- [Woman] Hello everyone,
welcome to today's live broadcast
on multiplex IHC and Novel
Monoclonal Antibodies
as Enabling Tools for the
Study of Tumor Immunology.
I'm (inaudible) from LabRoots
and I'll be your moderator
for today's event.
Today's webcast is presented by LabRoots,
the leading social media site
for science professionals
and sponsored by Cell
Signaling Technology.
Founded by research scientists in 1999,
Cell Signaling Technology is
a private family-owned company
headquartered in Danvers, Massachusetts.
With over 400 employees worldwide,
active in the field of applied
systems biology research
particularly as it relates to cancer,
CST understands the
importance of using antibodies
with high levels of sensitivity
and lot-to-lot consistency.
It's why they produce all
their antibodies in-house
and perform painstaking validations
for multiple applications.
And the same CST scientists
who produce antibodies
also provide technical
support for customers.
Helping them design
experiments, troubleshoot
and achieve reliable results.
See more at www.cellsignal.com.
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So let's get to today's presenter.
We're proud to welcome Dr. Matthew Silver.
Dr. Matthew Silver leads
he translational assay crew
at Cell Signaling Technology,
where he focuses on the
development of the both
the translational research
reagents and multiplex assays.
Matthew received his PhD in biochemistry
from the University of New Hampshire,
where he studied Neuroendocrine
regulation of reproduction.
His subsequent postdoctoral
studies were performed
at Wyeth Research within the
Department of Inflammation.
I'll turn it over to Dr. Matthew Silver.
- [Dr. Silver] Thank you very
much for the introduction.
Again my name is Matt Silver
and my presentation today
it's gonna be focused on
the use of multiplex IHC
and Novel Monoclonal Antibodies
to study Tumor Immunology.
So, my presentation is going
to be broken down into three sections.
I'm gonna start with an introduction
to multiplex IHC and how we're
performing multiplex IHC,
(inaudible) moving to IHC antibodies
that we've been developing
they're relevant to immunotherapy targets.
And I'll finish up with some information
on Panel development.
So, start with multiplex IHC
the rational for multiplex IHC
is straight forward, it comes down to
the simple fact that
there're a lot of biomarkers
and there tend to be a limited
amounts of biopsy material.
The benefits from multiplex
IHC, really come to the fact
that tissue samples are
small and typically rare
for example the fine needle aspirates.
It can ultimately acquire
more information per slide
and protection using a
multiplex IHC approach.
And while not necessarily
as good as what you get
with a traditional Chromogenic IHC,
it's important to note
that you certainly maintain
much better tissue context
than what you get with
other "grind-and-bind"
multiplex assays such
as next-gen sequencing,
PCR or mass spec.
So there's a lot of
benefits to multiplexing.
Of course, there are a
number of challenges as well.
For example, fluorochrome
spectral overlap can be an issue
that can limit the degree
to which you can multiplex.
Tissue autofluorescence,
which can come from a number of things,
including the fixation process
itself can wreak havoc with
the sensitivity of your assay.
You need good antibodies that work well
and formalin-fixed
paraffin-embedded tissues,
that's really key to the whole process.
But more than that, you
need good antibodies
that work well together
and a multiplex setting.
And then down the road
pathologist acceptance
will be an issue as multiplex IHC,
makes a transition at some point,
from a research,
translational research mode
into a potential clinical assay.
So, at CST we've been working
to make multiplex IHC routine,
which we feel comes down to
the combination of your imaging platform,
the detection reagents that you're using,
and of course best-in-class antibodies.
And specifically in which
the ways these different
areas address some of the
challenges I mentioned on
the previous slide.
The imaging platform that
you choose can help address
fluorochrome spectral overlap
and tissue autofluorescence.
Detection reagents
help with multiplexing
with signal amplification
to enable your your assays
through higher sensitivity,
and then best-in-class
antibodies speak for themselves.
We really need highly
validated quality antibodies
that work well in formalin-fixed
paraffin-embedded tissues.
So the solutions that
we've been working with
on the imaging side, we
have primarily been working
with multi spectral imaging
as a platform which enables us
to multiplex with five plus colors
and it also helps us
eliminate autofluorescence
that we get from the tissue.
You do not need to use
Multispectral imaging
if you're doing three color assay.
But we try to push the
colors up again to five plus
in which case the Multispectral imaging
is a key enabling technology.
The antibodies speak for themselves;
you need good antibodies
that work in formalin-fixed
paraffin-embedded tissues,
which would be CST antibodies.
And then on the detection region side,
we work with a couple
of different approaches.
We're using a tyramide
based multiplex approach,
which is a multiplexing approach
that provides a tremendous
amount of signal amplification.
And it does this in a
way that antibody species
and isotype independent.
And we also work with some
modified monoclonal antibodies
enable multiplexing using a secondary
based detection system.
So for the presentation
that I'm gonna do today,
I'm gonna focus in on
the detection regions
and primarily talk about
our use of tyramide.
So on the detection region side,
it's really the key
to multiplexing with signal amplification.
And so when you're talking
about doing a four color assay,
which to be clear would be,
three targets plus DAPI,
it's pretty straightforward.
For example, if you're going to look at
HER family member panel.
So looking at EGFR,
HER2, HER3 as an example,
it's pretty easy to find
the rabbit monoclonal
and a mouse IgG1 and a mouse IgG2a
to enable your multiplexing.
However we tend to run into trouble
when you move into a
five plus color assay.
And you can take a couple
of different approaches
you could look for alternative isotypes,
which in some cases will work out.
However, there's really
a limited number of
high quality rat or mouse
IgG3 or chicken antibodies.
And so if you're looking
to develop a broad
series of panels, this can become
a hurdle that can be
difficult to get over.
Alternatively, you can
use direct conjugates
in which case you're only limited by
the number of fluorochromes
if you have access to.
However, when working in tissue,
directly conjugated antibodies tend
to lack the sensitivity required
to really develop a robust panel.
And just as an example, the
data shown here on the right
are looking at our EGFR Staining.
Using our rabbit monoclonal
with the secondary antibody,
to standard primary secondary
based assay shown right here.
And the conjugated version of
the antibody is shown here on the left,
and this is looking at
A431 cells shown in red
versus MCF7 in blue.
And you can see while
you certainly can detect
signal with the conjugated antibody,
you take a dramatic hit in sensitivity
here measured by a mean
fluorescence intensity per cell.
So in some cases, this
might work out just fine
and you'll be able to detect your target.
However, in many the sensitivity
loss will will wreak havoc.
So the solutions that
we've been working with
again are these of tyramide
signal amplification
and modified monoclonal antibodies.
on the tyramide began as
a serial stain approach
that leads to a high degree
of signal amplification.
Where to modified monoclonal
is a parallel staining,
more traditional parallel staining,
which enables a moderate
degree of secondary based
antibody signal amplification.
And to give everyone a sense of
the difference in signal
intensity that you'd expect with
these different approaches,
on the left here we have
what you'd see with a conjugated antibody,
where you have your
epitope and your antibodies
that is conjugated to a certain
number of fluorochromes,
which typically be three or four,
and that provides signal
that you would detect.
You get amplification when
you're using secondary antibodies
now because
you have multiple conjugated
secondary antibodies
that are binding your primary,
so you increase the number
of fluorochromes per epitope.
When moving to the tyramide
signal amplification approach
on the other hand,
you have a very dramatic
degree of simplification
because this detection mechanism is based
on an enzymatic reaction.
tyramide itself is a small
phenol basic molecule,
they can conjugate to a
number of different things,
including haptens, or fluorochromes.
And then in a reaction
that's catalyzed by HRP, you
create a highly reactive free
radical form of tyramine,
which will create covalent bonds
with electron rich
regions in close proximity
to the reaction,
which typically would
be a tyrosine residues
on the antibodies on
and around the epitopes.
And so again, the Tyramidic approach leads
to dramatic amplification of your signal.
Which you need to control
very carefully, (but)
if handled properly can be
a very powerful technique.
And here's an example
looking at our ALk antibody,
D5F3 staining H3122 cells
which express the EML4-ALK translocation
and T47D cells which are AlK negatives,
using our standard IF
approach of using a primary
and secondary antibody up top here,
versus the tyramide
protocol on the bottom.
And you can see what
the Standard approach,
you can detect the signal,
although it's fairly weak
and it's (inaudible) the T47D cells.
But when you move to the
tyramide based approach,
you can see a very dramatic
increase in signal intensity
while maintaining the
negative signal in T47D cells.
On the right we have the quantification
of the signals looking at
MFI first intensity per cell.
For H3122 cells are in red,
T47D cells are in blue,
and you can see this
very dramatic increase
in signal intensity in H3122 cells
with a very minor increase
here and the T47D cells.
So two other important things
to point out in this slide.
In addition to the drastic
increase in signal to noise
and signal intensity,
the actual imaging
times are much faster at
the tyramine protocol,
where we have a 10
millisecond exposure compared
to 75 milliseconds for
the standard protocol.
And then we're also using
less antibody because of
the strong amplification
with the detection region.
So we're using the antibody one to 1000
with tyramide compared
to one to 250 with the
standard imaging approach.
So this is tyramide in cell pellets.
We've validated this
approach in tissues as well.
And so this next slide just
highlights a number of tissues
that we've stained using
the tyramide approach.
Simply that the illustrate the point
that this isn't just about staining cells,
and it's very much compatible on a tissue,
formalin-fixed paraffin-embedded
tissues as well.
So that's how TSA works
in the single plex setting
and it's perfectly suited
for multiplexing as well.
So the introduction of an
additional microwaving step,
or some steps that ultimately will disrupt
the antibody interaction with your sample,
we tend to use microwaving
because it fits into
the workflow very nicely
by repeating antigen retrieval.
But alternative heating
methods and acid stripping
are alternative approaches
to doing this as well.
So, the way this would work
is you have your epitope
with your primary and secondary antibody
and you go through your
detection reaction,
activating tyramine,
which becomes deposited
around your sample.
You then introduce a microwaving step,
which would wash away your
primary and secondary antibody
and your covalently bound
tyramide to fluorochrome
conjugate remains behind on your sample.
You then simply come in
with your next antibody
and the species and
isotype no longer matters
because your primary and secondary
from the previous step
have been washed away.
And now you include a tyramide conjugate
to an alternative fluorochrome.
So, initially it was Alexa 488.
As an example, you can
now come in with Alexa 647
and go through your your reaction,
another microwaving step and
simply repeat until you use
the number of fluorochromes
that you can handle on
the imaging side or the
number of fluorochromes
that you need for the
particular experiment
or panel that you are
currently working on.
So just a couple of comments about
the tyramide Signal
Amplification multiplex Protocol,
again as a serial stain.
So you have your initial
microwave treatment,
which is your energy retrieval step.
You go through your first
Stain and repeat microwaving
in your second stain
in the serial fashion.
The actual primary and
secondary antibodies
in tyramide reactions live
within each staining step.
It ultimately a fairly long procedure,
which it's about three hours per color
that you're working on.
And obviously, that can
translate to a very long day
if you're looking at a three
or four plus color essay,
but you can introduce an
overnight break in between
by incubating your slides
and blocking buffer
without consequence on
your signal intensity.
And so one thing that I
think is very much needed is
the development of automated protocols
and solutions for this
approach because again,
it is a very long protocol.
A couple comments on optimization,
one of the really important
steps to do going through
in development panels is
looking at position within
the staining series.
So on one hand, your
fluorochromeis currently bound
to your sample that as you go through
the multiple microwaving steps,
you can lose - sort of
chip away - some of your
conjugated fluorochrome.
And so you can experience some
signal loss as you go through
the microwaving steps.
And so you might want to
put your weaker antibodies
or weaker signals later
in the staining series.
However, you need to
balance that with the fact
that as you go through
multiple rounds of microwaving,
as you might imagine, you can
experience epitope damage.
And so it's really
important as you go through
and develop these panels
that you look at each
antibody each position across
the staining series to control
for potential signal
loss and Epitope damage.
So as an example, you're
looking at ROS1 staining
and a 3 Cell Pellet mix
that we've developed.
Which included 1/3 of the
cells of being HCC78 cells
which express ROS1.
When we look at ROS1 staining
in the first position,
you can see roughly 1/3
of the cell stainings
and example pointed out right there
probably look at ROS1
in the fourth position.
Now all we have is this one cell left in.
It took quite a bit of
scanning to find the cell
completely lost the signal
after third microwaving step.
So it's really important to go through
and get an understanding of how
your antibodies are gonna
perform in each step along
the staining sequence.
So, at this point, we
have a quick poll question
that we're going to put off,
if you could kindly
answer this poll question.
I'd greatly appreciate it.
With this in place with
your imaging platform
and detection reagents and
best in class antibodies.
It's now time to develop panels
when you have all these things in place.
And there are nearly
limitless number of panels
that are of interest.
A few of many panel concepts
are illustrated here
that we've been working
on in immunotherapy
looking at AlK, ROS1 and Met
or EGFR mutations lung cancer,
ER, PR and HER2 and breast cancer.
And HER Family member panels
are interested in signaling nodes panels,
and many others including chemoresistance
looking at ERCC1, RRM1 and TS.
For today's presentation,
I'm gonna focus in on
Immunotherapy looking at
(coughs)
a number of targets
or you ultimately can address questions
around Tumor infiltration,
Checkpoint control co-expression,
and basic Immune Cell characterization.
So just a very brief introduction.
I think as everyone's
aware, cancer immunotherapy
is an explosive field
and it's all about
empowering your immune system
to fight cancer.
And the concept here is
really focused on empowering
again your immune system to fight cancer.
For a long time, the field had struggles
with the question of
why doesn't your immune
system take care of
the problems of the tumor itself?
And obviously, part of the problem is
they're tumors are self-derived.
However, there are a lot of
changes as tumors evolves,
which leads to a great
number of neoantigens
that the immune system
should be able to hone in on.
And so one way to think about
it is the concept called
immunoediting which
consists of three phases
elimination, equilibrium and escape.
So in the beginning, the
immune system will challenge
the tumor in order to eradicate it.
However, when this isn't
perfectly successful,
the tumor and the immune system
find themselves in a balance
which is the equilibrium phase.
where the tumor is under constant pressure
from the immune system,
which drives selection of
immune resistance clone
and time these take over and this leads to
the escape phase where
tumors can propagate even in
the context of an intact immune system.
After years of research,
several mechanisms have been
identified that will the
assist immune system.
This includes manipulation
of immune checkpoint protein,
amongst other approaches like vaccination
and T cell engineering.
But today I'm gonna focus
in on immune checkpoints,
which typically help
maintain self-tolerance
and protect tissues
during immune response.
However, it's a dysregulation
of these checkpoint proteins
in tumors that can lead
to immune resistance.
And so there are a
number of these proteins,
many of which are depicted on this slide
where we have a T cell
depicted on the bottom
and an antigen presenting
cell depicted up top.
The T cell receptor is shown right here in
the middle of the slide.
Which when stimulated,
will activate T cells
which is only done in
the context of CD28
co-stimulation. And everything else
on the schematic plays
a role in fine-tuning
this response either in a
positive or negative way.
And so these are all
immune checkpoint proteins.
And essentially all of
these are being investigated
as possible drug targets,
where you'd either block an off signal
or stimulate an on signal.
So of course, it's an
enormous amount of investment
and effort currently going on
into the development of therapies
for these targets.
However, we recognized a few years back,
that there is a large gap in the field,
which is that there's a lack of
high quality commercially
available IHC antibodies
for these cancer immunotherapy targets.
And so we developed
the objective of developing
a comprehensive pipeline
primarily of Rabbit monoclonal antibodies,
where all of these projects and
efforts would be IHC driven.
So again, these targets aren't new
they've been around for a long time,
and there are a lot of
great flow antibodies,
but IHC has been a gap.
And then a key to all the development work
here that's been going
on here is collaboration
so working with key labs
in academia and pharma
to develop these IHC antibodies.
We initially had to prioritize
because there are a lot of
potential targets to go after.
And so we started with PD-L1
where again the development
objectives were IHC
and we're looking for human reactivity
due to lack of commercially
available PD-L1, IHC antibodies
a couple of years ago.
And so this this webinar is not
primarily focusing on antibody validation,
but just to give everyone
a sense of how we go about
developing antibodies.
We're often using western blots
for initial screens using
positive and negative lysates.
Before moving into IHC
similar positive and negative
formalin-fixed paraffin-embedded
tissues cell pellets,
and then we ultimately
move into IHC testing
using control and cancer tissues.
So starting with a Western,
here's some example of Western data,
looking at our PD-L1 antibody E1L3N.
Where we are testing lysates
from Karpas299, SUP-M2 and PC3.
Cross reactivity testing is a
really critical component to
the validation work.
So here we have a series
of transfection cell lines.
We've COS7 cells or Mock transfected
or transfected with
human PD-L1, PD-L2, PD-L3
or PD-L4.
And you can see that our
clone E1L3N only detects
PD-L1 and it's nice and clean and
the lanes containing the
other family members.
And then we have a Myc-Tag here on
the right show expression of all of
the different constructs.
We then move to IHC testing, here we have
Karpas-299 cells and PC3 cells
that were formalin-fixed paraffin-embedded
to see a nice staining
differential between
these two cell lines
where Karpas-299 cells express PD-L1
the untreated PC3 cells do not.
From here we move into human placenta,
which is a nice control for for PD-L1
we'd expect to see staining
in the syncitiotrophoblast layer of
the placenta which is the outer section,
which is staining here in brown,
but it should be negative
in the stroma and villi,
the internal parts of the structure,
which is nice and clean,
as you can see here.
And from there,
we move into screening a number of normal
and cancer sections.
Here is an example looking
at human lung cancer
that staining very nicely
with our with our antibody.
And then further validation
comes post release,
where the antibodies are used
by a number of investigators.
And here's an example
from a recent publication
it was demonstrating that our clone E1L3N
can bind PD-L1 in the tumor
as well as immune cells
in the Stroma as well
as Alveolar Macrophages
shown in panel C.
Which is an important point to show that
the antibodies can bind PD-L1
and a number of different cell contexts.
So in addition to the initial
clone that we released,
we have a number of
different PD-L1 antibodies
that we have been working on
and have recently released.
We have our PD-L1 clone E1J2J,
which binds PD-L1 in
the extracellular domain
as an example staining is shown here on
the top two tissue sections here,
human placenta and human colon cancer.
And we've also recently released
a mouse monoclonal antibody
clones 405.9A11, which
like E1L3N binds PD-L1
intracellular domain.
And so a similar set of example
data here on human placenta
and human lung cancer.
And to round out the PD-1,
PD-L1 family we've also recently
released a PD-1 IHC antibody,
the mouse monoclonal antibody clone EH33.
There's an example staining
shown here in human tonsil.
And we've also released our
PD-L2 antibody clone D7U8C.
And again, an example
staining Hodgkin's lymphoma.
And of course we're very
interested in targets outside of
the PD-1, PD-L1 family itself
is where number of antibodies
that we've validated for
IHC and released over
the last several months including
B7-H4 and B7-H3 antibodies
and new IHC antibody for VISTA,
which is an interesting
new immune checkpoint control protein
that's related to both B7
and CD28 family members
and TIM-3 was recently released as well.
And we're looking into
alternative spaces such as
immunometabolism as well,
which are IDO and CD73 antibodies.
So CD73 is important and
adenosine metabolism.
And then of course it's really important
to have good coverage
of immune cell markers
with a series of antibodies
that are coming out.
Looking at your standard
markers such as CD3 and FoxP3
and many others as well.
And so the point is, (coughs)
with a broad portfolio in place,
it's now possible to
start working on panels
in the immuno-oncology space.
And so as an example, I'm
gonna illustrate how we have
developed an immune
checkpoint control panel.
And this particular case,
we're gonna be focusing in on PD-L1 B7H4
and VISTA all of which are important
immune checkpoint control proteins.
And we're looking at these
in the context of CD8,
which is a marker of effector T cells,
and cytokeratin as well,
which is an epithelial cell marker.
And so for the next series of slides,
I'm going to run down our optimization.
And so how we set up
the panel's themselves
and then I'll show some data
on screening of a small cohort
of breast and ovarian
cancer patient samples
that we have performed.
So as I mentioned a couple of minutes ago,
there are a number of
important steps along
the way as you validate panels
which are illustrated on
the top of the slide here.
And it all begins with
IHC antibody development.
So developing antibodies,
requiring antibodies
that work well, that are validated for use
in formalin-fixed
paraffin-embedded tissues.
From there we move into
experiments titrating
the antibodies using
the TSA based detection.
This leads into order
optimization experiments.
So again, where in the staining series
does each antibody sit?
Ultimately this then leads to
antibody fluorochrome pairing,
before moving into control
cell pellets testing,
and then ultimately tissue testing.
So here we have on the left
our PD-L1 antibody titration
So again looking at Karpas-299
cells and PC3 cells.
We look using our clone E1L3N, we have
mean fluorescence intensity
per cell on the left,
and the dilution point across the x-axis.
And so we ultimately
walked the staining down
the titration series to
find the optimal balance
between signal and your Karpas-299 and PC3
which in our hands was one to 1400.
Next, we moved into order optimization,
again, looking at our PD-L1 clone
where you can see mean
fluorescence intensity per cell
again, on the y-axis on the left,
the signal to noise, the right
and across the x-axis,
we have the position within the series.
Where we have first all the
way to the fifth position.
And so here you can see
there's a dramatic impact of
the position on signal intensity
and signal to noise as well.
We're actually seeing an increase
in signal to noise as we go
deeper into the staining series.
Here we move into the antibody
fluorochrome comparison,
where again with mean
fluorescence intensity per cell on
the y-axis and across the x-axis we have
the different fluorochromes
that we have using these experiments,
which include Alexa 350,
FITC, Alexa 594, Cy3 and Cy5.
And you can see there is
a fairly dramatic impact
on signal intensity
based on the fluorochrome
that you using.
So this is actually really important stuff
where you can have a
big impact on balancing
the signals within your panel.
As you can imagine, this
is a nice opportunity
to pair your stronger antibodies
with weaker fluorochromes
and your weaker antibodies
or weaker signals
with stronger fluorochromes in an effort
to balance out the signal intensities
within the panels themselves.
So that was a quick rundown
using PD-L1 as an example.
So this table here shows the panel itself,
where we have VISTA and the first channel
with Cy3, which will be shown in yellow
and all the subsequent images.
And the second staining
point we have CD8 with Cy5,
which will be shown in red,
then B7-H4 the Alexa Fluor 594,
which will be shown in magenta.
This will be followed by PD-L1,
which we coupled with FITC,
which will be shown in green.
And then cytokeratin is in
the fifth spot with Alexa 350,
and this is going to be shown in cyan.
And we ultimately chose
PD-L1 in FITC despite
the fact that it wasn't
the strongest signals
you see here on the left.
And this was done ultimately
because of the need to balance the panel.
And and so we didn't need PD-L1
in the strongest possible channel,
we reserved that for the VISTA clone.
So an example of staining in
a controlled tissues is shown
here, this is human tonsil,
which we would expect
to see staining of all these markers,
where you can see PD-L1 expression
(coughs)
in PD-L1 expression in green,
which overlaps in part
with cytokeratin staining
which is here in cyan,
and you can see VISTA
positive cells and of course,
a number of the CD8 positive T cells.
And so with this in place, we moved
to staining a tissue cell
which consisted of 12
breast cancer sections,
and 22 ovarian cancer sections.
And I'm gonna show a couple of examples of
what we have seen.
And so here's an initial example
of breast cancer section,
that stands positive for all markers
where again we have PD-L1 in green
which is shown right here,
B7-H4 which is co-expressed
with cytokeratin, which
is shown here you can see
the B7-H4 in magenta
which is overlapping with
the cytokeratin in cyan.
And ultimately VISTA is expressed
in a series of sporadic cells,
which is an independent
cell compartment compared
to PD-L1 and B7-H4.
And then there's a number of
CD8 positive T cells within
this environment as well.
It's an important observation here is that
PD-L1, B7-H4 and VISTA are all expressed
in independent cell compartments,
where again we have B7-H4 co-expressed
with cytokeratin positive cells,
PD-L1 is in a completely
distinct set of cells,
which from our previous
additional cells staining
typically CD68 positive cells
and then VISTA is in a
completely independent
cell compartment as well.
As a note this ultimately
was an image composite
which is broken down.
Ultimately, when you going through
the image analysis into
independent components,
which are shown here and see ultimately
looking at each single independently
where we have DAPI,
PD-L1, B7-H4, cytokeratin,
VISTA and CD8 all broken
down and these come together
to merge into this composite image.
This is another example
of a breast cancer section
that we stain in this particular case,
the section was positive
for B7-H4 and VISTA
but negative for PD-L1,
so we're not detecting
any of the green signal.
Now moving into a couple of
example ovarian cancer sections,
similar to the first breast cancer section
that we looked at a couple of slides ago,
this case is positive for PD-L1,
shown here in this little
cell cluster standing green.
Again B7-H4 in magenta,
which is in cells that
coexpress cytokeratin.
And then we have VISTA
cells that are positive
and completely independent
cell compartment from
B7-H4 and PD-L1.
And another ovarian
cancer section shown here,
again this one is PD-L1 negative,
but it's positive for B7-H4
which is co-expressed again
with cytokeratin.
And then our VISTA cells are
in a completely different
cell department from our B7-H4,
cytokeratin positive itself.
So those are a few examples.
A quick summary of the
staining is shown here,
which is organized based on
breast cancer and ovarian cancer
and which markers are
positive for including VISTA
and the left column B7-H4 in the middle,
and PD-L1 here on the right.
And ultimately, within these panels,
we found that in the breast cancer 33% of
the cases are positive for PD-L1,
58% are positive for B7-H4 and
all had VISTA positive cells.
And interestingly, at least 75% of
the cases were positive for
two checkpoint control proteins
On the ovarian side we
saw 36% of the cases
staining positive staining for PD-L1,
whereas half of them
were positive for B7-H4
then 95% of the cases
were positive for VISTA.
And again 65% of the cases
were positive for At least
two immune checkpoint control proteins.
As we think this is a really
important tool to look at
the co-expression immune
checkpoint control protein
because of course, it'd
be very interesting
to know what immune
checkpoint control proteins
are expressed in a
particular patient's cancer.
And if you have multiple immune
checkpoint control proteins,
this might be an important indicator
that you need to have a
combination therapy approach.
So, in summary, multiplex IHC
panel has been established
to detect PD-L1, B7-H4
and VISTA along with CD8 and cytokeratin.
And in our initial evaluation
of breast and ovarian cancer,
we saw co-expression at least two
immune checkpoint control protein
in 75% of breast cancer patients
and 55% of ovarian cancer patients.
Importantly PD-L1 and VISTA
expression was observed
in independent cell populations,
while B7-H4 is in yet another
distinct cell compartment
which in this case is
co-expressed with cytokeratin.
And so we have a lot of follow
up panels in development
and so we're continuing
to build our portfolio
of immune checkpoint
and immunotherapy relevant markers.
Rabbit anti-mouse and
mouse anti-human antibodies
are a big focus as well.
And we're also looking at alternative
immune cell based immunotherapy targets,
like CAR-T targets.
For example EGFR V3 antibody,
an example shown here of
staining and Glioblastoma.
So a couple of example, follow up panels.
On the left here looking at breast cancer.
We have a panel looking at PD-L1
along with FoxP3 and CD8.
There's a lot of interest
in ratio of effector cells
to T-REG cells.
And so this panel is designed
towards looking at PD-L1
in the context of CD8 to FoxP3 ratio.
And another panel here on the right,
looking at PD-L1, along with CD3 and CD8.
So CD3 and CD8 are very important
basic immunology markers and
comprise the immuno score.
So it's looking at PD-L1
with these relevant markers as well.
And finally, here's an
example of human lung cancer
that was stained with PD-1 and PD-L1
along with CD3, CD8 and cytokeratin
and marking the nucleus with DAPI.
And we can see this nice example
up in the top right corner,
which blown up here,
looking at PD-L1 in magenta
which is staining in a
completely distinct compartment
from the set of
cytokeratin positive cells.
And you can see a number of
PD-1 positive cells green
and then around this
particular patch of cells.
So at this point, we're going
to ask one more full question.
And it's gonna appear on
your screens in a moment.
And with that, I'd like to thank everyone
for attending today and for
your time and attention.
And if you have any questions,
I'd be very happy to field them.
And the contact information is up on
the screen at this point.
Which can always follows with
me with questions as well.
Thank you.
- [Woman] Was an excellent
presentation Dr. Silver,
thanks for bringing
that information to us.
Before we get started with
all of your questions,
here's a quick reminder
of how to reach us today.
Questions can be sent via
the green Q and A button
at the lower left of
the presentation window.
We'll get to as many as we can.
Our first question comes from
Jose Malone, from (inaudible).
Jose asked, "Please perform
with tyramide amplifications"?
- [ Dr. Matthew] All the
experiments and images
that I showed today were performed
with a tyramide Signal
Amplification based approach.
(coughs)
- [Woman] Next we have Martin from MKI.
Martin asked, "IHC even
multiplexed as you have,
"seems to have so many limitations.
"Of course, the spatial
arrangement of cells
"can be very important and
add valuable information,
"the limitations on
multiplexing due to breaching
"or antibodies in the
(inaudible) themselves".
He says, "I'm not sure,
I'm not very sure why
"there is such limitation on multiplexing
"and why you need to
amplify the signal so much".
- [Dr. Matthew] The reason
that we tend to feel
that we need to amplify signal is due
to autofluorescence
that you'll see in a
number of different tissues
or all tissues, some of
course are worse than others.
Ideally, you'd be able to use
directly conjugated antibodies
and this would simplify
quite a number of things
and it would make
quantitation more accurate,
but the simple fact of the matter is due
to autofluorescence
you need amplifications
to rise above that noise.
And so, the again the use
of amplification is simply
to provide you with a signal intensity
to be able to detect your signal.
We're of course interested
in alternative approaches
to not to be stuck around the need
for such extreme amplification.
But at this point in time,
tyramide is a very nice solution
to get that job done.
- [Woman] Next we have Lisa (inaudible)
from UVA who asks,
"Are these tissues fixed with formalin"?
- [Dr. Matthew] Yes,
they're fixed with formalin,
the focuses of the work that we've done
and all the images that I've shown
have been on formalin-fixed
paraffin-embedded tissues.
So yes they are fixed.
- [Woman] Next we have
Krishna from MTI Research,
who asked, "Is this webcast is going
to be made available later"?
And I think
- [Dr. Sliver] Yes it is.
(laughs)
The answer that question is yes,
this webcast will be made available.
(inaudible)
- [Woman] Next, we have a
question from one viewer
who asked, "If there's a limitation,
"or is there a limit to
the number of targets
"that can be detected"?
"Are there any hardware
or software restrictions
"to multiplexing"?
- [Dr. Dr. Matthew] Absolutely.
The hardware is a very
important factor that dictates
the degree to which you can
multiplex as I mentioned,
we're primarily using
multispectral imaging
which enables us to look
at five plus colors.
I think getting up to eight colors
at this point is possible.
We don't quite have the
algorithms in place to do this.
If you're using standard
imaging approaches,
we can look at roughly
three colors plus DAPI.
But then you start to run
into a lot of problems
with the overlap of
fluorochrome emissioned
using a standard microscope.
So yes, the hardware
is an important factor
that dictates the degree
to which you can multiplex.
- [Woman] One other viewer asked,
"How do you go about
selecting a reliable antibody
"for an IHC"?
"Are there antibodies against targets
"that are particularly
difficult to work with"?
- [Dr. Matthew] The first
part of that question,
The you go about it really is you need
to identify high quality,
highly validated IHC antibodies.
And so well I'm probably a little biased,
I think the best place to go
is to CST for those antibodies.
And really what it comes down
to is, is you need to trust
the validation work that has been done.
And so if the antibody works really well
on your standard IHC experiment,
so basic Chromogenic IHC,
we found that these antibodies
translate over very nicely
to the TSA base multiplexing approach.
There's some more
validation work that needs
to be done often the optimal
work and concentrations change
because the detection
chemistries are different.
And the multiplexing workflow
is slightly different.
But ultimately, if you
have a good IHC antibody,
it will work well for this TSA
base multiplexing approach.
And then to the second
part of the question,
are there any targets that tend
to be particularly difficult?
There are certain antibodies
been certainly phospho.
Specific antibodies are challenging,
but that's not a problem
for the development of the IHC
antibody in the first place.
Once you've cleared that hurdle,
and we have a validated IHC antibody.
We haven't found any targets
that haven't worked well in this approach.
- [Woman] There are no more questions
I'd like once again
to thank Dr. Matthew silver
for his presentation.
Do you have any final comments for us?
- [Dr. Matthew] I do not,
I'd just like to thank
everyone again for their time.
- [Woman] I like to
thank our speaker today
for today's informative presentation,
as well as our sponsor
Cell Signaling Technology
for making it possible to
bring this presentation to you.
Today's webcast will be available
for on demand viewing through July 2016.
You will receive an email
from us alerting you
when it's available on demand
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You're welcome to foward
this announcement,
to any colleagues who
weren't able to join today.
Thanks for logging on
and participating in today's
broadcast (inaudible).
See you next time.
