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Co-localization of HER2/Neu, ER, PR, Ki67, and Cytokeratin on
a Triple Positive Breast Cancer Patient
Zhengyu
1
Pang ,
Judit
4
Zubovits
, Kashan
2
Shaikh ,
Dan
3
Wang , Alex
2
Corwin ,
Gina
3
Clarke ,
Sean
1
Dinn ,
Robert
1
Filkins ,
and Martin J.
3
Yaffe
1 Diagnostic and Biomedical Technologies, 2 Electrical Technologies & Systems, GE Global Research Center, Niskayuna, NY, 3 Ontario Institute for Cancer Research and
Sunnybrook Research Institute, University of Toronto, Toronto, ON
4 Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON
The treatment of breast cancer is based on the knowledge of estrogen (ER),
progesterone (PR) and HER2/Neu status of a tumour (HER2). However,
prediction of the effect of treatment based on these three biomarkers alone is
somewhat limited, thus motivating the inclusion of additional molecular
biomarkers. To this end, we developed and validated a multiplexing
technology to allow multiple biomarkers to be imaged on the same tissue
section and then analyze biomarker expression at the subcellular level
(nuclear, membrane, and cytoplasmic)
Images of background fluorescence were acquired before and after
the staining. Images were registered, background fluorescence
removed, segmented and analyzed at the single cell level.
Biomarker expression was quantified for subcellular compartments
such as nucleus, cytoplasm and membrane.
Introduction
GE Global Research Center (GRC) has developed proof of concept
instrumentation and the tools for labeling, multiplexing, imaging and
analysis of onco-proteins in fixed tissues. This multiplexing platform
enabled researchers at Sunnybrook Research Institute to study multiple
biomarkers on the same tissue section. Specifically we aimed to 1):
validate multiplexing technology by comparing to traditional histology; 2)
investigate co-localization of 3 established biomarkers in breast cancer,
namely, HER2/Neu, Estrogen receptor (ER), and progesterone
receptor (PR) along with epithelial biomarker (cytokeratin), proliferation
index biomarker (Ki67) and segmentation biomarker (NaKATPase)
HER2
ER
PR
R=0.158
Fig. 2. Integrated platform for multiplexing and imaging. Right is the microfluidic insert
Results and Discussion
R=-0.015
R=0.277
R=0.770
R=-0.083
R=0.079
R=-0.051
R=-0.002
Nuc.ER
Nuc.PR
Immunofluorescence images confirm the positivity of the
ER/PR/HER2 as diagnosed by traditional DAB staining and
confirmed by clinical pathologist at Sunnybrook (JZ). This
validated the multiplex platform for the use in the clinical pathology
lab. Next the co-localization of HER2/Neu, ER, PR along with
Ki67, cytokeratin at specific subcellular compartment was
investigated at the single cell level. As expected, ER, PR and Ki67
expression was localized to the nuclei, whereas cytokeratin and
HER2 staining was membranous (Fig.3).
R=0.012
Nuc.Ki67
Cyto.Cytokeratin
Fig. 4. Matrix plot of HER2, ER. PR, Ki67 and cytokeraitn
Taken together, multiplexing technology should provide
an important tool in research to understand molecular
mechanisms in cancers on an individual patient basis with
the potential role of guiding personalized therapy.
Materials and Methods
For Research Use Only
R=0.178
Mem.HER2
Fig. 1. Multiplexing of HER2, ER, and PR
An anonymized sample of triple positive breast cancer was retrieved from
the files of Sunnybrook Health Science Centre Department of Anatomic
Pathology. Multiplexing was performed using a proprietary microfluidic
system (Fig. 2) . Slides were stained with primary antibodies against
HER2/neu and ER, and then visualized using a secondary antibody
directly conjugated with fluorescent probes Cy3 and Cy5, respectively.
After bleaching the sample, antibodies against PR and Ki67 and directly
conjugated with Cy3 and Cy5 were subsequently used; and followed by
cytokeratin and NaKATPase direct conjugates (Fig. 1).
The matrix plot of these 5 biomarkers is shown in Fig. 4. The
correlation between PR and ER is very significant (R=0.77),
although there are some cells with low ER, but high PR. This
is consistent with the well-established correlation between
ER and PR expression. Correlations between Ki67 and ER/
PR are interesting. Cells cluster into two groups along the
marker axes. Cells with low Ki67 expression have high ER or
PR expression, and vice versa, suggesting that cells in the
proliferative state (high Ki67 expression) tend to lose the ER/
PR expression. There is a much less correlation between
Ki67 and HER2, as the membranous biomarker expression
is probably less dependent on the cell cycle.
Acknowledgement
Authors would like to thank Dr. Alberto Santamaria-Pang for
single cell analysis, Drs. Colin McCulloch and Yunxia Sui
for correlation study using R. This work is supported by
Molecular Imaging and Diagnostic Advanced Technology at
GE Global Research Center and a grant from Ontario
Institute for Cancer Research.
HER2 ER/PR Ki67
Memb. Cytoplasmic Nuclear
Fig. 3. Overlay images (left) and single cell segmentation (Right)
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December 2014 JB26535US