Circulation March 23, 2021 Issue

For this week's Feature Discussion, join author author Hannah
Valantine and Senior Associate Editor Biykem Bozkurt as they
discuss the Original Research Article "Cell-Free DNA to Detect
Heart Allograft Acute Rejection."


TRANSCRIPT BELOW


Dr. Carolyn Lam:


Welcome to Circulation on the Run, your weekly podcast summary
and backstage pass to the journal editors. I'm Dr. Carolyn Lam,
associate editor from the National Heart Center and Duke National
University of Singapore.


Dr. Greg Hundley:


And I'm Dr. Greg Hundley associate editor, director of the Pauley
Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn,
I think we've got an interesting feature this week.


Dr. Carolyn Lam:


Oh boy, we sure do. This one is one of those potentially practice
changing landmark papers. I'll give you a clue. It's about using
cell-free DNA to detect heart allograft acute rejection in
transplantation. Huge, huge, but you got to wait. Listen to the
summaries of this week's exciting issue first. Greg, you want to
go first?


Dr. Greg Hundley:


Yes. And I can't wait for that feature discussion. I think that's
going to be one of our top papers this year, but first onto some
of the summaries. So my first article comes to us from Dr. Pilar
Alcaide from Tufts University School of Medicine. So Carolyn,
despite the well-established association between T-cell mediated
inflammation and non ischemic heart failure, the specific
mechanisms triggering T-cell activation during the progression of
heart failure and the antigens involved are poorly understood. So
Carolyn, these authors hypothesize that myocardial oxidative
stress induces the formation of isolevuglandin modified proteins
that function as cardiac neoantigens to elicit CD4 positive
T-cell receptor activation, and then promote heart failure.


Dr. Carolyn Lam:


Oh, that's really interesting. Inflammation in heart failure is a
hot topic. Tell me more.


Dr. Greg Hundley:


So Carolyn, these authors discovered that TCR antigen recognition
increases in the left ventricle as cardiac dysfunction
progresses, and they identified a limited repertoire of activated
CD4 positive T-cell chronotypes in the left ventricle.
Mechanistically, cardiac pressure overload resulted in reactive
oxygen species dependent dendritic cell accumulation of
isolevuglandin protein adducts, which induced robust CD4 positive
T-cell proliferation.


Dr. Greg Hundley:


So collectively, Carolyn, these results demonstrate an important
role of reactive oxygen species induced formation of
isolevuglandin modify cardiac neoantigens that lead to TCR
dependent CD4 positive T-cell activation within the heart. And
therefore, these results help understand the relationship between
T-cell mediated inflammation and heart failure.


Dr. Carolyn Lam:


Wow. Super. Thanks, Greg. I'm moving all the way from basic
science now to talk about intensive lifestyle interventions,
which we know are first line approaches to effectively treat
obesity and manage the associated cardio-metabolic risk factors.
However, to date, whether effective lifestyle based obesity
treatment in primary care works, we need more data. And this is
what this paper provides.


Dr. Carolyn Lam:


This comes from Dr. Katzmarzyk and colleagues from Pennington
Biomedical Research Center in Los Angeles. And what they report
is the PROPEL Trial, which randomly allocated 18 clinics equally
to usual care or an intensive lifestyle intervention, and
subsequently enrolled 803 adults with obesity from participating
clinics. The usual care group continued to receive their normal
primary care, while the intensive lifestyle intervention group
received 24 months of high intensity lifestyle-based obesity
treatment in a program, embedded in the clinic setting and
delivered by health coaches in weekly sessions initially and
monthly sessions from months seven through 24.


Dr. Greg Hundley:


Well, Carolyn, sounds like a really practical study here. So what
were these results?


Dr. Carolyn Lam:


Yes, Greg, this was a pragmatic trial. And although pragmatic,
this lifestyle intervention was consistent with national
guidelines, and participants receiving the PROPEL intensive
lifestyle intervention lost significantly more weight over 24
months than those receiving usual care. Results also demonstrated
clinically relevant improvements in high density lipoprotein
cholesterol, total to HDL cholesterol ratio, metabolic syndrome
severity, and fasting glucose. The PROPEL model may therefore be
a viable option to deliver effective obesity and cardio-metabolic
risk factor treatment in primary care.


Dr. Greg Hundley:


Well, Carolyn, what an interesting article. A lot that we can
take home from that. Well, I'm going to switch and talk to you
about blood flow restoration and its effect on venous thrombosis
and vein wall injury. And this article comes to us from Dr.
Farouc Jaffer from the Massachusetts General Hospital at Harvard
Medical School. So Carolyn, up to 50% of patients with proximal
DVT will develop the post-thrombotic syndrome, which is
characterized by limb swelling and discomfort, hyperpigmentation,
skin ulcers, and impaired quality of life. While catheter based
interventions, enabling restoration of blood flow, have
demonstrated little benefit on post-thrombotic syndrome, the
impact on the acuity of the thrombus and mechanisms underlying
this finding remain obscure. So here, these authors in
experimental and studies, they examined whether restoration of
blood flow has a restricted time window for improving DVT
resolution.


Dr. Carolyn Lam:


Oh, very interesting, and potentially a significant clinical
implications, huh? Tell us about it.


Dr. Greg Hundley:


Well, Carolyn, there were two types of studies performed in mice
and those in human subjects in the ATTRACT pharmacomechanical
Catheter-Directed Thrombosis trial. So in the series of
experiments in mice, within a restricted therapeutic window,
restoration of blood flow improved DVT resolution. And then in
the human studies, the pharmacomechanical catheter directed
thrombolysis did not improve the PTS scores for patients having a
symptom onset to randomization or SOR time of less than four days
or greater than eight days. So therefore, further studies are
warranted to examine the value of time restricted restoration of
blood flow strategies to reduce post-thrombotic syndrome in
patients with deep venous thrombosis.


Dr. Carolyn Lam:


Interesting. Thanks. Greg. My next paper is related, also talking
about anticoagulants. And this time, the authors led by Dr.
Hijazi from Uppsala Clinical Research Center in Sweden evaluated
the risk benefit balance of antithrombotic therapy according to
kidney function in the AUGUSTUS Trial. As a reminder, in the
AUGUSTUS Trial resulted in less bleeding and fewer
hospitalizations than vitamin K antagonists, whereas aspirin
caused more bleeding than placebo in patients with atrial
fibrillation and acute coronary syndrome or PCI treated with a
P2Y12 inhibitor.


Dr. Greg Hundley:


Carolyn, thanks for reviewing for us the AUGUSTUS Trial results.
So what did they find in this study?


Dr. Carolyn Lam:


So what they did is they looked at patients with atrial
fibrillation and ACS and/or a PCI, and found that apixaban, as
compared to vitamin K antagonists, displayed a consistent safety
and efficacy profile, irrespective of kidney function, without
significant interaction and in accordance with the overall trial.


Dr. Carolyn Lam:


Next, they found that aspirin, relative to placebo, on top of
oral anticoagulation and a P2Y12 inhibitor resulted in more
bleeding, irrespective of kidney function again, and with an even
greater increase among those with a GFR more than 80.


Dr. Carolyn Lam:


These findings can help clinicians perhaps make informed
decisions on the antithrombotic therapy in patients with atrial
fibrillation and kidney disfunction, with ACS and/or a PCI.


Dr. Greg Hundley:


Very nice.


Dr. Carolyn Lam:


All right, Greg. Well, tell you what, let's go onto the other
papers in this issue. I would like to tell you about a letter to
the editor from Dr. Saleh on carotid atherosclerosis thickness, a
proxy for cardiovascular disease events. There's an ECG challenge
by Dr. Liu entitled, intriguingly, A Noteworthy
Electrocardiogram, and this really describes new SD segment
elevation and its differential diagnosis. To refresh, look up the
paper.


Dr. Greg Hundley:


Very nice, Carolyn. Well, I have a research letter to tell you
about from Dr. Lanz entitled One Year Outcomes of a Randomized
Trial, Comparing a Self-expanding to a Balloon Expandable
Transcatheter Aortic Valve. And then finally, Dr. Maron has a
very nice perspective piece entitled Exploring New and Old
Therapies for Obstructive Hypertrophic Cardiomyopathy Mavacamten
in Perspective. Well, Carolyn, I can't wait to get to this week's
feature discussion. How about you lead us through that?


Dr. Carolyn Lam:


Me too. Let's go, let's go. I could not be more thrilled to be
doing today's feature discussion. And I have to admit I'm feeling
very star struck because I'm with two of the women I think are
most at my own respect. And the first is Dr. Hannah Valantine,
and she is professor from Stanford University now and also at
NIH, and she's the corresponding author of today's incredible
paper. And the next guest is of course, Dr. Biykem Bozkurt,
senior associate editor of circulation from Baylor College of
Medicine.


Dr. Carolyn Lam:


Welcome, ladies. On the topic today, it's really landmark. We
could be talking about a new gold standard that may replace the
endocardial myocardial biopsy. Wow. So if I could just start off,
Dr. Valantine, could you please tell us about your study? What is
cell-free DNA?


Dr. Hannah Valantine:


Yes. Thank you. It's a wonderful opportunity to be doing this
podcast, and thank you for the interest on the technology. If you
can imagine, when you put an organ transplant, essentially what
you're doing is a genome transplant. You're transferring the
genome of the donor into the recipient.


Dr. Hannah Valantine:


Now, we all have single nucleotide polymorphisms, otherwise known
as SNPs, that are unique to the donor DNA, and that are unique to
the recipient DNA. So that once we put that organ in and there is
a teeny little bit of damage, little fragments of DNA come out of
the donor organ into the recipient circulation, and we can pick
that up, circulating in the plasma. And that's why we call it
donor derived cell-free DNA.


Dr. Hannah Valantine:


So, you know the SNPs that belong to the donor, and you know the
SNPs that belong to the recipient. You extract the DNA from the
plasma of the recipient and you sequence it. And bingo, you can
tell what the percentage of that cell-free DNA is coming from the
donor, and that is the basis of the test.


Dr. Carolyn Lam:


Oh my goodness. I love that explanation. It's so lucid, and it's
reminding me of what happened when I was pregnant. It's the same
technology that's used, I think, in prenatal testing, in oncology
in some cases, but this is the first time that you've shown it in
a multicenter approach in cardiac transplantations. So could you
please tell us about that?


Dr. Hannah Valantine:


Yes. Well, we first did this work in a single center when I was
at Stanford, where we developed the technology, myself and a
couple of colleagues in bio-engineering. But when you do a study,
as you know, in one center, doesn't mean it's necessarily
transferable or trans label into multicenter.


Dr. Hannah Valantine:


So when I went off to NIH, I transferred the technology there and
did something else that was rather unique. I put together a
consortium of the five local heart and lung transplant centers in
the DC area. And we enrolled patients from each of those five
centers into this study. And in the heart cohort, which is what
is reported here, we were able to take blood samples on a serial
basis. And there were 171 of them in the study. So, what happens
is that we genotype the donor and the recipient of each of those
170 patients at the beginning, before the transplants, so that we
could then know and monitor their cell-free DNA as they progress
at serial time points after the transplant. And that's the way we
were able to confirm the value of this test.


Dr. Hannah Valantine:


I can describe to you what the findings were. So what we found is
that the cell-free DNA started to rise a lot earlier, before the
heart biopsy showed a rejection. So it was just remarkable,
because of the serial samples, we were able to look back and say,
well, was there an elevation of the cell-free DNA before the
positive biopsy? And that was definitively the case.


Dr. Hannah Valantine:


As you know, there are two types of rejection, antibody,
mediated, rejection, and cellular driven rejection. And antibody
mediator rejection, quite frankly, is the Achilles heel of organ
transplantation because it's difficult to pick up and it occurs,
that means it's diagnosed late, and it's really resistant to
treat.


Dr. Hannah Valantine:


So what we found in this study is that the cell-free DNA was
elevated for at least a couple of months before the heart biopsy
actually showed the presence of antibody mediated rejection. And
that has significant implications for the management of patients.
And there are some other characteristics of the cell-free DNA
that distinguish cellular and antibody mediated rejection. That
is really important because the two types of rejection are
treated differently. That's a great excitement of the results of
this study.


Dr. Carolyn Lam:


Oh my gosh. I'm just tingling. My hairs are standing, just
listening to you explain that. I really think we have a true
liquid biopsy now for cardiac transplant rejection.


Dr. Carolyn Lam:


But Dr. Bozkurt, you're such an expert in heart transplantation.
Could you frame it for us, just how significant these findings
are?


Dr. Biykem Bozkurt:


This is transformative. So first, I would like to congratulate
Dr. Valantine and her team for pioneering and leading this
concept for such a long time. And now with this validations
study, for providing the framework for the future studies for
alternative strategies, implementation of how we're going to do
this, as the liquid biopsy in lieu of endomyocardial biopsy.


Dr. Biykem Bozkurt:


So the findings that I think are truly practice changing are,
yes, this study validates the ability to detect rejection. The
second very interesting finding is predict the rejection almost
three months before that we're able to detect it by
histopathology. Third, for the first time, being able to detect
antibody mediated rejection, as well as cellular. Fourth, being
able to eliminate the necessity in approximately 81% of the
patients with a very high negative predictive value.


Dr. Biykem Bozkurt:


Now, but I'm going to pose this question to Hannah. What is the
gold standard now? Because in the historical past, we used to
rely on histopathology to be diagnosed myocardial infarction.
Now, we know that Troponin-I is a driver or Troponin-T or cardiac
troponin. The profile of this is so, I would say, impressive,
both for its negative predictive value. And I do realize the
sensitivity and specificity is over 80%. And in the positive
predictive, when we use the biopsy as the gold standard, the
numbers are not as high as a negative predictive value, but if we
add the clinical, those who've had the LV dysfunction, those who
developed rejection subsequently, three months later, it is
performing quite well.


Dr. Biykem Bozkurt:


So what now is going to be the gold standard? I'm thinking, shall
we start calling things allograft injury and go and embrace the
injury drum  now with this profiling, and then trying to
determine whether we can intervene early and prevent rejection?
And I guess my question is, what's the next step?


Dr. Hannah Valantine:


Thank you for that lovely summary. This clearly, at this point, I
would say the gold standard should be the cell-free DNA because
in the study we switched it on its head. And we said, if the
cell-free DNA is the gold standard, then how sensitive is the
heart biopsy? And it wasn't very sensitive. And this is something
that we've known for many years.


Dr. Hannah Valantine:


And I'll tell you a little anecdote that has driven my passion
over the last 35 years to come up with a better diagnostic tool.
When I was first an assistant professor and using
echocardiography to study whether or not we could replace the
biopsy, I had a patient who in whom I noticed had diastolic
dysfunction. And my protocol was if we saw diastolic dysfunction,
we actually did a biopsy. And to cut a long story short, I
ordered these biopsies, and the biopsy was negative. And in
retrospect, I think he had antibody mediated rejection. But the
sad part of it is that after a few weeks I got a call from his
wife to inform me that she found him dead in bed.


Dr. Hannah Valantine:


And so this has really motivated me to really find something
better. And to your question, the cell-free DNA should be the
gold standard. The problem is that physicians are human, and
there is often takes a lot of time for adoption. So even though
the data speaks for itself, that adoption piece is a social,
psychological factor that to be overcome. But actually, we've had
a really interesting experience in the context of COVID-19 when
we were not able to do elective procedures, many centers have
reverted to the cell-free DNA technology, and guess what? The
patients are doing very well, as we documented in this study. So
I think we're going to see a huge paradigm shift in the
management of patients with cell-free DNA being the gold
standard.


Dr. Hannah Valantine:


But I think that's not all because you asked the very important
question about what's next and what's next about this research is
to figure out, using the same technologies, why it is that black
patients reject their organs so much more than white patients.
And in this study, I hope you noticed that 44% of the cohort are
African-American. And so it opens the door now to study this
area. And then the third implication is that we can use cell-free
DNA to actually look at other mechanisms and develop the
technology. For example, DNA methylation profiles will come on
board very shortly that can teach us more about where the damage
is happening and add to the diagnostic tools.


Dr. Biykem Bozkurt:


I have another burning question. The peripheral gene expression
profiles that also are available clinically to be able to
characterize these patients. How do you see the difference in the
cell-free DNA versus gene expression? And in this study, both the
donors and the recipients were genotyped. Do you see the
necessity of having to do so? Whether the current, you know, snip
analysis will allow us not having to genotype patients, which may
have an implication on the cost, as well as the practicality of
how to implement this on the clinician bedside.


Dr. Hannah Valantine:


Absolutely. As you know, there are now several ways to get around
having to genotype the donor by using an array of 260 or so SNPs
known that are common in the population. And you can use that.
And when you use that technology, it's equally sensitive. So that
is what is going to move this forward in terms of clinical
utility, so that we don't have to do that. So it's an extension
of the technology. I like to do the recipient and donor
genotyping because there's so much more research. So from the
research perspective, it's useful to continue this technology.
But for the application, yes, we're going to be able to do it
without the genotyping, and rapidly.


Dr. Hannah Valantine:


And even beyond that, there are now new technologies coming up
that we could use. For example, DVPCR, that could be translated
into more rapid, an even more rapid test. Right now, the test
takes about a day and a half to come back. My goal ultimately
would be to have a point of contact test that we could actually
use right there to make the diagnosis on a sample of the
patient's plasma. So lots of new things coming out. This is just
the beginning.


Dr. Biykem Bozkurt:


We're also excited about the possibility of the cell-free DNA,
being able to predict coronary artery vasculopathy and/or other
clinical events. So I'm sure your team is going to come up with
results in regards to the future prognostication regarding the
clinical events. So any, perhaps, prediction as to what we shall
see those who don't decay, especially after 28 days? I'm very
intrigued by those patients, which I think was a subgroup in your
study.


Dr. Hannah Valantine:


Yeah. So that's absolutely right. What we are seeing is that
those in whom the cell-free DNA remains relatively elevated,
because what we find is that immediately after transplant, in the
first 24 hours, the cell-free DNA diminishes and to a baseline
low level. And then when there is rejection, we see these spikes,
but when it's remains elevated, this might be a predictor of
ongoing injury, an injury to the vascular dithulium that then
sets up the familio for allograft vascular disease. And so we're
chasing that hypothesis. The trouble is that the end point takes
a while to develop, which is good for the patients. And so look
out for future studies that where we will look at the correlation
of the cell-free DNA as a predictor of allograft vasculopathy.


Dr. Hannah Valantine:


The other really interesting thing goes back to mechanism. You
mentioned the question of consistently elevated cell-free DNA.
And so I'm asking myself the question, whether this three
floating cell-free DNA can actually act as a trigger of the
immune response and therefore lead to damage and rejection. And
that again is research that's ongoing, which will have
significant implications for patient care. And it might actually
help us in understanding why it is that African-American patients
reject their organs. Because even those that are doing well in
this study, they had relatively higher levels of cell-free DNA
throughout the course, and did not decay as in the same pattern
as the white patients. So lots of exciting work to come.


Dr. Carolyn Lam:


Oh my goodness, this is just a mind blowing discussion. So might
not just be a marker, but even a target for rejection or
anti-rejection therapy. And as a trialist, I'm already thinking
ahead. Might we see future trials with raised cell-free DNA, but
no evidence of rejection on biopsy, and whether or not treating
these patients would actually improve outcomes?


Dr. Carolyn Lam:


So this is incredible. I wish we had all day to chat, and I think
I really cannot let this end without at least saying something
about Dr. Valantine, your work in diversity, and to just thank
you on behalf of everybody for really forging this, and
especially as part of Circulation. As you know, we have an issue
focused on disparities, and we're just incredibly privileged to
have you on the podcast today. Biykem, please would you add some
last words?


Dr. Biykem Bozkurt:


I second those sentiments. We're grateful to Dr. Valantine for
being a trailblazer for our transplant patients, for our
community, to enhance the diversity, and for scientific
excellence in all fronts. So thank you, Dr. Valantine.


Dr. Hannah Valantine:


Thank you very much. I'm humbled by your comments and very
appreciative, and thank you for the support of this work.


Dr. Carolyn Lam:


Thank you, audience, for listening today. You've been listening
to Circulation on the Run. Thank you for joining us, and don't
forget to join us again next week.


Dr. Greg Hundley:


This program is copyright of the American Heart Association 2021.