食管癌放疗患者生存相关遗传变异的研究

发布时间：2018-07-05 15:06

本文选题：食管鳞状细胞癌 + 单核苷酸多态　；参考：《北京协和医学院》2015年博士论文

【摘要】：目的：虽然食管癌患者单纯放疗、同步放化疗和根治术后预防性放疗已成为常规辅助治疗手段,但接受放疗的患者疗效和预后差异较大,具体原因还未被阐明。由于治疗前尚没有可靠方法鉴别出哪些患者从放疗中获益,所以在临床治疗中存在过度治疗或治疗不足的问题。本研究运用全基因组关联研究的方法,发现影响食管癌放疗患者预后的遗传变异。此外,由于ATM在电离辐射或某些抗癌药物导致的DNA损伤应答中发挥重要作用,我们还探讨ATM单核苷酸多态(SNP)是否与单纯放疗或同步放化疗的Ⅲ/Ⅳ期食管癌患者生存相关。方法：我们采用全基因组关联研究的策略,第一阶段,利用Affymetrix GeneChip Human Mapping 6.0 set芯片对282例Ⅱ/Ⅲ期食管癌根治术后预防性放疗患者进行全基因组生存相关的SNP研究；第二阶段,用Sequenom平台分型方法在243例独立样本中验证最有统计学显著性差异(P104)的SNP。最后在262例食管癌单纯放疗和388例单纯手术患者中验证这些生存相关SNP。此外,我们应用PCR-RFLP方法检测了412例单纯放疗或同步放化疗治疗的Ⅲ/Ⅳ期食管癌患者ATM基因4个标签SNP的基因型。采用Kaplan-Meier法分析不同基因型患者生存情况,Log-rank法进行单因素检验。采用多因素Cox比例风险回归模型分析评估每一个SNP与生存的关联。我们还进行一系列生化实验探讨影响食管癌放疗患者生存的SNP和基因功能。结果：525例根治术后预防性放疗食管癌患者中,304例(57.9%)患者死亡,中位生存期(MST)是30.0个月。运用Cox多因素回归模型校正年龄、性别、吸烟、饮酒以及病理分期,结果显示位于KIT基因上游约22 kb的rs11722325显著影响患者的生存(P=2.82×10-7)。rs11722325 AA、AC和CC基因型患者MST为46.0个月、29.0个月和19.0个月,多因素Cox加性模型分析携带C等位基因患者的死亡风险比A等位基因患者增高,HR和95% CI为1.54(1.30-1.81)。rs11722325与Ⅱ-Ⅳ期接受单纯放疗食管癌患者的生存显著相关(P=0.0057)；但在单纯手术Ⅰ-Ⅲ期食管癌患者中并未发现rs11722325显著影响患者生存。rs6554199与rs11722325连锁不平衡,连锁度r2为0.77,该位点位于KIT基因启动子区域,显著影响患者的无病生存期(P=0.0004)和总生存期(P=3.58×10-5).rs6554199 GG.GT和TT基因型食管癌根治术后放疗患者MST为44.0个月、28.0个月和20.0个月,多因素Cox加性模型分析携带T等位基因患者的死亡风险比G等位基因患者增高,HR和95% CI为1.50(1.27-1.78)。此外,rs6554199还与Ⅱ-Ⅳ期接受单纯放疗食管癌患者的生存显著相关(P=8.60×10-5)；但并未发现rs6554199显著影响单纯手术Ⅰ-Ⅲ期食管癌患者生存。生物化学实验表明rs6554199 GT遗传变异破坏了KIT基因启动子区rs6554199 G等位基因与核磷酸蛋白(NPM)核转录因子的特异性结合,从而抑制KIT mRNA和蛋白表达水平。KIT表达在食管癌组织中显著低于配对食管正常组织。当无X射线照射,食管癌细胞中KIT表达不影响食管癌细胞的增殖；X射线照射之后,KIT过表达抑制食管癌细胞增殖和集落形成。KIT过表达降低了由X射线引起的DSBs损伤修复能力,增加食管癌细胞放射敏感性。过表达KIT可以通过抑制β-catenin和Slug表达而上调E钙粘蛋白表达,从而抑制食管癌细胞的侵袭和迁移。此外,研究发现ATM基因rs664143和rs664677与单纯放疗或同步放化疗治疗食管癌患者生存显著相关,携带rs664143 AA或GA基因型患者MST 14.0个月,显著短于GG基因型患者MST 20.0个月,HR以及其95% CI为1.45(1.12-1.89)。携带rs664677 CC或TC基因型患者MST 14.0个月,显著短于携带TT基因患者MST23.5个月,HR以及其95% CI为1.57(1.18-2.08)。按临床分期和治疗方案分层分析发现rs664143和rs664677与食管癌放疗患者生存都相关。rs664143、rs664677、rs189037和rs373759 4个标签SNP与单纯放疗或同步放化疗治疗食管癌患者局部进展相关(rs664143,P=0.018;rs664677,P=0.014;rs189037,P=0.024;rs373759, P=0.040).但是这4个标签SNP与单纯手术治疗食管癌患者的生存都不相关。结论：本研究发现的位于KIT基因上游约22 kb的rs11722325和启动子区的rs6554199是食管癌放疗患者预后相关遗传因素。分子流行病学和功能研究相一致的结果强烈支持KIT rs6554199 GT变异是影响KIT基因表达的功能性遗传变异。KIT基因异常表达与放疗敏感性相关。此外,ATM基因上rs664143和rs664677与单纯放疗或同步放化疗中晚期食管癌患者局部进展和生存相关。这些遗传变异可能是预测食管癌放疗患者预后的生物标志物,将对指导食管癌个体化放疗具有潜在应用价值。
[Abstract]:Objective: Although radiotherapy, concurrent radiochemotherapy and prophylactic radiotherapy have become a routine adjuvant therapy for patients with esophageal cancer, the curative effect and prognosis of patients receiving radiotherapy are different, and the specific reasons are still unexplained. There is a problem of overtreatment or inadequate treatment. This study uses a whole genome association study to detect the genetic variation that affects the prognosis of patients with esophageal cancer radiotherapy. In addition, we also explore whether the ATM single nucleotide polymorphism (SNP) is associated with the important role of ATM in the DNA damage response caused by ionizing radiation or some anticancer drugs. Survival correlation of patients with stage III / IV esophageal cancer with radiotherapy alone or synchronous radiotherapy. Methods: We used the strategy of whole genome association study. In the first stage, we used the Affymetrix GeneChip Human Mapping 6 set chip to carry out complete genome survival related SNP study on 282 patients with stage II / III esophageal carcinoma after radical resection of esophageal cancer. In the second stage, the Sequenom platform classification method was used to verify the most statistically significant difference (P104) in 243 independent samples (SNP.). Finally, the survival related SNP. was verified in 262 cases of simple radiotherapy of esophageal cancer and 388 patients with simple surgery. We used PCR-RFLP method to test 412 cases of radiotherapy alone or synchronous radiotherapy. The genotype of 4 label SNP of ATM gene in patients with stage IV esophageal cancer. The survival of patients with different genotypes was analyzed by Kaplan-Meier method and single factor test was performed by Log-rank method. The association of each SNP with survival was evaluated by multifactor Cox proportional hazard regression model. We also conducted a series of biochemical experiments to influence the radiotherapy of esophageal cancer. SNP and gene function of the patient's survival. Results: of the 525 patients with prophylactic radiotherapy of the esophagus, 304 (57.9%) patients died and the median survival time (MST) was 30 months. The Cox multiple regression model was used to correct age, sex, smoking, drinking, and pathological staging. The results showed that the rs11722325 in the upstream of the KIT gene was about 22 kb in the upstream of the KIT gene. The patients' survival (P=2.82 x 10-7).Rs11722325 AA, AC and CC genotype patients were 46 months, 29 months and 19 months. The multiple factor Cox additive model was used to analyze the mortality risk of C allele patients than those of the A allele, HR and 95% CI were 1.54 (1.30-1.81) and stage II - IV received simple radiotherapy for esophageal cancer patients. There was a significant correlation of survival (P=0.0057), but in patients with simple operation I and III of the esophagus, rs11722325 had not been found to significantly affect the survival of the patients with.Rs6554199 and rs11722325 linkage disequilibrium, and the linkage R2 was 0.77. The loci were located in the KIT gene promoter region, which significantly affected the patient's disease-free survival (P=0.0004) and the total survival period (P=3.58 * 10-5). MST was 44 months, 28 months and 20 months after radical resection of.Rs6554199 GG.GT and TT genotypes. The mortality risk of T alleles in patients with T allele was higher than those of G alleles, HR and 95% CI were 1.50 (1.27-1.78). Besides, rs6554199 and stage II - IV received simple radiotherapy for esophageal cancer patients. The survival was significant (P=8.60 x 10-5), but it was not found that rs6554199 significantly affected the survival of patients with stage I - III of the esophagus. Biochemical experiments showed that rs6554199 GT genetic variation destroyed the specific binding of the rs6554199 G allele to the nuclear phosphoroprotein (NPM) nuclear transcription factor in the KIT gene promoter region, thus inhibiting KIT mRNA and The expression of protein expression level.KIT in esophageal cancer tissues was significantly lower than that of normal esophageal tissue. When no X ray irradiation, the expression of KIT in esophageal cancer cells did not affect the proliferation of esophageal cancer cells. After X ray irradiation, KIT overexpression inhibited the proliferation of esophageal cancer cells and the overexpression of colony forming.KIT, which reduced the DSBs damage caused by X rays. Overexpression of KIT can inhibit the expression of E cadherin by inhibiting the expression of beta -catenin and Slug, thus inhibiting the invasion and migration of esophageal cancer cells. In addition, the study found that the ATM gene rs664143 and rs664677 are significantly related to the survival of patients with esophageal cancer with simple radiotherapy or concurrent chemoradiotherapy for the survival of esophageal cancer patients. Patients with rs664143 AA or GA genotype were MST for 14 months, significantly shorter than GG genotype for 20 months, HR and 95% CI 1.45 (1.12-1.89). Rs664677 CC or TC genotypes were 14 months, significantly shorter than those who carried the gene patients for months, and 95% for 1.57. The analysis found that rs664143 and rs664677 were related to the survival of the patients with esophageal cancer,.Rs664143, rs664677, rs189037 and rs373759 4 Tags SNP related to the local progression of esophageal cancer patients with simple radiotherapy or synchronous radiotherapy (rs664143, P=0.018; rs664677, P=0.014; rs189037,). The survival of the patients with esophageal cancer was unrelated. Conclusion: the rs11722325 in the upstream of the KIT gene and the rs6554199 in the promoter region, located in the upstream of the KIT gene, are the genetic factors associated with the prognosis of the patients with esophageal cancer radiotherapy. The results of molecular epidemiology and functional studies strongly support the KIT rs6554199 GT variation which is the influence of the KIT gene table. The abnormal expression of.KIT gene is associated with radiation sensitivity. In addition, the ATM gene rs664143 and rs664677 are related to the local progression and survival of patients with advanced esophageal cancer in simple radiotherapy or concurrent radiotherapy and chemotherapy. These genetic variations may be a biomarker for predicting the prognosis of patients with esophageal cancer and will guide esophageal cancer. Individualized radiotherapy is of potential application value.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

【共引文献】