Expression Profiling In Acute and Chronic Cardiac Allograft Rejection

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Abstract

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. At five years post-transplantation, nearly 50% of survivors will have CAV. In the first year after transplantation, nearly 63% of patients experience at least one episode of cardiac rejection and approximately one third of these patients will have multiple episodes. The clinical symptoms of acute cardiac rejection 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 studies all have been studied, but have been unsuccessful, thus far, for either condition. Several methodologies have been studied including electrocardiography, echocardiography, nuclear imaging, and phosphorus spectroscopy without success. The current gold standard for the diagnosis of acute cellular rejection remains right ventricular endomyocardial biopsy. This is 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 to study acute cardiac allograft cellular rejection and CAV. Our group has developed and tested standard laboratory procedures for sample processing and if necessary amplification. We have established laboratory and bioinformatics infrastructure to support oligonucleotide microarray investigations. Two major local transplant programs (Johns Hopkins University and INOVA-Fairfax) have been or are currently collaborating. We have an IRB approved protocol and are currently actively enrolling patients (184 individuals enrolled to date). We hypothesize that large scale expression profiling of circulating peripheral blood mononuclear cells (PBMC, predominantly T lymphocytes) will identify genes that can serve as reliable biomarkers of acute and chronic cardiac cellular rejection. In the initial bench phase of the project, PBMCs are obtained from heart transplant recipients during periods of immunological tolerance of the allograft (no acute rejection) and immunologic intolerance of the allograft (acute rejection) and from heart transplant recipients with and without CAV to determine whether unique gene expression patterns are associated with each state. Other analytic tools that may be employed include proteomics, RT-PCR, Western blot, insitu-hybridization, immunohistochemistry, and histopathology. In the latter phase of the project we hope to translate these profiles into an acceptable bedside test for acute and chronic cardiac allograft cellular 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 were further processed to high density oligonucleotide microarray analysis. Since October 2011 through August 2012 we have processed an additional 44 samples to TRNA and processed 397 TRNA samples to an RNA stable state. The protocol continues to actively enroll subjects.