Almac Diagnostics. NGS Panels: From Patient Selection to CDx. Dr Katarina Wikstrom Head of US Operations Almac Diagnostics

Transcription

1 Almac Diagnostics NGS Panels: From Patient Selection to CDx Dr Katarina Wikstrom Head of US Operations Almac Diagnostics

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

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

4 The Almac Group The Almac Group 4

5 Global Group Presence Our locations Our depots Countries we have provided services to in the last 5 years

6 Specialised Services Biomarker Discovery and Development API Services; Biocatalysis; Small Molecules & Peptides Drug Product Development Analytical Services Clinical Services Clinical Technologies Commercial Manufacture and Pack 6

7 Unparalleled Expertise & Reach 5,000 Employees globally 25/25 Work with all top 25 BioPharma companies 5,400+ Client locations globally 15 facilities globally All phases Experience in all phases of drug development 290 Regulatory & client audits in Years experience 50% Contributed to ½ of FDA approved NME s in Network of 60+ in-country depots for clinical supply 7

8 Almac Diagnostics Almac Diagnostics is a global precision medicine company Providing: Discovery Development Commercialisation of complex diagnostic & companion diagnostic tests

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

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

11 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

12 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 for multiple biomarkers is not required Complexity leads to analytical validation and regulatory challenges

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

14 Illumina TruSight Tumor 170 Panel Overview Illumina enrichment based targeted NGS panel run on the NextSeq500 and NextSeq550 Covers 170 genes associated with common solid tumors Compatible with FFPE tissue and simultaneously analyses both RNA and DNA from a single sample Targets all coding exons in 170 genes including 55 genes for fusions and splice variants, 148 for SNVs and Indels, and 59 genes for CNV Capable of detecting somatic mutations as low as 5% mutant allele frequency, with >95% sensitivity and specificity

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

16 EORTC & Almac Diagnostics Collaboration (RUO) Almac Diagnostics has been selected by the European Organisation for the Research and Treatment of Cancer (EORTC) as its preferred partner for molecular profiling of cancer patient samples for SPECTA (Screening Patients for Effective Clinical Trial Access), a pan-european Cancer network. Using the Illumina TruSight Tumor 170 solid tumor NGS panel to analyse patterns of genomic variation within patient samples.

17 Illumina TruSight Tumor 170 Panel Almac are validating Illumina s TruSight Tumor 170 panel as a CLIA compliant & CE marked assay for Biomarker Led Clinical Trials and CDx Development Available January

18 Almac TruSight Tumor 170 CTA REAGENTS CONTROLS INSTRUMENTATION SOFTWARE INSTRUCTIONS Qiagen AllPrep DNA/RNA FFPE Kit Qiagen Rnase A TruSight Tumor 170 Kit for use with NextSeq NextSeq 500/550 v2 High Output 300 Cycle Kit PhiX Control v3 TST-170 DNA Controls TST-170 RNA Controls No Template Control PhiX Control NextSeq 500 NextSeq 550 Veriti Dx Focused Ultrasonicator Roche LightCycler 480 Bioanalyser Qubit Multimode Platereader Veriti Dx ThermoCycler Illumina BaseSpace Illumina TST-170 App Almac Fixed Seed Downsampling Almac TST-170 QC Software Wet Lab TST 170 SOPs Dry Lab TST 170 SOPs

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20 20 BaseSpace Sequence Hub BaseSpace Sequence Hub QC 6 Almac Custom Fixed Seed Downsampling BaseSpace App BaseSpace App TruSight Tumor 170 Data Upload to BaseSpace [Automated].Bcl conversion to FastQ [Automated] FastQ demultiplexing [Automated] Sequencing Run QC metric [Manual] Fixed Seed Downsampling [Manually triggered] Analyse DNA base libraries Analyse RNA base libraries (Manually triggered) IQC Test Result [END] QC 7 DNA Process control report [Pass/Fail] QC 8 Scope of Core Product DNA Output 1 Alignment stat Qc software TST 170 App QC Metrics Predicted Small Variants Predicted CNVs Alignment stat Qc software TST 170 App QC Metrics DNA Clinical Sample Report [automated] Generation of Additional alignment statistics; Reportable Range for DNA libraries Read Count for RNA Libraries [Manually triggered] QC 7 Fail IQC Test Result DNA output 1 Pass RNA & DNA PC assessment [automated] QC 8 Fail IQC Test Result DNA Output 1 LIMS Import data set LIMS Import data set Customer specific Variant filtering Customer specific Variant filtering Generate enrolment call/iqc Generate enrolment call/iqc Overall Enrolment Call Automatic PTR Pass RNA Output 1 Alignment stat QC software TST 170 App QC Metrics Predicted Fusions Alignment stat QC software TST 170 App QC Metrics RNA Clinical Sample Report [automated] Fail IQC Test Result RNA output 1 RNA Process control Report [Pass/Fail] Fail IQC Test Result RNA output 1 IBM Watson Interpretation

21 Analytical Validation Overview Establishment of Analytical Performance Characteristics including: Accuracy Precision Lower limit of Detection/Sensitivity Reportable Range Establishment of Quality Control metrics and limits Development and Validation of suitable Process Controls

22 Establishment of Analytical Performance Characteristics Accuracy Study Samples representative of each intended indication, GIAB samples, cell lines Path Review and DNA/RNA extraction TST-170 library prep and sequencing Haloplex library prep and sequencing Archer FusionPlex library prep and sequencing ddpcr on Bio-Rad QX100 Mutation Calls (DNA), CNV calls (DNA), Fusion Calls (RNA) Mutation Calls (DNA) Fusion Calls (RNA) CNV Calls (DNA) Determine % Agreement (Negative, Positive, Overall) by variant class

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

24 Establishment of Quality Control Metrics and Limits Used to ensure quality of processing and resulting data on which test result is derived Sample and Library QC o Minimum percentage tumour o Minimum input to library preparation o Minimum nucleic acid quality o Minimum acceptable library yield Sequencing QC o Allowable sequencer error rate o Minimum percentage of bases above Q30 o Cluster density o Sequencing run yield Alignment Statistics QC Small variant calling (DNA) o Percentage reportable range sequenced above coverage threshold Copy number calling (DNA) o Coverage Median Absolute Deviation o Median Bin Count CNV Target Fusion calling (RNA) o Minimum absolute mapped read count o Median Insert Size o Median CV of coverage greater than 1000X

25 Development and Validation of suitable process controls Process controls consisting of blended cell lines containing mutations detected by the panel generated (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 RNA and 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 assay delivery and reagent release

26 Almac TruSight Tumor 170 CTA - Data Analysis Overview Data analysis performed using Illumina s BaseSpace Application Raw data streamed direct from NextSeq instruments to BaseSpace Almac proprietary analysis pipelines deployed in BaseSpace used for custom QC and additional file formatting BaseSpace used for storage and sharing of raw files Analysis output small variants and CNV (DNA samples and splice/fusion variants (RNA samples) Output integrated into Almac LIMS for custom PTR generation

27 Almac TruSight Tumor 170 CTA - Bioinformatics Pipeline BaseSpace pipelines RNA DNA available for both RNAseq and DNAseq data Down-sample FASTQ Down-sample FASTQ Almac have added Adaptor Trimming Adaptor Trimming processing steps to ensure Alignment Mark Duplicates Alignment INDEL realignment process is deterministic to meet CLIA validation expectations Splice Variant Caller* Fusion Caller RNA Sample Metrics Copy Number Variant Caller Small Variant Caller (SNVs/MNVs) DNA Sample Metrics Almac have added Variant Annotation * Fusion Filtering Variant Filtering enhanced QC metrics to Fusion Merging Variant Annotation ensure data quality * Splice Variants considered RUO only as these are outside scope of current CLIA validation

28 CTA utilisation of IUO validated Almac TruSight Tumor 170 assay Almac will analytically validate the Illumina TruSight Tumor 170 assay, however trial specific activities will be 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 by FDA)

29 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

30 Example Almac Clinical Trial PTR for TruSight Tumor 170 Assay 30

31 31 IBM Watson for Genomics - Variant Interpretation Additional 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 Alterations Therapies with Clinical Trials FDA Approved Drugs The final report can be provided alongside the raw data to Pharma or Biotech clients

32 32 IBM Watson for Genomics Example Report

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

34 CDx Technical and Commercial Considerations Requirement to Focus Panel Further 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

35 CDx Early Development Lock DRUG DEVELOPMENT Clinical Trial Phase I / II Pivotal Clinical Trial DX DEVELOPMENT CTA Clinical Trial Assay Market-ready CDx DEV VAL USE DEV VAL USE

36 CDx Late Development Lock DRUG DEVELOPMENT Clinical Trial Phase I / II Pivotal Clinical Trial New Drug Application DX DEVELOPMENT CTA Clinical Trial Assay Market-ready CDx DEV VAL USE DEV USE VAL BRIDGE Premarket Approval

37 For More Information Please speak to Kevin Ellison or Debbie Yost at our World CDx stand Or Visit our Almac TruSight Tumor 170 web page and fill in a short expression of interest form

38 Thank You QUESTIONS? Dr Katarina Wikstrom Head of US Operations, Almac Diagnostics