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Quantitating multiple signaling pathway
proteins in preclinical and clinical studies
October 2010
SHARING OPTIONS:
The common need in cancer research and pharmaceutical drug
development is to reveal the configurations of active signaling pathways in
diseased tissues, to support target validation, trial design, patient
selection, response assessment and if trials are successful, the diagnostic
component of theranostics. Importantly, the predictive power of measurements of
signaling protein expression depends on the precision and accuracy of
tissue
analysis tools.
For example, many techniques deployed today, such as those
based on
microarray detection, or analysis of sample lysates, provide data that
are in fact averages from volumes of tissue, including many cells not of
interest. These methods blur out key proteomic information that reside at the
cellular level, and relate to the signaling states of individual cells.
Role of signal transduction pathways in cancer
During the course of tumor progression, cancer cells acquire
a number of characteristic alterations. These
include the capacity to
proliferate independently of exogenous growth-promoting or growth-inhibitory
signals, the tendency to invade surrounding
tissues and metastasize to distant
sites, the penchant for eliciting an angiogenic response and the ability to
evade mechanisms that limit cell
proliferation, such as inflammatory response,
apoptosis and replicative senescence. These properties reflect alterations in
key cellular signaling
pathways that in normal cells control cell proliferation,
motility, and survival.
Many of the
proteins currently under investigation as
possible targets for cancer therapy are signaling proteins that are components
of these pathways. The nature
of these signaling pathways and their roles in
tumorigenesis are the subject of intense study by pharmaceutical companies,
motivated by the hope that
progress in understanding these signaling pathways
will accelerate drug development. It is a complex research task to identify
relevant pathways,
understanding them and demonstrating correlation with
outcome. An additional level of complexity arises from the fact that it is
often the
interrelationship between pathway proteins and their localization
that help characterize the pathway, rather than the mere presence of a protein.
Example: Detecting phospho-epitopes of AKT, ERK and S6
These three pathway markers are widely studied and play a
vital role in cancer pathogenesis. In this
particular example, the goal is to
detect the activation of PI3K/AKT, RAS, and MEK signaling pathways.
AKT has recently been found to play a paradoxical role: on
one hand, it increases cancer cells' survival capability, while on
the other
hand, it blocks their motility and invasion abilities, thereby preventing
cancer from spreading [1]. It had been presumed that one could
promote cancer
cell death by inhibiting AKT that controls the synthesis of proteins involved
in proliferation. Yet now, with this added complexity, the
role of AKT must be
understood further, so as not to promote metastases by inhibiting AKT
expression.
Activation of the MEK pathway up-regulates ERK protein
levels, promoting cell division. This pathway is often up-regulated in human
tumors and
is thought to fulfill multiple roles in the acquisition of a complex
malignant phenotype. Accordingly, a specific blockade of the MEK pathway is
expected to result in not only an anti-proliferative effect, but also in
anti-metastatic and anti-angiogenic effects in tumor cells.
Recently,
potent small-molecule inhibitors targeting
components of the MEK pathway have been developed. Among them, BAY 43-9006 (Raf
inhibitor), and PD184352,
PD0325901 and ARRY-142886 (MEK1/2 inhibitors) have
reached the clinical trial stage. The combination of MEK pathway inhibitors and
conventional
anticancer drugs might provide an excellent basis for the
development of new chemotherapeutic strategies against cancer.
Finally, s6 is a ribosomal protein involved in translation
of mRNAs. It is thought to play an important role in controlling
cell growth
and proliferation.
Automated, multiplexed tissue cytometry
Detecting pathway markers using conventional histology or
immunofluorescence is a challenge, given the need to observe many markers
simultaneously (i.e., to multiplex) in
order to gain a full understanding of the pathways involved and the phenotypes.
Conversely,
conventional multiplexing techniques, such as microarrays or flow
cytometry, fail to provide the contextual information needed to confirm
intracellular
localization; also a requirement in order to confirm pathway
state. What is needed is simultaneous measurement of multiple proteins, on a
per-cell
basis, set in the context of the original anatomy.
New platform technologies now offer us the
opportunity to
access this level of information, by utilizing an effective, practical and
reliable platform for cytometric analysis of intact tissue
sections. This can
be conceptualized as "tissue cytometry." The platform supports preclinical and
clinical studies through the integration of
multiplexed immunohistochemical
(IHC) or immunofluorescent (IF) labeling strategies, robotic slide handling,
and automated multispectral image
acquisition and analysis. Multispectral
imaging systems and advanced image analysis software together provide the ideal
platform for this application.
The ideal imaging platform integrates: a) easy-to-implement
multiplexed staining protocols; b)
an automated slide analysis system that can
isolate marker signals from one another and from autofluorescence; and c)
pattern recognition-based image
analysis software for automatically segmenting
images and extracting quantitative data from cells of interest.
Multispectral imaging and automated image analysis
accelerates preclinical and clinical studies
Quantitative, independent and specific multi-label protocols
have been developed that in conjunction with
easy-to-use multispectral imaging
systems and advanced learn-by-example software, can greatly accelerate clinical
and preclinical studies
[2].
For example, today, approximately one-third of small-molecule
kinase inhibitors
in development or trial target pathways are associated with
EGFR activation. Analysis of EGFR activation in tumor xenographs is typically
done by
immunohistochemical staining of tissue sections for phosphor-epitopes
of EGFR. Samples are analyzed by eye by pathologists, either under the
microscope
or on the computer screen as digital slides.
Typically, pathologists can process slides at an
average
rate of 100 samples per day. A study of hundreds of samples takes days or weeks.
On the other hand, if samples are stained with multiple color
protocols that
help guide image analysis and provide internal controls, such as a stain for
total EGFR, slides can be analyzed automatically with image
analysis software.
Such software can then present segmentation results and associated marker
intensity scores to pathologists for review, modification
if necessary, and
final approval.
In benchmark studies, results have shown that an analysis
process
that takes many days, at 100 slides per day, can be reduced to hours,
at a rate of 200 to 300 slides per hour. The pathologists remain central to the
process by training the image analysis algorithms to identify important tissue
areas, and as a final quality control gate on image analysis
results.
In a recent study performed at one pharmaceutical company, a
trained image analysis
solution accurately segmented tissue into regions of
interest for 98 percent of samples in a large, 3,000-sample study.
There is another benefit to this approach, in addition to
increased productivity and shorter study durations. Data is more
consistent,
since stain intensity scores are based on measured signal levels from a digital
camera instead of human visual perception, which can vary
over time based on
changing ambient environments and is not well suited to capture the non-linear
signal levels inherent in chromogenic absorption.
Pharmaceutical companies are motivated by the hope that
progress in understanding signaling
pathway activity will accelerate drug development.
The tasks of revealing activated pathways, understanding their
interrelationships and determining
correlation with outcome are challenging.
The complexity inherent in signaling pathway activity can only be elucidated by
revealing key marker
localization and distribution within tumor cells, rather
than the mere presence of a protein independent of morphological context.
Combining multispectral imaging
with advanced image analysis tools to perform tissue cytometry rapidly and on a
large scale
and using many markers at once has proven to enable a better
understanding of the mechanism of disease and potentially better, more precise
avenues of
treatment.
Clifford C. Hoyt,
a founder of
Cambridge Research & Instrumentation Inc. (CRi) in Woburn,
Mass., joined the company in 1987 as a staff scientist. He has played a central
role in the development of many of CRi's core technologies, including
the
liquid crystal tunable filter for multispectral and polarized light imaging and
the integration of these core technologies into analytical
instruments for
applications such as in vitro fertilization, high-throughput drug screening,
stem cell research, in vivo small-animal imaging, live-
cell biology and
tissue-based immunohistochemical analysis. He holds 12 patents, has numerous
patents pending and is the author or co-author of 20
publications in technical
journals.
Darren
Lee
is vice president of marketing at CRi. He has more than 20 years of experience
in marketing management, business development and
engineering in the life
sciences and clinical diagnostics industries. He has held senior-level
management positions at Primera Biosystems, a molecular diagnostics company,
and Decision Biomarkers, a
life-science systems developer. He has also served
in senior management positions at PerkinElmer and Packard Bioscience.
References:
1. Yoeli-Lerner, Yiu, Rabinovitz,
Erhardt, Jauliac and
Toker: "Akt blocks breast cancer cell motility and invasion through the
transcription factor NFAT." Molecular Cell,
November 2005.
2. Levenson, Fornari and Loda: "Multispectral imaging and
pathology: Seeing and doing more." Expert Opinion on Medical Diagnostics, 2008. Back |
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