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Horizon DiagnosticsAddressing the Variability of Molecular Assays: The Need for Reference Standards
Jonathan FramptonDiagnostics Product Manager
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Undefined Cell Line Reference Materials
Working with a laboratory that were able to detect EGFR ΔE746-A750 using Sanger Sequencing down to LOD of 2%
EGFR ΔE746-A750Sample Description
Actual Allelic Frequency
1% 7%
2% 14%
5% 28%
7.5% 42%
10% 41%
15% 51%
100% 94%
Digital PCR highlighted that a “2% mutant” was actually a “14% mutant”
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Undefined Cell Line Reference Materials
HCC827 cell line containing EGFR ΔE746-A750
Cell Line EGFR Copy Number
HeLa Source 1 2.6
HeLa Source 2 2.7
HCC827 Source 1 15
HCC827 Source 2 38
Non-integer copy number suggests a heterogeneous cell line populationVariable EGFR copy number depending on the source material
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What is a Reference Standard
Independently/Externally validated reference material
Allows you to understand the sensitivity and limit of detection of your assay
Allows you to monitor the reproducibility of your molecular assay
Quantitative Reference standards are a critical part of the development and QC of molecular assays
Reference Standards will tell you if your assay is working today.
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Using Horizon Discovery’s Gene Editing Platform
“Wild type cell line”
Single Cell Dilute
Clonal wild type cell line Cell Line Validation
Gene Engineering Technology (GENESIS™)
Clonal mutant cell line Cell Line Validation
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Functional Genomics
Disease modelling
Reference Standards
> 40 different parental cell lines now targeted; 500 projects, covering 16 tissue types
Examples of what you can accomplish with GENESIS
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Cell Line Validation
Clonal heterozygousmutant cell line
Cell Validation Test Assay
Confirm identity of parental cell line STR and/or SNP6.0
Confirm integration in the correct locus gDNA locus specific PCR & Sanger Sequencing
Confirm expression of modified allele cDNA-PCR & Sanger Sequencing
Confirm clonality Droplet digital PCR, gDNA PCR & Sanger Sequencing
Confirm gene copy number Droplet digital PCR
Clonal wild type cell line
Fully validated genetically defined X-MAN isogenic cell line pair
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Mutations Engineered
E17K
Q209L
V600E
V600K
V600R
R132C
R132H
G719S
T790M
L858R
L861Q
ΔE746-A750
V617F
S252W
G12A
G12C
G12D
G12R
G12S
G12V
G13D
A146T
T315I
D835Y
L1601P
F1174L
R1275Q
F1245V
Q209L
Q61H
Q61K
Q61L
Q61R
D816V
R140Q
R172K
E542K
E545K
H1047R
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Quantitative Molecular Reference Standards
Genomic DNA Stoichiometric Dilutions
Mutant Wild type
FFPE Processing
FFPE Mixed RatioSections
Mutant Wild type
Genomic DNA Standards verified using Digital™ PCR
Stoichiometric dilutions are accurate down to 0.1% and 0.05%
Gene Mutation Allelic FrequencyB-Raf V600K 50% 10% 5% 1% 0.5% 0.1% 0.05%
K-Ras G12D 50% 10% 5% 1% 0.5% 0.1%
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FFPE Block Production
B-Raf 50% V600K
Prepare block and sectionsB-Raf 100% wild type B-Raf 5% V600KMix cells
9:1
Droplet Digital PCRBioRad QX100
Our proprietary FFPE technology allows us to accurately control:• Number of cells and cores per block• Homogenous cell distribution throughout the block• Allelic frequency• Section thickness & DNA content
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H+E
DNA
H+E Staining & DNA Extraction demonstrates the homogeneity and consistency of our FFPE technology
FFPE Block Consistency
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FFPE Block Allelic Frequency
Gene MutationB-Raf V600E 25% 5% 3.5% 1%B-Raf V600K 50% 5% 1% ---
Consistent allelic frequency throughout the FFPE Blocks
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Quantitative Multiplex DNA Reference Standard
Precise DNA dilutions
Digital PCR Analysis
Quantitative Multiplex DNA Reference Standard
X-Man Genetically Defined Mutant Cell Lines
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Quantitative Multiplex DNA Reference Standard
Permits validation of a wide range of multiplex platforms including Next Generation Sequencing (NGS)
Chromosome Gene Variant %7q34 BRAF V600E 10.5
4q11-q12 cKIT D816V 10.07p12 EGFR ΔE746 - A750 2.07p12 EGFR L858R 3.07p12 EGFR T790M 1.07p12 EGFR G719S 24.5
12p12.1 KRAS G13D 15.012p12.1 KRAS G12D 6.01p13.2 NRAS Q61K 12.53q26.3 PI3KCA H1047R 17.53q26.3 PI3KCA E545K 9.0
Initial offering covers 11 mutations – Available Now
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External Data – Ion Torrent
External partners using independent assays confirm accurate allelic frequencies
Source: Horizon Discovery Partner A Partner B Partner C
Platform: QX100™ Droplet Digital™ PCR System
AmpliSeq Cancer Panel
Ampliseq Cancer Hotspot Panel v2
Ampliseq Cancer Hotspot Panel v2
(Average of 8 runs)
Sequencing Depth N/A 3000-4000x Average 5000x 2000X
Gene Mutation Observed mutant ratio
BRAF V600E 10.2 9.9 9.1 10.3
KIT D816V 10.4 10.0 11.0 10.1
EGFR ΔE746 - A750 2.0 2.3 Not detected Not detected
EGFR L858R 2.7 2.7 2.1 2.4
EGFR T790M 0.9 0.8 Not detected Not detected
EGFR G719S 24.4 23.7 23.1 24.8
KRAS G13D 16.1 16.3 12.35 15.5
KRAS G12D 5.0 5.2 Not detected 5.1
NRAS Q61K 12.8 9.0 12.7 12.6
PIK3CA H1047R 18.6 16.7 16.8 17.9
PIK3CA E545K 8.9 3.2 8.4 8.8
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Gene Variant Freq %Core mutations
BRAF V600E 10.5cKIT D816V 10.0EGFR ΔE746 - A750 2.0EGFR L858R 3.0EGFR T790M 1.0EGFR G719S 24.5KRAS G13D 15.0KRAS G12D 6.0NRAS Q61K 12.5
PI3KCA H1047R 17.5PI3KCA E545K 9.0
Additional mutationsALK P1543S 33.0APC R2714C 33.0
FBXW7 G667fs 33.5FGFR1 P150L 8.5FLT3 S985fs 10.5FLT3 V197A 11.5IDH1 S261L 10.0MET V237fs 6.5
MLH1 L323M 8.5NOTCH1 P668S 31.5NTRK1 5'UTR 8.5
PDGFRA G426D 33.5ABL2 P986fs 8.0
BRCA2 A1689fs 33.0NF2 P275fs 8.0
ARID1A P1562fs 33.5CDX2 V306fs 41.5EP300 K291fs 8.0
NF1 L626fs 7.5
Expanded Datapack Multiplex Reference Standard
• Expanded digital PCR analysis• Covers 30x mutations• Additional (non-Oncology) mutations are
predicted to be present
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Generated the multiplex standards using the same background (RKO or SW48)
Base mutations present at >10% allelic frequency
Can be titrated up or down to analyse LOD of platform/panel
Gene Variant Freq %Core mutations
KRAS G12D 16.7KRAS G13D 16.7KRAS A146T 16.7
Additional mutationsALK P1543S 50.0APC R2714C 50.0
CTNNB1 S33Y 50.0EGFR G719S 33.3
FBXW7 G667fs 50.0NOTCH1 P668S 50.0PDGFRA G426D 50.0
Gene Variant Freq %Core mutations
EGFR L861Q 12.5EGFR ΔE746-A750 12.5EGFR L858R 12.5EGFR T790M 12.5
Additional mutationsFGFR1 P150L 50.0MLH1 L323M 50.0BRAF V600E 66.7CDG1 3’UTR 50.0
PI3KCA H1047R 50.0
EGFR & KRAS Titratable Multiplex Reference Standards
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FFPE DNA vs Normal DNA
Gene Variant dPCR gDNA NGS gDNA dPCR FFPE NGS FFPE
BRAF V600E 10.5 9.3 9.5 8.5KIT D816V 10 10.8 9 8.6
EGFR ΔE746 - A750 2 1.9 1.5 0EGFR L858R 3 2 2.5 0EGFR T790M 1 1 1 0EGFR G719S 24.5 28.3 26 16KRAS G13D 15 17.1 16.5 17.2KRAS G12D 6 7.4 5.5 6.4NRAS Q61K 12.5 10.7 12 8.2
PIK3CA H1047R 17.5 18.2 15.5 18.5PIK3CA E545K 9 9.9 10 7.3
PGM fails to detect the lower allelic frequencies in FFPE sample
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Custom Multiplex Options
Cover a range of mutations including:• Single nucleotide polymorphisms • insertion-deletions• 5’UTRs• Frameshifts
DNA or FFPE format available (Xenograft an option)
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Mutations Engineered
E17K
Q209L
V600E
V600K
V600R
R132C
R132H
G719S
T790M
L858R
L861Q
ΔE746-A750
V617F
S252W
G12A
G12C
G12D
G12R
G12S
G12V
G13D
A146T
T315I
D835Y
L1601P
F1174L
R1275Q
F1245V
Q209L
Q61H
Q61K
Q61L
Q61R
D816V
R140Q
R172K
E542K
E545K
H1047R
Your Horizon Contact:
Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 655 581 Email: [email protected] Web: www.horizondx.com
Dr Jonathan FramptonProduct [email protected]+44 (0) 1223 815299
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