The world leader in serving science

Xingwang Fang, PhD

Sr. Staff Scientist, Thermo Fisher Scientific

Xingwang.Fang@ThermoFisher.com

A Complete Workflow for Circulating DNA Isolation and Analysis

2

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

3

Challenges

Tissue sampling is invasive

Tissue sampling is limited

Advantages

Archive serial samples

Cost

Information on tumor heterogeneity

Tissue Sample Liquid Sample

Tissue Sampling versus Liquid Biopsy

5

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.

6

Total Exosome Isolation Reagents for Different Body Fluids

Cell Culture Media

Reagent

P/N 4478359

Blood Serum Reagent

P/N 4478359

Blood Plasma Kit

P/N 4484450

Urine Reagent

P/N 4484452 *Patent pending

Reagent for Other

Body Fluids

P/N 4484453

• Maximize recovery of intact exosomes for any type of downstream applications

• Easily isolate exosomes using the simple and reliable protocol

• Avoid time-consuming ultra-centrifugation

• Maximize flexibility– can be scaled up or down depending on the sample size

8

Simultaneous Detection of variants in CTCs, cfDNA and gDNA from a single blood sample

9

Components For Laboratory Use

Kit RUO

• Same formulation

• 50 rxns of 2 mL

• Launched in 2015

• PN: A29319 For 100 extractions of 4 mL plasma

Sample Prep for cell free DNA

Applied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit

10

GuSCN

Detergents

Beads

Bind

Wash

Elute

• GuSCN disrupts nucleosomal unit to release

cfDNA

• Detergents solubilize proteins

• Dynabeads™ MyOne™ SILANE selectively bind

cfDNA

• Streck tube required proteinase K treatment to

remove crosslinking

• Wash Solution removes residual proteins and other

purity

• 80% removal residual salts/detergents from Wash

Solution

• Release DNA in low salt buffer

The Principle of MagMAX cfDNA Isolation

11

Unbiased Recovery of circulating DNA

• Quantitative recovery of cfDNA (100-700bp)

• 100ng of 50bp DNA Ladder spiked into 4 mL Streck-tube plasma and eluted in 50 uL

• 50bp recovery ~50%. Can be increased by using more binding solution

• ≥100 bp recovery ~100%

• cfDNA enrichment due to less efficient elution of DNA >2kb.

12

3 reagents + Dynabeads™

Ship & store at room temp

Convenient

Fast

Lysis/Binding

10min

4x washes

10 min

Elution

10 min

Robust Scalable

Beads (uL) 40 50 60 70 75 60

L/B (mL) 5 4 4.5 5 5.5 6

cfDNA(ng) 43 43 44 44 42 45 45 46 43 43

4mL EDTA plasma. cfDNA yield vs beads / Lysis/Binding Soln usage.

cfDNA analyzed on 2% E-Gel and quantified with Qubit HS dsDNA assay. dsDNA spiked into 4 mL plasma.

DNA quantified with Qubit HS assay cfDNA yield quantified by Bioanalyzer

Lysis/Binding

5 min

3x washes

8 min

Elution

8 min

Up to 20 mL Up to 650 uL

Ease of Use

13

Thermo Scientific™ KingFisher™ Flex Thermo Scientific™

KingFisher™ Duo Prime

24 samples per run 6 samples per run

1.5 mL – 2.5 mL: 1 Lysis/Binding Plate/Row 33 min / run

3 mL – 5 mL: 2 Lysis/Binding Plates/Rows 38 min / run

6 mL – 7.5 mL: 3 Lysis/Binding Plates/Rows 43 min / run

Automation on KingFisher Platforms (1.5–7.5 mL)

14

MagMAX Protocol vs. Competitor

Kit Q

KingFisher Flex KingFisher Duo Prime

24 samples in 38 min 6 samples in 38 min

MagMAX cfDNA

15

Automation vs. Manual

--- manual

--- automated --- manual

--- automated MagMAX cfDNA

Manual vs Automation

using KingFisher Flex

10 mL Plasma

MagMAX automated using KingFisher Flex

Vs

Q manual

4 mL Plasma

--- MagMAX

--- Q --- MagMAX

--- Q

--- MagMAX

--- Q --- MagMAX

--- Q

Equivalent short DNA yield

Lower long DNA yield

Enriched cfDNA

16

MagMAX™ cfDNA isolation from 2nd trimester maternal urine

Urine protocol

IPA+ enhances

small DNA

recovery

Plasma protocol

Plasma protocol (+ preservative)

Urine protocol

Urine protocol (+ preservative)

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NSCLC plasma

Ion AmpliSeq™ Cancer Hotspot Panel v2

Equivalent Performance with Ion AmpliSeq Technology

18

Oncomine™ cfDNA Lung Assay

• New library method for targeted

sequencing of high value variants

• Increased sensitivity to detect rare

variants present at 0.1% in cfDNA

• 35 amplicon panel for Lung

targeting 157 hotspots in 11 genes

Oncomine™ cfDNA Lung Assay

ALK

BRAF

EGFR

ERBB2

KRAS

MAP2K1

MET

NRAS

PIK3CA

ROS1

TP53

19

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

• Requires as little as 10ng of DNA

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

Tumor cfDNA

Matched

Normal DNA

Tumor cfDNA

Optimized Designs for Improved cfDNA Performance

20

Variant calls from FFPE vs Plasma (late stage lung cancer)

Samples Variant FFPE Plasma

1 EGFR-L858R 71.42% 2.62%

2 TP53-R158L 51.89% 4.32%

3 MET-T1010I

KRAS-G12C

43.87%

34.62%

51.75%

0.28%

4 N/A No detection No detection

5 EGFR-L858R

MET-T1010I

TP53-Y220C

58.44%

41.93%

35.54%

7.28%

48.72%

1.93%

6 TP53-R158L 10.19% 1.26%

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Critical to Quality Specifications

22

X Thermo

Fisher

Customer’s Data—Purity, Yield, Scalability, and Automation

23

MagMAX Q Site # (depth high to low)

CV

MagMAX

WG Seq – coverage slightly higher

M Q

Conclusions: • MagMAX compared very

favorably to Q

• Very modest differences are

likely outweighed by

MagMAX’s ease-of-

automation

Targeted Seq – same depth, same CV @ high depth

Customer’s Data—NGS

24

Value of MagMAX cfDNA Isolation Kit

“The MagMAX cfDNA Isolation Kit demonstrated to have a

really easy and quick workflow compared to other

methods, with an excellent yield of cfDNA. We have

decided to implement it in our routine use.”

Beatriz Bellosillo, PhD

Hospital del Mar, Barcelona

25

“The MagMAX cfDNA Kit turned out to be a simpler

and faster method to isolate cfDNA from plasma.

Additionally, the method is better in differentially

isolated cfDNA from contaminant high molecular

weight genomic DNA.”

Value of MagMAX cfDNA Isolation Kit

José Luis Costa, PhD

IPATIMUP, Porto, Portugal

26

1.4%

0.4%

Sequencing cfDNA sample 4 (p.T790M) and Digital PCR verification

Data from José Luis Costa, iS3/IPATIMUP, Portugal

27

2nd-gen MagMAX

- Monosized beads

- Ready to use reagents

- Single elution

Circulating DNA

FFPE

RNA/DNA

Pathogen RNA/DNA

PCR Cleanup

gDNA

Size Selection

In the Future—MagMAX Sample Prep for Broad Applications

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FFPE Sample Prep Protocol Overview

+ DNA Binding Soln

+ Magnetic beads

DNA on

beads

RNA in

liquid

Wash Elute DNA

RNA, miRNA (and

residual DNA) on

beads

RNA,

miRNA on

beads

DNase

(optional)

Elute RNA

miRNA

Wash + RNA Binding Soln

+ Magnetic beads

Digested FFPE

29

DNA Yield and Size

FFPE

Tissue

FFPE

Cells

Filter Beads

10

15

20

25

Block4 MultiMixC Fusion Ctrl

Filter MagGAPDH, 83bp

Nanodrop Qubit

Block Method Avg SD 260/ 280 Avg SD

Block4 Filter 4.45 0.39 1.84 0.52 0.01

Mag 3.52 0.40 1.90 0.59 0.01

MultiMixC Filter 2.57 0.25 1.87 0.55 0.03

Mag 1.34 0.17 1.74 0.39 0.04

Fusion Ctrl Filter 4.43 0.12 1.92 0.68 0.02

Mag 2.54 0.51 1.90 0.54 0.13

Equivalent DNA yield and profile

30

RNA Yield and Size

Nanodrop Qubit

Block Method Avg SD 260/ 280 Avg SD

Block4 Filter 0.56 0.03 1.87 0.38 0.04

Mag 1.41 0.16 1.81 1.38 0.11

MultiMixC Filter 0.37 0.06 1.96 0.17 0.01

Mag 0.92 0.07 2.01 0.99 0.10

Fusion Ctrl Filter 0.52 0.08 1.93 0.38 0.13

Mag 1.94 0.25 1.84 1.91 0.34

20

22

24

26

28

30

32

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Block4 MultiMixC Fusion Ctrl

Filter Mag bAct, 171bp

Mag

Filter

0

5

10

15

20

10

15

20

25

30

35

40

Filter Mag Filter Mag Filter Mag

Block4 MultiMixC Fusion Ctrl

Avg

CTs

+RT -RT dCt

Minimal DNA contamination

GAPDH, 83bp

Higher RNA Yield by all measures

31

Th

erm

o F

ish

er

Sc

ien

tifi

c S

olu

tio

ns

Sample Collection

Template isolation

Variant Detection

Analysis

LiquidBiopsy Blood

Collection Kit

LiquidBiopsy

Instrument enrichment

KingFisher Duo Prime

KingFisher Flex

QuantStudio 3D Digital

PCR System

Ion Torrent platforms

Sequencing Analysis

Ion Reporter Software

v4.4

Ion AmpliSeq Cancer Hotspot Panel v2

Ion Ampliseq Research

Panels

Rare Mutation Analysis TaqMan Assays

Scalable Solutions for Liquid Biopsy Research

MagMAX cfDNA Isolation Kit

32

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

© 2016 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.

Thank you for your attention!