©2013 Promega Corporation.

Reliable Purification of Amplifiable DNA from FFPE Using the Maxwell® CSC Instrument

Doug Horejsh, Ph.D., Sr. Research Scientist - R&D

Outline

• Introduction to DNA purification from Formalin Fixed, Paraffin Embedded (FFPE) samples

• Challenges of qualifying the extracted DNA for downstream amplification

• Data demonstrating the successful and consistent purification of gDNA from breast, lung and colon FFPE samples.

Why Use FFPE Samples? Valuable Resource for Pro- & Retrospective Studies

3

• Provides opportunity to correlate disease state and tissue morphology with genotype (DNA) and/or gene expression (RNA)

• Common method for archival of pathology tissue specimens • Very common with cancer samples

• Some FFPE tissue archives date back 50 years or more

• Valuable tool for retrospective studies of disease

• Opportunities for MDx and CDx applications

Preservation of Samples by Formalin Fixing and Paraffin Embedding

4

Denatures protein, crosslinks with DNA

Fix tissue (formalin, neutral pH)

Dehydrate 1. Ethanol 2. Xylene (Toluene)

Paraffin embed (hot wax)

Sectioning and Storage 1. Microtome 2. Store at room temperature

Challenges

5

• Formalin fixation crosslinks proteins and nucleic acids • Nucleic acids are partially degraded/fragmented • Degree of fragmentation varies depending on:

• Sample type and age • Fixation conditions

• Formalin fixation effects downstream applications

• Crosslinking makes nucleic acid inaccessible to amplification enzymes • Formalin residuals inhibit enzyme activity during extraction and PCR • Degradation/fragmentation inhibits amplification

• Deparaffinization often necessitates use of xylene or other organics • Hazardous

Current Research Offerings for FFPE DNA Extraction Manual & Automated Extraction Options Available

Manual Kit

• ReliaPrep™ FFPE gDNA Miniprep System

Automated Systems

• Maxwell® 16 Instrument & Kits

• Maxwell® 16 FFPE Tissue LEV DNA Purification Kit

• Maxwell® 16 FFPE Plus LEV DNA

Purification Kit

For Research Use Only. Not for Use in Diagnostic Procedures

Maxwell® Instruments and Chemistry Multiple Instruments to Match Your Needs

IVD Instruments: Maxwell® CSC Instrument

• Blood gDNA Purification Kit currently available • FFPE gDNA Purification Kit (coming soon, today’s discussion) • IVD, 21 CFR Part 11 compliant

• Intended for In Vitro Diagnostic Use. This product is currently

only available in US.

Research Instruments: Maxwell® 16 Instruments

• Multiple purification kits available for a variety of sample types • Blood, plasma, serum, buffy coat, buccal swabs, tissue,

FFPE tissue, CSF, amniotic fluid, nasal washes • Available with (AS3000) or without (AS2000) sample tracking

and UV light for decontamination

• Intended for Research Use Only. Not For Use In Diagnostic Procedures.

LEV Kits Maxwell® CSC and Maxwell® 16

Research Instruments

Maxwell® Instruments and Chemistry Easy-to-Use, Particle Mover Automation

2 3 4 5 6 7

1 3 4 5 6 7

2 3 4 5 6 7

1 3 4 5 6 7 2

LEV Plunger

Cleared Lysate

1 3 4 5 6 7 8

3 4 5 6 7 2 8

1 4 5 6 2 8 2 1 4 5 6

Mixing

Capture

Binding

Washing &

Elution Elution Tube

(300µl) Elution Tube (30-100µl)

SEV Kits Maxwell® 16 Research

Instruments only

Maxwell® Instruments and Chemistry Improved Consistency with Less Hands-on Time

• Process 1 to 16 samples in 30-60 minutes depending on sample/kit

• Bench top automation provides:

• Consistent results

• High yields of gDNA or RNA depending on sample type

• Sample tracking available

• Configured for large (SEV, 200-400µl) or small (LEV, 30-100µl) elution volumes

• Compact footprint conserves valuable bench space

For Research Use For In Vitro Diagnostic Use

Easy Pre-processing 1. Scrape sections 2. Proteinase K solution and Incubation Buffer 3. Incubate the sample at 70°C overnight 4. Lysis Buffer 5. Transfer to Maxwell® 16 Kit cartridge 6. Run automated extraction protocol

• Maxwell® 16 FFPE Tissue LEV DNA Purification Kit (optimal purity) • Maxwell® 16 FFPE Plus LEV DNA Purification Kit (optimal yield) • For Laboratory Use

Maxwell® 16 Kits for DNA Purification from FFPE Optimized for Different Results

New Development Efforts: Maxwell® CSC FFPE DNA Extraction Kit • Optimize purity AND yield • Designed under Design Control and manufactured under cGMP • For In Vitro Diagnostic Use

Purification from Various FFPE Tissues

Maxwell® CSC System Nucleic Acid Extraction for IVD Use

11

This product is only available in the USA and is intended for in vitro diagnostic use.

Maxwell® CSC Instrument Clinical Sample Concentrator

• Manufactured under cGMP • Class I Exempt Medical Device (Clinical

Sample Concentrator ) • IQ / OQ available • Windows 7 Software: WiFi Capable /

Data Ready

For more information on the Maxwell® CSC Instrument, view the recorded webinar at: http://www.promega.com/resources/webinars/worldwide/archive/maxwell-csc-cgmp-compliant-automated-dna-extraction-from-blood/

Development of a Maxwell® CSC FFPE DNA Kit

Goal: Develop an IVD kit for use on the Maxwell® CSC Instrument

• What are the key metrics to measure success in DNA purification from FFPE samples?

• Purified DNA quantity and quality

• Once the metrics are identified, how will the purification chemistry be characterized and tested?

• Robust chemistry

• Development characterization

• Stringent product requirements

• Verified through extensive testing

Typical In-Line Measures of Yield and Quality Use to Predict Downstream Assay Success

• Factors that impact quality:

• Purification chemistry contaminants

• Cross-linked templates

• Fragmentation

• Concentration by:

• Absorbance

• Fluorescent dye

• Amplification (qPCR)

• Purity by:

• Absorbance ratio

• Internal positive control (IPC) to measure inhibition

• All are used to predict likelihood of success in downstream assays

Assess DNA Quantity/Quality by Absorbance Not Reliable for DNA Extracted from FFPE Samples

• Note the large peak at 225nm in the Chemistry B preparation

• Concentration and ratios are not useful at low absorbance levels

• Absorbance and amplifiability may correlate, but several other factors play a role

Chemistry A Chemistry B

Large peak at 225nm No peak at 225nm

gDNA Extraction from Matched Lung Tissue FFPE Slides

Assess DNA Quantity/Quality by Absorbance Not Reliable for DNA Extracted from FFPE Samples

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

breast breast colon colon lung lung

Chem A Chem B Chem A Chem B Chem A Chem B

ng/

ul

Tissue/method

Quantitation by Absorbance

0.0

1.0

2.0

3.0

4.0

5.0

6.0

breast colon lung

ng/

ul

Quantitation by Amplification

Chem A

Chem B

Large difference in quantitation • Absorbance at 260nm may not

be an accurate measure of amplifiable yield

Purity Ratios May Be More Predictive of Amplification Success

0.00

0.50

1.00

1.50

2.00

2.50

breast breast colon colon lung lung

Chem A Chem B Chem A Chem B Chem A Chem B

Tissue/Method

Purity by Absorbance Ratios Dark: 260/280 Light: 260/230

• These panels show that higher A260/230 ratios may correlate with amplification success

• Contribution of purification chemistry

• Study absorbance ratio against amplifiability

0.0

1.0

2.0

3.0

4.0

5.0

6.0

breast colon lung

ng/

ul

Quantitation by Amplification

Chem A

Chem B

No Correlation between A260/A230 and Amplifiability

• Input normalized to A260; Samples run using quantitative PCR

• Variety of chemistries tested across a single tissue type (breast) to include a range of A260/A230 ratios

• No correlation between A260/A230 and amplifiability • For QC, a more predictive test is needed

Is an Internal Positive Control (IPC) Assay a Better Alternative?

0

0.2

0.4

0.6

0.8

1

1.2

Sample 1 Sample 2

A260/A230 Ratio

0.57

0.20

0.1

1

Sample 1 Sample 2

Relative Amp for IPC

0

2

4

6

8

10

12

14

Sample 1 Sample 2

Target Amplification (ng/µl)

• The purity measure, A260/A230 ratio, fails to correlate with amplification • The internal positive control assay shows relative amplification that tracks

with amplification of a target in the FFPE sample

• Eluate inhibition is tested using a commercial internal positive control assay • Amplification target is added with master mix and purification eluates are

compared with no inhibition controls (water)

Many Factors Influence Amplifiability Yield, Purity, Inhibitors and Fragmentation

0.37

0.78

0.100

1.000

Sample 3 Sample 4

Relative Amp for IPC

0

1

2

3

4

5

6

Sample 3 Sample 4

Target Amplification (ng/µl)

• These two samples have similar concentrations and purity ratios • The IPC assay fails to track with amplification success for some samples

• Another factor appears to be playing a role here – fragmentation of DNA • Yield, purity ratios, inhibition, fragmentation are all involved

Chemistry Choice Affects Average gDNA Fragment Size

Chemistry A

Chemistry B

Highly fragmented, Less amplifiable, higher A260

More intact, More amplifiable

bp

Am

ou

nt

of

nu

cle

ic a

cid

• Important to note that fragmentation occurs as part of fixation

• Highly fragmented gDNA is less amplifiable, as the peak is about 100bp

• The difference in area under the curve >300bp is the advantage of Chem A over Chem B

Fragment Size Impacts Amplifiability

Chemistry A

Chemistry B

≈ 200bp

200bp

1 100

101 200

…

102 201

200 300

…

bp

Am

ou

nt

of

nu

cle

ic a

cid

Amplification of 100bp target is at best 50% with an average template fragment size of 200bp qPCR amplicon – 100bp Sanger seq – 250bp

Benefits of Automated FFPE DNA Extraction with the Maxwell® CSC DNA FFPE Kit

No xylene use equals improved safety

Easy to use system means fewer errors

Reduced hands-on time increases efficiency

High quality, less fragmented DNA improves downstream assays

= Reliable Amplification

Prototype Chemistry Identified Based on Initial Testing Shows Promise

The entire process was optimized to ensure and improve robustness

Samples • Adjacent slides from

multiple tissues • Relative comparison to

competitor chemistry

Method 1. Deparaffinization with

mineral oil 2. Lysis / Pro K treatment 3. Decrosslinking 4. RNase treatment 5. Purification on Maxwell® 6. Elution

Parameters Studied in Design Development (Evaluation of Robustness)

• Tissue input

• Lysis volume

• Pro K:

• Amount

• Temperature

• Lot variability

• De-crosslinking:

• Time

• Temperature

• Lysate carryover

• Cartridge components

• Elution volume

0

2

4

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12

14

16

0.25 curls 0.5 curls 1 curl 2 curls 3 curls

N = 4 per condition T-test p<0.01 for all r-squared = 0.979

Tissue Input: Number of Curls Scales with Recovery

• Direct correlation between amount of FFPE tissue input and amount of DNA recovered

• Additional starting material had no impact on qPCR inhibition as measured with an IPC (data not shown)

Elu

ate

DN

A C

on

cen

trat

ion

(n

g/µ

l)

Blue Dye Added to the Lysis/Pro K Solution to Improve pre-processing

Benefits of Dye Inclusion: Fewer pipetting steps Easier aqueous transfer Sample tracking into cartridge Potentially better performance

• Pipetted colorless aqueous layer from under mineral oil layer in prototype • Added inert blue dye to Lysis/Pro K step – no effect on chemistry

Dye included in final kit

Extraction Cartridges Pre-processed Samples with & without Dye

qPCR-based Quantitation

Transfer of Aqueous Phase

Decrosslinking Time & Temperature Assessment 4 Hours at 80°C Optimal for Breast Samples

28

• 4 slides each condition, samples not pooled

• 80°C for 4 hours produces most amplifiable DNA for both 100bp and 300bp amplicons

100bp Amplicon (pg/µl)

300bp Amplicon (pg/µl)

29

100bp Amplicon (pg/µl)

300bp Amplicon (pg/µl)

Decrosslinking Time & Temperature Assessment 4 Hours at 80°C Optimal for Colon Samples

• 4 slides each condition, samples not pooled

• 80°C for 4 hours produces most amplifiable DNA for both 100bp and 300bp amplicons

30

• 4 slides each condition, samples not pooled

• 80°C for 4 hours appears to be better for the 100bp amplicon, with minor effect on 300bp amplicon

100bp Amplicon (pg/µl)

300bp Amplicon (pg/µl)

Minor decrease

Best

Decrosslinking Time & Temperature Assessment Minor Decrease for Lung Samples at 80°C, 4 Hour

Decrosslinking Experiments Summary 4 Hours at 80°C Provided Best Overall Results

• 4-hour decrosslinking at 80°C improved performance (amplification of larger amplicons) for most tissues

• Majority of samples tested

• 4-hour decrosslinking had minor impact on performance when 1-hour decrosslinking was optimal

• Minority of samples tested

4-hour decrosslinking is used in the final kit format

No Inhibition Observed Using Internal Positive Control (IPC) Assay

• Eluate inhibition is tested using a commercial internal positive control assay • Amplification target is added with master mix and purification eluates are

compared with no inhibition controls (water)

• No inhibition seen in these eluates using a common commercial qPCR master mix

1.000 1.081 0.925 0.878

0.100

1.000

No inhib Breast Colon LungRe

lati

ve a

mp

lific

atio

n o

f IP

C

Sample eluate (exp n=6, PCR n=2)

• Another check-in compared with competitor to complete development and lock chemistry/process for verification

Promega vs. Competitor FFPE DNA Extraction More Amplifiable DNA Extracted with Promega Kit

549

1009

0

200

400

600

800

1000

1200

QiaAmp FFPE DNA Promega FFPE DNA

Co

pie

s /

mic

rolit

er

102bp Amplicon

Mounted Slides (5 micon): Breast Tumor Tissue, n=4

Competitor Promega

21

50

0

10

20

30

40

50

60

70

QiaAmp FFPE DNA Promega FFPE DNA

Co

pie

s /

mic

rolit

er

164bp Amplicon

Competitor Promega

Maxwell® CSC FFPE DNA Purification Protocol ~5 Hours of Preprocessing + 50 Minute Run Time

Step Action

1 Scrape entire paraffin sample into individual tube.

Spin full speed briefly in microfuge to bring material down.

2 Add 300µl of mineral oil to each sample

Vortex 10 seconds to mix

3 Incubate at 80°C for 2 minutes

Vortex to mix

4

Prepare a master mix of the Lysis buffer, Proteinase K and blue dye as shown below.

Reagent Amount/reaction Reactions (n+2) Total

Lysis Buffer 224µl 18 4032µl

Proteinase K 25µl 18 450µl

Blue Dye 1µl 18 18µl

5 Add 250µl of master mix to each sample tube, and vortex for 5 seconds.

6 Centrifuge at 10,000 x g for 20 seconds to separate layers. Gently mix aqueous phase with pipet to

break up pellet if present.

7 Incubate at 56°C for 30 min

8 Incubate at 80°C for 4 hours

9 Allow the sample to cool to room temperature. (~5 minutes)

10 Add 10µl of RNase A directly to the lysed sample in the lower phase. Pipet to mix

Incubate at room temp for 5 minutes

11 Spin sample full speed 2min. Add sample to cartridge, leaving pellet if present

12 Run Method

Similar Extraction Results from Ribbon Curl and Slide Samples

10

100

1000

10000

Breast Colon Lung

Me

an c

op

ies/

uL

RNaseP

TERT 164

Ribbon Curls Slides

10

100

1000

10000

Breast Colon Lung

RNaseP

TERT 164

No Increase in PCR Inhibitor Co-purification with Increasing Tissue Section Input

Group Number of samples

Mean Ct Mean Ct

No inhibition control 12 30.79 0.00

Double Tissue Input Samples 16 30.72 0.07

IPC Block 2 No Ct N/A

• Purifications done from either one or two sections to assess linearity • IPC assay was performed to ensure that no inhibitors were co-purified • Eluate DNA concentration is linear with input

Run Number of samples

Mean Ct Mean ΔCt

No inhibition control 12 30.73 0.00 Breast 16 30.63 0.11 Colon 16 30.71 0.02 Lung 16 30.66 0.08

IPC Block 2 No Ct N/A

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Single Section Double Sections

Me

an c

op

ies/

µL

RNase P

TERT 164

Eluates are Stable Under Multiple Storage Conditions

10

100

1000

10000

Breast Colon Lung

Me

an c

op

ies/

µl

Tissue

RNase P

TERT

• Eluates were stored for 1 week at 4°C or for 1 month at -20°C • 102bp and 164bp amplicons were tested

10

100

1000

10000

Breast Colon Lung

Tissue

RNaseP

TERT 164

1 Week at 4°C 1 Month at -20°C

Summary

• Interest in FFPE samples in the molecular diagnostics lab is rapidly increasing

• Molecular and Companion Diagnostic assays

• Target amplification-based metrics are the most useful quality control

• Absorbance, absorbance ratios, and IPC can be affected

• Assessment by qPCR ensures downstream success in PCR-based assays

• Promega has developed a new FFPE DNA kit for use on the Maxwell® CSC Instrument

• Kit was developed for in vitro diagnostic use in combination with the Maxwell® CSC Instrument

• Intended for use with breast, lung and colon FFPE samples

• Well controlled manufacturing processes – manufactured under cGMP

• Consistently extracts more amplifiable DNA than competitor kits

©2013, Promega Corporation.

Thank you for your time and attention Questions are welcome