Challenges and Opportunities in Developing Companion DiagnosticsJames L Sherwood

R&D Genetics, Personalised Healthcare and Biomarkers (ELRIG BIOMARKERS CONFERENCE, OXFORD)

Presentation OverviewTopics covered…

•What is AstraZeneca doing in PHC?• How we intend to achieve our goals

•Challenges to Diagnostic Development in oncology

• How sample type affect needs of diagnostic tests

•Opportunities in single gene testing• Example of commercialised kit

•Opportunities to further characterise precious samples from clinical trials

• Example of highly parallel mutation testing

Why are we working in PHC?

To deliver ‘meaningful’ medicines to patients

To benefit patients, helping to save and improve the quality of their lives

To ensure that new medicines will be approved and reimbursed

PHC is the right thing to do

• By using advanced science better to target new medicines to patients

• By improving medical outcomes and quality of care

• By giving patients confidence that they are receiving the best treatment for them

• By helping prescribers to find the right therapy first time

• By employing a clear PHC strategyto deliver evidence of meaningful effect, overcoming increasing payer demands

Our vision is…

• More than 50% of the AstraZeneca pipeline will be following a PHC approach

• At least two drug launches will be linked to companion diagnostic tests (or other methods to predict patients who will benefit most)

• PHC will be an integral part of our business

– Increased rates of successful launch and reimbursement

– Increased value and market access

By 2015…

1. Bain report 2011 (AstraZeneca internal report); 2. PharmaTimes April 2011

More than just catching up… We are aiming to take the lead

• Currently ranked among the top 5 companies in the PHC field1,2

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• Embed PHC at the heart of the R&D Strategy by creation of new Personalised Healthcare and Biomarkers function– Annual investment of US$30m in people,

skills and facilities

• Start drug development with a clear understanding of which patient groups will benefit and which will not

• Partner new drug development with biomarkers and companion diagnostics

• Utilise our dedicated Personalised Healthcare & Biomarkers function to…

Success, because we will….

Develop partnerships with diagnostic companies

Develop, validate and deliver registration-ready biomarkers and companion diagnostics

Support lifecycle management to help existing treatments achieve their potential

Building PHC science for the future

Diagnostic tests to detect genetic changes in cancer

Imaging in cancer, cardiovascular disease,

neuroscience and arthritis

Biomarkers associated with respiratory disease

Detecting changes in gene expression

Some Challenges.... some opportunities....

• Working in Genetics I will focus on tumour genetics.

• Specifically issues surround FFPE tissue

• Challenges for assay design

• Opportunities to exploit precious samples

• Personalised healthcare requires that biomarkers are measured in the samples that are available, in the case of Cancer usually archival blocks:

Analysis of Tumour Material from FFPE Blocks

1. Histopathology and dissection of tumour

2. Scraping

3. Extraction of DNA

Archival Tumour Sample

4. Sequencing of DNA 4. Other Molecular diagnostic analysis of DNA

Fresh Biopsy Sample

2. Snap frozen in liquid nitrogen

Mutation D

etecti on

Archival Tissue- a precious resource…….and a challenge for diagnostic tests.•FFPE Tissue highly variable

• Fixation• Age/ Oxidation• Storage• Variable block sizes

• ...even different paraffin waxes

• Inhibitors- both from fixation and biological e.g. Melanin

• Necrosis/calcification• Heterogeneity• Connective tissue/ fat

• Highly fragmented DNA• Low copy numbers of amplifiable

DNA• Not always readily available…

Robust validation

Innovative assay design

Effective collaboration

Challenges

Opportunitie

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Further challenges-Surrogate Tumour MarkersCirculating tumour cells and circulating free DNA (cfDNA)...

Michael Fleischhacker & Bernd Schmidt, Nature Medicine 14, 914 - 915 (2008)

Circulating tumour cells released to the circulation from the tumour

Circulating free tumour DNA released to the circulation

from the tumour (apoptosis/necrosis/?)

Opportunities- More accessible, less invasive, allows

serial sampling.- Most patients would agree to blood draw- Some technology available e.g. Inostics

Beaming

Challenges- Variable amounts of cfDNA between

patients/ tumour types/stages- Very low concentration in a very high

wild type DNA background.- Low detection rate using conventional

AS-PCR (~0-50% depending on tumour type and stage)

What challenges do FFPE or cfDNA present for technology in a clinical setting?

• Sensitivity- Mutant allele often present in background of wild type DNA (sequencing 20%, ARMS ~1%)

• Specificity- FFPE DNA fragmented ~200bp, inhibitors can cause false positive results, need for robust cut off criteria.

• Coverage of mutations e.g. G12/13 and Q61.

• How tissue intensive is it?

• Few handling steps- 5-6 handling steps not ideal in a clinical setting, contamination, time consuming, transpositions.

• Assay turnaround time- Sample reception, pathology, extraction, analysis, repeat analysis, reporting.

• Ease of use/ interpretation of results/ level of validation

Example: KRAS testing…

Factors affecting prospective & retrospective testing in clinical trials…

• Is further validation in biomarker matrix required e.g. Demonstrated functionality in cfDNA or different tumour type?

• LDT and RUO tests are subject to variation in manufacture, have QC procedures been put in place?

• Have you identified the risks and mitigated them?- E.g. Do you need a back up assay? Risk to supply chain?

Where will the test be used?• Is the pathology lab you are using aware you are using exquisitely

sensitive techniques?

Example: Use of Roche COBAS-KRAS Kit to analyse FFPE tissue

KRAS +/-

Overview of COBAS KRAS assay…

• The COBAS z480 KRAS assay covers 21 KRAS mutations over 3 codons. (G12/13 and Q61)

• Each kit provides enough reagents to analyse 24x2 samples.- All volumes are within ranges which can be aspirated and dispensed

accurately.

• No interpretation of results required.

• FFPE to result within one working day.

• The kit includes the DNA extraction kit.

• COBAS z480 platform becoming widespread in healthcare facilities.

COBAS Absolute Copy number Sensitivity

100 copy input 5%

100 copy input 1%

100 copy input 2%

100 copy input 0.5%

Claims 100 mutant copy minimum at 5% sensitivity- exceeds this comfortably

Sample Mutation Status

COBAS Test Result COBAS KRAS

120168B2 G12V Mutation Detected Codon 12/13126404A2 G12A Mutation Detected Codon 12/13125360A2 G12S Mutation Detected Codon 12/1342992A2 G12C Mutation Detected Codon 12/1373972A1 G12C Mutation Detected Codon 12/13124845B2 G13D Mutation Detected Codon 12/13126584A2 G13C Mutation Detected Codon 12/13125062A2 Q61R Mutation Detected Codon 61125359A2 G12S Mutation Detected Codon 12/13123877A2 G12A Mutation Detected Codon 12/13117225A2 WT Mutation not

DetectedN/A

120167B2 WT Mutation not Detected

N/A

120172B2 WT Mutation not Detected

N/A

120173B2 WT Mutation not Detected

N/A

122805A2 WT Mutation not Detected

N/A

122819A2 WT Mutation not Detected

N/A

122820A2 WT Mutation not Detected

N/A

122829A2 WT Mutation not Detected

N/A

122830A2 WT Mutation not Detected

N/A

122833A2 WT Mutation not Detected

N/A

Concordant in FFPE

COBAS KRAS Kit Summary...• DNA extraction superior (2.5x amplifiable DNA vs.

closest comparable kit)

• Sensitive and concordant in FFPE• Detects 100 copy mutant at 5% as claimed

• As sensitive as closest leading kit

• Already validated in CRC tissue and tests available in KRAS/ BRAF and EGFR

• BRAF test successfully launched as a IVD companion diagnostic for Vemurafinib

Further characterisation of clinical samples• More prospective Phase I & II trials which have a PHC

component and have response data.• An opportunity to explore precious data set....

www.wordle.netIllustration: Relative impact of various genes in CRC

Sequenom MassArray 4 in a Ph II NSCLC TrialBroad Spectrum Mutation Profiling•MALDI-TOF mass spectrometry based mutation detection

•Precious resource left over from NSCLC clinical trial

•Selected mutations in genes based on biological significance and frequency.

•14 genes 80+ positions (approx 160 known mutations)

•Fumagalli et al, BMC Cancer. 2010; 10: 101. A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes

High multiplexing, low samples usage...

• ~10 somatic mutations in each multiplex (8 multiplexes per sample)

• 5% sensitivity established by cell line admixtures

• Able to use very little template (400-500 copies avg. per multiplex*)

Uses a single mass modified base pair extension method to identify mutations

* Consult Sequenom on recommended template quantities

Screening SuccessExceptional success from FFPE

•DNA input ranged from <0.01ng to over 50ng

•100% assay success rate* (n=230)

•95%+ assay design success rate on first round

2 ARMS fail 1

69 ConcordantARMS/Sequenom

5 KRASG13/Q61 additional by Sequenom

Sequenom did not detect. (Less sensitive)

•NSCLC tested for KRAS mutations in a Phase II trial using Qiagen KRAS Therascreen Kit

•100% concordance where Sequenom & +’ve ARMS result available (Sequenom not as sensitive as ARMS & Qiagen KRAS kit interrogates G13D and not Q61)

•(n=230 NSCLC samples, only positive samples shown in Ven diagram. All samples negative by ARMS also negative by Sequenom in mutually assayed positions. Sequenom picked up 2xG13C and 3xQ61 mutations.)

Concordance with alternative technology...

Poster on display....

Co-OccurrenceWhat mutations occurred with KRAS?

Activating mutations in PIK3CA reduce sensitivity to MEK

inhibitors. Wee et al Cancer Res 2009;69:4286-

4293.

PTEN mutations may confer resistance to

MEK inhibitorsWee et al 2009

Activation of p16 may lead to down regulation of CDK4 and CcND

causing increased sensitivity to RAF inhibition

Gjoerup et al, J. Biol. Chem 1998;273(30):18812–18

Summary of Sequenom Approach

•These data may help explain some of the responses in a clinical setting and identify mutation profiles which respond well to therapeutics.

•Technique may assist in stratifying patients where public mutation data is sparse and assist in hypothesis generation when combined with response data.

•Employed in BATTLE trials to select which drug would benefit patient based on mutation status- demonstrated a significant improvement in patient outcome:

• Methodological Challenges for Personalized Cancer Therapies: Clinical Trials for Personalized Medicine Nat Rev Clin Onc. 2011;8(3):135-141

The BATTLE Trial: Personalizing Therapy for Lung Cancer. Cancer Discovery 1:44-53, 2011.

Overall Summary..

•AZ is committed to improving benefit to patients through its PHC strategy.

•Archival samples used in PHC present many challenges that need consideration.

•Need to fully validate technology in target sample matrix, identify risks and mitigate them especially when used in clinical setting.

•New technologies available that can help characterise your patient population more fully. Useful in early development and as lab developed tests (LDT’s) in product life cycle management.

Acknowledgements

• John Mills• John Morten• Gillian Ellison• Maria Orr• Helen Ambrose• Emma Donald• Gael McWalter

• Kyaw Aung (Paterson Institute)• Susanne Muller / Malcolm Plant (Sequenom)• Sean Tyacke/ Jane Turner (Roche Diagnostics)