本文選題：食管鱗狀細胞癌 + 單核苷酸多態(tài)　； 參考：《北京協(xié)和醫(yī)學院》2015年博士論文

【摘要】：目的：雖然食管癌患者單純放療、同步放化療和根治術后預防性放療已成為常規(guī)輔助治療手段,但接受放療的患者療效和預后差異較大,具體原因還未被闡明。由于治療前尚沒有可靠方法鑒別出哪些患者從放療中獲益,所以在臨床治療中存在過度治療或治療不足的問題。本研究運用全基因組關聯(lián)研究的方法,發(fā)現(xiàn)影響食管癌放療患者預后的遺傳變異。此外,由于ATM在電離輻射或某些抗癌藥物導致的DNA損傷應答中發(fā)揮重要作用,我們還探討ATM單核苷酸多態(tài)(SNP)是否與單純放療或同步放化療的Ⅲ/Ⅳ期食管癌患者生存相關。方法：我們采用全基因組關聯(lián)研究的策略,第一階段,利用Affymetrix GeneChip Human Mapping 6.0 set芯片對282例Ⅱ/Ⅲ期食管癌根治術后預防性放療患者進行全基因組生存相關的SNP研究；第二階段,用Sequenom平臺分型方法在243例獨立樣本中驗證最有統(tǒng)計學顯著性差異(P104)的SNP。最后在262例食管癌單純放療和388例單純手術患者中驗證這些生存相關SNP。此外,我們應用PCR-RFLP方法檢測了412例單純放療或同步放化療治療的Ⅲ/Ⅳ期食管癌患者ATM基因4個標簽SNP的基因型。采用Kaplan-Meier法分析不同基因型患者生存情況,Log-rank法進行單因素檢驗。采用多因素Cox比例風險回歸模型分析評估每一個SNP與生存的關聯(lián)。我們還進行一系列生化實驗探討影響食管癌放療患者生存的SNP和基因功能。結果：525例根治術后預防性放療食管癌患者中,304例(57.9%)患者死亡,中位生存期(MST)是30.0個月。運用Cox多因素回歸模型校正年齡、性別、吸煙、飲酒以及病理分期,結果顯示位于KIT基因上游約22 kb的rs11722325顯著影響患者的生存(P=2.82×10-7)。rs11722325 AA、AC和CC基因型患者MST為46.0個月、29.0個月和19.0個月,多因素Cox加性模型分析攜帶C等位基因患者的死亡風險比A等位基因患者增高,HR和95% CI為1.54(1.30-1.81)。rs11722325與Ⅱ-Ⅳ期接受單純放療食管癌患者的生存顯著相關(P=0.0057)；但在單純手術Ⅰ-Ⅲ期食管癌患者中并未發(fā)現(xiàn)rs11722325顯著影響患者生存。rs6554199與rs11722325連鎖不平衡,連鎖度r2為0.77,該位點位于KIT基因啟動子區(qū)域,顯著影響患者的無病生存期(P=0.0004)和總生存期(P=3.58×10-5).rs6554199 GG.GT和TT基因型食管癌根治術后放療患者MST為44.0個月、28.0個月和20.0個月,多因素Cox加性模型分析攜帶T等位基因患者的死亡風險比G等位基因患者增高,HR和95% CI為1.50(1.27-1.78)。此外,rs6554199還與Ⅱ-Ⅳ期接受單純放療食管癌患者的生存顯著相關(P=8.60×10-5)；但并未發(fā)現(xiàn)rs6554199顯著影響單純手術Ⅰ-Ⅲ期食管癌患者生存。生物化學實驗表明rs6554199 GT遺傳變異破壞了KIT基因啟動子區(qū)rs6554199 G等位基因與核磷酸蛋白(NPM)核轉錄因子的特異性結合,從而抑制KIT mRNA和蛋白表達水平。KIT表達在食管癌組織中顯著低于配對食管正常組織。當無X射線照射,食管癌細胞中KIT表達不影響食管癌細胞的增殖；X射線照射之后,KIT過表達抑制食管癌細胞增殖和集落形成。KIT過表達降低了由X射線引起的DSBs損傷修復能力,增加食管癌細胞放射敏感性。過表達KIT可以通過抑制β-catenin和Slug表達而上調(diào)E鈣粘蛋白表達,從而抑制食管癌細胞的侵襲和遷移。此外,研究發(fā)現(xiàn)ATM基因rs664143和rs664677與單純放療或同步放化療治療食管癌患者生存顯著相關,攜帶rs664143 AA或GA基因型患者MST 14.0個月,顯著短于GG基因型患者MST 20.0個月,HR以及其95% CI為1.45(1.12-1.89)。攜帶rs664677 CC或TC基因型患者MST 14.0個月,顯著短于攜帶TT基因患者MST23.5個月,HR以及其95% CI為1.57(1.18-2.08)。按臨床分期和治療方案分層分析發(fā)現(xiàn)rs664143和rs664677與食管癌放療患者生存都相關。rs664143、rs664677、rs189037和rs373759 4個標簽SNP與單純放療或同步放化療治療食管癌患者局部進展相關(rs664143,P=0.018;rs664677,P=0.014;rs189037,P=0.024;rs373759, P=0.040).但是這4個標簽SNP與單純手術治療食管癌患者的生存都不相關。結論：本研究發(fā)現(xiàn)的位于KIT基因上游約22 kb的rs11722325和啟動子區(qū)的rs6554199是食管癌放療患者預后相關遺傳因素。分子流行病學和功能研究相一致的結果強烈支持KIT rs6554199 GT變異是影響KIT基因表達的功能性遺傳變異。KIT基因異常表達與放療敏感性相關。此外,ATM基因上rs664143和rs664677與單純放療或同步放化療中晚期食管癌患者局部進展和生存相關。這些遺傳變異可能是預測食管癌放療患者預后的生物標志物,將對指導食管癌個體化放療具有潛在應用價值。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.1

【共引文獻】

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