Liquid Biopsy Research Applications Solutions · Liquid Biopsy Research Applications Solutions ......

©2014 Thermo Fisher Scientific. All rights reserved. 1 For Research Use Only. Not for use in diagnostic procedures. The world leader in serving science

Liquid Biopsy Research Applications Solutions Rosella Petraroli Senior Manager Global Market Development

©2014 Thermo Fisher Scientific. All rights reserved. 2 For Research Use Only. Not for use in diagnostic procedures.

Evolution of Cancer Classification

• Cancer classification has changed from anatomical to molecular

classification

• Molecular subtype–based stratification is a promising new strategy in

clinical research

From anatomical to molecular approach

• Breast Cancer

• Cervical Cancer

• Colorectal Cancer

• Liver Cancer

• Lung Cancer

• Ovarian Cancer

• Pancreatic Cancer

• Prostate Cancer

• Skin Cancer

• Stomach Cancer

• Thyroid Cancer

• ALK

• AKT1

• BRAF

• EGFR

• ERBB2

• KRAS

• NRAS

• MAP2K1

• PIK3CA

• RET

• ROS1

• Undefined


Overview of Tumor Molecular Evolution Over Time

• Primary tumor may not reflect current tumor molecular profile

• Treatment options may cause changes in tumor cells

*

* MRCA = Most recent common ancestor

Source: Yates LR, et al. (2012) Evolution of the cancer genome. Nat Rev Gen 13: 795–806.


Tissue Sampling versus Liquid Biopsy

Challenges

Tissue sampling is invasive

Tissue sampling is limited

Advantages

Archive serial samples

Cost

Information on tumor heterogeneity

Tissue Sample Liquid Sample


Multiple Sources of Tumor DNA in Blood

• Circulating Tumor Cell (CTC)

2 Main

DNA Targets:

CTCs & cfDNA

• Cell-free DNA (cfDNA)

• Exosomes

Crowley E, et al. (2013) Liquid biopsy: monitoring cancer genetics in the blood. Nat Rev Clin Onc 10: 472–484.


Liquid Biopsy Applications in Cancer Research

Research Studies

• Review of past therapeutic decision

• Correlation with tumor stage

• Tumor evolution at different time

points

• Review of tumor recurrence

Circulating Tumor Cells and Circulating Cell-free DNA

Research Applications

Modified source: Haber, DA, and Velculescu, VE. (2014). Blood-Based Analyses of

Cancer: Circulating Tumor Cells and Circulating Tumor DNA. Cancer Discovery 4:650–

661.


Liquid Biopsy Research Applications

• Sample stability

• Standardization

• Contamination between samples

• Limited material for molecular analysis

• Limit of detection below 1%

• Increase number of target mutations

Today’s Challenges


Ch

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Scalable Solutions for Liquid Biopsy Research

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Sample Collection

Template Isolation

Variant Detection

Analysis

Ion Torrent™

LiquidBiopsy™

Blood Collection Kit

Ion Torrent™

LiquidBiopsy™ Instrument

Applied Biosystems™ MagMAX™ Cell-Free DNA

Kits and Thermo Scientific™ KingFisher™ Flex

Magnetic Particle Processor


• Standardization

• Limited material for

molecular analysis

• Standardization

• Contamination


molecular analysis

• Limit of detection <1%

• Increase number of target

mutations

• Standardization


• Increase number of

target mutations

• False positive rate

Applied Biosystems™

QuantStudio ™ 3D Digital

PCR System

Ion Torrent™

sequencing platforms

Ion Reporter™

Software

CTC Enrichment

cfDNA Isolation


LiquidBiopsy Instrument Workflow Three Templates from a Single Blood Sample • Enables CTC enumeration and molecular

analysis of cfDNA, CTC and germline DNA

from a single blood sample

• Automates flow cell priming, blood sample

loading, target cell isolation and

immunofluorescence labeling of captured

cells

• Enables multiplexing: Up to 4 samples in ~3

hours


Comprehensive 2-day LiquidBiopsy Instrument Workflow Sample to Genomic Data

NGS

Analysis

Purify cfDNA

Germline WBC

Extracted CTCs

LiquidBiopsy

Instrument—

Enrichment

Sequencing

Analysis with

Ion Reporter

Software

Multiple template analysis of CTC, germline and cfDNA isolation from a single blood sample

Multiplex up to 4 samples in ~3 hours

CTC enumeration and genomic data

Invitrogen™

EVOS™ Floid™

Cell Imaging Station

Optional

Enumeration

Optional

verification

with dPCR


Research More Tumor Types with Multiple Supported Antibodies

The ability to enrich CTCs enables researchers to ask

which molecular profile should be serially analyzed

Recovery and

purity of target

cells can be

matched with

different reagents

Various cell

populations can

be recovered—

informative on

molecular level

Winer-Jones, JP, et al. (2014) Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel

CTC Flow Cell. PLoS ONE 9: e86717.


Consistent Number of Captured WBCs Enables More Accurate Variant Calls

• Performance enables detection of ~3

CTCs/7.5 mL blood sample at >1%

limit of detection

• A predictable WBC capture rate

leads to more accurate variant calls

• Normal and diseased research

samples show the equivalent

performance at cell capture 0

125

250

375

500

Non-t

arg

et

events

/mL

Non-target Cell Carryover

CD45 cells

captured/m

L blood =

22 ± 20

Mean non-

target cell

capture + 2

SD

198

+ = Captured WBCs is

22 cells/mL

At least 3 captured

CTCs

Verified 1% limit of

detection using Ion

Reporter Software*

* 1% LOD at 2,000x read depth using Ion PGM System


Excluded based on inclusion in germline

PIK3ca

PIK3ca

PIK3ca

PIK3ca

PIK3ca

TP53

PIK3ca

TP53

PTPN11 TP53

KIT

KDR

KDR

KIT

BRAF

KIT

TP53

TP53

KRAS

PTPN11

PTEN

KRAS

COSMIC mutations Uncategorized mutations

CTC cfDNA

PIK3ca

TP53 TP53

KDR

PIK3ca

PIK3ca

TP53

TP53

TP53

TP53

TP53

KRAS

ERBB2

KRAS KRAS

VHL

PTEN

PTEN

BRAF STK11

SMO

SMO

SMO FGFR3

VHL

KDR

ATM

RB1

RB1

FBXW7

FBXW7

FLT3 APC

AKT1 AKT1

AKT1

HNF1A PIK3ca

MET

cfDNA and CTCs are Complementary Templates


• Simple protocol

• 3 reagents + Applied Biosystems™ Dynabeads™ Magnetic Beads

• Ship and store at room temp

• Fast

• 30 to 45 min manual protocol

• Automated on KingFisher Flex (24 samples)

• Automated on Thermo Scientific™ KingFisher™ Duo Prime

Purification System (6 samples)

• Scalable

• Flexible input volumes from 0.5 mL to 10 mL plasma

• Specific

• Enrich for cfDNA fraction over gDNA or isolate all DNA

KingFisher Flex

MagMAX Cell-Free DNA Isolation

MagMAX cfDNA Kit


4 ml plasma

4 ml Buffer

ACL + carrier

RNA

ProK 60oC

30 min

Add 7.2

ml

incubate

on ice

Load 15.2 ml

vacuum

Wash x3

vacuum

Elute

nucleic acid

(20-150 ul)

56oC

10 min

drying

Manual cfDNA Workflow Comparison

4 ml plasma

+5 ml L/B

solution

+60 ul beads

10 min

shaking

room temp

pellet beads

on magnet

Resuspend

beads in

wash

solution

Transfer to

microfuge

tube Wash 2x 1 ml wash solution

Wash 2x 1 ml 80% ETOH Dry 3-5 minutes Elute DNA

50 ul

QIAamp™ Circulating Nucleic Acid kit

MagMAX™ Cell-Free DNA isolation kit

~15 minutes 10 minutes <10 minutes

35 minutes 1.5 hours

spin


MagMAX Cell Free DNA Kit versus Competitor

• Equivalent short DNA yield

• Lower long DNA yield

• Enriched cfDNA

--- MagMAX

--- Q --- MagMAX

--- Q

--- MagMAX

--- Q --- MagMAX

--- Q

4 mL plasma

Automated MagMAX cfDNA Kit versus Q Manual Procedure


Personalized Medicine: The Path Forward

McKinsey & Company, 2013

Genomic Research Ever Expanding List of Genes and Variants


• Requires as little as 10 ng of DNA

• Enables results with limited samples

• Enables detection of multiple biomarkers

• Delivers all of the biomarker results at the same

time

• Custom/pre-designed panels

• Custom Ion AmpliSeq™ Cancer Research Panels

• Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2

• Ion AmpliSeq Cancer Hotspot Panel v2

Ion AmpliSeq™

Technology

Identify Mutations - NGS Cancer Research Panels


140 bp Design for cfDNA Now Available at AmpliSeq.com

Custom designs for genes and variants


Optimized Designs for Improved cfDNA Performance

60 80 100 120 140

01

00

00

20

00

03

00

00

40

00

0CHPv2 Sample 4075 Run 48030

Insert length

Ave

rag

e d

ep

th

Tumor cfDNAMatched normal DNA

60 80 100 120 140

01

00

00

20

00

03

00

00

40

00

0

CHPv2 Sample 4067 Run 48054

Insert lengthA

ve

rag

e d

ep

th

Tumor cfDNAMatched normal DNA

01

00

00

20

00

03

00

00

40

00

00

10

00

02

00

00

30

00

04

00

00

• cfDNA exhibits shorter DNA fragments

• Shorter amplicons yield higher coverage with cfDNA samples

Avera

ge D

epth

0

10,0

00

20,0

00

30,0

00

40,0

00

Insert Length

60 80 100 120 140 60 80 100 120 140

02

00

00

40

00

06

00

00

Insert length

Co

ve

rag

e

140bp

175bp

Covera

ge

Insert Length

60 80 100 120 140

0

20,0

00

40,0

00

60,0

00

140 bp

175 bp

Tumor cfDNA

Matched

Normal DNA

Tumor cfDNA


Enabling 1% Limit of Detection with Ion Reporter Software

• Ion AmpliSeq Cancer Hotspot Panel v2

• Ion Reporter™ Software v4.4

• Detectable variant types: SNPs, Indels <10 bp, Indels not part of homopolymers ≥5

• Same performance was verified on normal, CTC and cell-free DNA samples across 20 archived samples

Hotspots de novo whole target region

Sample MegaMix 1% dilution MegaMix 1%

dilution Normal cfDNA

1% dilution Tumor cell lines

1% dilution

Type SNP/Indel SNP/Indel SNP SNP

Number 275/15 ~390/~15 12 14

Sensitivity >99%/>99% >97%/>99% >99% >99%

FP/per sample 0 1 1 1


Different Techniques Have Different Detection Limits

Technique Limit of

Detection

Optimal

Application

Sanger Sequencing

Pyrosequencing 10% Tumor tissue

Quantitative PCR 5% Tumor tissue

NGS 2% Tumor tissue

ARMS 1% Tumor tissue

Digital PCR, Beaming,

Droplet PCR

0.002%

cfDNA, CTC, rare

variant in tumor

tissue


Unique Value for Research in Peripheral Monitoring

Powerful tools for all stages of cancer research

• Sequence and identify somatic mutations with Ion Torrent platforms

• Orthogonal verification of low frequency somatic mutations (as low as 0.1%) is enabled with the

QuantStudio™ 3D Digital PCR System

We enable an end-to-end solution

for cancer research

Identify mutations Verify low

frequency

mutations

Ongoing analysis

of mutations

Identify new mutations

in metastatic research

samples

Solid tumor Peripheral blood


Wet-lab Verified Assays for Rare Allele Detection

• Research assays are designed to quantify mutations present at 0.1%

(many can go as low as 0.01%)

• All our assays are covered under our TaqMan Assays qPCR Guarantee Program*

• Assays available in 12-reaction and large-scale sizes

• More than 100 assays available

Wet-lab verified dPCR Rare

Mutation Analysis TaqMan™

Assays

QuantStudio 3D Digital PCR

System

Rare Mutation Analysis Software

Tools in Analysis SuiteTM

Software

*Terms and conditions apply. For complete details go to https://www.lifetechnologies.com/uk/en/home/brands/taqman/taqman-guarantee.html


KRAS G12V quantification by dPCR to 0.1%

A) Targeted mutation rate 10%

Mutation rate detected 10.384%

CI: 8.2235% - 13.076%

B) Targeted mutation rate 1%


CI: 0.722% - 2.507%

C) Targeted mutation rate 0.1%


CI: 0.064% - 0.310%

D) Wild-type control sample


Ch

alle

ng

es

Scalable Solutions for Liquid Biopsy Research

Th

erm

o F

ish

er

Sc

ien

tifi

c S

olu

tio

ns

Sample Collection

Template Isolation

Variant Detection

Analysis

Ion Torrent™

LiquidBiopsy™

Blood Collection Kit

Ion Torrent™

LiquidBiopsy™ Instrument

Applied Biosystems™ MagMAX™ Cell-Free DNA

Kits and Thermo Scientific™ KingFisher™ Flex

Magnetic Particle Processor


• Standardization


molecular analysis

• Standardization

• Contamination


molecular analysis


• Increase number of target

mutations

• Standardization


• Increase number of

target mutations

• False positive rate

Applied Biosystems™

QuantStudio ™ 3D Digital

PCR System

Ion Torrent™

sequencing platforms

Ion Reporter™

Software

CTC Enrichment

cfDNA Isolation


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

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher

Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche

Molecular Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent

Technologies, Inc. LiquidBiopsy is a trademark of Cynvenio Biosystems, Inc.

Liquid Biopsy Research Applications Solutions · Liquid Biopsy Research Applications Solutions ......

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