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

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.

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.

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.

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.

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%

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.

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.

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.

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.

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.

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.