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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
• 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
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
What are the problems with histologic classification of glioma?
Coons SW et al. (1997) Cancer; van den Bent, MJ. (2010) Acta Neuro
52% concordance rates
What are the problems with histologic classification of glioma?
• 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
• 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
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
• 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
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
• 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
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.
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