1. No category

Measurement of multiple biomarkers using a novel fluorescence immunohistochemistry multiplexing technique
Sean Dinn, Sudeshna Adak, Musodiq Bello, Anriban Bhaduri, Ali Can, Robert Filkins, Michael Gerdes, Fiona Ginty, Qing Li, Zhengyu Pang,
Christopher Sevinsky, Anup Sood, Maximilian Seel and Jun Xie
Molecular Imaging and Diagnostics Advanced Technology Program, General Electric Global Research Center, Niskayuna, NY
Technology Summary
Standard Approach
Dye Cycling
Stain
A
B
C
C
Y
B
Y
Stain
Inactivate dye
Digital Image-2
Inactivate dye
Y
A
Digital Image-1
Y
Stain
Standard detection of biomarkers in IHC involves
incubating tissue with an unlabeled primary antibody
followed by visualization with a labeled secondary
antibody (i.e. horseradish peroxidase+DAB (“brown
stain”), fluorescent dye)
The ability to measure multiple biomarkers in a
single tissue section is limited by the number of
available species that can be used without having
problems with cross-reactivity
The use of fluorescently labeled secondary
antibodies is limited by the number of resolvable color
channels
Our technology overcomes these shortcomings by
the use of a combination of fluorescently labeled
primary antibodies and a dye cycling strategy
Tissue is stained with a directly conjugated antibody,
then the dye signal is inactivated and the tissue is
ready for probing with another antibody
This procedure can be repeated many times and
combined with the use of several resolvable dyes
per round creates a powerful multiplexing technique
Y
Semi-quantitative detection and repeated tissue sampling
for multiple markers limit current diagnostics that use
chromogenic immunohistochemistry. We have developed
a fluorescence multiplexing process for single sections to
be analyzed repeatedly for molecular markers.
Multiplexing was achieved by staining and imaging of 2
markers, followed by fluorescence inactivation. These
channels can be reused for additional rounds of staining
and imaging, repeatable to a minimum of 10 cycles
without loss of tissue morphology or antigenicity.
Combining tissue markers with cell and molecular markers
allows localization and quantification of diagnostic
markers in defined tissue structures (epithelium, stroma,
nuclei, membranes, cytoplasm, blood vessels, etc.). This
technique is applicable to multiple applications including
biomarker discovery, proof-of-concept evaluation, patient
therapy selection and toxicological profiling. Multiplexing
greater then 10 channels on a single tissue represents a
major step toward understanding complex biological
pathways in tissue.
Digital Image-3…
etc
Inactivate dye
Y Y Y
Use a single dye over and
over. Co-register and
pseudocolor.
Limited by “resolvable”
color channels- 3-4.
Her2/neu breast carcinoma
H&E
b-Catenin
P53
a-actin
Pan cytokeratin DAPI
Overlay Overlay with H&E
P53 breast carcinoma
Progesterone Receptor Androgen Receptor Estrogen Receptor
Her2
Monochromatic single channel images are then pseudo-colored and registered
Integrated
Integrated Multidisciplinary Workflow
Multiplexing Method
Image Acquisition
Fluidics System
Compendium
Validated
Antibodies
Antibody
Work-up
Primary  Secondary
Signal/Background = 4.1
Direct Conjugate (7.6 D/
P) Signal/Background = 4.2
AR
Effect of Dye Inactivation on Markers
120
ER
p53
100
Image Analysis Overview
Compartmental Images
Clinical
Hypothesis
Comparable staining achieved with Cy3 anti-p53 direct conjugate
Pixel Intensity
Abstract
0x
80
No inactivation
10x inactivation
60
40
10x
20
0
AR
ER
p53
Multiplexing Results on Breast Cancer Tissue
Target Images
Background Detector
Optimization
Image Pre-processing
Cy3 Androgen Receptor
Multiplexing
Cy3 Inactivation
Cy3 pan cadherin
Reproducibility
Segmentation
1°2°
1°
Specificity
Dapi
Image Analysis Algorithms
Software Solutions for Tissue
Microarray Analysis
Cy3 Androgen Receptor
Cy5 Her2
Cy3 Pan Cadherin
Cytoplasm Detector
Autofluoresence
removal
Image Analysis
Epithelial Nuclei Detector
0.05
Registration
Biomarker Discovery Tools
Disease Prognosis Prediction
0.035
Segmentation
Scores
Novel Score
Algorithms
Goal is to develop a robust process that can start with a
clinical hypothesis andproduce high content analysis
Multidisciplinary team of biologists, chemists,
engineers and computer scientists
Process being implemented with multiple collaborators
For Research Use Only
0.04
© 2014 General Electric Company ― All rights reserved.
GE and the GE Monogram are trademarks of General Electric Company.
December 2014 JB26538US
Probability
Clinical Data Analysis
Protein Co-localization
Outcome prediction
Pathway analysis
Epithelial Nuclei
Stromal Nuclei
Membrane
Cytoplasm
Tissue
0.045
Cy5 Estrogen Receptor Cy5 p53 (negative) Cy3 pan cytokeratin
Cy3 a-actin
Distribution Score Generator
0.03
0.025
0.02
0.015
0.01
Statistical Analysis Toolbox
0.005
0
0
50
100
150
200
cMet Intensity
250
Cy3 b-catenin
Cy3 g-tubulin
Cy3 Vimentin
Cy3 cmyc
300
Conclusions
A novel platform has been developed to analyze multiple biomarkers on a single tissue section
Method used to inactivate fluorescent dyes does not effect antigenicity or specificity over a wide range of targets
Image analysis algorithms are used to segment markers into subcellular components to facilitate quantitative
analysis of protein levels and localization
Data analysis is used to predict important metrics such as patient stratification, survival analysis, disease
recurrence and toxicity effects