分子标志物在食管癌同期放化疗中的指导价值研究

发布时间：2018-04-28 12:59

本文选题：食管肿瘤 + 放化疗法　；参考：《山东大学》2017年博士论文

【摘要】：第一部分ERCC1、TYMS、RRM1、TUBB3基因在食管鳞癌中的表达[研究目的]本研究的目的是利用实时荧光定量RT-PCR技术,检验不能手术食管鳞癌患者肿瘤组织中的ERCC1、TYMS、RRM1、TUBB3四种基因mRNA表达水平,观察四种基因在食管癌中的表达情况,以指导不能手术食管鳞癌患者同期放化疗时化疗药物选择。[研究方法]1、标本获取:26例患者全部选自2010年12月1日至2013年11月1日在山东大学附属省立医院肿瘤研究治疗中心住院患者,全部为病理证实的食管鳞状细胞癌初诊患者。排除既往患其他恶性肿瘤,排除处于怀孕期或哺乳期女性患者。获取患者肿瘤组织,经10%福尔马林固定,石蜡包埋,取包埋肿瘤组织切片8张,每张切片厚度8-10μm。2、实验步骤:利用荧光定量RT-PCR技术对组织标本行ERCC1、TYMS、RRM1、TUBB3基因mRNA表达水平定量检测。(1)提取肿瘤组织中总RNA。(2)用超微量紫外线分光光度计测量RNA浓度。(3)逆转录合成cDNA。(4)相对定量RT-PCR反应。(5)结果判定。3、数据处理:采用2 —△△Ct方法处理RT-PCR检测所得的数值,待测基因的表达差异用Ct比较法来计算。△Ct (校准标本)1=待测基因Ct值1—内参基因Ct值1△Ct (待测标本)2=待测基因Ct值2—内参基因Ct值2△△Ct= △Ct (待测标本)2—△Ct (校准标本)1用2-△△Ct表示表达差异。4、统计方法:采用SPSS 15. 0统计学软件进行数据处理,组间差异采用χ2检验,多因素分析用Cox回归模型,P0. 05表示差异有统计学意义。[结果]1、26例患者ERCC1、TYMS、RRM1、TUBB3四种基因的mRNA表达水平如下:ERCC1基因高、中、低表达例数分别为3(11. 5%)、12(46. 2%)、11例(42. 3%);TYMS基因表达高、中、低例数分别为9(34. 6%)、15(51.7%)、2(7. 7%); RRM1高、中、低表达例数分别为9 (34. 60%)、15(51.7%)、2(7. 7%); TUBB3高、中、低表达例数分别为 12(46.2%)、10(38. 5%)、4(15. 4%)。患者中四种基因共表达情况:四种基因同时高表达1例,四种基因同时中表达3例,同时低表达0例:三种基因同时中表达5例;两种基因同时低表达5例;2、基因表达与临床特征之间的关系:食管鳞癌肿瘤组织浸润深度、肿瘤长度、临床分期及淋巴结转移状态与ERCC1、TYMS、RRM1、TUBB3四种基因mRNA表达水平无明显相关性。第二部分分子标志物指导下同期放化疗对食管鳞癌的临床疗效[研究目的]不能手术的食管鳞癌目前标准治疗模式是同期放化疗。食管癌同期放化疗后的常见失败模式既有局部复发也有远处转移,提高临床疗效是迫切需要解决的问题。食管的耐受性限制了放疗剂量提高。而化疗药物方面尽管紫杉类方案较PF方案近期疗效好,但长期生存期未见明显优势。因此这部分的研究目的是探讨利用分子标志物指导和筛选同期放化疗中的药物,与标准方案的同期放化疗比较,观察治疗疗效的差别。[研究方法]1、入组及放化疗方案:54例不能手术的食管鳞癌患者进入本前瞻性非随机对照临床研究,分实验组和对照组。所有患者均给予同期放化疗:放疗采用调强放疗技术,常规分割,处方剂量设定为95%PTV为60～66Gy,2.0Gy/次,30～33次。计划要求95%等剂量曲线覆盖90%以上PTV。化疗在放疗的第1、5周给予。实验组化疗药物根据ERCC1、TYMS、RRM1、TUBB3四种基因的mRNA表达水、选择。首先,排除mRNA高表达相对应的药物:如ERCC1高表达排除顺铂;如3种基因高表达,1个基因低表达或中表达,则只选择一种药物'与放疗同步,如4种基因均高表达则选西妥昔单.抗;2种基因高表达2种中表达,则选择中表达对应的药物。其次,根据基因低表达选择相对应的药物:如3或4个基因低表达,则化疗药物采用两药联合,同时据年龄、身体状况、合并糖尿病等基础疾病综合考虑,依次选择顺铂、氟尿嘧啶、多西紫杉醇、吉西他滨;2种基因同时低表达选择相对应的两药联合;1种低表达保证其对应药物入选;第三,4种基因同时中表达首选PF方案;2种或3种基因中表达时选择药物顺序是顺铂、氟尿嘧啶、多西紫杉醇、吉西他滨;第四,兼顾药物毒副反应,如吉西他滨加重肺毒性不用于胸段食管癌,合并糖尿病的患者慎用多西紫杉醇。药物用法用量:顺铂15mg/m2第1-5天,氟尿嘧啶500mg/m2第1-5天,吉西他滨1000mg/m2第1、8天,多西紫杉醇60mg/m2第1天,每28天重复;西妥昔单抗首次剂量400mg/m2(第1天),随后每周剂量250mg/m2,共7周。主要观察两组的客观有效率和生存情况。2、放化疗结束后1-2个月行近期疗效评价。根据治疗前后胸部CT、食管内镜结合食管钡餐造影,评价标准参照实体瘤疗效评价标准(RECIST v1.1)进行,分为完全缓解(CR)、部分缓解(PR)、无变化(SD)及病变进展(PD)。总生存时间(OS):指治疗开始时间至患者死于任何疾病、截止观察时间和最后随诊时间。3、统计方法:采用SPSS 15.0统计软件,两组临床资料可比性及不良反应比较采用χ2检验,采用Kaplan-mmeier方法计算生存率,Log-rank检验用来比较组间生存率的差异性。P0. 05为差异有统计学意义。[结果]1、治疗完成情况:实验组具体药物分布如下:顺铂/氟尿嘧啶(PF) 10例,单药顺铂(P) 7例,多西他赛//顺铂(DP) 4例,吉西他滨/顺铂(GP) 2例,单药多西他赛(D) 2例,单药西妥昔单抗(C225) 1例。治疗完成情况:实验组2例患者未完成放疗,2例实际放疗剂量48Gy (未完成原因:1例放射性肺炎,1例出现纵隔瘘);有4例患者仅完成1周期化疗(其中1例因出现3级放射性食管炎而终止化疗;3例因发生4级血液学毒性未完成化疗;这4例病例分别发生在DP方案组2例、PF方案1例、GP方案1例)。对照组中3例患者未完成全程放疗,实际照射剂量分别为40Gy、50Gy、56Gy (未完成放疗原因:2例出现食管气管瘘、1例出现放射性肺炎),有5例患者仅完成1周期化疗(其中4例因血液学毒性、1例因肺炎而终止)。实验组随访率100%,对照组失访1例,随访率96.4%。2、近期疗效:54例患者全部可评价疗效。实验组和对照组中出现CR、PR、SD的例数分别为17、5、3和14、10、2,总有效率(CR+PR)分别为84.6%和85.7%,无统计学差异(P=0.483)。3、复发与生存情况:实验组7例(26. 9%)复发,5例(19. 2%)出现远处转移,1例(3. 8%)出现第二原发肿瘤;对照组9例(32. 1%)复发,8例(28. 6%)出现远处转移。实验组的中位生存期为35. 5个月,对照组为25. 8个月。实验组患者的1、2、3年生存率分别为84. 1%、68. 1%、46. 30%,对照组的分别为71. 1%、59. 3%、27. 7%,实验组长期生存率明显高于对照组(P=0.047)。4、基因mRNA表达与生存之间的关系:ERCC1基因表达高低与患者的OS有明确的相关性,ERCC1基因表达高低组之间的OS有显著性差异(χ2=5.048;P=0.024),低表达患者生存期长,高表达患者生存期短。TYMS、RRM1、TUBB3基因mRNA表达高低组之间的OS无显著性差异。5、毒副反应:两组血液学和非血液学毒性反应无明显差异。实验组中放射性食管炎发生率较高,采用GP方案的2例患者均发生3级放射性食管炎,其中1例至随访截止期(已生存35个月)因食管狭窄一直仅能流质饮食。[结论]1、ERCC1、TYMS、RRM1、TUBB3四种基因的mRNA在食管鳞癌中的表达不一,ERCC1低表达多见,约50%;高表达相对较少,约10%患者出现;TYMS、RRM1、TUBB3基因高表达多见,低表达相对较少。ERCC1、TYMS、RRM1、TUBB3四种基因的mRNA表达与临床特征之间无明显相关性。2、ERCC1基因与患者的生存期有明确的相关性,ERCC1基因高低组之间的OS有显著性差异。3、实验组患者的1、2、3年生存率分别为84. 1%、68. 1%、46. 3%,对照组的分别为71. 1%、59. 30%,、27. 7%,实验组长期生存率明显高于对照组(P=0. 047)。本研究初步结果提示根据分子标志物筛选化疗药物与放疗同步治疗不能手术的食管癌长期生存率高,对于局部晚期食管癌的个体化治疗方案的选择提供了新的思路和依据,值得进一步临床研究。[研究意义]不能手术的食管鳞癌标准治疗方案为同期放化疗,但治疗疗效需要提高。目前食管癌分子靶向治疗尚未取得实质性进展。在肿瘤治疗进入精准治疗的今天,如何对不同食管癌患者给予个体化治疗是亟待解决的难题。我们借助ERCC1、TYMS、RRM1、TUBB3四种基因的mRNA表达高低,排除可能对食管癌不敏感的化疗药物,经小样本的前瞻性非随机的研究证实了其可行性,对以后此类患者的治疗有一定的指导意义。我们希望开展前瞻性、多中心、随机、对照研究以进一步验证我们的结果,实现不能手术的食管鳞癌的个体化同期放化疗。
[Abstract]:Part 1: expression of ERCC1, TYMS, RRM1, TUBB3 gene in esophageal squamous cell carcinoma [Objective] the purpose of this study was to examine the expression level of the four genes of ERCC1, TYMS, RRM1 and TUBB3 in the tumor tissues of patients with inoperable esophageal squamous cell carcinoma, and to observe the expression of the four genes in the cancer of the esophagus. Chemotherapy drug selection during concurrent chemoradiotherapy for patients with inoperable esophageal squamous cell carcinoma. [study method]1, specimen acquisition: all 26 patients were selected from December 1, 2010 to November 1, 2013 at the cancer research and treatment center of the affiliated Provincial Hospital of Shandong University. All of the primary patients with pathological confirmed squamous cell carcinoma of the esophagus were excluded from the past. Patients with other malignant tumors were excluded from pregnant or lactating women. The tumor tissue was obtained by 10% Faure Marin fixation, paraffin embedded and 8 pieces of embedded tumor tissue sections, each slice thickness was 8-10 m.2. The experimental procedure: the expression of mRNA, TYMS, RRM1, TUBB3 gene mRNA expression level by the fluorescent quantitative RT-PCR technique. (1) the total RNA. (2) in the tumor tissue was extracted with ultra micro ultraviolet spectrophotometer to measure the concentration of RNA. (3) reverse transcriptase cDNA. (4) relative quantitative RT-PCR reaction. (5) the result was.3, data processing: the values of RT-PCR detection were treated with 2 - Delta Delta Ct method. The difference of the expression of gene was calculated by Ct comparison method. Quasi specimen) 1= value 1 - Ct value 1 - internal reference gene Ct value 1 delta Ct (test specimen) 2= pending gene Ct value 2 - Ct value of internal reference gene Ct value 2 Delta Delta Ct= Delta Ct (the specimen) 2 Delta Ct (calibrated specimen) 1 use 2- Delta Delta Ct to express difference, statistical method: using 15 statistical software to carry out data processing, the difference between groups using chi 2 test, multiple causes The Cox regression model and P0. 05 showed that the difference was statistically significant. [results the mRNA expression levels of the four genes of ERCC1, TYMS, RRM1, and TUBB3 in]1,26 patients were as follows: the ERCC1 gene was high, middle, and low expression was 3 (11.5%), 12 (46.2%), 11 (42.3%), and the TYMS gene was high, and the low number was 9 (34.6%), 15 (51.7%), 2 (7.7%)); RRM1. The number of high, middle, and low expression cases was 9 (34.60%), 15 (51.7%), 2 (7.7%); TUBB3 high, middle, low expression number was 12 (46.2%), 10 (38.5%), 4 (15.4%). At the same time, 5 cases of low expression and 2, the relationship between gene expression and clinical characteristics: the infiltration depth, tumor length, clinical stage and lymph node metastasis state of esophageal squamous cell carcinoma have no significant correlation with the level of mRNA expression of ERCC1, TYMS, RRM1, TUBB3, and the clinical effect of radiotherapy and chemotherapy on esophageal squamous cell carcinoma under the guidance of the second part markers The standard treatment mode of esophageal squamous cell carcinoma that can not be operated on is a concurrent chemoradiotherapy. The common failure mode after concurrent chemoradiotherapy of esophageal cancer has both local recurrence and distant metastasis. It is an urgent problem to improve the clinical efficacy. The tolerance of the esophagus limits the dose of radiotherapy. The Taxus regimen was better than the PF regimen in the near future, but there was no obvious advantage in the long term survival period. Therefore, the purpose of this part is to explore the use of molecular markers to guide and screen the drugs in the concurrent chemoradiotherapy. Compared with the concurrent chemoradiotherapy of the standard regimen, the difference of therapeutic effect was observed. [study method]1, group and radiotherapy and chemotherapy scheme: 54 cases were not. Patients with surgical esophageal squamous cell carcinoma entered this prospective non randomized controlled clinical study, divided into experimental and control groups. All patients were given concurrent chemoradiotherapy: radiotherapy using intensity modulated radiation therapy, routine segmentation, and prescribed dose of 95%PTV 60 to 66Gy, 2.0Gy/ times, 30~33 times. The plan required 95% equal dose curves to cover more than 90% PTV. chemotherapy The experimental group was given at week 1,5 of the radiotherapy. The experimental group was selected according to the mRNA of four genes of ERCC1, TYMS, RRM1 and TUBB3. First, eliminate the high expression of mRNA, such as the high expression of ERCC1, and the high expression of the 3 genes, the low expression of 1 genes or the middle surface, then only one drug was chosen to synchronize with the radiotherapy, such as 4 genes. High expression of the high expression of cetuximab. Resistance; high expression of 2 genes in 2 kinds of expression, then the choice of the corresponding drug. Secondly, according to the low expression of gene selection of the corresponding drug: such as 3 or 4 genes low expression, then the combination of two drugs, according to age, physical condition, combined diabetes and other basic diseases, in turn choice Cisplatin, fluorouracil, docetaxel, gemcitabine; 2 genes at the same time low expression of the corresponding two drug combinations; 1 low expressions to ensure their corresponding drug selection; third, 4 genes are the first choice for the first choice of PF scheme; the 2 or 3 genes are expressed in cisplatin, fluorouracil, docetaxel, gemcitabine; fourth, Drug side effects such as gemcitabine, such as gemcitabine aggravating lung toxicity, are not needed for thoracic esophageal cancer, and docetaxel is used carefully in patients with diabetes. Drug usage: Cisplatin 15mg/m2 1-5 days, fluorouracil 500mg/m2 day, 1000mg/m2 1,8 days of gemcitabine, first days of docetaxel 60mg/m2, repeated every 28 days; the first agent of cetuximab. 400mg/m2 (first days), followed by a weekly dose of 250mg/m2, for a total of 7 weeks. The objective efficiency and survival of the two groups were observed, and the short-term efficacy was evaluated for 1-2 months after the end of radiotherapy and chemotherapy. According to the chest CT, esophagus endoscopy combined with the esophagus barium meal, the criteria for evaluating the curative effect of solid tumor (RECIST v1.1) were evaluated according to the criteria (RECIST v1.1). Total remission (CR), partial remission (PR), no change (SD) and disease progression (PD). Total survival time (OS): refers to the beginning of the treatment to the patient died of any disease, the cut-off time and the final follow-up time.3, statistical methods: using SPSS 15 Statistical Software, two groups of clinical data comparison and adverse reactions compared to the use of x 2 test, use Kaplan-mmeier Methods to calculate the survival rate, Log-rank test was used to compare the difference of.P0. 05 between group survival rates. [results]1, treatment completion: the distribution of specific drugs in the experimental group was as follows: cisplatin / fluorouracil (PF), 7 cases of single drug cisplatin (P), 4 cases of dcisround / / cisplatin (DP), 2 cases of gemcitabine / cisplatin (GP), and single drug DV. 2 cases (D) and 1 cases of single drug cetuximab (C225). Treatment completion: 2 patients in the experimental group did not complete radiotherapy, 2 cases of actual radiation dose 48Gy (1 cases of radioactive pneumonia, 1 cases of mediastinal fistula); 4 patients completed 1 cycles of chemotherapy (1 cases were terminated by chemotherapy for 3 level radioactive esophagitis; 3 due to 4 blood. " There were 4 cases in the DP regimen 2 cases, the PF scheme 1 cases, the GP scheme 1 cases respectively. 3 patients in the control group had not completed the whole course radiotherapy, the actual radiation dose was 40Gy, 50Gy, 56Gy (not completed the radiotherapy cause: 2 cases of esophagotracheal fistula, 1 cases of radionuclide pneumonia), and 5 patients completed only 1 cycles chemotherapy only ( Among them, 4 cases were caused by hematological toxicity and 1 cases were terminated because of pneumonia. The follow-up rate of the experimental group was 100%, the control group lost 1 cases, the follow-up rate was 96.4%.2, the curative effect of 54 patients was all evaluated. The number of cases of CR, PR, SD in the experimental group and the control group were 17,5,3 and 14,10,2 respectively, the total effective rate (CR+PR) was 84.6% and 85.7%, respectively (P=0.48). 3).3, recurrence and survival: in the experimental group, 7 cases (26.9%) had recurrence, 5 cases (19.2%) had distant metastasis, 1 cases (3.8%) had second primary tumors, 9 cases (32.1%) recurred and 8 (28.6%) appeared distant metastasis in the control group. The median survival time of the experimental group was 35.5 months and the control group was for 26.9% months. The 1,2,3 year survival rate of the experimental group was respectively 8.1%, 46.30%, and the control group were 71.1%, 59.3%, 27.7%. The long-term survival rate of the experimental group was significantly higher than that of the control group (P=0.047).4, the relationship between gene mRNA expression and survival: the ERCC1 gene expression was clearly correlated with the patient's OS, and the OS between the high groups of ERCC1 gene expression was significantly different (x 2=5.048; P=0.024) and low expression. Patients with long survival time, high expression of patients with short survival time.TYMS, RRM1, TUBB3 gene mRNA expression between high and low OS no significant difference between.5, toxic and side effects: two groups of Hematology and non hematological toxicity of no significant difference. The incidence of radioactive esophagitis in the experimental group was higher, and 2 patients with GP program all had 3 levels of radioactive esophagitis, In 1 cases, 1 cases were followed up for 35 months (35 months of survival). [conclusion]1, ERCC1, TYMS, RRM1, TUBB3, the expression of mRNA in the esophageal squamous cell carcinoma is different, the low expression of ERCC1 is more common, about 50%; the high expression is relatively low, about 10% patients appear; TYMS, RRM1, TUBB3 genes are highly expressed, low expression is relatively less.ERC. There was no significant correlation between the mRNA expression of the four genes of C1, TYMS, RRM1, TUBB3 and the clinical characteristics of.2. The ERCC1 gene had a definite correlation with the survival period of the patients. The OS had a significant difference between the high and low groups of ERCC1.3, and the 1,2,3 year survival rate of the experimental group was 84.1%, 68.1%, 46.3% respectively, and the control group was 71.1%, 59.30%, 27.7% respectively. The long-term survival rate of the experimental group was significantly higher than that of the control group (P=0. 047). The preliminary results of this study suggest that the long-term survival rate of esophageal cancer which can not be operated on according to the molecular markers is high. It provides a new idea and basis for the selection of individualized treatment schemes for locally advanced esophageal cancer. It is worth further clinical study. Study. [research significance] the standard treatment of esophageal squamous cell carcinoma can not be operated for the same period of radiotherapy and chemotherapy, but the therapeutic effect needs to be improved. At present, the molecular targeting therapy of esophageal cancer has not made substantial progress. Today, how to treat different esophageal cancer patients with individualized treatment is an urgent problem. The mRNA expression of four genes of ERCC1, TYMS, RRM1, and TUBB3 is high and low, which excludes the chemotherapeutic drugs that may not be insensitive to esophageal cancer. The feasibility is confirmed by a small sample prospective and non random study. It is of certain guiding significance for the treatment of such patients. We hope to open a prospective, multicenter, randomized, controlled study to further test Our findings are individualized concurrent chemoradiotherapy for inoperable squamous cell carcinoma of the esophagus.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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