Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection

Abstract

Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga–base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy.

Cell-Free DNA: An Upcoming Biomarker in Transplantation

“…In heart transplant recipients, De Vlaminck and coworkers reported a mean plasma ddcfDNA fraction of 3.8% on the first day posttransplant, which declined within 1 week to a baseline level of 0.06% for the following year during stable graft conditions (45). Other studies reported plasma ddcfDNA fractions of approximately 0.9% in cardiac recipients with stable graft function (34,42) ( Table 3).…”

“…While amplification of Y-chromosome genes in the recipient's plasma and urine has been widely used in gender-mismatched transplantations (21,22,30,31,(34)(35)(36)(37)(38)(39), this strategy is limited to about 25% of the transplant population. A more universal approach is the use of informative genetic polymorphisms whereby a particular allele differs or is absent in the donor genome compared to the recipient genome; in addition to the quantification of donor-specific human leukocyte antigen deoxyribonucleic acid (HLA DNA) (40) and copy number deletion polymorphisms (41), single nucleotide polymorphisms (SNPs) can be targeted to determine the percentage of ddcfDNA by counting donor and recipient's bases at informative SNP sites with digital droplet polymerase chain reaction (ddPCR) (42)(43)(44) or massive parallel shotgun sequencing (MPSS) (34,45).…”

“…Low fraction of total cfDNA (0.06-3.5%) (34,42,45) qPCR or ddPCR of Y-chromosome genes (22,34,(37)(38)(39) (56) cfDNA isolation:…”

Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

“…It is not only human cells that shed their nucleic acids into the blood: DNA from plant-based foods has been detected (15), and other life forms such as viruses, bacteria, and fungi release their DNA and RNA into the blood, a phenomenon which has been exploited to determine the presence of infectious disease (12,16) and to measure alterations of the virome due to pharmacological immunosuppression (17). There are roughly an order of magnitude more nonhuman cells than nucleated human cells in the body (18,19); combining this observation with the average genome sizes of a human, bacterium, and virus (Gb, Mb, and kb, respectively) suggests that approximately 1% of DNA by mass in a human is derived from nonhost origins.…”

“…The existence of circulating nucleic acids in blood has been known since the mid-20th century (10), but only in the last few years has the advent of high-throughput sequencing led to clinical diagnostics based on these nucleic acids [also known as cell-free DNA (cfDNA) or RNA], including detecting fetal abnormalities (11), transplanted organ rejection events (12,13), and signatures of cancers (14). It is not only human cells that shed their nucleic acids into the blood: DNA from plant-based foods has been detected (15), and other life forms such as viruses, bacteria, and fungi release their DNA and RNA into the blood, a phenomenon which has been exploited to determine the presence of infectious disease (12,16) and to measure alterations of the virome due to pharmacological immunosuppression (17).…”