Abstract
Diagnosis of subclinical renal rejection for early treatment can be difficult due to the stable serum creatinine levels. Although regarded as the gold standard, biopsy is not deemed ideal for cases where continuous monitoring is required due to its invasiveness. Here, we present a case report of a renal transplant recipient with a stable serum creatinine level but elevated donor-derived cell-free DNA (5.1%) who was monitored for rejection and response to treatment, guided by donor-derived cell-free DNA testing during an extended period. Antibody testing revealed de novo donor-specific antibodies (A11, mean florescence intensity of 1600) that were confirmed by allograft biopsy as subclinical antibody-mediated rejection. The patient was treated with rituximab, and the therapeutic efficacy was assessed every 6 months with donor-derived cell-free DNA and biopsy analysis. Eight months after treatment, a decrease in donor-derived cell-free DNA levels was observed (3.61%), which approached reference levels (<1%). Twenty-eight months after the first treatment, donor-derived cell-free DNA increased, and biopsy analysis of last donor-derived cell-free DNA monitoring time showed antibody-mediated rejection, which subsequently decreased following the second rituximab treatment. Subsequent follow-ups revealed the donor-derived cell-free DNA level was stabilized after the second treatment. This finding suggested that donor-derived cell-free DNA could serve as a valuable diagnostic marker for continuous subclinical antibody-mediated rejection monitoring and for evaluation of treatment responses.

Key words : Donor-specific antibody, Evaluation of treatment response, Subclinical antibody-mediated rejection

Introduction
Subclinical rejection is diagnosed in ~29% of kidney transplant patients and is characterized as asymptomatic with stable serum creatinine levels.¹ Reliance solely on clinical signs may interfere with an accurate diagnosis. Allograft biopsy, especially protocol biopsy, is considered a crucial diagnostic method but is not recommended for cases where continuous monitoring is required because of its invasiveness and the potential risks involved. Moreover, assessment of the efficacy of a treatment regimen for subclinical rejection presents its own challenges due to the absence of clear clinical indicators.² Donor-derived cell-free DNA (ddcfDNA) has been reported to be closely associated with antibody-mediated rejection (ABMR) and serves as an auxiliary diagnostic tool for ABMR.^3-5 Hence, although biopsy is not considered ideal for continuous monitoring of the therapeutic effect of an administered drug against ABMR owing to its invasive nature,^6,7 ddcfDNA could serve as a safer and more reliable surveillance tool to monitor graft status and to design personalized therapies. However, few studies have reported on the use of ddcfDNA to diagnose subclinical ABMR and to assess its effectiveness in monitoring the therapeutic efficacy of an administered drug. In this case report, we present a renal transplant patient who was diagnosed with subclinical ABMR through ddcfDNA monitoring and subsequently underwent treatment guided by serial ddcfDNA testing. This study was approved by the Ethics Committees of Jinling Hospital, Nanjing University School of Medicine. Informed consent was obtained from each of the participants.

Case Report
A 64-year-old man underwent kidney transplant in 2015 and exhibited smooth postoperative recovery with normalization of serum creatinine, despite having 4 human leukocyte antigen (HLA) mismatches with the donor. The donor kidney used for this study was obtained from a patient with confirmed brain death, informed consent was obtained from the donor’s family before organ procurement, and the procedure followed the 1975 Helsinki Declaration. However, 1 year after transplant, our patient was diagnosed with renal allograft artery stenosis, accompanied by a 25% increase in serum creatinine levels. After administration of interventional dilation treatment, the patient’s serum creatinine levels returned to reference levels. Subsequently, we modified the immunosuppression regimen from tacrolimus combined with mycophenolate mofetil (MMF) and methylprednisolone pulse therapy (MPPT) to rapamycin combined with MMF and MPPT. Notably, the graft function remained stable throughout the follow-up period.
In 2018, the ddcfDNA test was implemented for clinical purposes in China. The patient was subjected to ddcfDNA testing, which showed elevated levels (5.1%); however, the patient’s serum creatinine level remained stable. Subsequently, the HLA antibody test indicated the presence of donor-specific antibodies (DSA) (A11, mean florescence intensity = 1530). A subsequent graft biopsy confirmed the diagnosis of ABMR (according to Banff 2019) with 40% of peritubular capillaries (PTC) showing C4d positivity (Figure 1A, 1-4). The patient was treated with a combination of 200 mg of rituximab and intravenous immunoglobulin with an immunosuppression regimen that included the switch to tacrolimus combined with MMF and MPPT. During follow-up, ddcfDNA levels were assessed in the first month after treatment and subsequently every 6 months, in addition to regular monitoring of renal function and drug concentration, biopsy analysis, and HLA antibody tests. Clinical treatment decisions were guided by observation of ddcfDNA levels and biopsy analysis.
One month after rituximab treatment, the patient showed an increased level of ddcfDNA during the initial examination. Despite the appearance of DSA (A11), the patient’s graft function remained stable. Therefore, the treatment plan was not altered. After 6 months, there was a significant decrease in ddcfDNA from 5.71% to 3.61%. Subsequently, ddcfDNA was assessed every 6 months, which was observed to consistently decrease over time. At 24 months after treatment, ddcfDNA decreased to 1.03%, which approached reference levels (<1%). At this point, a repeat biopsy of the transplanted kidney was performed, which revealed a decrease in pathological features of rejection with resolution of tubulitis. The C4d staining changed from an original positive result in 40% of PTC to a negative result; however, glomerulonephritis classified as mild (grade 1) and PTC (>10%) remained positive (Figure 1A: 2-2, 2-3, and 2-4).
After a surveillance duration of 8 months (during which the lowest level of ddcfDNA was 1.03%), we observed that ddcfDNA had increased by 48.5% during the sixth assessment of ddcfDNA. Considering that the last ddcfDNA assessment in combination with biopsy analysis (8 months earlier) indicated ABMR, we promptly administered rituximab at a dose of 200 mg, which resulted in a subsequent decrease in ddcfDNA. The patient’s status was monitored with follow-up sessions for the next 26 months, during which stable ddcfDNA levels and negative antibody status were consistently observed (Figure 1B).
Although most studies have reported a ddcfDNA level of <1.0% as indicative of stability and nonrejection, we observed a substantial decline in ddcfDNA after the first combination treatment with MMF, intravenous immunoglobulin, and rituximab; this observation, along with the subsequent stabilization after the second phase of treatment with rituximab, likely indicated a restoration of graft health.

Discussion
As a biomarker of renal allograft injury, ddcfDNA is closely associated with ABMR; however, reports on the predictive role of ddcfDNA in subclinical rejection are limited. Our study presents evidence to suggest that subclinical rejection may be detected in patients with stable renal function by monitoring ddcfDNA levels and can be confirmed with biopsy. This case study reveals that ddcfDNA could be an important diagnostic tool and may facilitate the early detection and long-term monitoring of subclinical ABMR.
Evaluation of the efficacy of treatment for rejection is clinically challenging, especially in cases of subclinical rejection for which routine indicators of rejection are stable. Moreover, traditional observation methods, such as serum creatinine levels, DSA changes, and repeated biopsies, have limited value for assessment of the treatment outcomes for subclinical rejection. Considering that serum creatinine levels remain stable in these cases, it is difficult to accurately evaluate these effects before and after treatment. Only a small percentage of patients exhibit decreased DSA levels after treatment, and the correlation between such changes and treatment effectiveness is mostly weak.⁸ Repeated biopsies are invasive and challenging and incur potential risks.
In our patient, short-term reexamination showed an initial increase in ddcfDNA after the first treatment, which subsequently decreased during the following 6 months, and it has been established that rituximab takes some time to exert its effect on rejection.^9,10 Such observations may also reflect the responsiveness of ddcfDNA during active rejection episodes, because, at the time of the second ddcfDNA assessment, rejection activity continued to increase (Figure 1B). After 20 months of rituximab treatment, ddcfDNA decreased to 1.03%, approaching reference levels. In addition, the patient underwent repeat allograft biopsy. The disappearance of tubulitis may indicate a favorable response to treatment, whereas the PTC score changed from PTC2 to PTC1 following the Banff 2019 criteria,¹¹ and the C4d score changed from positive (40%) to negative. Overall, the changes in ddcfDNA levels were consistent with the pathological indicators, suggesting a positive response to treatment and an improvement to the health status of the graft.
Compared with biopsy, which is a procedure characterized by difficulty to obtain timely data on graft status and thereby potentially impede precise treatment, ddcfDNA offers the potential to monitor therapy progression in a continuous noninvasive manner, due to quick and easy accessibility to the patient’s sample for precise analysis and thereby facilitate determination of the optimal treatment dosage to maintain patient stability.
During the follow-up visits with our patient, ddcfDNA levels remained fairly stable, but any increase was considered a sign of potentially active rejection episodes, which led to a decision to administer rituximab. Of note, we did not perform a new biopsy (in August 2020), because a 6-month interval is not considered a sufficient period to warrant an additional biopsy. After treatment, however, ddcfDNA levels decreased, which indicated further improvement in renal health in response to rituximab treatment. Although the ddcfDNA levels throughout the treatment and monitoring period did not fall below the 1% mark, which is proposed to be indicative of graft stability and renal health restoration, the substantial decrease of ddcfDNA from 5.71% to 1.03% and subsequent stabilization following treatment indicates its potential utility to monitor graft status. Therefore, we propose that ddcfDNA could serve as a reliable biomarker to monitor subclinical rejection and to evaluate recipients’ responses to therapy.

Conclusions
The detection of ddcfDNA is beneficial for identification of subclinical ABMR and evaluation of therapeutic efficacy against subclinical rejection. Therefore, measurement of ddcfDNA may have potential diagnostic and evaluative value for subclinical rejection. This study had limitations, as there were no further confirmations using biopsy after rituximab treatment to confirm the procedure and its role to decrease ABMR. Moreover, only a single patient was involved in this case study, representing a limitation with regard to error margin and the absence of a control component. As a result, we recommend that future clinical studies on long-term renal graft surveillance and treatment response consider population size in an effort to improve comparative assessment on the reliability of ddcfDNA as a biomarker of allograft status. Nonetheless, this case report supports the most recently published reports on the utilization of ddcfDNA in kidney transplant subclinical rejection monitoring.

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