QIAGEN Complete Solutions for Liquid Biopsy Molecular …-+QIAGEN+en+Bi… · PowerPoint Style Guide, 07.10.2015. ... data interpretation Small amount of starting material Short TAT

Sample to Insight

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Market Development Manager, Advanced Molecular Pathology

QIAGEN

QIAGEN Complete Solutions for Liquid Biopsy Molecular Testing

Christopher Swagell, PhD

Agenda

QIAGEN – Solid Tumor Testing and Liquid Biopsy

Sample Collection – PAXgene ccfDNA

Sample Extraction – QIAamp Circulating NA

Plasma EGFR Testing – therascreen

Next Generation Sequencing - GeneReader

Sample to Insight

PowerPoint Style Guide, 07.10.2015


Personalized Healthcare Portfolio - therascreen

Lung

Brain• MGMT

• IDH1 & IDH2

Melanoma• BRAF

• NRAS

Sample to Insight

Lung• EGFR

• ALK

• KRAS

Gastro-intestinal• KIT

• PDGFRA

Colorectal• KRAS

• NRAS

• BRAF

• PIK3CA

• UGT1A1

• MLH1

RAS

Market leading portfolio for solid tumor testing

ARMS Scorpions qPCR

� High sensitivity � Selective

Pyrosequencing

EGFR, KRAS, NRAS, BRAF etc.

Mutation testing

QIAGEN solutions for Molecular Pathology – by technology

GeneReader NGS Workflow

� Sequence information

� Sequence information

Sample to Insight

� Selective mutation detection

� Ease of use� One-step

procedure

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� Complex mutation testing with simple data interpretation

� Small amount of starting material

� Short TAT

� Completely integrated sample to report workflow

� Highly powerful bioinformatics

Co-development projects with industry leaders

Global pharma partnerships and selected co-development projects with a total of 15 master framework agreements signed

Sample to Insight

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PHC/Oncology Development Pipeline

Disease# Assays

On-Market# Development

Projects# Projects w/ Liquid Biopsy

# PharmaPartner

LungNSCLC

3-410

(9 x PMA, 1 x CE) 2 9

Colon/GastricCRC/GIST

72

(2 x PMA)1 2

GynecologicalBreast/ Ovarian

-3

(2 x PMA, 1 x CE)1

3

Brain 22

(1 x PMA, 1 x CE)- 2

Sample to Insight

Glioma2

(1 x PMA, 1 x CE)- 2

GenitourinaryProstate/Bladder

-3

(2 x PMA, 1 x CE)1 3

SkinMelanoma

21

(1 PMA)- 1

BloodLeukemiaLymphoma

> 152-3

(1 PMA, 1 CE)- in disc.

> 20 145> 30

QIAGEN offers Sample to Insight Solutions for Liquid Biopsy

Based on either NGS or (RT-)PCR

Sample collection

&

Sample Extraction

PCR-Setup(Real Time)

PCR

Data Analysis &

Interpretation

PCR Workflow (e.g. known somatic mutation or pathway analysis)

PAXgene Blood ccfDNA Tubes

Manual Sample Extraction

Sample to Insight

& Stabilization

Extraction& Isolation

Pre-Amplification

/Library

Preparation

NGS Run(Sequencing)

Data Analysis &

Interpretation

NGS GeneReader Workflow (e.g. identification of known / novel mutations)

7Innovation is in the blood,

Automated Sample Extraction

Report

Free circulating nucleic acids

RNA and DNA from dead cells shed into the bloodstream, can contain cancer-related mutations.

Exosomes

Tiny microvesicles found in body fluids that transport RNA between cells.

What is Liquid Biopsy?

A minimal invasive technology for detecting signs of cancer and other diseases

Sample to Insight

Circulating tumor cells

Tumor cells shed from a tumor into the bloodstream carrying genetic information.

Tissue samples

FFPE tissue samples of tumor extracted from the patient’s body through an invasive procedure.

Comprehensive portfolio offering proven ways to access nucleic acids

Innovation is in the blood,

Critical Points Along the Workflow – ccf DNA

� hemolysis of blood cells increases Wildtype gDNAbackground � Sample requires rapid processing and/or stabilization

� Very low concentration of

Blood draw(venipuncture)

Extract circulating nucleic acids:QIAamp Circulating NA Kit,

Separate plasma

Sample to Insight

From Sample to Insight: QIAGEN solutions for Liquid Biopsy applications 9

Real-time PCR

Sequencing library prep

Next-generation sequencing

� Very low concentration of ccfDNA (1-50 ng DNA/ml plasma)

� Efficient large volume extraction required

� Low concentration and highly fragmented DNA

� efficient and sensitive downstream processing

Optional DNA modification (e.g., bisulfite treatment)

QIAamp Circulating NA Kit,QIAsymphony Virus/Pathogen Kit

AgendaSlide created with Agenda Assistant






Sample to Insight

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Blood sample processing - Workflow

Draw whole blood in cfDNA

BCT (ccfDNA PAXgene)

Spin @ 1900g for plasma

separation

Spin @ 16,000g

Carefully save

supernatant

Spin @ 1900g for

plasma separation

Draw EDTA whole

blood1-2h 7-10 days

Sample to Insight

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Store at -80 °C

Thaw plasma

Extraction of

cfDNA

Spin @ 16,000g

Supernatant:

plasma w/o cell

debris and reduced

gDNA background

PAXgene Blood ccfDNA Tube (RUO)

Sample Collection and Handling

CE-IVD ccfDNA PAXgene tubes expected in early 2017

Sample to InsightDisclaimer:

For Research Use Only. Not for use in diagnostic procedures.

Distributed by

PAXgene Blood ccfDNA Tube (RUO) - Features

Stabilization of extracellular levels of ccfDNA

Non-crosslinking preservation

Minimal hemolysis – maximum yield from plasma

BD Vacutainer® Plastic Tube with Hemogard™ Safety Closu re

Minimized risk of tube breakage

Sample to Insight

Enhanced safety for healthcare and laboratory personnel

Consistent blood draw volume

Integrated Preanalytical Workflow

Seamless integration into manual or automated extraction technology

QIAamp Circulating Nucleic Acid Kit

QIAsymphony DSP Circulating DNA Kit

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1,50

2,00

2,50

Hemolysis

EDTA

Streck

PAXccfDNA

Prevention of Red Blood Cell Lysis During Transport and Storage

PAXgene Blood ccfDNA stabilization reagent prevents the hemolysis of erythrocytes

Sample to Insight

Picture kindly provided by

0,00

0,50

1,00

t0 t1 t3 t6 t8 t10

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Blood cell stabilization prevents apoptosis

PAXgene Blood ccfDNA stabilization reagent prevents the apoptosis of blood cells

Reduces wildtype DNA background

Sample to Insight

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Sample to Insight

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QIAGEN´s solutions for cfDNA extraction

Current solution (manual):

QIAamp Circulating NA Kit

≤5 ml plasma input | 24 samples | 3 hours | for IVD use

QIAGEN market leaders in sample extraction

Sample to Insight

In development (automated):

QIAsymphony Circulating DNA KitUsing new beads and chemistry

1-4 ml plasma input

96 samples | 6 hours (hands-off) | for IVD use

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ccfDNA isolation considerations – Fragmentation

1000

1500

2000

geno

me

equi

v./

sam

ple

x100

0 QIAamp circulating NA Kit 66 bp

Standard blood kit 66 bp

QIAamp Circulating Nucleic Acid Kit: Cat. No. 55114

Sample to Insight

0

500

sample 1 sample 2 sample 3

geno

me

equi

v

Distributed by

� Improved recovery of fragmented DNA

� Ultimately results in better sensitivity

QIAamp Circulating Nucleic Acid Kit:







Sample to Insight


therascreen EGFR Plasma Testing - Assay

Intended use

Sample to Insight

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Intended use

� The therascreen EGFR Plasma RGQ PCR Kit is a real-time PCR test for the qualitative detection of mutations of the EGFR gene derived from plasma from whole blood collected in EDTA tubes of NSCLC patients.

Covers the most frequent and clinically validated mutations

� exon 19 deletions

� L858R point mutation

� T790M point mutation (resistance mutation)

therascreen EGFR Plasma Testing - Workflow

60 min 160 min 15 min

Sample to Insight

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Total < 4 hrs

Case Study 1Title only (a)

� 74 yo, male, non-smoker

� Health degradation and dyspnea

� Tumor upper lobe right lung and plerual effusion

� L858R mutation detected in bothlung biopsy and plasma

� After 2 month TKI treatment

Sample to Insight

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� Health deterioration and weightloss

� No reduction in tumor size

� L858R remained stable in plasma

� T790M mutation detected plasma

� Identification of resistancemutation during patient

progression

Marcq et al, J Thorac Oncol. 9 e49-e50 (2014) Detection of EGFR Mutations in the Plasma of Patients with Lung Adenocarcinoma for Real-Time Monitoring of Therapeutic Response to Tyrosine Kinase Inhibitors?

Case Study 2Title only (a)

� 63 yo, female, never-smoker

� Cough and weight loss

� Large tumor upper lobe right lungand single bone metastasis

� ex19Del mutation detected in bothlung biopsy and plasma


� 50% reduction in lung tumor

Sample to Insight

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� 50% reduction in lung tumor

� EGFR mutation becameundetectable in plasma

� After 11 months TKI treatment

� Progression of tumor and appearance of ex19Del mutationdetected in plasma

� Dissapearance of EGFR mutationmay be a marker of TKI response

Marcq et al, J Thorac Oncol. 9 e49-e50 (2014) Detection of EGFR Mutations in the Plasma of Patients with Lung Adenocarcinoma for Real-Time Monitoring of Therapeutic Response to Tyrosine Kinase Inhibitors?







Sample to Insight

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QIAGEN NGS Workflow Solution - GeneReader

QIAGEN GeneReader – Launched December 2015

Sample to Insight

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The first truly complete NGS workflow

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Expertise and service you can count on

5Actionable

InsightsFlexibility to fit your

needsGuaranteed results

with predictable costs

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2016: GeneReader NGS System development program

GeneReader NGS System development

Expanding range of sample types• Liquid biopsy , fresh and frozen tissue

o Driving sensitivity down to <1%

Solid Tumour Panel

Sample to Insight

Expanding Sample to Insight capabilities

o Driving sensitivity down to <1%o Liquid Biopsy Summer launch

Broadening test content to lung and breast• Expand oncology panels, other applications

o Enabling CNV & Fusion detection

Expanding System capabilities• Further laboratory platform integration and

automation (LIMS capability)

A T G C A

T A C G T

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Gracias

Gracias por su atención

Sample to Insight

Customer presentation:therascreen EGFR Plasma RGQ PCR Kit 27

Distributed byQIAamp Circulating Nucleic Acid Kit:

ccfDNA PAXgene tubes:

Documents

QIAGEN Complete Solutions for Liquid Biopsy Molecular …-+QIAGEN+en+Bi… · PowerPoint Style Guide, 07.10.2015. ... data interpretation Small amount of starting material Short TAT