Acute cardiac allograft cellular rejection remains a significant source of morbidity and mortality within the first year after heart transplantation. Afterwards, cardiac allograft vasculopathy (CAV), as a result of chronic vascular rejection, is the major cause of morbidity and mortality. Within the first year post-transplantation, almost two-thirds of recipients will experience at least one rejection episode and approximately one third of these patients will have multiple episodes. At five years post-transplantation, nearly 50% of survivors will have CAV. The clinical symptoms of acute cellular rejection (ACR) are relatively nonspecific (fatigue, dyspnea, low grade fever). Most CAV patients remain asymptomatic until they develop serious problems such as myocardial infarction, heart failure, ventricular dysrhythmias or sudden cardiac death. No widely accepted noninvasive method exists for the accurate diagnosis of acute or chronic cardiac rejection. Noninvasive methods such as electrocardiography, echocardiography, and nuclear imaging all have been studied, but have been unsuccessful, thus far, for either condition. The current gold standard for the diagnosis of ACR remains right ventricular endomyocardial biopsy (EBx), an invasive method of diagnosis subject to morbidity and random sampling and interpretation error. Likewise, the gold standard for diagnosing CAV is cardiac catheterization with intravascular ultrasound, an invasive procedure also subject to morbidity. We are applying functional genomics, detection of donor DNA, and peptidomics to study ACR and CAV. By correlating putative biomarkers with clinical, histological, and imaging based evidence of allograft disease we hope to build a database comprised of genomic and peptidomic data relevant to the immunologic relationship between the donor organ and recipient. Our group has developed and tested standard laboratory procedures for sample processing and if necessary transcriptome amplification. We have established laboratory and bioinformatics infrastructure to support oligonucleotide microarray investigations. Published reports have established that detection of donor DNA in recipients blood can serve as a diagnostic tool of graft injury. The level of donor DNA measured as percentage of circulating cell-free donor DNA (%ccfdDNA) has the potential to accurately diagnose acute rejection with a high sensitivity and specificity, possibly at times earlier than the diagnosis obtained by conventional EBxs. The ability of cell free DNA to diagnose graft injury early opens a new window to re-examine markers of rejection. These markers are traditionally evaluated using biopsy results, often positive late during rejection. %ccfdDNA offers an opportunity to better characterize our analyses. In collaboration with the NHLBI Laboratory of Transplantation Genomics, we are embarking on biomarker discovery using our collected samples and their recently developed genomic approaches. Peptidomics has emerged as a viable and promising diagnostic tool and has been applied to diseases such as tuberculosis and chemotherapy-induced heart disease. This tool, has been used to identify peptide biomarkers of diagnostic and prognostic significance in these disease entities. In collaboration with the Peptidomic Nanoengineering Core at The Houston Methodist Hospital Research Institute we are embarking on biomarker discovery using our collected samples and their recently developed peptidomic techniques. Blood and urine specimens were obtained serially from heart transplant recipients during periods of immunological tolerance of the allograft (no ACR) and immunologic intolerance of the allograft (ACR) and from heart transplant recipients with and without CAV. The samples will be analyzed to determine whether unique gene and/or protein/peptide expression patterns are associated with each state. In the latter phase of the project we hope to translate these profiles into an acceptable test for acute and chronic cardiac allograft rejection. In addition to developing a biomarker approach to the diagnosis of rejection in cardiac transplant patients, expression profiling has the potential to identify immunoregulatory pathways that can serve as new targets for immunosuppressive therapy (rational drug development). In 2010, we processed 64 samples for high-density oligonucleotide microarray analysis. In the latter part of 2010, a second batch containing 168 samples was processed to TRNA, the required step before going to microarrays. In 2011, an additional 139 samples were processed to TRNA and 89 samples further processed for high-density oligonucleotide microarray analysis. In October 2011 through August 2012 reporting period we processed 44 samples to TRNA and processed 397 TRNA samples to a RNA stable state. In the 2012 - 2013 reporting period we processed an additional 30 samples to TRNA. In the 2013-2014 reporting period we obtained an additional 17 blood samples temporally related to 17 heart biopsies and processed 64 prior samples to TRNA. In 2014-2015, we obtained 15 blood samples temporally related to 15 heart biopsies. Over the current reporting period (201516) we shared a subset of our plasma (1,205) and urine samples (74) with the NHLBI Laboratory of Transplantation Genomics. One hundred and one of the plasma samples have been analyzed to date for cell free DNA. The remainder await external validation of biopsy specimens to confirm rejection status before proceeding further. The urine samples are being analyzed to determine the physical properties of cell-free DNA in urine to aid in protocol development. In addition, during the 2015-16 reporting period we entered into a collaborative biomarker discovery study with the Peptidomic Nanoengineering Core at The Houston Methodist Hospital. The goal is to identify markers of graft injury as well as markers that can identify the cause of injury. Our protocol has created at NIH a bio-bank of samples from transplant patients with well-characterized clinical phenotypes. Under a Materials Transfer Agreement executed on August 14, 2015, we are in the process of sharing a subset (639) of our plasma samples with the Houston group. We are waiting for external validation of biopsy specimens to confirm rejection status before shipping samples. During the 2015-16 reporting period the protocol was closed (Oct 29, 2015) to new transplant candidates with a total enrollment of 187 subjects. At that time there remained 4 subjects we were following from the transplant waitlist who had not been transplanted. Two were inactive and two were awaiting transplant. As of December 15, 2015 we no longer obtain samples with biopsies from those we follow that have been transplanted. In addition, as of December 15, 2015 we no longer follow the subjects who remain on the transplant wait list as it is doubtful they will be transplanted and obtain 1 year of follow-up data before we finish the analyses in progress. We continue to follow clinical information (i.e. survival) and also continue to recruit healthy volunteers to match our remaining unmatched patients. The protocol has obtained a minimum of one year of sample collections from all patients being actively followed that have been transplanted. We have collected blood and urine samples during 621 EBxs. On average from each biopsy time point we stored 5 plasma tubes, 4 serum tubes, 1 tube of RNA, and 1 urine sample. Two abstracts were presented at the 2016 International Society for Heart and Lung Transplantation 36th Annual Meeting (Urine Cell-Free Donor-Derived DNA after Heart Transplantation. Journal of Heart & Lung Transplantation 35(4S):S16, 2016; Reproducibility of Genomic Data Using Standards-Cell Free DNA to Monitor Rejection after Heart Transplantation. Journal of Heart & Lung Transplant 35(4S):S161, 2016.
| Agbor-Enoh, Sean; Tunc, Ilker; De Vlaminck, Iwijn et al. (2017) Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation. J Heart Lung Transplant : |
