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Gaining New Insights Through IF Multiplexed Staining and Analysis Tyna Hope, Ph.D. P.Eng Biomarker Imaging Research Laboratory October 5, 2017
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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Assessing more from Tissue Sections
October 5, 2017
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Gaps with More Common Methods
October 5, 2017
• 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
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MxIF
October 5, 2017
HER2/neu + ER +
PgR
HER2/neu
Cytokeratin
Ki67PgRER
ER + PgR + Ki67 +
CytokeratinDAPI
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Sequential Stain and Bleach
Cy3-Ab1 Cy5-Ab2
Acquire background
AF Image
Stain slide with
Ab1 and Ab2
Acquire IF imageBleach
Fluorophor
>60 proteins
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Multiplexing Method
Multiplexing with GE’s MxIF
October 5, 2017
• Formalin fixed and paraffin
embedded tissue samples.
• Automatic sequential stain-image-
bleach approach utilizing direct
antibody-fluorophore conjugation.
• We are also able to stain and
bleach manually by use the imaging
equipment.
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CD3
+
CD4
CD4
+
CD8
CD3 +
CD4 +
CD8
CK + CD4 + CD8CD3 – Red
CD4 – Green
CD8 – Blue
Cytokeratin – purple
Application in Ovarian Cancer Research
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ER PR HER2 Ki67 P21 CD4 CD8
Application in Breast Cancer Research
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Our First Experiment
• Breast cancer biomarker panel: ER, PR, Ki67, HER2
• Segmentation markers: Dapi, cytokeratin, NaKATPase, S6
• First generation equipment
October 5, 2017
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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
October 31, 2017
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Calibration and Validation Data
TMA Patients,cores
Cores
included
Biomarkers
AS2000 55,3 144 ER and PR
AS3000 50,3 136 Ki67 and Her2
AS4000 50, 2 99ER,PR,Ki67,HE
R2
Tissue microarrays were intended to be used for calibration
(AS2000,AS3000) and validation (AS4000) during model development.
Omitted cores are due to missing tissue.
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Technique Development
We had to change the protocol to
prevent false positive signals.
Antibody species cross-reaction
caused the nuclear staining ki67 to
appear to stain the membrane.
Cy5
Her2
Bleached
Ki67PR
ER
Dan Wang, et al., AIMM 2015 August 5Wang 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 2015 Aug 5.
Cy3
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Technique Development
Cy5
Her2
BleachedBleached
Ki67PR
ER
Dan Wang, et al., AIMM 2015 August 5
ER
Ki67HER2/neuPgRER
Ki67HER2/neuPgR
200 um
Validation of IF staining protocol using IHC stained serial sections.
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Discussion
Plan: Develop methods on calibration data and assess on validation data.
Reality: We had limit our assessment to ER and HER2 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
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Results
Models were developed on ER and HER2 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% (94.3% accuracy), 95% CI (0.89, 0.98).
•HER2 model: OOB 4.6% (95.4 % accuracy)
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Companion Software
October 5, 2017
Visualize as H&E
Nuclear, cytoplasm, membrane
segmentation
Obtain quantitative information on each cell.
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In-house Developed tools
October 5, 2017
ER PgR Ki67
+ - - +
- + - +
- - + +
- - - +
- + + +
+ - + +
+ + - +
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In-house Developed Tools
October 5, 2017
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In-house Developed Tools
October 5, 2017
Tumour A
microenvironment
Tumour A
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Tumour B
microenvironment
Tumour B
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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.
October 5, 2017
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Finishing Touches
October 5, 2017
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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: [email protected]
October 5, 2017
