Digital Pathology in Clinical Trials

1. Digital Pathology in Clinical Trials Arnold B. Gelb, M.D. Senior Staff Pathologist Quest Diagnostics Clinical Trials

2. Potential Conflict of Interest Statement • Employee of Quest Diagnostics • Owner of stock in Quest Diagnostics • No financial interest in any imaging company

3. Introduction Image Analysis Clinical Trials Adjudication Digital Archiving

4. Quest Diagnostics Clinical Trials: Overview One of the world's largest Central Laboratories, working to accelerate clinical drug development • Scientific Expertise • > 900 in-house scientists and pathologists covering all therapeutic areas • Extensive test menu and consultative capabilities • Full service Biomarkers and Anatomical Pathology capabilities • Laboratory Management • GCP/ICH operating philosophy • Extensive resources, knowledge and experience in handling and managing clinical specimens, logistics, and data in regulatory compliant environment • Global Reach • Quest owned central lab facilities in North America, Europe, and India • Affiliate laboratories in China, Singapore, and Australia • >4,000 cities in 60 countries • >99% assays performed in-house.

5. PC PC Viewing Stations running ImageScope (unlimited) Site B Site A Internet ScanScope XT’s ScanScope XT’s slide scanners slide scanners Redundant Fail-Safe Server DSR analysis algorithms DSR analysis algorithms Redundant Fail-Safe Server Spectrum web application RAID Spectrum web application RAID slide storage slide storage central servers Datacenter network connections RAID central database digital slides firewalls internal networks Digital Pathology Systems and Networks

6. InScape™

7. Layout

8. Case Details Page

9. The Digital Slide Image

10. Digital Slide Viewing Software There is a toolbar at the top. Slide information such as the slide barcode or the slide ID can be seen in the title bar. Zoom tool is present The thumbnail and label can be turned off in the View toolbar.

11. Image Analysis Window

12. Introduction Image Analysis Clinical Trials Adjudication Digital Archiving

13. IHC Biomarkers • Over 50 assays developed and validated, including esoteric and novel markers, e.g. • Apoptosis • Cell cycle control • mTOR pathway • erbB receptor inhibition • IGF pathway • Angiogenesis • CD antigens

14. Quantitative Analysis of IHC Biomarkers • Identify and quantify morphology and intensity • Subcellular localization – algorithm selection • Nuclear, membranous or cytoplasmic • Tissue (tumor) specific classification • Manual selection of representative tumor regions by pathologist • Automatic feature extraction (pattern recognition) • Enables analysis of invasive tumor from entire image (LANL)

15. Image Analysis Algorithms • May need to adjust intensity of staining • Scanning typically based on % transmittance • Customize or “tune” algorithm to a laboratory’s unique staining process • Controls • Future: batch to batch “tuning” • Validation • Sufficient number of (de-identified) slides across percentage range and intensity scores • 3 blind evaluation arms

16. “Tuning”  Nuclear Segmentation Original Image Normal Segmentation Over Segmented Under Segmented

17. Control Slide

18. Validation Manual Slide Scoring Validate Algorithm Performance Digital Slide Scoring Image Analysis

19. pHH3 • Histone H3 has a key role in the folding and inter-association of the chromatin fiber. • Phosphorylation of Histone H3, at serine-10 and -28, coincides with the induction of mitotic chromosome condensation. • H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation.

20. Digital assessment of the nuclear stain: Index = 13.7% pHH3

21. RXRA • Members of the RXR family, including RXRα, β and γ, are activated by 9-cis retinoic acid, that is expressed in both liver and kidney. • RXRα is an orphan nuclear receptor encoded on chromosome 9q34.2. • It plays roles in a variety of processes including embryonic patterning and organogenesis, cell proliferation and differentiation.

22. RXRA

23. pFOX03a • The forkhead family of transcription factors mediate signaling via a pathway involving IGF-1R, PI3K and Akt. • The gene is located on chromosome 6q21. • Spliced transcript variants encoding the same protein have been observed. • In the presence of survival factors, Akt phosphorylates FOXO3a, leading to its retention in the cytoplasm. • Survival factor withdrawal leads to its dephosphorylation, nuclear translocation, and target gene activation. • Within the nucleus, it triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

24. pFOX03a

25. pLIMK • LIMK1 and LIMK2 are serine/threonine kinases that are involved in actin cytoskeletal reorganization, cell growth regulation and tumor suppression via cofilin phosphorylation downstream of distinct Rho-dependent protein kinase signaling pathways. • Both are activated by various extracellular stimuli, including stromal cell-derived factor-1a and insulin, through activation of small GTPases and their downstream kinases, ROCK and PAK1. • Both LIMK 1 and 2 have been related to tumorigenesis and, thus, are studied in conjunction with anti-cancer therapies that target this pathway.

26. pLIMK

27. Integrins • Heterodimers composed of noncovalently associated transmembrane α and β subunits. • 16 subunits yield over 20 receptors. • Roles in cell adhesion, cytoskeletal organization, and signaling. • Involved in cell growth, differentiation, migration and apoptosis.

28. An Integrin • Digital assessment of membrane staining component: • 3+ cells = 54.7% • 2+ cells = 23.4% • 1+ cells = 21.9%

29. Introduction Image Analysis Clinical Trials Adjudication Digital Archiving

30. Adjudication • Advantages of digital pathology for global clinical trials • Avoids time delays and costs of shipping slides • Read slides from different locations • Education of satellite groups in trial host countries • Ensure high level of agreement/ consistency • Conferencing between central lab pathologist(s) and sponsor’s study pathologist(s) or experts • Fairer resolution than a simple majority or expert rule • Digital substitute for glass slides • When a clinical trial host country has export limitations • Primary diagnosis  H&E (breast cancer) in progress

31. Adjudication: Global Clinical Trials Laboratories

32. Adjudication - Validation • FDA • Validate as much as one can ahead of time • Awaiting completion of FDA approval process • Validation • Ideally study and tissue specific validation • Compare performance of digital to glass slides • Can compute intra- and inter-observer kappa coefficients

33. Introduction Image Analysis Clinical Trials Adjudication Digital Archiving

34. Digital Archiving • Static images → whole slides • Third party multi-format image support • Third party information systems • Scan Types • Bright field: dry, oil • Fluorescence • Advantages • Circumvents problems of slide breakage or stain fading • Real-time access possible

35. Digital Archiving • Length of storage • Duration of study • On server • Long-term • Meet minimum retention requirements • FDA – 5 years post application (21 CFR §58.195) • But when is it submitted by sponsor? • CAP – 10 years for glass slides • Default – 15 years • Removable media/ hard drives • Costs of storage tend to decrease over time • Other • Use in submission of data to FDA • Digital sides more compelling evidence than static images • e.g., hepatitis C

36. Key Points • Image Analysis • Customize staining and algorithms • Validation • Software advances → entire-slide analysis • Adjudication • Decrease TAT • Harmonization • Conferencing • Digital Archiving • Real-time access • Long term storage • Regulatory submission

37. Acknowledgements • Stephanie Astrow • Marc Edwards • Vicki Freeman • Ron Luff • Maribeth Raines

38. Thank you very much for your time and attentionArnold B. Gelb, M.D.arnold.b.gelb@questdiagnostics.com(661) 799-6079