Dr. John Friedewald serves as Medical Director of Kidney Transplantation at Northwestern Memorial Hospital and is a Professor of Medicine and Surgery at Northwestern University Feinberg School of Medicine. As a transplant nephrologist, Dr. Friedewald is interested in improving the outcomes and management of patients receiving a kidney transplant.

After obtaining a medical degree from Columbia University College of Physicians & Surgeons, Dr. Friedewald completed an internal medicine residency and nephrology fellowship at Johns Hopkins Hospital. He’s a three-time recipient of the Feinberg Teaching Award, which recognizes outstanding faculty members at the Northwestern University Feinberg School of Medicine.

On November 16, 2023, Dr. Friedewald participated in a Eurofins Transplant Genomics webinar titled “Using Gene Expression Profile Biomarkers as an Immunosuppression Guide in Post-Transplant Care.” Along with Dr. Ram Peddi of California Pacific Medical Center and Dr. Michael Marvin, a transplant surgeon at Geisinger Medical Center, Dr. Friedewald reviewed several case studies involving the use of gene expression profile tests to help inform immunosuppression management.

The Need for Novel Biomarkers in Transplant Care

Surgeons performed more than 40,000 organ transplants in 2022, giving patients of all ages and backgrounds new leases on life. Although transplantation has immediate benefits, there’s always a risk of rejection, prompting researchers to look for novel ways to manage immunosuppression.

In the first 35 years of her life, Amy Silverstein received two heart transplants. The first one failed due to rejection, while the second led to her premature death due to metastatic lung cancer. Silverstein believes that she developed cancer after long-term use of the steroids, antimetabolites, and calcineurin inhibitors used to prevent rejection.

Writing about her experience for The New York Times, Silverstein explains, “Organ transplantation is mired in stagnant science and antiquated, imprecise medicine that fails patients and organ donors…Standing on the edge of death now, I feel compelled to use my experience in the transplant trenches to illuminate and challenge the status quo.”

The Challenges of Immunosuppression Management

Many researchers share Silverstein’s views on the need to balance the risks and benefits of anti-rejection therapy, including Dr. Friedewald. During the webinar, he described it as a “Goldilocks problem” of too much immunosuppression, not enough immunosuppression, or just the right amount of immunosuppression.

He compares immunosuppression to keeping a car in the middle of the road. With too much immunosuppression, the car may veer right. Too little immunosuppression, and the car veers left and heads toward rejection. Dr. Friedewald acknowledges that transplant professionals still don’t have great tools to determine if patients are “driving off the road.”

Subclinical Disease Detection

Part of the problem is that with current monitoring tests, renal allograft damage often doesn’t become evident until the patient’s serum creatinine level increases, resulting in a corresponding decrease in their glomerular filtration rate. Therefore, it’s extremely important to look for signs of subclinical rejection before significant, irreversible damage occurs.

When treated, patients with subclinical T-cell mediated rejection have outcomes similar to those of patients without rejection, but that doesn’t mean TCMR is unimportant. Clinicians can and should work to detect and treat it. Fortunately, subclinical rejection has been getting more attention in the literature, with multiple journals publishing studies related to TCMR detection and the long-term outcomes of patients with TCMR.

In these studies, patients with subclinical rejection experienced the following outcomes:

More de novo antibodies
Worse fibrosis on a 2-year biopsy
Decreased kidney function
More episodes of clinical rejection
Worse graft survival at 5 years

Biomarker Discovery and Development

Oncologists have long understood the importance of detecting subclinical disease. After all, no one would suggest delaying treatment for breast cancer until after it metastasizes. In the transplant field, however, not everyone believes in subclinical rejection, which has delayed progress in developing better options for immunosuppression management.

During the webinar, Dr. Friedewald introduced convincing evidence that subclinical rejection identified by surveillance biopsies is linked to poor outcomes in as little as 2 years after a transplant. He’s been working with gene expression profile biomarkers to distinguish normal tissue samples from samples showing signs of subclinical rejection.

Gene Expression Testing

Gene expression tests look for patterns in thousands of genes, which helps researchers understand how cells are functioning at a specific point in time. In Dr. Friedewald’s clinical trial, researchers examined tissue samples from surveillance biopsies and labeled each one TX (“Transplant eXcellent”) or SubAR (“Subclinical Acute Rejection”).

TX: The biopsy was normal, and the patient had normal/stable kidney function.
SubAR: The biopsy showed signs of pathologic rejection, and the patient had normal/stable kidney function.

After classifying the tissue samples, researchers developed the TruGraf® assay to create a reference population. TruGraf is a minimally invasive test designed to measure the levels of differentially expressed genes in the blood of kidney transplant recipients. In the case of TruGraf, it’s 120 genes that are either upregulated or downregulated between the TX and SubAR groups, making it easier to identify patients who are likely to be adequately immunosuppressed.

Test Validation

Once the research team discovered the target gene set, they locked the relative weights of each gene, making it possible to compare any new sample to the reference population. To avoid unnecessary surveillance biopsies, they set the negative predictive value between 80% and 90%, which corresponds to a very low likelihood of rejection in patients with TX samples.

TruGraf achieved a positive predictive value of nearly 50%, or double the PPV of doing random biopsies, specificity of 75% and 71%. A higher PPV allows clinicians to make better decisions about which patients to biopsy and when.

Donor-Derived Cell-Free DNA (dd-cfDNA) in Transplant Monitoring

Donor-derived cell-free DNA (dd-cfDNA), or DNA fragments that originate from cells undergoing injury and cell death, is also useful for monitoring transplant patients. It degrades into nucleosome units containing approximately 166 bases before it’s filtered from the blood by the kidneys and liver. Dd-cfDNA also has a short half-life — around 30 minutes.

Using rapid genotyping with the Transplant Rejection Allograft Check® (TRAC), it’s possible to distinguish donor cfDNA (“donor-derived cfDNA”) from host/recipient DNA. If the ratio of donor-derived cfDNA to host cfDNA increases, it signals injury to the graft. In Dr. Friedewald’s clinical trial, dd-cfDNA performed about as well as TruGraf, achieving a PPV of 56% and an NPV of 84%. When combined, TruGraf and dd-cfDNA performed better than either test alone, with PPV increasing to 81% and NPV increasing to 88%.

Clinical Applications and Case Studies

After presenting data from his clinical trial, Dr. Friedewald discussed the applications of using TruGraf in combination with dd-cfDNA.

Case Study #1

The first case involved a 65-year-old man with diabetes who underwent a living unrelated transplant 13 years ago. He was on tacrolimus/MMF immunosuppressive therapy and had a history of the following:

Former smoker
Squamous cell carcinoma 4 years ago
Tacrolimus levels ranging from 6 to 8
Nagging skin cancers
Serum creatinine of 1.1
No proteinuria

The patient had a TruGraf result of TX and a TRAC result of 1.1%. Dr. Friedewald decided to increase his MMF dosage from 500 milligrams twice per day to 750 milligrams twice per day and repeat the cfDNA test in 1 month. After 30 days, the man still had a TruGraf result of TX, and his cfDNA level decreased to 0.28%.

Approximately 6 months later, the man had a positive biopsy on one of the lymph nodes in his neck. Dr. Friedewald decreased his MMF dosage and adjusted the goal for his tacrolimus level. In February 2023, the man had a TruGraf result of Not-TX, prompting another adjustment of his immunosuppressive regimen. Repeat testing showed a TruGraf TX result and a downward trend in his dd-cfDNA level.

Case Study #2

The second case study focused on a 24-year-old woman who received a transplant from a deceased donor about 10 years ago and had no history of rejection. Her creatinine remained stable at 1.0 to 1.2 milligrams per deciliter. At one point, she developed viral infections, prompting a reduction of her tacrolimus target and daily MMF dosage.

After the medication adjustment, the patient expressed concern about her creatinine level, which had increased from 1.1 to 1.2. Dr. Friedewald recommended TruGraf and TRAC to look for signs of subclinical rejection. She had an elevated TRAC level of 4% and a TruGraf result of Not-TX. Due to the high PPV of both tests together, she underwent a biopsy to determine the extent of any ongoing rejection.

The biopsy showed chronic active antibody-mediated rejection with transplant glomerulopathy and interstitial inflammation. Oddly, she had no significant proteinuria. Dr. Friedewald’s team decided to administer IVIG with the hope of treating the rejection and the viral infections at the same time. After changing mycophenolate to sirolimus, they repeated the TRAC and TruGraf tests at 4 weeks and 8 weeks.

Unfortunately, this course of treatment was complicated by another viral respiratory infection and severe thrombotic microangiopathy. Once she recovered, she resumed immunosuppressive therapy with a low dose of tacrolimus, Belatacept, Eculizumab, a low dose of MMF, and a low dose of prednisone. Her next TruGraf test showed a TX result, and her TRAC decreased to 0.6%, which is below the threshold of positive. Since then, her creatinine has stabilized, and she’s doing well with the current therapy.

Using Biomarkers to Guide Treatment Adjustments

Biomarker testing is noninvasive, so it’s useful for understanding a patient’s risk of acute rejection. Based on the results of TRAC and TruGraf testing, clinicians may be able to adjust immunosuppressive regimens to prevent subclinical rejection from progressing.

The Future of Biomarker Application

The future of biomarker testing is bright, especially in the field of transplant medicine. Ideally, researchers will continue to identify urine and blood biomarkers capable of detecting changes at the molecular level. Using such biomarkers, it may be able to make accurate predictions regarding the likelihood of rejection or develop customized treatment regimens for transplant patients.

For now, more clinical trials are needed to collect data and determine the reliability of biomarker testing for these purposes. Researchers also need to educate healthcare professionals on the use of these tests and their potential to improve patient outcomes.Transplant Genomics aims to improve transplant outcomes for recipients through noninvasive treatment options. Learn more today.