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CME

Lung Transplant Rejection: Micro Learnings on Advances in Surveillance Technology

  • Authors: Brian Keller, MD, PhD; Sean Agbor-Enoh, MD, PhD; Deborah Jo Levine, MD, FCCP, FEST
  • CME Released: 4/22/2022
  • Valid for credit through: 4/22/2023
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    Physicians - maximum of 0.50 AMA PRA Category 1 Credit(s)™

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Target Audience and Goal Statement

This activity is intended for pulmonologists, transplant surgeons, and other HCPs who work with patients who have received a lung transplant.

The goal of this activity is to improve knowledge around current standards for acute and chronic rejection monitoring and increase understanding of the clinical trial data on donor-derived cell-free DNA (dd-cfDNA) for transplant rejection monitoring.

Upon completion of this activity, participants will:

  • Have increased knowledge regarding the
    • Mechanisms of acute lung transplant rejection
    • Mechanisms of chronic lung transplant rejection
    • Limitations of current standards in posttransplant surveillance
    • Clinical trial data on dd-cfDNA as a biomarker for lung transplant rejection
    • Role of emerging technologies in differentiating posttransplant infection from rejection


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Faculty

  • Brian Keller, MD, PhD

    Associate Professor
    Department of Internal Medicine
    Division of Pulmonary, Critical Care and Sleep Medicine
    The Ohio State University College of Medicine
    Columbus, Ohio

    Disclosures

    Disclosure: Brian Keller, MD, PhD, has the following relevant financial relationships:
    Consultant or advisor for: CareDx
    Speaker or member of speakers bureau for: CareDx
    Research funding from: CareDx, Natera, Zambon Company S.P.A.

  • Sean Agbor-Enoh, MD, PhD

    Lab Chief
    NIH Lasker Clinical Tenure Track Investigator and NIH Distinguished Scholar
    Laboratory of Applied Precision Omics
    Division of Intramural Research
    National Heart, Lung, and Blood Institute
    National Institutes of Health
    Bethesda, Maryland
    Assistant Professor
    Lung Transplantation Program
    Pulmonary and Critical Care Medicine
    The Johns Hopkins Hospital
    Johns Hopkins School of Medicine
    Baltimore, Maryland
    Participation by Dr Agbor-Enoh does not constitute or imply endorsement by the Johns Hopkins University or the Johns Hopkins Hospital and Health System.

    Disclosures

    Disclosure: Sean Agbor-Enoh, MD, PhD, has no relevant financial relationships.

  • Deborah Jo Levine, MD, FCCP, FEST

    Professor/Clinical Medical Director
    Lung Transplantation
    Director
    Pulmonary Hypertension Center
    The University of Texas Health Science Center at San Antonio
    San Antonio, Texas

    Disclosures

    Disclosure: Deborah Jo Levine, MD, FCCP, FEST, has no relevant financial relationships.

Editors

  • Iwona Misiuta, PhD, MHA

    Medical Education Director, Medscape, LLC

    Disclosures

    Disclosure: Iwona Misiuta, PhD, MHA, has no relevant financial relationships.

  • Yoji Yamaguchi, MA, ELS

    Scientific Content Manager, Medscape, LLC

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  • Amanda Jett, PharmD, BCACP

    Associate Director, Accreditation and Compliance, Medscape, LLC

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    Disclosure: Amanda Jett, PharmD, BCACP, has no relevant financial relationships.

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


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CME

Lung Transplant Rejection: Micro Learnings on Advances in Surveillance Technology

Authors: Brian Keller, MD, PhD; Sean Agbor-Enoh, MD, PhD; Deborah Jo Levine, MD, FCCP, FESTFaculty and Disclosures

CME Released: 4/22/2022

Valid for credit through: 4/22/2023

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Activity Transcript

Chapter 1: Post-Lung Transplant Care: Acute Rejection

Brian Keller, MD, PhD: Hello, my name is Brian Keller, and I'm from The Ohio State University. Today, I would like to overview the types of acute rejection that can occur following lung transplant. Acute rejection is a common and important complication in patients after lung transplantation. Up to 50% of patients will experience an episode of acute rejection in the first year after transplant. It is also important because acute rejection is associated with an increased risk of chronic lung allograph dysfunction (CLAD). There are generally 2 types of acute allograph rejection: acute cellular rejection (ACR) and antibody-mediated rejection (AMR). A risk factor that has been identified for ACR is the increasing number of human leukocyte antigen (HLA) mismatches between a donor and recipient. The risk factors for AMR are less well defined, but investigations continue. 

ACR is diagnosed by transbronchial biopsy and histopathologic grading. The grading of ACR was revised in 2007 from the original 1996 International Society for Heart and Lung Transplant (ISHLT) Standardization of Acute Rejection Grading. It is defined by 2 grades of rejection: the A grade, which represents perivascular rejection, and the B grade, which represents lymphocytic bronchiolitis (LB). A grade rejection is scored on scale of 0 to 4, where A0 represents no rejection; A1 represents minimal rejection identified by isolated mononuclear cell infiltrates around small veins; A2, mild acute rejection, is identified by an expanding the numbers of mononuclear cell infiltrates with expansion into the interstitium; A3, moderate rejection, shows expansion into the interstitium that continues, as well as the involvement of interalveolar macrophages; finally, in A4, severe acute rejection, we start to see fibrin deposition, organizing pneumonia, endothelialitis, and eosinophils.

With small airways, inflammation, or LB, grade B0 represents no LB. Grade B1R and B2R represent the revised versions of B1/B2 rejection from the original classification, with B1R representing low-grade rejection, patchy mononuclear cells surrounding small airways. Grade B2R, or high-grade LB, shows enhanced mononuclear cell infiltrates, as well as invasion into the epithelial layer and disruption of that layer with associated apoptosis and necrosis. BX is an ungradable sample, either because small airways were not included or because artifacts prohibit the appropriate interpretation of the B grade rejection in that sample.

AMR is diagnosed using 5 standard diagnostic criteria, including allograph dysfunction, the presence of donor-specific antibodies (DSAs), appropriate lung histology, including capillaritis, acute lung injury with or without diffused alveolar damage, and with or without endothelialitis, and complement deposition, complement 4d (C4d) stain. Other causes need to also be excluded for the diagnosis of AMR.

AMR classification is through the separation of a clinical AMR picture vs a subclinical AMR picture with the differentiation being the presence of allograph dysfunction, defined as alterations of pulmonary physiology, gas exchange properties, radiologic features, or deteriorating functional performance. For the purpose of this talk, I'm going to focus on the clinical AMR. Clinical AMR can be defined or divided [by] definitions of diagnostic certainty. With definite AMR, including the presence of allograph dysfunction and all 3 of the criteria of the presence of DSA, histology [is] consistent with AMR and C4d staining presence. Probable AMR includes allograph dysfunction and 2 of the 3 criteria, and possible AMR includes only 1 of the criteria, along with allograph dysfunction.

Treatment of AMR is more complicated than treatment of ACR, involving multimodal treatment targeting steps along the pathway thought to be involved with the development of production of DSAs, which includes the removal of circulating antibodies, the targeting of B cells and plasma cells, inhibition of complement-mediated pathways, and newer therapeutics that target novel pathways. 

In summary, acute rejection is an important and common complication following lung transplantation. ACR is a cell-mediated process and is associated with CLAD development, specifically the bronchiolitis obliterans syndrome form of CLAD. The number of mismatches in HLA between the donor and recipient is a risk factor for the development of ACR, and ACR is diagnosed with transbronchial biopsy. Treatment is generally with corticosteroids, although in severe forms of ACR, antithymocyte globulin, alemtuzumab, or extracorporeal photopheresis may be used to enhance immunosuppression.

AMR, as its name suggests, is an antibody-mediated process. It, too, is associated with CLAD development, although risk factors for AMR development are not fully known. Diagnosis is based on criteria, including allograph dysfunction, DSAs, histopathologic findings consistent with AMR, and the deposition of C4d. Treatment is generally a multimodal approach targeting production of antibodies, removal of circulating antibodies, and the complement deposition pathway. 

Thank you for participating in this activity, and please go on to answer the question that follows.

Chapter 2: Post-Lung Transplant Care: Chronic Lung Allograft Dysfunction

Brian Keller, MD, PhD : Hello. My name is Brian Keller and I'm from the Ohio State University. In this section, we will discuss chronic forms of post-lung transplant rejection.

Chronic lung allograph dysfunction is a term used to describe a decline in lung function that is evident beyond the first year of transplant and persists for 3 or more weeks. CLAD is important because it is a significant cause of morbidity and mortality in lung transplant recipients and the most common cause of death in patients beyond the first year. It is relatively common, with an incidence of about 50% at 5 years and 67% at 10 years in lung transplant recipients. Treatment is difficult; there is currently a paucity of therapies proven to be broadly effective in treating CLAD.

Numerous studies have identified risk factors for the development of CLAD, and these include the number and severity of episodes of acute rejection and lymphocytic bronchiolitis; certain infections, such as cytomegalovirus, Aspergillus, Pseudomonas and other bacterial, fungal or noncytomegalovirus (CMV) viral infections; medication noncompliance; the number of human leukocyte antigen mismatches between the donor and recipient; and the presence of gastroesophageal reflux disease (GERD).

The diagnosis of CLAD is a spirometric- and chest radiographic imaging-based diagnosis. What we are looking for is an initial greater-than-10% decline in forced expiratory volume in 1 second (FEV1) and or forced vital capacity (FVC) from baseline. This leads to a diagnosis of possible CLAD, and investigators should institute additional searches for non-CLAD causes of allograph dysfunction, such as acute infection, ACR, AMR, and other factors that may cause allograph dysfunction. If the decline in allograph function persists for greater than 3 weeks but up to 3 months and is associated with an additional 20% decline in FEV1, the diagnosis is upgraded to probable CLAD. When the decline in allograph function persists for greater than 3 months and is more than 20% from baseline, a diagnosis of definite CLAD is made, and this should be confirmed by spirometry, total lung capacity (TLC), and chest computed tomography (CT) diagnosis. This would help to identify the phenotype of CLAD.

In general, there are 4 CLAD phenotypes. I'm showing here the 2 most common forms. Bronchiolitis obliterans syndrome (BOS) represents 60% to 75% of all CLAD cases. BOS is identified by an obstructive ventilatory defect with air trapping and the absence of pulmonary infiltrates. Restrictive allograph syndrome (RAS) represents 25% to 35% of CLAD cases. It generally is associated with a worse prognosis than BOS and is also differentiated from BOS in the presence of pulmonary infiltrates. When patients do not fall cleanly into the BOS or RAS categories, they may fall into the other 2 categories, which include a mixed form, where patients have evidence of obstruction and restriction as well as opacities on their CT imaging; or they may fall into an undefined category where they don't fit nicely into any of the 3 previous phenotypes.

Recently, CLAD staging has been updated. In 2002, CLAD was diagnosed purely based on BOS, as RAS had not yet been identified. In 2019, the CLAD staging was updated to include not only BOS but also RAS. And now, CLAD is staged on a score of 0 to 4, where CLAD 0 represents normal allograph function, greater than 80% of a patient's baseline, and CLAD 1 through 4 represent progressively more severe reductions in allograph function from a patient's baseline. Currently, a patient may be staged as CLAD 1 BOS phenotype or, say, CLAD 2 RAS phenotype.

In summary, CLAD is an important complication following lung transplantation and occurs with a fairly high frequency of up to 50% at 5 years. The 2 main forms of CLAD include BOS, which is common and is associated with an obstructive ventilatory defect and the absence of pulmonary infiltrates. Risk factors for BOS that have been identified include ACR, LB, gastroesophageal reflux disease, and infections with Pseudomonas and Aspergillus. There are no proven therapies for BOS.

Restrictive allograph syndrome is less common than BOS but carries a worse prognosis. It is associated with a restrictive ventilatory defect and the presence of pulmonary infiltrates on chest CT. Risk factors for RAS are not as clearly described, but factors that have been linked to RAS include ACR, diagnosis of DSAs, AMR, and eosinophilia in the bronchoalveolar lavage (BAL) and blood. Again, there are no proven therapies for RAS.

Thank you for participating in this activity, and please go on to answer the question that follows.

Chapter 3: Monitoring for Acute Rejection and Infection: Challenges of Our Current Techniques

Deborah Jo Levine, MD, FCCP, FEST : Good afternoon. My name is Debbie Levine. I'm from the University of Texas. Welcome to section 3, "Monitoring the Post-Lung Transplant Recipient." Lung transplant is the best therapy for patients with end-stage organ disease for which there is no other medical or surgical therapy available. Although we have had success in increasing the numbers of lung transplants over the last decade, this slide from the International Society for Heart and Lung Transplantation in 2019 shows us that our survival really has been pretty stagnant.

The median survival in 2019 was still only 6.5 years. The reason for this decrease in survival after lung transplant within the first year is infection. The leading cause of death after 1 year posttransplant is chronic lung allograft dysfunction. Now, there are many risk factors for patients developing CLAD throughout their transplant journey. There are both immune and nonimmune risk factors, acute cellular rejection, acute antibody-mediated rejection, donor-specific antibodies -- all immune causes or risk factors for CLAD.

However, primary graft dysfunction, reflux, cytomegalovirus (CMV) and other infections, including other viruses, fungi, and bacteria, are all nonimmune risk factors for CLAD. These are all areas also that are important in acute graft dysfunction. Early identification of graft dysfunction is imperative to help prevent or delay CLAD. Clinical assessment, imaging, pulmonary function testing (PFT), bronchoscopy with histopathologic evaluation -- all of these are used either alone or in combination to identify and monitor acute graft injury. But what remains challenging is that the sensitivity, the specificity, and the reliability of the diagnoses are not high for any one of these strategies alone. Because of that, most centers are using some type of surveillance monitoring, as rejection, and even infection, can be often asymptomatic and sometimes without functional decline.

Let's go through these one by one. Can we depend on acute clinical findings to help make a specific diagnosis of acute graft dysfunction? When you look at symptoms, dyspnea, cough, and malaise, all of these are very nonspecific symptoms and really can apply to any of the 3 diagnoses. For example, for ACR, there's a very low sensitivity and specificity for lower grades of rejection. Signs, including low-grade temperature, hypoxemia, are also very nonspecific. Because it's common to be asymptomatic and no signs and because they're all nonspecific, we cannot reliably differentiate between all of these clinical presentations.

What about imaging? Can imaging help discriminate between diagnoses? Well, we know that diagnostic performance is limited. In fact, 50% of biopsy-proven ACR may have normal findings. These patients may be getting sicker with normal radiographic findings. When the imaging, whether it be a chest x-ray or a CAT scan is abnormal, it can be one of many nonspecific findings, whether it be reticular nodular, interstitial, or airspace lesions, all of these can occur in infection or rejection. Patients can have effusions. Again, all of these are very poor in differentiating or discriminating between diagnoses.

What about pulmonary function tests (PFTs)? We use PFTs routinely, both at home, with home spirometry, and in the clinic. They're actually the best screening tool for identifying early graft dysfunction. It's noninvasive. It's a safe and reproducible test, but again, the sensitivity and specificity are lacking. In fact, you can have decreased forced expiratory volume in 1 second (FEV1). You have a decreased flow and you might not know why, but then again, you could have a stable or improving FEV1, and that does not exclude acute rejection or infection. The challenges in PFTs are nonspecific presentation and the fact that there are multiple overlying etiologies.

Clinical and functional parameters can really support a presumptive diagnosis of infection or rejection. But these presumptive diagnoses may be very, very hazardous. If you look at some of these patients with some of these noninvasive tests, rejection from the nonspecific clinical parameter can actually be potentially hazardous. For example, you may miss an infection if you are thinking of rejection. If you were thinking of rejection and you gave increased immunosuppression, you can actually make a patient worse. Bronchoscopy with biopsy for histopathological determination, as well as collection of bronchoalveolar lavage (BAL) and cytology, as well as microbiologic analysis, can really help to hone down on these differential diagnoses.

How do we do that? Surveillance bronchoscopy is done in multiple centers throughout the world. It's the most frequent monitoring approach, with up to 80% of centers doing surveillance bronchoscopies. These are bronchoscopies performed at regular intervals, usually within the first 2 years. However, there's no standardization of utility, protocol, or implication of these biopsies. They're not without risk. You can be at risk with biopsies for pneumothorax bleeding, respiratory failure, as well as pneumoniaRemember, biopsy is the gold standard for diagnosis in ACR, and biopsy is 1 of several characteristics for diagnosis in AMR.

What are the challenges then in histology? Well, histological changes are often nonspecific, and you may have overlapping diagnoses. Then, reading or interpreting these biopsies is variable across centers. There are multiple studies looking at the variability in concordance between readers. There can also be histological rejection, whether it be infection, aspiration, drug toxicity, acute lung injury, all of these can mimic rejection and make the diagnosis difficult to make. Again, you might not have an alternate diagnosis. You also may have a concurrent diagnosis. 

There are several characteristic features that make a diagnosis of AMR. Circulating DSA, graft dysfunction, the histology of AMR and then positive complement 4d (C4d) staining. What are the challenges of some of these? Well, let's look at DSA. The difficulty in identifying DSA and actually having concordance across centers is real. Recognizing the clinical significance of these specific DSAs is even more challenging. Understanding how to best monitor these DSAs between centers and intracenter is still ongoing. 

C4d staining is a marker of complement binding in the biopsy. It's used in kidney transplant and heart transplants where it's used to really help recognize AMR. We haven't enjoyed the same success in lung transplant. The role of C4d deposition in the diagnosis of AMR and lung transplant has proven to be challenging. C4d is difficult to identify in biopsies. We know that C4d-negative cases have similar outcomes as those with positive cases. These become challenging because not everybody is evaluating for it. When they do, it's difficult to find.

Concluding this section, I really wanted to make the point that acute rejection and infection may lead to acute graft dysfunction, but more importantly, these are risk factors for CLAD, which can lead to increased morbidity and mortality posttransplantation. The progressive understanding of the pathogenesis, early identification, monitoring management of acute graft dysfunction may help us to delay or even prevent CLAD. Most importantly, the low sensitivity and specificity of these existing tools that we have do reflect the need for more collaboration between centers, but also to help us look for newer diagnostic platforms. In section 5 of this course, we're going to be talking about some of these newer diagnostic platforms that may help us identify these issues earlier.

Thank you very much, and I look forward to seeing you in section 5.

Chapter 4: Current Evidence of Noninvasive Monitoring: Donor-Derived Cell-Free DNA as a Biomarker for Injury

Sean Agbor-Enoh, MD, PhD: Hello. My name is Sean Agbor, and I'm from the National Institutes of Health (NIH) with a joint appointment at Johns Hopkins. Today, in this chapter, I would like to give an overview of the current evidence supporting the use of cell-free DNA (cfDNA) to monitor lung transplant rejection. 

So, here is an overview of the potential clinical utility of cfDNA with evidence from cohort and natural history studies. I will talk about cfDNA as part of routine clinical care, and then give an overview of potential upcoming clinical utility studies that test the benefit of cfDNA. The graph below gives you potential clinical uses of cfDNA as proposed by existing evidence from cohort and natural history studies.

What are cfDNA? These are short fragments of DNA that are released into the circulation when cells die. They're actually quite abundant. In 1 mL of plasma, there is approximately about 100 billion fragments of cfDNA. In a setting of transplantation then, you have DNA from 2 sources, DNA from the recipient and DNA from the donor organ. And so the DNA coming from that donor, which we call donor-derived cell-free DNA (dd-cfDNA), can easily then be quantified by various methods. Existing techniques can use shotgun sequencing, digital droplet polymerase chain reaction (PCR), or quantitative PCR to monitor and quantify dd-cfDNA by targeting the single-nucleotide polymorphisms or the DNA differences between the donor and the recipient.

Does this work? Well, let me show you some evidence. This is one of the many studies that have been published recently orchestrated by the Genome Research Alliance for Transplantation (GRAfT) consortium. In this study, they recruited lung transplant patients before transplantation and monitored them after transplantation with a collection of serial plasma samples. The plasma samples were then used to measure dd-cfDNA using the shotgun sequencing technique. Concurrently, the patients undergo a biopsy, which is standard of care. The study also therefore collected biopsy results, adjudicated the biopsy results for acute rejection. And the authors then asked the question, what is the performance of cfDNA in this study in detecting transplant rejection?

And here is what they found. Number 1, they showed that cfDNA measurement is quite reproducible between laboratories. The graph that I'm showing you here is for a patient who got acute rejection. The 2 lines that you can see there shows you cfDNA that was measured at the NIH and at Stanford with replicate samples. As you can see, based on those 2 lines, the graphs are literally superimposable on each other, indicating that the measurement of cfDNA is quite reproducible.

The next question the study authors ask is, how does cfDNA correlate with measures of allograft injury? In lung transplantation, patients are monitored very carefully to look for any organ dysfunction. The 2 methods that are used to monitor organ are biopsy of that organ or measurement of the lung function. In these graphs here, I have 2 graphs here; the graph to your left, look at cfDNA in relation to measurements of rejection, while the graft to your right look at cfDNA in regards to measurement of lung function, which is done by pulmonary function testing, which is routine clinical care.

First, I'll take your attention to the graph to your left. Here is the x-axis. You could see patients with different grades of acute cellular rejection (ACR). And as you go from rejection grade 0 to grade 1 to grade 2, you have increasing levels of cfDNA, indicating that cfDNA levels correlate with the severity of rejection measured on histopathology. If I turn your attention to the curve on your right, as you measure allograft dysfunction with pulmonary function testing, you go from no allograft dysfunction to mild or to moderate and severe. Again, you have this stepwise increase in your cfDNA. This indicate that cfDNA correlates with existing measures of allograft dysfunction.

Now, does cfDNA actually indeed detect rejection? Here I have 2 graphs. The y-axis on the graph to your left is your levels of dd-cfDNA in percentage, and on the x-axis are different clinical measurements. Controls in this case are patients without rejection. ACR are patients with ACR, AMR, and then acute rejection, the combined samples for both ACR and AMR. As you could see here, ACR, AMR, or acute rejection, they all show higher cfDNA levels compared to controls. The graph to your right gives you a sense of the performance, sensitivity, and specificity for cfDNA to detect AMR, ACR, or acute rejection. As you could see from those graphs, this is a receiver operator curve characteristic analysis, indicating that cfDNA reliably detects AMR, ACR, or acute rejection.

The power of cfDNA is captured in this slide. Here, I'm showing you an x-axis where time 0 is the time the patient either shows clinical evidence of rejection, or when the biopsy shows evidence rejection. The minus numbers that you see are the number of months prior to that diagnosis of rejection. As you can see, at time 0, your cfDNA level is elevated. Two to 4 months prior to that diagnosis of rejection, you have elevations of cfDNA, indicating that cfDNA does not only detect rejection, it detects it before the patient shows any clinical symptoms or the biopsy shows any evidence of rejection.

Now, next, I will show you the utility of cfDNA as part of routine clinical care. This is a study that was orchestrated during the COVID-19 pandemic. During the pandemic, many centers stopped doing biopsies of their patients to prevent them from coming to the hospital. So, in this study, the authors convinced multiple centers to use cfDNA in monitoring these patients. This is a multicenter retrospective cohort study that included 175 patients from whom 380 cfDNA measurements were done. The authors showed that cfDNA levels were higher when patients got acute lung allograft dysfunction (ALAD), which could be either infection or rejection. The patients showed higher levels of cfDNA during ALAD compared to controls when they had no infection or rejection.

During the study, the authors, using cfDNA, were able to prevent 82.7% of biopsies, which is done through bronchoscopy, during the study surveillance period. cfDNA levels greater than 1% is an indication of underlying ALAD, or acute lung allograft dysfunction. That threshold of 1% has a sensitivity of about 73.9%, a specificity of 87.7%, a positive predictive value of 43%, and a negative predictive value of 96.5%, again indicating that cfDNA reliably detects rejection, as we have observed in cohort studies.

In summary, I've shown you evidence indicating that cfDNA may be a reliable marker to monitor patients, not just for acute rejection, but also for infection. There are multiple other uses of cfDNA, as indicated by existing cohort studies. That cfDNA could also be used to monitor patients for treatment response, and it can be used to dose immunosuppression appropriately. The upcoming clinical utility trials will give us a clear sense of the potential clinical utility of cfDNA in the monitoring of lung transplant patients. 

Thank you for participating. 

Chapter 5: Identifying Acute Graft Dysfunction: Overcoming Challenges

Deborah Jo Levine, MD, FCCP, FEST : Good afternoon. My name is Debbie Levine from the University of Texas, and today we're going to be talking about identifying acute graft dysfunction, overcoming challenges.

Chronic lung allograft dysfunction (CLAD) is made up of multiple risk factors, both immune and nonimmune. ACR, AMR, and donor-specific antibodies, or DSA, are all immune risk factors for CLAD. But there's many nonimmune risk factors as well, whether it be primary graft dysfunction (PGD), reflux, cytomegalovirus(CMV), or other infections, including other community-acquired respiratory viruses, fungi, or bacterial infections. So all of these are risk factors for CLAD, but they also can cause acute graft injury. Acute rejection, infection, and many of these others may lead to acute graft dysfunction. But the most important fact about identifying these early are that they're risk factors for CLAD, which can lead to increased morbidity and mortality after 1 year posttransplant. So really, understanding the pathogenesis, the identification, monitoring, and management of acute graft dysfunction with all of these etiologies can help us delay or even prevent CLAD. And that's what we're talking about today.

So, what are the challenges limiting accurate detection or diagnosis of acute graft injury? Well, look, the first challenge is that, as we've talked about, there's multiple etiology, whether it be acute rejection, whether it's CLAD, or whether it's infection or others, it's the heterogeneous entity with multiple etiologies. What about the second challenge? The second challenge that limits diagnosis is that there's variable approaches between centers to monitoring and evaluation, so we may not all get to the same diagnosis based on our approach. And then challenge 3 is, how do we get more accurate diagnostics for earlier diagnosis? We now know we have multiple current techniques, but how do we take that next step beyond to using newer techniques to help us with an earlier diagnosis? We have multiple current diagnostic and monitoring tools, but they all have their challenges, either they're overlapping diagnoses, they may have interpretation variability, and then there's always multiple diagnostic tools that have a low sensitivity or specificity.

All of these are challenges in making the correct diagnosis, but these challenges reflect the need for better understanding of our current techniques. They also reflect the need for more standardized monitoring and the need for additional diagnostics. So let's take these challenges one by one.

We all use, in lung transplant, donor-specific antibodies (DSAs) to help us identify any antibodies that could lead to acute AMR, and also CLAD. However, we may use a different assay in validating the actual antibody, we may quantitate them differently, and we may look at different antibodies as being important or not important based on the qualification. How we use these antibodies, in terms of identifying risk, as well as managing patients, is important. So DSA truly reflects the need for better understanding of how we can all use our current techniques in a more standardized manner and in a better way.

What about challenge 2? Well, let's use histopathology as an example to help us reflect on the need for more standardized monitoring. Histopathology has gone through multiple iterations and formulations of definition, diagnosis, and standardizing the nomenclature. As time has gone on, we have focused the identification and criteria to help the community come together in a more unified diagnosis for rejection.

What about challenge 3? Challenge 3 is really looking at the need, in all cases, for additional diagnostics for what we have now. Early identification would allow proactive strategies to delay progression. It helps us in risk stratification, adjustment of immunosuppression, as well as in patient surveillance and management.

So when you talk about the need for additional diagnostics, all you have to do is look towards the literature for multiple new techniques that are being developed, whether it be in tissue, like molecular microscope or nanotechnology, in serum, donor-derived cell-free DNA (dd-cfDNA), or even nonhuman leukocyte antigen (HLA) antibodies, or even in the BAL or bronchial wash.

Let's look at a few of these. What about the molecular microscope? These molecular techniques are informed on transbronchial biopsies. These look at both the injury patterns and rejection and certain phenotypes that occur within each biopsy to help us better develop our sense of acute rejection vs chronic rejection vs infection. Performing transbronchial biopsies is an opportunity to look at gene signatures in the biopsies. And again, looking at acute rejection, lymphocytic bronchitis, or even CLAD. It's looking at different microarray platforms in tissue. 

dd-cfDNA has demonstrated promise in identifying lung injury, and actually can help to serve as an objective noninvasive method in monitoring graft dysfunction and outcomes. It actually can serve as a biomarker in terms of screening, risk assessment, diagnosing, and monitoring for acute graft dysfunction. 

Accurate early identification and monitoring of acute graft injury is imperative to improve graft and recipient survival. Newer platforms help to provide the opportunity for clinical use in the future to use together with our current markers with some promising results.

Thank you so much, and please feel free to look at all of the other sections in this course. Thank you.

This transcript has been edited for style and clarity.

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