Aug2013 horizon dx engineered cell line reference materials
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Aug2013 horizon dx engineered cell line reference materials
- 1. Horizon Diagnostics Addressing the Variability of Molecular Assays: The Need for Reference Standards Jonathan Frampton Diagnostics Product Manager
- 2. 2 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-A750 Sample 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”
- 3. 3 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 population Variable EGFR copy number depending on the source material
- 4. What is a Reference Standard 4 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.
- 5. 5 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
- 6. Functional Genomics Disease modelling Reference Standards 6 > 40 different parental cell lines now targeted; 500 projects, covering 16 tissue types Examples of what you can accomplish with GENESIS
- 7. 7 Cell Line Validation Clonal heterozygous mutant 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
- 9. Quantitative Molecular Reference Standards 9 Genomic DNA Stoichiometric Dilutions Mutant Wild type FFPE Processing FFPE Mixed Ratio Sections Mutant Wild type
- 10. Genomic DNA Standards verified using Digital™ PCR Stoichiometric dilutions are accurate down to 0.1% and 0.05% Gene Mutation Allelic Frequency B-Raf V600K 50% 10% 5% 1% 0.5% 0.1% 0.05% K-Ras G12D 50% 10% 5% 1% 0.5% 0.1%
- 11. 11 FFPE Block Production B-Raf 50% V600K Prepare block and sections B-Raf 100% wild type B-Raf 5% V600K Mix cells 9:1 Droplet Digital PCR BioRad 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
- 12. 12 H+E DNA H+E Staining & DNA Extraction demonstrates the homogeneity and consistency of our FFPE technology FFPE Block Consistency
- 13. FFPE Block Allelic Frequency 13 Gene Mutation B-Raf V600E 25% 5% 3.5% 1% B-Raf V600K 50% 5% 1% --- Consistent allelic frequency throughout the FFPE Blocks
- 14. 14 Quantitative Multiplex DNA Reference Standard Precise DNA dilutions Digital PCR Analysis Quantitative Multiplex DNA Reference Standard X-Man Genetically Defined Mutant Cell Lines
- 15. 15 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 10.0 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 1p13.2 NRAS Q61K 12.5 3q26.3 PI3KCA H1047R 17.5 3q26.3 PI3KCA E545K 9.0 Initial offering covers 11 mutations – Available Now
- 16. 16 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
- 17. 17 Gene Variant Freq % Core mutations BRAF V600E 10.5 cKIT D816V 10.0 EGFR ΔE746 - A750 2.0 EGFR L858R 3.0 EGFR T790M 1.0 EGFR G719S 24.5 KRAS G13D 15.0 KRAS G12D 6.0 NRAS Q61K 12.5 PI3KCA H1047R 17.5 PI3KCA E545K 9.0 Additional mutations ALK P1543S 33.0 APC R2714C 33.0 FBXW7 G667fs 33.5 FGFR1 P150L 8.5 FLT3 S985fs 10.5 FLT3 V197A 11.5 IDH1 S261L 10.0 MET V237fs 6.5 MLH1 L323M 8.5 NOTCH1 P668S 31.5 NTRK1 5'UTR 8.5 PDGFRA G426D 33.5 ABL2 P986fs 8.0 BRCA2 A1689fs 33.0 NF2 P275fs 8.0 ARID1A P1562fs 33.5 CDX2 V306fs 41.5 EP300 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
- 18. 18 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 16.7 KRAS G13D 16.7 KRAS A146T 16.7 Additional mutations ALK P1543S 50.0 APC R2714C 50.0 CTNNB1 S33Y 50.0 EGFR G719S 33.3 FBXW7 G667fs 50.0 NOTCH1 P668S 50.0 PDGFRA G426D 50.0 Gene Variant Freq % Core mutations EGFR L861Q 12.5 EGFR ΔE746-A750 12.5 EGFR L858R 12.5 EGFR T790M 12.5 Additional mutations FGFR1 P150L 50.0 MLH1 L323M 50.0 BRAF V600E 66.7 CDG1 3’UTR 50.0 PI3KCA H1047R 50.0 EGFR & KRAS Titratable Multiplex Reference Standards
- 19. 19 FFPE DNA vs Normal DNA Gene Variant dPCR gDNA NGS gDNA dPCR FFPE NGS FFPE BRAF V600E 10.5 9.3 9.5 8.5 KIT D816V 10 10.8 9 8.6 EGFR ΔE746 - A750 2 1.9 1.5 0 EGFR L858R 3 2 2.5 0 EGFR T790M 1 1 1 0 EGFR G719S 24.5 28.3 26 16 KRAS G13D 15 17.1 16.5 17.2 KRAS G12D 6 7.4 5.5 6.4 NRAS Q61K 12.5 10.7 12 8.2 PIK3CA H1047R 17.5 18.2 15.5 18.5 PIK3CA E545K 9 9.9 10 7.3 PGM fails to detect the lower allelic frequencies in FFPE sample
- 20. 20 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)
- 22. 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: info@horizondx.com Web: www.horizondx.com Dr Jonathan Frampton Product Manager j.frampton@horizondiscovery.com +44 (0) 1223 815299
Notas do Editor
- Who am I? What is the purpose of the presentation?
- Heterogenous samples (mixed clones)
- Are you using internal kit controls? Have you made controls yourself from a cell line?
- Can perform standard KI and KO approaches with enhanced precision over any technique. Can uniquely spatial, inducible and (soon) conditional genomic alterations. Examples of this are current projects modelling amplifications and translocation (EML4-Alk / Crizotinib Dx Standard)
- Are you using internal kit controls? Have you made controls yourself from a cell line?