State of the Art in Molecular Testing and Current Diagnostic Challenges
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1 State of the Art in Molecular Testing and Current Diagnostic Challenges Conversations in Oncology in Shanghai, China Mike Zhu, MD/PhD Amoy Diagnostics Co., Ltd BI Symposium
2 Headline Update on molecular testing for target therapy Progress of blood-based molecular testing Emerging biomarker testing for immunotherapy 2
3 Pretreated Most Current Therapies in NSCLC Require Detailed Pathological Diagnosis Almost all recently introduced therapies require, or potentially require, a detailed pathological diagnosis First-line Overall Survival in NSCLC and Biomarkers 2004 Pemetrexed: NSQ 2014 Nintedanib + docetaxel: NSQ 2014 Ramucirumab + docetaxel 2015 Pembrolizumab: PD-L1 +ve 2015 Nivolumab: NSQ 2015 Nivolumab: SQ Median OS, months Erlotinib Docetaxel ~8 10 ~10 12 ~ ~12+ New biomarkers? Personalised strategies = Potential to improve outcomes 1994 Vinorelbine Median OS, months ~ Platinum-based doublet 2006 Bevacizumab + chemotherapy: NSQ ~ Pemetrexed + chemotherapy: NSQ 2001 Docetaxel + chemotherapy ~ Nab-paclitaxel + chemotherapy ~12+ Median OS >20 months with targeted therapies for patients with EGFR mutations Biomarkers in routine clinical use for adenocarcinomas 3
4 Actionable Molecular Targets of NSCLC Histologies Mutations in Adenocarcinoma 1 Chinese Adenocarcinoma 2 Mutations in Squamous Cell Carcinoma 1 1. Rosell R and Karachaliou N. Lancet. 2016;387: Zheng D Oncotarget. 2016;7:
5 NSCLC Variants and Targeted Therapies FDA-Approved Therapies Therapies Approved in Other Cancers Drugs in Development Adapted from Presentation of David Spigel at ASCO Tsao AS et al. J Thorac Oncol. 2016;11:
6 Technical Solution for Tissue-Based Tumor Mutation Testing Provided by Amoy Dx. 6 6
7 2016 EMQN Report: ARMS as the #1 Method for Tumor Mutation Testing ARMS ARMS Provided by Amoy Dx. 7
8 EGFRm/ALKf/ROS1f Testing 3-in-1 Giotrif 1-3 slides 4.5 hours 1.5 hours Chemo Fast TAT: <7 hours Info of 3 targets Quick decision Provided by Amoy Dx. 8
9 Headline Update on molecular testing for target therapy Progress of blood-based molecular testing Emerging biomarker testing for immunotherapy 9
10 Noninvasive Molecular Profiling with Liquid Biopsies Three major clinical applications: 1. Initial detection of actionable driver mutations 2. Monitoring response to therapy and predicting outcome 3. Identification of resistance mutations in patients failing targeted therapy Crowley et al. Nat Rev Clin Oncol 2013;10: Adapted from the presentation of Alice Shaw at ASCO
11 EGFRm in ctdna Can Predict Efficacy of EGFR-TKIs IFUM study (Gefitinib) 1 ENSURE study (Erlotinib) 2 AURA study (Osimertinib) 3 1. Douillard et al. J Thor Oncol 2014;9: ; 2. Wu, et al. Ann Oncol. 2015;26: ; 3. Oxnard et al. J Clin Oncol. 2016;34:
12 EGFRm in ctdna Can Predict Efficacy of EGFR-TKIs (continued) LUX-Lung 3 (Afatinib) LUX-Lung 6 (Afatinib) Wu et al. Br. J Cancer. 2017;116:
13 Clearance of Plasma EGFR Mutations Predicts Outcome to Osimertinib Probability of progression-free survival Patients with EGFR TKI resistance, T790M +vs at baseline Plasma samples collected at baseline and 6 weeks postosimertinib Plasma genotyping by BEAMing digital PCR Response as per plasma status at 6 weeks PFS based on clearance or detection of EGFR mutations in plasma samples taken 6 weeks post initiation of osimertinib Clearance of 6 weeks EGFR- (n=92) EGFR+ (n=51) 0.2 Detectable 6 weeks Thress KS et al. ASCO Poster Time from first dose (months) 13
14 Dynamic Monitor for Response and Progression 80% Clin PD Mol CR/PR Mol PD Zheng et al Scientific Report (modified, for reference only)
15 Studies for Resistance Mechanism Identification 1. Thress et al. Nat Med. 2015;21: Arulananda S et al. J Thorac Oncol. 2017;12: Li A et al. Clin Cancer Res. 2017;23: Presented by Shuhang Wang at ESMO
16 Characteristics of cfdna/ctdna: Highly & Regularly Fragmented Purified plasma DNA from 6 patients 1 Size distribution of plasma DNA from patients 2 1. Jahr et al. Can Res 2001:61: Heitzer et al. Clin Chem 2015;61:
17 Challenge in ctdna EGFRm Detection 80.00% 60.00% 40.00% E19-Dels L858R 80.00% N=93 (100%) 60.00% N= % (100%) 80.00% 60.00% 40.00% T790M N=129 (100%) 20.00% 20.00% 20.00% 0.00% % % 2.50% 2.00% 1.50% 1.00% 0.50% 0.00% Samples 2.50% 2.00% 1.50% 1.00% 0.50% 0.00% N=26(28%) N=25 (25%) N=53 (40%) Samples 2.00% 1.50% 1.00% 0.50% 0.00% Samples 17 AmoyDx internal data, for reference only.
18 ARMS Methods for ctdna EGFRm Detection ADx ARMS 1 Qiagen ARMS 2 Plasma Tumor Tissue Tumor Tissue Total Plasma EGFR + EGFR - EGFR + EGFR - Total EGFR EGFR EGFR EGFR Total Total Sensitivity: 67.5%, Specificity: 100% Sensitivity: 65.7%, Specificity: 99.8% Roche Cobas 3 EGFR TKI-sensitive mutations Sensitivity: 75%, Specificity: 96% CfDNA EGFR + EGFR - Total Tumor tissue EGFR Tumor tissue EGFR Total Liu et al. J Clin Path. 2013;66: ; 2. Douillard et al. Br J Cancer. 2014;110:55-62; 3. Mok et al. Clin Cancer Res 2015;21:
19 ddpcr for Detection of ctdna EGFR E19-Dels/L858R (Retrospective) Sensitivity:81.8%; Specificity:98.4%. Sensitivity:80%; Specificity:95.8%. 19 Zhu et al. J. Mol Dia 2015;17:
20 ddpcr for Detection of ctdna EGFR and KRAS Mutations in ansclc (Prospective) Sacher et al. JAMA Oncol 2016;2:
21 dpcr for Detection of ctdna EGFR T790M 1 Oxnard et al. J Clin Oncol 2016;34: Presented by Alice Shaw at ASCO
22 Super-ARMS for ctdna EGFRm Detection Super-ARMS 0.2% sensitivity Improved designing Optimized PCR reaction Upgrade of ADx-ARMS Increased sensitivity Compatible with ADx-ARMS qpcr platform ARMS 1% sensitivity Provided by Amoy Dx. 22
23 EGFRm Detection: ctdna by Super- ARMS vs Tissue by ARMS EGFRm Status Tissue by ADx-ARMS + - Total ctdna by ADx-Super- ARMS Total Sensitivity: 82%; Specificity: 100% Li et al. PlosOne 2017;12:e
24 Prediction of EGFR-TKI Efficacy by EGFRm in ctdna by Super-ARMS 1 st gen of EGFR-TKIs 1 3 rd gen of EGFR-TKI 2 1. Li et al. PlosOne 2017;12:e ; 2. Zhou et al. ESMO Poster 1331P. 24
25 Initial Detection of Actionable Mutations: NGS Profiling of ctdna in ansclc Biopsy and plasma mutation types under final threshold setting 1 Presented by Alice Shaw at ASCO ,3 Sensitivity:73%; Specificity:86%. Ultra-deep NGS assay to monitor plasma EGFR from patients in TRIGGER trial 4 AmoyDx Internal Data, for reference only 5 N=69 mean coverage of 20,230 ± 1410 sequences per sample Sensitivity:74%; Specificity: 100% 1. Uchida J et al. Clin. Chem. 2015;61: Mack P et al. WCLC Abstract Dagogo-Jack I et al. ASCO Abstract Marchetti A et al. J Thorac Oncol. 2015;10: AmoyDx internal data, for reference only 25
26 Key Technologies for Blood Testing Method Strength Weakness ARMS SuperARMS ddpcr Simple and fast High specificity Platform widely available Simple and fast High sensitivity: >80% High specificity Platform widely available High sensitivity Quantification Known mutations only Sensitivity: 65~70% only Known mutations only Known mutations only Low level of multiplexing NGS Known and unknown mutations No limitation of number of markers Complicated technology and procedure Significant loss of original DNA templates Provided by Amoy Dx. 26
27 Choice of methods for EGFRm blood testing 27
28 1st Scenario for Molecular Testing of NSCLC 28
29 2 nd Scenario for Molecular Testing of NSCLC 可手术切除 确诊 NSCLC 患者 不可手术切除 80%EGFR 突变阳性病人 血液 EGFR 检测 EGFR- ( 可获取组织 ) ( 无法获取组织 ) EGFR+ 组织 EAR 同检其他治疗 EGFR-TKIs EAR 全阴 ALK+ ROS1+ EGFR+ 其他检测或治疗 ALKi ROS1i EGFR-TKIs 复发 复发 血液 EGFR 检测 T790M+ T790M- 第三代 EGFR-TKIs 复发 其他检测或治疗 29
30 Future Applications: Personalized ctdna Monitoring in Adjuvant Setting TRACERx Study Using personalized liquid biopsies to track dynamic changes in ctdna during treatment and predict early response vs relapse in the adjuvant setting Abbosh et al. Nature. 2017;545:
31 Number of targets Technological Solutions for Tumor Mutation Detection >100 Tissue Blood 100 NGS 10 1 ARMS Super ARMS ddpcr 1% 0.2% 0.1% Sensitivity 31 Provided by Amoy Dx.
32 Headline Update on molecular testing for target therapy Progress of blood-based molecular testing Emerging biomarker testing for immunotherapy 32
33 PD-1/PD-L1 Inhibition: Established Immunotherapy with Significant ORR Gap ORR of single agent (%) 1 Remaining Gap in ORR Presented By Vamsidhar Velcheti at 2017 ASCO. 1. Michot et al. Eur J Cancer. 2016;54:
34 Higher Response Rate in Highly Selected Patient Population Overall survival (%) Biomarkers for patient selection: EGFRm ve ALKf ve PD-L1 exp >50% Summary of response in the intention-to-treat population Variable Objective response Pembrolizumab Group (N=154) Chemotherapy Group (N=151) Number of patients Percent (95% CI) KEYNOTE 024: Pembrolizumab vs Chemotherapy in 1 st line NSCLC (36.8 to 53.0) 27.8 (20.8 to 35.7) OS in intention-to-treat population Pembrolizumab Chemotherapy Hazard ratio for death, 0.60 (95% CI, ) P= Month Reck et al. N Engl J Med 2016;375:
35 Major Randomized Phase II/III Trials of PD-1/PD-L1 Inhibition vs Chemotherapy: Impact of PD-L1 Expression Presented by Jacek Jassem at ESMO
36 PD-L1 as Biomarker is Confounded by Expression Heterogeneity and Dynamics Presented by Aaron Mansfield at ASCO
37 % Tumour Cell Staining Technical Challenges in PD-L1 IHC as Biomarker for Patient Selection % Immune Cell Staining BMS Merck Roche AstraZeneca Immunotherapy Nivolumab Pembrolizumab Atezolizumab Durvalumab Ab clone C3 SP142 SP263 Platform Dako Dako Ventana Ventana Cell type to score Tumor cell Tumor cell Tumor cell and immune cell Tumor cell 临床研究中的阈值 1%, 5%, or 10% 1%, 50% TC0/1/2/3 or IC0/1/2/3 25% C SP142 Tumor Cell SP263 SP Samples Presented in WCLC C SP142 SP263 Immune Cell Samples 37
38 Looking Beyond PD-L1: Tumor Mutation Burden (TMB) 38 Alexandrov LB et al. Nature 2013;500(7463):
39 PFS by TMB Subgroup (CheckMate 026: Nivolumab in 1st L NSCLC) High TMB Low/Medium TMB Solange P, et al. at AACR 2017, Abstract CT
40 Association of TMB in Blood with Atezolizumab Efficacy in 2 nd Line NSCLC (OAK and POPLAR) Gandara D et al. ESMO Abstract 1295O. 40
41 btmb Level Predictive for PFS Benefit But Not OS Benefit Gandara D et al. ESMO Abstract 1295O. 41
42 Challenges in Using TMB as Biomarker for Patient Selection Kowanetz M et al. Oral presentation at WCLC Hellman M. Oral presentation at TAT Congress Solange P, et al. at AACR 2017, Abstract CT082.
43 Conclusions Molecular biomarker testing for target therapy has been well established: from tissue to blood testing, from single gene to panel, from PCR to NGS. Liquid biopsies are now in clinical practice for NSCLC full management: therapeutic decision-making, monitoring real-time response, and identifying resistance. Questions remaining include frequency of serial monitoring, when to switch, how to use data to decide treatment combination/sequencing. Reliable PD-L1-based diagnostics still remains a challenge for immunotherapy patient selection, more and better markers are needed. Tumor mutational burden measurement could be another promising approach. 43
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