基因组学在癌症精准诊疗的应用 Hai Yan, MD, Ph.D Distinguished Professor Duke 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 dismal Wen 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
Well differentiated Oligodendroglioma Anaplastic Oligodendroglioma Glioma precursor Oligoastrocytoma Anaplastic Oligoastrocytoma Diffuse Astrocytoma Anaplastic Astrocytoma SECONDARY GBM WHO GRADE Louis DN et al. (2007) IARC PRIMARY GBM de novo 90% Glioblastoma II III IV 10%
What are the problems with histologic classification of glioma? It is challenging: o Heterogeneity o Overlapping features Oligodendroglioma Oligoastrocytoma oligo like Astrocytoma astro like
What are the problems with histologic classification of glioma? 52% concordance rates Coons SW et al. (1997) Cancer; van den Bent, MJ. (2010) Acta Neuro
What are the problems with histologic classification of glioma? It is inaccurate: o Primary vs. secondary GBM Primary GBM de novo 62 years old Clinical History: <3 months Secondary GBM progressive 45 years old Clinical history: 4 5 years
Molecular Classification of Glioma Need for new, more objective molecular markers: Novel therapeutic targets & personalized therapy Define Molecular Subclasses More accurate diagnostic & prognostic information Better understanding of gliomagenesis
Molecular Classification of Glioma
IDH1 and IDH2 mutations in gliomas Yan et al., NEJM, 2009 Yan et al., NEJM, 2009
From Genome to Therapy IDH1and IDH2 mutations Structure of mutant IDH1 Yan et al., NEJM, 2009 Morris et al., MCP, 2010
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 881 IDH1/2 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 Distance from ATG 150 100 50 TERT CCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGG GGGAGGGCCCAGGGGCCGGGTCGGGGGAGGCC C250T C228T de novo Ets binding site de novo Ets binding site CCCTTCCGGGTCCCCGGCCCAGCCCCTTCCGG GGGAAGGCCCAGGGGCCGGGTCGGGGAAGGCC Wild type Mutant Allele Ets Binding site: TTCCGG TERT expression Telomerase activation Primary GBM (IV) Oligodendroglioma (II III) Adapted from Huang et al. (2013) Science; Horn et al. (2013) Science;
TERT/IDH classification of diffuse glioma Simpler genomic classifiers using hotspot mutations >80% of diffuse gliomas Killela PJ and Reitman ZJ et al. (2013) PNAS; Killela PJ et al. (2014) Oncotarget.
Prognostic relevance: TERT/IDH classification of glioma Oligodendroglioma GBM Astrocytoma Oligoastrocytoma TERT mut/idh mut TERT mut/idh wt TERT wt /IDH mut TERT wt /IDH wt Survival probability based on histologic diagnosis Survival probability based on TERT/IDH status
Problem with mutation detection Current approach: DNA sequencing lacks sensitivity Example biopsy Limit of detection: o 20% mutant allele fraction o 40% tumor cellularity 30% tumor 70% normal tissue DNA extraction DNA Sequencing Analysis of TERT promoter mutations Result: TERT promoter wildtype False negative
Validation of TERT promoter/idh WT cases 43 cases previously identified as TERT/IDH WT by Sanger sequencing Already enriched for >70% tumor cellularity Tested using qpcr approach TERT promoter IDH1/2 TERT promoter IDH1/2 Diffuse and Anaplastic Astrocytoma (Grade II III), n=114 Glioblastoma (Grade IV), n=207 19 %75 % 73% 11%
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 Histology A WT C228T GBM B WT C228T GBM C WT C228T GBM D WT C228T Anaplastic Astrocytoma E WT C228T GBM F WT C228T GBM G WT C228T GBM Case ID IDH1 Sanger IDH1 qpcr Histology H WT R132H Astrocytoma K 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 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 ; Charles Swanton et. al, NEJM, 2016
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
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 ) 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 10 个基因的重排分析 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 活检穿刺, 肺癌组织 88 panel 检测,EGFR L858 外周血 ctdna panel 未检出突变 检测术前脑脊液 ctdna panel : 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 ctdna panel :EGFR L858R 0.718%/ T790M 0.61% 发病 : 头痛 特罗凯 特罗凯 手术 2014.11 2014.12 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 无 ; 肺部肿瘤缩小 MRI 肺CT 脑15.7 脑部肿瘤 胸椎出现肿瘤 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 脑部肿瘤进展, 其他部位肿瘤消失
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 这能找到那些最有可能从 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 (Immunerelated 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 血清中肿瘤蛋白标志物检测 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 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% D.T. Le, B. Vogelstein et. al, NEJM, 2015
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.
CanSelect TM 88 Panel 含有 MMR 基因和 MSI 指标 76 基因的突变分析 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 14 个基因重排 ALK BCR ETV1 ETV6 KLL ROS1 PDGFRA BCL2 EGFR FTV4 EWSR1 RARA TMPRSS2 RET 13 个基因的拷贝数变化 5 个微卫星不稳定性指标 ALK EGFR ERBB3 FGFR2 KIT MYC PDGFRA ERBB2 FGFR1 FGFR3 MET MYCN RET BAT-25 BAT-26 NR-21 NR-24 MONO-27
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.