本文選題：食管腫瘤 + 放化療法��； 參考：《山東大學》2017年博士論文

【摘要】：第一部分ERCC1、TYMS、RRM1、TUBB3基因在食管鱗癌中的表達[研究目的]本研究的目的是利用實時熒光定量RT-PCR技術,檢驗不能手術食管鱗癌患者腫瘤組織中的ERCC1、TYMS、RRM1、TUBB3四種基因mRNA表達水平,觀察四種基因在食管癌中的表達情況,以指導不能手術食管鱗癌患者同期放化療時化療藥物選擇。[研究方法]1、標本獲取:26例患者全部選自2010年12月1日至2013年11月1日在山東大學附屬省立醫(yī)院腫瘤研究治療中心住院患者,全部為病理證實的食管鱗狀細胞癌初診患者。排除既往患其他惡性腫瘤,排除處于懷孕期或哺乳期女性患者。獲取患者腫瘤組織,經(jīng)10%福爾馬林固定,石蠟包埋,取包埋腫瘤組織切片8張,每張切片厚度8-10μm。2、實驗步驟:利用熒光定量RT-PCR技術對組織標本行ERCC1、TYMS、RRM1、TUBB3基因mRNA表達水平定量檢測。(1)提取腫瘤組織中總RNA。(2)用超微量紫外線分光光度計測量RNA濃度。(3)逆轉(zhuǎn)錄合成cDNA。(4)相對定量RT-PCR反應。(5)結(jié)果判定。3、數(shù)據(jù)處理:采用2 —△△Ct方法處理RT-PCR檢測所得的數(shù)值,待測基因的表達差異用Ct比較法來計算�！鰿t (校準標本)1=待測基因Ct值1—內(nèi)參基因Ct值1△Ct (待測標本)2=待測基因Ct值2—內(nèi)參基因Ct值2△△Ct= △Ct (待測標本)2—△Ct (校準標本)1用2-△△Ct表示表達差異。4、統(tǒng)計方法:采用SPSS 15. 0統(tǒng)計學軟件進行數(shù)據(jù)處理,組間差異采用χ2檢驗,多因素分析用Cox回歸模型,P0. 05表示差異有統(tǒng)計學意義。[結(jié)果]1、26例患者ERCC1、TYMS、RRM1、TUBB3四種基因的mRNA表達水平如下:ERCC1基因高、中、低表達例數(shù)分別為3(11. 5%)、12(46. 2%)、11例(42. 3%);TYMS基因表達高、中、低例數(shù)分別為9(34. 6%)、15(51.7%)、2(7. 7%); RRM1高、中、低表達例數(shù)分別為9 (34. 60%)、15(51.7%)、2(7. 7%); TUBB3高、中、低表達例數(shù)分別為 12(46.2%)、10(38. 5%)、4(15. 4%)。患者中四種基因共表達情況:四種基因同時高表達1例,四種基因同時中表達3例,同時低表達0例:三種基因同時中表達5例;兩種基因同時低表達5例;2、基因表達與臨床特征之間的關系:食管鱗癌腫瘤組織浸潤深度、腫瘤長度、臨床分期及淋巴結(jié)轉(zhuǎn)移狀態(tài)與ERCC1、TYMS、RRM1、TUBB3四種基因mRNA表達水平無明顯相關性。第二部分分子標志物指導下同期放化療對食管鱗癌的臨床療效[研究目的]不能手術的食管鱗癌目前標準治療模式是同期放化療。食管癌同期放化療后的常見失敗模式既有局部復發(fā)也有遠處轉(zhuǎn)移,提高臨床療效是迫切需要解決的問題。食管的耐受性限制了放療劑量提高。而化療藥物方面盡管紫杉類方案較PF方案近期療效好,但長期生存期未見明顯優(yōu)勢。因此這部分的研究目的是探討利用分子標志物指導和篩選同期放化療中的藥物,與標準方案的同期放化療比較,觀察治療療效的差別。[研究方法]1、入組及放化療方案:54例不能手術的食管鱗癌患者進入本前瞻性非隨機對照臨床研究,分實驗組和對照組。所有患者均給予同期放化療:放療采用調(diào)強放療技術,常規(guī)分割,處方劑量設定為95%PTV為60～66Gy,2.0Gy/次,30～33次。計劃要求95%等劑量曲線覆蓋90%以上PTV�；熢诜暖煹牡�1、5周給予。實驗組化療藥物根據(jù)ERCC1、TYMS、RRM1、TUBB3四種基因的mRNA表達水、選擇。首先,排除mRNA高表達相對應的藥物:如ERCC1高表達排除順鉑;如3種基因高表達,1個基因低表達或中表達,則只選擇一種藥物'與放療同步,如4種基因均高表達則選西妥昔單.抗;2種基因高表達2種中表達,則選擇中表達對應的藥物。其次,根據(jù)基因低表達選擇相對應的藥物:如3或4個基因低表達,則化療藥物采用兩藥聯(lián)合,同時據(jù)年齡、身體狀況、合并糖尿病等基礎疾病綜合考慮,依次選擇順鉑、氟尿嘧啶、多西紫杉醇、吉西他濱;2種基因同時低表達選擇相對應的兩藥聯(lián)合;1種低表達保證其對應藥物入選;第三,4種基因同時中表達首選PF方案;2種或3種基因中表達時選擇藥物順序是順鉑、氟尿嘧啶、多西紫杉醇、吉西他濱;第四,兼顧藥物毒副反應,如吉西他濱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。藥物用法用量:順鉑15mg/m2第1-5天,氟尿嘧啶500mg/m2第1-5天,吉西他濱1000mg/m2第1、8天,多西紫杉醇60mg/m2第1天,每28天重復;西妥昔單抗首次劑量400mg/m2(第1天),隨后每周劑量250mg/m2,共7周。主要觀察兩組的客觀有效率和生存情況。2、放化療結(jié)束后1-2個月行近期療效評價。根據(jù)治療前后胸部CT、食管內(nèi)鏡結(jié)合食管鋇餐造影,評價標準參照實體瘤療效評價標準(RECIST v1.1)進行,分為完全緩解(CR)、部分緩解(PR)、無變化(SD)及病變進展(PD)。總生存時間(OS):指治療開始時間至患者死于任何疾病、截止觀察時間和最后隨診時間。3、統(tǒng)計方法:采用SPSS 15.0統(tǒng)計軟件,兩組臨床資料可比性及不良反應比較采用χ2檢驗,采用Kaplan-mmeier方法計算生存率,Log-rank檢驗用來比較組間生存率的差異性。P0. 05為差異有統(tǒng)計學意義。[結(jié)果]1、治療完成情況:實驗組具體藥物分布如下:順鉑/氟尿嘧啶(PF) 10例,單藥順鉑(P) 7例,多西他賽//順鉑(DP) 4例,吉西他濱/順鉑(GP) 2例,單藥多西他賽(D) 2例,單藥西妥昔單抗(C225) 1例。治療完成情況:實驗組2例患者未完成放療,2例實際放療劑量48Gy (未完成原因:1例放射性肺炎,1例出現(xiàn)縱隔瘺);有4例患者僅完成1周期化療(其中1例因出現(xiàn)3級放射性食管炎而終止化療;3例因發(fā)生4級血液學毒性未完成化療;這4例病例分別發(fā)生在DP方案組2例、PF方案1例、GP方案1例)。對照組中3例患者未完成全程放療,實際照射劑量分別為40Gy、50Gy、56Gy (未完成放療原因:2例出現(xiàn)食管氣管瘺、1例出現(xiàn)放射性肺炎),有5例患者僅完成1周期化療(其中4例因血液學毒性、1例因肺炎而終止)。實驗組隨訪率100%,對照組失訪1例,隨訪率96.4%。2、近期療效:54例患者全部可評價療效。實驗組和對照組中出現(xiàn)CR、PR、SD的例數(shù)分別為17、5、3和14、10、2,總有效率(CR+PR)分別為84.6%和85.7%,無統(tǒng)計學差異(P=0.483)。3、復發(fā)與生存情況:實驗組7例(26. 9%)復發(fā),5例(19. 2%)出現(xiàn)遠處轉(zhuǎn)移,1例(3. 8%)出現(xiàn)第二原發(fā)腫瘤;對照組9例(32. 1%)復發(fā),8例(28. 6%)出現(xiàn)遠處轉(zhuǎn)移。實驗組的中位生存期為35. 5個月,對照組為25. 8個月。實驗組患者的1、2、3年生存率分別為84. 1%、68. 1%、46. 30%,對照組的分別為71. 1%、59. 3%、27. 7%,實驗組長期生存率明顯高于對照組(P=0.047)。4、基因mRNA表達與生存之間的關系:ERCC1基因表達高低與患者的OS有明確的相關性,ERCC1基因表達高低組之間的OS有顯著性差異(χ2=5.048;P=0.024),低表達患者生存期長,高表達患者生存期短。TYMS、RRM1、TUBB3基因mRNA表達高低組之間的OS無顯著性差異。5、毒副反應:兩組血液學和非血液學毒性反應無明顯差異。實驗組中放射性食管炎發(fā)生率較高,采用GP方案的2例患者均發(fā)生3級放射性食管炎,其中1例至隨訪截止期(已生存35個月)因食管狹窄一直僅能流質(zhì)飲食。[結(jié)論]1、ERCC1、TYMS、RRM1、TUBB3四種基因的mRNA在食管鱗癌中的表達不一,ERCC1低表達多見,約50%;高表達相對較少,約10%患者出現(xiàn);TYMS、RRM1、TUBB3基因高表達多見,低表達相對較少。ERCC1、TYMS、RRM1、TUBB3四種基因的mRNA表達與臨床特征之間無明顯相關性。2、ERCC1基因與患者的生存期有明確的相關性,ERCC1基因高低組之間的OS有顯著性差異。3、實驗組患者的1、2、3年生存率分別為84. 1%、68. 1%、46. 3%,對照組的分別為71. 1%、59. 30%,、27. 7%,實驗組長期生存率明顯高于對照組(P=0. 047)。本研究初步結(jié)果提示根據(jù)分子標志物篩選化療藥物與放療同步治療不能手術的食管癌長期生存率高,對于局部晚期食管癌的個體化治療方案的選擇提供了新的思路和依據(jù),值得進一步臨床研究。[研究意義]不能手術的食管鱗癌標準治療方案為同期放化療,但治療療效需要提高。目前食管癌分子靶向治療尚未取得實質(zhì)性進展。在腫瘤治療進入精準治療的今天,如何對不同食管癌患者給予個體化治療是亟待解決的難題。我們借助ERCC1、TYMS、RRM1、TUBB3四種基因的mRNA表達高低,排除可能對食管癌不敏感的化療藥物,經(jīng)小樣本的前瞻性非隨機的研究證實了其可行性,對以后此類患者的治療有一定的指導意義。我們希望開展前瞻性、多中心、隨機、對照研究以進一步驗證我們的結(jié)果,實現(xiàn)不能手術的食管鱗癌的個體化同期放化療。
[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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.1

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