Utility and limitations of the use of donor-derived cell-free DNA in kidney transplantation

Abstract

In recent years, the use of new biomarkers in different phases of the diagnosis and treatment of several diseases has allowed substantial improvement in clinical practice. The use of donor-derived cell-free DNA (dd-cfDNA) in organ transplantation has led to significant progress in the treatment of post-transplant outcomes, particularly after kidney transplantation. In addition, the use of dd-cfDNA in organ transplantation has led to significant advancements in post-transplant outcome monitoring. The aim of this study is to review many of the recent studies on the use of this biomarker and to evaluate its most relevant advantages and limitations. dd-cfDNA is released from several types of cells of the transplanted organ, most often from endothelial cells and this happens in the case of organ damage, most often rejection. Its presence in the bloodstream of the recipients is an important sign of graft damage; its principal advantage is in the avoidance of invasive tools such as renal biopsy. Additionally, several studies reported that the finding of dd-cfDNA in the serum may precede histological abnormalities; its utility in the diagnosis of subclinical rejection is extremely important. Among the principal limitations of this tool are the difficulty in distinguishing different forms of graft damage. According to several studies this tool has several limitations in diagnosing T-cell mediated rejection. In addition, particular care should be taken in distinguishing dd-cfDNA from recipient-derived cfDNA.

Key Words: Biomarkers; Donor-derived cell-free DNA; Antibody mediated rejection; T cell mediated rejection; Subclinical rejection; Advantages and disadvantages

Core Tip: New biomarkers have allowed to improving our knowledge in clinical medicine, in the field of transplantation in particular. Donor-derived cell-free DNA has proven to be one of the best. Such donor DNA is delivered from the damaged graft into the bloodstream of the recipient. The finding of donor-derived cell-free DNA is a clear sign of damaged graft, more often involving the endothelial cells. Several pathologies may be the cause, most often antibody-mediated rejection. Less frequent, T-cell mediated rejection. Major advantage of such technique is to avoid invasive procedures as renal biopsy.

INTRODUCTION

Medical knowledge has evolved from experience-based medicine to evidence-based medicine and now to precision medicine. We are currently in this latter phase, largely owing to the development of new technologies and, in particular new biomarkers. In the field of transplantation, there is a pressing need for new biomarkers, particularly non-invasive biomarkers, because our primary therapeutic approach, in addition to kidney allografts, is immunosuppression (along with medications to mitigate the side effects of immunosuppression). For decades, we have been delivering our therapy with indiscriminate monitoring tools, waiting for clinically evident rejection to occur due to insufficient immunosuppression, or for clinically evident infection or malignancy due to excessive immunosuppression. A biomarker can be objectively measured and evaluated as an indicator of a biological process, a pathological process or a pharmacological response to a specific treatment. Biomarkers can be studied and evaluated through proteomics, genomics, transcriptomics and metabolomics[1]. They are particularly useful in patient’s follow-up after organ transplantation and are predictive in terms of sensitivity and specificity. Among others, new relevant biomarkers applied to the study and evaluation of transplanted kidneys include donor-derived cell-free DNA (dd-cfDNA), the genetic profile of the recipient, and RNA biomarkers in the urine. Owing to its precision and reliability, dd-cfDNA represents a breakthrough in kidney transplantation, as will be specified in the following chapters. This study describes the principal characteristics of dd-cfDNA, along with its clinical utility, advantages and limitations.

CHARACTERISTICS AND ORIGIN OF DD-CF DNA

Under normal conditions, approximately 80% of plasma cell-free DNA (cfDNA) is derived from the apoptosis and necrosis of white blood cells, whereas the remaining fraction is derived from the liver and other organs[2,3]. The fraction value of dd-cfDNA refers to the proportion of dd-cfDNA in relation to the total cfDNA. Both absolute and fractional values have advantages and disadvantages, as outlined in Table 1.

Table 1 Comparison of advantages and disadvantages between absolute and fractional values.

Metric	Advantages	Disadvantages
Absolute value	Not influenced by fluctuations in WBC - derived cfDNA levels	Poor comparability between different experimental platforms
	Not affected by variations in the extent of inflammation in different pathological conditions	Long-term storage of plasma/urine samples, even within the same experimental platform, can also impact results
	Not influenced by recipient cell lysis during blood drawn
Fractional value	Not influenced by total cfDNA degradation and extraction reagents	Prone to fluctuations in WBC levels, such as during rejection detection in infection states
	Strong compatibility between different experimental platforms

WBC: White blood cell; cfDNA: Cell-free DNA.

The presence of dd-cfDNA in the blood of the recipient after kidney transplantation is attributable to any damage to the transplanted organ, which leads to an increase in dd-cfDNA in the recipient’s bloodstream, particularly if the damage affects the endothelial cells[4]. Indeed, dd-cfDNA serves as a specific marker of endothelial cell damage, and its elevation may be ascribed to organ rejection, infections or drug-induced damage[5]. Several methods are available to determine the amount of dd-cfDNA in recipient plasma. These methods generally involve variation in polymerase chain reactions (PCRs), including real-time quantitative PCR, droplet digital PCR and massively parallel sequencing (next generation sequencing).

In practice, two methods are used to measure dd-cfDNA, AlloSure and Prospera. A study by Melancon et al[6] reported no significant difference between the two methods in terms of sensitivity, specificity or positive and negative predictive values. Table 2 presents the main studies that have examined these methods in the context of rejection[7-11]. The sensitivity was 0.45 for the Allosure method and 0.54 for Prospera, the specificity was 0.84 for AlloSure and 0.69 for Prospera, and the area under the curve receiver operating characteristic was 0.73 for AlloSure and 0.74 for Prospera.

Table 2 Studies assessing plasma donor-derived cell-free DNA for the diagnosis of rejection using commercially available assays.

Ref.	Assay used	Patient number	Threshold (%)	Sensitivity/specificity	PPV/NPV
Bloom et al[7]	AlloSure	102	1	59/85	61/84
Sigdel et al[8]	Prospera	193	1	88.7/72.6	52/95
Huang et al[9]	AlloSure	63	0.74	79/72	77/75
Bu et al[42]	AlloSure	1092	0.5	78/71	50/90
Bromberg et al[32]	Prospera	424	1	79/85.3	32.6/97.9

PPV: Positive predictive value; NPV: Negative predictive value.

Both methods have technical limitations, such as interassay variability in detecting low-frequency dd-cfDNA and handling of donor-recipient genetic mismatches, sex mismatched transplants or differences in body weight. Standardized protocols, multicenter validation studies and meta-analyses are recommended. The kinetics of dd-cfDNA have been well described by Jaikaransingh et al[12]. Gielis et al[13] reported an exponential decline in dd-cfDNA levels in the initial period after kidney transplantation. Shen et al[14] reported similar results, including in recipients with delayed graft function. Factors associated with higher concentrations of dd-cfDNA include deceased donor allografts and delayed graft function. After the initial period, dd-cfDNA reaches baseline levels and an elevation may indicate transplant damage. However, a study by Schütz et al[15] revealed a slow increase in dd-cfDNA after a prolonged period post transplantation which was attributed to a gradual decline in recipient cfDNA due to the action of recipient lymphocytes at lower calcineurin levels.

Abnormalities in dd-cfDNA may arise from various causes and can occur at different periods post- transplantation. Acute rejection is a significant cause of dd-cfDNA abnormalities and will be extensively discussed in a separate chapter. According to Chen et al[16] and Shen et al[14], patients are affected by polyomavirus (BK virus) nephropathy. This is true even in patients who are not experiencing concurrent rejection, potentially due to tubulitis and interstitial inflammation. Additionally, patients with urinary tract infections have higher levels of cfDNA, but in these cases, this is related to increased recipient cfDNA resulting from white blood cell infiltration. Another infectious cause of higher absolute levels of dd-cfDNA is the B19 parvovirus infection.

Ischemia and reperfusion injury increases dd-cfDNA levels, which normalize within 10 days postsurgery[14], rendering their measurement ineffective for identifying rejection. However, dd-cfDNA measurement is useful for monitoring treatment effects in cases of acute rejection. A rapid decrease in dd-cfDNA related to the reduction in inflammatory cells following methylprednisolone boluses is also observed independently of rejection therapy. The significance of dd-cfDNA in cases of rejection treated with methylprednisolone pulses is related to treatment efficacy, provided that samples are taken during stable periods.

Kyeso et al[17] studied the effect of kidney biopsy on dd-cfDNA levels. Owing to the short half-life of dd-cfDNA, elevated levels may be observed up to 2 hours after biopsy. A particular condition leading to higher dd-cfDNA levels is dual kidney transplantation[18]. This phenomenon is thought to be related to the fact that dual transplantation often involves marginal kidneys not suitable for single kidney transplantation, which are more prone to apoptosis, resulting in higher dd-cfDNA levels. Other factors that may alter the different fractions of dd-cfDNA included pretransplant blood transfusions and gestation. Therefore, these conditions should be evaluated when dd-cfDNA is used as a diagnostic biomarker[19,20].

DD-CFDNA AND THE DIAGNOSIS OF REJECTION

dd-cfDNA is extremely important in diagnosing kidney rejection, even in phases where histological alterations are not yet present and patients appear clinically stable. It is essential to recall the origin of blood and urinary dd-cfDNA after solid organ transplantation. After solid organ transplantation, dd-cfDNA circulates in the recipient’s blood, accounting for only a small fraction of total cfDNA (recipient plus donor-derived). Small cfDNA fragments from the blood can be filtered through the glomerular barrier and appear in the urine, referred to as “transrenal DNA”. Following kidney transplantation, high-molecular weight dd-cfDNA from cells is shed from the donor-derived urinary tract and subsequently appears in the urine[4]. Table 3 provides an overview of different cfDNA biomarkers[21-25].

Table 3 Overview of different cell-free DNA biomarkers.

Biomarker	Characteristics and clinical utility
Total (plasma/serum cfDNA)	Donor plus recipient-derived cfDNA
	Reflects total cell damage, not limited to the allograft[21]
	Possible release of recipient-derived cfDNA by recipient’s immunological effector cells activated during rejection[22]
Donor-derived plasma/serum cfDNA	Directly interrogates graft integrity[23]
	Increased in cases of graft damage[24]
Urinary cfDNA	Tr-DNA
	Molecules crossing the kidney barrier and appearing in urine as a soluble fraction[25]
	Reflects increased burden of tissue injury and apoptosis[24]
	Donor-derived or recipient derived

cfDNA: Cell-free DNA; Tr-DNA: Transrenal DNA.

The use of dd-cfDNA in the diagnosis of rejection, particularly subclinical rejection, remains controversial. In a recent study by Aubert et al[26] 2882 kidney allograft recipients were enrolled from 14 transplant centers to document the efficacy of dd-cfDNA in diagnosing kidney rejection. The study revealed that dd-cfDNA correlated with antibody-mediated rejection (ABMR) with a P value greater than > 0.0001 and with T cell-mediated rejection (TCMR), with a P-value less than < 0.0001. The authors compared the mean values of dd-cfDNA in different rejection scenarios and in other conditions affecting the transplanted kidney. The results are shown in Table 4.

Table 4 Donor-derived cell-free DNA values in different conditions affecting the transplanted kidney.

Conditions	dd-cfDNA values
Mixed rejection	2.85% ± 0.68%
Acute TCMR	2.03% ± 1.13%
Active ABMR	1.15% ± 0.15%
Chronic active ABMR	1.09% ± 0.15%
Chronic active TCMR	0.59% ± 0.17%
BKV associated nephropathy	0.44% ± 0.06%
FSGS	0.40% ± 0.06%
Glomerulitis without rejection	0.45% ± 0.06%
Isolated IFTA	0.36% ± 0.02%

dd-cfDNA: Donor-derived cell-free DNA; TCMR: T-cell mediated rejection; ABMR: Antibody-mediated rejection; BKV: BK virus; FSGS: Focal segmental glomerulo sclerosis; IFTA; Interstitial fibrosis, tubular atrophy.

The authors concluded that the inclusion of dd-cfDNA levels enhances diagnostic accuracy. Furthermore, in the same study, the authors reported that dd-cfDNA is predictive of subclinical rejection in stable patients. This study agrees with that of Sigdel et al[8], who analyzed dd-cfDNA using single nucleotide polymorphism to distinguish patients with rejection from those with a different disease. The capacity of dd-cfDNA to distinguish rejection is reported in Table 5.

Table 5 Donor-derived cell-free DNA and diagnosis of rejection.

Conditions	Value
Acute rejection (median)	2.32%
Non-acute rejection (median)	0.47%
Area under curve	0.87
Sensitivity	88.7%
Specificity	72.6%
Positive predictive value	52%
Negative predictive value	95.1%

A new and interesting study by Kim et al[27] compared the ability of dd-cfDNA and donor-specific antibodies (DSA) measurements to predict allograft rejection and severe microvascular inflammation in kidney transplant recipients. The results of this study are shown in Table 6. The findings indicated that the dd-cfDNA test is a predictive tool for biopsy proven rejection (BPR) and microvascular inflammation, and its performance in BPR prediction improved when it was combined with DSA for BPR. Overall, the value of dd-cfDNA in the diagnosis of TCMR remains controversial. Some studies, such as that of Bloom et al[7], demonstrate little significance, whereas others indicate significance, principally using absolute values. According to Oellerich et al’s study[28], when diagnosing borderline TCMR, the significance of absolute dd-cfDNA values is considerably better than that of fractional values. A major limitation of dd-cfDNA measurement is its inability to reliably differentiate between different types of graft damage. Notably, several studies have demonstrated its limited sensitivity in diagnosing TCMR.

Table 6 Diagnostic performance of donor-specific antibodies, donor-derived cell-free DNA and combination of donor-specific antibodies and donor-derived cell-free DNA for detecting rejection, antibody-mediated rejection and severe microvascular inflammation.

Characteristics	Rejection		ABMR		Severe MVI
	Sensitivity (%) (95%CI)	Specificity (%) (95%CI)	Sensitivity (%) (95%CI)	Specificity (%) (95%CI)	Sensitivity (%) (95%CI)	Specificity (%) (95%CI)
DSA	62.5	63.3	90.0	83.3	50.0	78.0
dd-cfDNA	93.8	58.3	90.0	51.9	92.9	56
dd-cfDNA (> 1.0%)	50	83.3	60	81.5	78.6	90
DSA or dd-cfDNA (> 0.4%)	100	50	100	44.4	92.9	46
DSA and dd-cfDNA (> 0.4%)	56.3	91.7	80	90.7	50	88

ABMR: Antibody-mediated rejection; MVI: Microvascular inflammation; CI: Confidence interval; DSA: Donor-specific antibodies; dd-cfDNA: Donor-derived cell-free DNA.

Recently, two meta-analyses addressed the ability of dd-cfDNA measurement to diagnose either ABMR or TCMR. The first meta-analysis[29] examined seven studies[28,30-33] using the meta-analysis of observational studies in epidemiology guidelines[34]. In these studies, dd-cfDNA levels were higher in patients with ABMR compared than in stable patients, whereas patients with TCMR presented dd-cfDNA levels that did not differ from those of stable patients. The second meta-analysis reported similar findings[35]. In conclusion, higher dd-cfDNA levels were observed in patients with ABMR, but dd-cfDNA levels did not differ between patients with TCMR and those without rejection. Both meta-analyses suggest that dd-cfDNA could be a useful marker for diagnosing ABMR but probably not TCMR.

The differing results among all the aforementioned studies in terms of the capacity of dd-cfDNA measurement to be used to diagnose ABMR and TCMR may be explained in part by the fact that ABMR involves microvascular injury, leading to the release of dd-cfDNA from endothelial cells[36], whereas TCMR primarily involves interstitial injury with infiltration of recipient immune cells[37]. Hence, it is important to study the different components of dd-cfDNA and recipient-derived cfDNA.

Additionally, dd-cfDNA is a relevant prognostic tool. An important study by Stites et al[38] analyzed 79 patients with TCMR1A or borderline rejection, and examined long-term effects following documented rejection. This study assessed the percentage change in the estimated glomerular filtration rate (eGFR), the development of DSAs and the rates of recurrent rejection. Specifically, patients were divided into those with dd-cfDNA < 0.5% and those with dd-cfDNA > 0.5%. The results are reported in Table 7.

Table 7 Patients with donor-derived cell-free DNA > 0.5% were at increased risk of recurrent rejection, donor-specific antibodies detection, and estimated glomerular filtration rate decline over the following 3-6 months.

Characteristics	Statistics	Low (dd-cfDNA < 0.5%)	High (dd-cfDNA > 0.5%)	P value
dd-cfDNA value (%)	Mean (SD)	0.25 (0.087)	1.76 (1.40)	-
	Median	0.21 (0.19-0.29)	1.40 (0.87-2.02)	-
	Min, max	0.19, 0.49	0.52, 2.02	-
Change in eGFR (%)	Mean (SD)	-0.40 (18.149)	-0.84 (11.98)	0.0040
	Median	0.00 (-0.92, 4.76)	-7.50 (-16.22, -1.39)	-
	Min, max	-70.73, 33.33	-37.50, 32.65	-
Presence of DSAs, n (%)	-	1/37 (2.7)	17/42 (40.5)	< 0.0001
Recurrent rejection, n (%)	-	0/37 (0.0)	9/42 (21.4)	0.0028

dd-cfDNA: Donor-derived cell-free DNA; DSA: Donor-specific antibodies; eGFR: Estimated glomerular filtration rate; Min: Minimum; Max: Maximum.

A reduction in eGFR, an increase in DSAs and an increased recurrence rate of rejection were found to be significantly greater in patients with dd-cfDNA > 0.5%. The use of dd-cfDNA in diagnosing subclinical or borderline rejection in patients without clinical signs is essential to prevent worse graft outcomes, as documented by a study by Nankivell et al[39] involving 551 transplant recipients. Following these patients for 60 months, Nankivell et al[39] reported that unrecognized subclinical rejection was later associated with kidney dysfunction and reduced graft survival. Similar findings have also been reported in patients who are more susceptible to subclinical rejection and poorer kidney outcomes, such as pediatric kidney transplant recipients[40] and patients treated with therapeutic protocols that include rapid steroid withdrawal[41]. In these patient groups, monitoring dd-cfDNA levels may be a useful approach.

A longitudinal monitoring study using dd-cfDNA was conducted by Bu et al[42]. The study, called allografts with longitudinal surveillance reports data from 1094 kidney transplant recipients whose dd-cfDNA levels were determined three times after transplantation. As in other previous studies[43,44], eGFR decline, the development of de novo DSAs and allograft rejection rates were evaluated. Overall, the allografts with longitudinal surveillance study confirmed the data already reported by Stites et al[38], stressing the importance of determining dd-cfDNA levels, which were associated with worse allograft outcomes when the value higher than 0.5%. All the cited studies document that there is a relationship between dd-cfDNA presence and diagnosis of rejection and long-term outcomes, however it is not easy to establish a cost-effectiveness in this setting. To date ongoing clinical trials as Kidney Allograft Outcomes AlloSure Registry (KOAR) and Prospera Kidney Transplant Active Rejection Assessment Registry (ProActive) are not able to give safe answers to this question. KOAR is an observational study to evaluate safety and efficacy outcomes in renal transplant recipients in whom post-transplant care is managed using AlloSure test. The ProActive registry is a longitutinal multi-center study for patients using Prospera, an allograft rejection test. Both trials are active, but still now do not give answer to the proposed question. Another novel biomarker not discussed in this study is the gene expression profile (GEP)[45]. A pertinent question is whether the combination of GEP and dd-cfDNA measurement improves the diagnosis of subclinical rejection in kidney transplant recipients. Park et al[46] addressed this problem by studying 428 surveillance biopsies in 208 patients enrolled in a clinical trial (clinical trials in organ transplantation 08)[45]. The results are summarized in Table 8. The authors concluded that the combination of both methods enhances the detection of subclinical rejections. Furthermore, they reported that GEP identified more cellular rejections, whereas dd-cfDNA measurement was more effective in recognizing ABMR.

Table 8 Summary of diagnostic metrics to detect subclinical acute rejection.

Diagnostic performance	GEP alone	dd-cfDNA alone	Positive = GEP + dd-cfDNA
Sensitivity	0.43	0.47	0.20
Specificity	0.85	0.88	0.98
Positive predictive value	0.47	0.56	0.81
Negative predictive value	0.82	0.84	0.80
Accuracy	0.75	0.78	0.80

GEP: Gene expression profile; dd-cfDNA: Donor-derived cell-free DNA.

BENEFITS; PITTFALLS AND OPEN QUESTIONS REGARDING THE USE OF DD-CFDNA AS A BIOMARKER

A study by Osmanodja et al[47] highlighted the importance of using both absolute and relative dd-cfDNA measurements together to achieve higher test performance, particularly in patients with chronic active ABMR. A very recent study by Graver et al[5] reported the main advantages and pitfalls associated with the use of different subspecies of cfDNA. Additionally, several unanswered questions remain. Table 9 outlines the main points concerning the clinical application of dd-cfDNA. Let us first discuss the potential benefits. Dd-cfDNA is a noninvasive blood biomarker that can be used in the diagnosis of rejection. This represents a significant advantage, as renal biopsy was previously considered the gold standard for diagnosing rejection, despite its associated risks, sampling errors and difficulty to monitoring the transplant over time[48,49].

Table 9 Summary points regarding donor-derived cell-free DNA use in clinical practice.

Potential benefits	Pitfalls	Unanswered questions
Noninvasive blood biomarker	Fractional quantification affected by changes in rd-cfDNA	Clinical utility and cost-effectiveness
Applicable to all solid organ transplants	Does not exclude (early) TCMR (if rd-cfDNA normal)	Ideal surveillance testing schedule
Elevations may occur up to 30 days before histological changes	Does not reliably discriminate between normal histology and interstitial fibrosis/tubular atrophy	Significance of normal level in presence of histological inflammation
Absolute quantification of dd-cfDNA not affected by changes in rd-cfDNA	Elevated in non-rejection pathologies associated with tissue injury or immunological risk (BKN, CNI toxicity)	Superiority of assay-specific optimal diagnostic threshold vs deviation from patient baseline
Avoidance of protocol biopsy if graft function stable and dd-cfDNA low	Not recommended for use in early posttransplant period	Superiority of quantitative/continuous vs qualitative/binary measurements
Avoidance of unnecessary biopsies	Not recommended for use for 24 hours post-biopsy	Superiority of fractional vs absolute quantification
Non-invasive diagnosis of acute rejection	Confounded in pregnancy	Role of urinary dd-cfDNA
Assessment of response to rejection treatment	Confounded in some repeat and multi-organ transplants	Role within a panel of biomarkers
Indicator for treatment of chronic active ABMR	-	-

rd-cfDNA: Recipient derived cell-free DNA; TCMR: T-cell mediated rejection; dd-cfDNA: Donor-derived cell-free DNA; BKN: BK nephropathy; CNI: Caòlcinerin inhibitor; ABMR: Antibody-mediated rejection.

dd-cfDNA measurement is applicable to all solid organ transplants with potential variation due to organ size. Another advantage is that dd-cfDNA elevation may occur up to 30 days prior to histological changes, this allows for early diagnosis and treatment[50]. Absolute quantification of dd-cfDNA is not influenced by a potential increase in recipient-derived-cf-DNA due to conditions such as recipient infections[51]. In cases of stable graft function with persistently low dd-cfDNA levels, protocol biopsies and unnecessary biopsies may be avoided. Conversely, in cases of graft dysfunction with very high dd-cfDNA levels, acute graft rejection can be diagnosed in a noninvasive way. The response to rejection therapy can be monitored with repeated dd-cfDNA measurements[52]. However, the use of cf-DNA presents several disadvantages, including the following. Fractional quantification is significantly affected by changes in recipient derived-cfDNA, which may be related to factors such as urinary tract infections, methylprednisolone pulse therapy, and vigorous physical activity potentially leading to false-positive or false-negative results[53].

As previously discussed, dd-cfDNA measurement cannot reliably be used to detect early TCMR and cannot distinguish between normal histology and interstitial fibrosis/tubular atrophy. dd-cfDNA levels may be elevated in nonrejection pathologies such as BK virus nephropathy or calcineurin inhibitor uses. dd-cfDNA measurement should not be performed in the early posttransplant period or shortly after renal biopsy as its levels may be elevated in these conditions. Additionally, pregnancy, repeat transplants or multiorgan transplants may yield confounding results. Other factors as recipient obesity, ethnicity or comorbidities could influence dd-cfDNA, but to date they have not been adequately studied. It is recommended that future studies will analyze and control such variables. Given all these data, one of the major challenges is interpreting dd-cfDNA results in the appropriate clinical context.

Table 10 shows the relationships among the clinical context, dd-cfDNA levels, interpretation of the results and their management. Several questions remain unanswered regarding the use of dd-cfDNA as a biomarker. The manner in which dd-cfDNA should be used to influence outcomes changes following rejection has yet to be clarified[54], and the cost of incorporating dd-cfDNA measurement into clinical care, particularly when serial measurements are used, remains unclear[55]. The optimal testing schedule has not been determined, including the frequency of testing post-transplantation and how to utilize dd-cfDNA to monitor the clinical response following acute rejection treatment. Two studies are currently examining this issue: The evaluation of patient outcomes from the KOAR, which investigates the use of an assay, currently in use, and the ProActive, which evaluates a different commercial assay.

Table 10 Interpretation of donor-derived cell-free DNA results according to the clinical context.

Clinical context	dd-cfDNA result	Interpretation	Management
Acute graft dysfunction	High	Rejection likely	Biopsy to confirm
	Low	Rejection unlikely	Biopsy to exclude TCMR and other pathologies
Stable graft function	High	Rejection likely	Proceed with protocol biopsy
	Low	Rejection unlikely	Avoid protocol biopsy
Chronic graft dysfunction	High	Chronic AMR likely	Consider biopsy to guide treatment
	Low	Chronic AMR unlikely	Consider biopsy to detect other pathologies
Rejection undergoing treatment	High	Ongoing rejection	Continued treatment of rejection
	Low	Resolution of rejection	Clinical monitoring

dd-cfDNA: Donor-derived cell-free DNA; TCMR: T-cell mediated rejection; AMR: Antibody mediated rejection.

It is also challenging to understand the clinical significance of patients with allograft histological inflammation but normal levels of dd-cfDNA. In such cases, traditional allograft biopsy regains its clinical significance. There is ongoing debate regarding whether absolute or fractional quantification of dd-cfDNA is preferable, and it is likely that measuring both is advantageous. In kidney transplantation, dd-cfDNA is released from damaged graft tissue into the recipient’s bloodstream and, in some cases, into the urine. Another point of discussion is the relevance and significance of urinary (transrenal) dd-cfDNA. Several cases of acute rejection with elevated urinary dd-cfDNA have been documented[56]. Further research is needed to better understand the significance of dd-cfDNA.

Determining which of the new biomarkers is most effective for studying graft rejection and graft outcomes remains an open question. All are likely useful, but the already cited study by Park et al[46] clearly indicates that the combination of dd-cfDNA and GEP enhances the diagnosis of subclinical rejection. An important point before concluding is represented by preanalytical considerations on circulating cfDNA. First is an optimal blood sampling, which in turn is different whether serum or plasma, the type of anticoagulant and the collection tube. Second is the handling of blood samples between drawing and processing. This point should consider the influence of blood sample storage conditions on dd-cfDNA concentration and the influence of blood sample storage conditions on dd-cfDNA fragmentation. Third point is the handling of plasma samples between blood processing and nucleic acid extraction[57]. These protocols should be standardized such as has been made for liquid biopsy guidelines in oncology[58].

CONCLUSION

dd-cfDNA is one of the most recent biomarkers in transplantation research, alongside GEP and urinary cytokines. dd-cfDNA is released from the allograft into the graft recipients and represents a small proportion of the total cfDNA. In the case of a stable kidney graft, dd-cfDNA constitutes 0.5% of the total cfDNA. Higher levels of dd-cfDNA following kidney transplantation are associated with graft damage, most frequently ABMR. Allograft dysfunction with normal levels of dd-cfDNA is generally not attributed to allograft rejection, as may be confirmed by renal biopsy. Studies on kidney transplant recipients have demonstrated that dd-cfDNA is a useful tool for detecting subclinical rejection often with an as yet normal histology.

Similarly, dd-cfDNA is an important tool for assessing the efficacy of treatment for rejection. Further research is needed to elucidate all the relationships between the clinical aspects of transplanted recipients and the varying levels of both fractional and total dd-cfDNA. Future multicenter studies and clinical trials on detecting dd-cfDNA in transplant patients with suspected graft rejection are still needed. The methods used in such studies are vital and any samples obtained should be in line with the biopsy results. Table 11 represents five clinical trials of dd-cfDNA measurement in transplantation downloaded from the ClinicalTrials.gov database. Finally, it is useful to conclude with some take-home messages as follows: (1) dd-cfDNA is released from the allograft into the blood of solid organ transplant recipients; (2) dd-cfDNA accounts for < 0.5% of total plasma cfDNA in kidney transplant recipients without injury of the graft; (3) Elevated dd-cfDNA in the case of allograft dysfunction indicates allograft pathology, particularly ABMR, upon allograft biopsy; (4) Normal dd-cfDNA levels in the case of allograft dysfunction does not indicate rejection, especially antibody mediated rejection; and (5) Preliminary studies in kidney transplant recipients with stable graft function, and in kidney transplant recipients receiving treatment for rejection, indicate the utility of dd-cfDNA in these conditions.

Table 11 Ongoing clinical trials.

NCT number	Title	Condition	Actual enrolment	Recruitment status	Location	Age of participants
NCT03765203	Utility of a novel dd-cfDNA test to detect injury in renal post-transplant patients	Kidney transplant failure	175 participants	Completed	United States	Child, adult, older adult
NCT04271267	Cell-free DNA as a biomarker after lung transplantation	Lung transplant recipients	125 participants	Completed	No data	Adult, older adult
NCT02424227	Non invasive blood test to diagnose acute rejection after kidney transplantation	Kidney transplant recipients	401 participants	Active, not recruiting	United States	Child, adult
NCT01985412	Non-invasive sequencing-based diagnosis of rejection	Cardiac transplant rejection; lung transplant rejection	65 participants	Completed	United States	Child, adult
NCT02109575	Quantitative detection of circulating donor specific DNA in organ transplant recipients	Cardiovascular disease; acute rejection of cardiac transplant; cardiac transplant rejection; heart transplant failure and rejection	No data	Active, not recruiting	United States	Child, adult, older adult

NCT: National Clinical Trial.