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

2

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

Liquid Biopsy 2014 3

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


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

5 Liquid Biopsy 2014

Can we push sensitivity to allow detection at early stages?

Sample & Assay Technologies How to overcome challenges


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

7

New and existing products

Liquid Biopsy 2014

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

Liquid Biopsy 2014

8

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


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


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 Questions

Improved microbiome sequencing through host DNA removal

Sample & Assay Technologies Technological Life Cycle of LB applications

Title, Location, Date 17

Dynamic field developing quickly


18

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

Liquid Biopsy 2014

qiagen liquid biopsy: solutions and visions

Documents