analysis of chemotherapy in esophageal cancer collaborative groph. Individual patient data-based meta-analysis assessing pre-operative chemotherapy in resectable oesophageal carcinoma [J]. J Clin oncol,2007,25(18-suppl):4512.
[139]Raja SG. Does neoadjuvant chemotherapy improve survival in patients with resectable thoracic oesophageal cancer [J]. Interact Cardiovasc Thorac Surg,2007,6(5):661.
[140]汪楣,谷铣之,黄国俊.食管癌术前放射治疗的前瞻性临床研究[J].中华放射肿瘤学杂志,2001,10(3)：168-172.
[141]Sydney JA, William Duncan, Marc Gignoux. Preoperative Radiotherapy in Esophageal Carcinoma:A meta-analysis using Individual Patient Data (Oesophageal Cancer Collaborative Group)[J]. Int J Radiation Oncology Biol Phys,1998,41(3):579-583.
[142]安丰山,黄金球,谢映涛.食管癌新辅助放化疗的前瞻性临床研究[J].中华肿瘤杂志,2003,25(4)：376-379.
[143]Burmeister BH, Smithers BM, Fitzgerald L, et al. A randomized phase III trial of preoperative chemoradiation followed by surgery (CR-S) versus alone (S) for localized respectable cancer of the esophageal [J]. Proc Am Soc Clin Oncol,2002,21:518.
[144]Ioannis GK, Gail RW, Kristian F. Neoad juvant Treatment for resectable Cancer of the Esophageal and the gastroesophageal Junction: A meta analysis of randomized clinical trials [J]. Annals of Surgical Oncology,2003,10(7):754-761.
[145]John DU, Hari VB, Chris JB. A meta-analysis of randomized controlled trials that compared neoadjuvant chemotherapy and surgery to surgery alone for respectable esophageal cancer [J]. The American of surgery,2002,188(3):274-279.
[146]Tepper J, Krasna MJ, Niedzwiecki D, et al. Phase Ⅲ trial of trimodality therapy with cisplatin, fluorouracil, radiotherapy, and surgery compared with surgery alone for esophageal cancer: CALGB 9781[J]. J Clin Oncol,2008,26(7):1086-1092.
[147]肖泽芬,杨宗贻,梁军.食管癌根治术后预防性放射治疗的临床价值[J].中华放射肿瘤学杂志,2002,24(6)：608-611.
[148]Robccca W, R malthaner. Combined chemotherapy and radiotherapy (without surgery) compared with radiotherapy alone in localized carcinoma of the esophagus [J]. The Cochrane Library,2006(1):CD002092.
[149]Cooper JS, Guo MD, Herskovic A, et al. Chemoradiotherapy of locally advanced esophageal cancer. Long-term Follow-up of a prospective randomized trial (RTOG85-01) [J]. JAMA,1999,281(17):1623-1627.
[150]Conroy T, Yataghene Y, Etienne PL, et al. Definitive chemoradiotherapy (CRT) with FOLFOX4 or 5-FU-cisplatin as first line treatment for patients (pts) with inoperable esophageal cancer (IEC):Final results of a randomized phase Ⅱ study. J Clin Oncol[J], 2007,25(18-suppl):4532.