Aug2014 horizon dx

Redefining Reference Standards

Dr Jonathan FramptonProduct Manager

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Disclaimer

This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or apply for or enter into any contract or make any other commitment whatsoever in relation to, securities. Although reasonable care has been taken to ensure that the facts stated in this Presentation are accurate and that the opinions expressed are fair and reasonable, the contents of this Presentation have not been formally verified by Horizon Discovery plc (the “Company”) or any other person. Accordingly, no representation or warranty, expressed or implied, is made as to the fairness, accuracy, completeness or correctness of the information and opinions contained in this Presentation and no reliance should be placed on such information or opinions. Further, the information in this Presentation is not complete and is subject to updating, revision, further verification and amendment. Neither the Company, nor any of its subsidiaries, nor any of its respective members, directors, officers or employees nor any other person accepts any liability whatsoever for any loss howsoever arising from any use of such information or opinions or otherwise arising in connection with this Presentation.

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Impact of formalin treatment on template and assay performance

Goal:

To assess the effect of formalin on genomic DNA and the on assay performance for somatic variant detection.

1. Created matched mutant and wild type cell lines

2. Generated precisely defined HDx™ mixed cell pellets

3. Formalin treat one pellet from each pair

4. Extract genomic DNA from both pellets

5. Quantitate gDNA 6. Assess allelic frequency by dPCR

Defined HDx™ cell line mixture

No Formalin Treatment

Formalin Treatment

DNA Extraction DNA Extraction

DNA Quantification

DNA Quantification

DNA quantification of formalin-compromised gDNA

Three methodologies employed to perform quantitation:• Quantifluor Assay • Agilent Tapestation • Nanodrop

Observations:

1. There is variation in the concentration of DNA from matched pairs (overestimation in formalin vs no formalin).

2. The Nanodrop data shows a greater overestimation of concentration in formalin vs no formalin samples from matched pairs.

Mutant detection on formalin-compromised DNA by digital PCR

Non-compromised DNA

Formalin-compromised DNA[bp]

Sample Expected Genotype Formalin Treatment

Mutant Allelic Frequency Measured

1 5.0% B-Raf V600E - 5.2

2 5.0% B-Raf V600E + 5.5

3 2.5% B-Raf V600E - 2.7

4 2.5% B-Raf V600E + 3.7

5 1.0% B-Raf V600E - 1.0

6 1.0% B-Raf V600E + 1.3

7 0.5% B-Raf V600E - 0.6

8 0.5% B-Raf V600E + 0.6

9 0.2% B-Raf V600E - 0.2

10 0.2% B-Raf V600E + 0.4

TapeStation analysis (above) and digital PCR genotyping (below) of matched pairs.

Expected and measured allelic frequenciesObservations:Begin to see the subtle variation in variant calling between formalin vs no formalin matched pairs.

Implications:Artifacts – eg effects of formalin on DNA by deamination affect variant calling, potentially by increasing the mutant to wild type ratio. Sample Expected Genotype Formalin

TreatmentMutant Allelic

Frequency Measured

1 5.0% Mutant - 5.2

2 5.0% Mutant + 5.5

3 2.5% Mutant - 2.7

4 2.5% Mutant + 3.7

5 1.0% Mutant - 1

6 1.0% Mutant + 1.3

7 0.5% Mutant - 0.6

8 0.5% Mutant + 0.6

9 0.2% Mutant - 0.2

10 0.2% Mutant + 0.4

Mut

ation

Fre

quen

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Impact of Formalin treatment on wild type samples

Formalin Intensity

1. Utilised clonal wild type cell line2. Treated cell pellets with four different

formalin conditions3. Analyzed allelic frequency by digital PCR

Sample Expected Genotype Mutant Allelic Frequency Measured

1 0% Mutant 0.04%

2 0% Mutant 0.04%

3 0% Mutant 0.07%

4 0% Mutant 0.15%

Sample preparation may interfere with assay sensitivity and specificity

Mut

ation

Fre

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Case Study 2. Tru-Q NGS Reference Standards

EGFR mutants

K-Ras mutants

B-Raf mutants

N-Ras mutants

PIKCA mutants

Quantification by Droplet Digital PCR

C Blend 110 mutations

at 5%


at 5%


at 5%

A Blend40 Mutations

@ 1.3%

B Blend 120 Mutations

at 2.5%

B Blend 220 Mutations

at 2.5%


at 5%

14 Additonal Biomarkers

1.3%20 copies per μl




Dilution Series with wild type

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Source:Horizon

DiagnosticsPredicted %

Horizon DiagnosticsObserved %

Partner

Platform: N/AQX100™ Droplet

Digital™ PCR System

Ion Torrent

Gene Mutation

BRAF V600M 4.0 4.4 3.5

EGFR T790M 4.2 3.9 4.3

EGFR L858R 4.2 4.2 3.5

EGFR L861Q 4.2 4.1 3.6

KIT D816V 5.0 5.4 6.4

KRAS G12A 5.0 5.7 4.9

KRAS G12R 5.0 5.2 4.6

NRAS Q61K 5.0 4.9 3.3

Case Study 2. Data for Tru-Q NGS Reference Standards

Specific and Sensitive down

to 5% allelic frequency

Horizon DiagnosticsPredicted %


Partner

N/AQX100™ Droplet


Ion Torrent

2.0 2.2 2.1

2.1 2.0 2.1

2.1 2.0 2.3

2.1 2.1 1.8

2.5 2.6 3.2

2.5 3.0 2.5

2.5 2.9 2.6

2.5 2.5 2.5

Horizon DiagnosticsPredicted %


Partner

N/AQX100™ Droplet


Ion Torrent

1.0 1.0 1.9

1.0 1.1 missing

1.0 1.1 missing

1.0 1.0 missing

1.3 1.3 1.5

1.3 1.4 missing

1.3 1.3 missing

1.3 1.2 missing

Specific and Sensitive down

to 2.5% allelic frequency

Not sensitive to detect down

to 1% for all variants

5% blend 2.5% blend 1.3% blend

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G12V

Horizon Diagnostics’ suite of reference material includes standards for the increasing number of ‘rare’ mutations being targeted for cancer therapeutics, which by definition are hard to find in clinical samples.

E17K

Q209L

V600E

V600K

V600R

R132C

R132H

G719S

T790M

L858R

L861Q

ΔE746-A750

V617F

S252W

G12A

G12C

G12D

G12R

G12S

G12V

G13D

T315I

D835Y

L1601P

F1174L

R1275Q

F1245V

Q209L

Q61H

Q61K

Q61L

Q61R

D816V

R140Q

R172K

E542K

E545K

H1047R

EML4/ALK V600M

V600G

Δ1836

ABL1 AKT1 ALK B-Raf cKIT EGFR FGFR2

FLT3 GNAQ GNA11 IDH1 IDH2 JAK2 K-Ras

NOTCH1 MET N-Ras MLL PI3K

Y1253D MLL/ENL

PTEN

ΔEX6/EX7

ROS1

ROS1

RUNX1

RUNX1/RUNX1T1

Q61H

A146T

Mutation Coverage

Healthcare

Aug2014 horizon dx