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Dr Jonathan FramptonProduct Manager, Diagnostics
The Effects of Formalin on Clinical Diagnostics
Twitter: @HorizonDX_news
Dr Hadas AmitProduct Development, Diagnostics
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Dr. Jonathan Frampton, PhD
Product Manager, Diagnostics
In his role, Jonathan works closely with a broad range of European, North American and EMEA oncology-focussed Quality Assurance Schemes with the goal of driving the standardization and normalization of molecular assays across the globe. Jonathan holds a PhD from University of Sussex in Genomic DNA Damage and Stability and has extensive product development experience through previous roles including Cambridge-based antibody company Abcam.
Presenters
What is the impact of assay failure in your laboratory and how do you monitor for it?
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3
FFPE Sample
Cancer Patient DNA Extraction Diagnosis
Driving better treatment for cancer patients
Application of Companion Diagnostics
Pre-Analytical Analytical
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Biopsy DNA Quantification
StorageDNA Extraction
DNA Quantity & Quality
Pre-analytical Sample Handling and Processing
FFPE Processing
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DNA Sample AnalysisActionable
Decision
Quality of Diagnostic Result
Sample preparation
Analytical Processing and Reporting
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Impact of Sample Processing and Formalin Treatment
Pre-analytical
Analytical
DNA Quantification
Decreased DNA Yield
Decreased DNA Quality
QuantificationErrors
DNA
Cross-linking
Oxidative damage
Methylation
FragmentationNecrosisSample
Apoptosis
DNA ExtractionFormalinTreatment
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Sample Analysis
DNA QuantificationErrors
False PositiveMutations
False NegativeMutations
Assay Sensitivity
Assay Specificity
Impact of Formalin Treatment on Clinical Diagnostics
InaccurateActionable
Decision
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Dr. Hadas Amit
Senior Scientist, Diagnostics
Hadas is a senior scientist with a strong interest in the future of personalised medicine with focus on advances in cell free tumor DNA (cfDNA). Hadas is leading the research and development of Formalin-Compromised Reference Standards at Horizon Diagnostics.
Presenters
“Wild type cell line”
Single Cell Dilution
Clonal mutant cell line
Pre-EngineeringCell Line Validation
SNP 6.0
Sanger Sequencing
Digital PCR
RT-PCR
Post-EngineeringCell Line Validation
Gene Editing Platform
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How are HDx Reference Standards Manufactured and Validated?
Genomic DNA Stoichiometric Dilutions
Mutant Wild type
Dilutions are accurate down to 0.05%
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Analyzed Allelic Frequency Down to 0.05%
Goal:
To assess the effect of formalin on genomic DNA and the on assay performance for somatic variant detection.
Defined BRAF V600E Cell Line Mixture
No Formalin Treatment
Formalin Treatment
DNA Extraction DNA Extraction
DNA Quantification
DNA Quantification
Digital PCR Analysis
Digital PCR Analysis
Impact of Formalin Treatment on Template and Assay Performance
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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.
DNA Quantification of Formalin-Compromised DNA
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Goal:
To assess the effect of formalin on genomic DNA and the on assay performance for somatic variant detection.
Defined BRAF V600E Cell Line Mixture
No Formalin Treatment
Formalin Treatment
DNA Extraction DNA Extraction
DNA Quantification
DNA Quantification
Digital PCR Analysis
Digital PCR Analysis
Impact of Formalin Treatment on Template and Assay Performance
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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
Digital PCR genotyping 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:Artefacts – eg effects of formalin on DNA by deamination affect variant calling, potentially by increasing the mutant to wild type ratio.
Sample Expected Genotype Formalin
Treatment
Mutant Allelic
Frequency
Measured
1 5.0% BRAF V600E - 5.2
2 5.0% BRAF V600E + 5.5
3 2.5% BRAF V600E - 2.7
4 2.5% BRAF V600E + 3.7
5 1.0% BRAF V600E - 1
6 1.0% BRAF V600E + 1.3
7 0.5% BRAF V600E - 0.6
8 0.5% BRAF V600E + 0.6
9 0.2% BRAF V600E - 0.2
10 0.2% BRAF V600E + 0.4
Mutant Detection on Formalin-Compromised DNA by Digital PCR
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Formalin Intensity
1. Utilized 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% EGFR T790M 0.04%
2 0% EGFR T790M 0.04%
3 0% EGFR T790M 0.07%
4 0% EGFR T790M 0.15%
Sample preparation may interfere with assay sensitivity and specificity
Impact of Formalin Treatment on Wild Type Samples
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EGFR Formalin-Compromised HDx Reference Standards
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Sample Expected Genotype Formalin
Treatment
1 3.0% EGFR T790M -
2 3.0% EGFR T790M +
3 0.75% EGFR T790M -
4 0.75% EGFR T790M +
5 0.13% EGFR T790M -
6 0.13% EGFR T790M +
7 0% EGFR T790M -
8 0% EGFR T790M +
Using blinded samples of Formalin-Compromised HDx Reference Standards to develop new assays and assessing limit of detection
Using blinded samples of Formalin-Compromised HDx Reference Standards to develop new assays and assessing limit of detection
EGFR Formalin-Compromised HDx Reference Standards
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Sample Expected Genotype Formalin
Treatment
Mutant Allelic Frequency
Measured
SD Positive
replicates
1 3.0% EGFR T790M -1.26%
2.34%
0.38
0.68
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2 3.0% EGFR T790M +2.66%
1.26%
0.77
0.38
8
8
3 0.75% EGFR T790M -0.18%
1.03%
0.15
0.21
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4 0.75% EGFR T790M +0.78%
0.34%
0.64
0.20
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5 0.13% EGFR T790M -0.42%
0.19%
0.37
0.11
2
3
6 0.13% EGFR T790M +0.21%
0.00%
0.06
0.00
5
0
7 0% EGFR T790M -0.00%
0.00%
0.00
0.00
0
0
8 0% EGFR T790M +0.00%
0.00%
0.00
0.00
0
0
Formalin-Compromised Quantitative Multiplex HDx Reference Standards developed for NGS platform control and sensitivity of detection
Formalin-Compromised Quantitative Multiplex HDx Reference Standards
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Chromosome Gene MutationMutant allelic
frequency
7q34 BRAF V600E 10.5%
7p12 EGFR ΔE746 - A750 2.0%
7p12 EGFR L858R 3.0%
7p12 EGFR T790M 1.0%
7p12 EGFR G719S 24.5%
12p12.1 KRAS G13D 15.0%
12p12.1 KRAS G12D 6.0%
12p12.1 NRAS Q61K 12.5%
12p12.1 PI3KCA H1047R 17.5%
12p12.1 PI3KCA E545K 9.0%
Defined Quantitative MultiplexCell Line Mixture
No Formalin Treatment
Formalin Treatment
DNA Extraction DNA Extraction
DNA Quantification
DNA Quantification
Digital PCR Analysis
Digital PCR Analysis
Highly characterised reference standards: Fragmentation levels, DNA quantification and defined allelic frequency
Formalin-Compromised Quantitative Multiplex HDx Reference Standards
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DNA fragmentation DNA amplifiability
C 1 2 3
Formalin-Compromised Quantitative Multiplex HDx Reference Standards
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Chromosome Gene Mutation
Horizon
Expected
AF
7q34 BRAF V600E 10.5%
7p12 EGFR ΔE746 - A750 2.0%
7p12 EGFR L858R 3.0%
7p12 EGFR T790M 1.0%
7p12 EGFR G719S 24.5%
12p12.1 KRAS G13D 15.0%
12p12.1 KRAS G12D 6.0%
12p12.1 NRAS Q61K 12.5%
12p12.1 PI3KCA H1047R 17.5%
12p12.1 PI3KCA E545K 9.0%
Mutant calling of reference standards on IonTorrent™ confirms the sensitivity of the platform for this particular work flow
Formalin-Compromised Quantitative Multiplex HDx Reference Standards
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Chromosome Gene Mutation
Horizon
Expected
AF
Non-Compromised
DNA
Formalin-
Compromised DNA
Medium Intensity
Formalin-
Compromised DNA
Severe Intensity
HD500 HD-C750 HD-C751
7q34 BRAF V600E 10.5% 10% 10% 10%
7p12 EGFR ΔE746 - A750 2.0% No call No call 2%
7p12 EGFR L858R 3.0% 2% 4% 2%
7p12 EGFR T790M 1.0% No call No call No call
7p12 EGFR G719S 24.5% 24% 18% 25%
12p12.1 KRAS G13D 15.0% 13% 12% 16%
12p12.1 KRAS G12D 6.0% 8% 3% 14%
12p12.1 NRAS Q61K 12.5% 11% 7% 11%
12p12.1 PI3KCA H1047R 17.5% 22% 23% 23%
12p12.1 PI3KCA E545K 9.0% 8% 5% 13%
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Impact of Formalin Treatment on Clinical Diagnostics
FFPE Sample
Cancer Patient DNA Extraction Diagnosis
FFPE ReferenceStandard
Formalin-Compromised DNA
ReferenceStandard
Routine validation of
your workflow
Verification of the robustness of your assay
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Sensitivity of your Assay
HD500HD700
Formalin Intensity
HD200
Robustness and Sensitivity of your Workflow
HD751HD750
FFPE
DNA
Robustness of your Assay
How to Test the Robustness and Sensitivity of your Workflow and Assay
Your Horizon Contact:
Horizon Discovery Group plc, 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
Jonathan Frampton
