【作者】 张娜；

【导师】 邱雪杉；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的头颈癌被认为是第七大常见肿瘤,其中鳞状细胞癌（Squamous cellcarcinoma of the head and neck,HNSCC）占头颈癌的90%。手术、放疗或手术联合放疗是头颈癌治疗的基础。近年来,随着放化疗联合治疗模式的逐步建立,晚期病人生存率和局部控制率有了一定程度的提高。然而在头颈癌放化疗治疗中仍然存在一些问题,如:放化疗毒副作用大、肿瘤对放化疗不敏感、治疗后病人复发率高、5年生存率低等。迄今为止,头颈癌放化治疗和疗效预测仍是肿瘤治疗的一个难题。肿瘤特异性分子的靶向治疗具有特异性强、在有效杀灭肿瘤细胞的同时不损伤正常组织的优点,它是针对可能导致细胞癌变的环节,从分子水平逆转这种恶性生物学行为,从而抑制肿瘤细胞生长的生物治疗模式。其中表皮生长因子受体（Epidermal growth factor receptor,EGFR）是当前分子靶向治疗研究的热点。EGFR是ErbB酪氨酸激酶受体。该家族共有4个成员即EGFR/ErbB1,HER2/ErbB2,HER3/ErbB3及HER4/ErbB4。ErbB与特异的配体结合形成配体-受体二聚体,随后发生自身磷酸化,激活其下游主要信号通路,调节细胞功能,如细胞增殖、凋亡、血管形成、细胞粘附等,可导致细胞癌变,进而发生浸润及转移等。研究表明,80%-90%头颈鳞癌患者的EGFR为阳性。EGFR的过度表达与预后差、转移快、生存期短和放化疗不敏感等密切相关。EGFR的靶向药物主要有两类:一类为单克隆抗体,如西妥昔单抗（Cetuximab,Erbitux,IMC-C225）,它是特异性阻断EGFR的IgG1单克隆抗体,针对受体胞外配体连接区,阻止配体与受体的结合而抑制受体的激活。另一类为小分子酪氨酸激酶抑制剂,如易瑞沙（Gefitinib,Iressa,ZD1839）,主要抑制受体胞内酪氨酸激酶活性区。EGFR靶向药物只起到抑制肿瘤的作用,不能杀伤肿瘤细胞,其有效率在10%左右。因此,靶向治疗联合放化疗可能有利于提高疗效,减少副作用,是肿瘤治疗的发展趋势。目前,在晚期头颈鳞癌治疗中,有学者报道Cetuximab联合放疗或化疗提高了疗效。但Gefitinib联合放疗是否也有相似的疗效;Cetuximab联合化疗（Cisplatin）及常规放疗是否能提高疗效;以及对它们疗效的预测,国内外未见报道。本实验利用10株头颈鳞癌细胞克隆模型,分别比较单药治疗、联合治疗对细胞生存活性的影响,同时检测了10株HNSCC细胞的ErbB家族成员蛋白表达及信号通路关键因子,预测EGFR抑制剂在头颈鳞癌治疗中的疗效,为进一步提高头颈鳞癌治疗疗效,合理应用靶向治疗提供重要线索。方法一、研究对象新建的10株人头颈鳞癌细胞系,标记为UT-SCC（本文简写为SCC）。SCC-8,SCC-9,SCC-11,SCC-19a,SCC-29,SCC-34和SCC-38来源于喉癌（其中SCC-9源于颈转移,SCC-11源于复发病例）,SCC-24a1,SCC-24a2和SCC-40来源于舌癌。二、研究方法1、细胞培养:细胞系传代于15-30代之间。10%热灭活胎牛血清（fetal bovine serum,FBS）DMEM培养基贴壁生长,孵育箱培养,0.01%胰蛋白酶（trypsin）及0.02%K-EDTA消化传代,每周传代1-2次,细胞维持在对数生长状态。2、克隆形成率分析法:Ham’s F-12（15%FBS）培养液。96孔培养板每孔细胞的接种数根据细胞植盘率（Plating efficiency,PE）调整。加干预因素,孵育箱培养4周,倒置显微镜下观查,每孔活细胞≥32个的孔作为阳性孔而计数。3、Western blot方法:检测人HNSCC细胞ErbB家族成员蛋白表达和活性。细胞裂解液以相同蛋白量上样,SDS-聚丙烯酰胺凝胶电泳,分别进行一抗、二抗杂交,ECL显色。4、RT-qPCR方法:检测人HNSCC细胞ErbB家族成员mRNA表达。（1） Primer Express software设计引物和Taqman探针。（2）利用Trizol试剂提取细胞总RNA。（3）重组质粒pEBS7-EGFR作为标准品,actin为对照。三、实验分组1、96孔培养板克隆形成率分析法检测HNSCC细胞对三种药物（Cetuximab,Gefitinib,Cisplatin）的敏感性（IC70和IC50）。实验设细胞对照组和药物（5种不同浓度）处理组。2、96孔培养板克隆形成率分析法检测HNSCC细胞对Cetuximab或Gefitinib联合放疗疗效。Cetuximab/Gefitinib+Radiation:（1）单纯放射组:分0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy和7.5 Gy单次照射。（2）放射+药物组:放射剂量同上。Cetuximab或Gefitinib IC70。（3）放射+药物组:放射剂量同上。Cetuximab或Gefitinib IC50。3、96孔培养板克隆形成计数法检测HNSCC细胞对Cetuximab联合顺铂（Cisplatin）及放疗疗效。Cetuximab+Cisplatin+Radiation:（1）单纯放射组:分0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy和7.5 Gy单次照射。（2）放射+化疗组:放射剂量同上。Cisplatin IC70。（3）放射+化疗+药物组:化放疗方法同（2）,Cisplatin IC70,CetuximabIC70.（4）放射+化疗+药物组:化放疗方法同（2）,Cisplatin IC70,CetuximabIC50。四、疗效评价与统计分析1、细胞植盘率（PE）:PE=-In（阴性孔数量/全部孔数量）/每孔使用细胞数量。2、药物敏感性:Cetuximab,Gefitinib,Cisplatin的敏感性用IC50,IC70表示,即抑制50%,30%克隆形成所需的浓度。3、联合治疗疗效评价:（1）细胞存活分数（Survival fraction,SF）=（阳性孔数量/每孔使用细胞数量）×控制组每孔使用细胞数量/控制组中阳性孔数量。适用于线性二次（LQ）模型F=exp[-（αD+βD²）],拟合剂量生存曲线,（D表示剂量,α和β为剂量效应系数,α:为单击所产生的细胞死亡,即细胞存活曲线的初斜率;β:由于损伤累积而导致细胞死亡）。（2）通过数值整合获得曲线下面积（Area under the curve,AUC）值等同于平均致死（失活）剂量,AUC=1/α+（1.0）(D_F-1)。联合放疗AUC率=（药物+放疗）AUC/放疗AUC;联合放化疗AUC率=（药物+放疗+化疗）AUC/放疗AUC。（3）联合治疗疗效评价采用细胞存活分数（SF）和AUC率的比较分析,比较分析采用t检验。药物剂量对超相加作用的影响采用单因素方差分析（ANOVA）。Cetuximab或Gefitinib联合放疗疗效和Cetuximab联合化疗（Cisplatin）及放疗的疗效评价采用“相加效应”（P＞0.05）即药物+放疗（或放化疗）的附加效应;“超相加效应”（P＜0.05）即大于药物+放疗（或放化疗）的附加效应亦即协同效应。4、相关性分析:ErbB表达与Cetuximab或Gefitinib药物敏感性（IC50）及放疗敏感性（AUC）的相关性采用Spearman统计分析,P＜0.05为有相关性。结果一、EGFR抑制剂及联合放化疗对头颈鳞癌治疗疗效1、10株HNSCC细胞的药物敏感性（1） Cetuximab,Gefitinib,Cisplatin IC70值变化范围:分别为0.22-4.08nM;0.036-4.8μM;0.038-0.22μg/ml。（2） Cetuximab,Gefitinib,Cisplatin IC50值变化范围:分别为0.58-8.2 nM;0.15-8.4μM;0.054-0.31μg/ml。（3） 10株HNSCC细胞对Cetuximab最敏感性的细胞系为SCC-24a2,SCC-40;10株HNSCC细胞对Gefitinib最敏感性的细胞系为SCC-24a2,SCC-19a;7株HNSCC细胞对Cisplatin最敏感性的细胞系为SCC-8,SCC-34。2、联合治疗疗效评价（1） Cetuximab联合放疗疗效评价:Cetuximab联合放疗的克隆形成率分析法结果显示,Cetuximab IC50剂量组对SCC-24a2疗效呈超相加效应（P＜0.05）,显著优于单纯放疗;对其它细胞系呈相加效应（P＞0.05,其中SCC-40的P=0.054）,优于单纯放疗。Cetuximab IC50剂量组联合放疗治疗中作用最敏感的两株细胞（SCC-24a2,SCC-40）与Cetuximab单药治疗中最敏感的两株细胞相一致。Cetuximab IC70剂量组对7株HNSCC细胞系均呈相加效应（P＞0.05）,优于单纯放疗。（2） Gefitinib联合放疗疗效评价:Gefitinib联合放疗的克隆形成率分析法结果显示,Gefitinib IC70和IC50剂量组对SCC-9,SCC-24a2,SCC-29,SCC-34和Gefitinib IC50剂量组对SCC-40疗效呈超相加效应（P＜0.05）,对其它细胞系呈相加效应（P＞0.05）,优于单纯放疗。（3） Cetuximab联合化疗（Cisplatin）及放疗疗效评价:Cetuximab联合化疗（Cisplatin）及放疗的克隆形成率分析法结果显示,Cetuximab IC70和IC50剂量组对SCC-24a和SCC-40疗效呈超相加效应（P＜0.05）;超相加效应的两株与Cetuximab单药疗效中最敏感的两株细胞系相同;对其它细胞系呈相加效应（P＞0.05）,优于单纯放疗或放化疗的疗效。二、检测EGFR信号通路关键因子分析其与药物疗效相关性1、Westerm blot方法检测检测人HNSCC细胞ErbB家族成员蛋白表达和活性:10株HNSCC细胞有不同程度的EGFR,ErbB2,ErbB3和pEGFR,pErbB2,pErbB3,pAkt,pErk表达。无ErbB4表达。2、RT-qPCR方法检测结果检测人HNSCC细胞ErbB家族成员mRNA表达:9株HNSCC细胞的4种基因有不同程度表达,高低依次为EGFR,ErbB2,ErbB3,ErbB4。3、ErbB表达与Gefitinib敏感性分析:ErbB3表达水平与HNSCC细胞对Gefitinib IC50抵抗性呈正相关性（P=0.02,r=0.71）。ErbB信号通路中pErbB2水平与Gefitinib IC50抵抗性呈正相关性（P=0.02,r=0.71）。4、ErbB表达与Cetuximab敏感性分析:EGFR蛋白表达水平与HNSCC细胞对Cetuximab IC50的抵抗性呈负相关（P=0.048,r=-0.64）。5、ErbB表达与HNSCC细胞放射敏感性分析:pErbB3表达与放疗平均致死剂量（AUC）呈正相关（P=0.0045,r=0.81）。结论1、HNSCC体外研究表明,Cetuximab或Gefitinib联合放疗的疗效优于单独放疗（相加效应）,Gefitinib（5株超相加效应）对HNSCC细胞的疗效优于Cetuximab（1株超相加效应）。在Cetuximab联合放疗中,Cetuximab IC50剂量组作用最敏感的两株细胞与Cetuximab单药治疗中最敏感的两株细胞相一致。在Cetuximab联合Cisplain及放疗中,使用Cetuximab（IC70/IC50）和低剂量Cisplatin（IC70）可以提高疗效（相加效应）,优于单纯放疗或放化疗,并且超相加作用的两株细胞与Cetuximab单药治疗中最敏感的两株细胞相一致。结果提示Cetuximab的敏感性可以预测Cetuximab联合放疗或联合放化疗的疗效。2、pErbB2和ErbB3表达水平与HNSCC细胞对Gefitinib抵抗性呈正相关;EGFR蛋白表达水平与HNSCC细胞对Cetuximab的抵抗性呈负相关;pErbB3表达与HNSCC细胞对放疗抵抗性呈正相关。提示pErbB2和ErbB3可预测Gefitinib抵抗性,EGFR可预测Cetuximab的敏感性,pErbB3可预测放疗抵抗性。更多还原

【Abstract】 IntroductionHead and neck cancer is the seventh most common cancer worldwide with 90% cases being squamous cell carcinomas（HNSCC）.Surgery and radiotherapy are the corner stones in the treatment of HNSCC.In recent years,concurrent chemoradiotherapy has gradually been established,the overall survival and the locoregional control of patients with locoregionally advanced HNSCC have been improved. However,there are still some problems in treatment regimes,such as the increased cytotoxic effects,the treatment resistance,the high recurrence rate and the low survival rate of 5 years.Thus,effectiveness of the treatment regime and predictions of the curative effect remain difficult questions for HNSCC.The cancer target therapy has some advantages with specificities to the tumor cells, strong anticancer effects and minimal toxicities to normal tissues.Targeted anticancer therapy is a biological therapy.It is based on a procedure that may cause the transformation of normal cells into cancer cells.It interferes with this procedure on a molecular level,thereby inhibiting tumor growth.One of the most promising strategies is the targeted anticancer therapy based on the epidermal growth factor receptor （EGFR）.EGFR is ErbB receptor of the tyrosine kinase.The ErbB family contains four members:EGFR/ErbB1,HER2/ErbB2,HER3/ErbB3 and HER4/ ErbB4.Specific ligand binds to separate receptors by formation of active dimmers.This procedure is followed by auto-phosphorylation,activates a series of complex cellular signal pathways,regulates biological function of cells,such as cell proliferation,apoptosis, angiogenesis,cell adhesion and can lead to the development of cancer in abnormal events,further to invasiveness and metastasis.EGFR is overexpression in 80%-90%of SCC.Overexpression of the EGFR is correlated with a poor prognosis,a high recurrence rate,a low survival rate and resistance to chemotherapy and radiotherapy.Two predominant classes of EGFR inhibitors have been developed.On the one hand,monoclonal antibodies（mAbs）,such as Cetuximab（Erbitux,IMC-C225）,which is an IgG1 monoclonal antibody,blocks phosphorylation and activation of EGFR by binding with high affinity to the extracellular domain of EGFR.On the other hand, small molecule tyrosine kinase inhibitors（TKIs）,such as Gefitinib（Iressa,ZD1839）, which inhibits the intracellular kinase domain of EGFR.The function of targeting agent is only put tumor in the control,which can not kill the tumor.The rate of effectiveness of a majority of molecular targeting agents is approximately 10%.Exploring a targeting agent combined with chemo-radiotherapy is a treatment entailing less side effects than a traditional chemo-radiotherapy,making it a worthwhile object of study in the development of a cancer treatment.Some studies using cetuximab have shown enhanced efficacy in treatment of locoregionally advanced HNSCC when combined to radiotherapy or chemotherapy at present.Whether gefitinib has similar efficacy as part of radiotherapy is currently less well characterized.There is only little information on the potential enhancing cytotoxic effect when combining cetuximab with cisplatin-based chemoirradiation and predicting effective treatment in HNSCC.Using 10 established cell lines,we examined the effects of cetuximab and gefitinib as single agent and in a combined treatment with radiotherapy and subsequently compared them with one another.We examined the effects of ombining cetuximab with cisplatin-based chemoirradiation.The present study was performed to test the expression of the ErbB family members,and analyze prediction factors for curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm on HNSCC in vitro.We hope to provide a new paradigm to further improve the therapeutic ratio in the treatment of HNSCC.Methods1.Cell linesTen recently established HNSCC cell lines were used,established in the University of Turku as described UT-SCC（Abbreviate as SCC）.SCC-8,SCC-9, SCC-11,SCC-19a,SCC-29,SCC-34 and SCC-38 are SCCs of laryngeal origin （SCC-9 from a neck metastasis and SCC-11 from recurrence）.SCC-24a1,SCC-24a2 and SCC-40 are SCCs from tongue.2.Methods（1） Cell culture:The cells were maintained in passage 15-30 by passing weekly or bioweekly in DMEM medium（10%FBS）.Cells in mid-logarithmic growth were incubated at +37°C,were attached to the wall of the culture flask,and were used for the experiments.（2） Clonogenic growth assay:The cells were harvested with trypsin-EDTA, counted,and suspended in Ham’s F-12 medium containing 15%FBS.The number of cells plated per well was adjusted to the plating efficiency（PE） of the cell line.The cells were incubated at +37°C for 4 weeks after indicated interference factors, whereafter the positive number of wells containing coherent,living colonies,consisting of 32 cells or more,were counted using an inverted phase-contrast microscope.（3） For protein expression and phosphorylation analysis of ErbB family member and key marks of EGFR signal pathway by Western blotting:cells were lysed and sample of total protein was separated on SDS-PAGE gels.Bound secondary antibody was visualized by ECL.（4） For mRNA expression of ErbB receptors by RT-qPCR:①The primers and Taqman probes designed using Primer Express software②Total RNA was extracted from cultured cells using Trizol③Construction of recombinant pEBS7- EGFR as a criterion,β-actin as an internal control.3.Experimental design（1） The IC70 and IC50 of drug（Cetuximab,Gefitinib,Cisplatin） sensitivity was determined using the 96-well plate clonogenic assay based on limiting dilutions.Each analysis with one cell line was divided into a negative control group and drug groups with different concentration.（2） The 96-well plate clonogenic assay was used to examine cetuximab or gefitinib combined with radiation on HNSCC in vitro.For example:cetuximab or gefitinib + radiation a= radiation group（fraction dose:0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy and 7.5 Gy）b= radiation + drug group（same radiation dose with above mention,IC70 of cetuximab or gefitinib）c= radiation + drug group（same radiation dose with above mention,IC50 of cetuximab or gefitinib）（3） The 96-well plate clonogenic assay was used to examine cetuximab,cisplatin combined with radiation on HNSCC in vitro.For example:cetuximab + cisplatin + radiationa= radiation group（fraction dose:0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy and 7.5 Gy）b= radiation + chemotherapy group（same radiation dose with above mention, cisplatin IC70）c= radiation + chemotherapy + drug group（same radiation dose with above mention,cisplatin IC70 and cetuximab IC70）d= radiation + chemotherapy + drug group（same radiation dose with above mention,cisplatin IC70 and cetuximab IC50）4.For evaluation of curative effects and data analysis（1） The PE was calculated using the formula -In（number of negative wells / total number of wells） / number of cells plated per well.（2） The drug（cetuximab,gefitinib,cisplatin） sensitivity was determined by the IC70 and IC50 values of each cell line,resulting in 30%and 50%inhibition in clonogenic survival.（3） The evaluation of curative effects to combined-modality paradigm①Fraction survival data as a function of the radiation dose with or without the indicated cetuximab and gefitinib doses were found to fit in the linear quadratic equation.A microcomputer program was used to fit data to F=exp[-（αD+βD²）].②The area under the curve（AUC） value,equivalent to mean inactivation dose, was obtained by numerical integration.The AUC-ratio（AUC for cetuximab or gefitinib + radiation +/- chemotherapy /AUC for radiation） and surviving fraction after the indicated doses of drugs were used to compare the effect of the drugs together with irradiation with the effect of irradiation alone.③Statistical comparisons were made using Student’s t-test.The influence of the drug concentrations on the amount of supra-additive effect was tested by 1-way analysis of variance（ANOVA）.The type of interaction was described by the terms.A additive,corresponding to the effect being equal with the calculated effects of the drug and radiation +/- chemotherapy.A supra-additive effect would indicate more than the effect of the additive.（4） For associations between ErbB expression and sensitivity to gefitinib, cetuximab and radiation were analyzed using Spearman correlation coefficient.Results1.For evaluation of curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm on HNSCC in vitro（1） The drug sensitivity of 10 HNSCC cell lines①The IC70 concentration of cetuximab,gefitinib,cisplatin vary between 0.22 and 4.08 nM;0.036 and 4.8μM;0.038 and 0.22μg/ml.②The IC50 concentration of cetuximab,gefltinib,cisplatin vary between 0.58 and 8.2 nM;0.15 and 8.4μM;0.054 and 0.31μg/ml.③SCC-24a2 and SCC-40 were the most sensitive cell lines to cetuximab in the 10 HNSCC cell lines.SCC-24a2 and SCC-19a were the most sensitive cell lines to gefitinib in the 10 HNSCC cell lines.SCC-8 and SCC-34 were the most sensitve cell lines for cisplatin in 7 HNSCC cell lines.（2） The evaluation of curative effects to combined-modality paradigm of 7 HNSCC cell lines①Cetuximab combined with radiationThe effect of cetuximab in combination with radiation was additive（The effect of SCC-24a2 with IC50 concentration was additive,P=0.054）,which is superior to radiation alone.The effect of SCC-24a2 with IC50 concentration was supra-additive （P＜0.05）.②Gefitinib combined with radiation The effect of gefitinib in combination with radiation was additive,which is superior to radiation alone,except for SCC- 9,SCC- 24A2,SCC- 29,SCC- 34,and SCC- 40 with IC50 concentration,which were supra-additive（P＜0.05）.③Cetuximab,cisplatin combined with radiationThe effect of simultaneous cetuximab,cisplatin combined with irradiation was at least additive,which is superior to radiation alone.SCC-40 and SCC-24a presented a supra-additive fashion in the concomitant exposure with cisplatin and both cetuximab concentrations.The two cell lines that reacted in a supra-additive manner to cetuximab combined with chemoirradiation were among the most sensitive to treatment with cetuximab as a single agent.2.For prediction of curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm by testing key marks of EGFR signal pathway on HNSCC in vitro（1） For protein expression and phosphorylation analysis by Western blotting:Most HNSCC lines expressed variable amounts of EGFR,ErbB2,ErbB3 and pEGFR, pErbB2,pErbB3,pAkt,perk,whereas no signal was detected for ErbB4 in any of the 10 cell lines.（2） For ErbB mRNA expression by RT-qPCR:At mRNA level,the SCC cells expressed mostly EGFR followed by ErbB2,ErbB3 and ErbB4.（3） Association of ErbB expression with sensitivity to gefitinib:The association between high ErbB3 expression and high IC₅₀ for gefitinib reached statistical significance（P = 0.02）.The association between pErbB2 levels and IC₅₀ for gefitinib was significant（P = 0.02）.（4） Association of ErbB expression with sensitivity to cetuximab:EGFR protein expression reached a significant level of association with sensitivity to cetuximab（P = 0.048）.（5） The cellular expression of phosphorylated ErbB3 was associated with resistance to radiation measured as AUC（P = 0.0045）. Conclusions1.Concomitant use of cetuximab or gefitinib with radiation is superior to radiation alone and the gefitinib was superior to cetuximab in down-regulating HNSCC cell clonogenic growth when combined with radiation in HNSCC in vitro.The two cell lines in the most sensitive to cetuximab with IC50 concentration combined with radiation were the most sensitive to treatment with cetuximab as a single agent. Cetuximab was combined with chemo-radiotherapy,in which merely a low dose of cisplatin and cetuximab were used in order to achieve a significant curative effect in HNSCC in vitro.The effect of simultaneous cetuximab,cisplatin combined with irradiation was at least additive,which is superior to radiation alone.The two cell lines in a supra-additive manner to cetuximab combined with chemoirradiation were the most sensitive to treatment with cetuximab as a single agent.2.The pErbB2/ErbB3 protein expression reached a significant level of association with resistance to gefitinib.EGFR protein expression reached a significant level of association with sensitivity to cetuximab.The cellular expression of pErbB3 was associated with resistance to irradiation.Increased pErbB2/ErbB3 signaling may predict resistance to gefitinib,EGFR protein expression may predict sensitivity to cetuximab,and pErbB3 protein expression may predict resistance to irradiation in HNSCC.更多还原