1. Bridging the Divide- Using Digital Pathology to Guide Ultrastructural Pathology Evaluations. GD Gagne, JH Decker and JA Fagerland. Preclinical Safety, Abbott Laboratories

2. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Overview Digital pathology at Abbott Ultrastructural pathology: Identification of pigment in liver and adrenal tissues Evaluating biomarkers of glomerular injury using TEM, laser capture microdissection Image analysis applications

3. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Digital Pathology at Abbott 2008- Digital pathology system (120-slide) installed in Pathology Department, Lake County, IL (LC) 2009 –120-slide system installed in Ludwigshafen, Germany Pathology Department – 6-slide scanner installed in LC Ultrastructural Pathology/Investigative Toxicology facility –Added whole-slide image analysis capability, including pattern recognition software Applications: multisite slide sharing, global pathology teleconferencing, pathologist-to-pathologist consultation

4. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Ultrastructural Pathology Single global EM facility (LC) Transmission electron microscopy (TEM) evaluation used to further characterize histopathology findings TEM sections:1-2 mm 2 LM sections: ≤8 cm 2

5. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Bridging the divide between LM and TEM Digital pathology system allows: –consultation between electron microscopist and pathologist to assure changes seen by histopath are present in sections taken for TEM –comparison of H&E-stained histological sections for LM with toluidine blue-stained semithin sections for TEM –interactively review by personnel at different sites –example: Identification of pigment in liver and adrenal gland in a 12-month toxicity study in cynomolgus monkeys

6. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Evaluation of Pigment in Liver and Adrenal Tissues Twelve month toxicity study in cynomolgus monkeys Accumulation of pigment noted by LM in liver and adrenal gland from monkeys administered high dose of test compound (had not been seen in previous monkey tox studies) Liver and adrenal tissue was retrieved from formalin and sent to Abbott for EM evaluation Questions: –Nature of the pigment –Similarity to pigment seen in rats dosed with same compound

7. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Liver, high dose monkey Images from digital slides of H&E and semithin sections

8. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Liver, high dose monkey, Kupffer cell,TEM images

9. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Liver, high dose monkey, endothelial cells

10. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Adrenal gland, high dose monkey. Semithin section image from digital slide.

11. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Adrenal gland, zona reticularis, high dose monkey

12. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Evaluating biomarkers of glomerular injury Purpose of Study- To determine if urinary biomarkers can provide a sensitive and accurate measurement of receptor tyrosine kinase (RTK) inhibitor-induced glomerular injury RTK inhibitors have been shown to cause proteinuria and glomerular injury (visible by EM) in experimental animals and humans in clinical trials TEM used to –characterize glomerular changes –correlate changes to biomarker measurements

13. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott ProteinMechanism of Release into UrineInjury Site Albumin Freely filtered through the glomerular basement membrane, reabsorbed (and a fraction degraded) by the proximal tubule Glomerular and tubular injury Lipocalin-2 (NGAL) Expressed and released in response to injury Glomerular and tubular injury OsteopontinExpressed and released in response to injury Glomerular and tubular injury KIM-1 (Kidney Injury Molecule-1) Expressed and released in response to injuryTubular injury Biomarker Characteristics Courtesy Y. Yang

14. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Individual Animal Biomarker Results- (specimens examined by TEM) Total Albumin (ug) Total Osteopontin (ug) Total Lipocalin (ng) Total KIM-1 (ug) 1001 a 870.61404.88.5 1003 a 175.618.11896.19 4001 b 3247.2201.615389.920.4 4005 b 4456.973.14506.211.6 a: Vehicle b: RTK Inhibitor, dosed for 7 days at 10 mg base/kg/day Increase in albumin and lipocalin, but not KIM-1 suggests glomerular injury

15. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Compare H&E to semithin section at identical mags

16. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Locate features on EM monitor while viewing semithin section slide image (High dose kidney: Glomerulus) Semithin sectionTEM Image

17. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott High Dose kidney: Glomerulus

18. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Conclusion TEM evaluation revealed glomerular changes consistent with RTK inhibition: –Damage to capillary endothelium, loss of fenestrations –Subendothelial electron-dense deposits –Accumulation of electron-dense granules (protein) in podocytes TEM supported the biomarker results that indicated glomerular injury

19. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Laser capture microdissection (LCM) of glomeruli Collect glomeruli for gene expression analysis to correlate with biomarker changes Use LCM to collect tissue Use digital pathology system to document efficiency of laser capture

20. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Prepare stained tissue section (H&E, immunostains, etc.) Align cap (with thermoplastic transfer film) over area of interest Under microscope, locate cells to be dissected Fire laser pulse to embed cells in thermoplastic film of cap Lift cap to remove microdissected cells Place cap in microfuge tube with appropriate extraction reagents for molecular analysis Laser Capture Microdissection

21. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Documentation of Laser Capture Microdissection of Glomeruli Before LCMAfter LCM

22. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Image Analysis Quantification of hepatic lipid on liver sections Select 5 random fields (5x magnification) from each specimen, using whole slide image Detect lipid droplets with MetaMorph ® software, using color, size, and shape discriminators to detect lipid droplets as white, round objects. Report total detected area. Measure total liver area. Calculate “Area fraction lipid” using a spreadsheet.

23. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Feature extraction-lipidTissue area threshold Original ImageThresholded Lipid Area Measurement

24. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Hepatocyte Lipid Quantification

25. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Next Step Apply pattern recognition software to identify and measure areas of vacuolated hepatocytes on whole slide images

26. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Quantitation of percent human hepatocytes in sections of chimeric liver Human hepatocytes Mouse hepatocytes Anti-human mitochondria antibody used to identify human hepatocytes in chimeric mouse liver [PXB-mouse, PhoenixBio]

27. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Morphometric Thresholding on Whole Slides ControlTreated Blue = Mouse origin Yellow = Human origin

28. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Liver Chimerism by Morphometry Control Treated Treatment caused an unexpected reduction in percentage of human hepatocytes in chimeric liver

29. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Summary Applications of digital pathology and whole slide imaging presented: –Ultrastructural pathology Correlation of light microscopy and electron microscopy observations Use of TEM and LCM for biomarker validation –Image analysis Hepatic lipid quantitation Quantitation of human hepatocyte fraction in whole-slide images of chimeric livers

30. Bridging the Divide Pathology Visions 2009 Jerry Gagne © 2009 Abbott Acknowledgements Cellular Molecular and Experimental Toxicology –Yi Yang –Wayne Buck –Andrew Lisowski –Rita Ciurlionis –Eric Blomme Pathology –Carmen Nasarre –George Foley Abbott Antiviral Research –Tami Pilot-Matias –Christine Collins –Susan Lacy –Teresa Ng Investigative Toxicology –David Cugier