肺癌耐药分子标志与个体性化疗.ppt

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1、肺癌耐药分子标志与个体性化疗,徐 萌暨南大学附属第一医院肿瘤科,肺癌耐药分子标志：药理遗传学和药理基因组学的研究表明与肺癌多药耐药相关的基因及其异常信号传导通路。个体性化疗：根据肺癌患者药理遗传学和基因组学特点，采用特异和最佳的化疗药物方案，提高化疗疗效，尽量降低化疗副反应，最大延长患者生存期。,肺癌化疗耐药的复杂性 肺癌耐药逆转药物的局限性肺癌个体性化疗的实践性典型病例,以铂类药物为基础的联合化疗比单一化疗效果好，是中晚期肺癌一线治疗的金标准;紫杉醇、诺维本、多西紫杉醇、吉西他滨;在过去三十年中，非小细胞肺癌的中位生存时间只延长了大约三个月;中晚期非小细胞肺癌经验性化疗疗效停滞于平台期，致力

2、于寻找各种敏感的化疗耐药分子标志和高效的耐药逆转剂具有广泛的应用前景。,Corey Langer 2000;Breathnach et al 2001;Schiller et al 2002,一.肺癌化疗耐药的复杂性,What is the Scope of the Problem?New Cancer Cases&Deaths 2001,CA Cancer J Clin.51:23,2001,*Vast majority of deaths due to chemoresistance,*,DRUG ACCUMULATION IS REDUCED IN DRUG-RESISTANT CELL

3、S,Resistant cells,Sensitive cells,Why Does Chemo Fail?,耐药参与机制主要有：(1)多药耐药基因(MDR1)及其编码的细胞膜P-糖蛋白表达增加；(2)多药耐药相关蛋白(MRP)表达增加；(3)谷胱甘肽解毒酶系统活性增强；(4)DNA拓扑异构酶活性降低或结构异常；(5)DNA损伤的修复能力增强等,?,耐药分类(1)药理耐药（pharmacological resistance）：由机体对药物的影响所导致的耐药，如药物进入机体代谢增强或活化障碍、肿瘤血供不足，药物组织穿透力差，导致细胞有效浓度降低。(2)生化耐药（biochemical resi

4、stance）：肿瘤细胞的遗传性及生化特性发生复杂的变化，致使细胞通过不同途径对药物产生耐药性。(3)凋亡耐药（apoptosis resistance）：大部分抗肿瘤药物引起细胞死亡是通过细胞凋亡，而促凋亡基因的缺失或抗凋亡基因的过度表达都将相应地导致肿瘤细胞对化疗药物产生耐药性。(4)微环境耐药(microenvironment resistance)：肿瘤细胞的存活和生长有赖于器官微环境，器官微环境可以通过调节不同耐药基因表达来影响肿瘤细胞对化疗药物的敏感性。,Reduced apoptosisAltered cell cycle checkpointsIncreased metabol

5、ism of drugsIncreased or altered targetsIncreased repair of damageCompartmentalization区室作用,MECHANISMS OF RESISTANCE TO ANTI-CANCER DRUGS,Decreaseduptake,Increasedefflux,与肿瘤多药耐药性相关的ABC转运体,多药耐药性（Multidrug Resistance,MDR）：意义：肿瘤细胞在抗肿瘤药物长期和反复作用下 对其出现耐药性的同时，而且对其它多种 结构乃至作用机制不同的抗肿瘤药物也产 生交叉抗性。Biedler首先描述了MDR

6、表型（20世纪70年代）现已发现100余种与MDR相关的转运体（2007年）,“疏水真空清除模型”（Hydropobic vaccum cleaner model）,要点：MDR转运体的2TMDs结构域形成一个药物通道，利用水解ATP提供的能量把药物（包括所有被修饰的疏 水性药物）转运到细胞外，这种药泵功能使胞内药物 浓度维持在低水平，从而导致肿瘤细胞产生耐药性。证据：Rosenberg et al.(1997)对纯化的P-gp的二维晶体电镜 3-D重构（2.5nm），发现分子中间有直径约5nm的漏 斗状结构，对MDR转运体的药物通道模型假设提供 较直接的实验证据。,相关临床现象：mdr1表达

7、水平高的正常组织器官发生的肿瘤中，检测到的mdr基因的表 达水平亦高，而且这种肿瘤在临床上 往往对化疗药物也不敏感，即表现抗性。反之亦然。证明：mdr1基因编码的P-gp的表达水平与肿瘤耐药相关。,P-gp是一种具有ATP酶活性药物排出泵（drug efflux pump）,overexpers in ritro/in rivo：P-gp 能转运多种结构 和功能不相关的药物（Mr：300-2000Da）：阿霉素（doxorubibin）柔红霉素（daunorubicin）放线菌素（antinomycin D）依托泊苷（etoposide）秋水仙素（colchicines）长春新碱（vincri

8、stine）氨茴环素（anthracyclines）表鬼臼毒素（epipodophyllotoxins）,MRP 的性质和功能（1）M.W.170KDa plus(N-端连接的糖基)约190KDa。（2）分布：在正常细胞中：内膜系统，如内质网、高尔基体和运输囊泡膜等。在肿瘤细胞中：主要在细胞质膜上，少部分在细胞质中多靠近细胞核。（3）MRP1不仅可直接外排药物，负责药物的 胞内隔离，使药物不能与靶点结合，导致耐药性。MRP1的药物抗性：如对：蒽环素（anthracyclins）生物碱(Vinka alkaloids)表鬼臼毒素(epipodophyllotoxins)米托蒽醌(mitoxant

9、rone),P-gp与MRP：相似：基本结构，转运功能，药物抗性 不同：P-gp:主要转运天然的和阳离子性 的疏水化合物 MRP:主要转运阴离子性的化合物，转运的药物常是与谷胱甘肽 和其他阴离子的缀含物，或 是与谷胱甘肽一起协同转运,MRP基因表达增强是肺癌原发性耐药发生的早期标记物，也是肺癌耐药的主要机制，肺癌细胞在化疗药物诱导条件下的耐药表型改变都是为了抵抗化疗药物杀灭，保护自身生存。徐萌.恶性肿瘤化疗及其对策.第一版.北京：军事医学科学出版社，2002.90-117.Xu Meng，et al.J Tumor Marker Oncology，2004，19(4):235-241.,肿瘤细

10、胞MDR的机理的复杂性：耐药表型可因肿瘤类别和细胞分化阶段的 不同而异，甚至有明显差别；不同肿瘤对不同的化疗药物的反应和机理 不同；不同的肿瘤对同一种化疗药物的反应和机 理也可能不同。,耐药的肿瘤干细胞肿瘤形成中经过多次突变形成肿瘤细胞的异质性，其中少量的细胞具有很强的增殖能力，被称为肿瘤干细胞（tumor stem cell）。肿瘤的发生是由于肿瘤干细胞的增殖,治疗的目的必须是确定和杀灭肿瘤干细胞，由于肿瘤干细胞的增殖能力有限，比肿瘤细胞对化疗药物更具耐药性,体内残余的耐药的肿瘤干细胞足以使肿瘤复发或转移。以肿瘤干细胞为靶向的治疗疗效更为显著。,Cancer Stem Cell-Lung C

11、ancer支气管肺癌干细胞BASC,BASC has self-renewal capacity.BASC proliferates in airway damage,regeneration and tumor progression.,Cell 2005;121:823-835,K-ras conditioning mouse,limiting dilution assay,Stem Cells-Drug Resistance,High levels of ABC drug transportersQuiescenceCapacity for DNA repairAccumulation

12、of mutations,Nature Review Cancer 2005;5:275-284,Nature Review Drug Discovery 2006;5:219-234,Resistance-ABC Transporter,Targeting ABC Transporter,Nature Review Drug Discovery 2006;5:219-234,Cancer Stem Cell-high drug efflux capacity,Cancer stem cell has ABC transporters with drug-efflux capacity.,Pr

13、oc.Natl.Acad.Sci.USA 101,1422814233(2004),human tumor cell lines,SP:side population,JF&IMR 32:NB cell lines,SP,Non-SP,control,fluorescence,Hoechst 33342-stained flow cytometry,SP,Non-SP,Stem Cell-Chemoresistance,Cancer stem cell has higher levels of drug resistance proteins.,Cancer Res 2000,60:4403-

14、4411,MRP:multiple resistance associated proteinLRP:lung resistance related protein,MFI:mean fluorescence intensity,semiquantitative RT-PCR,flow cytometry,钙离子通道阻滞剂、环孢菌素、钙调蛋白抑制剂、抗疟药等；另外细胞因子、类固醇激素、单克隆抗体、免疫毒素、特异性双抗、反义寡聚核苷酸、结合白蛋白的药物都可逆转肿瘤的耐药性 目前研究最多的还是维拉帕米、环孢素A及其衍生物 全身性应用逆转剂副作用较大，可产生心脏损害，诱发黄疸，免疫抑制 目前在临床应

15、用受到很大的限制,二.既往耐药逆转药物的局限性,100bp标志物 对照组 MRP siRNA组 ADM组 MRP siRNA加ADM组,图1 转染siRNA后肺癌细胞MRP mRNA表达水平,由于临床肺癌患者有原发性耐药以及体内肿瘤细胞耐药倍数较低，目前还没有一种逆转化疗多药耐药方法在临床广泛应用，主要原因有:现有逆转剂老药新用，逆转耐药不是其主要功能，临床毒副作用大；生物逆转剂在体内不稳定，难以作用到靶点；临床耐药往往数种机制同时参与，只对单一机制起作用的逆转剂难以发挥显著的效应。缺乏大样本的循证医学依据。,Overall Survival,Progression free Survival

16、,引自Kurbacher CM,Cree IA,Brucker.Anticancer Drugs.1998,9:51-57,传统化疗和ATP-TCA指导的化疗的生存率比较,卵巢癌术后,三 肺癌个体性化疗的实践性,乳腺癌易感基因(BRCA1):铂类，抗微管类药物,预后？错配切除修复酶(ERCC1):铂类药物 微管蛋白(-tubulin):抗微管类药物 核糖核苷还原酶M1(RRM1):嘧啶类抗代谢(吉西他滨)抗血管生成化疗,Gene expression according to histology in NSCLC,Clinical data from M Skrzypski,E Jassem,

17、J Jassem,铂类药物,Platinum based doublets are standard for patients with advanced NSCLC and good performance status.40%-60%of patients NSCLC progress during platinum based therapy,while some have excellent responses.Schiller,Harrington et al.;N Engl J Med,2002Platinum doublets have a marginally increase

18、d RR(17%)vs third generation non-platinum doublets but OS is not improved.DAddario,Pintilie et al.;J Clin Oncol,2005,Clinically important mediators of DNA repair for platinum based damage,Essential NERDNA unwinding:ERCC2(XPD)Incision of DNA:ERCC1(XPF)Transcription coupled NERBRCA1Base Excision Repai

19、rXRCC1,General mechanisms of platinum resistance,DetoxificationInhibitors of apoptosisDNA methylationChanges in influx/effluxIncreased DNA repair capacityRabik and Dolan;Cancer Treat Rev,2006,Established mediators of platinum resistance in NSCLC,BRCA1ERCC1Germline polymorphismsTumor expression by mR

20、NAImmunohistochemistryPolymorphisms in ERCC2&XRCC1,BRCA1,In a breast cancer cell line low BRCA1 mRNA expression increased sensitivity to cisplatin and etoposide,but increased resistance to paclitaxel and vincristine.In a BRCA1-negative cell line,reconstitution of wild-type BRCA1 led to a 20-fold inc

21、rease in cisplatin resistance and,in contrast,in a 100010 000-fold increase in sensitivity to antimicrotubule drugs.Low BRCA1 mRNA levels in sporadic breast cancer were associated with a higher frequency of distant metastases.Taron,Rosell et al.;Hum Mol Genet,2004,BRCA1 mRNA expression levels and su

22、rvival in NSCLC patients treated with neo-adjuvant gemcitabine&cisplatin.,Total 55 pts;Bottom:N=15,Middle=28,Top=12;p=0.01 Taron,Rosell et al.;Hum Mol Genet,2004,Two strands of evidence for customizing therapy based on BRCA1 mRNA levels in SCAT,Among several DNA repair genes,BRCA1 is the most reliab

23、le predictive marker of chemotherapy outcome in stage IV and stage III NSCLCIn early-stage chemonaive NSCLC patients,BRCA1 is the best prognostic marker of survivalBRCA1 has been selected as the marker for assigning chemotherapy in the SCAT experimental armLow BRCA1 gem/cisIntermediate BRCA1 doc/cis

24、High BRCA1 doc,Resected NSCLC pN1/pN2,Q 2&3 BRCA1,Q 4 BRCA1,Gem/Cis,Docetaxel,Docetaxel/Cis,Q 1 BRCA1,EGFR mutations in adenocarcinomas patients stratified by invasive gene signature(CSF-1,EGFR&CA IX)&tumor size,Spanish Customized Adjuvant Therapy in completely resected N1&N2 NSCLC,CONTROL,EXPERIMEN

25、TAL,Docetaxel/Cis,Customized adjuvant chemotherapy in stage II-IIIA NSCLC,Spanish Lung Cancer Group,Survival to neoadjuvant gem/cis according to BRCA1 expression in stage III NSCLC,HAZARD RATIO,q4,q2+q3,q1,Taron,Rosell,Felip,et cla.Hum Molec Genet 2004,Outcome according to BRCA1,ERCC1&RRM1 mRNA in e

26、arly NSCLC,Clinical data from R Bartolucci,F Puma,R Farabi,Gene expression analysis was performed in frozen tumor samples from 126 completely resected Polish NSCLC patients.,NR=not reached*Survival data is not available for some patients.Gene amplification was not successfully performed in all sampl

27、es for all genes.,Rosell et al;ASCO 07,Increased BRCA1 mRNA:an independent prognostic variable in completely resected chemonaive NSCLC patients,ERCC1,Essential component of NERAssessed by:Functional germ-line polymorphismsExpression levels by mRNAExpressional levels by IHC,Polymorphisms of ERCC1 and

28、 survival in cisplatin treated NSCLC.,ERCC1单核苷酸多态性预测铂类药物的疗效及患者预后:ERCC1研究比较多的单核苷酸多态性位点主要是Asn118Asn（C/T），第118位密码子的同义突变。携带有C/C基因型的非小细胞肺癌患者对铂类化疗疗效比携带T/T 或C/T 基因型患者好，总体生存的时间长。Asn118Asn（C/T）单核苷酸位点在不同人种中分布不同，这可能是解释不同的研究小组在不同人种中所做研究结果不同。在亚洲人种中，目前发表的研究结果支持C/C基因型是预测铂类敏感的指标。Isla,Sarries et al.;Ann Oncol,2004,E

29、RCC1 mRNA levels in advanced NSCLC treated with gemcitabine-cisplatin.,56 patients with advanced NSCLC.Low vs high ERCC1 mRNARR:52%and 36%(p=NS),MS:15 months and 5 months(P 0.001)Brabender et al.;Clin Cancer Res,2002,Rosell,et.al.;ASCO;2005,Rosell,et.al.;ASCO;2005,ERCC1 by immunohistochemistry in re

30、sected NSCLC,761 patients from the IALT trial who had tissue available1867 patients with completely resected stages I-III NSCLC randomized to observation or cisplatin plus etoposide or vinorelbine.Immunostaining with monoclonal antibody to ERCC1 interpreted by two blinded pathologists.Score calculat

31、ed by multiplying intensity of staining with proportion of positive nuclei.Median score defined high vs low ERCC1 expression.Olaussen,Dunant et al.;N Engl J Med,2006,ERCC1 in IALT Results,ERCC1 was more likely to be positive in:Squamous cell carcinomaAge 55For the study population as a whole,ERCC1 h

32、ad no prognostic value.Olaussen,Dunant et al.;N Engl J Med,2006,Olaussen,Dunant et al.;N Engl J Med,2006,ERCC1阴性的患者术后辅助铂类联合化疗能明显提高患者的生存（OR 0.65;95%CI:0.50-0.86;p=0.002），然而ERCC1阳性的患者对术后含铂类方案却不能获益（OR 1.14;95%CI:0.84-1.55;p=0.40）。,Low genotypic group docetaxel/cisplatin,High genotypic groupdocetaxel/ge

33、mcitabine,RANDOMIZE,Genotypic armERCC1 levels,1:2,docetaxel/cisplatin,Control arm,low ERCC1 mRNA,high ERCC1 mRNA,Customizing cisplatin-based chemotherapy on quantitative ERCC1 mRNA expression:a phase III randomized trial in NSCLC,Cobo et al.JCO 2007,Customizing cisplatin-based chemotherapy on quanti

34、tative ERCC1 mRNA expression:a phase III randomized trial in NSCLC,Cobo et al.JCO 2007,IALT Bio(Olaussen et al,NEJM 06),ERCC1 is a prognostic factor in CT-nave patients,Diagnostic testing to guide treatment decisions,Filipits,M.et al.J Clin Oncol;25:2735-2740 2007,Fig 2.Kaplan-Meier analyses of over

35、all survival according to treatment(A)in patients with p27Kip1-negative tumors and(B)in patients with p27Kip1-positive tumors,NSCLC patients with p27-negative tumors benefit from adjuvant cisplatin-based chemotherapy,Prognostic and Predictive Importance of p53 and RAS for Adjuvant Chemotherapy in No

36、nSmall-Cell Lung Cancer,the prognostic and predictive value of p53 gene/protein aberrations using tumor samples from JBR.10,a North American phase III intergroup trial that randomly assigned 482 patients with completely resected stage IB and II nonsmall-cell lung cancer(NSCLC)to receive four cycles

37、of adjuvant cisplatin plus vinorelbine or observation alone.Results:Of 253 patients,132(52%)were positive for p53 protein overexpression.Untreated p53-positive patients had significantly shorter overall survival than did patients with p53-negative tumors).However,these p53-positive patients also had

38、 a significantly greater survival benefit from adjuvant chemotherapy compared with patients with p53-negative tumors.Mutations of p53 and RAS genes were found in 124(31%)of 397 and 117(26%)of 450 patients,respectively.Conclusion:p53 protein overexpression is a significant prognostic marker of shorte

39、ned survival,and also a significant predictive marker for a differentially greater benefit from adjuvant chemotherapy in completely resected NSCLC patients.Journal of Clinical Oncology,Vol 25,No 33(November 20),2007:pp.5240-5247,RRM1和ERCC是靶向化疗的敏感性分子指标。可根据基因表达高低选择个体化疗方案，RRM1高表达者建议避免使用吉西他滨，ERCC1阴性者可考虑

40、使用铂类药物治疗，可根据下列四种表达情况知道临床化疗：ERCC1(+)/RRM1()多选择紫杉醇/吉西他滨化疗方案；ERCC1(+)/RRM1(+)可选择长春瑞滨/紫杉醇方案；ERCC1(-)/RRM1()可选择铂类/吉西他滨方案；ERCC1(-)/RRM1(+)可选择铂类/紫杉醇方案。,Molecular signatures in resected patientsPotential clinical implications,Prognosis assessmentObjective;selection of patients for adjuvant CT:Adjuvant chemo

41、therapy in patients with high-risk gene signatureMolecular signatures should be validated in adjuvant therapy trialsHow to integrate molecular signatures with single gene prognostic and predictive markers should be explored,肺癌抗血管生成化疗,Adapted from Poon RT,et al.J Clin Oncol 2001;19:120725,新生血管生成在肿瘤发展

42、过程的不同阶段所扮演的角色,恶变前期,恶性肿瘤,肿瘤生长,血管侵袭,微转移处于休眠状态,明显的转移,(肿瘤无血管),(血管新生开始),(肿瘤形成血管),(肿瘤细胞进入血管内),(远道种植),(再次形成新生血管),传统的化疗用药方法在理论上可对抗内皮细胞的增殖，但不能起到持续阻断血管生成的作用。密集式低剂量给药可能会增加化疗药对再生内皮细胞的抗血管生成效应以及对肿瘤细胞的促凋亡效应。这种剂量低、间歇期短、能持续应用的给药方式称抗血管生成化疗,是迄今肿瘤化疗新的治疗模式之一。小剂量(最大耐受量的1/101/3)、长时间连续或高频率(如每周13 次)给药模式。,抗血管生成化疗,靶细胞的选择:血管内皮

43、细胞 VS 肿瘤细胞,血管内皮细胞基因组较为稳定,不易产生耐药性;正常成熟组织毛细血管内皮细胞处于静止状态,而肿瘤血管内皮细胞增殖活跃,出现许多相对特异的标记分子,表达较正常静止内皮细胞可高达50倍以上,是潜在的抗肿瘤血管靶向分子;一个内皮细胞可支持50100肿瘤细胞的生长。因此针对血管内皮细胞比直接针对肿瘤细胞更为有效;针对肿瘤血管治疗策略具有抗瘤的广谱性。重组内皮抑素具有抑制肿瘤组织新生血管生成和直接抑制肿瘤细胞生长及迁移的双重抗肿瘤活性。,抗血管生成化疗药物筛选标准差异的细胞毒性：抑制内皮细胞剂量应低于对肿瘤细胞干扰内皮细胞功能：紫杉醇明确的作用机制：抑制血管生成具体环节体内抑制血管生成

44、：如紫杉醇,抗血管生成化疗显示较强的临床应用价值:为常规化疗不敏感或(和)复发患者的治疗开辟新领域,由于无明显骨髓毒性,也为因体力不支、状态不佳等因素不宜接受常规化疗的患者提供新的治疗方法;不存在耐药,可用于常规化疗后耐药患者;与传统抗血管生成抑制剂联合疗效更佳;与常规化疗、放疗联合可能会使疗效进一步提高。,四.典型病例,张X，男，57岁,右上肺低分化腺癌并多发性骨转移T3N2M1，期，MRP(+),p-gp(+),ERCC1(-),RRM1(+).,治疗前,治疗后,小结与展望,MDR分子机制深入研究将实现对肺癌的发生机制、诊断及化学治疗的突破性。肺癌患者化疗耐药分子标志基因与化疗疗效之间关系能够尽早得到建立。根据肺癌患者化疗耐药分子标志，精确选择并制定特异化疗方案，科学调整个体化剂量，实现肺癌个体化性化疗方案疗效的新飞跃。,谢谢,