1. Practical Approaches to Tumor Xenograft Analysis Frank Voelker, Flagship Biosciences LLC Trevor Johnson, Flagship Biosciences LLC Veronica Traviglone, Infinity Pharmaceuticals Igor Deyneko, Infinity Pharmaceuticals

2. www.flagshipbio.com Outline Introduction Anatomy of a Xenograft Defining the Approach to the Analysis Analytical Strategies Examples of Cases Presenting FACTS for Target Tissue Analysis

3. www.flagshipbio.com General Anatomy of a Xenograft CD-31 Stain for Microvessels

4. www.flagshipbio.com Chronic Inflammation and Peripheral Adipose Tissue Adjacent tissue frequently contains host responses such as inflammation, adipose tissue or adnexal structures that need to be excluded from the analysis

5. www.flagshipbio.com Extensive Necrosis and Degeneration Necrosis and neoplastic cells in various stages of degeneration are common features of a xenograft. CD31 Immunostain

6. www.flagshipbio.com Host Response of Vascular Ingrowth Demonstrated with CD31 Immunostain Even the smallest microvessels may be enclosed in adventitial tissue as an extension of stromal ingrowths

7. www.flagshipbio.com Compare H&E and IHC Stains H&ECD31

8. www.flagshipbio.com Compare Corresponding Areas of H&E and CD31 Stains Compare microscopic characteristics of an H&E- stained section with comparable regions of an IHC CD31-stained section This will provide valuable perspective regarding IHC target tissue staining and will allow more accurate identification of tissue classes. H&ECD31

9. www.flagshipbio.com Significant Xenograft Tissue Components  Neoplastic Cells  Tissue Biomarker  Connective Tissue Stroma  Necrosis and Degeneration  Cystic or Secretory Vacuoles  Artifactual Space Which of these do you want to include in your analysis?

10. www.flagshipbio.com Some Examples of Analytical Endpoints Neoplastic Cell Area Total Xenograft Area Marker Area or Score Neoplastic Cell Area A function of neoplastic cell abundance versus necrosis and/or stromal prominence Immunohistochemistry expression of marker metabolism within neoplastic cell population Number of Neoplastic Nuclei Neoplastic Cell Area An indication of mean neoplastic cell size Targeted Cell Number Total Xenograft Area Cell frequency within measured area Number of Microvessels Neoplastic Cell + Stromal Area Microvessel analysis

11. www.flagshipbio.com What aspects of a marker determine treatment effect? ( Nwp/Ntotal)x(100) + Np/Ntotal)x(200) + Nsp/Ntotal)x(300) = “H Score” (For a maximum of 300) Staining variation in tumors or other tissues frequently raises the question of whether to measure either percent area or average intensity of an immunostain. In some cases, a solution to this problem is to measure score as an output convention encompassing both percent area and stain intensity.

12. www.flagshipbio.com 12 Manual Use of Positive and Negative Pen Tools Similar IHC staining of fibronectin and secretion droplets in this xenograft tumor with subsequent poor differentiation by the Genie™ classifier required the use of the negative pen tool to assist in quantitating fibronectin using the IHC deconvolution algorithm.

13. www.flagshipbio.com Manual Use of Positive and Negative Pen Tools Use of a 21UX Cintiq Wacom drawing board facilitates the use of positive and negative pen tools in manually delineating critical features of the xenograft.

14. www.flagshipbio.com Using Genie™ to Segregate Neoplasm from Nontarget Tissue in a Xenograft Stained for Phospho-Histone 3 Central regions of the xenograft contain an interdigitating pattern of necrosis and connective tissue trabeculae too complex for manual exclusion. Accurate segregation of neoplasm (green) from necrosis and connective tissue (red) was accomplished using Genie™

15. www.flagshipbio.com The Rationale Behind Histology Feature Recognition Primary ImageGenie PreprocessingAnalytical AlgorithmAnalytical Result Using software to preprocess an image with the purpose of segregating target tissue components from nontarget tissue. Aperio Genie™ Primary Image PreprocessingFeature RecognitionPostprocessing Visiopharm Subsequent analysis yields accurate data only from the target tissue component, and omits erroneous nonspecific results from nontarget tissue. Feature recognition is valuable in xenograft analysis when target and nontarget regions are too intricately interwoven for manual exclusion.

16. www.flagshipbio.com Strategy for Analyzing Successive Samples 1234567891011121314151617181920 Progression of analysis through sample series Red = 1 st Classifier Blue = 2 nd Classifier Green = 3 rd Classifier * = Points when a new classifier was developed during the analysis *** Xenograft neoplasms within a study are surprisingly heterogeneous even though derived from the same source, and it is difficult and time consuming to derive a common pattern recognition classifier appropriate for all neoplasms within a study. It is more expeditious and also yields more accuracy to develop new classifiers as the analysis progresses.

17. www.flagshipbio.com Consistent Analysis of Study Samples Using Genie™ Subsequent Uniform Analysis of Segregated Target Tissue for area/intensity Even though multiple classifiers are constructed for successive samples, the final analyses of target tissue components will be accurate and comparable if the same algorithm threshold values are constantly maintained.

18. www.flagshipbio.com Using Genie™ to Segregate Neoplasm from Nontarget Tissue in a Xenograft Stained for Phospho-Histone 3 Higher magnification reveals accurate separation of target from nontarget tissue. The grayed-out nontarget tissue class consists of a mixture of connective tissue trabeculae and necrotic tumor cells.

19. www.flagshipbio.com Assessing Fibronectin in a Xenograft Using Several Software Programs Assessing the intensity and quantity of fibronectin as a marker in xenograft stroma Aperio Deconvolution Mark-up Visiopharm Mark-up Original IHC Image

20. www.flagshipbio.com 20 Assessing Angiogenesis in a Xenograft Using Several Software Programs Use of the microvessel analysis algorithm to assess angiogenesis in a xenograft neoplasm in a mouse Microvessel analysis provides important information regarding potential antineoplastic effects of pharmaceutical compounds Original CD31-Stained Image Aperio Microvessel Algorithm Mark-up Visiopharm Mark-up

21. www.flagshipbio.com Using the Microvessel Analysis Algorithm to Count Cyclin B1-Positive Target Cells in a Xenograft Use of the microvessel analysis algorithm to assess macrophage populations in mouse xenograft neoplasms Threshold algorithm parameters are modified to accommodate the smaller size and shape characteristics of the cells

22. www.flagshipbio.com Use of Visiopharm™ Software to Analyze a Marker Separately in Cytoplasm and Nuclei of a Neoplasm Discriminating between nuclear and cytoplasmic regions of a neoplasm allows separate biomarker intensity measurement for both nuclear and cytoplasmic markers (analysis with Visiopharm software).

23. www.flagshipbio.com Distinctive or Special Staining Facilitates Target Tissue Pattern Recognition H&EH&E Classifier CD31CD31 Classifier First pass accuracy is much greater for the H&E-stained section than for the hematoxylin- counterstained IHC section Current histology pattern recognition programs often have difficulty distinguishing target tissue profiles because of low contrast /nonspecific counterstaining

24. www.flagshipbio.com Feature Analysis on Consecutive Tissue Sections (FACTS) 4. QC and pathologist review 3. Image and ROI registration 2. Automated feature recognition 1.Consecutive tissue sectioning

25. www.flagshipbio.com Image and ROI Registration Register images with <3% Feature Error Across multiple consecutive slides –Not a trivial problem given the potential histological and biological differences between tissue sections and stains Several registration techniques currently used throughout CT/MRI, CT/PET, CT radiology, ultrasound, and multispectral imaging –Typically elastic, morphing, because of image warping –Heavy use of image features or fiducials Type of registration for best performance in FFPE tissue is heavily dependent on the application 3. Image and ROI registration

26. www.flagshipbio.com Typical Image Registration Process Feature detection –Distinctive objects to be aligned Feature matching –Finding the association between the distinct objects Transform model estimation –The mapping functions used to align the subsequent image to the reference image –May be non-elastic (much simpler) or elastic Image re-sampling and transformation –Applying the mapping functions where non-integer coordinates can be correctly interpolated

27. www.flagshipbio.com Non-Elastic Image to Image Registration Perform color deconvolution to focus on a common Stain, typically Hematoxylin –Helps reduce potential errors from strongly stained “positive” features Use slide features and image intensities to adjust rotation and translation of selected images or regions u(x,y) -> v(x,y) Rotation: x’ = xcos(θ) - ysin(θ) y’ = xsin(θ) + ycos(θ) Translation:x -> x’ y -> y’

28. www.flagshipbio.com Example of Slide Registration

29. www.flagshipbio.com Annotated Feature to Feature Registration More complex due to biological and histological differences between serial sections Usually Elastic to count for these differences –Important to note that the actual images are never morphed, just the annotations Let the application determine the method Phase Registration: ℮xp(2 ∏ i (u x’ + v y’)) = ƒ(f) ƒ(g) / | ƒ(f) ƒ(g) | Mutual Information: MI(X,Y) = H(Y) – H(Y | X) = H(X) – H(Y) – H(X,Y) where H(X) = Ex(log(P(X))

30. www.flagshipbio.com Wavelet Transform: Decompose image into collection of appropriate wavelet (Spline, Haar, Etc.) Filter image along rows and columns (high and low pass) Find frequency coefficients and apply differential measurements Annotation Feature to Feature Registration (cont.)

31. www.flagshipbio.com Example of Feature Registration

32. www.flagshipbio.com Acknowledgments Dr. Steven Potts, Flagship Biosciences LLC Dr. David Young, Flagship Biosciences LLC Ms. Charlotte Aagaard Johnson, Visiopharm Inc. Mr. Rob Diller, Flagship Biosciences LLC Mr Erik Hagendorn, Flagship Biosciences LLC Questions? Email contact: frank@flagshipbio.com trevor@flagshipbio.com