QIAGEN liquid biopsy: solutions and visions
Transcript of QIAGEN liquid biopsy: solutions and visions
Sample & Assay Technologies
QIAGEN liquid biopsy: solutions and visions
Marco Polidori, Manuel Frietsch November 24th
Sample & Assay Technologies QIAGEN liquid biopsy: solutions and visions
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Agenda
Overview Liquid Biopsy areas
Application challenges
Workflow challenges
New and current sample prep solutions
QIAGEN Roadmap overview
New automated solution for cfDNA: overview and data
Sample & Assay Technologies What Liquid Biopsy means for QIAGEN
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Liquid biopsy is divided into 3 major domains
Free circulating Nucleic acids
DNA
miRNA
Circulating Tumor Cells (CTC)
Exosomes
Total RNA
DNA
Sample & Assay Technologies Challenges
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Each application comes with its own challenges
Free circulating DNA
Very Low concentration
(dependent on burden)
Not all tumors shed DNA
DNA is highly fragmented
Background of WT DNA
Exosomes
Low abundance
Challenging to enrich
Mostly RNA (RNAseq?)
Clinical value not clear yet
CTC
Very low abundance
EMT
Difficult to analyse
Mostly in metastatic stage
Sample & Assay Technologies Workflow challenges
Sample collection and storage:
Amount of blood taken
Stabilization to keep WT background to a minimum
Weight, temperature and fragility (hidden costs)
Sample preparation
Sample volume
Yield & purity
Standardization/automation
Assay
Sensitivity & specificity
Compatibility with low analyte amount
Concordance to tissue derived DNA
Data Analysis
PCR vs. NGS
Actionable vs. junk data
Data protection
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Can we push sensitivity to allow detection at early stages?
Sample & Assay Technologies How to overcome challenges
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What are the main needs
Higher sensitivity
Standardised sample preparation
Clinically validated biomarkers
Comprehensive and cost effective NGS workflow
Meaningful data interpretation
How can we achieve that?
Easier preparation of plasma and cfDNA specific stabilisation
High sample volumes (4-10ml plasma)
Improved sample prep (including automation)
Partnerships with clinical research institutions and Pharma
Nanoscale library prep
Combine WTA/WGA with cfDNA and CTCs
State of the art Bioinformatics (curated content)
Sample & Assay Technologies QIAGEN Liquid Biopsy sample prep
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New and existing products
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QIAamp circulating NA Kit
Up to 5ml plasma/serum
Gold standard for yield and purity
>200 publications since launch (2009)
exoRNeasy Kits
Specifically purify total RNA from exosomes and other EVs
High input volume (up to 4 ml)
NGS tested
Repli-g Single Cell Kit
Minimum bias WGA/WTA
From as little as a single cell
Complete genome / transcriptome coverage
Sample & Assay Technologies QIAGEN roadmap
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Sample
stabilization
Data
analysis/
interpretation
Assays Sample
enrichment
Sample
preparation
Pre-
amplification
PAXgene
QIAamp
QIAsymphony
Repli-g
(WGA/WTA)
GeneRead
cancer panels
therascreen
Cancer
workbench
Ingenuity VA
Improve stability of
cells and preserve
cfDNA
Automated
solutions
Next generation
sample prep
Increase sensitivity
for low input
amounts
Develop clinical
assays with
partners and
improve research
tools
Meaningful
information
exoRNeasy
Sample & Assay Technologies Evolution of cfDNA extraction
QIAGEN Solutions
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Current solution (manual):
QIAamp DSP Circulating NA Kit
≤5 ml plasma input | 24 samples | 3 hours | for IVD use
Current solution (automated)
QIAsymphony Custom Protocol & Kit (#1074536)
4 ml plasma input | 24 samples | 3½ - 4h hours (hands-off) |
performance acceptable for EDTA but not for Streck plasma
In development (automated):
QIAsymphony DSP Circulating DNA Kit
Using new Anion-exchange bead chemistry
4 ml input | plasma from EDTA and Streck tubes
96 samples | 6 hours (hands-off) | IVD use |
Design Goal: performance equivalent to QIAamp Kit
Sample & Assay Technologies
Spindler K G et al. Clin Cancer Res 2012;18:1177-1185
Conclusions: KRAS analysis in plasma is
a viable alternative to tissue analysis.
Quantitative levels of cfDNA and pmKRAS
are strongly correlated and hold promise of
clinical application.
Correlation between KRAS mutations and ccfDNA
9 ml blood (EDTA tube)
1.2 ml plasma
QIAsymphony Circulating NA Kit (#1074536)
TheraScreen K-Ras mutation kit
Sample & Assay Technologies Technological Life Cycle of LB applications
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Dynamic field developing quickly
Sample & Assay Technologies
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Example: plasma circulating DNA and therascreen
Manual and automated large-volume extraction of circulating nucleic acids
Sample & Assay Technologies Novel exoRNeasy Kit from QIAGEN
Detection of somatic mutations from CRC in exRNAs
Patient with KRAS G12D positive colorectal cancer (CRC)
2 ml pre-filtered plasma
exRNA isolation using the exoRNeasy Serum/Plasma Maxi Kit
Targeted re-sequencing on an Illumina MiSeq
Over 10% of all reads that matched to the KRAS gene carry the c.35 G>A /
p.G12D mutation previously identified in the primary tumor
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