Cover story
OF TRANSPLANT
REJECTION
Researchers are uncovering and testing biomarkers to help guide treatment and improve long-term outcomes for organ transplant patients CELIA HENRY ARNAUD, C&EN WASHINGTON
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n TV medical dramas, a common suspenseful story line follows a person in need of an organ transplant waiting to find a donor. A happy ending ensues when the organ arrives, packed in ice and ready for surgical insertion. To be sure, organ transplants save lives. More than 33,000 organs were transplanted in the U.S. in 2016, the most recent year for which data are available, according to the United Network for Organ Sharing. In the real world, though, after a transplant, a recipient’s immune system fights against the organ, recognizing it as foreign. As many as 15% of people receiving kidney transplants, for instance, experience acute rejection, meaning that the immune system sends in its troops and causes inflammation within the first year. For those patients, the story line isn’t tied up neatly.
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Rejection episodes like these are usually treatable and reversible. So “rejection” doesn’t necessarily mean the transplanted organ—or graft, as doctors like to call it— is lost. Tissue damage can occur during these episodes, however, and if it happens enough times or is not treated quickly enough, the organ fails. To ensure prompt treatment, physicians need a way to monitor organ function and diagnose rejection. The only method available to them now is direct biopsy of the transplant. In the case of a kidney transplant, the typical schedule for routine monitoring is to take biopsies of the organ at one week, one month, three months, six months, one year, and yearly after that. Rather than poke a needle into an organ to sample cells and tissues—a risky venture—doctors would prefer to noninvasively measure molecular markers in a person’s blood and urine to monitor recovery. These markers, typically nucleic acids and proteins, could help indicate when a transplanted organ has started to fail, help guide treatments, minimize the number of biopsies needed, and ultimately improve transplant success rates.
The anatomy of rejection Not all transplant rejection is the same. It can be divided into two major categories, distinguished by the mechanism through which it occurs and to a lesser extent by the length of time since transplant. Acute rejection typically results from immune cells called T cells infiltrating the organ and causing local inflammation that damages the organ. To stave off acute rejection, patients take drugs that suppress immune function. If an episode happens anyway, it can be treated with
high doses of steroids. Acute rejection episodes typically occur in the first year after transplant, but if a recipient stops taking the immunosuppressants, they can occur months or even years after the transplant. Chronic rejection, in contrast, is primarily an antibody-mediated process. During chronic rejection, the person’s immune system continually attacks the transplanted organ. Eventually the damage builds up enough that the organ stops working. A patient experiencing chronic rejection doesn’t display symptoms until the situation is too advanced to do anything about it. No treatment for the condition yet exists. Biomarkers that indicate how a person’s immune system is functioning could help guide doctors in prescribing the correct dose of immunosuppressants for individual transplant patients to help ward off acute rejection. And they might someday help develop a treatment for chronic rejection. “Most transplants are treated the same,” says Peter S. Heeger, a transplant immunologist at Icahn School of Medicine at Mount Sinai. At most centers, he says, patients receive therapy to dial down the immune system at the time of the transplant. After the transplant, they receive multiple immunosuppressant drugs. “Because we don’t have a tool to measure immune function, we usually give all patients the same amount of medication,” says Jamil R. Azzi, an expert in kidney transplants at Brigham & Women’s Hospital and Harvard Medical School. Physicians typically “wait six months and slowly lower the dose of one of the drugs. They don’t really measure anything. They just wait and see what happens,” Heeger says. Such an approach means that some people receive more drug than they need,
In brief Thousands of organs are transplanted every year. Those organs are constantly under attack from the body’s immune system. Although short-term outcomes are good, long-term survival of transplanted organs remains below 60%. Molecular biomarkers in blood and urine can provide physicians with information about immune status and organ function. But physicians don’t yet know whether making treatment decisions on the basis of those biomarkers will improve outcomes.
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that we’re on the right path,” she adds. “By the time the creatinine is up, a few whereas others receive less. Both of these Mas’s team is hunting for biomarkweeks, months, or years have passed, and options can have negative consequences. ers that signal immune status and graft now you have a severely scarred kidney,” Too much drug suppresses the immune response to injury. The researchers are Azzi says. Just as a fire can smolder and system too much, leaving a patient susestablishing a composite score calculation burn without producing a flame, there can ceptible to infections. Side effects of that includes clinical characteristics such be “smoldering rejection that we’re not overprescribing immunosuppressants detecting with current biomarkers, leading as donor age and recipient sex along with are a major factor in the premature death messenger RNA and microRNA molecular to chronic damage,” he says. of transplant recipients. At the other exmarkers that can be used to determine Because creatinine has been the best treme, too little drug can pave the way for biomarker available for kidney dysfunction whether the organ is functioning properly. rejection and organ loss. Physicians want “The composite score represents a holistic for the past few decades, “we’re not makto avoid both of these situations and tailor approach rather than a single marker,” ing the progress that other fields like canthe amount of immunosuppressants that Mas says. “The human transplant model is cer are making,” Mas says. That field has patients receive. been more successful in identifying appro- too complex to assume that a single bioBut the only way that physicians currently have for determining whether they should priate biomarkers.“But now I’m optimistic marker will provide accurate information about the graft status.” adjust a patient’s drug dose is to One of the furthest advanced take a biopsy. “There are complipanels of RNA biomarkers cations that come from putting comes from the lab of Manikkam a needle into an organ with lots Biomarkers need to go through many stages before Suthanthiran at Weill Cornell of vessels,” Azzi says. Beyond the they can be used in clinical practice. In the case of Medical College. The scientists risk of bleeding, there’s the time, transplant rejection, candidate biomarkers are still there test for acute rejection in expense, and patient anxiety to waiting for prospective clinical trials. Ater those kidney transplant patients by contend with, he says. trials, the biomarkers will still need to go through quantitatively measuring the ex“We’re hoping for a nonincommercialization and inal implementation. None of pression of genes in cells isolated vasive test that we can do frethe transplant biomarkers have yet reached that stage. from the recipients’ urine. quently,” Azzi says. “If there’s The team carried out a mulany signal with this test showing PHASES TIMELINE ticenter trial with a set of eight higher probability for ongoing In the past, discovery genes (N. Engl. J. Med. 2013, rejection—even mild—then we Discovery took five to 10 years. DOI: 10.1056/NEJMoa1215555). A biopsy.” Biomarkers that signal (Screen tissue, blood, Newer technologies statistical analysis of the data rerejection could offer such a and urine samples for allow it to happen in vealed that the scientists could diagnostic. biomarker candidates) months to several years. use the expression levels of as “With the advancement of few as three of the genes to caltechnology, we envision that culate an acute rejection score. we’ll be able to develop biomarkThey used the score to corers that not only tell us the diagExisting samples from rectly identify patients who nosis but also the severity of the patient cohorts with Validation went on to have an acute redisease,” Azzi says. known end points allow (Test to ensure jection episode. “About 20 to validation to happen biomarkers agree 40 days before rejection, you quickly. In the absence across platforms can see that the score starts to of such samples, and among go up,” Suthanthiran says. “The Current biomarkers fall short multicenter trials need research groups at molecular rejection seems to of that goal. For example, in the to be performed, which multiple centers) precede the clinical rejection.” case of kidney function, physican take several years. The biomarker panel could cians for decades have been limsave patients from having united to creatinine as a biomarker. necessary biopsies. In the future, Inside the body, the level of creSuthanthiran wants to conduct atinine—a breakdown product of trials to see whether the informuscle—remains fairly constant. Clinical trial mation from the biomarkers can It’s the kidneys’ job to filter (Conduct be used to make decisions about the chemical from the body, so prospective, the amount of immunosuppreschanges in the amount of creatiCan take several years. multicenter trials in sants a patient receives. nine in urine can signal problems which biomarkers Azzi’s team is assessing with the organs’ function. Valeria are used to make whether extracellular vesicles Mas, director of the Translational treatment decisions) in urine called exosomes can Genomics Transplant laboratory be used to detect kidney transat the University of Virginia, plant rejection (ACS Nano 2017, says creatinine is an inaccurate DOI: 10.1021/acsnano.7b05083). biomarker. Yes, it can be detectThese exosomes are shed by ed noninvasively in a patient’s Commercialization cells and therefore reflect the urine without doing a biopsy. Unknown and content and constituents of But its levels increase only after implementation those cells. For example, the major damage to the kidneys has protein CD3, a cell surface reoccurred.
The long road to transplant biomarkers
Today’s biomarkers
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ceptor, is unique to T cells. If CD3 appears in exosomes in urine, T cells have infiltrated the transplanted kidneys, Azzi says. “This means you have T cells attacking the kidney” and acute rejection has begun. Azzi plans to use exosomes to identify other proteins or genes that can be combined in a biomarker panel to monitor the organ. “We envision not only diagnosing rejection. We want to diagnose different kinds of rejection. We want to diagnose the severity of rejection. We want to diagnose the state of the transplanted kidney itself.”
Transplant stats The odds for long-term transplant success remain stubbornly low, as shown for some of the most-transplanted organs.
33,610 Number of all organ transplants in the U.S. in 2016
In search of validation One hurdle to identifying protein-based biomarkers in tissue samples is that the groups working in this area use different proteomics methods to carry out the screening, says Katrin Kienzl-Wagner, a surgeon at Medical University of Innsbruck who also conducts research to identify biomarkers of transplant rejection. “Everybody is applying a slightly different experimental setup, so it makes it difficult to compare data from different groups,” Kienzl-Wagner says. “Maybe the biggest hurdle is that the patient populations in the proteomics studies that have been done so far are rather small. If you really want to validate a marker, you need large study cohorts, which would mean that all transplant centers from Europe or the U.S. or even worldwide would have to do the same experiment.” Most biomarkers are being identified from gene expression profile or peptide-based proteomics studies. Neil L. Kelleher, a mass spectrometrist at Northwestern University, is instead working with doctors at Northwestern’s Comprehensive Transplant Center to identify proteoforms in blood that can serve as biomarkers of acute rejection. A proteoform is a specific molecular form of a protein produced from a human gene. For example, a proteoform could be a protein that’s had a particular functional group, added to it—a posttranslational modification. Or it could be a protein with a slight sequence variation compared with the gene from which it was produced. These seemingly small differences can dictate significant changes in protein function and correlate strongly with organ rejection. But they can be lost during the processing steps of conventional proteomics methods that involve digesting whole proteins into peptides. So Kelleher’s team searches for proteoform biomarkers with so-called top-down proteomics methods, which allow the researchers to work with intact proteins.
19,060 7,841 55% 56% Number of kidney transplants in the U.S. in 2016
Number of liver transplants in the U.S. in 2016
Proportion of kidneys transplanted in the U.S. in 2006 that survived at least 10 years
Proportion of livers transplanted in the U.S. in 2006 that survived at least 10 years
3,191 58%
Number of heart transplants in the U.S. in 2016
2,327 30%
Proportion of hearts transplanted in the U.S. in 2006 that survived at least 10 years
Proportion of lungs transplanted in the U.S. in 2006 that survived at least 10 years
Number of lung transplants in the U.S. in 2016
Sources: United Network for Organ Sharing, Scientific Registry of Transplant Recipients JANUARY 29, 2018 | CEN.ACS.ORG | C&EN
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To identify potential biomarkers, Kelleher and his student Timothy Toby isolated immune cells from the archived frozen blood samples of liver transplant recipients. They’re interested in proteins that have different forms in healthy transplant patients versus those undergoing rejection, Kelleher says. Josh Levitsky, a liver transplant physician at Northwestern’s Comprehensive Transplant Center who collaborates with Kelleher, emphasizes that they’ve been using frozen but still viable immune cells. “We’re seeing what proteins and proteoforms are being produced at the time of rejection,” he says. “It feels most representative of what’s going on physiologically.” They found that most of the proteoforms expressed at different levels in healthy transplant patients and in patients who had experienced acute rejection were variants that differed from their expected sequence. The most significant of the proteoforms were variants of a protein known as CXCL4, which is thought to play roles in inflammation and wound repair. The variants were more abundant in healthy recipients, suggesting they might be protective (Am. J. Transplant. 2017, DOI: 10.1111/ajt.14359). Rejection biomarkers are typically identified for a specific organ type. But by doing a meta-analysis of eight independent data sets collected from transplant patients, Minnie Sarwal, now at the University of California, San Francisco, and coworkers identified a common rejection module, or CRM: a set of 11 genes whose expression is elevated during acute rejection in four organ types—kidney, lung, heart, and liver (J. Exp. Med. 2013, DOI: 10.1084/jem.20122709). One drawback is that the data sets came from tissue biopsy analysis rather than blood or urine analysis, so the CRM might not ultimately apply to the type of diagnostics doctors seek. Still, when the researchers tested their CRM against a set of kidney tissue samples, they found that the samples from organs that had been acutely rejected correlated with a higher CRM score (PLOS One 2015, DOI: 10.1371/ journal.pone.0138133).
Driving toward the clinic Researchers agree that a major hurdle to getting transplant biomarkers to the clinic is a lack of funding. A perceived lack of funding notwithstanding, the U.S. National Institutes of Health is actually supporting clinical trials through the Clinical Trials in Organ Transplantation,
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or CTOT, consortium. Sponsored by the National Institute of Allergy & Infectious Diseases, CTOT has a mission to run studies that improve short-term and long-term outcomes for transplant recipients. CTOT, established in 2004, is now in its third funding cycle. So far, CTOT has doled out more than $135 million. Since its inception, the consortium has undertaken 21 clinical trials, 12 of which are now closed, eight of which are currently active, and one of which is in development. “Part of the goal is to identify reliable biomarkers and then, once you have them,
And one of the biggest needs is improving the long-term outcomes. Short-term organ-survival rates for all organs have improved, but 10-year survival rates have remained stubbornly stuck below 60%. For lung transplants, that number is lower—closer to 30%. To try to bring those survival rates up, the University of Virginia’s Mas wants researchers to focus less on acute rejection and more on the process that leads to chronic rejection. Even though doctors prescribe strong immunosuppressants, “the immune system always finds a way to
“It’s hard to do studies that are 10 years long.” —Peter S. Heeger, transplant immunologist, Icahn School of Medicine at Mount Sinai to use them to treat people on the basis of the presence or absence of the biomarker,” says Mount Sinai’s Heeger, who is a member of CTOT’s steering committee. For example, Suthanthiran’s gene panel was tested in a CTOT trial. And the CRM developed by Sarwal and colleagues will be tested as a biomarker panel in future NIH-funded clinical trials that are currently in the planning stages, says Tara K. Sigdel, a member of Sarwal’s group. The aim of CTOT’s studies varies depending on the transplanted organ being considered. For instance, the outcomes for lung transplants are significantly worse than for other transplants, Heeger says. “We have to understand and fix that,” he says. With that goal in mind, one CTOT trial is an observational study of about 800 lung transplant patients. The study is collecting blood and biopsy samples, as well as fluid obtained from rinsing lung tissue. “They’re looking for what are the best markers and trying to understand why the organs are rejected,” Heeger says. But ultimately, physicians need biomarkers that can improve outcomes. For that to happen, trials need to be done in which some patients are treated on the basis of the results of biomarker tests and other patients receive the current standard of care. “We now have a series of biomarkers that seem to be reproducibly able to detect acute injury to the transplant,” especially for the kidneys, Heeger says. “What we need to do now is design studies to test whether treating a patient based on those biomarkers is going to change the outcomes.”
respond to the graft, even when we cannot see any clinical manifestation.” The focus needs to be on avoiding that immune response that doctors aren’t seeing clinically but is leading to chronic rejection, she adds. “Monitoring the graft is going to be the solution.” Heeger agrees that more attention needs to be paid to long-term outcomes, but “it’s hard to do studies that are 10 years long,” he says. Instead researchers need a surrogate end point. “Let’s say I want to know who’s going to lose their graft in 10 years and then identify a biomarker that’s going to tell me that. I can’t wait 10 years to find out.” But if researchers can find a marker at the two-year point that accurately predicts what will happen after 10 years, they may be able to adjust treatment to ward off graft loss. But the hurdle—and perhaps a reason that there have been so few studies—is that even with a biomarker for chronic rejection, which is usually antibody based, no good treatments exist, Heeger says. “We don’t have good drugs to ward off antibody responses,” he adds. “And people who develop antibodies often end up doing worse.” As eager as he is for biomarkers to succeed, Azzi says that as a physician he won’t use any biomarker unless he believes that it has predictive value and will improve outcomes. “I need more data to do that,” he says. “We have our work cut out for us,” Heeger says. “It’s always nice to identify new things, but we’ve already identified and validated a number of biomarkers. It’s time to determine if acting on them improves outcomes.” ◾
