Transcript PowerPoint-Präsentation

Liquid Biopsies
Analysis of circulating cell-free
tumor-DNA in plasma
Anna Buder
Institute of Cancer Research
Department of Medicine I
Medical University of Vienna
Lung Cancer International
Preceptorship Vienna
20th-21st june 2016
Activating EGFR mutations in adenocarcinoma
• EGFR mutations in 10-15% of adenocarcinomas from Caucasian patients
Sensitivity to EGFR tyrosine kinase inhibitors (TKIs)
• 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 vs. resistance mutations
Activating EGFR mutations
occur in exons 18-21
(tyrosine kinase domain)
Acquired resistance caused
by T790M mutation in > 50%
of patients
Third generation TKIs
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
Rociletinib
AZD9291
Mutations are highly specific
Pre-Cancer
Cell
Cancer
Cell
Mutations
Normal
Cells
No Mutations
Access to somatic mutations
Tumor Tissue:
Blood / Body fluids:
- FFPE
- cell-free DNA
- Frozen Tissue
- Circulating tumor cells (CTCs)
- Exosomes
Circulating cell-free tumor-DNA
in plasma:
• DNA fragments of 120-200bp
• Half-life of ~ 2 hours
• Minimally invasive access
• Specific to tumor
Origin of cell-free DNA
540 bp
360 bp
180 bp
Jahr, S. Cancer Res, 2001
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)
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
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
1.Blood Collection
2.Blood Collection
3.Blood Collection
Further Blood
Collections
Plasma Preparation
cfDNA extraction
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
First Conclusions
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
Thank you for your attention!