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 diagnostics@almacgroup.com 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

katarina.wikstrom@almacgroup.com