liquid biopsy: overcome challenges of circulating dna with automated and standardized extraction...

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Automation of ccfDNA isolation using the QIAsymphonyDr. Marco Polidori

Global Product Manager

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Legal disclaimer

• QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

• For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

at www.QIAGEN.com or can be requested from QIAGEN

Technical Services or your local distributor.

Automation of ccfDNA isolation using the QIAsymphony

http://www.qiagen.com/

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Agenda

Introduction

Challenges of ccDNA isolation

ccDNA isolation methods

Validation data

Application data

Summary

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4Automation of ccfDNA isolation using the QIAsymphony

Free circulating Nucleic acids• DNA• mRNA• miRNA

Exosomes• Total RNA• DNA• Protein

Circulating Tumor Cells• Enumeration• Genotyping• Gene expression

The main areas of Liquid Biopsy

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Liquid Biopsy: Game changing potential


From sequencing an entire fetal genome using maternal plasma…

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Liquid Biopsy: Game changing potential


…to groundbreaking oncology studies

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Application areas


New paradigm: Less invasive

bio3400.nicerweb.com

Zeit.de

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QIAGEN Sample to Insight Solutions for ccfDNA


QIAamp Circulating Nuecleic Acid Kit

QIAsymphony free circulating DNA Kit

GeneRead DNAseq cancer panel v2

CLC cancer workbench

Ingenuity Variant Analysis

QIAseq Ultra Low Input Library prep Kits

Any sequencerPAXgeneccfDNA Tubes (in development)

Sample Collection

Sample Isolation Amplification Library

preparation SequencingData analysis

& interpretation

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Blood draw (venipunctur

e)Separate plasma

(Logistics)

Extract circulating nucleic acids:

QIAamp Circulating NA Kit,QIAsymphony Circulating NA Ki

Real-time PCR digital PCR

therascreen assays

Next-generation sequencing

SamplePre-

analytical workflow

Analytical workflow Results

Circulating nucleic acids: Analysis workflow

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Critical points along the workflow


Real-time PCR digital PCR

Sequencing library prep

Next-generation sequencing

• gDNA background due to hemolysis Stabilization

• Very low concentration of ccfDNA Efficient large volume prep

• Low concentration efficient and sensitive downstream processing

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

Blood draw (venipunctur

e)

Extract circulating nucleic acids:QIAamp Circulating NA Kit,QIAsymphony ccfDNA Kit

Separate plasma

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Sample handling: An example


Mutant allele frequency variation and concentration example

Chetan Bettegowda et al. 2014 doi:10.1126/scitranslmed.3007094

Amount of mutated fragments vary strongly from <100 to >100,000 per 5 ml.

Amount of ctDNA also depends on cancer type and tumor burden!

Effectively, between 10 and 20 ml of blood should be taken for ccfDNA studies

http://stm.sciencemag.org/search?author1=Chetan+Bettegowda&sortspec=date&submit=Submit



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Store at -80 °CThaw plasma

Extraction of cfDNA

Draw whole blood in Cell-Free DNA BCT (Streck)

Spin @ 300g for plasma separation

Spin @ 16,000g

Supernatant: plasma w/o cell debris and

reduced gDNA background

Carefully save supernatant

Spin @ 1900g for plasma separation

Draw EDTA whole blood

1-2h 14 days

Blood sample processing

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Sample stabilization: PAXgene project


No elevated levels of free DNA using PAXgene ccfDNA Tubes*

t1 t3 t6 t8 t10 t1 t3 t6 t8 t10EDTA PAXccfDNA

0

50

100

150

200

250

300

350 18S rDNA qPCR assay

66 bp amplicon

500 bp amplicon

ratio

tx/t0

*product under development

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EDTA t0 PAXccfDNA t0 EDTA t10 PAXccfDNA t10

35 100 200 300 400 500 700 2000 10380 [bp]

Sample stabilization: PAXgene project

Efficient long-term stabilization of cells and extraction of ccfDNA

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In development (automated):QIAsymphony Circulating DNA KitUsing new beads and chemistry 2 or 4 ml input | plasma from EDTA and Streck tubes 96 samples | 6 hours (hands-off) | IVD use


Current solution (manual): QIAamp Circulating NA Kit≤5 ml plasma input | 24 samples | 3 hours

2009

MBA 2015

IVD 2016

Evolution of cfDNA extraction

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QIAsymphony SP instrument: Workflow

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Binding buffer

Proteinase K

Plasma

Magnetic beads

Binding at RT

Bead separation

Elution in 60 µl

Wash step 1

Wash step 2

Binding at RT

Bead separation

Wash step 3

2 ml protocol

4 ml protocol

QIAsymphony SP instrument: Workflow

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Size distribution of extracted ccfDNA from plasma


Set-up of experiment:• ccfDNA extracted from 4 ml

maternal plasma using:• QIAamp Circulating NA kit as

manual reference method (red)

• QIAsymphony® Circulating DNA Chemistry (blue)

• 1 μl eluate subjected to Agilent High Sensitivity DNA Kit (5-500 pg/μl)

Set-up of experiment:• ccfDNA extracted from 4 ml

plasma using:• QIAamp Circulating NA kit as

manual reference method (red)

• QIAsymphony® Circulating DNA Chemistry (blue)

• 5 ng eluate subjected to library prep (ThruPLEX-FD Prep Kit; Rubicon Genomics)

• 1 μl purified eluate from library prep subjected to Agilent DNA 7500 Kit

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•Qubit™ dsDNA HS Assay Kit: 0.23-0.56 ng/µl

•GeneRead Library Prep I Core Kit: Input 10 µl (2.3-5.6 ng DNA)

•Quantification of ccfDNA by qPCR; 10 nM required for Illumina Sequencing applications (green)

•MiSeq NGS run using 5 nM (5 µl) Calculation of mapped reads distributed on chromosomes

Results: Compatibility of ccfDNA eluates in library prep and NGS

End repair A-addition Adapter ligation

Cleanup and size selection

HiFi library amplification (optional)

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4 ml plasma

QS Circulating DNA Kit

QIAamp Circulating NA Kit

RT-PCR: 18S-66 bp

Qubit dsDNA HS Assay Kit

Results: Detection of ccfDNA comparing RT-PCR & Qubit™

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4 ml plasma


RT-PCR: 18S-66 bp (∑20 µl) 2 µl - 4 µl - 8 µl

2 ml plasma 6 ml plasma

Results: Linearity for 2-6 ml plasma input

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Fill up to 2 ml 2 ml transferred

Liquid Level Detection (LLD)

FIX (no LLD)

Transfer of 1-2 ml plasma (dead volume required)

Results: Variations in sample input volume

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LoD

LoB

Set-up of experiment:

• 4 ml female plasma spiked with male plasma from 2-1000 µl

• DNA extracted from 4 ml plasma using the QS Circulating DNA Kit

• Male DNA yield determined by real-time PCR (DYS-14, SRY1) using the QIAGEN QuantiTect® Multiplex PCR Kit

• Results for SRY1 (upper figure) and DYS14 (lower figure) are depicted as copies per ml plasma

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Results: Sensitivity for low copy recovery

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Set-up of experiment: • Urine samples from 10 healthy donors; 3-4 ml urine as sample input• Circulating DNA yield determined by real-time PCR (18S coding

sequence, 66 bp amplicon) using the QIAGEN QuantiTect® Multiplex PCR Kit

• Results were calculated as target copies per ml plasma and compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit

Extraction of ccfDNA from 4 ml urine

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cut-off

4 ml plasma


QIAamp Circulating NA Kit

QuantYfeX® assay PrenaTest®

Early Access Kit: Customer feedback

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Set-up of experiment: • plasma samples from 9 clinical donors; 3.0-3.5 ml

plasma as sample input• Circulating DNA yield determined by Qubit™ dsDNA HS

Assay Kit used with the Qubit® 2.0 Fluorometer: Results were calculated as ng DNA per µl eluate from 3.0-3.5 ml plasma input and compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit

• 12 µl eluate was subjected to library prep (Ion AmpliSeq™Library Kit 2.0, Thermo Fisher)

• Subsequently 3 nM of each ccfDNA was transferred to the pool which was diluted to 12 pM for targeted NGS. Calculated mutation frequency was compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit

1:w/o, 2:TP53 exon5, 3:KRAS exon2, 4.1:EGFR exon19, 4.2:EGFR exon20, 5:w/o, 6:EGFR exon19, 7:w/o, 8.1:EGFR exon21, 8.2:EGFR exon20, 8.3:TP53 exon3, 9: w/o

Detection of caner ccfDNA from clinical samples

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Summary


• Proper tube choice and handling can minimize gDNA background

• Use at least 2-5 ml plasma (or other biofluids) whenever possible to maximize sensitivity

• The QIAsymphony Circulating DNA Kit isolates ccfDNA with the same or higher efficiency than QIAamp Circulating NA reference

• The QIAsymphony handles 96 samples in about 6 hours hands free starting directly with plasma/serum

• Input volumes for the QIAsymphpny Circulating DNA Kit can be ramped up to 6 ml

• Expected launch of IVD certified version in October 2016, already available as MBA (non-IVD)

Some take home points

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Katharina BellerMartin HorlitzThorsten VossManuel FrietschKevin MatthaeiStephan RachwalAgata StoltmannRita KistAnnabelle SchubertAnnette NoconSandra HammerschmidtNicole HoffmannStephanie AngenendtPatricia WeideDagmar HeroldYun Kyung LeeAndrea KloseGaby BrockerhoffSilke SonnwaldNan FangHolger Wedler

Many thanks to…

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Marco Polidori, [email protected]

Tel: +49 2103 29 11441

Questions?

Thank you for attending

Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US

Call: +49 2103-29-12400 for EU

Email: [email protected]

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Automation of ccfDNA isolation using the QIAsymphony 30

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liquid biopsy: overcome challenges of circulating dna with automated and standardized extraction...

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