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Dr Jonathan FramptonProduct Manager, Diagnostics
The Clinical Application, Development and Validation of Cell-free DNA Assays/Platforms
Twitter: @HorizonDX_news #cfDNA
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Presenters
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.
#cfDNA
What is the impact of assay failure in your laboratory and how do you monitor for it?
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Application of Liquid Biopsies
Blood/Plasma SampleCancer Patient DNA Extraction Diagnosis
Driving better treatment for cancer patients
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Sample Handling and Processing
Blood/PlasmaSample
DNA QuantificationStorageDNA Extraction
DNA Quantity & Quality
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Analytical Processing and Reporting
DNA Sample Analysis Actionable Decision
Quality of Diagnostic Result
Sample preparation
#cfDNA
What is the impact of assay failure in your laboratory and how do you monitor for it?
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Challenges Faced by cfDNA Assay Implementation or Development
Blood/Plasma SampleCancer Patient DNA Extraction Diagnosis
Sample collectionHandling
DNA FragmentationContamination
DNA RecoveryDNA quantification
DNA/Biomarker lossDNA Fragmentation
False negativesFalse positivesSpecificitySensitivity
DNA qualityDNA quantity
Variable
Impact
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Presenters
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 cfDNA Reference Standards at Horizon Diagnostics.
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“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
How are Reference Standards Manufactured and Validated?
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Genomic DNA Stoichiometric Dilutions
Mutant Wild type
Dilutions are accurate down to 0.05%
Analyzed Allelic Frequency Down to 0.05%
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We are now working with over 700 hospitals, kit developers, proficiency schemes and pathology labs worldwide…
Improving Cancer Treatment Through Translational Research & Clinical Intervention
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Cell-free DNA Reference Standard Case Studies
Case Study 1 – EGFR
Multiplex standard down to 0.05%
Validated by ddPCR
Case Study 2 – Multiplex cancer panel
Multiplex standard containing >40 validated mutationsHighlights sensitivity and
specificity of NGS platforms
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Case Study 1: Cell-free DNA EGFR Multiplex Reference Standard
T790M
G719S
ΔE746-A750L858R
L861Q Wild type
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Analytical Processing and Reporting
How good is digital PCR for detecting mutations at 0.05%?
DNA Sample Analysis Actionable Decision
Sample preparation
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EGFR Multiplex Sample #6 Wild Type
Effective mutant allele frequency 0.05% 0%
Expected Observed Expected Observed
Total CopiesEGFR
Mutant
ΔE746-A750 25 23 0 2.0
G719S 25 20 0 12.0
L858R 25 31 0 1.0
L861Q 25 32 0 1.0
T790M 25 44 0 16.0
Total Copies EGFR 50000 50380 50000 49700
Reference Standards demonstrate the sensitivity and accuracy of a “cfDNA workflow”
Case Study 1: Cell-free DNA 0.05% EGFR Multiplex Reference Standard
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Pause for questions
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Case Study 2: Tru-Q HDx™ Reference Standards for Next Generation Sequencing
Quantification by Droplet Digital PCR
A Blend40 Mutations
@ 1.3%
B Blend 120 Mutations
at 2.5%
B Blend 220 Mutations
at 2.5%
C Blend 110 mutations
at 5%
C Blend 210 mutations
at 5%
C Blend 310 mutations
at 5%
C Blend 410 mutations
at 5%
EGFR mutants
K-Ras mutants
B-Raf mutants
N-Ras mutants
PIKCA mutants
14 Additonal Biomarkers
1.3%20 copies per μl
Quantification by Droplet Digital PCR
Quantification by Droplet Digital PCR
Quantification by Droplet Digital PCR
Dilution Series with wild type
1 copy per μl
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Analytical Processing and Reporting
How good is NGS for detecting mutations at 1%?
DNA Sample Analysis Actionable Decision
Sample preparation
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Case Study 2: Analysing PGM Workflow down to 1%
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
Specific and Sensitive down
to 5% allelic frequency
Horizon DiagnosticsPredicted %
Horizon DiagnosticsObserved %
Partner
N/AQX100™ Droplet
Digital™ PCR System
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 %
Horizon DiagnosticsObserved %
Partner
N/AQX100™ Droplet
Digital™ PCR System
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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What’s Coming Next?
Blood/plasma sample
Urine sampleHDx™ Reference Standard
HDx™ Reference Standard
HDx™ Reference Standards will confirm your specificity and sensitivity
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What are the Outstanding Questions?
What is the impact of assay failure in your laboratory and how do you monitor for it?
What sensitivity do you want to achieve?
What mutations are you looking at?
How can we support your workflow
#cfDNA Last Slide
What to do Now?
Molecular Lab Next Generation Sequencing Lab
Kit, Assay, Platform Developer?
HDx™ Reference StandardsEvery Assay, Every Run, Every Confidence
Contact me at [email protected]
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 Product [email protected]+44(0) 1223 655 580