【作者】 左志贵；

【导师】 傅传刚；

【作者基本信息】 第二军医大学 ， 普通外科学（专业学位）， 2013， 博士

【摘要】 一、研究背景结直肠癌是常见的消化道恶性肿瘤之一，目前在全世界恶性肿瘤的发病率中占第3位，随着经济发展导致饮食结构的改变，我国结直肠癌发病率呈逐年升高的趋势。根据结直肠肿瘤分期的不同，结直肠癌5年生存率介于10-90%之间。直肠是结直肠肿瘤最易发部位，在西方国家中直肠癌约占结直肠癌总发病率40%，而我国却占总发病率的60%左右，并以腹膜返折平面以下的中低位直肠癌占大多数。由于直肠癌深处盆腔，与结肠癌相比，直肠癌术后局部复发率高、远期生存率低，而局部进展期直肠癌则预后更差，其5年生存率位于20-40%之间。因此，如何有效预防直肠癌术后局部复发和提高直肠癌患者总生存率和无病生存率一直是直肠癌研究的重点。自20世纪80至90年代美国进行的多个肿瘤研究项目显示直肠癌术后放化疗可以减少局部复发率和提高总体生存率以后，手术、放疗和化疗组成了直肠癌综合治疗的三大支柱，20世纪后期手术联合术后放化疗是局部晚期直肠癌患者的经典治疗方案。而近10年来多个大样本多中心临床研究表明术前放化疗与术后相比可以达到提高保肛率、R0切除率及进一步减少局部复发率的作用，因此术前放化疗作为局部进展期直肠癌辅助治疗的重要手段目前建议在术前进行，术前放化疗联合手术已经成为局部进展期直肠癌治疗的标准方案，目前多学科综合治疗在提高局部进展期直肠癌总体治疗效果中的作用已经得到公认。根据局部进展期直肠癌患者对术前放化疗的敏感程度制定个性化治疗方案及在放疗过程中联合使用化疗及分子靶向治疗以进一步提高局部进展期中低位直肠癌术前放化疗效果则是目前直肠癌临床研究的热点。EGFR通路在肿瘤放疗抵抗中的作用既往已有相关研究报道，但EGFR信号向下转导有两条途径，通路具有多节点性，目前尚无相关研究同时探讨K-ras/B-raf/PIK3CA突变与结直肠癌放疗敏感性的关系，我们从细胞学水平探讨EGFR通路多节点与放疗敏感性的关系。由于表皮生长因子与放疗抵抗相关，而氟尿嘧啶目前广泛应用于术前放疗增敏，目前尚无相关研究西妥昔单抗与氟尿嘧啶联合对放疗效果的影响，因此我们分别从细胞水平、结直肠癌肿瘤细胞裸鼠模型并对临床研究进行系统回顾探讨了西妥昔单抗联合氟尿嘧啶对结直肠癌细胞放疗敏感性的影响。二、研究内容第一部分表皮生长因子通路与结直肠癌细胞放射敏感性的关系目的探讨表皮生长因子受体(EGFR)表达及其下游通路的关键基因K-ras/B-raf/PIK3CA突变状态对结直肠癌细胞放射敏感性的影响。方法对9个人结直肠癌细胞系应用RealTime RT-PCR技术及Western blotting检测EGFR在mRNA水平及蛋白水平的表达高低，同时检测这9个人结直肠癌细胞系K-ras、B-raf、PIK3CA的突变状态，对这9个结直肠癌细胞系行2Gy剂量射线照射，对照射后的细胞行克隆形成实验明确各细胞株放射敏感特性，照射48小时后行Hoechst33258染色凋亡形态学观察，采用流式细胞术检测细胞凋亡率、细胞周期比例，统计分析结直肠癌细胞EGFR表达及K-ras、B-raf、PIK3CA突变状态与放疗敏感性的关系并分析放疗抵抗及敏感的可能机制。结果相关性分析显示结直肠癌细胞系EGFR表达与放疗抵抗成正相关（r=0.717, P=0．030），EGFR高表达与放疗抵抗相关，而PIK3CA突变与放疗敏感相关(t=2.401，P=0．047)，K-ras、B-raf突变与放疗抵抗及敏感无关。照射后48h凋亡检测显示放疗敏感细胞系(HCT116)总凋亡率随放射剂量增加明显增加(P<0．05)，而放疗抵抗细胞系(HT29)总凋亡率只有放射剂量明显较大才增加，细胞周期检测显示放疗敏感系(HCT116)的G1/G0期比例随放疗剂量增加显著降低(P<0．05)，而放疗抵抗细胞株细胞系(HT29)G1/G0期比例在放疗剂量增加时无明显变化（P＞0.05）。结论人结直肠癌细胞系EGFR高表达与放疗抵抗显著相关，PIK3CA突变与放疗敏感显著相关，EGFR通路多位点同时参与了结直肠癌细胞的放疗抵抗和敏感作用，EGFR通路参与结直肠癌细胞放疗抵抗机制可能与抑制肿瘤细胞凋亡、增加肿瘤细胞在G1/G0期停顿相关。第二部`分放疗对直肠癌EGFR表达影响与放疗敏感性和预后关系目的分析放疗前后局部进展期中低位直肠癌患者癌组织EGFR蛋白水平表达的变化，探讨放疗前后EGFR蛋白表达变化与病理消退及预后的关系，进一步全面研究局部进展期中低位直肠癌患者新辅助治疗后病理消退的临床及病理影响因素。方法对2002年1月至2009年12月期间122例局部进展期中低位直肠癌患者接受新辅助治疗后行手术的临床资料及术后病理消退分级进行回顾性研究，对其中46例患者放疗前后直肠癌组织病理标本进行EGFR免疫组织化学检测蛋白表达高低，Logistic回归分析与直肠癌新辅助治疗后病理消退相关的临床及病理影响因素，生存分析及Log-rank检验探讨EGFR表达变化及其他临床病理因素与预后的关系，COX多因素回归分析与新辅助治疗后直肠癌患者长期生存相关的独立影响因素。结果122例患者中明显病理消退52例（42.62%），其中完全病理消退11例（9.02%），病理消退不明显70例（57.38%）。同步放化疗患者病理明显消退率为54.05%（40/74），高于单纯放疗患者病理明显消退率25%（12/48）（X2=10.5，P=0.002），长程放疗方案患者病理明显消退率为60%（30/50），高于短中程放疗患者病理明显消退率30.56%（22/72）（X2=10.465，P=0.005），放疗总量大于4000cGy患者病理明显消退率为60.42%（29/48），高于放疗总量小于等于4000cGy患者病理明显消退率31.08%（23/74）（X2=10.889，P=0.002），放疗后EGFR表达增加病理明显消退率为23.03%（3/13），低于放疗后EGFR表达不变或减少患者病理明显消退率63.64%（21/33）（X2=7.769，P=0.005），多因素Logistic回归分析显示同步放化疗、长程放疗及放疗后EGFR蛋白表达不变或减少是局部进展期直肠癌患者术前新辅助治疗后病理消退分级的三个独立影响因素(P＜0.05)。Kaplan-Meier生存分析显示本组患者总体生存率65.8%，生存分析及Log-rank检验结果显示组织学类型（P=0.025）、CEA水平（P=0.032）、肿瘤浸润深度（P=0.022）、淋巴结转移（P＜0.001）、随访过程中远处转移（P=0.002）、放疗前EGFR蛋白高表达（P=0.024）及放疗后EGFR表达增加（P=0.007）对患者长期生存有显著影响，总体生存率差异有统计学意义（P＜0.05），COX比例风险回归模型对单因素分析有意义的各因素行多因素回归分析发现淋巴结转移、随访过程中远处脏器转移以及放疗前后EGFR蛋白表达变化均是影响患者生存的独立因素（P=0.024、P=0.042、P=0.023），因此放疗后EGFR蛋白表达增加、术后标本淋巴结转移及随访过程中远处脏器转移是导致新辅助治疗后直肠癌患者长期生存减少的三个独立影响因素，其中随访过程中远处脏器转移及淋巴结转移比放疗前后EGFR蛋白表达变化对生存影响更为密切。结论局部进展期中低位直肠癌患者行术前放化疗可以引起EGFR蛋白表达的变化，局部进展期中低位直肠癌患者中行同步放化疗、长程放疗方案及放疗后EGFR蛋白表达未增加者临床疗效优于单纯放疗、短中程放疗及放疗后EGFR蛋白表达增加者，放疗后EGFR蛋白表达增加不仅与局部进展期直肠癌患者放疗抵抗相关，而且预示接受术前放疗中低位直肠癌患者的预后不良。第三部分西妥昔单抗联合氟尿嘧啶对结直肠癌细胞放疗敏感性影响的实验研究及系统回顾目的分析评估表皮生长因子拮抗剂西妥昔单抗联合氟尿嘧啶对结直肠癌细胞及中低位直肠癌患者术前放疗效果的影响。方法根据人结直肠癌细胞系RKO在体外培养过程中给予不同干预措施分为2Gy射线照射组、氟尿嘧啶联合2Gy射线照射组、西妥昔单抗联合2Gy射线照射组、氟尿嘧啶及西妥昔单抗联合2Gy射线照射组，对各组细胞行CCK8实验检测不同干预后的细胞增殖情况，干预48小时后各组细胞行Hoechst33258染色凋亡形态学观察，采用流式细胞术检测干预48小时后各组细胞凋亡率、细胞周期比例，统计分析不同干预方式对RKO结直肠癌细胞系增殖的影响，利用RKO行裸鼠成瘤，对成瘤裸鼠同样分为四组分别给予2Gy射线照射、氟尿嘧啶联合2Gy射线照射、西妥昔单抗联合2Gy射线照射、氟尿嘧啶及西妥昔单抗联合2Gy射线照射。检索PubMed，EMBASE，ISI数据库及Cochrane图书馆，对2012年12月前所有将西妥昔单抗用于术前放疗增敏的临床研究进行系统回顾。结果西妥昔单抗组及氟尿嘧啶组各自单独联合放疗均明显提高了放疗对肿瘤细胞的生长抑制作用，差异有统计学意义（P＜0.05），但是氟尿嘧啶联合西妥昔单抗与各自单用相比对提高放疗的效果不明显，抑制率比较差异无统计学意义（P＞0.05）。西妥昔单抗及氟尿嘧啶各自与2Gy放射线联合均明显提高了2Gy放射线单独照射的凋亡率，而氟尿嘧啶与2Gy放射线联合提高2Gy放射线单独照射的凋亡率更加明显，使用西妥昔单抗与氟尿嘧啶联合对提高2Gy放射线单独照射的凋亡率并不比氟尿嘧啶联合对提高2Gy放射线单独照射的凋亡率更明显，西妥昔单抗与2Gy放射线联合明显降低了G1/G0期肿瘤细胞的比例（P＜0.05），而氟尿嘧啶与2Gy放射线联合则导致细胞周期检测出现一个明显的凋亡峰，Hoechst33258检测显示研究组（2Gy照射+氟尿嘧啶组及2Gy照射+氟尿嘧啶组+西妥昔单抗组）均出现典型的细胞凋亡形态学改变。裸鼠成瘤实验显示2Gy照射+氟尿嘧啶组，2Gy照射+西妥昔单抗组，2Gy照射+氟尿嘧啶组+西妥昔单抗组各组瘤体重量均明显低于对照组，差异有统计学意义（P＜0.05），而2Gy照射组虽然瘤体重量低于对照组，但差异无统计学意义（P＞0.05）。结果还显示2Gy照射+氟尿嘧啶组瘤体重量明显低于2Gy照射+西妥昔单抗组，差异有统计学意义（P＜0.05），虽然2Gy照射组瘤体重量与对照组相比及2Gy照射+西妥昔单抗组的瘤体重量与2Gy照射组相比均无统计学差异（P＞0.05），但是2Gy照射+西妥昔单抗组的瘤体重量与对照组相比有统计学差异（P＜0.05）。共14项I/II期临床研究纳入522例患者探讨了西妥昔单抗对局部进展期中低位直肠癌患者术前放疗增敏，pCR率0-20%，西妥昔单抗联合放疗总pCR为10.73%，低于氟尿嘧啶联合放疗总13.5%。结论西妥昔单抗提高放疗敏感性主要通过改变细胞周期，减少引起对放疗损伤逃逸的G1/G0期细胞的比例，而氟尿嘧啶提高放疗敏感性主要通过增加细胞凋亡，氟尿嘧啶与西妥昔单抗相比增加放疗敏感性更加显著。氟尿嘧啶、西妥昔单抗各自单药均明显增加了2Gy照射对裸鼠移植瘤的生长抑制作用，但氟尿嘧啶对放疗的增敏作用强于西妥昔单抗，动物实验及临床研究的系统回顾均显示西妥昔单抗联合氟尿嘧啶并没有提高氟尿嘧啶单独对放疗敏感性的增加。更多还原

【Abstract】 As one of the most common malignant cancer，incidence of the colorectal carcinoma iscurrently accounting for No.3worldwidely. Just below the peritoneal fold, the major rectalcancer occurred in the middle and lower part of the rectum.Because of the specificanatomy and biology of rectal cancer, patients with rectal cancer has more local recurrencerate,low long-term survival rate and poor prognosis comparing with patients with coloncancer. Historically, the combination of postoperative radiotherapy (RT) and5-Fu-basedchemotherapy has been shown to reduce local recurrences and improve survival for locallyadvanced rectal cancer, moreover,improvements in surgical technique have dramaticallylowered the incidence of locally recurrent disease in the last few decades, intact removal ofthe entire mesorectum (total mesorec-tal excision or TME) in cancers of the mid or lowerthird of the rectum was pioneered by Heald has resulted in local recurrence rates lowerthan5-10%. Although radical resection is the mainstay of treatment in patients with locallymoderate and low rectal cancer,the stategies have been changing in the late decades.The last two decades have witnessed the development of a variety of preoperative RTand chemoradiotherapy (CRT) schedules designed to optimize the sequence of treatmentmodalities and the most appropriate scheduling of RT and5-Fu-based CRT.Severalmulticenter,randomized,phase Ⅲ clinical trials have confirmed that fluoropyrimidine(5-fluorouracil or Capecitabine) concomitant to preoperative radiation improved localcontrol compared with either preoperative radiation alone or postoperativechemoradiotherapy (CRT).The advantages of preoperative over postoperative RT includeenhanced effectiveness in well-oxygenated tissue, downstaging of advanced tumors.Thetheoretical superiority of preoperative versus postoperative combined modality therapy hasbeen confirmed by the German rectal cancer trial,preoperative CRT following by surgery isthe standard regimen for locally advanced rectal cancer today.Overexpression of epidermal growth factor receptor is frequently associated with poortumor response to preoperative chemoradiation and poor prognosis.Cetuxiamb,ananti-EGFR chimeric human-mouse monoclonal antibody, can enhanced the radiosensitivityof colorectal cancer cells in some experimental studies.Nowadays the preoperativeconcurrent chemoradiation with5-fluorouracil and radiation has been extensively applicated in locally advanced rectal cancer.Increasing evidences show that addingcetuximab to the combination effect of5-fluorouracil and radiation has a increasingtherapeutic potential, It has been speculated that combination of cetuximab(monoclonalantibody of EGFR) might improve the effect of5-Fu-based chemoradiotherapy.Part I Relationship between epithermal growth factor receptorexpression and K-RAS/PIK3CA mutation status with radiosensitivity ofcolorectal carcinoma cell lines in vitroObjective To investigate the effect of epithermal growth factor receptor(EGFR)expression and the key gene in the pathway K-ras/B-raf/PIK3CA mutation status on theradiosensitivity of human colorectal carcinoma(CRC) cell lines in vitro. Methods RealtimeRT-PCR and Western blotting was used to measure EGFR expression at mRNA and proteinlevel in nine human CRC cell lines, and K-ras/B-raf/PIK3CA mutation status of each CRCcell line was also identified，respectively.48hours after treated with irradiation at a singledose of2Gy, the cell viability was measured by clonogenic survival assay，the rate of cellapoptosis and cell cycle distribution were tested by flow cytometry, and the cellmorphology was observed with hoechst33258dying to analyze the correlationship betweenEGFR expression and radiosensitivity of CRC cell lines, then further explore the potentmechanism. Results A significant correlationship between EGFR expression at mRNA orprotein level and survival fraction of2Gy（SF2）was observed in correlation tes（tr=0.717,P=0．030）,the correlation was also identified significantly between the mutation status ofPIK3CA and radiosensitivity, while mutation status of K-RAS and B-RAF were notcorrelated with radiosensitivity.48hours after exposing to irradiation，the apoptosis rate ofradiosensitive cell line (HCT116) was significantly increased in a dose dependent manner(P<0．05), while the apoptosis rate of radioresistant cell line (HT29) was significantlyincreased only when radiation dose above6Gy. The ratio of G0/G1phase was reducedsignificantly with the increase of radiation dose in radiosensitive cell line(HCT116,P<0．05),while the trend was not oberserved in radioresistant cell line (HT29,P＞0.05). Conclusions Overexpression of EGFR is closely related to radioresistant ofhuman CRC cell lines, and mutation status of PIK3CA is significantly correlated with theradiosensitivity of CRC cells,but mutation status of K-ras、B-raf is not correlated with theradiosensitivity of CRC cells．The inhibition of apoptosis and G0/G1arrest seemed toinduce the radioresistant of CRC cell lines. Part II Predictive and prognostic value of the changes of epidermalgrowth factor receptor(EGFR) expression in locally advanced rectalcancer after preoperative chemoradiotherapyObjective To observe the changes of EGFR protein expression in locally advancedrectal cancer after preoperative chemoradiotherapy and study the predictive and prognosticvalue of the changes, simultaneous analysize the clinical and pathological influencingfactors of good pathological tumor regression grades after chemoradiotherapy.MethodsThe specimens including clinical and pathological of122patients with mid-low locallyadvanced rectal cancer receiving preoperative chemoradiotherapy between January2002and December2009were analysized retrespectively,only forty-six patients has matchedspecimens including biopsy specimens preoperative radiotherapy and postoperativesurgical specimens.The tumor regression grades (TRG) were detected by hematoxylin andeosin staining in the paraffin-embeded specimen, EGFR protein expression were detectedby immunohistochemistry．Taking TRG as the criteria of radiosensitivity, chi-square testand multivariate logistic regression were used to analyze the relationship between EGFRprotein expression before and after preoperative chemoradiotherapy in rectal cancer andradiosensitivity, logistic regression analysis was used to evaluate the influencing factorsassociated with clinical efficacy after neoadjuvant therapy in locally rectal cancer,while theKaplan-Meier survival analysis and multivariate COX regression were used to analyze therelevance between EGFR protein expression and prognosis.Results Fifty-twopatients(42.62%) reached good TRG,including11patients(9.02%) with pathologicalcomplete response,seventy patients(57.38%) were poorly responsive to radiation.Patients accepting with concurrent preoperative chemoradiotherapy had a good responserate54.05%(40/74）,significantly higher than that of patients with preoperativeradiotherapy alone(25%,12/48）（X2=10.5,P=0.002),the good response rate afterlong-course radiation therapy was60%（30/50）, significantly higher than that of patientswho underwent short or medium course radiation30.56%（22/72）（X2=10.465，P=0.005）,the good response rate after radiotherapy dose＞4000cGy was60.42%（29/48）,significantly higher than that of patients underwent radiotherapy dose≤4000cGy31.08%（23/74）（X2=10.889,P=0.002）, the good response rate in patients who hadincreased EGFR expression during preoperative chemoradiotherapy was23.03%（3/13）,significantly lower than that of patients who had no increased EGFR expression63.64%（21/33）（X2=7.769，P=0.005），multivariate Logistic regression analysis showed concurrent preoperaticve chemoradiation,long-course radiation therapy and no increasedEGFR expression during preoperative chemoradiotherapy were independently associatedwith the clinical efficacy in mid-low locally advanced rectal cancer after neoadjuvanttherapy(P＜0.05). Kaplan-Meier survival curve analysis showed ratio of122patients inthis group of preoperative radiotherapy of locally advanced rectal cancer patients with5-year survival rate was65.8%, Kaplan-Meier survival curves and Log-rank test showedthat histological types(P=0.025）,CEA levels （P=0.032）,depth of tumor invasion（P=0.022）,lymph node metastasis（P＜0.001),distant organ metastasis duringfollow-up(P=0.002),higher EGFR protein expression before radiotherapy（P=0.024）andincreased EGFR expression during preoperative chemoradiotherapy（P=0.007） affectedsurvival of patients, but including patinets’ age, sex, CA199levels, gross types of tumor,tumor differentiation, tumor diameter, tumor invasion of the intestinal wall circumferencewere not associated with prognosis (P>0.05);COX Multivariate regression analysisshowed that lymph node metastasis（P=0.024）,distant organ metastasis（P=0.042）duringfollow-up and the changes of EGFR expression（P=0.023） were independent factorswhich affecting the survival of patients, in which distant organ metastasis during follow-upand lymph node metastasis impact on survival more closely than the changes of EGFRexpression. Conclusions Concurrent preoperative chemoradiotherapy， long-courseradiation therapy and no increase EGFR expression during preoperativechemoradiotherapy provides higher good response rate，preoperative chemoradiotherapyinduced the changes of epidermal growth factor receptor expression，moreover, thechanges of epidermal growth factor receptor expression were associated with theradiosensitivity and prognosis of patients with locally advanced rectal cancer．Part III Combination effect of cetuxiamb with5-fluorouracil to theradiosensitivity of colorectal cancer cells:an in vitro and in vivoexperimental study and systematic review of clinical trialsObjective To assess whether blockade of EGFR with cetuximab can enhance thepreoperative combination effect of5-fluorouracil and radiation in rectal cancer on basis ofan in vitro and in vivo experimental study and systematic review of clinical trials.MethodsHuman CRC cell lines RKO were irradiated with radiation(4Gy) alone, combing with5-fluorouracil, cetuximab or both agents(5-fluorouracil and cetuximab).The cellularproliferation were evaluated by CCK8assay,the cell apoptosis and cell-cycle distribution were investigated using FCM.The inhibitory effect on the growth of RKO xenogrsfts wasassessed in athymic nude mice. Fourteen I/II phase clinical trials which studied thecombination effect of cetuximab and5-fluorouracil on preoperative chemoradiation ofrectal cancer were reviewed after we searched PubMed, EMBASE, ISI databases and theCochrane library before December,2012.Results CRC cell lines RKO treated withcetuximab and irradiated at4Gy predominantly exhibited G0/G1phase arrest in comparisonwith those in the control cells(p=0.006).An evidently higher apoptosis rate on irradiation at4Gy was observed in5-fluorouracil-treated or both agents-treated (5-fluorouracil andcetuximab) cells compared with that in the control and cetuximab-treated cells(P＜0.001).Decreased cell proliferation and increased cell death were further supported byHoechst33258staining,which observed a lot of apoptotic cells on basis of nuclearmorphology changes such as chromatin condensation and nucleus fragmentation.In nudemice bearing RKO xenografts, one agent(5-fluorouracil or cetuximab) plus radiationsignificantly inhibited the tumor growth over radiation alone(P＜0.001), disappointing,theinhibitory rate of both agents(5-fluorouracil and cetuximab) plus radiation was not higherthan that of5-fluorouracil plus radiation(P＞0.05),but the inhibitory rate of5-fluorouracilalone plus radiation was significantly higher than that of cetuximab alone plus radiation(P＜0.001). Fourteen I/II phase trials of preoperative chemoradiation with cetuximab inrectal cancer were identified in systematic review. A total of522patients were identifiedwho received cetuximab in combination with radiotherapy and5-fluorouracil orcapecitabine preoperatively.The pCR rate ranged from0to20%. The overall pooled pCRfor cetuximab-based chemoradiation was10.73%(56/522),which was lower comparedwith an overall pCR rate of13.5%with5-Fu-based chemoradiotherapy.Conclusions5-Fluorouracil or cetuximab alone or both agents can enhance the radiosensitivity of rectalcancer, but5-fluorouracil showed the better effect and cetuximab adding to5-fluorouracilcan not manifest the synergistic effect in experimental study and systematic review ofclinical trials,on the whole,it seems that the EGFR inhibitor cetuximab lacks the value ofapplying to the concurrent preoperative chemoradiation in locally advanced rectal cancer.更多还原