1、1210 Breakthrough Technologies2015液体活检3循环肿瘤DNA（ctDNA）循环RNA（Circulating RNA）循环肿瘤细胞（CTC）外泌体（Exosome）4循环游离核酸(circulating free nucleic acids)是一种存在于动植物和人的体液中的细胞外游离状态核酸。目前已经在血浆、血清、尿液、前列腺液、支气管灌洗液、唾液、脑脊液、胃液、胆汁、淋巴液、腹腔液及粪便中检测到了循环游离核酸。已发现的游离循环核酸包括循环游离DNA和循环游离RNA。51.肿瘤细胞的坏死或凋亡2.循环血或微转移灶中肿瘤细胞溶解3.肿瘤细胞分泌DNA进入血液循环1
2、.机体正常细胞的死亡和凋亡2.肿瘤侵袭致周围细胞、组织变性而释放DNA入血3.微生物来源DNA肿瘤患者血中存在DNA 酶抑制剂，抑制血浆DNA 降解，从而使DNA在血中更容易富集。来自肿瘤细胞：来自非肿瘤细胞：6含量较低：约占总cfDNA的 1-90%，含量与肿瘤类型、分级及是否转移等多种因素相关。片段较小：在100300bp之间。包含整个肿瘤基因组信息：突变、缺失、重排、多态性等遗传改变；甲基化、微卫星不稳定性等表观遗传改变；基因拷贝数变异等。781.作为一个广谱肿瘤标记物：cfDNA的含量及完整性2.作为肿瘤遗传/表观遗传信息的载体：突变、缺失、重排、多态性等遗传改变；甲基化、微卫星不稳定
3、性等表观遗传改变；基因拷贝数变异等3.特异性的肿瘤标记物：具有遗传或表观遗传特征ctDNA的检测最常用的是采用扩增某个基因（如-ACTIN，ALU,LINE1）的方式，用来自正常细胞的genome DNA 作为标准品，基于扩增基因与总DNA在样品和标准品中比例关系是一致的这一理论，计算cfDNA的浓度（ng/ml）或基因组拷贝数。DNA染料，荧光直接定量技术9 血液采集和加工:血浆优于血清，尽快分离后低温保存；cfDNA提取：无论采用哪种方法，第一步都是提取血浆ctDNA，因此所用提取方法和试剂的不同就会造成巨大差别；标准品：gDNA 片段与cfDNA片段大小不一致；以高度重复序列或多拷贝基因
4、为检测对象，理论上可提高检测灵敏度。但存在高度重复序列或多拷贝基因在个体间拷贝数存在差异的弊端 Realtime PCR的敏感性10l 通过特殊的技术工艺处理，可以直接用血浆或血清进行定量PCR反应，不影响PCR反应，扩增效率与提纯DNA相比一致，减少了DNA提取步骤造成实验误差。l 以高度重复序列LINE1为测定靶基因为测定对象不仅提高检测灵敏度，而且用于DII计算，重复性好。11DNA的完整性(Integrity)：是指循环DNA中长DNA片段与短DNA片段的比值；是有别于非肿瘤人群是肿瘤患者血液ctDNA的另一个显著特征。Cancer Res.J2003;63:3966-3968DNA的
5、完整性指数（DII）检测示意图13 Real time or end-point PCR:Mutant allele-specific PCR,ARMS,ARMS-Scorpion；PNA-PCR,Cold PCR,PNA-ARMS Next Generation Sequencing:全基因组测序、全外显子测序、靶向重测序、全基因甲基化测序等 Digital PCR:BEAMing，Digital droplet PCR Chip-based TechniqueTechniqueSensitivityOptimal ApplicationSanger sequencing 20%Tumor
6、tissueMassively Parallel Sequencing1-2%Tumor tissueQuantitative PCR1-5%Tumor tissueNext Generation Sequencing0.1-1%Tissue or ctDNABEAMing/Digital PCR 0.01%ctDNA,rare variants in tumor tissueBEAMing技术：结合了数字PCR以及流式技术。其方法是每一类DNA分子都会专一的与磁性珠相连接，然后DNA分子之间的差异可以通过流式细胞仪检测荧光标记来做出评估。这种方法是基于小珠(Bead)、乳浊液(Emulsio
7、n)、扩增(Amplification)、磁性(Magnetic)，这四个主要组分来构建的，所以被称作为BEAMing。151.不受组织取材异质性的影响，或能反映肿瘤组织异质性N Engl J Med.2012 Mar 8;366(10):883-92172.可实时反映体内肿瘤细胞的遗传改变：肿瘤发展过程中和肿瘤治疗过程中肿瘤细胞的遗传信息发生改变，手术或活检标本无法反映这种改变肺腺癌患者治疗过程中血浆 DNA L858R EGFR突变检测A.术后肺内转移综合治疗，肝脏出现转移灶后，血浆DNA检出EGFR L858R突变B.化 疗 前 血 浆 D N A 检 出L858R EGFR突变检测，化
8、疗缓解后，血浆DNA L858R EGFR突变消失Journal of Thoracic Oncology,2012,7(9):1369-1381183.含量丰富，取材方便，多无限次取材，无创或微创ctDNA的含量足以进行PCR扩增和突变检测，文献报道癌症患者血浆cfDNA的含量平均在100ng/ml左右。以此量推算，每毫升血浆中约有基因组的拷贝数为1.5104个（每个癌细胞约含DNA 6.6pg）；而循环肿瘤细胞的数量在010个左右/7.5ml全血。ctDNA液体活检的劣势：混有非肿瘤来源的cfDNA，无法确定ctDNA在其中的比例，其中突变DNA可能占的比例非常小，需要更灵敏的检测技术1.
9、作为肿瘤标记物的应用2.作为肿瘤遗传/表观遗传信息载体的应用19理论上最广谱的肿瘤标记物，与组织类型无关，与表达水平无关Bettegowda et al.,Sci Transl Med 2014Comparison between the three studied groups as regards cfDNA plasma level.Association Between cfDNA and VariableHistopathological Parameters21222324Analysis of Circulating Tumor DNA to Monitor Metastatic
10、 Breast CancerSarah-Jane Dawson,N Engl J Med.2013 Mar 28;368(13):1199-209N Engl J Med.2013 Mar 28;368(13):1199-209.Comparison of Circulating Tumor DNA,CA15-3,and Circulating Tumor Cells as Blood-Based Biomarkers更好的检测灵敏度更好的检测灵敏度N Engl J Med.2013 Mar 28;368(13):1199-209.Comparison of Circulating Tumor
11、 DNA,CA15-3,and Circulating Tumor Cells as Blood-Based Biomarkers更好的检测灵敏度更好的检测灵敏度27和肿瘤负荷有更好的相关性和肿瘤负荷有更好的相关性最早对治疗做出反应最早对治疗做出反应28cfDNA CTC CA15-3更准确的预后效果更准确的预后效果Plasma DNA integrity as a biomarker for primary and metastatic breast cancer and potential marker for early diagnosisDharanija Madhavan,et al
12、.Breast Cancer Res Treat(2014)146:1631742930KaplanMeier curves fora progression-free(PFS in MBC patients using cfDI orlog2cfDNA concentration as the predictor variableBreast Cancer Res Treat(2014)146:163174ctNDA含量和完整性与PFS的关系KaplanMeier curves forb overall survival(OS)in MBCpatients using cfDI orlog2
13、cfDNA concentration as the predictor variableBreast Cancer Res Treat(2014)146:163174ctNDA含量和完整性与OS的关系指导靶向治疗：选择敏感个体；分析耐药机制；评价治疗效果；更改治疗方案肿瘤分子诊断：早期诊断；转移诊断，分类诊断33 FASTACT-2：一个多中心、随机、安慰剂对照、双盲的吉西他滨加铂类序贯厄洛替尼或安慰剂作为IIIB/IV期 NSCLC 患者一线治疗方案的III 期临床研究。患者1:1随机分组，接受6个周期的吉西他滨(1,250 mg/m2 intravenously on days 1 an
14、d 8 of a 4-week cycle)加铂类(carboplatin 5 AUC,or cisplatin 75 mg/m2 intravenously on day 1 of a 4-week cycle),然后在每周期的1528天给予厄洛替尼(150 mg/day orally;erlotinib arm)或安慰剂(placebo arm)。34研究开始7天内（Baseline），第三个周期开始的第一天（C3）和疾病进展三个时间点取血分离血清或血浆3536在基点血液和组织样本的突变检测的结果的一致率是88%(209/238)。敏感性和特异性分别是75%(72/96)和96%(137/
15、142，阳性预测值 94%(72/77)，阴性预测值85%(137/161)。组织和血液EGFR突变检测结果比较3777例，GC+E组 PFS 13.1个月，GC+P组 6.0 个月 HR,0.22;95%confidence interval(CI),0.140.33,P0.0001),两组 OS 分别是 29.3 个月 和 18.8 个月(HR,0.54;95%CI,0.350.83,P=0.0044).137例，GC+E组 PFS 6.2个月，GC+P组 6.1 个月 HR,0.83;95%CI,0.651.04,P=0.1076),两组 OS 分别是 15.3 个月 和 13.6 个月
16、（HR,0.94;95%CI,0.721.22,P=0.6449）.同时有组织和血液EGFR突变检测结果的病例38GC+E组（31例）PFS 12.8个月，GC+P组（36例）6.0 个月 HR,0.25;95%CI,0.140.47,P 0.0001),两组 OS 分别是 29.3 个月 和 21.4个月(HR,0.59;95%CI,0.301.15,P=0.1202).仅有cfDNA的67例GC+E组PFS 5.5个月，GC+P组5.9个月 HR,0.85;95%CI,0.601.19,P=0.3398),两组 OS 分别是 13.0 个月 和 13.6个月(HR,1.07;95%CI,0
17、.731.56,P=0.7387).仅有cfDNA的166例仅有血液EGFR突变检测结果的病例3940在全部治疗病人（E+P）在厄洛替尼治疗病人（E）这表明EGFR突变ctDNA的存在与否可作为肺癌疗效预测的标志物4142Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasmaNat Commun.2015 Nov 10;6:8839.研究了GMB 和肺癌、乳腺癌脑转移脑脊液中基因组的改变，与血浆进行对比检测方
18、法：hybridization capture-based massively parallel targeted sequencing（基于杂交捕获的大规模并行测序）and/or exome sequencing,droplet digital PCR(ddPCR)431.脑脊液比血浆更好地代表CNS 肿瘤的基因改变442.脑肿瘤患者脑脊液ctDNA的动态变化反映肿瘤治疗进程45F3463.CSFs ctDNA的检测有助于软脑膜癌的诊断4.不同转移部位的基因变异分析有助于追溯转移克隆起源Nat Commun.2015 Nov 10;6:8839.F1b49唾液ctDNA检测Schematic
19、 showing the shedding of tumor DNA from head and neck cancers into the saliva or plasma.Tumors from various anatomic locations shed DNA fragments containing tumor-specific mutations and HPV DNA into the saliva or the circulation.The detect ability of tumor DNA in the saliva varied with anatomic loca
20、tion of the tumor,with the highest sensitivity for oral cavity cancers.The detect ability in plasma varied much less in regard to the tumors anatomic location.Sci Transl Med.2015 Jun 24;7(293):293ra104.Correlation of EGFR Mutation Status between Plasma and Saliva Am J Respir Crit Care Med.2014 Nov 1
21、5;190(10):1117-26.1896 年 Ashworth 首次提出循环肿瘤细胞(circulating tumor cell,CTCs)的概念:指自发或因诊疗操作进入外周血循环的肿瘤细胞。指自发或因诊疗操作进入外周血循环的肿瘤细胞。PLoS One.2014 Apr 3;9(4):e93173.处于EMT状态的肿瘤易播散CTCCTC中间质样表型的细胞占大多数CTC间质样标记物表达上调CTC中处于间质样表型的细胞易成转移灶Cancer Cell.2013 Mar 18;23(3):272-3.53Current concept of cellular and molecular cha
22、racteristics of circulating tumor cells(CTCs)Nat Rev Cancer.2014 Sep;14(9):623-31.CTCs 富集技术富集技术Nat Rev Cancer.2014 Sep;14(9):623-31.CTCs 检测技术检测技术Nat Rev Cancer.2014 Sep;14(9):623-31.功能研究：成瘤能力、迁移情况等。Science.2014 Jul 11;345(6193):216-20.57药物筛选，个性化治疗Science.2014 Jul 11;345(6193):216-20.59Giuliano et al
23、.Breast Cancer Research 2014,16:44061治疗后不同时间点检测病人血清中CTC数目，结果显示治疗的整个过程中CTC可以作为预后评估的重要指标J Clin Oncol 26:3213-3221.治疗过程中CTC的动态变化也可以作为预后评估的重要指标J Clin Oncol 26:3213-3221.Potential of single CTCs analysis to evaluate tumour heterogeneity and disease evolution.Nat.Rev.Clin.Oncol.21 Jan,2014CTCs监测疗效监测疗效CTCs
24、 clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events.Although rare in the circulation compared with single CTCs,CTCs clusters have 23-to 50-fold increased metastatic potential.Cell.2014 Aug 28;158(5):1110-22CTCs clusters与肿瘤转移与肿瘤转移 The Presence of CTCs C
25、lusters in Patients with Breast and Prostate Cancer Correlates with Poor Prognosis.Plakoglobin Is Required for CTCs Cluster Formation and Contributes to Breast Cancer MetastasisCell.2014 Aug 28;158(5):1110-22Nature Reviews Clinical Oncology 10,472-484.ctDNActDNACTCCTCNon-invasiveEasy to store/suitab
26、le for retrospective studiesAnalysis of DNAClinical assays availableEnumeration onlyAnalysis of miRNAAnalysis of RNA+/-Analysis of protein68SubjectAdvantagesLimitationsCTCs Visualization of intact cells for morphological identification of a malignant phenotype Relevance for the metastatic process an
27、d disease progression Allow functional in vitro/in vivo assays(e.g.,xenotransplantation of CTCs into immunodeficient mice)Opportunity for molecular characterization at both cellular and sub-cellular level(e.g.,genomic analysis of single CTCs)Allows immuno-labeling based approaches Complementary with
28、 ctDNA:CTCs can survive current chemotherapy and might indicate failure of therapeutic interventions Potentially influence changes in treatment modalities Low abundance and fragility Require extremely sensitive and specific analytic methods False-negative(due to epithelial-to-mesenchymal transition)
29、and false-positive results Heterogeneity of the CTC populations(e.g.,detection of CTCs with tumor-initiating capacity)Multiplicity of technologies used for CTC isolationCell-free ctDNAMore sensitive for detection of disease burdenComplementary with CTCs for detection of minimal residual disease afte
30、r surgery or therapy with curative intent Might predict acquired drug resistance Potentially influence changes in treatment modalities False-negative and false-positive results(e.g.,no specific isolation of tumor DNA unless the detection of tumor-specific mutations;or mutations of tumorassociated ge
31、nes in normal tissue of aging patients and in frequent benign diseases)No functional assays Lack of standardization of preanalytical conditions(e.g.,dilution and contamination of ctDNA with normal DNA from dying blood cells after blood collection)Genome Med.2013 Aug 23;5(8):73.These small membrane v
32、esicles carry signals to distant parts of the body,where they can impact multiple dimensions of cellular life.Clotilde Thry TheScientist July 1,2011 Zhang and William“Exosomes and Cancer:A Newly Described Pathway of Immune Suppression”Clinical Cancer Research 2011Camussi et al.“Exosome/microvesicle-
33、mediated epigenetic reprogramming of cells”J.Am.Cancer Research 2011三、外泌体71Exosomes are small vesicles that are released by almost every cell type to the extracellular environment.Contrary to other types of extracellular vesicles,exosomes have endocytic origin and are formed as intraluminal vesicles
34、(ILVs)by inward budding of the limiting membrane of late endosomes or multivesicular bodies(MVBs)J Cell Biol.2013 Feb 18;200(4):373-83.Diagram depicting a typical cancer-cell derived exosome-S-S-S-S-TetraspaninsCD9,CD81,CD63Adhesion MoleculesIntegrins1,2,4ICAM-1ComplementRegulatorsCD59,CD55,CD46Chol
35、esterolPSMHC MoleculesMHC Class-IMICA,MICB,ULBP-1,etcTumourAssociatedAntigensMUC-1,Her-2/neuMesothelinGrowth FactorsTGF1hsp70miRNAmRNAhsp70,hsc70,hsp90 etcActinGAPDHCeramidemedicine.cf.ac.ukJ Cell Biol.2013 Feb 18;200(4):373-83.Exosomes research workflow:from sample to insightSample collection&Analy
36、te enrichmentSample IsolationAmplificationqPCRData Analysis&InterpretationmRNAmiRNAlncRNAexoRNeasy Serum/Plasma KitRTPreAMP cDNA KitRT2 PCRSystemFree data analysis tool Ingenuity Pathway AnalysisexoRNeasy Serum/Plasma KitmiScript P r e A M P PCR KitmiScript PCR SystemFree data analysis tool Ingenuit
37、y Pathway AnalysisexoRNeasy Serum/Plasma KitRT lncRNA PreAMP PCR KitRT2 lncRNAPCR SystemFree data analysis tool75Vesicle typesExosomesEctosomes,or shedding microvesiclesApoptotic bodiesOriginEndolysosomal pathway;intraluminal budding into late endosome and fusion of multivesicular body with cell mem
38、braneCell membrane;outward buddingCell membrane;outward blebbing of apoptotic cell membrane Size(nm)30100501,0005002,000Microscopic appearanceaRound-shaped,cup-shapedRound-shaped,irregular-shapedand electron-denseHeterogeneousProtein markersTetraspanins(CD63,CD9,CD81),alix and TSg101Integrins,select
39、ins,and CD40 ligandHistonesContentsCytosolic and membrane proteins including receptors and MHC molecules;mRNA,miRNA;lipid raftsCytosolic and membrane proteins,including receptors;mRNA,miRNANuclear fragments,cell organelles76Body fluid derived exosomes as a novel template for clinical diagnostics外泌体可
40、以从多种体液当中被分离出来，比如：乳汁、唾液、血清、血浆、恶性胸水以及尿液。人类唾液、尿液以及羊水中包含的外泌体人类唾液、尿液以及羊水中包含的外泌体When four esRNA samples were subjected to RT-PCR analysis,both specific CD24 and GAPDH sequences could be amplified(Figure 3A).Journal of Translational Medicine,2010,9(1):1-9Front Oncol.2014 May 27;4:127.将人类乳腺癌外泌体与正常细胞混合注入到小鼠体
41、内后可以引起肿瘤79外泌体将外源蛋白质、RNA导入肿瘤细胞，促进细胞存活及增殖80外泌体内蛋白含量与宫颈癌分级相关Gynecologic Oncology 110(2008)1321外泌体内特定miRNA含量与宫颈癌分级相关Gynecologic Oncology 110(2008)1321外泌体促进原位肿瘤发生转移 影响血管新生 重塑间质细胞 重塑细胞间质Cancer Res;71(11)June 1,2011外泌体促进肿瘤肝转移小龛的形成Nat Cell Biol.2015 Jun;17(6):816-26.Nat Cell Biol.2015 Jun;17(6):816-26.外泌体促进
42、肿瘤肝转移小龛形成的示意图Traffic.2011 Dec;12(12):1659-68.Traffic.2011 Dec;12(12):1659-68.Exosome-based Cyber-war Between Cancer and the Immune SystemMunich et al.OncoImmunology Oct 2012Yu et al.Journal of Immunology Dec 2007Tumor exosomesIL-6 and Stat3 BlockingDC differentiationABCMarleau et al.J.Translational Medicine 2012去除肿瘤外泌体改善肿瘤治疗效果去除肿瘤外泌体改善肿瘤治疗效果FedExosomes:Engineering Therapeutic Exosomes that Truly DeliverMarcus and Leonard,Parmaceuticals(2013)
