4.Hai Yan Oct 28 2016 Qingdao Thermo fisher...2015.8 2015.11 2015.12 2016.1 2016.3 2016.7.27 检查全身发现肺部原发肿瘤，无脑部肿瘤 15.3 脑部肿瘤消失；肺部肿

基因组学在癌症精准诊疗的应用

Hai Yan, MD, Ph.DDistinguished ProfessorDuke Medical Center

目录

• 癌症基因组学研究进展

• 癌瘤内异质性研究

• 癌症液态活检临床应用

• 癌症免疫治疗分子标记

目录





Glioma: a major health challenge

• Malignant glioma is an incurable disease and fatal

• 80% of all primary malignant brain tumors

• Clinical management is aggressive:• Surgical resection• Radiotherapy• Chemotherapy

• However prognosis remains dismalWen PY and Kesari S. (2008) NEJM; Louis DN, et al. (2007) WHO Classification of Tumors of the Central Nervous System

Brain Tumor Histogenesis

Daniel J. Brat MD, PhD

Louis DN et al. (2007) IARC

Well‐differentiatedOligodendroglioma

DiffuseAstrocytoma

OligoastrocytomaGlioma precursor

WHO GRADE II III IV

AnaplasticOligodendroglioma

AnaplasticAstrocytoma

AnaplasticOligoastrocytoma

GlioblastomaPRIMARYGBMde novo

SECONDARYGBM

90%10%

What are the problems with histologic classification of glioma?

• It is challenging:

o Heterogeneity

o Overlapping features

Oligodendroglioma

Astrocytoma

Oligoastrocytoma

“astro”‐like

“oligo”‐like


Coons SW et al. (1997) Cancer; van den Bent, MJ. (2010) Acta Neuro

52% concordance rates


• It is inaccurate:

o Primary vs. secondary

GBM• 62 years old• Clinical History:• <3 months

• 45 years old• Clinical history:

• 4‐5 years

Primary GBMde novo

Secondary GBMprogressive

Molecular Classification of Glioma

Need for new, more objective molecular markers:

DefineMolecular Subclasses

Novel therapeutic targets & personalized therapy

Better understanding of gliomagenesis

More accurate diagnostic & prognostic information

Molecular Classification of Glioma

IDH1 and IDH2 mutations in gliomas

Yan et al., NEJM, 2009

Yan et al., NEJM, 2009

From Genome to Therapy

Morris et al., MCP, 2010Yan et al., NEJM, 2009

Structure of mutant IDH1IDH1and IDH2 mutations

IDH mutation based therapies

IDH inhibitors clinical trials

AG‐221 IDH2 inhibitor Phase 1,2,3 AML

AG‐120 IDH1 inhibitor Phase 1

AML, Glioma, chondrosarcoma and cholangiocarcinoma

AG‐881IDH1/2 inhibitor (penetrate BBB) Phase 1

AML, Glioma, chondrosarcoma and cholangiocarcinoma

RESIST: Patients with IDH1 Positive Recurrent Grade II Glioma Enrolled in a Safety and Immunogenicity Study of Tumor‐Specific

TERT promoter mutations result in telomerase activation

‐50 ‐100 ‐150

CCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGG GGGAGGGCCCAGGGGCCGGGTCGGGGGAGGCC

C250T

de novo Ets binding site

Wild type

CCCTTCCGGGTCCCCGGCCCAGCCCCTTCCGG GGGAAGGCCCAGGGGCCGGGTCGGGGAAGGCC

C228T

Mutant Allele

de novo Ets binding site

TERT

TERT expression

Distance from ATG

Telomerase activation

Ets Binding site: TTCCGG

Adapted from Huang et al. (2013) Science; Horn et al. (2013) Science;

Primary GBM (IV)

Oligodendro‐glioma (II‐III)

TERT/IDH classification of diffuse glioma

Killela PJ and Reitman ZJ et al. (2013) PNAS; Killela PJ et al. (2014) Oncotarget.

• Simpler genomic classifiers using hotspot mutations >80% of diffuse gliomas

Prognostic relevance: TERT/IDH classification of glioma

Survival probability based on histologic diagnosis

Oligoastrocytoma

Oligodendroglioma

Astrocytoma

GBM

Survival probability based on TERT/IDH status

TERT wt /IDH wt

TERT mut/IDH mut

TERT wt /IDH mut

TERT mut/IDH wt

Problem with mutation detection

• Current approach: DNA sequencing lacks sensitivity

• Limit of detection:

o 20% mutant allele fraction

o 40% tumor cellularity30% tumor 70% normal tissue

Example biopsy

DNA extractionDNA Sequencing Analysis of TERT promoter mutations

Result: TERT promoter wildtypeFalse negative

Validation of TERT promoter/IDHWT cases

• 43 cases previously identified as TERT/IDH WT by Sanger sequencing

• Already enriched for >70% tumor cellularity

• Tested using qPCR approach

Glioblastoma (Grade IV), n=207TERT

promoter

IDH1/2

73%11%

Diffuse and Anaplastic Astrocytoma (Grade II‐III), n=114TERT

promoter

IDH1/2

19%75%

qPCR Validation results

• 16% of cases had low % (<5%) TERT promoter mutations.

• 4.6% of cases had low % IDH1 R132H mutations.

• Genotypes largely match up with expected histologies

• All of these were WT by sequencing • A normal, non‐enriched population is

likely to have many more false negatives

Case ID TERT Sanger TERT qPCR HistologyA WT C228T GBMB WT C228T GBMC WT C228T GBM

D WT C228T Anaplastic Astrocytoma

E WT C228T GBMF WT C228T GBMG WT C228T GBM

Case ID IDH1 Sanger IDH1 qPCR HistologyH WT R132H AstrocytomaK WT R132H Astrocytoma

World Health Organization IARC Blue Book Classification of CNS Tumors and will be used in clinics internationally

对NSCLC的认识由组织分型向基因分型发展

• 基因突变与药物敏感性直接相关

EGFR基因 Gly719X突变, 第19号外显子缺

失，Leu858Arg突变, Leu861Gln突变

• 基因突变与患者基本耐药性相关

EGFR第20号外显子插入

• 基因突变与患者获得性耐药相关

• EGFR Thr790Met突变, Asp761Tyr突变,

Leu747Ser突变, Thr854Ala突变

Pao and Girard, Lancet Oncol, 2011

2015年七月易瑞沙通过FDA审批

• 适应症及其应用易瑞沙是一种络氨酸激酶抑制剂，FDA批准其作为携带EGFR第19号外显子缺失或

第21号外显子L858R突变的，转移性非小细胞肺癌患者的一线治疗药物。 (1)

局限性：无EGFR第19号外显子缺失或第21号外显子L858R错义突变，但携带有

EGFR其它类型突变的患者使用易瑞沙的安全性和有效性尚不明确。 (1)

• 一组亚组分析显示：

带有EGFR基因突变的肿瘤患者，使用吉非替尼进行靶向治疗的患者的无进展生存

期（PFS）显著高于使用化疗的患者。 (HR 0.48, 95% CI 0.36 to 0.64, p<0.0001),

而不带有EGFR突变的肿瘤患者，使用化疗治疗的患者的PFS显著高于使用吉非替尼

进行靶向治疗的患者 (HR 2.85, 95% CI 2.05 to 3.98, p<0.0001).

目录





肺癌克隆进化及肿瘤内部异质性研究

Charles Swanton et. al, NEJM, 2016

肺腺癌进化轨迹模式图

早期trunk突变（EGFR激活突变、

TP53、BRAF、KRAS)驱动肿瘤发生，

为肿瘤早期主要突变类型；肿瘤进展过

程中逐渐出现多种branch突变的发生和

扩增，肿瘤内部形成多个subclone，互

相协同和拮抗，成为继发耐药突变（R）

或转移复发（M）事件的主要细胞群体；

治疗过程肿瘤内部异质性克隆更替

Alice T. Shaw et. al, NEJM, 2016

目录





肿瘤液态活检

液态活检监测复发和耐药

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 10 | AUGUST 2013 |

液态活检技术在NSCLC领域的探索性研究

2014年9月，易瑞沙（Iressa）血液ctDNA伴随诊断获欧盟批准

2015年2月，CFDA批准Iressa/ Tarceva中文说明书的变动：如果肿瘤标本不可评估，可使用从血液（血浆）标本中获得的循环肿瘤DNA

2016年6月，FDA批准首个基于EGFR基因突变“液态活检”方法——Roche cobas ® EGFR Mutation Test v2，用于检测非小细胞肺癌（NSCLC）患者EGFR外显子19缺失和外显子21的 L858R替代突变，以及T790M耐药突变

ctDNA作为一种新型的肿瘤标志物，通过检测其与肿瘤发生和靶向

药物相关的基因突变信息，实现伴随诊断、指导用药和疗效监控

ctDNA作为一种新型的肿瘤标志物，通过检测其与肿瘤发生和靶向

药物相关的基因突变信息，实现伴随诊断、指导用药和疗效监控

结直肠癌-3DPCR临床应用

2011年7月~2015年11月，230例手术切除的II

期CRC患者的1046份血浆样本进行ctDNA检测

与MRD、复发风险研究；

患者肿瘤组织进行15panel检测以明确每个患者

特异的ctDNA突变基因检测目标；术后4~10周

进行初次采血，后续2年每3个月进行间隔采血，

进行ctDNA突变和CEA蛋白水平检测

Tie J. Sci Trans Med. 2016 术后非辅助化疗患者ctDNA突变情况与复发频率相关性研究

肿瘤液态活检

ctDNA检测技术比较分析

技术检出限检测区域技术特点

Sanger >10% 全基因序列双脱氧链终止法

qPCR 1% 已知位点荧光素标记

ARMS PCR 0.1% 已知位点扩增阻滞突变

NGS 0.1-1% 全基因序列半导体测序

数字PCR 0.01% 或更低已知位点微滴反应

ctDNA Panel - 涵盖63个基因

58个基因的突变分析（*检测全部编码区）

10个基因的重排分析

ABL1 CTNNB1 GNA11 MAP2K1 RB1

AKT1 DNMT3A GNAQ MET° RET

ALK* EGFR* GNAS MLH1 SMAD4

APC ERBB2° HNF1A MPL SMARCB1

AR ERBB4*° HRAS* MYC SMO

ATM ESR1* IDH1 NPM1^ SRC

BRAF* EZH2 IDH2 NRAS* STK11*

CDH1 FBXW7 JAK2* PDGFRA* TERT

CDK4*° FGFR1° JAK3 PI3CA* TP53*

CDK6*° FGFR2 KDR PIK3R1 VHL

CDKN2A FGFR3 KIT* PTEN*

CSF1R FLT3 KRAS* PTPN11

ALK BRAF NTRK1 PDGFRB RET

BCR EGFR PDGFRA RARA ROS1

数字PCR的原理

3DPCR技术在NSCLC领域的探索性研究

针对脑转移癌的液态活检技术

案例：肺癌脑转移的液态活检

2014.11 2014.12

发病：头痛

2015.8 2015.11 2015.12 2016.1 2016.3 2016.7.27

检查全身发现肺部原发肿瘤，无脑部肿瘤

15.3 脑部肿瘤

消失；肺部肿瘤缩小

16.1.2脑部肿瘤重新出现。

脑部肿瘤明显缩小，其他部位肿瘤消失

手术

胸椎出现肿瘤

特罗凯特罗凯

脑部肿瘤进展，

其他部位肿瘤消失

MRI

肺

CT

脑

15.7脑部肿瘤

无；肺部肿瘤缩小

活检穿刺, 肺癌组织88 panel检测，EGFR L858

检测

术前脑脊液 ctDNApanel： EGFR L858R 16.49%/ T790M 5.67%; 术中脑部癌组织 88panel： EGFR L858R 26.3%/ T790M 10.7%术后脑脊液 ctDNApanel：EGFR L858R 0.718%/ T790M 0.61%

外周血ctDNApanel未检出突变

目录





FDA批准KEYTRUDA一线治疗非小细胞肺癌

在肿瘤表达高水平PD-L1的非小细胞肺癌患者身上，KEYTRUDA与常规化疗相比能够提高生存率

在新适应症下，KEYTRUDA现在能取代化疗，作为一线疗法治疗那些高表达PD-L1的转移性非小细胞肺癌患者

表明在非小细胞肺癌中检测PD-L1表达量的重要性。这能找到那些有可能从KEYTRUDA治疗中受益的患者

Multiple biomarkers predict response to PD-1

• Ligand expression on tumor (PD-L1/2)

• Immunogenic microenvironment (Immune-

related gene expression signature)

• Increased antigen presentation due to high DNA

mutation load (DNA mismatch repair deficiency,

DNA polymerase mutation, mutational load)

Mutational load as a predicative biomarker for Pembrolizumab

43

错配修复完整型的结直肠癌组：基本没有CEA水平下降者；

错配修复缺陷型结直肠癌组：70%（7/10）的患者在治疗后CEA水平大幅下降；

错配修复缺陷型的其他癌症组：75% (3/4) 的患者生物标志物(CEA、CA19-9或CA-125) 水平下降超过70%

血清中肿瘤蛋白标志物检测

D.T. Le, B. Vogelstein et. al, NEJM, 2015

肿瘤

标志

物水

平变

化（

%）

每条线代表一位患者对应标志物水平与自身在Pembrolizumab治疗前的基线进行对比

Correlation of mutational load calling by WES and targeted sequencing panels

• Comparable response predication for WES, FM and

Caris. Targeting sequencing panels can be used to

precisely infer total exonal non-synonymous

mutational burden.

CanSelectTM 88 Panel含有MMR基因和MSI指标

ABL1 CSF1R FANCF GNAS MLH1 PALB2 SMAD4

AKT1 CTNNB1 FANCG HNF1A MPL PDGFRA SMARCB1

ALK DDR2 FANCL HRAS MSH2 PDGFRB SMO

APC EGFR FBXW7 IDH1 MSH6 PIK3CA SRC

ATM ERBB2 FGFR1 IDH2 MTOR PMS2 STK11

BRAF ERBB4 FGFR2 JAK2 NF1 PTCH1 TERT

BRCA1 EZH2 FGFR3 JAK3 NF2 PTEN TP53

BRCA2 FANCA FLT3 KDR NOTCH1 PTPN11 TSC1

BRIP1 FANCC FOXL2 KIT NPM1 RB1 TSC2

CDKN2A FANCD2 GNA11 KRAS NRAS RET VHL

CDH1 FANCE GNAQ MET NTRK1 ROS1

ALK BCR ETV1 ETV6 KLL ROS1 PDGFRA

BCL2 EGFR FTV4 EWSR1 RARA TMPRSS2 RET

ALK EGFR ERBB3 FGFR2 KIT MYC PDGFRA

ERBB2 FGFR1 FGFR3 MET MYCN RET

BAT-25 BAT-26 NR-21 NR-24 MONO-275个微卫星不稳定性指标

76基因的突变分析

14个基因重排

13个基因的拷贝数变化

Conclusion

• Biomarker development is the key to cancer precise

diagnosis and guiding personalized treatment

• Digital technology, including NGS, 3DPCR, based biopsy will

be widely implemented in clinical practice.

Documents

4.Hai Yan Oct 28 2016 Qingdao Thermo fisher...2015.8 2015.11 2015.12 2016.1 2016.3 2016.7.27 检查全身发现肺 部原发肿瘤，无 脑部肿瘤 15.3 脑部肿瘤 消失；肺部肿

4.Hai Yan Oct 28 2016 Qingdao Thermo fisher...2015.8 2015.11 2015.12 2016.1 2016.3 2016.7.27 检查全身发现肺部原发肿瘤，无脑部肿瘤 15.3 脑部肿瘤消失；肺部肿