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 Services
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Biomarker Discovery and Development
API Services; Biocatalysis;
Small Molecules& Peptides
Drug Product
Development
Analytical Services
Clinical Technologies
Clinical Services
Commercial Manufacture
and Pack
Almac Diagnostics
Almac Diagnostics is a global precision medicine company
Providing:
• Discovery• Development• Commercialisation
of complex diagnostic & companion diagnostic tests
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 for
multiple 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 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
Almac Diagnostics & Illumina TruSight® Tumor 170 • Almac Diagnostics were selected as Illumina’s Beta
testing site for the TruSight Tumor 170 assay in 2016.
• 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.
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.
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 2018
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INSTRUCTIONSSOFTWAREINSTRUMENTATIONCONTROLS
Almac TruSight ® Tumor170 CTA
REAGENTS
• 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
Laboratory Process Overview
Data Analysis Overview
IQC Test Result[END]
Data Upload to BaseSpace
[Automated]
.Bcl conversion to FastQ[Automated]
Analyse DNA base librariesAnalyse RNA base libraries
(Manually triggered)
BaseSpace® App TruSight Tumor 170BaseSpace® Sequence Hub
FastQ demultiplexing[Automated]
BaseSpace® Sequence Hub
IQC Test ResultDNA output 1
Sequencing Run QC metric
[Manual]
Fixed Seed Downsampling [Manually triggered]
Almac Custom Fixed Seed Downsampling BaseSpace® App
IQC Test ResultDNA Output 1
QC 6
Alignment stat Qc software
TST 170 App QC Metrics
Predicted Small Variants
Predicted CNVs
Alignment stat QC software
TST 170 App QC Metrics
Predicted Fusions
Alignment stat Qc software
TST 170 App QC Metrics
Alignment stat QC software
TST 170 App QC Metrics
Fail
Pass
Fail
IQC Test ResultRNA output 1
Fail
IQC Test ResultRNA output 1
Fail
DNA Clinical Sample Report[automated]
RNA Clinical Sample Report
[automated]
QC 7
QC 7
RNA & DNA PC assessment[automated]
QC 8
QC 8
DNA Output 1
RNA Output 1
Customer specific Variant
filtering
LIMS Import data set
Generate enrolment
call/IQC
Customer 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[Manually triggered]
DNA Process control report
[Pass/Fail]
RNA Process control Report
[Pass/Fail]
Establishment of Analytical Performance Characteristics
Confidential
TST-170 library prep and sequencing
Path Review and DNA/RNA extraction
Mutation Calls (DNA), CNV calls (DNA),
Fusion Calls (RNA)
Determine % Agreement (Negative, Positive, Overall) by variant class
Samples representative of each intended indication, GIAB samples, cell lines
Haloplex library prep and sequencing
Archer FusionPlex library prep and sequencing
ddPCR on Bio-Rad QX100
Mutation Calls (DNA) Fusion Calls (RNA) CNV Calls (DNA)
Accuracy Study
• 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
Establishment of Analytical Performance Characteristics
Establishment of Quality Control Metrics and Limits
Confidential
Used to ensure quality of processing and resulting data on which test result is derived
Sample and Library QCo Minimum percentage tumouro Minimum input to library preparation o Minimum nucleic acid qualityo Minimum acceptable library yield
Sequencing QCo Allowable sequencer error rateo Minimum percentage of bases above Q30o Cluster densityo Sequencing run yield
Alignment Statistics QCSmall 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 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 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
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
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)
• 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
NGS Panel Test Delivery
Example Almac Clinical Trial PTR for TruSight® Tumor 170 Assay
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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
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IBM Watson for Genomics
Example Report
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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
Clinical Trial Phase I / II Pivotal Clinical Trial
CDx – Early Development Lock
DRUGDEVELOPMENT
DXDEVELOPMENT
CTAClinical Trial
Assay
Market-ready CDx
DEV VAL USE
DEV VAL USE
Clinical Trial Phase I / II Pivotal Clinical Trial
DEV VAL USE
DEV VAL
USE
BRIDGE
New Drug Application
Premarket Approval
DRUGDEVELOPMENT
DXDEVELOPMENT
CTAClinical Trial
Assay
Market-ready CDx
CDx – Late Development Lock
Almac Diagnostics TruSight Tumor 170 Web Page
www.almacgroup.com/diagnostics/trusighttumor170
This webpage houses all the relevant information. We will also be providing future updates including 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 in speaking to someone please email [email protected] and we will call you to discuss your requirements.
Where do I find more information?
Thank You
www.almacgroup.com/diagnostics
QUESTIONS?
Dr Katarina WikstromHead of US Operations, Almac Diagnostics