Acute graft rejection occurs in 30 to 45 percent of patients following heart transplantation, representing one of the highest rates among solid organ transplants. Patients recovering from heart transplantation surgery can expect a monthly routine endomyocardial biopsy to monitor signs of graft rejection, particularly in the first year post transplant.
While endomyocardial biopsy is considered the gold standard for diagnosis of acute rejection, it is both invasive and costly. It is clear that an alternative approach is needed to improve outcomes for patients and reduce the burden on the healthcare service.
An end to invasive procedures
The current standard of care for post-transplant patients involves endomyocardial biopsies and HLA donor-specific antibody testing. It can, however, be difficult to correlate the development of donor-specific antibodies post transplant to a clinical picture of rejection.
Donor-derived cell-free DNA (dd-cfDNA) has been evaluated as a potential early marker of allograft injury and poses a real alternative to invasive procedures.
What is dd-cfDNA?
dd-cfDNA refers to DNA fragments originating from the transplanted organ that are released into the recipient's bloodstream – primarily due to apoptosis and, to a lesser extent, necrosis during acute injury. These fragments are typically short, at 160 to 180 base pairs, and have a short half-life, ranging from 30 minutes to a few hours, providing a biological snapshot of the body’s cellular activity.
In a stable and well-functioning allograft, there is a baseline level of cellular turnover resulting in minimal release of dd-cfDNA. The exact baseline fraction differs depending on a variety of factors including graft type, recipient body size and BMI, and time since transplant. Recipient age can also have a bearing, though pediatric dd-cfDNA levels are not well documented.
A low dd-cfDNA level is reassuring, while an elevated level signals something happening that warrants further investigation. Observational studies have demonstrated that the dd-cfDNA has a high negative predictive value of 97 percent, making this an ideal rule-out test, without subjecting patients to an invasive biopsy.
The complexity comes with defining an abnormal dd-cfDNA result. Definitions differ across laboratories and transplant programs, where different cut-offs have been used in trials to improve specificity and sensitivity to detect rejection. Further work is required to standardize this.
The patient and clinical case
Sarah Peacock – consultant clinical scientist at Addenbrooke’s Histocompatibility and Immunogenetics (H&I) lab – understands the value of switching from invasive catheter lab procedures to a simple blood test. Together with Royal Papworth Hospital, she is developing a pilot study investigating the use of dd-cfDNA for post-transplant heart monitoring.
“The pivot to a minimally invasive blood test would remove the need for patients to undergo monthly invasive endomyocardial biopsies,” explains Peacock. “Blood draws could eventually take place at patients’ local GP surgeries – an approach that is supported by both consultants and patients at Royal Papworth.”
The lab workflow
Peacock's study highlights the importance of using a high-quality cell-free DNA extraction method. Currently, dd-cfDNA assessments generate a percentage figure representing the donor-derived fraction. To calculate this, they either use pre-transplant samples or make assumptions about which DNA markers originate from the donor versus the recipient. To ensure the accuracy of this final percentage, samples must be free from contamination with high molecular weight DNA.
For the Royal Papworth study, the researchers are using the Promega Maxwell extraction instrument in conjunction with the Maxwell Rapid ccfDNA extraction kit, which they found beneficial due to the speed of the extraction, simple setup protocol and small footprint of instrument.
Of the various analytical methods available for measuring dd-cfDNA, Peacock's study uses next-generation sequencing (NGS), which enables highly sensitive discrimination of donor-derived fragments using targeted or genome-wide approaches suitable for scalable, high-throughput testing. SNP- and indel-based panels will be used to report a fractional percentage of dd-cfDNA.
Barriers to adoption
“The key barriers to adoption of dd-cfDNA are financial and structural rather than scientific, together with the lack of a framework for quality assurance,” says Peacock. The utility of dd-cfDNA in the UK's National Health System (NHS) is currently under review by the National Institute for Health and Care Excellence (NICE). The NICE appraisal involves weighing up cost-effectiveness and patient benefits.
“The challenge in financial assessment here is that each NHS trust operates independently,” explains Peacock. “The potential savings need to be understood at a whole-system level, rather than attributed to individual departments, which is difficult when NHS finances are already under significant pressure.”
Studies published in the literature have mainly used dd-cfDNA results generated in centralized vendor-led laboratories. However, it is essential that decentralized dd-cfDNA testing, within the framework of quality assurance, occurs within local laboratories to determine analytical and clinical validity.
Peacock believes the change in government has brought some hesitancy around innovation due to the change in direction within the NHS. “That said, the clinicians are enthusiastic, patients are supportive, and the expertise is there,” she adds. “The key hurdles are simply getting the finances approved and establishing a robust quality assurance framework. Once the service is up and running, samples could be processed at Addenbrooke from other centers across the country. That would reduce costs due to the larger sample volumes, and batching for downstream testing would significantly reduce the cost per sample.”
The path forward
Despite widespread acceptance of dd-cfDNA as a biomarker for allograft injury in the US and Europe, the UK has not yet included it in clinical guidelines. Sarah believes this is due in part to financial barriers, and if these were removed, dd-cfDNA testing could become part of routine cardiac transplant follow-up care.
Literature supports this approach, and clinicians are enthusiastic both locally and at other centers. The H&I labs are well-positioned to take this forward, given the existing equipment, infrastructure, and knowledge of each patient's pre-transplant status and donor details.
Much of the existing data on dd-cfDNA has come from the United States, from testing carried out in centralized commercial laboratories. Because this is vendor-led research, it needs to be treated with some caution. If more data could be generated directly from UK H&I centers, it would likely give NHS decision-makers greater confidence that this technology could work within the NHS model.
The Royal Papworth pilot study represents an important step towards establishing a UK evidence base for dd-cfDNA testing, independent of the vendor-led data that has dominated the field to date. With clinician and patient support already in place, and the necessary laboratory expertise and infrastructure available, the scientific case is strong.
If in addition to this, the ongoing NICE evaluation can demonstrate both the patient benefits and the system-level cost savings dd-cfDNA testing could realistically become a routine part of cardiac transplant monitoring across the NHS – sparing hundreds of patients from unnecessary invasive procedures each year.
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