Author + information
- Mats Hidestrand, PhD,
- Aoy Tomita-Mitchell, PhD,
- Pip M. Hidestrand, MD,
- Arnold Oliphant, PhD,
- Mary Goetsch, MS,
- Karl Stamm, MS,
- Huan-Ling Liang, MD,
- Chesney Castleberry, MD,
- D. Woodrow Benson, MD, PhD,
- Gail Stendahl, DNP,
- Pippa M. Simpson, PhD,
- Stuart Berger, MD,
- James S. Tweddell, MD,
- Steven Zangwill, MD and
- Michael E. Mitchell, MD∗ ()
- ↵∗Children’s Hospital of Wisconsin, 9000 West Wisconsin Avenue, MS B730, Milwaukee, Wisconsin 53226
To the Editor:
Approximately 20,000 cardiac transplant recipients currently reside in the United States. Rejection remains a major cause of graft failure and requires lifelong surveillance. The current gold standard for monitoring rejection is catheter-based endomyocardial biopsy (EMB), which is associated with risk and expense (1). Donor-specific cell-free deoxyribonucleic acid (DScf-DNA) has been proposed as a marker for cellular injury caused by rejection (2). Shotgun whole-genome sequencing (WGS) has been used to detect DScf-DNA (3). The complexity and cost of the analysis required by WGS limits its application as a surveillance tool. We have employed targeted quantitative genotyping to determine the %DScf-DNA. The targeted approach relies on selected highly-informative genomic regions and potentially provides a rapid (24 to 48 h) cost-efficient (significantly less than whole genome sequencing) method suitable for clinical surveillance. We applied this method to detect DScf-DNA in pediatric cardiac transplant recipients in a prospective blinded study.
Cardiac transplant recipients followed at the Herma Heart Center at the Children’s Hospital of Wisconsin were invited to participate. Blood samples (5 ml) were collected in 3 clinical scenarios:
1. Scheduled surveillance EMB from asymptomatic heart transplant recipients in the catheterization laboratory immediately before scheduled surveillance EMB.
2. Unscheduled diagnostic EMB from symptomatic heart transplant recipients before unscheduled diagnostic EMB.
3. Rejection from heart transplant recipients with biopsy- proven rejection (> The International Society for Heart and Lung Transplantation grade 2R or antibody mediated rejection 1) before initiation of treatment, during treatment, and at 1 week.
Clinical, laboratory, cardiac catheterization, and echocardiographic data were recorded. Anticoagulated blood was collected to measure cell-free deoxyribonucleic acid (cf-DNA) levels. Plasma separation, cf-DNA extraction, and quantification of total cf-DNA were carried out as previously described (4). Genomic DNA for genotyping was prepared from 1 buffy coat of each recipient, and donor DNA was obtained from the Blood Center of Wisconsin. Determination of the %DScf-DNA in recipient plasma was performed using Digital Analysis of Selected Regions (DANSR, Ariosa Diagnostics, San Jose, California) (5). Genotyping of donor and recipient genomic DNA was carried out by the same assay. Loci are informative when recipient genotypes are homozygous and donor genotypes are either heterozygous or homozygous for the other allele. The minor allele frequency for informative loci was modeled as a binomial distribution. The %DScf-DNA was defined as the peak from this modeling. Summary statistics included median and range. Unpaired samples (i.e., rejection group vs. surveillance group) were compared using a Mann-Whitney U test. Rejection samples were compared with a Friedman analysis of variance. A Pearson correlation summarized correlations. A p value <0.05 was considered significant.
Fifty-three samples from 32 patients were analyzed.
The cf-DNA levels were obtained from 26 patients undergoing 38 scheduled surveillance EMBs (Fig. 1A). Thirty-two (84%) samples contained <1% DScf-DNA. No patient with DScf-DNA <1% had pathological rejection. DScf-DNA levels exceeded 1% in 6 samples, and the highest percentage of DScf-DNA was associated with asymptomatic biopsy-proven rejection. The remaining 5 samples had negative biopsies.
Seven samples were obtained from 6 patients before unscheduled diagnostic EMB to rule out rejection based on clinical criteria (Figs. 1B and 1D). Six samples had DScf-DNA levels >1%, and 1 sample contained DScf-DNA <1%. Four of the 6 were associated with biopsy-proven rejection; the other 2 patients had significant coronary artery vasculopathy on angiography. The single symptomatic patient with a low percentage of DScf-DNA had high levels of total cf-DNA (Fig. 1D), implying that the dominant pathology was global rather than confined to the donor organ. This patient was diagnosed with culture-positive sepsis, the accompanying EMB was negative for rejection, and coronary angiography was normal.
Four patients with biopsy-proven rejection were analyzed. All pre-treatment samples collected at diagnosis contained DScf-DNA >1% (sensitivity 100%). Following intravenous immunosuppressive therapy, all patients demonstrated significantly decreased %DScf-DNA. Interestingly, 3 to 4 days after discontinuing augmented immunosuppression, the %DScf-DNA rebounded in 3 of 4 patients (Fig. 1C). The DScf-DNA was compared with other candidate noninvasive laboratory variables (B-natriuretic peptide, troponin, and C-reactive protein) as well as echocardiographically determined left ventricular ejection fraction in predicting rejection on biopsy; DScf-DNA had the highest sensitivity and specificity (100% and 84%, respectively).
DScf-DNA may be sufficiently sensitive to detect rejection and injury to the donor organ earlier than currently available methods. Levels of DScf-DNA fall consistently by 1 week post-transplant, which may allow for noninvasive detection of rejection in the vulnerable early post-transplant period (data not shown). A sensitive noninvasive rejection monitoring method could decrease the number of biopsies needed over a lifespan, thereby considerably decreasing complications, discomfort, and cost. We were able to detect all rejection episodes, including both cellular and antibody-mediated rejection, at the earliest onset and even before clinical indicators of disease. However, these results are based on a limited sample size. A larger validation study is needed.
In summary, targeted quantitative genotyping was employed to determine circulating levels of DScf-DNA in pediatric heart transplant recipients. The percentage of DScf-DNA was elevated in all patients diagnosed with rejection. Further, all patients with DScf-DNA levels <1% were shown by biopsy and clinical parameters to be negative for rejection (negative predictive value 100%). Targeted quantitative genotyping of circulating DScf-DNA constitutes a sensitive, rapid, and cost-effective noninvasive tool potentially suitable for rejection surveillance as an alternative to EMB.
Please note: This study was funded by the Department of Surgery, New Faculty Pilot Grant Program at the Medical College of Wisconsin and the Clinical and Translational Science Institute (CTSI) pilot grant program, Milwaukee, Wisconsin. Digital Analysis of Selected Regions (DANSR) was run free of charge by Ariosa Diagnostics. Drs. Mats Hidestrand, Pip Hidestrand, and Goetsch own stock in Ariosa Diagnostics. Dr. Oliphant is an employee of Ariosa Diagnostics. Dr. Simpson has served as a paid consultant to Roche. Drs. Tomita-Mitchell and Mitchell own stock in and are cofounders of Ariosa Diagnostics. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- American College of Cardiology Foundation
- Pophal S.G.,
- Sigfusson G.,
- Booth K.L.,
- et al.
- Snyder T.M.,
- Khush K.K.,
- Valantine H.A.,
- Quake S.R.