Cell-free DNA diagnostics in transplantation utilizing next generation sequencing

Cell-free DNA diagnostics in transplantation utilizing next generation sequencing

  • Post category:Review Article
  • Reading time:8 mins read

Introduction

Cell-free DNA (cfDNA) is a type of degraded DNA that is released largely by apoptotic cells. Circulating cfDNA (Cir-cfDNA) provides a real-time window into the physiology and pathophysiology of cells or tissues due to its rapid turnover. cfDNA is a non-invasive biomarker for disease, infection, and tissue injury that may be isolated from a range of human body fluids including blood, urine, saliva, lymph, breastmilk, bile, spinal, and amniotic fluid. Furthermore, detectable cfDNA can come from both self and non-self sources, including foetal tissue, microbial and viral DNA, as well as transplanted stem cells and solid organ allografts.

Although everyone has a baseline level of cfDNA, which is mostly derived from ongoing cell turnover, the quantity and content of cfDNA might vary. The use of Next Generation Sequencing (NGS) to examine cell-free DNA (cfDNA) as a transplant diagnostic is an important step toward enhancing the accuracy of allograft health monitoring after transplantation. Interrogation of cfDNA provides a potent biomarker for disease and tissue harm that is also less intrusive.

cfDNA may be extracted from a variety of bodily fluids and examined using bioinformatics to determine where it came from. Donor-derived cfDNA monitoring in transplantation is a useful method for detecting active allograft damage, infection, and rejection at the time of donation. Multiple cfDNA detection and interrogation approaches are now being studied for clinical validity.

They have the potential to provide minimally invasive, quantifiable, and repeatable measurements of transplant damage across organ types. This review will explore our current understanding of Cir-cfDNA characteristics and current practices for isolation and interrogation using next generation sequencing (NGS) platforms. In the field of solid organ transplantation, detection of donor derived circulating cfDNA (dd-Cir-cfDNA) is being explored as a non-invasive measure of allograft injury Fig. 2.

Characteristics of circulating cfDNA

The concentration of Cir-cfDNA in healthy individuals is low and a recent study of 84 healthy subjects revealed a range of 1–10 ng/ml in plasma with an average of 6 ng/ml reflecting a genomic DNA equivalent of 1000 cells. Patients with autoimmune disease, cancer, recent transplants, or pregnancy exhibit elevated Cir-CFDNA  reflecting their altered medical state and/or tissue injury.

Collection and processing of Cir-cfDNA

The sensitivity needed for Cir-cfDNA interrogation in many clinical settings requires considerable attention to technical details surrounding sampling and processing procedures. Plasma is preferred over serum due to this contamination of genomic DNA from leukocytes during the clotting process. Longer processing times did not significantly interfere with the detection of the targeted Cir-CFDNA. Cir-cf DNA was extracted from between 0.4 and 2.0 ml of plasma using a magnetic beads-based cfDNA isolation kit yielding 1.7 to 30.8 ng/ml of nucleic acid. NGS libraries were prepared using a broad range of cfDNA input (2.5–105.5 ng) with acceptable results in the lower concentrations.

Detection of donor derived Cir-cfDNA as a transplant rejection diagnostic

Random shotgun sequencing allows data interrogation for pathogen-derived sequences and extends the scope of Cir-cfDNA monitoring to include infectious disease detection. For lung recipients elevated dd-Cir-cfDNA correlated with clinically diagnosed cytomegalovirus infection in serum or lung lavage fluid with an area under the curve (AUC) = 0.91. High frequency clinical and subclinical infections were also detected involving polyomavirus, human herpesviruses (HHV4-8) and adenovirus as well as bacterial and fungal infections, some of which are not routinely tested for post-transplant.

Detection of acute rejection in the early subclinical stages, prior to an injury sufficient to yield dysfunction, and early identification of chronic rejection lesions would provide opportunities for intervention in the hopes of improving long-term allograft survival. Independently validated and applied the Stanford Shotgun NGS method using a 1% threshold for dd-Cir-cfDNA and longitudinal samples from lung and heart recipients.

Rejection prevalence and rejection type varied between studies and different% dd-Cir-cfDNA positive thresholds were used depending on the study endpoint. Negative predictive values (NPV) were high and ranged from 83 to 100% across studies and organ types; however, the positive predictive values (PPV) were lower in all cases and varied widely between studies. Given the low PPV and current limitations in our understanding of how to interpret and whom and when to monitor for dd-Cir-cfDNA, this assay should not be considered a stand-alone diagnostic.

Deciphering the cellular origins of Cir-cf DNA

Future improvements for Cir-cfDNA diagnostics in transplantation will include the ability to trace this biomarker back to the exact cell types that have been injured and released DNA. This advancement would broaden the utility of Cir-cfDNA as a mulit-purpose diagnostic in transplantation, beyond detecting rejection alone. Cir-cfDNA monitoring may also allow early detection of malignancies in transplant patients. This could advance the diagnosis of graft-versus-host disease following hematopoietic stem cell transplantation or multi-visceral transplants by detecting elevated recipient tissue injury in the gut and skin.

Conclusion

The addition of dd-Cir-cfDNA detection by NGS as a noninvasive transplant diagnostic provides a significant opportunity to improve the detection of rejection, infection, and recurrent disease. Current commercial assays that quantitate major (recipient) and minor (donor) Cir-cfDNA fractions are available. Further studies are needed to clinically validate specific endpoint thresholds and determine optimal use with companion diagnostics provided by infectious disease and histocompatibility laboratories.

Importantly, dd-Cir-cfDNA assays hold the potential for non-invasive monitoring of allograft health, providing highly sensitive injury surveillance in real time, and facilitating early detection with the possibility of intervention prior to clinical dysfunction. Integrated with clinical parameters, histocompatibility and infectious disease diagnostics, and allograft function tests, the measurement of dd-Cir-cfDNA increases our arsenal of tools to improve transplant outcomes.

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