HORIZON DISCOVERY Controlling Chaos in Oncology Testing 16 th March 2016 Brian Burke PhD
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About Horizon Discovery Cell Line Builders Reference Standards 3
Value for money? Sensitivity? Data Analysis? Patient Safety THE PATIENT? Reimbursement Guidance and Proficiency Cost of Drug Development FDA CLIA CAP Pharma 4
Precision Medicine Development Pipeline Research & Development Assay Development Sample Screening Patient Stratification Clinical Studies Phase II & III CLIA Studies Patient Stratification On Market Companion Diagnostics Proficiency Testing/Ring Trials Acquisition of limited clinical samples Clinical Samples are heterogeneous and not quantitative Clinical Trial time lines and use of multiple partners/geographies can lead to drift in assay performance Outsourcing can make managing variation more difficult Unnecessary and sustained variability can affect Study performance New drugs rely on successful and effective adoption of diagnostics to ensure reimbursement Proficiency Testing benefits from samples that are quantitative and that permit preanalytical and analytical evaluation of diagnostic workflows CONFIDENTIAL 5 Research Use Only
Why are we here? Key Observations Assays are currently developed in silos There is no quick way to benchmark assays or platforms What makes a good assay in a development lab does not always translate In spite of the importance of diagnostics to Precision Medicine, they still remain an add on in many cases 6
WHY DO WE NEED REFERENCE STANDARDS?
Would you fly in a plane with an altimeter accurate to 10%? Heathrow 25m +/- 2.5m A Bumpy Landing Guangxi 690m +/- 69m Crash Landing 8848 m Tenzing 2860m +/- 286m Never Even Close!! 8
Percentage of Incorrect Results External Quality Assessment 40 EGFR Genotyping Errors External Quality Assessment 2014 35 30 25 20 15 10 5 0 EGFR Sample Tested Only 70% of laboratories passed the proficiency test. False -negatives and false-positives were the main sources of error. European Molecular Quality Network (EMQN) 9
Molecular Diagnostic Assay Biomarker Negative Biomarker False Negative No treatment No side-effects No treatment Shortened patients life Treatment with no benefit Patient suffers side-effects Biomarker False Positive Treatment Extended patients life Biomarker Positive As we push the limits of technology, the risk of false results will increase 10
Sources of Variability Tumour sample As lower allelic frequencies increase in clinical relevance, workflows become more fallible Reference Materials Action Analysis DNA extraction Variant Calling/ Confidence Scoring DNA Quantification Library Preparation Sequencing Alignment/Mapping 11
HOW DO WE BUILD REFERENCE STANDARDS?
Not All Controls are Created Equal! Horizon uses highly characterised cell lines to build reference standards This gives us highly defined building blocks to create reference standards which reflect the biology of the patient. Better still, these reference standards are consistent and evergreen! Horizon has developed manufacturing processes that allow for consistent, long term production of reference standards NORMAL CELL NORMAL CELLS MUTANT CELLS SINGLE MUTATION Products Services Drugs MUTATED CELL PATIENT IN A TEST TUBE 13
Formats available PRE-ANALYTIC ANALYTIC cfdna Representative genomic DNA fragmented to 160-170bp Validated allelic frequencies to 0.01% Genomic DNA Precise allelic frequencies from 0.1-50% Extensively validated 50ng/μl FFPE Precise allelic frequencies from 0.1-50% Validated quantities of DNA in FFPE FFPE IHC Validated by Quantitative Digital Pathology (QDP) Quantified levels of protein expression (low to high) Formalin Compromised DNA Three tiers of formalin treatment: low, medium and high Validated by Tapescreen assay Multiplex DNA for NGS Multiple mutations in a single sample Extensively validated 50ng/μl FFPE FISH Hard-to-source biomarkers available Renewable source of positive and negative cells FFPE RNA Hard-to-source biomarkers available Consistent and reproducible 14
Covering all Diagnostic Modalities NGS Oncology Panels Q-Seq Coverage of TruSeq, AmpliSeq, and more Real Time PCR EGFR, KRAS, BRAF Exomes Genome in a Bottle Immuno-Oncology PD-L1 Liquid Biopsies Cell free DNA Reference Standards RNAseq NEW NSCLC Fusion Control. 15
NEXT GENERATION SEQUENCING
NGS: Massive Opportunity vs Massive Variability Accuracy ranked highest in importance for users of next-generation sequencing systems. NOTE: Based on a survey of 108 end users. SOURCE: Frost & Sullivan 17
One Sample Three Platforms - 80% Agreement # SNPs (% of SNPs detected) Platform #1 Platform #2 Platform #3 SOURCE: GIAB Slides 2014 18
One Data Set Five Bioinformatic Workflows - 60% Agreement O Rawe et al. Genome Medicine 2013, 5:28 SOURCE: GIAB Slides 2014 19
Horizon NGS Philosophy Deployment NGS technology is one of the key advances in the diagnostic space, promising greater breadth of data with more tangible pricing points HDx led the adoption of a more direct approach to assessing the performance of NGS platforms in the oncology space HDx enables key variables such as allele frequency, effects of formalin or genotype specific effects to be investigated directly Horizon is now extending this to structural variants and increasing customization around formalin 20
Multi-Gene Multiplex Case Study for IonTorrent / Ampliseq gdna Source: Horizon Discovery Partner A Partner B Partner C Partner D Horizon Discovery Partner D FFPE Platform: QX100 Droplet Digital PCR System AmpliSeq Cancer Panel Ampliseq Cancer Hotspot Panel v2 Ampliseq Cancer Hotspot Panel v2 (Average of 8 runs) Ampliseq Panel QX100 Droplet Digital PCR System Ampliseq Sequencing Depth N/A 3000-4000x Average 5000x 2000X N/A Gene Mutation Expected Observed mutant ratio Observed mutant ratio BRAF V600E 10.5 10.2 9.9 9.1 10.3 9.3 9.5 8.5 KIT D816V 10.0 10.4 10.0 11.0 10.1 10.8 9 8.6 EGFR ΔE746 - A750 2.0 2.0 2.3 Not detected Not detected 1.9 1.5 0 EGFR L858R 3.0 2.7 2.7 2.1 2.4 2 2.5 0 EGFR T790M 1.0 0.9 0.8 Not detected Not detected 1 1 0 EGFR G719S 24.5 24.4 23.7 23.1 24.8 28.3 26 16 KRAS G13D 15.0 16.1 16.3 12.35 15.5 17.1 16.5 17.2 KRAS G12D 6.0 5.0 5.2 Not detected 5.1 7.4 5.5 6.4 NRAS Q61K 12.5 12.8 9.0 12.7 12.6 10.7 12 8.2 PIK3CA H1047R 17.5 18.6 16.7 16.8 17.9 18.2 15.5 18.5 PIK3CA E545K 9.0 8.9 3.2 8.4 8.8 9.9 10 7.3 Difficulty in detecting the lower allelic frequencies in FFPE sample 21
Case Study Data for Tru-Q NGS Reference Standards 5% blend 2.5% blend 1.3% blend Gene Source: Platform: Mutation Horizon Diagnostics Predicted % N/A Horizon Diagnostics Observed % QX100 Droplet Digital PCR System Partner Ion Torrent 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 Horizon Diagnostics Predicted % N/A Horizon Diagnostics Observed % QX100 Droplet Digital PCR System Partner 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 Diagnostics Predicted % N/A Horizon Diagnostics Observed % QX100 Droplet Digital PCR System Partner 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 5% allelic frequency Specific and Sensitive down to 2.5% allelic frequency Not sensitive to detect down to 1% for all variants 22
Clinical Validation Using Horizon s NGS Process Standard Case Study: CLIA Validation of AltheaDx Oncology Panel 150 (AOP 150) using HDx Reference Standards Concordance with dpcr, average six IonTorrent PGM NGS runs 23
Routine Assay Monitoring Results from a commercially-available NGS-based Oncology Panel evaluated using Horizon s Quantitative Multiplex Reference Standard (QMRS) in FFPE format; collected over a period of 8 months. 24
MOVING AWAY FROM THE INVASIVE
ctdna Low Copy Number Reference Standard Five Mutant Multiplex in six formats developed containing all the following EGFR mutations: T790M L861Q G719S L858R ΔE746- A750 Six multiplex reference standards defined by copy number: 1. 25 Mutant copies in 5,000 total EGFR copies (0.5% Mutant Allele Burden) 2. 25 Mutant copies in 50,000 total EGFR copies (0.05% Mutant Allele Burden) 3. 50 Mutant copies in 5,000 total EGFR copies (1.0% Mutant Allele Burden) 4. 50 Mutant copies in 50,000 total EGFR copies (0.1% Mutant Allele Burden) 5. 100 Mutant copies in 5,000 total EGFR copies (2.0% Mutant Allele Burden) 6. 100 Mutant copies in 50,000 total EGFR copies (0.2% Mutant Allele Burden) 26
ctdna Assay Verification Down to 0.05% Allelic Frequency 25 Mutant copies in 50,000 total EGFR copies EGFR Multiplex #1 #2 Wild Type Effective mutant allele frequency 0.5% 0.05% 0% Expected Observed Expected Observed Expected Observed ΔE746-A750 25 24 2.5 2.3 0 0.2 Mutant Copies per 5000 Total Copies G719S 25 28 2.5 2.0 0 1.2 L858R 25 26 2.5 3.1 0 0.1 L861Q 25 25 2.5 3.2 0 0.1 T790M 25 27 2.5 4.4 0 1.6 Concentration (Total Copies EGFR per µl) 500 525 5000 5038 5000 4970 Manufacturing performance is excellent in spite of limitations of some probe sets 27
Fragmented K-Ras and N-Ras Reference Standards Gene Mutation Allelic Frequency K-Ras G12C 50% 5% 1% 0.1% N-Ras Q61K 50% 5% 1% 0.1% K-Ras G12C N-Ras Q61K WT 50% 5% 1% 0.1% 50% 5% 1% 0.1% 28
Assay development and validation Fragmented Multiplex Reference Standards Multiplex 1 Multiplex 2 Gene Mutation Allelic Frequency EGFR L861Q 7.1% K-Ras G12C 10% N-Ras A146T 10% PI3K3CA E545K 10%% Gene Mutation Allelic Frequency EGFR 746-750 7.7% EGFR T790M 7.7% N-Ras G12D 10% N-Ras Q61K 10% N-Ras K117N 10% 29 29
Fragmented K-Ras and N-Ras Reference Standards Gene Mutation Allelic Frequency K-Ras G12C 50% 5% 1% 0.1% N-Ras Q61K 50% 5% 1% 0.1% 30
What do the cfdna Standards look like? Expected Allelic Frequency (AF%) Gene Variant 5% Multiplex I 1% Multiplex I 0.1% Multiplex I 100% WT Multiplex I EGFR L858R 5.00 1.00 0.10 0.00 EGFR ΔE746 - A750 5.00 1.00 0.10 0.00 EGFR T790M 5.00 1.00 0.10 0.00 EGFR V769 - D770insASV 5.90 1.00 0.10 0.00 KRAS G12D 6.30 1.30 0.13 0.00 NRAS Q61K 6.30 1.30 0.13 0.00 NRAS A59T 6.30 1.30 0.13 0.00 PIK3CA E545K 6.30 1.30 0.13 0.00 31
cfdna Reference Standards 5% - 0.1% 1% Multiplex I cfdna Reference Standard assessed using Droplet Digital PCR (blue), Ion Torrent (orange) and MiSeq (grey). All assays utilized amplicon-based enrichment. Figure 1: Example trace showing the fragment sizes collected by D1000 DNA ScreenTape assay, comparing cfdna HDx Reference Standards (Red and Green traces) to cfdna extracted from human plasma (Blue trace). cfdna from human plasma was provided by CareDx,Inc. Leftmost peaks-internal marker for the assay. Rightmost peaksfragmented materials. 32
HDx Reference Standards Every Assay, Every Run, Every Confidence MDx/NGS Labs Support workflow analysis and setup Provision of positive reference standard for everyday run control ISH Labs FISH and IHC applications External reference standard to ensure workflow integrity Universal across time, across platforms, across operators and across geographies 33
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