Applied Precision Omics

APO aims develop novel approaches to detect and treat lung transplant rejection. For children and adult with advanced lung diseases, transplantation is often the only treatment. Unfortunately, half of these patients will die within 5 – 6 years after transplant because of transplant rejection. Transplant rejection is detected by analyzing a biopsy of the transplanted organ. Obtaining biopsy samples require anesthesia and is quite an invasive procedure. Additionally, biopsy has low sensitivity and usually picks up rejection at late stages when treatments options are limited and often with limited benefits. Could early detection and treatment of rejection save the transplanted organ from rejection? The focus of APO is to investigate whether early detection and treatment of rejection improves transplant survival. APO has developed an approach that picks up transplant rejection 2 – 3 months before biopsy. APO is now exploring whether these novel genomic approaches risk stratify other pulmonary patients for long-term outcomes. APO is also planning a clinical trial to assess whether early detection and treatment of rejection improves survival in lung transplant patients and also engaged in additional studies to understand molecular mechanisms of transplant rejection.

Below highlights the three main components of our lab: 

1) Clinical Trial

Investigation of self-identified racial and ancestral genetic differences in lung transplantation long-term outcomes:

We hypothesize that individuals of non-European ancestry experience inferior lung transplantation outcomes when compared to those of European ancestry. In our cohort of patients, we examine demographic and clinical characteristics, as well as differential levels of cell free DNA, to explore outcomes of allograft failure, acute cellular rejection, and chronic lung allograft dysfunction in individuals of European and non-European ancestry. Via genome sequencing of both lung transplant recipients and donors, we examine genetic ancestral markers that are associated with graft rejection and dysfunction. The goal of this research is to better inform pre- and post-transplant clinical management and further refine donor-recipient organ matching to improve long-term outcomes and survival in lung transplant patients.

Post-Lung Transplant Immunosuppression and Molecular Allograft Injury

Maintenance of immunosuppression after solid-organ transplant is essential in order to prevent short-and long-term complications such as acute cellular rejection and chronic lung allograft dysfunction. Monitoring blood trough levels of immunosuppressive medications, such as tacrolimus, is commonly used to assess adequacy of immunosuppression. Significant patient-to-patient variability exists with regards to drug pharmacokinetics and metabolism, specifically in Black or African American and other minority patients. Medication dosing and blood trough goal targets are specific to institution and clinical practice. I am interested in examining the potential for leveraging sensitive biomarkers, such as donor-derived cell free DNA (ddfcfDNA), to augment immunosuppression monitoring, specifically in patients with equivalent immunosuppressive blood levels, but who go on to have disparate outcomes.

2) Cohort Studies

GRAfT

The Genomic Alliance for Transplantation (GRAfT), established by Dr. Hannah Valantine in 2015, is a consortium of NHLBI and 8 heart and lung transplant programs located in 5 hospitals within geographic proximity of the National Institutes of Health (NIH), directed by Dr. Hannah Valantine. The clinical sites for patient recruitment include Johns Hopkins University (JHU), University of Maryland Medical Center (UMMC), Inova Fairfax Hospital (Inova), Virginia Commonwealth University (VCU), and Medstar Washington Hospital Center (Medstar). These sites collect longitudinal bio-samples and clinical data in heart- and lung-transplant recipients for the GRAFT consortium. The resources for the GRAfT-GTD study provide support at all clinical sites for patient recruitment, enrollment and bio-sample collection, while also supporting the laboratory of transplant genomics at NHLBI to perform measurements of ddcfDNA (donor derived - cell free DNA), computational analysis, host and manage the biorepository of clinical data and all bio-samples, and the data coordinating center for GRAfT at NHLBI. GRAFT utilizes a shared leadership model, with Dr. Pali Shah (Director of the Johns Hopkins (JHU) lung-transplantation program, and Dr. Hannah Valantine (transplant cardiologist, NHLBI Senior Investigator and Director of GRAfT consortium) who together oversee extramural activities. The GRAfT-GTD study prospectively collects longitudinal bio-samples and clinical data in heart- and lung-transplant recipients to examine the performance of donor-derived cell-free DNA to diagnose acute rejection and to stratify patients at risk for chronic rejection. Extramural sites enroll patients and collect biospecimen and clinical data. National Heart Lung Blood Institute (NHLBI) hosts the database-coordinating center, biorepository, and Laboratory of Transplant Genomics (LoTG) directed by Dr. Hannah Valantine. The Valantine LoTG develops and validates assays to conduct mechanistic studies at the NIH and extramural collaborating labs. Intramural and extramural investigators collaborate in preparation of all manuscripts.

The research nurse coordinator is tasked with communicating with the  site coordinators and the data managers on behalf of the team. They address any issues the sites may have and make sure the sites are aware of any changes made to the protocol or any factors affecting the study. They ensure the sites are in compliance with the IRB regulations. They reconcile the samples received against the sample projections and against the invoices received. The also ensure that the enrollment logs are up to date for better sample follow up. They also follow up on data entry into CTDB to make sure it is current. The nurse coordinator helps pull reports from CTDB for the team members as needed. They work with the protocol navigators to make sure that changes made to the protocol are well in cooperated and that staff members who require licensing to perform their roles have current certificates. They update the delegation log and during continuing reviews, they ensure that the records sent to the IRB are current. The coordinator works with the PI in editing the protocol and ensures that the protocol protects the research participants.

Laboratory technicians and nurse coordinators at each site will undergo training on all relevant standard operating procedures prior to the start of the study at their site. All biosamples including whole blood, plasma, urine, tissue, PBMC, Bronchoalveolar lavage (BAL) cell and supernatant will be frozen and shipped to NIH. Materials for sample collection, processing, and shipment will be provided by the NIH.  All sample information will be entered into the NHLBI biospecimen inventory (BSI) data base system. Center for Human Immunology (CHI)-approved standard operating procedures will be used for sample collection and processing.

Samples will be used for a cell-free DNA assay, biomarker discovery, microbiome analyses, proteomics and immunologic assessment. Additional aliquots will be stored for future IRB-approved research projects. For cell-free DNA assay, DNA collected before transplantation from donor and recipient will be genotyped to identify single nucleotide polymorphisms (SNP). After transplantation, DNA isolated from recipient blood will be sequenced. The data will be matched with pre-transplant genotyping data to assign the proportion of donor DNA. Even though cell free DNA will be sequenced with potential to obtain detail genetic information of the donor and recipient, we plan to use genotype and sequencing data only to quantifying the amount of donor DNA and not identifying the genome sequences. A modification will be submitted for IRB review should there be a need to analyze detailed genetic information.  

The lead bioinformatician provides the bioinformatics support for implementation of research vision, establishes internal and external collaborations necessary to execute bioinformatics projects. They develop and optimize the bioinformatics pipelines for the research projects using high-throughput genomic data. They document the bioinformatics data analysis methods for the presentation, scientific manuscripts and grant application. Their project is defining genomic markers of race/ancestry, implementing and optimizing methods for determining genetic distance between donors and recipients, and informatics analysis of all mitochondrial DNA studies.

GRAfT Lungevity

At this meeting we have discussed the GRAfT Lungevity study protocol. This is an NHLBI study to assess the clinical utility of early treatment of impending AMR. Furthermore, we have discussed the study protocol outline towards standardizing practice across participating centers. A second goal of this meeting was to invite leaders of reputed transplant programs to discuss the GRAfT Lungevity Study Design and get their insights, particularly on the design and potential hurdles at their center.

3) Research

Basic and Translational Research

Liquid biopsy in transplant patients and mechanisms for allograft dysfunction

Lung transplant patients have the shortest survival due to high incidence allograft failure and ineffective treatment. The current diagnostic method to detect transplant rejection and monitoring allograft health is limited to invasive and less-sensitive biopsy. Circulating cell-free DNA (cfDNA), released from dying cells, emerged as a non-invasive potential diagnostic biomarker in diverse pathological conditions. We leveraged this novel technology and demonstrated early detection of allograft rejection and predict long-term outcomes in transplant patients using donor-derived cfDNA (ddcfDNA). We aimed to validate the use of ddcfDNA as a diagnostic biomarker to detect and differentiate subclinical rejection phenotypes and post-therapy monitoring in a large clinical trial study. Additionally, we aimed to delineate molecular pathways responsible for allograft rejection, different rejection phenotypes, poor response to therapy, and chronic allograft dysfunction using whole-genome methylome sequencing and single-cell genomics approches. This will pave the way for personalized therapeutic options for patients failed to respond the standard treatment.

Our current research investigates self-identified racial and ancestral genetic differences in lung transplantation long-term outcomes. We hypothesize that individuals of non-European ancestry experience inferior lung transplantation outcomes when compared to those of European ancestry. In our cohort of patients, we examine demographic and clinical characteristics, as well as differential levels of cell free DNA, to explore outcomes of allograft failure, acute cellular rejection, and chronic lung allograft dysfunction in individuals of European and non-European ancestry. Via genome sequencing of both lung transplant recipients and donors, we examine genetic ancestral markers that are associated with graft rejection and dysfunction. Additionally, we are interested whether donor-recipient racial and genetic graft discordance may affect long-term transplantation outcomes. The goal of this research is to better inform pre- and post-transplant clinical management and further refine donor-recipient organ matching to improve long-term outcomes and survival in lung transplant patients.

Antibody mediated rejection (AMR) has been identified as a major source of allograft dysfunction in lung transplantation, and case series have shown that patients with this form of rejection have 50-70% mortality after developing it. Despite these numbers, there is a population of patients that respond to treatment and go on to recover from this type of rejection. Given the expedited decline to allograft dysfunction and death at the time of AMR, it is important to identify which patients are responding to treatment to assess if an alteration in treatment regimen is needed. A validated definition of response to pulmonary AMR treatment would help to guide clinical care post-AMR and will help to improve the clinical outcomes of lung transplant patients overall. The proposed definition from the ISHLT contains criteria for graft function, DSA titers, and overall pathology. We hypothesize that coupling donor derived cell-free DNA levels (ddcDNA) with graft function, DSA titers, and pathological findings can serve as a predictive model for responders to AMR treatment. Utilizing the Graft cohort of lung transplant patients, we are investigating 2 main research questions:

1. What are the differences in lung transplantation patients that respond and do not respond to AMR treatment?
2. Can donor derived cell-free DNA be utilized to predict responders and non-responders to pulmonary AMR treatment?

Alloimmune response remains a major drive for development of acute and chronic rejection. Conventionally, the alloimmune response is thought to be primarily driven by mismatched human leukocyte antigens (HLA). However, acute rejection has been reported in the absence of HLA-directed alloimmune responses. A factor completely overlooked is the differences in mitochondrial DNA (mtDNA)  and protein mismatch between transplant donor and recipient. mtDNA differs in size and shape from the nuclear genome and is evolutionarily similar to bacterial DNA. Because of such similarities to bacteria, circulating mtDNA and other mitochondria damage-associated molecular patterns (DAMPs) have been associated with activating immune responses. In a transplant setting with dual-genomes (donor and recipient have different DNAs), leakage of allograft mitochondrial peptides and DNA possibly sets the stage for inflammation and allo-immune activation. Therefore, we hypothesize that mtDNA and DAMPs, especially the ones derived from the donor mitochondria, trigger allo-specific immunity in solid-organ transplant recipients. To study the role of mitochondria in solid-organ transplantation we use a protocol to isolate and enrich mtDNA that takes advantage of the unique mitochondrial features, such as hypomethylation and genome circularity, and genomic sequencing and bioinformatics pipeline to analyze the variants between donor and recipient mtDNAs. Recently, we have begun to apply the latest advancements in digital PCR to quantify the cell-free mtDNA levels and describe its behaviors in post-transplantation. The aim of these studies is to understand the biological and clinical impact of cell-free mtDNA and mitochondria DAMPs in solid-organ transplantation.

Any transplanted organ can get damaged and cause the release of cellular content into the circulatory system. Once a part of the heart tissue gets damaged, cells can undergo regular cell death, but also non-regular cell death. Especially if the latter occurs it allows for the cellular contents to spill in the interstitial space from where these contents can diffuse into the circulatory system for clearance.  Therefore, cell-free DNA, peptides, exosomes, and cell-free mitochondria DNA are detected in the blood of solid organ transplants.

From all the cellular debris circulating in the blood, of particular interest and importance are the circulating mitochondria DNA and DAMPs. Because of their bacterial origins mitochondria DNA and mitochondria DAMPs can be recognized by the immune system as foreign antigens and mount further immune responses against the damaged organ.

Donor-derived cell-free DNA obtained in the urine has the ability to act as a clinical biomarker to monitor organ transplant health similar to that of plasm donor-derived cell-free DNA. The hypothesis was tested by collecting plasma and urine samples concurrently then measuring ddcfDNA and analyzing the physical characteristics of the cfDNA. Identifying urine ddcfDNA as a clinical biomarker has several benefits.  First, it is less risk for the patient as they will not need to have blood drawn.  Second, the patient can provide samples from the comfort of their home reducing cost of transport to the hospital or clinic. Following questions were solved. What are the physical characteristics of urine cfDNA? How do they compare to plasma cfDNA? Is there a relationship between urine cfDNA and plasma cfDNA? What is urine ddcfDNA like during organ transplant rejection time-points? 

Research Related with a Disease

1. COVID-19

Currently, our research focused on three areas: a) early detection of allograft rejection, post-therapy monitoring and predicting long-term outcomes in lung transplant patients, b) tissue damage mapping in COVID-19 patients, and c) elucidating molecular mechanisms associated with poor outcomes both in lung transplant and COVID-19 patients using cutting-edge molecular techniques and computational approaches.

Tissue damage mapping in Covid-19 patients

The clinical spectrum of COVID-19 ranges from mild disease to severe multiorgan failure and death. The sources of tissue injury contributing to such variation remain poorly defined. We demonstrated a strikingly high cfDNA level in COVID-19 patients. Continued studies aim to further investigate the sources of tissue injury that correlate with different clinical trajectories and outcomes using deconvolution of cell/tissue-specific cfDNA methylomes. Moreover, COVID-19 causes tissue damage in a tissue types without direct viral infection. The triggers for such injury remain poorly defined. With our collaborator Dr. Star research group, we aimed to investigate whether the excessive cfDNA released in COVID-19 patients act act as a damaged associated molecular pattern to induce systemic and uncontrolled inflammation and thereby contributing to the multiple-tissue injury and organ failure.

2. PAH

Pulmonary arterial hypertension (PAH) is a rare but lethal disease, characterized by progressive narrowing and occlusion of the small pulmonary arteries, strain on the right side of the heart and eventual death from right heart failure. At time of diagnosis, the majority of patients have advanced functional impairment. Thus, there is a need for a better understanding of PAH pathogenesis and improved diagnostics. The NIH clinical center PAH section is collaborating with the Laboratory of Applied Precision Omics to investigate the ability of plasma cell free DNA (cfDNA) to accurately stage severity of disease and/or predict clinically relevant outcomes. Preliminary results were presented at the American College of Cardiology 68thAnnual Scientific Session (J Am Coll Cardiol, 73(9, S1): S1897, 2019).  In PAH patients, plasma cfDNA was found to be elevated compared to controls (n = 7) and correlated with disease severity (mild PAH n = 7, severe PAH n = 8). cfDNA may add prognostic power to current PAH risk calculators and may help guide management decisions. Based on preliminary results, we entered into an Material Transfer Agreement with PAH programs at Allegheny General Hospital and Tufts Medical Center. We received over 200 plasma samples with clinical data for analysis.

Affiliated Training Program

Feedback and comments from our lab members for our affiliated training program: 

Argit: "The post-baccalaureate training in the laboratory of transplantation genomics and laboratory of precision omics serves as a great opportunity to gain cutting edge research skills for both, clinician (MD) and physician-scientist (MD/PhD), aspiring students. As a new established laboratory in the NIH community, research projects in the lab are vast and allow a student to get immersed in the dynamics of the translational research from the clinical design in the hospital to the biological experiments in the laboratory. This interdisciplinary aspect of the lab gave me the opportunity to design my own project, to collaborate with scientists across the country and within the NIH, and to present at international conferences. I believe the skills learned in the lab will serve as the foundation for my upcoming training as a physician-scientist."

Ananth: "I am currently a pulmonary and critical care medicine fellow at University of Maryland and NHLBI/NIH. After completing my clinical training at the University of Maryland, I am now engaging in the research portion of my fellowship in the Laboratory of Applied Precision Omics."

Cedric: "I joined Dr. Valantine and Dr. Agbor’s lab as a graduate awarded the Postbaccalaureate Intramural Research Training Award (IRTA). The lab came to my attention as I explored possibilities for my gap year before starting medical school in the fall of 2020. I wanted to gain translational research experience and to add data analysis tools to my skillset; along with broadening my understanding of cutting edge scientific techniques. Not only has the lab been able to provide me with a robust experience to accomplish the aforementioned goals, but it has provided a dynamic and caring lab community which has allowed me to grow in so many ways as a future physician scientist. I have felt at home in the lab since day one and have been encouraged to utilize the expertise of the other lab members to learn not just about the various components of the lab, but the tangential research and clinical implications. Dr. Valantine and Dr. Agbor do a great job of encouraging you to take autonomy in your experience, while guiding you in contributing to the team overall and developing as a future scientist yourself. The same motivating energy can be seen in every lab member, which contributes to the unmatched lab culture. Given the breadth of the work and implications of this lab, I would highly recommend this lab to any future scientist."

Temesgen: "I am a PhD student in Cell and Molecular Biology at Howard University and predoctoral research fellow in the Laboratory of Dr. Sean Agbor-Enoh at the NHLBI. I did my master's degree in Immunology at the University of Gondar. I studied basic immunoregulatory genes that governs antigen presentation capacity to T cells. Currently, I am working on Tissue Origin of DNA Methylation project to: (1) elucidate the molecular mechanism of transplant rejection and (2) identify cell-free DNA methylation markers in plasma for early detection of tissue injury. Working under Dr. Agbor-Enoh give me the opportunity to interact with well-rounded professionals and integrate diverse ideas and perspectives pertaining to the various global issues"