Development of Circulating Tumor DNA Title of presentation Arial Bold 30pt in White Biomarkers Secondary title 22pt using Arial Next in White Generation Sequencing Brian Dougherty PhD, MBA Translational Genomics Lead AZ R&D Boston Speaker title Event title Arial 14pt in White 00 Month Year Location Arial 14pt in White
Mission of AstraZeneca Translational Science Disease Biology Patient Selection Idea Pre-Clinical Translational Clinical Diagnostics Regulatory Commercial Drug Back- Translation Companion Diagnostics 2
Talk Overview NGS Background The Right Patient for the Right Drug Tumor BRCA1/2 mutations and olaparib (PARP inhibitor) response in ovarian cancer Drug Mechanism of Resistance Plasma circulating tumor DNA and predicted resistance to next-generation EGFR inhibitors in lung cancer 3
NGS Background The Right Patient for the Right Drug Tumor BRCA1/2 mutations and olaparib (PARP inhibitor) response Drug Mechanism of Resistance Plasma circulating tumor DNA and next-generation EGFR inhibitors 4
Foundation Medicine-AstraZeneca: Translational Science Collaboration
PFS for BRCA1/2 Mutated Patients Proportion of patients progression-free Number at risk Olaparib BRCAm Placebo BRCAm 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 Olaparib BRCAm Placebo BRCAm BRCAm (n=136) Olaparib Placebo Events: total pts (%) 26:74 (35.1) 46:62 (74.2) Median PFS, months 11.2 4.3 HR=0.18 95% CI (0.11, 0.31); P<0.00001 3 6 9 12 15 Time from randomization (months) 74 59 33 14 4 0 62 35 13 2 0 0 82% reduction in risk of disease progression or death with olaparib
PFS by BRCAm Status Proportion of patients progression-free Number at risk Olaparib BRCAm Placebo BRCAm Olaparib BRCAwt Placebo BRCAwt 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 Olaparib BRCAm Placebo BRCAm Olaparib BRCAwt Placebo BRCAwt BRCAm (n=136) BRCAwt (n=118) Olaparib Placebo Olaparib Placebo Events: total pts (%) 26:74 (35.1) 46:62 (74.2) 32:57 (56.1) 44:61 (72.1) Median PFS, months 11.2 4.3 5.6 5.5 HR=0.18 95% CI (0.11, 0.31); P<0.00001 3 6 9 12 15 Time from randomization (months) 74 59 33 14 4 0 62 35 13 2 0 0 57 44 17 9 2 0 61 35 10 4 1 0 HR=0.53 95% CI (0.33, 0.84); P=0.007 BRCAmut on Olaparib BRCAwt on Olaparib BRCAwt, wild-type (includes patients with no known BRCAm or a mutation of unknown significance)
NGS Background The Right Patient for the Right Drug Tumor BRCA1/2 mutations and olaparib (PARP inhibitor) response Drug Mechanism of Resistance Plasma circulating tumor DNA and next-generation EGFR inhibitors 8
The Challenge of Sequencing Clinical Oncology Samples Oncology clinical specimens FFPE: low quantity tissue, low quality, de-amination artifacts Plasma: low concentration, sheared DNA, variable amounts of tumor DNA Plasma ctdna Increasing use in clinic Liquid biopsy (blood, not biopsy) faster/cheaper than CTC s serial time points Challenges limited DNA amounts for assays limited sensitivity (absolute number of tumor molecules in plasma) number of tumor molecules in plasma decrease with successful treatment NGS methods development more challenging NGS analysis more complex (e.g., calculating amplifications) Specialized ctdna NGS CROs nice technology but assay price-points make outsourcing too expensive, especially given multiple samples/patient 9 www.sysmex-inostics.com
Plasma Circulating Tumor DNA (ctdna) Sequencing Genotyping AKT1 EGFR MET PTEN ALK ERBB2 MTOR PTGS2 BRAF HRAS NRAS RB1 CDKN2A KIT PDGFRA STK11 www.cruk.cam.ac.uk CTNNB1 KRAS PIK3CA TP53 Targeted gene panels WGS Exome
Rapid Identification of Resistance Mutations Treatment Mutation copies/ml plasma Tumor Mutation (follows tumor burden) Resistance Mutation(s) 6-15 + months 11
Lessons in Acquired Resistance to AZD9291 Back-Translation: Starting with the Patient Cell Lines Tumor Explants Patients Found: KRAS Amp NRAS Amp, Mut RAS-family Amp MET Amp? EGFR C797S HER2 Amp MET Amp RAS mutations never found in EGFRm lung cancer patients Didn t Find: EGFR C797S HER2 Amp? KRAS NRAS Eberlein et al, Cancer Research 2015 Thress et al, Nature Medicine 2015 12
13 EGFR C797S Resistance Mutation
C797S Mutation Inhibits AZD9291 and CO-1686 Drug response in engineered Baf3 cells 14
Plasma ddpcr Identifies C797S Cases (6/19) At least 3 molecular subtypes of T790M+ acquired resistance Acquired C797S, T790M+ No acquired C797S, T790M+ No acquired C797S, loss of T790M 15 T790M negative at baseline
Three molecular subtypes of acquired resistance in T790M+ patients (serial plasma sampling) 1. T790M retained, plus C797S 2. T790M retained, no C797S 3. T790M lost, no C797S Patient #1 Patient #9 Patient #12 Patient #3 Patient #8 Patient #13 Weeks on AZD9291 Weeks on AZD9291 Weeks on AZD9291
NGS of Matched Tumor/Plasma Reveals Heterogeneity of Drug Resistance Heterogeneity of drug resistance second allel Patient #4: 2nd mutation in matched plasma Patient #5: 2nd mutation in matched plasma 17
Plasma ctdna WGS and Clinical Drug Resistance Complementary methods for determining resistance mutations - Targeted gene panels and exomes to find mutations at high sensitivity - WGS for genome-wide copy number and structural alterations ERBB2 (chr. 17) Inner circles: 6 independent libraries, 1X Outer Circle: merge (6X) Post-treatment NSCLC patient plasma sample: EGFR and HER2 Amp (no MET Amp) EGFR (chr. 7)
Closing Remarks NGS revolutionizing cancer translational medicine Rapid growth of plasma sample collection in the clinic Driving need for development of robust, inexpensive plasma DNASeq assays Plasma DNASeq enables disease detection disease progression response to therapy emergence of drug resistance serial time points pan-tumor patient profiling data CDx: genotyping now, NGS in the near future 19
Acknowledgements Thank you to the patients and families involved in AstraZeneca trials Dan Stetson Aleksandra Markovets Zhongwu Lai Justin Johnson Ambar Ahmed Dave Whitston Sakina Saif Alla Bushoy Miika Ahdesmaki Hedley Carr Ken Thress Darren Hodgson Jeff Brown Carl Barrett Geoff Oxnard Cloud Paweletz Pasi Jänne 20