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CME / ABIM MOC

Micro-Lessons in Heart Transplant Monitoring

  • Authors: Shelley Hall, MD; Josef Stehlik, MD, MPH; Jon A. Kobashigawa, MD
  • CME / ABIM MOC Released: 4/22/2022
  • Valid for credit through: 4/22/2023
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Target Audience and Goal Statement

This activity is intended for cardiologists, transplant surgeons, and other healthcare providers who work with patients who have received a heart transplant.

The goal of this activity is that learners will have improved knowledge of current recommendations for monitoring for rejection, including use of available noninvasive technologies, after heart transplantation.

Upon completion of this activity, participants will:

  • Have increased knowledge regarding the
    • Advantages and limitations of endomyocardial biopsy (EMB)
    • Donor-derived cell-free DNA (dd-cfDNA) in heart transplant rejection monitoring
    • Gene expression profiling (GEP) in heart transplant rejection monitoring
    • Other noninvasive testing options for acute rejection
    • Research into biomarkers for heart transplant rejection surveillance


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Faculty

  • Shelley Hall, MD

    Chief of Transplant Cardiology & Mechanical Support/Heart Failure
    Clinical Assistant Professor (Affiliated)
    Texas A&M Health Science Center, College of Medicine
    Chair, UNOS Cardiac Committee
    Chair, Thoracic and Critical Care Council of Practice, AST
    President, Texas Chapter, ACC
    Baylor University Medical Center
    Dallas, Texas

    Disclosures

    Consultant or advisor for: Abbott; Abiomed; CareDx; Medtronic; Natera
    Research funding from: Abbott; Abiomed; CareDx; Medtronic; Natera; Novartis

  • Josef Stehlik, MD, MPH

    Christi T Smith Professor Medicine
    University of Utah Health
    Division of Cardiovascular Medicine
    Salt Lake City, Utah

    Disclosures

    Consultant or advisor for: Medtronic; Natera; Sanofi; Transmedics
    Research funding from: Natera

  • Jon A. Kobashigawa, MD

    DSL/Thomas D. Gordon Professor of Medicine
    Director, Advanced Heart Disease Division
    Director, Heart Transplant Program
    Associate Director, Smidt Heart Institute
    Associate Director, Comprehensive Transplant Center
    Cedars-Sinai Medical Center
    Los Angeles, California

    Disclosures

    Consultant or advisor for: CareDx; Novartis; Sanofi
    Research funding from: CareDx; CSL Behring; Sanofi; TransMedics

Editors

  • Joy P. Marko, MS, APN-C, CCMEP

    Senior Medical Education Director, Medscape, LLC 

    Disclosures

    Disclosure: Joy P. Marko, MS, APN-C, CCMEP, has no relevant financial relationships.

  • Frederick Stange, DO

    Scientific Content Manager, Medscape, LLC  

    Disclosures

    Disclosure: Frederick Stange, DO, has no relevant financial relationships. 

Compliance Reviewer

  • Susan L. Smith, MN, PhD

    Associate Director, Accreditation and Compliance, Medscape, LLC

    Disclosures

    Disclosure: Susan L. Smith, MN, PhD, has no relevant financial relationships.

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This activity has been peer reviewed and the reviewer has no relevant financial relationships.


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CME / ABIM MOC

Micro-Lessons in Heart Transplant Monitoring

Authors: Shelley Hall, MD; Josef Stehlik, MD, MPH; Jon A. Kobashigawa, MDFaculty and Disclosures

CME / ABIM MOC Released: 4/22/2022

Valid for credit through: 4/22/2023

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Activity Transcript

Chapter 1: Heart Transplant Rejection: What Are We Looking For?

Shelley Hall, MD: Welcome to Micro Lessons in Heart Transplant Monitoring. Today, we'll be covering heart transplant rejection. I'm Dr Shelley Hall from Baylor University Medical Center in Dallas, Texas, and I'll be starting with chapter 1, Heart Transplant Rejection: What Are We looking For?

Now, in the old days of transplants, we were really dependent upon our clinical exam skills, and these are 2 acronyms that we would utilize to try and identify signs and symptoms of transplant rejection. You can see they're mainly heart failure symptoms or signs, swelling, shortness of breath, fatigue, jugular vein distention (JVD). All of these were looking for signs that the graft was failing. My favorite was D, disturbance in mood. If you've met a transplant patient, they all have a disturbance in mood, whether they're having rejection or not. But that's all we had at the beginning.

What we do know from repetitive analyses, and this is from the ISHLT registry, is that after surviving the first year, if you had a history of rejection that required therapy, your survival was impaired as to those who never had a treated rejection.

But what do we mean by rejection? There are multiple types of rejection. The 3 most common being cellular-mediated, antibody-mediated, and chronic vascular rejection. So let's look a little bit into each of these.

The first is cellular rejection. Now, this is detected by our endomyocardial biopsy, our surveillance biopsy, meaning we're doing this without symptoms provoking us. It rarely has signs and symptoms because we catch these on just these random biopsies. These are some slide examples of what they look like. And the old saying is “blue as bad.” The more blue you see, which is the white cells invading the red, the muscle, the worse the level of rejection. But the reality in today's modern transplant medicine, this is less than 1 episode per patient per year, and it's rarely fatal. As a matter of fact, the bulk of these can be in out-patient bolus steroids, either in pill form or IV formulation, and don't even require a hospitalization.

The more troublesome rejection that we deal with now in transplant is antibody mediated rejection (AMR), previously referred to as humoral. This is when the graft is injured by activation of the complement system by recipient-created antibodies. Now, this type of rejection is more likely to produce graft dysfunction with the associated heart failure symptomatology. This can occur months, but even years following the transplant, so you're never completely out of the woods for AMR, whereas cellular rejection, it's usually in the first 6 months after transplant.

The way we diagnose this is also through the biopsy, and we look at the pathological signs like capillary endothelial swelling, demonstrated in the upper right and middle slides, we look at inflammation of those capillaries or the macrophages invading the vessel wall. And then with special stains, we can identify specific complement deposition in the capillaries. And that's where your green slide that shows the immunofluorescent, or the lower right hand slide shows staining for C4d.

But AMR is a continual process. So we can potentially detect that there's going to be a potential from AMR by detecting donor-specific antibody development in the recipient -- that's not done anything to the graft yet, or with the biopsies, we can see the injury pattern in the cells, but no symptoms, or the graft can start to deteriorate from injury and they develop symptoms. But even if we attack this and treat it, we can still be left with chronic abnormalities of graft vasculopathy, chronic graft dysfunction, and restrictive physiology.

And as we move towards that chronic disease, the transplant vasculopathy, this is an accelerated form of coronary artery disease. The incidence is actually pretty common, 10% at 1 year and 40% by 5 years. But there are lots of different ways to define this. This is the leading cause of cardiac death in our transplant recipients after a first year, either in the form of sudden death, heart failure, or silent myocardial infarction. There's usually no angina, remember, this heart is denervated now. Now, depending about how you diagnose it, the upper panel of images is from intravascular ultrasound (IVUS) and you can detect intimal thickening there without any changes in the angiography, that's the middle panel, where you just see some slight little bumps. And then the pathology slide at the bottom show what happens left unchecked and will obliterate the lumen. If are you already getting angiographic lesions, very poor survival after that level of diagnosis.

Now, how we detect this is important if we detect it by IVUS, which is the top right panel, you can detect smooth intimal thickening with no evidence by angiography, and many feel that IVIS a superior method for detecting. And you can see that they can detect vasculopathy 75% of patients at 3 years. If we move to just using angiography, that is when we start to see stenotic lesions, that has a very poor survival and poor prognosis, and many advocate that we should be screening with in IVUS as a result. And in the bottom panel is a pathological sample. Now, that's what happens when it's left unchecked or uncontrolled. It essentially obliterates the lumen.

So where have we been with transplant medicine? Well, we started by identifying rejection by signs and symptoms when the graft was failing. Our modern therapies now, with endomyocardial biopsy is we're looking at cellular invasion and signs of destruction. But we want to get to the beginning before we hurt the graft.

So in summary, what is rejection? Well, the definition is evolving as our technology advances. As a transplant professional, I want to know what's happening to the graft earlier, less invasively and faster than ever before. Today's module will review the current and future technologies that could allow us to help detect rejection earlier, or even avoid rejection altogether in our patients. I want to thank you for listening and please move on to the next chapter.

Chapter 2: Endomyocardial Biopsies: Benefits and Challenges of the Current Standard

Josef Stehlik, MD, MPH: Welcome, everybody. My name is Joseph Stehlik and my task today is to talk about endomyocardial biopsy, its benefits and challenges.

In the early days of heart transplantation in the late 1960s and early 1970s, after the technical aspects of the heart transplant procedure were worked out, a challenge remained how to best diagnose rejection and distinguish it from infection, the 2 frequent complications early after transplant, since there was not an easy way to obtain endomyocardial tissue. This has changed as Dr. Philip Caves at Stanford has proposed the use of a percutaneous bioptome that would take us to the intraventricular septum and allow us to take small pieces of the myocardial tissue. Since then, this bioptome has been developed a little bit more. We now have disposable flexible bioptomes. Our preferred access is right internal jugular vein, as it's the straightest shot towards the septum. But we, of course, can also use access through left internal jugular veins, subclavian veins, and femoral veins through a long bioptome. Here are the key advantages and limitations of biopsy. Advantages are that we get a tissue diagnosis, so we really get as close as we can it to the graft, the biopsy can be done percutaneously and can be done repeatedly. The limitations include sampling error, rejection often is focal, so we need to take multiple samples, there is intra and intro observer variability of the interpretation of the histopathology, and there are some complications and cost associated with this invasive procedure. This table outlines the major and minor complications. I would like to just highlight a few that we pay a lot of attention to. One would be RV perforation, leading to hemopericardium and tamponade that requires pericardiocentesis, and also injury to the tricuspid valve. We really try to stay away from the tricuspid valve apparatus to prevent that. So while the complications are not frequent, it is important to know that these complications and others listed on the slide can happen during the procedure. In addition to the technical aspects of biopsy that has really helped in the care of patients after heart transplantation, is standardized grading of rejection. This slide shows the International Society for Heart and Lung Transplant standardized grading of acute cellular rejection. This was an update from 2005 that divides the rates of rejection as 1R mild, just paravascular and interstitial infiltrates, grade 2, moderate acute cellular rejection, where we have more than 2 foci of infiltrate with associated myocyte damage, and then severe rejection that in addition has myocyte damage, edema, hemorrhage, and/or vasculitis.

A number of immunosuppressive medications have been developed to prevent and treat acute cellular rejection, and in most patients with cellular rejection, we are able to control this pretty readily. This might not be the case for antibody-mediated rejection, another type of rejection that was first described to the University of Utah in 1988. Since then, we also already have standardized approach to grading after several iterations. This is from 2013. This describes PMR1, either 1H, a result that shows histopathologic changes of AMR that include endothelial swelling and macrophages in the capillaries, PMR1I in patients that only have immunopathologic changes, including complement deposition in the vasculature, PMR2, that has both histopathologic and immunopathologic changes, and PMR3, or severe pathologic AMR that also has interstitial hemorrhage, capillary fragmentation, and mixed inflammatory infiltrates in addition to the changes described above. Now, in summary, I would like to say that endomyocardial biopsy is a reference standard for the diagnosis of rejection. We do have standardized grading of acute cellular and antibody-mediated rejection that enables us to protocolize treatment approaches. And while non-invasive approaches to assist graft health reduce our reliance on surveillance endomyocardial biopsies, we still use biopsies early after transplant for surveillance, and we often use biopsy in patients who present with graft dysfunction to diagnose rejection in a forecast biopsy. Well, thank you for your attention, and let's now move to the next chapter.

Chapter 3: Donor-Derived Cell-Free DNA: Mechanism, Available Evidence, and Future Research

Josef Stehlik, MD, MPH: Welcome everybody. My name is Josef Stehlik, and I will be talking about donor-derived cell-free deoxyribonucleic acid (DNA) in heart transplantation. We will review the mechanism, available evidence and future research. Now we all have circulating cell-free DNA in our bodies. Cell-free DNA is a fragmented, double-stranded DNA that originates from cell apoptosis and necrosis, and the circulating half-life is only about 90 minutes.

Cell-free DNA is used in clinical care today. Initially, the use was in prenatal diagnostics, where fetal cell-free DNA is detected in the mother's circulation and testing for genetic abnormalities can be made. More recently, it is used in oncology as a biomarker of early recurrence of cancer by detecting tumor-derived cell-free DNA. And most recently, its uses are in solid organ transplantation. This graphic shows the cell-free DNA that circulates in a solid organ transplant recipient's blood. We will have cell-free DNA of 2 genotypes, the recipient's genotype and the donor's genotype that comes from the allograft.

Here, I show the evolution of the test used to detect cell-free DNA. Initially, we could detect cell-free DNA only if there was a sex mismatch between the donor and the recipient as the testing was based on the presence or absence of the X and Y chromosomes. Then, whole genome shotgun sequencing of the donor and recipient was used to confidently determine whether the cell-free DNA originated in the recipient or in the donor allograft. Currently, the tests being used do not require donor and recipient genotyping as highly informative single nucleotide polymorphisms are being used.

Next, I will review some data that linked donor derived cell-free DNA, expressed as a proportion, with acute rejection. This initial study from Stanford University has shown that the proportion of donor-derived cell-free DNA increases with mild rejection compared to no rejection and further increases in moderate to severe rejection. A receiver operating characteristics curve is shown on the right and shows favorable performance as a test with high negative predictive value and favorable positive predictive value.

A more recent study was sponsored by the NIH, and again used whole genome shotgun sequencing, was done in multi-center fashion by the graft investigators. This extends the previous finding to show us that there is a rapid drop off of cell-free DNA in the first 2 weeks after transplantation. On the middle graphics, you will see that the proportion of donor-derived cell-free DNA increases integrated response with mild to moderate acute cellular rejection (ACR) and also in acute antibody-mediated rejection (AMR). And again, receiver operating characteristic curve on the right. Further, the investigators shared that their increase in cell-free DNA happens weeks to months before antibody-mediated rejection and a couple of weeks before acute cellular rejection, and the increase seems to be higher in antibody mediated rejection.

There are a couple tests that are now available and approved for clinical care, they use next-generation sequencing and highly informative single nucleotide polymorphisms. The AlloSure® test uses 266 SNPs, no donor and recipient genotyping is required and the test is clearly validated. We see again association with acute cellular rejection and antibody-mediated rejection. Another clinically available test is the Prospera™ kidney and Prospera™ heart test. It uses next-generation sequencing, 13,000 single nucleotide polymorphisms, no donor or recipient genotyping is required and the blood does not need to be centrifuged or shipped on ice. Again, the test is clearly validated. The Prospera™ kidney ROC is shown on the right on top, the Prospera™ heart test characteristics have been presented by the company and publication in scientific journal is pending. For completeness, I will just include the myTAIHEART® test and the Viracor TRAC® test, which are not currently available for clinical use.

Well, in summary, what do we know about donor-derived cell-free DNA in heart transplantation? We do know that the proportion of donor derived cell-free DNA is associated with acute rejection; it has been shown in multiple studies. We do know that the receiver operating characteristics are favorable such that the negative predictive value is very high, and there is favorable positive predictive value. We do have some outstanding questions and that is what is the true clinical utility of this test in heart transplantation, such that most studies so far have been limited to observational data? How do we discriminate ACR vs AMR and how do non-rejection events as cardiac allograft vasculopathy, left ventricular (LV) dysfunction without rejection, time since transplant affect the clinical characteristics of the test for diagnosing rejection. Finally, could use of absolute donor-derived cell-free DNA concentration be a better biomarker than just a proportion of donor-derived cell-free DNA?

As far as future directions, I think the key aim should be to conduct prospective studies to firmly establish the clinical utility of this test in heart transplantation in both rejection surveillance, but maybe even more importantly in the future for treatment decisions. Thank you very much for your attention and let's move to the next chapter.

Chapter 4: Gene Expression Profiling: Mechanism, Available Evidence, and Future Research

Shelley Hall, MD: Welcome to chapter 4, Gene Expression Profiling, where we'll review the mechanism, some trial evidence, and future research in this field. I'm Dr. Shelley Hall, Chief of Transplant Cardiology at Baylor University Medical Center in Dallas, Texas.

So, what do we know? Well, there are a lot of various pathways for cellular rejection. Cellular inflammatory mediators, different types of genes that respond to steroids, all play a role in cellular rejection, and this is where gene expression profiling came about. It looked at a collection of pertinent genes that elevated or lowered in ACR, or acute cellular rejection, and they came up with this combination by looking at nearly 300 genes to begin with. And then they developed what's called the gene expression profiling test or the AlloMap® test. They'd narrow this down to 20 genes, 11 reporting genes that represent those different molecular pathways, and 9 control genes used to try and standardize the test.

It's important that we understood how these genes reflect some go up, some go down, some are responding to steroids ... But the important things is they weren't influenced by our common immunosuppressive medicines or calcineurin inhibitors or mTOR inhibitors. They can be affected by infections, especially CMV. In the early days, we didn't think that was possible, but research has validated that infection can alter the score. They created a scoring system ranging from 0 to 40, and the lower the score, the lower, the probability of acute cellular rejection. This started in the early 2000s and then was validated through the CARGO trial.

Now, this is the average scoring that was identified in all of these different trials, and they're essentially typically a stable patient in the high 20s. You’ll notice they were in the lower 20s at the beginning at those first couple of months after transplant. That's because they're more immunosuppressed. As most programs ween down the steroids, you notice that the score starts to rise gradually to the late 20s.

This was validated first on the right panel of greater than 6 months out. We started with the lowest risk patients to analyze this. And you can see that by then, when there are much lower scores, you have a 98.9% negative predictive value for severe rejection at a score of 34, compared to those earlier months, as you can see in the left panel, 2 to 6, where the mean score that gives you the 98.5% is 28. So this is important to understand that this score will evolve over time after transplant and the “threshold levels” will rise of accordingly.

Now, this is where they first started to compare the 2 patients, EMB being biopsy protocols, or GEP, gene expression profiling, using that to manage the patients, and they were randomized. This was the IMAGE trial, and they showed that at 2 years, they did the same -- their survival was the same and they both had the same primary endpoint. So this really opened the doors that, do we really have to do 20, 25 biopsies a year to a patient to safely manage them, certainly in the low risk patient populations?

As a result of all of this, this was put into the ISHLT guidelines. The gene expression profiling can be used to rule out the presence of ACR, of grade 2R or greater in appropriate low-risk patients between 6 months and 5 years. This was actually one of the higher class recommendations. There are very few class 1 recommendations in the transplant literature. As a matter of fact, there's only one with a level of evidence A, and the bulk or level of evidence C. So this was a very strong recommendation to our transplant community.

Another variable that came out of this was called the AlloMap® variability score, and what this means is, is there a difference to the patient whose threshold scores are nice and flat and steady, vs the patient that's up and down like a yo-yo? Well, it turns out there is, and that makes sense. Somebody who's nice and flat, their immune system is cool, there's steady use of their medications, and therefore, it turns out, less likelihood of rejection. The yo-yo patient may be having problems with compliance and they're up and down again with their medicines, their immune system is getting to crack through, and they are at a higher risk of rejection.

Now, when you receive a score from this, you have now both of those pieces of information from the report. On the top panel, you can see the AlloMap® score and it plots out all of the values that the patients undergone and you can visually almost see that variability, and then the lower panel actually calculates that variability for you. And so you have both of those pieces of information when you're trying to analyze a patient. This identifies the various aspects that you'll see and how to read the report. You can see that the solid black circles are the ones that are used to calculate the variability score that's listed below.

Gene expression profiling was the absolute first major breakthrough for non-invasive surveillance of cell rejection in cardiac transplantation, and its power is its strong negative predictive value for cellular rejection. Now, the variability score can be useful in predicting future events or patients that you need to pay closer attention to. This test has no validation in antibody-mediated rejection. We still do not have a way to comment on that for the gene expression profiling testing, and therefore it should be one tool in the armamentarium of patient care for surveillance of low-risk patients. Thank you and please move on to the next chapter.

Chapter 5: Additional Noninvasive Biomarkers

Jon A. Kobashigawa, MD: Hello, I'm Dr. Jon Kobashigawa, and I've been asked to speak on additional noninvasive biomarkers. So I've decided to center in on 3 very common biomarkers that people ask me about: high-sensitivity troponins, T-cell activation, and donor-specific antibodies. Let's start with high-sensitivity troponins. I can tell you that in the past, people used to report that, yes, troponins were correlated to acute cellular rejection. But there are some issues and some contention in the literature as well. Well, this is probably one of the largest studies to look at this troponin. 170 patients had 883 serum high-sensitivity troponin results paired to a routine surveillance endomyocardial biopsy. Now, 6% of the biosignificant ACR (acute cellular rejection) and the ROC analysis approximated the null hypothesis, meaning there were no connections here. Sub-analysis including repeated high-sensitivity cardiac troponin levels in a single individual and early less than 3 months of the endomyocardial biopsies also showed no diagnostic utility of this high-sensitivity cardiac troponin measurement. So in conclusion, in the largest publicized study to date, there was no association between high-sensitivity cardiac troponin concentration and the presence of significant acute cellular rejection on endomyocardial biopsy.

All right, let's go on now to the next topic, and that is T-cell immune function testing. So how is this actually done? Well, we take the patient's serum and we take the whole blood and we add iron beads to this blood sample. Why? Because the CD4 cells take up these iron beads. And then we add phytohemagglutinin (PHA), and the lymphocytes become stimulated. They're incubated, and then we use a magnet just to pull the CD4 cells to the side, and then we wash these cells and then add detergents for cell lysis to release the ATP. And the ATP is then given some detection reagents, and then we measure the ATP through a light photometer. Now, how does this all work? Well, think about this. If you have a lot of immunosuppression on board, then the T cells are more or less quiescent. And even if you simulate with PHA they still don't make too much ATP at all. So if you make very little ATP, that means you're over immunosuppressed. All right. Now, let's take on the other hand, if you have no immunosuppression on board and you are given PHA and the lymphocytes are stimulated, you make a lot of ATP. Therefore, when you have a lot of ATP, you are under immunosuppressed and you can have rejection.

How does this all work out in terms of the literature? Well, there's little contention here as well. On the left is a study by Gupta and colleagues. It's a retrospective study, 125 patients, 182 tests. And basically what he did was they compared the stable patients to those 14 patients with clinical events, 9 infections, 4 acute rejections, 1 heart failure, and there was no significant difference among the ATP scores. So they concluded that T cell immune assay had limited utility as an adjunct to routine clinical evaluation in assessing the risk of infection or rejection in cardiac transplant patients. But as you can see, the numbers were very small.

Now, we had done a study back in 2010, and this again was a retrospective study, but much larger patient population -- 296 patients, 864 immune monitoring assay scores, 818 stable patients were compared to 46 patients with clinical events. There were 38 subsequent episodes of infection. And as you can see here, the infection episodes had a lower ATP score, 187, compared to the stable patients who had 280 ATP scores. The P-value is highly significant. That just suggests that yes, these patients were over immunosuppressed, and that's why they had the infectious complications. Unfortunately, we only had 8 subsequent episodes of treated rejections, but numerically, those numbers were higher at 327 vs the stable patients at 280, P-value was not significant. But quite interestingly, 3 of the patients had severe rejection, what we call hemodynamic compromise rejection, and their ATP scores were much higher at 491. So we concluded that the noninvasive use of T cell immune function appears to predict infection in heart transplant patients, the association between high IM scores and rejection is inconclusive, but suggestive from our study. We'll need a larger number of patients to make this valid.

This slide actually shows the benefit of the immune monitoring and what we actually did with our data is to look at the odds ratio for rejection. Now let's take the blue line. The blue line represents the odd ratio for an infection. And as you can see, if you have very little ATP score, you have a lot of high risk for infection. And as the curve goes from left to right, you actually have increasing ATP scores and your risk for infection goes down. Now to the contrary, if you look at the red line, if that's the odds ratio for rejection, and if you have too much ATP that means you're under immunosuppressed and you have a high risk for rejection. And as you go from right to left, in terms of the red line, that's your decreasing risk for rejection. Now, where the 2 lines cross actually is your sweet spot. That's where you really want to have your patients. Why? Because that is the lowest risk for infection and rejection. If you go 2 standard deviations out from that black arrow, our level that we are comfortable with are 200 to 550 ATP scores. That's where we like to keep our patients, and I believe that's where you'll find that you'll decrease the risk for infection and rejection.

And finally, let me just now turn to our last topic and that's donor specific antibody. Now, de novo donor specific antibody, meaning DSA, develops posttransplant in 10 to 30% of patients in most studies, both pre-formed and de novo donor specific antibody are associated with increased incidence of antibody-mediated rejection, coronary artery vasculopathy, and heart allograph loss. De novo DSA against anti-HLA class II, specifically HLA-DQ antigens, are at particularly high risk for heart allograft loss. And that is a big problem that we see ... also high risk for cardiac allograft vasculopathy as well. One of the big questions about antibodies or donor specific antibodies are whether or not they correlate to pathology or biopsy proven antibody-mediated rejection. Now, pathologic AMR maybe associated with either circulating donor specific antibodies or non-HLA antibodies. AMR associated with donor specific antibodies is associated with heart allograft dysfunction and allograft loss. Now, one of the big issues about transplantation is that you have a lot of patients waiting for heart transplant who have circulating antibodies that may include donor specific antibodies. Now, these highly sensitized patients with pre-formed complement-binding donor-specific antibodies can be treated with eculizumab. And these patients had a significant reduction in biopsy-proven rejection, supporting the potential use of complement inhibitors for heart transplantation at high immunologic risk for antibody-mediated rejection. Stay tuned more to follow on that.

Thank you so much for your attention and please move onto the next chapter.

Chapter 6: Emerging Technologies: What Is Under Investigation?

Jon A. Kobashigawa, MD: Hi, I'm Dr. Jon Kobashigawa, and I've been asked to speak on emerging technologies: what's under investigation?

Well, let me get to microRNA. Well, microRNAs are implicated in T and B cell differentiation, clonal proliferation, vascular permeability, macrophage activation, toll receptor signaling. A lot of words, but in essence, what actually microRNA are, they are inhibitory RNA. In other words, they suppress many of these pathways. Now, what's really cool about microRNA is that they are very stable. The mature microRNA ... basically, you can find them in the serum, but you can also find them in pathology in the paraffin blocks. And so, has microRNA actually been used in heart transplantation to detect rejection? And the answer is, yes.

This is a study by Dong Van Huyen, et al. back in 2014 and they looked at myocardial and serum microRNA analysis. They evaluated 14 microRNA by PCR in 113 patients. And actually 50% of them had rejection. Now, what you see on the right side is your figure, and these are the 7 microRNA. And what's interesting here is that the microRNA actually detected not only rejection, but also the type of rejection: antibody-mediated rejection or T-cell-mediated rejection. And each of the microRNA had different expressions, either up or down, but significantly different from stable patients.

Well, what's actually happening into the biopsy sample itself? Well, in terms of the donor. And as it turns out, of those 7 microRNA, 4 of those 7 microRNA you could actually detect in the biopsy sample or in the donor itself, the donor heart. And as you see on the right side, these are the slides from the paraffin blocks that actually show pickup of those mRNA. And they were mRNA 10, 31, 42 and 155. So these are quite interesting that you may have a little window from the periphery to the donor heart itself to detect rejection.

So we conclude microRNA are promising biomarkers in solid organ transplantation, potential ability to diagnose and distinguish subtypes of rejection. Provides novel molecular targets for future drug development efforts. Stay tuned. More to follow on this.

All right, let me move on to exosomes and heart transplantation. So what are exosomes? Well, all cells release extracellular vesicles. Now these exosomes are cell to cell transit systems and they mediate both short paracrine and long-distance intracellular communications. They're found nearly in all bodily fluids. What's really interesting here is that exosomes have been found to detect not only rejection in solid organ transplantation, but also cancer in various bodily parts as well.

The paper to your left is from Richard Hu and colleagues, and basically looked at circulating donor heart exosome profiling, which enabled non-invasive detection of antibody-mediated rejection. Now, these are exosomes that are coming from the donor heart, which is quite interesting that they could even tell the difference between donor and recipient exosomes. Now the right side was looking at serum exosome protein profiling for the noninvasive detection of cardiac allograph rejection. This is interesting because certain proteins can represent rejection as well.

What was the take-home points? Well, characterizing of circulating exosomal proteome suggest that heart transplant and allograph rejection alter the circulating exosome protein content. Exosomal protein analysis could be a novel approach to detect and monitor acute transplant rejection and lead to the development of predictive and prognostic biomarkers.

What we can conclude from this is that, well, it's still a nascent field, very early translational biology is still, and animal models still needs to be performed. Now some promising results with perhaps detecting rejection with either protein or microRNA in these vesicles content from circulating exosomes. Might be a way to do liquid biopsy of the graft, just doing a simple blood test. Standardization of purification is necessary. And currently we're too early to really apply this clinically. Again, stay tuned. More to follow on this as well.

And let me finally end with digital pathology. When you look at an endomyocardial biopsy basically is just the pathologist using their eyeballs to look at the biopsy sample. As we all know, too, there is only a 67 concordance rate, even among expert pathologists to detect rejection. So there's a lot of problems in terms of artifact that may look like rejection. Now, these digitalized slides are data. Digitalized image equals transformation to a matrix. Computer algorithms enable interrogation of image data. We can extract unstructured pixel data, create featured maps of morphologic biomarkers, analyzed to correlate biomarkers with an outcome. When I say morphologic biomarkers, we can actually look at some cytokines as well, proteins that are in the slides themselves that the naked eye can't really detect, but yes, this digital pathology can do that.

Well, what happens when we have this slide? Well, we can look at this biopsy slide of rejection in heart transplantation, and we can turn this slide into this, which is a very complex matrix. There are many advantages of this computational method, highly reliable, same input, same output. Easily disseminated, modular in nature, high throughput, self-improvement potential, and discovery of novel biology. Why do we say self-improvement? Artificial intelligence machine learning is applied to this as well. So certainly it can get better over time as well.

Has this actually been done? Yes, actually. Eliot Peyster and Ken Margulies from the University of Pennsylvania has actually done quite a bit of work with this digital pathology. What they found is that using endomyocardial biopsy tissues, they found hidden information that the grading scale, meaning the pathology read, misses. They used PD-L1, which is a cytokine FoxP3, which is a representative of T-cell regulatory cells. CD68 was just microphages. And they can predict clinical rejection severity. If you look at the right, they can actually predict future rejection risk as well.

So take-home points from digital pathology. Digital pathology for immune profiling can improve diagnostic and prognostic value of histology samples. It can also uncover novel biology of potential therapeutic value, and who knows? Maybe the endomyocardial biopsy can be the true gold standard used in digital pathology.

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