Relationship Between EGFR And Radiosensitivity And Combination Effect Of Cetuximab With5-fu To Radiosensitivity Of Colorectal Cancer

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.