液體活檢的研究進(jìn)展與應(yīng)用課件 (2)

1、液體活檢研究進(jìn)展及應(yīng)用山東省腫瘤醫(yī)院/山東省放射腫瘤學(xué)重點(diǎn)實(shí)驗(yàn)室宋現(xiàn)讓 研究員2016.06.1712015 十大突破技術(shù)22015 十大突破技術(shù)10 Breakthrough Technologies2015液體活檢3血液/血清/血漿活檢循環(huán)腫瘤DNA（ ctDNA ）循環(huán)RNA（Circulating RNA）循環(huán)腫瘤細(xì)胞（ CTC ）外泌體（ Exosome）4一、循環(huán)游離核酸(circulating free nucleic acids)循環(huán)游離核酸(circulating 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、

2、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。5（一）腫瘤患者cfDNA的來源腫瘤細(xì)胞的壞死或凋亡循環(huán)血或微轉(zhuǎn)移灶中腫瘤細(xì)胞溶解腫瘤細(xì)胞分泌DNA進(jìn)入血液循環(huán)機(jī)體正常細(xì)胞的死亡和凋亡腫瘤侵襲致周圍細(xì)胞、組織變性而釋放DNA入血微生物來源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。來自腫瘤細(xì)胞：來自非腫瘤細(xì)胞：6（二）循環(huán)腫瘤DNA生物學(xué)性質(zhì)含量較低：約占總cfDNA的 1-90%，含量與腫瘤類型、分級(jí)及是否轉(zhuǎn)移等多種因素相關(guān)。片段較?。涸?/p>

3、100300bp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。7（三）從ctDNA檢什么？8作為一個(gè)廣譜腫瘤標(biāo)記物：cfDNA的含量及完整性作為腫瘤遺傳/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)最常用的是采用擴(kuò)增某個(gè)基因（如-ACTIN，ALU, LINE1）的方式，用來自正常細(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

4、的濃度（ng/ml）或基因組拷貝數(shù)。DNA染料，熒光直接定量技術(shù)（四）cfDNA/ctDNA的檢測(cè)方法1. cfDNA含量的檢測(cè)9cfDNA 含量檢測(cè)的關(guān)鍵點(diǎn)血液采集和加工: 血漿優(yōu)于血清，盡快分離后低溫保存；cfDNA提?。簾o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Realtime PCR的敏感性10解決cfDNA提取誤差：免DNA提取的定量PCR方法檢測(cè)ctDNA含量通過

5、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。112. ctDNA完整性的檢測(cè)DNA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。Cancer Res. J2003;63:3966-3968DNA的完整性指數(shù)（DII）檢測(cè)示意圖133.ctDNA遺傳/表觀遺傳信息的檢測(cè)Real time or end-poin

6、t PCR: Mutant allele-specific PCR, ARMS, ARMS-Scorpion； PNA-PCR, Cold PCR, PNA-ARMSNext Generation Sequencing: 全基因組測(cè)序、全外顯子測(cè)序、靶向重測(cè)序、全基因甲基化測(cè)序等Digital PCR: BEAMing，Digital droplet PCRChip-based Technique直接探針檢測(cè)，無需擴(kuò)增cfDNA檢測(cè)敏感性TechniqueSensitivityOptimal ApplicationSanger sequencing 20%Tumor tissueMassive

7、ly 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技術(shù)：結(jié)合了數(shù)字PCR以及流式技術(shù)。其方法是每一類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)記來做出評(píng)估。這種方法是基于小珠(Bead)、乳濁液(Emulsion)、擴(kuò)增(Am

8、plification)、磁性(Magnetic)，這四個(gè)主要組分來構(gòu)建的，所以被稱作為BEAMing。15（五）ctDNA液體活檢的優(yōu)勢(shì)1. 不受組織取材異質(zhì)性的影響，或能反映腫瘤組織異質(zhì)性N Engl J Med. 2012 Mar 8;366(10):883-92172. 可實(shí)時(shí)反映體內(nèi)腫瘤細(xì)胞的遺傳改變：腫瘤發(fā)展過程中和腫瘤治療過程中腫瘤細(xì)胞的遺傳信息發(fā)生改變，手術(shù)或活檢標(biāo)本無法反映這種改變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DNA檢出L858R EGFR突變檢測(cè)，

9、化療緩解后，血漿DNA L858R EGFR突變消失Journal of Thoracic Oncology,2012,7 (9):1369-1381183. 含量豐富，取材方便，多無限次取材，無創(chuàng)或微創(chuàng)ctDNA的含量足以進(jìn)行PCR擴(kuò)增和突變檢測(cè)，文獻(xiàn)報(bào)道癌癥患者血漿cfDNA的含量平均在100ng/ml左右。以此量推算，每毫升血漿中約有基因組的拷貝數(shù)為1.5104個(gè)（每個(gè)癌細(xì)胞約含DNA 6.6pg）；而循環(huán)腫瘤細(xì)胞的數(shù)量在010個(gè)左右/7.5ml全血。ctDNA液體活檢的劣勢(shì)：混有非腫瘤來源的cfDNA，無法確定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

10、術(shù)（六）循環(huán)腫瘤DNA臨床應(yīng)用作為腫瘤標(biāo)記物的應(yīng)用作為腫瘤遺傳/表觀遺傳信息載體的應(yīng)用19作為TM理論上最廣譜的腫瘤標(biāo)記物，與組織類型無關(guān)，與表達(dá)水平無關(guān)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

11、Monitor Metastatic 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更好的檢測(cè)靈敏度N Engl J Med. 2013 Mar 28;368(13):1199-209. Comparison of

12、 Circulating Tumor DNA, CA15-3, and Circulating Tumor Cells as Blood-Based Biomarkers更好的檢測(cè)靈敏度27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)28cfDNA CTC CA15-3更準(zhǔn)確的預(yù)后效果Plasma DNA integrity as a biomarker for primary and metastatic breast cancer and potential marker for early diagnosisDharanija Madhavan, et al.Breast Cancer R

13、es 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含量和完整性與PFS的關(guān)系KaplanMeier curves forb overall survival (OS) in MBCpatients using cfDI orlog2cfDNA con

14、centration as the predictor variableBreast Cancer Res Treat (2014) 146:163174ctNDA含量和完整性與OS的關(guān)系2. 作為遺傳/表觀遺傳信息載體指導(dǎo)靶向治療：選擇敏感個(gè)體；分析耐藥機(jī)制；評(píng)價(jià)治療效果；更改治療方案腫瘤分子診斷：早期診斷；轉(zhuǎn)移診斷，分類診斷33 FASTACT-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?；颊?:1隨機(jī)分組， 接受6個(gè)周期的吉西他濱 (1,250 mg/m2 intravenously

15、on days 1 and 8 of a 4-week cycle)加鉑類 (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天給予厄洛替尼(150 mg/day orally; erlotinib arm) 或安慰劑 (placebo arm)。34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3536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

16、是75% (72/96) 和96% (137/142，陽性預(yù)測(cè)值 94% (72/77) ，陰性預(yù)測(cè)值85% (137/161)。 組織和血液EGFR突變檢測(cè)結(jié)果比較3777例，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, 0.54; 95% CI, 0.350.83, P=0.0044). 137例，GC+E組 PFS 6.2個(gè)月， GC+P組 6.1 個(gè)月 HR, 0.83; 95%CI,0

17、.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, 0.721.22, P= 0.6449）. 同時(shí)有組織和血液EGFR突變檢測(cè)結(jié)果的病例38GC+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). 僅有cfDNA的67例GC+E組PFS 5.5個(gè)月， GC+P組5.9個(gè)月 HR

18、, 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). 僅有cfDNA的166例僅有血液EGFR突變檢測(cè)結(jié)果的病例39cfDNA EGFR 突變的動(dòng)態(tài)變化40在全部治療病人（E+P）在厄洛替尼治療病人（E）C3時(shí)ctDNA EGFR 突變狀態(tài)的變化與PFS和OS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4142Cerebrospinal fluid-derived circulating tumour DNA better

19、 represents the genomic alterations of brain tumours than plasmaNat Commun. 2015 Nov 10;6:8839.研究了GMB 和肺癌、乳腺癌腦轉(zhuǎn)移腦脊液中基因組的改變，與血漿進(jìn)行對(duì)比檢測(cè)方法：hybridization capture-based massively parallel targeted sequencing（基于雜交捕獲的大規(guī)模并行測(cè)序 ） and/or exome sequencing, droplet digital PCR (ddPCR)431. 腦脊液比血漿更好地代表CNS 腫瘤的基因改變44

20、2. 腦腫瘤患者腦脊液ctDNA的動(dòng)態(tài)變化反映腫瘤治療進(jìn)程45F3463. CSFs ctDNA的檢測(cè)有助于軟腦膜癌的診斷4.不同轉(zhuǎn)移部位的基因變異分析有助于追溯轉(zhuǎn)移克隆起源Nat Commun. 2015 Nov 10;6:8839.F1b49唾液ctDNA檢測(cè)Schematic 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

21、tumor-specific mutations and HPV DNA into the saliva or the circulation. The detect ability of tumor DNA in the saliva varied with anatomic location 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 loca

22、tion.Sci Transl Med. 2015 Jun 24;7(293):293ra104.唾液與血漿中ctDNA EGFR基因突變Correlation of EGFR Mutation Status between Plasma and Saliva Am J Respir Crit Care Med. 2014 Nov 15;190(10):1117-26. 二、循環(huán)腫瘤細(xì)胞（CTC ）1896 年 Ashworth 首次提出循環(huán)腫瘤細(xì)胞(circulating tumor cell, CTCs)的概念:指自發(fā)或因診療操作進(jìn)入外周血循環(huán)的腫瘤細(xì)胞。PLoS One. 2014 Ap

23、r 3;9(4):e93173. CTC 與 EMT（上皮細(xì)胞間質(zhì)化）處于EMT狀態(tài)的腫瘤易播散CTCCTC中間質(zhì)樣表型的細(xì)胞占大多數(shù)CTC間質(zhì)樣標(biāo)記物表達(dá)上調(diào)CTC中處于間質(zhì)樣表型的細(xì)胞易成轉(zhuǎn)移灶Cancer Cell. 2013 Mar 18;23(3):272-3. 53循環(huán)腫瘤細(xì)胞Current concept of cellular and molecular characteristics of circulating tumor cells (CTCs) Nat Rev Cancer. 2014 Sep;14(9):623-31.CTCs 富集技術(shù)Nat Rev Cancer.

24、2014 Sep;14(9):623-31.CTCs 檢測(cè)技術(shù)Nat Rev Cancer. 2014 Sep;14(9):623-31.體外培養(yǎng)循環(huán)腫瘤細(xì)胞 功能研究功能研究：成瘤能力、遷移情況等。Science. 2014 Jul 11;345(6193):216-20.57體外培養(yǎng)循環(huán)腫瘤細(xì)胞 藥物篩選藥物篩選，個(gè)性化治療Science. 2014 Jul 11;345(6193):216-20.CTC的檢測(cè)方法舉例 CellSearch系統(tǒng)59CTC數(shù)目與腫瘤轉(zhuǎn)移相關(guān)Giuliano et al. Breast Cancer Research 2014, 16:440CTC數(shù)目與腫瘤預(yù)

25、后相關(guān)61CTC數(shù)目與腫瘤進(jìn)展相關(guān)（1）治療后不同時(shí)間點(diǎn)檢測(cè)病人血清中CTC數(shù)目，結(jié)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)J Clin Oncol 26:3213-3221.CTC數(shù)目與腫瘤進(jìn)展相關(guān)（2）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.Potential of single CTCs analysis to evaluate tumour heterogeneity and disease evolution.Nat. Rev. Clin. Oncol. 21 Jan, 2014CTCs監(jiān)測(cè)療效CTCs

26、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與腫瘤轉(zhuǎn)移 The Presence of CTCs Clu

27、sters 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):1110-22CTCs and ctDNANature Reviews Clinical Oncology10,472-484.Comparison of ctDNA and CTCsctDNACT

28、CNon-invasiveEasy to store / suitable for retrospective studiesAnalysis of DNAClinical assays availableEnumeration onlyAnalysis of miRNAAnalysis of RNA+/-Analysis of protein68Summary of the detection of CTC vs. ctDNASubjectAdvantagesLimitationsCTCs Visualization of intact cells for morphological ide

29、ntification of a malignant phenotype Relevance for the metastatic process and 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 a

30、nalysis of single CTCs) Allows immuno-labeling based approaches Complementary with 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 sp

31、ecific analytic methods False-negative (due to epithelial-to-mesenchymal transition) 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

32、 of disease burdenComplementary with CTCs for detection of minimal residual disease after 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 tum

33、or DNA unless the detection of tumor-specific mutations; or mutations of tumorassociated genes 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

34、dying blood cells after blood collection)Differences Between CTC and ctDNA analysisGenome Med. 2013 Aug 23;5(8):73. These small membrane vesicles 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 W

35、illiam “Exosomes and Cancer: A Newly Described Pathway of Immune Suppression” Clinical Cancer Research 2011Camussi et al. “Exosome/microvesicle-mediated epigenetic reprogramming of cells” J. Am. Cancer Research 2011三、外泌體71（一）外泌體的產(chǎn)生Exosomes are small vesicles that are released by almost every cell ty

36、pe to the extracellular environment. Contrary to other types of extracellular vesicles, exosomes have endocytic origin and are formed as intraluminal vesicles (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. 1.

37、 結(jié)構(gòu)Diagram depicting a typical cancer-cell derived exosomemedicine.cf.ac.uk2. 成分J Cell Biol. 2013 Feb 18;200(4):373-83. 3. 產(chǎn)生Exosomes research workflow: from sample to insightSample collection& Analyte enrichmentSample IsolationAmplificationqPCRData Analysis &InterpretationmRNAmiRNAlncRNAexoRNeasy S

38、erum/Plasma KitRTPreAMP cDNA KitRT2 PCRSystemFree data analysis tool Ingenuity Pathway AnalysisexoRNeasy Serum/Plasma KitmiScript PreAMP PCR KitmiScript PCR SystemFree data analysis tool Ingenuity Pathway AnalysisexoRNeasy Serum/Plasma KitRT lncRNA PreAMP PCR KitRT2 lncRNAPCR SystemFree data analysi

39、s tool4. 研究方法755.體液中小囊泡的區(qū)別Vesicle typesExosomesEctosomes, or shedding microvesiclesApoptotic bodiesOriginEndolysosomal pathway; intraluminal budding into late endosome and fusion of multivesicular body with cell membraneCell membrane; outward buddingCell membrane; outward blebbing of apoptotic cell

40、membrane Size (nm)30100501,0005002,000Microscopic appearanceaRound-shaped, cup-shapedRound-shaped, irregular-shapedand electron-denseHeterogeneousProtein markersTetraspanins (CD63, CD9,CD81),alix and TSg101Integrins, selectins, and CD40 ligandHistonesContentsCytosolic and membrane proteins including receptors and MHC molecul