1. Anna Buder Institute of Cancer Research Department of Medicine I Medical University of Vienna Liquid Biopsies Analysis of circulating cell-free tumor-DNA in plasma Lung Cancer International Preceptorship Vienna 20 th -21 st june 2016

2. EGFR mutations in 10-15% of adenocarcinomas from Caucasian patients Response rate of 58-74% Median PFS of 10-14 months Identification of oncogenic driver mutations in adenocarcinoma has become a standard procedure in diagnostic testing Activating EGFR mutations in adenocarcinoma Sensitivity to EGFR tyrosine kinase inhibitors (TKIs)

3.  Activating EGFR mutations occur in exons 18-21 (tyrosine kinase domain)  Acquired resistance caused by T790M mutation in > 50% of patients Activating EGFR mutations vs. resistance mutations

4. Acquired resistance is caused by T790M mutation in > 50% of patients Third generation TKIs target EGFR-activating mutations & T790M mutation Osimertinib (AZD9291) - AstraZeneca Rociletinib (CO-1686) - Clovis Olmutinib (HM61713) - Boehringer Ingelheim Third generation TKIs

6. AZD9291 Rociletinib

7. Mutations are highly specific Pre-Cancer Cell Cancer Cell Normal Cells Mutations No Mutations

8. Access to somatic mutations Tumor Tissue: - FFPE - Frozen Tissue Blood / Body fluids: -cell-free DNA -Circulating tumor cells (CTCs) -Exosomes

9. Circulating cell-free tumor-DNA in plasma: DNA fragments of 120-200bp Half-life of ~ 2 hours Minimally invasive access Specific to tumor

10. Jahr, S. Cancer Res, 2001 Origin of cell-free DNA 180 bp 360 bp 540 bp

11. Plasma: 91 % Water 7 % Proteins Metabolites (traces) Cell-free DANN (traces) Cellular Components: 2-3 % White Blood Cells 2-3 % Platelets 2-3 % 90% Red Blood Cells Circulating tumor cells (trace)

12. Technology for analysis of circulating ctDNA Digital PCR Individual point mutations, deletions Only known mutations Sensitivity dependent on specific mutation & assay optimization Fast and highly reproducible results Low cost Minimal bioinformatic expertise Next generation sequencing Evaluation of genomic regions by PCR or capture-based methods Genomic amplifications, rearrangements, aneuploidy, whole- exome sequencing High false discovery rate Turnaround time ≥ 1-2 days

13. Liquid Biopsy-Noninvasive Detection of Response and Resistance in EGFR-mutated NSCLC Blood is collected every 1-3 months for continuous monitoring of T790M resistance mutation & activating mutations Further Blood Collections 1-3 months 1-3 months 1-3 months 1.Blood Collection 3.Blood Collection 2.Blood Collection

14. Plasma Preparation cfDNA extraction PCR-based mutational testing

15. Plasma genotyping using ddPCR Oxnard GR et al., Clin Cancer Res 20, 1698-1705, 2014 Cell-free tumor-DNA is extracted from plasma Sample is partitioned into droplets, each containing 0 to 1 molecules of target DNA PCR is performed in each droplet Droplets containing mutant and wild-type DNA emit different colored signals and can be analyzed & quantified

16. Liquid biopsy is an appropriate method to identify actionable alterations and to select therapy Plasma ddPCR detects EGFR T790M with high specificity and high sensitivity Plasma ddPCR is a powerful tool for early detection of resistance mechanisms Liquid biopsy could replace tissue biopsy in the future First Conclusions

17. Thank you for your attention!