肺癌耐药分子标志与个体性化疗课件

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整理ppt肺癌耐药分子标志与个体性化疗徐 萌整理ppt整理ppt整理ppt整理ppt整理ppt 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。 整理ppt 肺癌耐药分子标志：药理遗传学和药理基因组学的研究表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整理ppt整理ppt以铂类药物为基础的联合化疗比单一化疗效果好，是中晚

2、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整理ppt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What is the Scope of the Problem? New Cancer Cases & Deaths 2001CA C

3、ancer J Clin.51:23, 2001*Vast majority of deaths due to chemoresistance*整理pptWhat is the Scope of the ProblResistant cellsTopotecanSensitive cellsMitoxantroneDRUG ACCUMULATION IS REDUCED IN DRUG-RESISTANT CELLS Resistant cellsSensitive cells整理pptResistant cellsTopotecanSensitWhy Does Chemo Fail?耐药参与

4、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?整理pptWhy Does Chemo Fail?耐药参与机制主要有：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 resistance）：肿瘤细胞的遗传性及生化特性发生

5、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。 整理ppt耐药分类整理pptReduced apoptosisAltered cell cycle checkpointsIncreased metabolism of

6、drugsIncreased or altered targetsIncreased repair of damageCompartmentalization区室作用MECHANISMS OF RESISTANCE TO ANTI-CANCER DRUGSDecreaseduptakeIncreasedefflux整理pptReduced apoptosisMECHANISMS OF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

7、也产 生交叉抗性。Biedler首先描述了MDR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）整理ppt与肿瘤多药耐药性相关的ABC转运体多药耐药性（Multidr“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

8、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。整理ppt“疏水真空清除模型” 要点：MDR转运体的2TMDs结构相关临床现象：mdr1表达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。整理ppt相关临床现象：mdr1表达水平高的正常组织器官发生的肿瘤中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

9、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（vincristine） 氨茴环素（anthracyclines） 表鬼臼毒素（epipodophyllotoxins）整理pptP-gp是一种具有ATP酶活性药物排出泵（ drug efMRP 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

10、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(mitoxantrone)整理pptMRP 的性质和功能整理pptP-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

11、含物，或 是与谷胱甘肽一起协同转运整理pptP-gp与MRP：整理pptMRP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. 整理pptMRP基因表达增强是肺癌原发性耐药发生的早期标记物，也是肺癌肿瘤细胞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

12、，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。整理ppt肿瘤细胞MDR的机理的复杂性：整理ppt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。 整理ppt耐药的肿瘤干细胞整理pptMDR: The maj

13、or obstacle to successful cancer chemotherapyChemotherapyChemotherapy整理pptMDR: ChemotherapyChemotherapy整Cancer Stem Cell-Lung Cancer支气管肺癌干细胞BASCBASC has self-renewal capacity.BASC proliferates in airway damage, regeneration and tumor progression.Cell 2005;121:823-835K-ras conditioning mouselimiting

14、dilution assay整理pptCancer Stem Cell-Lung Cancer支Stem Cells-Drug ResistanceHigh levels of ABC drug transportersQuiescenceCapacity for DNA repairAccumulation of mutationsNature Review Cancer 2005;5:275-284整理pptStem Cells-Drug ResistanceHighNature Review Drug Discovery 2006;5:219-234Resistance-ABC Tran

15、sporter整理pptNature Review Drug Discovery 2Targeting ABC TransporterNature Review Drug Discovery 2006;5:219-234整理pptTargeting ABC TransporterNaturCancer Stem Cell-high drug efflux capacityCancer stem cell has ABC transporters with drug-efflux capacity.Proc. Natl. Acad. Sci. USA 101, 1422814233 (2004)

16、human tumor cell linesSP: side populationJF & IMR 32: NB cell lines SPNon-SPcontrolfluorescenceHoechst 33342-stained flow cytometrySPNon-SP整理pptCancer Stem Cell-high drug efStem Cell-ChemoresistanceCancer stem cell has higher levels of drug resistance proteins.Cancer Res 2000, 60:4403-4411MRP: multi

17、ple resistance associated proteinLRP: lung resistance related proteinMFI: mean fluorescence intensitysemiquantitative RT-PCRflow cytometry整理pptStem Cell-ChemoresistanceCanc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

18、害，诱发黄疸，免疫抑制 目前在临床应用受到很大的限制二. 既往耐药逆转药物的局限性整理ppt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表达水平整理ppt100bp标志物 对照组 MRP s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

19、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。整理ppt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050100150200250020406080100ATP-TCA指导的化疗 (n = 39)传统化疗(n = 17)p= 0,0009Weeks% Patients050100150200250020406080100ATP-TCA指导的化疗 (n = 39)传统化疗(n = 17)p = 0,0016Weeks% PatientsOverall SurvivalProgression free Survival引自Kurbacher CM, Cree IA,

20、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整理ppt050100150200250020406080100ATP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)抗血管生成化疗整理ppt三 肺癌个体性化疗的实践性乳腺癌易感基因(BRCA1):铂Gene expression according to histology in

21、 NSCLCSquamous cell carcinomaAdenocarcinomaP*Median (range)Median (range)ERCC11.41 (0.45-7.34)0.72 (0.23-2.45)0.0001MZF10.62 (0.06-6.72)0.25 (0.03-1.49)0.0001Twist10.37 (0.30-76.01)2.50 (0.14-19.16)0.0001RRM12 (0.6-6.9)1.2 (0.4-2.9)0.0001TRX2.13 (0.40-11.88)0.91 (0.31-7.94)0.0001Tdp11.7 (0.6-7.3)1.3

22、 (0.1-2.6)0.02NFAT0.4 (0.1-2.3)0.5 (0.1-1.8)0.65BRCA14.26 (0.55-18.48)1.50 (0.09-8.08)0.0001BubR116.3 (1.4-90)7 (0.8-25)0.0001Clinical data from M Skrzypski, E Jassem, J Jassem整理pptGene expression according to h铂类药物Platinum based doublets are standard for patients with advanced NSCLC and good perfor

23、mance 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 increased RR (17%) vs third generation non-platinum doublets but OS is not improved.DAddario, Pintil

24、ie et al.; J Clin Oncol, 2005整理ppt铂类药物Platinum based doublets arClinically important mediators of DNA repair for platinum based damageEssential NERDNA unwinding: ERCC2 (XPD)Incision of DNA: ERCC1 (XPF)Transcription coupled NERBRCA1Base Excision RepairXRCC1整理pptClinically important mediatorsGeneral m

25、echanisms of platinum resistanceDetoxificationInhibitors of apoptosisDNA methylationChanges in influx/effluxIncreased DNA repair capacityRabik and Dolan; Cancer Treat Rev, 2006整理pptGeneral mechanisms of platinumEstablished mediators of platinum resistance in NSCLCBRCA1ERCC1Germline polymorphismsTumo

26、r expression by mRNAImmunohistochemistryPolymorphisms in ERCC2 & XRCC1整理pptEstablished mediators of platiBRCA1In 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

27、, reconstitution of wild-type BRCA1 led to a 20-fold increase 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 e

28、t al.; Hum Mol Genet, 2004整理pptBRCA1In a breast cancer cell lBRCA1 mRNA expression levels and survival 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整理pptBRCA1 mRNA expression levels

29、aTwo strands of evidence for customizing therapy based on BRCA1 mRNA levels in SCATAmong several DNA repair genes, BRCA1 is the most reliable predictive marker of chemotherapy outcome in stage IV and stage III NSCLCIn early-stage chemonaive NSCLC patients, BRCA1 is the best prognostic marker of surv

30、ivalBRCA1 has been selected as the marker for assigning chemotherapy in the SCAT experimental armLow BRCA1 gem/cisIntermediate BRCA1 doc/cisHigh BRCA1 doc整理pptTwo strands of evidence for cuResected NSCLC pN1 / pN2Q 2 & 3 BRCA1Q 4 BRCA1Gem/CisDocetaxelDocetaxel/CisQ 1 BRCA1 EGFR mutations in adenocar

31、cinomas patients stratified by invasive gene signature (CSF-1, EGFR & CA IX) & tumor size Spanish Customized Adjuvant Therapy in completely resected N1 & N2 NSCLC CONTROLEXPERIMENTALDocetaxel/Cis整理pptResected Q 2 & 3 BRCA1Q 4 BRCACustomized adjuvant chemotherapy in stage II-IIIA NSCLCSpanish Lung Ca

32、ncer Group整理pptCustomized adjuvant chemotheraBRCA1RR (95%CI)Pq10.170 (0.04-0.66)0.010q2+q30.400 (0.16-1)0.052q41-Survival to neoadjuvant gem/cis according to BRCA1 expression in stage III NSCLCHAZARD RATIO NMedian95% C.IPq1=14NR-0.014*q2+q3=2837.8010.6-65q4=1212.700-28.7q4q2+q3q1Taron, Rosell, Felip

33、, et cla. Hum Molec Genet 2004整理pptBRCA1RR (95%CI)Pq10.170 (0.04-Outcome according to BRCA1, ERCC1 & RRM1 mRNA in early NSCLCClinical data from R Bartolucci, F Puma, R Farabi整理pptOutcome according to BRCA1, ERGene expression analysis was performed in frozen tumor samples from 126 completely resected

34、 Polish NSCLC patients. NR=not reached*Survival data is not available for some patients. Gene amplification was not successfully performed in all samples for all genes. Rosell et al; ASCO 07Increased BRCA1 mRNA: an independent prognostic variable in completely resected chemonaive NSCLC patients 整理pp

35、tGene expression analysis was pERCC1Essential component of NERAssessed by:Functional germ-line polymorphismsExpression levels by mRNAExpressional levels by IHC整理pptERCC1Essential component of NEPolymorphisms of ERCC1 and 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

36、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 整理pptPolymorphisms of ERCC1 and surERCC1 mRNA levels in advanced NSCLC treat

37、ed 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 55For the study population as a whole, ERCC1 had no prognostic value.Olaussen, Dunant et al.; N Engl J Med, 2006整理pptERCC1 in IALT ResultsERCC1 wasOlaussen, Dun

38、ant et al.; N Engl J Med, 2006ERCC1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）。 整理pptOlaussen, Dunant et al.; N EnLow genotypic group docetaxel / cisplatinHigh genotypic groupdocetaxel / gemcitabine RANDOMIZEGenotypic a

39、rmERCC1 levels1:2docetaxel / cisplatinControl armlow ERCC1 mRNAhigh ERCC1 mRNACustomizing cisplatin-based chemotherapy on quantitative ERCC1 mRNA expression: a phase III randomized trial in NSCLCCobo et al. JCO 2007整理pptLow genotypic group docetaxel Customizing cisplatin-based chemotherapy on quanti

40、tative ERCC1 mRNA expression: a phase III randomized trial in NSCLCCobo et al. JCO 2007整理pptCustomizing cisplatin-based chIALT Bio (Olaussen et al, NEJM 06) ERCC1 is a prognostic factor in CT-nave patients整理pptIALT Bio (Olaussen et al, NEJMTumor tissue availableTumor tissue not availableNever smoker

41、SmokerEGFR mutationserlotinibmRNA levels (QPCR)BRCA1ERCC1MZF1MAD2BubR1Q1 gem/cisQ2 & Q3 doc/cis Q4 docSerum DNANever smokerSmokerMSP66yrsCHFREGFR mutationsPBCserlotinibSNP 60 yrMet/Metdoc/cisgem/cisgem/cis14-3-3Diagnostic testing to guide treatment decisions整理pptTumor tissue availableTumor tiFilipit

42、s, M. et al. J Clin Oncol; 25:2735-2740 2007Fig 2. Kaplan-Meier analyses of overall survival according to treatment (A) in patients with p27Kip1-negative tumors and (B) in patients with p27Kip1-positive tumorsNSCLC patients with p27-negative tumors benefit from adjuvant cisplatin-based chemotherapy整

43、理pptFilipits, M. et al. J Clin OncPrognostic and Predictive Importance of p53 and RAS for Adjuvant Chemotherapy in NonSmall-Cell Lung Cancerthe prognostic and predictive value of p53 gene/protein aberrations using tumor samples from JBR.10, a North American phase III intergroup trial that randomly a

44、ssigned 482 patients with completely resected stage IB and II nonsmall-cell lung cancer (NSCLC) to receive four cycles 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 sig

45、nificantly shorter overall survival than did patients with p53-negative tumors). However, these p53-positive patients also had 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%) o

46、f 397 and 117 (26%) of 450 patients, respectively. Conclusion: p53 protein overexpression is a significant prognostic marker of shortened survival, and also a significant predictive marker for a differentially greater benefit from adjuvant chemotherapy in completely resected NSCLC patients. Journal

47、of Clinical Oncology, Vol 25, No 33 (November 20), 2007: pp. 5240-5247整理pptPrognostic and Predictive Impo 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。 整理ppt RRM1和ERCC是靶向化疗的敏感性分子指标。可 Molecular signatures in resected patientsPotential clinical implicationsPrognosis assessmentObjective; selection of patients for adjuvant CT