液體活檢的研究進(jìn)展與應(yīng)用（PCR培訓(xùn)班）

1、2018-5-14山 東 省 腫 瘤 防 治 研 究 院 Shandong Cancer Hospital山東大學(xué)附屬山東省腫瘤醫(yī)院 Affiliated to Shandong University1. 概述2. 循環(huán)腫瘤DNA（ctDNA）3. 循環(huán)腫瘤細(xì)胞（CTC）4. 外泌體（Exosome）宋現(xiàn)讓 研究員2018年5月14日2015液體活檢一個(gè)相對(duì)于組織活檢的概念，是指以體 液為檢材獲得腫瘤生物信息的過程。10 Breakthrough Technologies可用作液體活檢的檢材：血液、尿液、胸水、腹水、唾液、腦脊液等。液體活檢檢什么？循環(huán)腫瘤DNA，循環(huán)腫瘤細(xì)胞（CTC），外泌體

2、( Exosome）液體活檢201510 Breakthrough Technologies1. 不受組織取材異質(zhì)性的影響N Engl J Med. 2012 Mar 8;366(10):883-9212018-5-142. 可實(shí)時(shí)反映體內(nèi)腫瘤細(xì)胞的生物信息：腫瘤發(fā)展過程中和腫瘤治療過程中，腫瘤細(xì)胞的生物信息發(fā)生改變，手術(shù)或活檢標(biāo)本無(wú)法反映這種 改變3. 取材方便，多或無(wú)限次 取材，無(wú)創(chuàng)或微創(chuàng)肺腺癌患者治療過程中血漿DNA L858R EGFR突變檢測(cè)A.術(shù)后肺內(nèi)轉(zhuǎn)移綜合治療，肝 臟 出 現(xiàn) 轉(zhuǎn) 移 灶 后 ， 血 漿DNA檢出EGFR L858R突變B . 化 療 前 血 漿 D N A 檢

3、 出L858R EGFR突變檢測(cè)，化療組織活檢：取材困難，有創(chuàng)，無(wú)法多次取材或即時(shí)取材。組織活檢信息不能反映當(dāng)下腫瘤生物信息。液體活檢：無(wú)創(chuàng)或微創(chuàng)，可根據(jù)需求多次或無(wú)限次取材。Journal of Thoracic Oncology,2012,7(9):1369-1381緩解后，血漿 DNA L858REGFR突變消失1. 不能確定其來(lái)源；2. 不能反映腫瘤的組織結(jié)構(gòu)、腫瘤細(xì)胞與間質(zhì)細(xì)胞的位置關(guān)系、相互作用；3. 混有非腫瘤來(lái)源的cfDNA，無(wú)法確定ctDNA在其中的比例，其中突變DNA可能占的比例非常小，需要更靈敏的檢測(cè)技術(shù)來(lái)自腫瘤細(xì)胞：1. 腫瘤細(xì)胞的壞死或凋亡循環(huán)游離核酸(circula

4、ting free nucleic acids) 是一種存在于動(dòng)植物和人的體液中的細(xì)胞外游離狀態(tài)核酸。目前已經(jīng)在血漿、血清、尿液、前列腺液、支氣管灌洗液、唾液、腦脊液、胃液、膽汁、淋巴液、腹腔液及糞便中檢測(cè)到了循環(huán)游離核酸。已發(fā)現(xiàn)的游離循環(huán)核酸包括 循環(huán)游離DNA和循環(huán)游離RNA。2. 循環(huán)血或微轉(zhuǎn)移灶中腫瘤細(xì)胞溶解3. 腫瘤細(xì)胞分泌DNA進(jìn)入血液循環(huán)來(lái)自非腫瘤細(xì)胞：1. 機(jī)體正常細(xì)胞的死亡和凋亡2. 腫瘤侵襲致周圍細(xì)胞、組織變性而釋放 DNA入血3. 微生物來(lái)源DNA腫瘤患者血中存在DNA 酶抑制劑，抑制血漿DNA 降解，從而使DNA在血中更容易富集。22018-5-14含量較低：約占總cf

5、DNA的 1-90%，含量與腫瘤類型、分級(jí)及是1. 作為一個(gè)廣譜腫瘤標(biāo)記物： cfDNA的含量及完整性否轉(zhuǎn)移等多種因素相關(guān)。2. 作為腫瘤遺傳/表觀遺傳信息的載體： 突變、缺失、重排、多態(tài)性等遺傳改變；甲基化、微衛(wèi)星不穩(wěn)定性等表觀遺傳改變；基因拷貝數(shù)變異 等片段較?。涸?00300bp之間。包含整個(gè)腫瘤基因組信息： 突變、缺失、重排、多態(tài)性等遺傳改變；甲基化、微衛(wèi)星不穩(wěn)定性等表觀遺傳改變；基因拷貝數(shù)變異 等。3. 特異性的腫瘤標(biāo)記物：具有遺傳或表觀遺傳特征 ctDNA的檢測(cè)血液采集和加工: 血漿優(yōu)于血清，盡快分離后低溫保存；最常用的是采用擴(kuò)增某個(gè)基因（如 -ACTIN，ALU, LINE1）的

6、方式，用來(lái)自正常細(xì)胞的 genome DNA 作為標(biāo)準(zhǔn)品，基于擴(kuò)增基因與總DNA在樣品和標(biāo)準(zhǔn)品中比例關(guān)系是一致的這一理論，計(jì)算cfDNA的濃度（ng/ml）或基因組拷貝數(shù)。cfDNA提取：無(wú)論采用哪種方法，第一步都是提取血漿 ctDNA，因此所用提取方法和試劑的不同就會(huì)造成巨大差別；標(biāo)準(zhǔn)品：gDNA 片段與cfDNA片段大小不一致；以高度重復(fù)序列或多拷貝基因?yàn)闄z測(cè)對(duì)象，理論上可提高檢測(cè)靈敏度。但存在高度重復(fù)序列或多拷貝基因在個(gè)體間拷貝數(shù)存在差異的弊端DNA染料，熒光直接定量技術(shù)：自體液中提取 cfDNA后，DNA結(jié)合熒光染料染色，然后直接用熒光光度計(jì)測(cè)定。Realtime PCR的敏感性DNA

7、的完整性(Integrity)：是指循環(huán)DNA中長(zhǎng)DNA片段與短DNA片段的比值；是有別于非腫瘤人群是腫瘤患者血液 ctDNA的另一個(gè)顯著特征。ll通過特殊的技術(shù)工藝處理，可以直接用血漿或血清進(jìn)行定量 PCR反應(yīng)，不影響PCR反應(yīng)，擴(kuò)增效率與提純DNA相比一致，減少了DNA提取步驟造成實(shí)驗(yàn)誤差。以高度重復(fù)序列LINE1為測(cè)定靶基因?yàn)闇y(cè)定對(duì)象不僅提高檢測(cè)靈敏度，而且用于 DII計(jì)算，重復(fù)性好。DNA的完整性指數(shù)（DII）檢測(cè)示意圖Cancer Res. J2003;63:3966-396832018-5-141. ASP/ARMSReal time or end-point PCR: Mutan

8、t allele-specific PCR,ARMS, ARMS-Scorpion ； PNA-PCR, Cold PCR, PNA-ARMS；QPCR+HRM利用3-末端引物錯(cuò)配可以顯著降低 PCR擴(kuò)增效率的特點(diǎn)，設(shè)計(jì)3-末端與突變位點(diǎn)完全匹配的引物，突變的模板得到選擇性擴(kuò)增，而未突變的DNA因?yàn)橐锊黄ヅ鋽U(kuò)增很少，達(dá)到富集突變的目的，因此又被稱為擴(kuò)增阻滯突變富集系統(tǒng)（ amplification refractorymutation system, ARMS ）。Digital PCR: BEAMing，Digital droplet PCRSequencing: Sangers sequ

9、ence, Pyrosequencing,Next Generation Sequencing: 全基因組測(cè)序、全外顯子測(cè)擴(kuò)增后可以通過電泳分析或 Realtime PCR直接獲得結(jié)果。目前有三種常用的ARMS+Realtime PCR方法，一種是蝎形MGB探針，DS公司（目前屬Q(mào)iagen）；另一種是蘇州為真的 T-ARMS，第三種是廈門艾德的AD。序、靶向重測(cè)序、全基因甲基化測(cè)序等該方法也可用于等位基因多態(tài)性分析。2. TaqMan SNP Genotyping Assays3. Realtime PCR+HRMHigh Resolution Melt Curve With SingleP

10、oint MutationHRM Analysis Data Difference Plot . Samples curves are subtracted from a referencecurve. Green=Wild type, Red=homozygous, Blue=heterozygousRaw data of HRMcurve42018-5-144. Cold-PCRPrinciple of Cold PCRSanger sequencingpyrosequencing5. PNA-mediated PCRPeptide nucleic acids(PNA) 是一種以核酸為骨架

11、的核酸肽， PNA探針能與單鏈DNA或RNA雜交，由于沒有電荷排斥，其親和力優(yōu)于DNA探針。與野生型DNA負(fù)鏈完全匹配的PNA探針阻止擴(kuò)增。MALDI-TOF genotyping technologiesTaqMan genotyping technologiesCOLD-PCR improves mutation detection in downstream assays .Principle of PNA-mediated PCRSensitivity of PNA-mediated PCR6. PyrosequencingConcentration of mutationPNACla

12、mp KRAS MutationConventional sequencingStepsequencing primer is hybridized toserves as template, and incubated with the enzymes, DNA polymerase, ATP1100%Aa single-stranded PCR amplicon thatasulfurylase, luciferase, and apyrase as well as the substrates, adenosine 5phosphosulfate (APS), and luciferin

13、.Step2The first deoxribonucleotide triphosphate (dNTP) is added to the reaction.DNA polymerase catalyzes the incorporation of the deoxyribo-nucleotidetriphosphate into the DNA strand, if it is complementary to the base in thetemplate strand. Each incorporation event is accompanied by release of20%10

14、%pyrophosphate (PPi) innucleotidea quantity equimolar to the amount of incorporatedStep3ATP sulfurylase converts PPi to ATP in the presence of adenosine 5phosphosulfate (APS). This ATP drives the luciferase-mediated conversion ofluciferin to oxyluciferin that generates visible light in amounts that

15、areproportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected byseen as peak in the raw data output ( Pyrogram). The height of each peak(light signal) is proportional to the number of nucleotides incorporated.StepApyrase,a charge coupled device (CCD) camer

16、a anda41%0%a nucleotide-degrading enzyme, continuously degradesunincorporated nucleotides and ATP. When degradation is complete, anothernucleotide is added.StepAddition of dNTPs is performed sequentially. It should be noted thatdeoxyadenosine alfa-thio triphosphate (dATPS) is used as substitute for5

17、athe natural deoxyadenosine triphosphate (dATP) since it is efficiently used bythe DNA polymerase, but not recognized by the luciferase. As the processcontinues, the complementary DNA strand is built up and the nucleotidesequence is determined from the signal peaks in the Pyrogram trace.52018-5-14Fe

18、atures of PyrosequencingApplication of PyrosequencingSequence analysisMutation detectionKnown positionsUnknown positions within hotspotsSequence identificationMicrobial typingCloned DNA resequencingmtDNAl As easy as PCR, as reliable as Sangers sequencinglIt is less time-consuming and less costly tha

19、n realtime PCRlQuantitative mutation analysisPoint mutationsTransgeneticsExpression profilingOligo IDInsertions/deletionsMultiple mutationsMultiplexinglFor unknown mutation and is mutation tolerantMicrosatelliteQuantificationAllele frequency assessmentSNP frequencyGenetic variationSNPs and DIPsDi-,

20、tri-, and tetra allelicMultipleTri/tetra allelic SNP frequencyIndel frequencyMultiplexingCpG methylationHaplotypeGene copy numberLoss of heterozygosityPolyploid genomesExpression analysesOut-of-phaseAllele-specific PCR7. NGS高通量測(cè)序技術(shù)不僅可以進(jìn)行大規(guī)模基因組測(cè)序，還可用于基因表達(dá)分析、非編碼小分子RNA的鑒定、轉(zhuǎn)錄因子靶基因的篩選和 DNA甲基化等。高通量測(cè)序技術(shù)有三大

21、優(yōu)點(diǎn)是傳統(tǒng) Sanger測(cè)序法所不具備的：第一，它利用芯片進(jìn)行測(cè)序，可以在數(shù)百萬(wàn)個(gè)點(diǎn)上同時(shí)閱讀測(cè)序；第二，高通量測(cè)序技術(shù)有定量功能，樣品中DNA被測(cè)序的次數(shù)反映了樣品中這種 DNA的豐度；第三、利用傳統(tǒng)Sanger測(cè)序法完成的人類基因組計(jì)劃總計(jì)耗資 27億美元，而現(xiàn)在利用高通量測(cè)序技術(shù)進(jìn)行人類基因組測(cè)序，測(cè)序成本只需 1千美金。局限性是檢測(cè)靈敏度和測(cè)序深度相關(guān)。一般來(lái)說(shuō)， NGS在腫瘤體細(xì)胞突變檢測(cè)時(shí)，檢測(cè)靈敏度為 10%；已知的與腫瘤相關(guān)驅(qū)動(dòng)基因數(shù)量有限，疾病表型和基因型的關(guān)系還有賴于生物信息的解讀，目前 NGS應(yīng)用于腫瘤細(xì)胞突變檢測(cè)的標(biāo)準(zhǔn)化和質(zhì)量控制尚未形成共識(shí)。NGS又稱大規(guī)模平行測(cè)序（

22、 MPS），包含多種可以一次性產(chǎn)生大量數(shù)字化基因序列的測(cè)序技術(shù)，是繼 Sanger測(cè)序的革命性進(jìn)步，采用平行測(cè)序的理念，能夠同時(shí)對(duì)上百萬(wàn)甚至數(shù)十億個(gè) DNA分子進(jìn)行測(cè)序，實(shí)現(xiàn)了大規(guī)模、高通量測(cè)序的目標(biāo)。不同廠家的產(chǎn)品測(cè)序原理不同，主要分為邊合成邊測(cè)序（ Sequencing bysynthesis，SBS）、基于“DNA簇”和“可逆性末端終結(jié)（Reversible Terminator）大規(guī)模平行測(cè)序、 4色熒光標(biāo)記寡核苷酸的連續(xù)連接反應(yīng)測(cè)序和半導(dǎo)體芯片測(cè)序。8. 數(shù)字PCR（ddPCR）將反應(yīng)體系分成20000200000個(gè)油包水液滴，分別進(jìn)行反應(yīng)，用不同探針分別檢測(cè)野生型和突變型基因的擴(kuò)增

23、情況。62018-5-14TechniqueSensitivityOptimal ApplicationSanger sequencing20%Tumor tissueMassively Parallel Sequencing1 - 2%Tumor tissueQuantitative PCR1-5%Tumor tissueNext Generation Sequencing0.1-1%Tissue or ctDNActDNA, rare variants in tumortissueBEAMing / Digital PCR 0.01%BEAMing技術(shù)：結(jié)合了數(shù)字PCR以及流式技術(shù)。其方法

24、是每一類 DNA分子都會(huì)專一的與磁性珠相連接，然后 DNA分子之間的差異可以通過流式細(xì)胞儀檢測(cè)熒光標(biāo)記來(lái)做出評(píng)估。這種方法是基于小珠 (Bead)、乳濁液(Emulsion)、擴(kuò)增(Amplification)、磁性(Magnetic)，這四個(gè)主要組分來(lái)構(gòu)建的，所以被稱作為BEAMing。常用基因檢測(cè)方法的基因序列覆蓋度與靈敏度NGS檢測(cè)血液樣本與組織樣本 EGFR突變檢測(cè)的一致性多組織檢測(cè)血液檢測(cè)合計(jì)陽(yáng)性（+）陰性（-）EGFR 19del 陽(yáng)性（+）EGFR 19del 陰性（-）EGFR L858R 陽(yáng)性（+）EGFR L858R 陰性（-）合計(jì)14401436覆蓋度3201321335

25、673733100ARMS數(shù)字PCR(ddPCR)6個(gè)NGS檢測(cè)為陰性，金標(biāo)準(zhǔn)檢測(cè)為陽(yáng)性的樣本，為 II期-IIIa期患者RT-PCR少組織檢出陽(yáng)性，血液也檢出陽(yáng)性，兩者一致率達(dá) 94%。其中6個(gè)NGS檢測(cè)陰性的樣本為II期-IIa期患者低高靈敏度72018-5-14血液T790M耐藥突變的“假陽(yáng)性”IV 克服腫瘤組織異質(zhì)性-典型臨床案例PlasmaTumor (ARMS)(BEAMing)T790M+111T790M-1818例患者T790M檢測(cè)血液(+) 組織(-)，經(jīng)過其他方法學(xué)再次檢測(cè)血液標(biāo)本（ddPCR或者cobas V2）T790M+T790M-474018例患者中，14例患者再次

26、血液檢測(cè)也是T790M陽(yáng)性發(fā)現(xiàn)大多數(shù)患者檢測(cè)結(jié)果不一致出現(xiàn)的原因在于腫瘤異質(zhì)性，而非BEAMING血檢為假陽(yáng)性除T790M耐藥突變之外，血漿ddPCR檢測(cè)EGFR，KRAS均沒有假陽(yáng)性，陽(yáng)性預(yù)測(cè)值為100%T790M耐藥突變的檢測(cè)結(jié)果尤其受腫瘤異質(zhì)性的影響，組織陰性是否是真陰性，血液陽(yáng)性是否是假陽(yáng)性 ?Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in AdvancedLung Cancer JAMA Oncol. 2016 Apr 7.Geoffre

27、y R.et al. J Clin Oncol. 2016, 34 (28), 3375-3382.1. 作為腫瘤標(biāo)記物的應(yīng)用2. 作為腫瘤遺傳/表觀遺傳信息的應(yīng)用Bettegowda et al., Sci Transl Med 2014Comparison between the three studiedgroups as regards cfDNA plasma level.Association Between cfDNA and VariableHistopathological Parameters82018-5-14Analysis of Circulating Tumor D

28、NA to Monitor Metastatic Breast CancerSarah-Jane Dawson, N Engl J Med. 2013 Mar 28;368(13):1199-209更好的檢測(cè)靈敏度更好的檢測(cè)靈敏度Comparison of Circulating Tumor DNA, CA15-3, and CirculatingTumor Cells as Blood-Based BiomarkersComparison of Circulating Tumor DNA, CA15-3, and CirculatingTumor Cells as Blood-Based B

29、iomarkersN Engl J Med. 2013 Mar 28;368(13):1199-209.N Engl J Med. 2013 Mar 28;368(13):1199-209.和腫瘤負(fù)荷有更好的相關(guān)性最早對(duì)治療做出反應(yīng)更準(zhǔn)確的預(yù)后效果cfDNACTCCA15-392018-5-14Plasma DNA integrity as abiomarker for primary andmetastatic breast cancer andpotential marker for earlydiagnosisDharanija Madhavan, et al.Breast Cancer

30、 Res Treat (2014) 146:163174ctNDA含量和完整性與PFS的關(guān)系ctNDA含量和完整性與OS的關(guān)系KaplanMeier curves forb overall survival (OS) in MBCpatients using cfDI orlog2cfDNA concentration asthe predictor variableKaplanMeier curves fora progression-free (PFS inMBC patients using cfDI orlog2cfDNA concentration asthe predictor v

31、ariableBreast Cancer Res Treat (2014) 146:163174Breast Cancer Res Treat (2014) 146:163174FASTACT-2： 一個(gè)多中心、隨機(jī)、安慰劑對(duì)照、雙盲的吉西他濱加鉑類序貫厄洛替尼或安慰劑作為 IIIB/IV期 NSCLC 患者一線治療方案的III 期臨床研究。患者1:1隨機(jī)分組， 接受6個(gè)周期的吉西他濱 (1,250 mg/m2intravenously on days 1 and 8 of a 4-week cycle) 加鉑類(carboplatin 5 AUC, or cisplatin 75 mg/m2

32、 intravenously onday 1 of a 4-week cycle), 然后在每周期的1528天給予厄洛替尼(150 mg/day orally; erlotinib arm) 或安慰劑 (placebo arm)。102018-5-14研究開始7天內(nèi)（Baseline），第三個(gè)周期開始的第一天（ C3）和疾病進(jìn)展三個(gè)時(shí)間點(diǎn)取血分離血清或血漿GC+E組 PFS 13.1個(gè)月，GC+P組 6.0 個(gè)月 HR, 0.22;95% confidence interval (CI),0.140.33, P0.0001), 兩組OS 分別是 29.3 個(gè)月 和 18.8個(gè)月 (HR, 0.

33、54; 95% CI,GC+E組 PFS 6.2個(gè)月，GC+P組 6.1 個(gè)月 HR,0.83;95%CI,0.651.04, P =0.1076), 兩組 OS 分別是15.3 個(gè)月 和 13.6 個(gè)月（HR, 0.94; 95% CI,在基點(diǎn)血液和組織樣本的突變檢測(cè)的結(jié)果的一致率是88% (209/238)。 敏感性和特異性分別是 75% (72/96)和96% (137/142，陽(yáng)性預(yù)測(cè)值 94% (72/77) ，陰性預(yù)測(cè)值85% (137/161)。0.350.83, P=0.0044).0.721.22, P = 0.6449）.僅有cfDNA的67例僅有cfDNA的166例GC+

34、E組（31例） PFS 12.8個(gè)月， GC+P組（36例）6.0 個(gè)月 HR,0.25; 95% CI,0.140.47, P 0.0001), 兩組 OS 分別是 29.3 個(gè)月 和21.4個(gè)月 (HR, 0.59; 95% CI,0.301.15, P= 0.1202).GC+E組PFS 5.5個(gè)月，GC+P組5.9個(gè)月 HR, 0.85;95% CI, 0.601.19, P=0.3398), 兩組 OS 分別是13.0 個(gè)月 和 13.6個(gè)月(HR,1.07; 95% CI,0.731.56, P =0.7387).112018-5-14在全部治療病人（E+P）在厄洛替尼治療病人（

35、E）這表明EGFR突變ctDNA的存在與否可作為肺癌療效預(yù)測(cè)的標(biāo)志物Nature. 2013 May 2;497(7447):108-12.IPASS：吉非替尼First-SIGNAL：吉非替尼WJTOG 3405 ：吉非替尼NEJGSG002：吉非替尼OPTIMAL：埃羅替尼獲得性耐藥EURTAC：埃羅替尼LUX-Lung 3：阿法替尼LUX-Lung 6：阿法替尼1. Langer CJ.J Clin Oncol. 2013;20;31(27):3303-3306.2. Yu HA, et al. Clin Cancer Res. 2013;19(8):2240-2247.Clinica

36、Chimica Acta 411 (2010) 16111624Cerebrospinal fluid-derived circulating tumour DNA better represents thegenomic alterations of brain tumours than plasma制定標(biāo)準(zhǔn)檢測(cè)循環(huán)腫瘤DNA的甲基化通過PCR檢測(cè)基因甲基化通過10個(gè)基因驗(yàn)證分析循環(huán)腫瘤DNA的甲基化程度用來(lái)鑒定血液中的循環(huán)腫瘤DNA-檢測(cè)早期轉(zhuǎn)移乳腺癌Cancer Res 2014;74:2160-2170.Nat Commun. 2015 Nov 10;6:8839.122018-5-1

37、4Cerebrospinal fluid-derived circulating tumour DNA better represents thegenomic alterations of brain tumours than plasmaDetection of somatic mutations and HPV in the saliva and plasma ofpatients with head and neck squamous cell carcinomasSchematic showing the shedding of tumor DN A fromhead and nec

38、k cancers into the saliva or plasma . Tumorsfrom various anatomic locations shed DNA fragmentscontaining tumor-specific mutations and HPV DNA intothe saliva or the circulation. The detect ability of tumorDNA in the saliva varied with anatomic location of thetumor, with the highest sensitivity for or

39、al cavity cancers.The detect ability in plasma varied much less in regard tothe tumors anatomic location.Nat Commun. 2015 Nov 10;6:8839.Sci Transl Med. 2015 Jun 24;7(293):293ra104.Correlation of EGFR Mutation Status between Plasma and Saliva1. ctDNA作為一個(gè)廣譜腫瘤標(biāo)記物：含量和完整性2. ctDNA作為液體活檢檢材：遺傳信息，表觀遺傳信息Am J

40、Respir Crit Care Med. 2014 Nov 15;190(10):1117-26.腫瘤細(xì)胞突破基底膜入血形成循環(huán)腫瘤細(xì)胞是腫瘤進(jìn)展的重要標(biāo)志，是腫瘤形成和轉(zhuǎn)移的早期事件。PLoS One. 2014 Apr 3;9(4):e93173.132018-5-14研究背景與超聲、影像、MRI和穿刺相比，外周血CTC檢測(cè)簡(jiǎn)便、無(wú)創(chuàng)，能直觀、實(shí)時(shí)監(jiān)測(cè)腫瘤進(jìn)展；2004年FDA批準(zhǔn)CellSearch CTC檢測(cè)系統(tǒng)，乳腺、前列腺癌、結(jié)直腸癌 檢測(cè)；2012年8月SFDA 批準(zhǔn)CellSearch 系統(tǒng)用于轉(zhuǎn)移性乳腺癌的檢測(cè)。Current concept of cellular and

41、 molecular characteristics of circulating tumor cells (CTCs)Nat Rev Cancer. 2014 Sep;14(9):623-31.Figure1Seeding of Established Tumors by CTCs (A)Adiagram of contralateral seeding experiment. Unlabeled andGFP/luciferase-expressing breast cancer cells were injected into contralateral No.a “donor tumo

42、r,”.2mammary glands asa“recipient tumor” andFigure 2 Preferential Tumor Seeding by Metastatic Cell Progenies (A) Comparison of seeding activity of highly and poorlymetastatic cells. Contralateral seeding experiments were performed with the GFP/luciferase-expressing parental cancer celllines, or w.Ki

43、m MY, et al. Cell. 2009, 24;139(7):1315-26.Kim MY, et al. Cell. 2009, 24;139(7):1315-26.142018-5-14Figure3 Tumor Attraction and Infiltration Functions (A) Unlabeled MDA231 cells were injected into a mammary gland No. 2. When tumorsbecame palpable, LacZ/GFP/luciferase-expressing MDA231-LM2 cells were

44、 introduced into the circulation by intracardiac inj.Cheuk T. et al. Cell, 2009, 139 (7):1226-8Kim MY, et al. Cell. 2009, 24;139(7):1315-26.Kim MY, et al. Cell. 2009, 24;139(7):1315-26.CTC clusters have 23- to 50-foldincreased metastatic potential.Aceto N. et al. Cell. 2014, 158(5):1110-22.Aceto N.

45、et al. Cell. 2014, 158(5):1110-22.1. 在血流剪切力作用下、被破壞、死亡被吞噬細(xì)胞清除2. 被免疫系統(tǒng)殺傷、清除3. 形成轉(zhuǎn)移灶：穿出血管形成轉(zhuǎn)移灶 /栓塞微血管形成瘤栓進(jìn)而形成轉(zhuǎn)移灶4.成為靜止細(xì)胞在骨髓中潛伏下來(lái)Aceto N. et al. Cell. 2014, 158(5):1110-22.152018-5-141. 上皮狀態(tài)癌細(xì)胞（E）、間質(zhì)狀態(tài)癌細(xì)胞（ M），中間狀態(tài)、CSC-like 癌細(xì)胞共存2. 單個(gè)細(xì)胞（single cell）和細(xì)胞團(tuán)/簇(Clustering cell）或CTM共存3. 形態(tài)完整CTC、癌細(xì)胞裸核、癌細(xì)胞碎片共存4.

46、CTC包裹plateletsNat Rev Cancer. 2014 Sep;14(9):623-31.CTCs 檢測(cè)技術(shù)Nat Rev Cancer. 2014 Sep;14(9):623-31.Isolation of rare circulating tumour cells in cancerpatients by microchip technologyNature Reviews/Clinical Oncologies2014, 11:129-144Sunitha Nagrath, et al. Nature, 2007 Dec, 450: 1235162018-5-14Yu M.

47、 et al. Science. 2014, 345(6193):216-20.Nature Methods 2015, Aug 15Ex vivo culture of CTCs for individualized drug testing藥物篩選，個(gè)性化治療Yu M. et al. Science. 2014, 345(6193):216-20.Science. 2014 Jul 11;345(6193):216-20.Giuliano et al. Breast Cancer Research 2014, 16:440172018-5-14治療后不同時(shí)間點(diǎn)檢測(cè)病人血清中 CTC數(shù)目，結(jié)

48、果顯示治療的整個(gè)過程中 CTC可以作為預(yù)后評(píng)估的重要指標(biāo)。治療過程中CTC的動(dòng)態(tài)變化也可以作為預(yù)后評(píng)估的重要指標(biāo)J Clin Oncol 26:3213-3221.J Clin Oncol 26:3213-322CTCs clusters與腫瘤轉(zhuǎn)移CTCs監(jiān)測(cè)療效CTCs clusters arise from oligoclonal tumor cell groupings and not from intravascularaggregation events. Although rare in the circulation compared with single CTCs,CTCs

49、clusters have 23- to 50-fold increased metastatic potential.Potential of single CTCs analysis to evaluate tumour heterogeneity and disease evolution.Nat. Rev. Clin. Oncol. 21 Jan, 2014Cell. 2014 Aug 28;158(5):1110-22The Presence of CTCs Clusters in Patients with Breast and Prostate Cancer Correlates

50、 withPoor Prognosis. Plakoglobin Is Required for CTCs Cluster Formation and Contributes toBreast Cancer MetastasisCell. 2014 Aug 28;158(5):1110-22Nature Reviews Clinical Oncology 10, 472-484.182018-5-14SubjectAdvantagesLimitations Visualization of intact cells for morphological identificationof a ma

51、lignant phenotype Relevance for the metastatic process and diseaseprogression Low abundance and fragility Require extremely sensitive and specificanalytic methodsctDNACTC Allow functional in vitro/in vivo assays (e.g.,xenotransplantation of CTCs into immunodeficient mice) Opportunity for molecular c

52、haracterization at both cellularand sub-cellular level (e.g., genomic analysis of single CTCs) Allows immuno-labeling based approaches Complementary with ctDNA: CTCs can survive currentchemotherapy and might indicate failure of therapeuticinterventionsNon-invasive False-negative (due to epithelial-t

53、o-mesenchymal transition) and false-positiveresultsCTCsEasy to store / suitable for retrospectivestudies Heterogeneity of the CTC populations (e.g.,detection of CTCs with tumor-initiating capacity) Multiplicity of technologies used for CTCisolationAnalysis of DNAClinical assays availableAnalysis of

54、miRNAAnalysis of RNAEnumeration only Potentially influence changes in treatment modalities False-negative and false-positive results (e.g.,no specific isolation of tumor DNA unless thedetection of tumor-specific mutations; ormutations of tumorassociated genes in normal+/-More sensitive for detection

55、 of disease burdenComplementary with CTCs for detection of minimal residual tissue of aging patients and in frequent benignAnalysis of proteinCell-free disease after surgery or therapy with curative intent Might predict acquired drug resistance Potentially influence changes in treatment modalitiesdi

56、seases) No functional assays Lack of standardization of preanalyticalconditions (e.g., dilution and contamination ofctDNA with normal DNA from dying blood cellsafter blood collection)ctDNAGenome Med. 2013 Aug 23;5(8):73.Genome Med. 2013 Aug 23;5(8):73.細(xì)胞外囊泡(EVs)：u 細(xì)胞-細(xì)胞直接接觸u 分泌性分子MicrovesiclesExosom

57、esApoptotic bodiesu 外泌體（Exosome）是指細(xì)胞凋亡過程中，細(xì)胞萎縮、碎裂，形成的有膜包圍的含有核和細(xì)胞質(zhì)碎片的小體RaposoG , and Stoorvogel W . J Cell Biol 2013;200:373-383192018-5-14TetraspaninsCD9, CD81, CD63VesicletypesApoptoticbodiesExosomesMicrovesiclesMHC MoleculesMHC Class-IAdhesion MoleculesEndolysosomal pathway;intraluminal budding in

58、to lateendosome and fusion ofmultivesicular body with cellmembraneIntegrins 1, 2, 4Cell membrane;Cell membrane; outward outward blebbingActinmRNAMICA, MICB,ULBP-1, etcICAM-1Originbuddingof apoptotic cellmembraneGAPDHSize (nm)30100501,0005002,000MicroscopicRound-shaped, irregular-shapedand electron-d

59、ensemiRNARound-shaped, cup-shapedHeterogeneousappearanceahsp70, hsc70,hsp90 etcComplementRegulatorsTumourAssociatedAntigensProteinmarkersTetraspanins (CD63, CD9,CD81),alix and TSg101Integrins, selectins, andCD40 ligandHistonesCD59, CD55,CD46Cytosolic and membrane proteinsincluding receptors and MHCm

60、olecules; mRNA, miRNA; lipidraftsCytosolic andMUC-1, Her-2/neuMesothelinmembrane proteins,including receptors;mRNA, miRNANuclear fragments,cell organellesContentshsp70Growth FactorsTGF1Diagram depicting a typical cancer-cell derived exosomeMembrane-associated (triangles) andtransmembrane proteins (r