Manual assessment of biomarker expression is associated with significant inter- and intra reader variability. In some cases there are also limitations when it comes to sensitivity and specificity of manual biomarker assessment. In one example to the left, the “pure” contribution of inter-reader variability associated with Ki67 assessment was quantified across 20 tumors and 126 participating labs. In that study, it was demonstrated how image analysis can be used to significantly reduce inter-reader variability. In a another study, the National Danish Validation study of Her2, it was demonstrated how improved sensitivity/specificity of quantitative HER2 protein expression wrt gene amplification lead to significant cost savings in reflex testing. By automating aspects of stain quality control, it will become scalable to he point where EQA organizations may be able and willing to offer more frequent – perhaps even on-demand – proficiency testing and calibration services. It is possible that objective and quantitative standards will contribute to improve compliance with protocol recommendations. In clinical multi-center trials it will be easier to standardize and monitor data from each center. And it is our hope tha larger diagnostic pathology labs will be able to benefit from such a method by closely monitoring drift in staining quality for biomarkers.

1. HORIZON DISCOVERY
Blueprints to Blue Sky – Analyzing
the Challenges and Solutions for
IHC Companion Diagnostics
Hannah Murfet, MCQI CQP, BSc, DipQ
Martin Kristensson ,M.Sc.

2. 2
Presenter
Hannah Murfet (MCQI CQP, BSc, DipQ)
Product Quality Manager, Horizon Discovery
Hannah is a medical biochemist turned quality professional with a strong interest
in the future of quality and regulatory management with focus on advances in
medical technology. Hannah is involved with the Chartered Quality Institute
predominantly as Vice Chair of the Next Generation Network and first appointed
young representative to the Advisory Council, through which she advocates the
role quality management in business.

3. 3
Presenter
Martin Kristensson, M.Sc.
Director of Sales & Project Manager (Digital Pathology), Visiopharm
Martin has been with Visiopharm since 2011 where he has worked in various
aspects of the business including technical support, professional services, as an
application scientist, and most recently as the Director of Sales for Digital
Pathology. His expertise in Project Management, was a key component in
initiating Visiopharm's clinical validation projects, and is continuously supports
pathology laboratories in the transition to digital pathology, including on-site
validation projects covering image analysis for diagnostics.

4. 4
The Impact of Biomarkers
Biomarkers are changing drug
development
1990’s 2013
1 Drug :
1 Assay Multiple Drugs :
Multiple Assays
FOR RESEARCH USE ONLY

5. 5
The Impact of Biomarkers
New biomarkers are increasing
complexity
Different tumor indications and combination of influencing markers
Different IHC Antibody Clones
Different staining protocols and platforms
Different clinical decision points
Different assessment methods
Challenged by limitations in IHC precision
Challenged by limited biopsy tissue
Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439878.pdf
FOR RESEARCH USE ONLY

6. 6
Challenges Facing the Clinical Pipeline
• Clinical Trial time lines and use of
multiple partners/geographies can lead
to drift in assay performance
• Outsourcing to CROs can make
managing variation more difficult
• Unnecessary and sustained variability
can affect study performance
• Manual evaluation methods leading to
the potential of variation
• New drugs rely on successful and
effective adoption of diagnostics to
ensure reimbursement
• Proficiency Testing – ensure
standardization and accuracy of
diagnostic testing are met.
• Clinical Samples are not readily available
Research & Development
• Assay Development
• Sample Screening
• Patient Stratification
Clinical Studies
• Phase II & III
• CLIA Studies
• Patient Stratification
On Market
• Companion Diagnostics
• Proficiency
Testing/Ring Trials
FOR RESEARCH USE ONLY

7. 7
The Regulatory Challenge
New biomarker complexities impact the regulatory
challenge
Safety and efficacy determined in clinical trial with
emphasis on marker positive
Multiple tests have the potential to change the
selected group for each test
For new tests, bridging studies become necessary to
support new safety and efficacy
Potential for mismatched approved drug/device
combinations
*Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439878.pdf
FOR RESEARCH USE ONLY

8. 8
The Impact of Biomarkers
What is PD-L1?
Biomarker with a major role in suppressing anti-
tumor immunity
Variation in tumor expression and response
Pre-analytical and analytical variables
Differences in application of assays therefore clear
stratification will be necessary
Open questions remain on utility of PD-L1 as a
predictive marker
*Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439878.pdf
FOR RESEARCH USE ONLY

9. 9
The Impact of Biomarkers
The PD-L1 Challenge
4-8 drugs in development
Parallel development programs
Variation in trial design
Multiple companion diagnostics –
different test for each drug
Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439878.pdf
FOR RESEARCH USE ONLY

10. 10
Introduction to Blueprint and Blue Sky
*Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439878.pdf
Blueprint
Proposal –
Industry
group*
Blue Sky –
Development
of novel IHC
tools
FOR RESEARCH USE ONLY

11. 11
Bluesky Development
Unlimited supply mechanism (cell line derived)
Reproducible production pathway
Independent from tissue archives
The same material source can be used for multiple
tests
On-slide controls
Assessment using QDP
Reference
Material
FOR RESEARCH USE ONLY

12. 12
FACTORS INFLUENCING DATA QUALITY
Tissue preparation (e.g. fixation)
Staining- protocols & sufficiency
Reading and interpretation
Bluesky Development – the devil is in the data…
FOR RESEARCH USE ONLY
Knowledge generation processTissue processing
and sampling
Tissue staining Imaging ConclusionReading and
interpretation
Data analysis
Dependencies
Sensitivity, Specificity, Reproducibility

13. 13
Reading and Interpreting Biomarker Expression
FOR RESEARCH USE ONLY

14. 14
Reading and Interpreting Biomarker Expression
Improving reproducibility
Example Ki67 Data: NordiQC
NEG 2+ 3+ TOTAL
NEG 217 217
2+ 46 5 2 53
3+ 0 1 41 42
263 6 43 312
Site III, Denmark HER2-CONNECT
Manual
Reading
TOTAL
N 156
%Agreement 84.29%
95% C.I. 80%-88%
2 cores per patient
Improving diagnostic accuracy
Example HER2, reduces 2+ with 70% compared to humans.
Reduce cost of reflex testing w. 4.000 EUR per 100 patients.
Data European Validation Study
FOR RESEARCH USE ONLY

15. 15
Potential Uses of the Method
Proficiency Testing and
Calibration (EQA)

16. 16
IHC Bluesky Application: Key Questions
Does your assay accurately and reproducibly
measure what you say?
Does the assay actually identify a biological
difference of clinical significance?
Does your validation show evidence to support
improvements in the clinical workflow?
How do you monitor for your IHC analytical
performance?
FOR RESEARCH USE ONLY

17. 17
IHC Bluesky Application: Utility of HDx Reference Standards
Reference
Material
Performance can be assessed when optimizing a
protocol or platform
Guide the evaluation of antibodies
Protocols can be assessed for use of the same
antibody clone
Concordance or discordance can be assessed to
determine reproducibility
FOR RESEARCH USE ONLY

18. 18
Case Study – PD-L1 HDx Reference Standard Development
1 2 3 4 5
A
B
Cell Signalling antibody - Clone E1L3N
FOR RESEARCH USE ONLY

19. 19
Case Study – Assessing the Negative and Positive
Reference Standards by QDP
Percentage positive H-Score
FOR RESEARCH USE ONLY

20. 20
Case Study – PD-L1 Positive Reference Standard
10X
40X
FOR RESEARCH USE ONLY

21. 21
Case Study – PD-L1
FOR RESEARCH USE ONLY

22. 22
Case Study – PD-L1 External Evaluation
1 2 3 4 5
A
B
Cell Signaling antibody - Clone E1L3N
FOR RESEARCH USE ONLY

23. 23
Case Study – Highlighting variability in the workflow
The same cell
signalling antibody
and same
concentration
+ve core
Supposed to be the -ve core
FOR RESEARCH USE ONLY

24. 24
IHC Blueprint Proposal
Goal
• A package of information upon which analytical comparison of various diagnostic assays
may be conducted.
• This includes understanding the analytical performance of different PD-L1 biomarker
assays.
• Study to be designed through collaboration with industry stakeholders and independent
third party.
Samples
• Mix of sample types that are representative of target patient populations.
• Focus on NSCLC and PD-L1 IHC Testing.
Staining
• Diagnostics stakeholders to stain IUO assays
Evaluation
• Company pathologists and independent third party
• Published results
Source: FDA-AACR-ASCO Public workshop on March 24 2015.
http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM439440.pdf
FOR RESEARCH USE ONLY

25. 25
Collaboration is Key

26. 26
Collaboration is Key
Contact us at
technical@horizondiscovery.com

27. Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Your Visiopharm Contact:
Hannah Murfet, MCQI CQP, BSc, DipQ
Product Quality Manager
h.murfet@horizondiscovery.com
Martin Kristensson, M.Sc.
Director of Sales/Project Manager
mkr@visiopharm.com