Molecular Approaches to Transplant Monitoring; Is the Horizon Here?

Transplantation is a remarkable treatment that gives a “second chance” of life to patients with end-stage organ failure. Unfortunately, this exposes the transplanted organ or allograft to the recipient’s immunity, predisposing the allograft to acute rejection, a dreadful complication, and a major risk factor for allograft loss. To preserve the allograft’s health, providers carefully monitor their patients to maintain immunosuppression adequacy, toward balancing the risk of rejection and infection. Monitoring approaches vary substantially among providers and transplant programs. Generally, monitoring encompasses biopsy of the allograft for histopathology and light microscopy to assess rejection, measurement of the allograft function (creatinine for kidney, pulmonary function test for lung, liver function test for liver, echocardiography for heart), measurement of immune activity with donor-specific antibody (DSA) testing, and assessment of immunosuppression adequacy by measuring immunosuppression drug blood concentrations. Assessment of allograft function or immunosuppression drug concentrations uses noninvasive testing approaches. Thus, these tests are generally implemented as surveillance, wherein patients undergo testing at predetermined posttransplant intervals independent of symptoms or clinical suspicion of allograft dysfunction. When patients present with signs for allograft dysfunction, additional clinical indication testing is performed. Biopsy, on the other hand, is an invasive procedure associated with procedure-related complications. Further, biopsy samples are analyzed by routine histopathology and light microscopy, which have limited sensitivity and precision to adequately detect and phenotype rejection and nonrejection complications. As a result, practice varies; some centers perform surveillance plus clinically indicated biopsies. Other centers only perform clinically indicated biopsies. In lung transplantation, for example, 70% of centers in the USA perform surveillance plus clinically indicated biopsies while 30% of centers perform only clinically indicated biopsies. The true benefit of these different monitoring approaches remains undefined. Fortunately, in the last few decades, novel molecular approaches have been introduced; many show promising results with benefits that address the limitations of biopsy plus conventional histopathology. In this Q&A, we focus on 2 prototype tools that recently received approval for patient use by the Centers for Medicare and Medicaid Services of the United States: plasma-based donor-derived cell-free DNA (dd-cfDNA) and “molecular biopsy,” the latter to indicate gene expression profiling of biopsy tissue samples using microarray and other platforms. Five experts discuss the potential use of these novel tools to monitor solid organ transplant patients. Michael Oellerich: dd-cfDNA is a minimally invasive quantitative biomarker for the detection of graft injury. As organ transplants are also genome transplants, dd-cfDNA opens up the possibility to monitor allograft health. In case of graft cell death, nucleosomes are released into the bloodstream as cfDNA. The clinical validity of dd-cfDNA to detect or exclude rejection and other graft injuries has been documented in more than 50 studies. Increases of dd-cfDNA levels were found in transplant recipients several days or even up to 3 months before clinical manifestation of acute rejection. There has been discussion that increased dd-cfDNA levels may be a trigger of inflammation. Early detection of in particular subclinical antibody-mediated rejection enables adapted therapeutic interventions and may improve outcomes. This needs to be further investigated in controlled studies with different dd-cfDNA tests. In kidney transplantation, dd-cfDNA may be useful to early detect antibody-mediated rejection in DSA positive patients. An adequate diagnostic performance of dd-cfDNA is suggested by currently published studies for detection of acute rejection [ROC area under curve (AUC) = 0.81]. Clinical sensitivity and specificity are around 80% and 76%, respectively. The high negative predictive value of 90% suggests that dd-cfDNA is useful to exclude rejection. Therefore, dd-cfDNA could be helpful to avoid unnecessary biopsies triggered by increased plasma creatinine. After successful rejection treatment, dd-cfDNA rapidly declined in liver, heart, or kidney transplant recipients. There are several limitations of dd-cfDNA. Increases of dd-cfDNA are not rejection specific. Other sources of graft injury associated with increase of dd-cfDNA include for example, pyelonephritis, acute tubular necrosis, and BK-virus nephropathy in kidney transplant recipients. In general, patients with advanced interstitial fibrosis tubular atrophy showed only a relatively small increase of dd-cfDNA and a discrimination of values from patients with normal histology was not possible. During stages of active disease progression, dd-cfDNA levels might be increased in interstitial fibrosis tubular atrophy. The detection of T cell-mediated rejection seems to be influenced by the employed test procedure. False-negative results may be due to the use of relatively long amplicons (100–130 bp) in the assay used. dd-cfDNA test results should be interpreted in context of all available relevant clinical data and diagnostic findings to achieve individualized transplant patient therapy with the potential to reduce graft loss and to save costs. Based on currently available evidence regarding clinical validity, dd-cfDNA seems to be useful for surveillance of transplant recipients and for clinically indicated testing. Further optimization of dd-cfDNA testing for effective clinical use is an ongoing challenge. Philip Halloran: Surveillance: no. Clinically indicated: cautious yes, if a probabilistic algorithm to guide the use of the information to avoid biopsy can be defined based on continuous numbers, not cutoffs. The utility of commercially available dd-cfDNA tests in surveillance is limited by high cost. Repeated use of an expensive surveillance test in stable patients is probably not justified without indications. dd-cfDNA has not been shown to actually reduce biopsies and to create savings, and it may be producing unnecessary biopsies. We need to know what to do with the many ambiguous values for dd-cfDNA in the commercial assays because they have the potential to trigger unnecessary biopsies and investigations. We need good algorithms that state the probability of rejection over the whole range of dd-cfDNA values. Michael Keller: Plasma dd-cfDNA is a noninvasive quantitative molecular biomarker that increases in the setting of allograft injury. Recent evidence suggests that the rise in dd-cfDNA may precede clinical manifestations of allograft injury by several months. Several observational cohort studies performed in kidney, heart, and lung transplant recipients indicate that dd-cfDNA displays acceptable performance characteristics for the detection of acute rejection—both acute cellular rejection and antibody-mediated rejection. In particular, dd-cfDNA demonstrates a high negative predictive value for acute rejection, providing the potential to effectively “rule out” the majority of acute rejection events. These characteristics make plasma dd-cfDNA appealing as a method of surveillance monitoring for underlying allograft injury. Despite a paucity of high-quality evidence supporting its use, most heart and lung transplant recipients undergo routine surveillance biopsy as standard of care to screen for acute rejection. As a screening tool, these biopsies provide procedural risks, are costly, and are often inconvenient. In addition, analysis of histopathology has high interobserver variability in the pathologic grading of biopsy samples. Dd-cfDNA provides a safe, accurate, and minimally invasive method of screening patients who may benefit most from proceeding to biopsy. A high-quality, randomized control trial comparing a dd-cfDNA method of surveillance monitoring in comparison to traditional surveillance biopsy in heart and lung transplant recipients would be especially helpful to further validate its use. In addition, further validation of specific threshold values to indicate the presence of acute rejection would be beneficial. Based on the available evidence, it is reasonable to utilize dd-cfDNA for surveillance monitoring in select patient populations such as heart and lung transplant recipients at high risk for procedural complications from biopsy or during periods of time when the performance of surveillance biopsy is limited, such as during periods of the COVID-19 pandemic. Iwijn De Vlaminck: One promising feature of dd-cfDNA is that it is an early marker of graft injury. It’s therefore reasonable to integrate dd-cfDNA in surveillance testing and not just clinically indicated testing. There are practical limitations that need be overcome, however, including the current high cost of the assay. Michael Oellerich: Both approaches, fractional and absolute determination of dd-cfDNA in kidney transplant recipients, have strengths and weaknesses. Fractional determination has the advantage that it is less sensitive to preanalytical variables (e.g., DNA extraction efficiency). Measurements can be easily compared between different studies. Fractional determination is insensitive to changes in the rate of degradation of cfDNA in blood circulation. It has, however, the disadvantage that it is affected by changes in recipient cfDNA (e.g., by infection or exercise). Leukocytosis or leukopenia can alter dd-cfDNA fraction as recipient cfDNA accounts for the major part of the denominator in fractional quantification. This can result in false-negative or false-positive results. During long-term surveillance dd-cfDNA fraction increases due to a decrease of total cfDNA so that the threshold would have to be adapted. The decline of total cfDNA with time after kidney transplantation is presumably due to a decrease in apoptosis rate for white blood cells as immunosuppressant drug doses are tapered off. Absolute quantification (cp/mL) has the advantage that it is not affected by changes in recipient cfDNA by other factors and that there is no influence on the threshold due to total cfDNA decline over time. In clinical practice, the combination of fractional and absolute determination seems to provide the most comprehensive diagnostic information in kidney transplant recipients. Philip Halloran: Absolutely. The use of ratios is never desirable when both the numerator and the denominator can change. Michael Keller: Plasma dd-cfDNA is traditionally reported as a percentage, representing the fraction of donor to donor + recipient cfDNA. While this method may help account for differences between the relative sizes of recipients and donor allografts, there are inherent limitations to this approach. Due to its presence in the denominator, levels of recipient cfDNA in the plasma will influence %dd-cfDNA. Recipient cfDNA may rise in a number of conditions irrespective of the status of the allograft including in the setting of sepsis, multiorgan failure, extreme exercise, and leukocytosis. This may make the interpretation of %dd-cfDNA difficult under conditions that alter the levels of recipient cfDNA. It also raises questions surrounding the validity of specific threshold values of %dd-cfDNA in the detection and diagnosis of various types of allograft injury. Established threshold values for %dd-cfDNA may carry more weight if validated in stable controls and performed on otherwise stable outpatient transplant recipients. However, it may be difficult to interpret %dd-cfDNA in the setting of systemic illness or nonallograft related organ failure, such as in hospitalized patients. Measurements of the absolute amount of plasma dd-cfDNA are not influenced by recipient cfDNA. However, differences in the sizes of allografts—particularly in lung transplant where total tissue mass may differ between individuals to a considerable degree—may impact the ability to interpret prespecified threshold values for the detection of allograft injury. Observational cohort studies in kidney transplant patients have demonstrated similar performance characteristics between absolute and %dd-cfDNA for the detection of acute rejection. There are strengths and weaknesses of both methods, however, developing a diagnostic algorithm using a combination of the 2 methods may be the optimal approach. Iwijn De Vlaminck: It’s clear that a readout of the relative proportion of donor DNA can be confounded by changes in recipient DNA that are not related to the health of the transplant organ. The absolute burden of donor DNA in blood does not have that issue, and may therefore provide a more robust readout of transplant health, but this will need to be confirmed in clinical studies. Measurements of the absolute amount of donor DNA may prove more difficult to standardize and may be sensitive to changes in the rate of clearance of cfDNA from the blood circulation. Perhaps models that integrate both the absolute and relative burden of cfDNA will prove to be optimal. Philip Halloran: Surveillance: no for kidney, surveillance biopsies are not justified for kidney; yes for heart because histology kappa values are poor. Clinically indicated: cautious yes. The clinician needs the quantitative assessments that only molecular assessment can provide, although we need a probabilistic algorithm to guide the use of the information to avoid biopsy based on continuous numbers not cutoffs. Iwijn De Vlaminck: The molecular biopsy is a great technology that enables detailed profiling of different pathways of transplant rejection. It represents an important advance in transplant medicine, but because it relies on invasive tissue biopsies it has a limited role to play as a surveillance tool, and is more relevant for clinically indicated testing. An ideal test would achieve the resolution in cellular pathology of the molecular biopsy but from blood rather than from tissue. Angela Wu: Most of our work has been focused on monitoring the interactions between microbes and their hosts in a clinical setting, whether these microbes are pathogenic or not. We have found that in addition to monitoring immunosuppression, the metagenomic composition of “molecular biopsy” samples can also be highly informative as to the health status of the patient. When used in combination with dd-cfDNA, host-associated biomarkers, and other clinical measures, this metagenomic information can enhance the accuracy of diagnosis and outcome prediction. Based on our investigations, “molecular biopsy” results can be strongly affected by different practices at different centers. In particular, if considering additionally the microbial component, the environmental microbes can be hugely different at different sites, affecting analysis outcomes and downstream decision-making. To ensure robustness of the sample handling and results, it is preferable to perform sample processing at a central location. Additional molecular based methods for preserving sample integrity and reducing loss/degradation of cfDNA are also being developed and streamlined to improve the logistical challenges associated with sending samples to central facilities. Michael Oellerich: For routine implementation of dd-cfDNA testing there are a number of challenges. The laboratory requires specific instrumentation, such as a ddPCR or a next generation sequencing instrument as well as specialized laboratory professionals. Standard clinical chemistry assay validation is necessary for these tests and the development of external quality management programs is recommended. Furthermore, clinical practice recommendations have to be developed. Centralized reference laboratories may have the advantage of lower testing costs and high quality. Individual laboratories in transplant centers have the advantage of shorter turnaround times of dd-cfDNA test results for hospitalized patients if the test would have to be used for actual decision-making. For out-patients, services from centralized reference laboratories would allow for a broader A monitoring should be in clinical outcome reasonable the cost for dd-cfDNA monitoring would be relatively reducing graft it is to be that costs for a to or could be substantially using dd-cfDNA testing and other relevant approaches the molecular biopsy. Philip Halloran: if at The performance of molecular assays as has been in as shown by the measurement of DSA using and and (e.g., This is true when the results use algorithms for results on a different assays should generally be central the of and practices are and for on an ongoing Michael Keller: the assays and plasma dd-cfDNA should be performed in centralized to maintain assay reduce in results, and The and of the technology to the assays may its implementation in many transplant centers. in methods and assay also a when implemented at centers. While the assay at a centralized may increase turnaround this may not present an in several clinical such as surveillance monitoring or to Iwijn De Vlaminck: The molecular biopsy is a great technology that enables detailed profiling of different pathways of transplant rejection. It represents an important advance in transplant medicine, but because it relies on invasive tissue biopsies it has a limited role to play as a surveillance and is more relevant for clinically indicated testing. An ideal test would achieve the resolution in cellular pathology of the molecular biopsy but from blood rather than from tissue. Angela Wu: As our work has focused on the metagenomic of which both donor and microbes and Monitoring of microbial and in cfDNA has been demonstrated as a to immunosuppression adequacy and also the need for There are 2 major that can be further investigated in this detection and monitoring of specific microbes of and surveillance of microbial and as a for the For the it is that are and could be from donor to recipient the transplanted in some In this or detection with methods may be detection of cfDNA or from these of and lower cost. However, assays from other such as in the case of or to The of an between total cfDNA sequencing and can be further sequencing or sequencing that uses to potential by to specific could be a to ensure both specificity and In addition, current commercial assays which have relatively and long if cfDNA can be into the detection by also DNA this could also improve detection Michael Oellerich: dd-cfDNA may be helpful to assess necessary with and to provide information for optimization of It can guide in patients to immune of immunosuppressant is often necessary to and dd-cfDNA may be helpful to detect and improve immunosuppressant DSA development that is a risk factor for antibody-mediated rejection and graft loss in kidney concentrations were associated in patients with increased dd-cfDNA values immune dd-cfDNA is to drug monitoring that but is a of graft due to conventional have In kidney transplant recipients, for example, a of graft can be present by the time a rise in plasma is plasma is not specific for allograft injury. Due to a of validation studies for monitoring immune activity or expression of are not used. biopsies are clinically and associated with a major with dd-cfDNA seems to be useful in patients on who are at increased risk of complications from biopsies. dd-cfDNA is more practical than biopsies to detect in immune This is especially important in kidney transplant patients with high and high immune DNA is a diagnostic for monitoring transplant patients immunosuppression Philip Halloran: that they detect rejection. Monitoring would be and and these can be in indication biopsies by central molecular testing. dd-cfDNA active rejection, so in a that this a of Michael Keller: such as the and are generally to achieve concentrations in solid organ transplant recipients. Due to the of various factors and drug the doses to achieve these concentrations may vary between levels may provide adequate immunosuppression for some patients other patients may of acute rejection at the variability and in concentrations In a ability to detect of acute rejection as a marker of immunosuppression however, of the of immunosuppression is to the of acute rejection in the As a quantitative biomarker of underlying allograft dd-cfDNA may allow for the of to achieve a of immunosuppression that is individualized to the recipient and the of acute rejection. While promising in further studies are to and validate its role for this use. Iwijn De Vlaminck: no that quantitative of immunosuppression are in transplant dd-cfDNA, however, provides only an indication of that the immune cell of immune cells or the burden of in blood may provide a more readout of immunosuppression adequacy in the Angela Wu: studies of dd-cfDNA that focus not only on the of the DNA but also additional such as or microbial composition are all additional information that the utility of cfDNA as a for organ health, disease and immune In the combination of these approaches will allow to assay for monitoring and as well as There is no that the of these types of assays and assessments are informative and can improve but the of information needs to be with the clinical need and costs to be in a Additional that can this information from cfDNA at cost and with of will be to mass Michael Oellerich: dd-cfDNA will be helpful to achieve immunosuppression in transplant recipients with the potential to reduce graft loss. Additional is to of dd-cfDNA with other such as and as well as a gene to further improve diagnostic randomized clinical utility studies are necessary to show that dd-cfDNA changes in treatment will improve outcomes. Philip Halloran: The for the commercial assays is that values indicate probability of active rejection, and high values are due to active antibody-mediated rejection or T cell-mediated rejection are values are and resolution on such values should guide is we need more probabilistic information a result is the time in a patient with no and in a patient with for biopsy and suspicion of rejection. Michael Keller: dd-cfDNA is an with the potential to provide a of useful clinical including the detection of allograft an assessment of to treatment of acute rejection, monitoring of the adequacy of immunosuppression, and Further validation of its clinical utility and into routine clinical care may the of solid organ transplant into an of precision and care The with other molecular approaches demonstrates further that expression of such as molecular diagnostic may molecular that in the detection and discrimination of various types of allograft may also provide further into in which in dd-cfDNA are not by evidence of allograft injury. While the may be further is necessary to several to its interpretation and clinical Iwijn De Vlaminck: dd-cfDNA is a marker of graft tissue injury. 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