1、液体活检在肠癌精准医疗中的应用,张波北京大学医学部病理学系,Global and Spanish Identifying codes: 2015-06-POLN-9XPMA8; Date of Approval: 02/07/15; 2015-06-POLN-9XPMLD; Date of Approval: 02/07/15Prescribing information is available on request,结直肠癌的发生率和死亡率,http:/globocan.iarc.fr/Pages/fact_sheets_population.aspx,卡培他滨单药,美国,依立替康 (+5
2、-FU+叶酸),依立替康 (+5-FU+亚叶酸),奥沙利铂(+5-FU+亚叶酸),卡培他滨联合治疗,BEV + 氟嘧啶为基础的CT,BEV + 氟嘧啶为基础的CT,BEV + CT,西妥昔单抗 +化疗(EGFR/KRAS 野生型),帕尼单抗 + FOLFOX(EGFR/KRAS 野生型),欧盟,奥沙利铂(+5-FU + 亚叶酸),瑞戈非尼三 / 四线,阿柏西普 (二线),西妥昔单抗 + FOLFIRI(KRAS野生型),BEV: 贝伐单抗; CT: 化疗; EGFR: 表皮生长因子受体FU: 氟尿嘧啶; KRAS: 克尔斯滕大鼠肉瘤病毒癌基因同源物,西妥昔单抗 +化疗(EGFR/RAS 野生型
3、),2014,无分子标记物,KRAS标记物,RAS标记物,mCRC治疗方案的更新-从KRAS到RAS,分子标记物的发展史,Metastatic colorectal cancer patient triage to anti-EGFR treatment in clinical practice,结直肠癌分子靶向治疗相关检测,瑞格菲尼,西妥昔/帕尼单抗,Aspirin,结直肠癌分子检测概略,IHC: MMR,Lynch syndrome,预防：LS；治疗：5-FU；预后：MSI；,靶向治疗；预后,K-RAS突变,KRAS mutations in various cancer types,KR
4、AS recurrent mutations in cancers,KRAS突变在原发及转移结肠癌灶,K-RAS基因突变检测,Cetuximab (西妥昔) and panitumumab (帕尼) are monoclonal antibodies that bind to the extracellular domain of EGFR, thereby inhibiting downstream signaling and resulting in decreased cell proliferation and migration.K-RAS for predicting respon
5、se to anti-EGFR antibodies, cetuximabor panitumumab. codons 12 of the K-RAS gene almost never benefited from treatment with these antibodies. However, 1520% of patients with wild-type K-RAS show an objective response with antibody alone and 3540%, when treated with cetuximab and irinotecan.G13D may
6、be an exception. administration of cetuximab to patients with this mutation was associated with a significantly better outcome than that seen in patients with other types of K-RAS mutations.,Anti-epidermal growth factor receptor (EGFR) treatment of Metastatic colorectal cancer (mCRC) patients,瑞格菲尼,西
7、妥昔/帕尼单抗,KRAS基因突变检测的意义,KRAS基因突变与anti-EGFR靶向治疗：G13D例外：需进一步证实；However, codon 12 and 13 mutations may differ in terms of their clinical impact. Indeed, evidence derived retrospectively in a small cohort (n=32) of chemotherapy-refractory mCRC patients suggests that patients with tumors harboring G13D mut
8、ations (the third most frequent KRAS mutation in CRC) may benefit from anti-EGFR antibody therapy.KRAS基因突变的预后意义；,KRAS检测的有关问题,质控：与敏感性定量： a very low frequency of KRAS mutations could not impair anti-EGFR therapies activity.肿瘤异质性：KRAS突变克隆；,Clinical impact of minor mutant subpopulations KRAS突变定量化,a very
9、 low frequency of KRAS mutations could not impair anti-EGFR therapies activity.,结直肠癌治疗与Extended RAS检测,KRAS (exon 2 codons 12/13) be a negative predictive biomarker for EGFR-directed monoclonal antibodiesamong patients with colorectal cancer.Extended RAS analysis including additional RASmutations (KR
10、AS exons 3/4 and NRAS exon 1/2/3/4). Extended RAS analysis should be considered before initiating anti-EGFR therapy to patients of metastatic CRC.KRAS exons 2/3/4 and NRAS exon 1/2/3/4,In addition to testing for mutations in KRAS exon 2 (codons 12and 13) as recommended previously, before treatment w
11、ith anti-EGFR antibody therapy, patients with mCRC should have their tumortested for mutations in: KRAS exons 3 (codons 59 and 61) and 4 (codons 117 and146) NRAS exons 2 (codons 12 and 13), 3 (codons 59 and 61), and4 (codons 117 and 146).,Extended RAS analysisExtended RAS: KRAS (beyond exon 2) and N
12、RAS,KRAS:Exon 2 codon (12 and 13), exon 3 (codon 61)and exon 4 (codons 117 and 146); NRAS:exon 2 (codons 12 and 13), exon 3 (codon 61), and exon 4 (codons 117 and 146);,Ras family,Ras mutation and activation,PRIME study, primary end points of (PFS and OS) efficacy results according to RAS mutation s
13、tatus,PRIME study,FIRE 3 study,FIRE 3 study,KRAS检测的拓展,KRAS基因拷贝数(KRAS gene copy number, GCN) ：An increase in KRAS gene copy number (GCN) has been associated with a more active mutation-like phenotype. KRAS GCN is a small subset (2%) of wild-type tumors (0.67%) and mutually exclusive with KRAS mutatio
14、ns. high CGN of wild-type KRAS may not respond to cetuximab administration, and may also be acquired during treatment with EGFR inhibitors.KRAS基因12,13密码外突变：61,146;KRAS同源基因突变：Neuroblastoma-RAS (NRAS) status: NRAS mutation rate in CRC is 35% and most mutations occur in codon 61, rather than codon 12 o
15、r 13. mutations in NRAS and KRAS are mutually exclusive. NRAS mutations have a significantly lower response rate than wild type.,Dynamic monitoring and drugs using,Circulating tumor markers,RAS检测: 一线治疗决策的重要因素,1. Van Cutsem E, et al. Ann Oncol 2014;25 (suppl 3): iii1iii92. NCCN clinical practice guid
16、elines; Colon Cancer, Version 2.2015. Available at www.nccn.org/professionals/physician_gls/pdf/colon.pdf. (accessed April 2015)3. Erbitux SmPC June/2014; 4. Vectibix SmPC February/2015,2014/2015指南推荐,Cetuximab and panitumumab are approved in patients with RAS wt mCRC.3,4 Cetuximab and panitumumab ar
17、e not indicated for the treatment of patients with mCRC whose tumors have RAS mutations or for whom RAS tumor status is unknown3,4,肠癌原发灶与转移灶：基因突变不一致,原发灶,转移灶,N=107对,53%,51%,21%,21%,13%,6%,6%,12%,56%,50%,21%,19%,10%,5%,5%,9%,Kopetz S, et al. ASCO 2014 (Abstract no. 3509); adapted from updated informat
18、ion presented at meeting:http:/meetinglibrary.asco.org/content/94598?media=sl (accessed June 30 2015),既往治疗过的CRC原发灶与转移灶不一致,原发灶和转移灶切除之间的任何化疗都导致:2.7倍高的不一致率(95% CI 1.36.0, p=0.005),90,80,70,60,50,40,30,20,10,0,0,1,2+,14%,31%,30%,化疗线数,突变配对-所有检测基因,突变数,Kopetz S, et al. ASCO 2014 (Abstract no. 3509); adapte
19、d from updated information presented at meeting:http:/meetinglibrary.asco.org/content/94598?media=sl (accessed June 30 2015),CRC肿瘤基因异质性,Sottoriva A, et al. Nat Genet 2015;47:209216,15例肠癌样本的349个腺体高度肿瘤内基因异质性（ITH）,KRAS检测的方法,As a general rule, a mutation frequency of 40% and a cluster of three mutation
20、types (p.G12D, p.G12V and p.G13D) in primary tumors and metastases can be considered benchmarks for routine KRAS analyses.KRAS mutational status by direct sequencing;High-resolution melting analysis;TheraScreen kit;KRAS mutational status by cobas;Strip Assay;Pyrosequencing;Next-generation sequencing
21、;,CTC,Adapted from Fleischhacker M, Schmidt B. Nat Med 2008;14:914915,肿瘤特异性变化（如突变）,肿瘤,肿瘤细胞释放DNA,循环肿瘤DNA,正常DNA,CTC,血管,生物标志物检测创新技术: 液体活检,*The liquid biopsy RAS IVD is awaiting a CE mark and, therefore, it is not currently being marketed,检测方法：BEAMing，qtPCR，cold PCR，ARMS,Digital PCR （数字PCR）,PCR扩增前对样品进行微
22、滴化处理突变型和野生型特异性的探针绝对定量灵敏度0.1%,二代测序仪(部分),Human genome = 3 Gigabases (Gb),生物信息学分析数据库比对,1、所有类型突变及比例2、拷贝数变化3、外来基因组4、全基因组变化,ASCO 2014默克雪兰诺与Sysmex Inostics签订合作协议,默克-Sysmex：液体活检技术*,WCGC 2015首次公布一致性数据: Scott R, et al. WCGC 2015 (Abstract No. P-273),ECC 2015增加一致性数据: Jones FS, et al. ECC 2015 (Abstract No. 201
23、2),中国：液体活检* 科研用试剂盒已上市，BEAMing平台待搭建,2015年2月第一个RAS液体活检中心启动,*Launch of a CE-marked IVD for RAS using liquid biopsies by Sysmex Inostics, in collaboration with Merck KGaA (Darmstadt, Germany), is expected in 2015 (RUO kit already available),Schematic of BEAMing,Photograph of a typical microemulsion,Dens
24、ity plots of flow-cytometric data obtained from BEAMing,Density plots of BEAMing with genomic DNA or RT-PCR products as template,Detection and validation of variants present in a minor fraction of the DNA population,配对的血和组织 标本来自31例III/IV期CRC患者,*cfDNA血检测使用OncoBEAM 33-mutation RAS panel (Sysmex Inosti
25、cs)使用诊断时获得的原发灶或转移灶FFPE肿瘤标本标准检测患者的RAS突变，同时用液体活检方法测血标本也是突变标准检测患者的RAS野生，同时用液体活检方法测血标本也是野生,液体活检*对比标准组织检测：RAS检测高一致率,1. Scott R, et al. WCGC 2015 (Abstract No. P-273),*Launch of a CE-marked IVD for RAS using liquid biopsies by Sysmex Inostics, in collaboration with Merck KGaA (Darmstadt, Germany), is expe
26、cted in 2015 (RUO kit already available),ECC 2015荟萃分析: 液体活检*和组织检测mCRC的RAS突变具有高一致率,Jones FS, et al. ECC 2015 (Abstract No. 2012),纳入的两项研究使用OncoBEAM Expanded RAS panel来检测血浆RAS突变，Sanger或焦磷酸测序检测原发灶或转移灶FFPE标本中的RAS突变 组织标本与血标本配对比较mCRC病例: 46例新诊断患者和22例进展/复发患者,RAS突变: 血 (55%) vs 组织标本(57%),*Launch of a CE-marked
27、 IVD for RAS using liquid biopsies by Sysmex Inostics, in collaboration with Merck KGaA (Darmstadt, Germany), is expected in 2015 (RUO kit already available),其他KRAS血检测研究：特异性高，敏感性差别较大,Li Y et al. Expert Rev Anticancer Ther. 2015 Jun;15(6):715-25,敏感性高的方法多采用BEAMing或定量PCR,最初西妥昔单抗治疗有效，后进展,诊断获得性耐药用BEAMing
28、法定量分析血里KRAS(Q61H) 突变DNA,液体活检未来的潜在用途*,Misale S, et al. Nature 2012;486:532536,Detection of circulating KRAS mutant DNA in a single patient with acquired resistance to cetuximab therapy; threshold percentage of mutation unknown;BEAM: beads, emulsification, amplification, and magnetics,*The liquid biop
29、sy RAS IVD is awaiting a CE mark and, therefore, it is not currently being marketedPD, progressive disease,液体活检发现mCRC患者抗EGFR治疗获得性耐药机制,1. Bettegowda C, et al. Sci Transl Med 2014;6:224ra24;2. Siravegna G, et al. Nat Med 2015;21:795801;,mCRC患者循环肿瘤DNA中发现抗EGFR治疗获得性耐药突变1,mCRC患者血检测发现抗EGFR耐药相关基因改变2,液体活检测出的
30、mCRC耐药突变*,*KRAS only,*Launch of a CE-marked IVD for RAS using liquid biopsies by Sysmex Inostics, in collaboration with Merck KGaA (Darmstadt, Germany), is expected in 2015 (RUO kit already available)ddPCR, droplet digital PCR; NGS, next-generation sequencing; PCR, polymerase chain reaction,1. Diaz
31、L, et al. Nature 2012;486:537540; 2. Misale S, et al. Nature 2012;486:532536; 3. Morelli M, et al. Ann Oncol 2015;26:731736; 4. Misale S, et al. Sci Transl Med 2014;6:224ra26; 5. Siravegna G et al. Nat Med 2015;21:795801,液体活检*: 快速和最小创伤的检测,1. Diehl F, et al. Proc Natl Acad Sci USA 2005;102:1636816373
32、;2. Diehl F, et al. Nat Med 2008;14:985990,*Launch of a CE-marked IVD for RAS using liquid biopsies by Sysmex Inostics, in collaboration with Merck KGaA (Darmstadt, Germany), is expected in 2015 (RUO kit already available),KRAS突变等位基因与抗EGFR治疗的关系,肿瘤负荷,CEA,KRAS p.Q61L,突变等位基因(%),肿瘤负荷(基线%)或CEA(ng/ml101),
33、基线,1st CT扫描,1线PD,Folfoxiri+Panit,Panit,Folfiri,节律化疗,2010,12,24,2011,02,21CT扫描:PR,2011,06,23,最后抗EGFR治疗,2011,07,01,2011,10,06,2011,11,07,肿瘤负荷,CEA,KRAS p.Q61L,突变等位基因(%),肿瘤负荷(基线%)或CEA(ng/ml101),基线,1st CT扫描,1线PD,Folfoxiri+Cetux,Cetux,Folfiri+Bev,Bev,2011,12,15,2012,04,12CT扫描:CR无可预测疾病,2013,01,24最后抗EGFR治疗,
34、2013,08,22,2014,06,19CT扫描:PD,Folfox+Bev,肿瘤负荷,CEA,KRAS p.Q61L,突变等位基因(%),肿瘤负荷(基线%)或CEA(ng/ml101),基线,1st CT扫描,1线PD,手术,手术,Folfoxiri+Cetux,Folfoxiri+Cetux,FolFOX+Bev,Bev,2012,04,19,2012,06,08CT扫描:PR,2012,08,162012,10,11,手术：原发病灶和肝转移灶,2013,06,18,2013,08,21,2014,07,03,FolFOX+Bev,2013,06,13最后抗EGFR治疗,2013,10,
35、22,手术：肝转移灶,肿瘤负荷,CEA,KRAS p.Q61L,突变等位基因(%),肿瘤负荷(基线%)或CEA(ng/ml101),基线,1st CT扫描,1线PD,手术,Folfoxiri+Panit,FolFOX+Bev,Bev,2011,04,04,2011,06,15CT扫描:PR,手术：结肠转移灶,2012,06,28,2012,10,05,2014,04,23,2012,03,07最后抗EGFR治疗,2013,09,17,2012,12射频治疗,疾病进展期患者接受抗EGFR治疗时，循环DNA出现KRAS突变等位基因，停止治疗后KRAS突变等位基因衰减,*CEA:癌胚抗原;Panit
36、:帕尼单抗;Cetux:西妥昔单抗;Bev:贝伐单抗;CR:完全缓解;PR:部分缓解;PD:疾病进展,AOUP-CRC04,AOUP-CRC01,AOUP-CRC03,AOUP-CRC06,Siravegna G, et al. Nat Med. 2015 Jul;21(7):795-801.,是否能持续监测，避免突变，找到最可能从西妥昔单抗再给药中获益的患者?*13,1. Diaz LA, et al. Nature 2012;486;537540 2. Misale S, et al. Nature 2012;486:5325363. Figure adapted from Vilar E
37、, Tabernero J. Nature 2012;486:4824834. Erbitux SmPC June/2014,不同的治疗,西妥昔单抗,治疗时间,RAS mutant ctDNA levels,初始RAS野生型mCRC患者., 可能在西妥昔单抗治疗过程中发生RAS突变,换方案治疗后RAS突变可能检测不到,患者对西妥昔单抗再治疗敏感？,FIRE-4研究: II期随机对照西妥昔单抗再治疗(德国)1,Available at 1. http:/www.aio-portal.de/index.php/studien.html;,预计完成时间: 2022年1月主要终点: 西妥昔单抗三线治疗
38、OS前瞻性研究敏感和耐药标记物,液体活检微创检测标本易获取避免肿瘤异质性影响动态监测部分平台敏感性较低临床疗效相关性数据缺乏平台或检测成本高,组织检测金标准与临床疗效相关性被证实成本低，易开展有创检测标本可能丢失肿瘤异质性获得性突变,组织检测 vs. 液体活检,RAS检测意义：转化为患者最佳治疗决策,液体活检* 提供及时准确的RAS检测结果，转化为患者最佳治疗决策9,10,目前,未来,1. Van Cutsem E, et al. J Clin Oncol 2015;33:692700; 2. Erbitux SmPC June 2014; 3. Heinemann V, et al. Lan
39、cet Oncol 2014;15:10651075; 4. Stintzing S, et al. ESMO 2014 (Abstract No. LBA11), updated information presented at the meeting: https:/ (accessed June 30 2015); 5. Lenz H-J, et al. ESMO 2014 (Abstract No. 501O); updated information presented at the meeting: https:/ (accessed June 30 2015); 6. Van C
40、utsem E, et al. Ann Oncol 2014;25(suppl 3):iii1iii9; 7. NCCN Colon Cancer Guidelines Version 2.2015; 8. Schwartzberg LS et al. J Clin Oncol 2014;32:22402247; 9. Diehl, F et al. Proc Natl Acad Sci 2005;102:1636816373; 10. Diehl F, et al. Nat Med 2008;14:985990,Cetuximab is approved in patients with RAS wt mCRC.3 Cetuximab is not indicated for the treatment of patients with mCRC whose tumors have RAS mutations or for whom RAS tumor status is unknown3*The liquid biopsy RAS IVD is awaiting a CE mark and, therefore, it is not currently being marketed,
