Gaining New Insights Through IF Multiplexed Staining and Analysis. Tyna Hope, Ph.D. P.Eng Biomarker Imaging Research Laboratory October 5, 2017

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1 Gaining New Insights Through IF Multiplexed Staining and Analysis Tyna Hope, Ph.D. P.Eng Biomarker Imaging Research Laboratory

2 Assessing more from Tissue Sections

3 Gaps with More Common Methods Most common practice is to assess via individual IHC on sequential serial sections (different cells) Multispectral methods are limited in the number of markers, because of the need to separate the colour stains in the same pixel

4 MxIF HER/neu ER PgR Ki67 Cytokeratin DAPI ER + PgR + Ki67 + Cytokeratin HER/neu + ER + PgR 4

5 Sequential Stain and Bleach Acquire background AF Image Cy-Ab Cy5-Ab Stain slide with Ab and Ab Bleach Fluorophor >60 proteins Acquire IF image 5

6 Multiplexing Method Formalin fixed and paraffin embedded tissue samples. Automatic sequential stain-imagebleach approach utilizing direct antibody-fluorophore conjugation. Multiplexing with GE s MxIF We are also able to stain and bleach manually by use the imaging equipment. 6

7 Application in Ovarian Cancer Research CD + CD4 CD4 + CD8 CD + CD4 + CD8 CK + CD4 + CD8 CD Red CD4 Green CD8 Blue Cytokeratin purple 7

8 Application in Breast Cancer Research ER PR HER Ki67 P CD4 CD8 8

9 Our First Experiment Breast cancer biomarker panel: ER, PR, Ki67, HER Segmentation markers: Dapi, cytokeratin, NaKATPase, S6 First generation equipment 9

10 Goals Determine if the signal correlated with the known grade of the patient Determine if the signal correlated with the pathologists scores Assess co-localization of biomarkers 0 October, 07

11 Calibration and Validation Data TMA Patients, cores Cores included Biomarkers AS000 55, 44 ER and PR AS000 50, 6 Ki67 and Her AS , 99 ER,PR,Ki67,HE R Tissue microarrays were intended to be used for calibration (AS000,AS000) and validation (AS4000) during model development. Omitted cores are due to missing tissue.

12 Technique Development Cy Cy5 We had to change the protocol to prevent false positive signals. Her ER Antibody species cross-reaction caused the nuclear staining ki67 to appear to stain the membrane. Bleached PR Ki67 Dan Wang, et al., AIMM 05 August 5 Wang D, Pang Z, Clarke GM, Nofech-Mozes S, Liu K, Cheung A, Filkins RJ,Yaffe MJ. Ki-67 membranous staining biologically relevant or an artifact of multiplexed immunofluorescent staining. Applies Immunohistochemistry & Molecular Morphology 05 Aug 5.

13 Technique Development ER PgR HER/neu Ki67 Cy5 Her ER ER PgR HER/neu Bleached Ki67 Bleached PR 00 um Ki67 Dan Wang, et al., AIMM 05 August 5 Validation of IF staining protocol using IHC stained serial sections.

14 Discussion Plan: Develop methods on calibration data and assess on validation data. Reality: We had limit our assessment to ER and HER in the calibration set. WHY? Cross-reactivity meant PR and Ki67 signals were questionable for calibration data set Validation signal magnitudes were much different than the calibration set 4

15 Results Models were developed on ER and HER from the calibration data set only. We found an association between the image signal and pathologists IHC score. Complex models, using groups of decision trees Scale and intensity invariant features Used out-of-bag (OOB) error rate (like cross-validation) ER model: OOB 5.7% (94.% accuracy), 95% CI (0.89, 0.98). HER model: OOB 4.6% (95.4 % accuracy) 5

16 Companion Software Visualize as H&E Obtain quantitative information on each cell. 6 Nuclear, cytoplasm, membrane segmentation

17 In-house Developed tools ER PgR Ki

18 In-house Developed Tools 8

19 9 In-house Developed Tools Tumour A microenvironment Tumour A Tumour B microenvironment Tumour B

20 Quantification of relationships Our goal is to describe the spatial arrangement of types of cells making up the tumour and/or the microenvironment. This may help us understand the heterogeneity of cell populations within a patient and across patients. 0

21 Finishing Touches

22 Biomarker Imaging Research Laboratory Lab Members Martin Yaffe, Ph.D., C.M. Principle Investigator Gordon Mawdsley, BSc, FCCPM, P.Phys. Lab Manager Alison Cheung, PhD Research Associate Yulia Yerofeyeva, M.Sc. Program Manager Kela Lui, MD Immunohistochemistry Technologist Dan Wang. M.Sc. Immunohistochemistry Technologist Taha Rashed BHc (MLS), MLT Lead Medical Laboratory Technologist Rachel Peters, AIMLS (UK), FIMLS (UK), ART (Can) Medical Laboratory Technologist Adebayo Adeeko, FIScT, PG Dip., MLA/T Medical Laboratory Technician Mayan Murray, M.Eng., EIT Research Engineer Reach us: