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Almac DiagnosticsNGS Panels:“From Patient Selection to CDx”Dr Katarina WikstromHead of US OperationsAlmac Diagnostics

Overview Almac Diagnostics Overview Benefits and Challenges of NGS Panels for Subject Selection NGS Panel Case Study – TST-170 Companion Diagnostic (CDx) Development Considerations

Overview Almac Diagnostics Overview Benefits and Challenges of NGS Panels for Subject Selection NGS-Seq Panel Case Study – TST-170 CDx Development Considerations

Specialised ServicesBiomarkerDiscovery andDevelopmentAPI Services;Biocatalysis;Small Molecules& ctureand Pack4

Almac DiagnosticsAlmac Diagnostics is a global precision medicinecompanyProviding: DiscoveryDevelopmentCommercialisationof complex diagnostic & companion diagnostictests

Almac Lab Infrastructure Global Diagnostics RUO & CLIA Laboratories– Craigavon,UK– Durham, NC, USA– Partnership in China – CLIA, CAP and CLEP accredited ISO 17025, ISO 15189 and ISO 13485 accredited Comply with GLP, GCP and GCLP

Overview Almac Diagnostics Benefits and Challenges of NGS Panels for Subject Selection NGS Panel Case Studies CDx Development Considerations

Benefits of NGS Panels for Subject Selection– Suitable for both DNA (e.g. SNVs, indels) & RNA (e.g. fusions, gene expression) assessment– Potential to capture complex biology– Powerful biomarker discovery tool– Potential to report multiple biomarkers using a single sample and wet lab process

Challenges of using NGS Panels for Subject Selection– Design and justification of broad panels requires careful consideration– Cost and throughput implications if biomarker discovery or panel use formultiple biomarkers is not required– Complexity leads to analytical validation and regulatory challenges

Overview Almac Diagnostics Benefits and Challenges of NGS Panels for Subject Selection NGS Panel Case Study – Illumina TruSight Tumor 170 CDx Development Considerations

Illumina TruSight Tumor 170 Panel Overview Illumina enrichment based targeted NGS panel run on theNextSeq500 and NextSeq550 Covers 170 genes associated with common solid tumors Compatible with FFPE tissue and simultaneously analysesboth RNA and DNA from a single sample Targets all coding exons in 170 genes including 55 genes forfusions and splice variants, 148 for SNVs and Indels, and59 genes for CNV Capable of detecting somatic mutations as low as 5%mutant allele frequency, with 95% sensitivity andspecificity

Almac Diagnostics & Illumina TruSight Tumor 170 Almac Diagnostics were selected as Illumina’s Betatesting site for the TruSight Tumor 170 assay in 2016. This facilitated early access to the assay prior tocommercial release. Beta testing successfully completed. Almac Diagnostics have built up a vast amount ofexperience with the assay in a short space of time thatPharma partners can benefit from. Currently providing the assay as an RUO panel formultiple Pharma partners.

EORTC & Almac Diagnostics Collaboration (RUO)Almac Diagnostics has been selected by theEuropean Organisation for the Research andTreatment of Cancer (EORTC) as its preferredpartner for molecular profiling of cancer patientsamples for SPECTA (Screening Patients forEffective Clinical Trial Access), a pan-EuropeanCancer network. Using the Illumina TruSight Tumor 170 solid tumor NGS panel to analysepatterns of genomic variation within patientsamples.

Illumina TruSight Tumor 170 PanelAlmac are validating Illumina’s TruSight Tumor 170 panel as a CLIA compliant & CE marked assay forBiomarker Led Clinical Trials and CDx Development – Available January 201814

Almac TruSight Tumor170 CTAREAGENTS Qiagen AllPrep DNA/RNA FFPE Kit Qiagen Rnase A TruSight Tumor 170 Kit for usewith NextSeq NextSeq 500/550 v2 High Output300 Cycle Kit PhiX Control v3CONTROLS TST-170 DNA ControlsTST-170 RNA ControlsNo Template ControlPhiX ControlINSTRUMENTATION NextSeq 500NextSeq 550Veriti DxFocused UltrasonicatorRoche LightCycler 480BioanalyserQubitMultimode PlatereaderVeriti Dx ThermoCyclerSOFTWAREINSTRUCTIONS Illumina BaseSpace Illumina TST-170 App Almac Fixed SeedDownsampling Almac TST-170 QCSoftware Wet Lab TST 170 SOPs Dry Lab TST 170 SOPs

Laboratory Process Overview

Data Analysis OverviewQC 6BaseSpace Sequence HubData Upload toBaseSpace[Automated].Bcl conversion to FastQ[Automated]BaseSpace Sequence HubAlmac Custom Fixed Seed DownsamplingBaseSpace AppBaseSpace App TruSight Tumor 170Fixed Seed Downsampling[Manually triggered]Analyse DNA base librariesAnalyse RNA base libraries(Manually triggered)Sequencing Run QCmetric[Manual]FastQ demultiplexing[Automated]IQC Test Result[END]DNA Processcontrol report[Pass/Fail]QC 7Scope of Core ProductQC 8DNA Output 1Alignment statQc softwareTST 170 App QCMetricsPredicted SmallVariantsPredictedCNVsAlignment statQc softwarePassFailTST 170 AppQC MetricsDNA ClinicalSample Report[automated]FailLIMS Impo rtdata setGeneration of Additional alignment statistics;Reportable Range for DNA librariesRead Count for RNA Libraries[Manually triggered]IQC Test ResultDNA output 1OverallEnrolmen t CallLIMS Impo rtdata setQC 8TST 170 AppQC MetricsFailIQC Test ResultRNA output 1PredictedFusionsAlignment statQC softwareRNA Processcontrol Report[Pass/Fail]Cus tomerspecific Variantfilterin gGenerateenrolmen tcall/IQCRNA Output 1PassAlignment statQC softwareGenerateenrolmen tcall/IQCIQC Test ResultDNA Output 1RNA & DNAPC assessment[automated]QC 7Cus tomerspecific Variantfilterin gTST 170 AppQC MetricsFailIQC Test ResultRNA output 1RNA Clinical SampleReport[automated]IBM Watson InterpretationAutomatic PTR

Establishment of Analytical Performance CharacteristicsAccuracy StudySamples representative ofeach intended indication,GIAB samples, cell linesTST-170 libraryprep and sequencingMutation Calls (DNA),CNV calls (DNA),Fusion Calls (RNA)Path Review and DNA/RNA extractionHaloplex library prepand sequencingArcher FusionPlex libraryprep and sequencingMutation Calls (DNA)Fusion Calls (RNA)ddPCR on Bio-RadQX100CNV Calls (DNA)Determine % Agreement (Negative, Positive, Overall) by variant classConfidential

Establishment of Analytical Performance Characteristics Precision of the Almac TruSight Tumor 170 assay determined by repeat sample runs utilisingdifferent operators, reagent lots, equipment lines and days Lower limit of detection determined using cell line titrations and synthetic fusion RNA transcriptspike titrations Reportable range of the assay determined by in silico analysis of regions of the panel consistentlysequenced above coverage threshold at defined per sample read budget

Establishment of Quality Control Metrics and LimitsUsed to ensure quality of processing and resulting data on which test result is derivedSample and Library QCo Minimum percentage tumouro Minimum input to library preparationo Minimum nucleic acid qualityo Minimum acceptable library yieldSequencing QCo Allowable sequencer error rateo Minimum percentage of bases above Q30o Cluster densityo Sequencing run yieldConfidentialAlignment Statistics QCSmall variant calling (DNA)o Percentage reportable range sequenced above coveragethresholdCopy number calling (DNA)o Coverage Median Absolute Deviationo Median Bin Count CNV TargetFusion calling (RNA)o Minimum absolute mapped read counto Median Insert Sizeo Median CV of coverage greater than 1000X

Development and Validation of suitable process controls Process controls consisting of blended cell lines containing mutations detected by the panelgenerated (SNV, indel, CNV and fusion) Cells blended to ensure small variants are present at frequencies mimicking somatic mutations Multiple process controls generated for DNA based processing and RNA based processing. One RNAand one DNA processing control included per run FFPE Cell blocks created to control entirety of laboratory processing from sectioning to result Controls assessed for stability during AV to ensure reproducible results to facility use during assaydelivery and reagent release

Almac TruSight Tumor 170 CTA - Data Analysis Overview Data analysis performed using Illumina’s BaseSpaceApplication Raw data streamed direct from NextSeq instruments toBaseSpace Almac proprietary analysis pipelines deployed inBaseSpace used for custom QC and additional fileformatting BaseSpace used for storage and sharing of raw files Analysis output – small variants and CNV (DNA samplesand splice/fusion variants (RNA samples) Output integrated into Almac LIMS for custom PTRgeneration

CTA utilisation of IUO validated Almac TruSight Tumor 170 assay Almac will analytically validate the Illumina TruSight Tumor 170 assay, however trial specific activities willbe required before the assay can be used for prospective clinical testing Trial specific activities include:–Design control relevant to the clinical trial – intended use specific–Regulatory activities (pre-sub, IDE application, country/state approval applications)–Agree clinically actionable variants and logic for clinical trial enrolment–Trial specific reporting software (integration of the results with patient information & PTR generation)–Additional mutation specific AV studies (if required)

NGS Panel Test Delivery Critical considerations include:– Standardization of input tissue– QC metrics– Process controls– Clarity of reporting– Turnaround time Quality assurance must be maintained and monitored through the pre-analytical,analytical and post analytical phases of testing Require the correct assay result for the correct subject at the correct time

25Example Almac Clinical Trial PTR for TruSight Tumor 170 Assay

26IBM Watson for Genomics - Variant InterpretationAdditional Powerful Mutation Reporting and Insight: Almac Diagnostics are currently in discussions to offer this reporting through Illumina TruSight Tumor 170 assay Watson can be uploaded with clinical trial patient data from Illumina Base Space vcf files Watson checks against latest data upload from Biomarkers and Trials (updated monthly) Watson then runs the data and produces a powerful report in less than two minutes showing:––– Actionable AlterationsTherapies with Clinical TrialsFDA Approved DrugsThe final report can be provided alongside the raw data to Pharma or Biotech clients

27IBM Watson for GenomicsExample Report

Overview Almac Diagnostics Benefits and Challenges of NGS Panels for Subject Selection NGS Panel Case Studies CDx Development Considerations

CDx Technical and Commercial Considerations Throughput Requirements–Manual vs Automated Processing–Size of Panel and Capacity of Platform Service-based Test Delivery or Distributed Kit Footprint of NGS Platform Supply Agreements Process Controls Data Analysis–Logistics–Reporting relevant results and requirement for data masking

CDx – Early Development LockDRUGClinical TrialPhase I / IIDEVELOPMENTCTADXDEVELOPMENTClinical TrialAssayMarket-readyCDxDEVVALDEVPivotal Clinical TrialUSEVALUSE

CDx – Late Development LockDRUGDEVELOPMENTCTADXDEVELOPMENTClinical TrialAssayMarket-readyCDxDEVVALClinical TrialPhase I / IIPivotal Clinical TrialUSEUSENew DrugApplicationBRIDGEDEVVALPremarketApproval

Where do I find more information?Almac Diagnostics TruSight Tumor 170 Web 0This webpage houses all the relevant information. We will also be providing future updatesincluding adding the validation data to the web page for viewing.If you have an upcoming clinical trial in the next 3-6 months and are interested now inspeaking to someone please email diagnostics@almacgroup.com and we will call you todiscuss your requirements.

Thank YouQUESTIONS?Dr Katarina WikstromHead of US Operations, Almac acgroup.com/diagnostics

LIMS Import data set Generate enrolment call/IQC Cus tomer specific Variant filtering LIMS Import data set Generate enrolment call/IQC Overall Enrolment Call Automatic PTR. Pass IBM Watson Interpretation. Scope of Core Product. Generation of Additional alignment statistics; Reportable Range for DNA libraries Read Count for RNA Libraries .