Donor-derived circulating cell-free DNA as a rejection biomarker and modulator of immunosuppression following liver transplantation: A randomized controlled trial

A. Background & rationale

Despite advancements in immunosuppressive therapies, acute cellular rejection still occurs in 10-40% of liver transplant patients. Although liver biopsy is the gold standard for diagnosing rejection, it is invasive, costly, associated with complications, causes discomfort, and has a long turnaround time. Immediately following transplantation, biopsies may be challenging due to low platelet counts, coagulopathy, and the presence of ascites.¹ Moreover, conventional liver function tests and biomarkers such as bilirubin and liver enzymes are nonspecific for rejection, as they can be influenced by various factors such as sepsis and biliovascular complications, highlighting an urgent need for noninvasive, reliable tools to monitor graft rejection and tailor immunosuppressive therapy.²

Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising noninvasive biomarker for monitoring liver transplant rejection. These small fragments of DNA, released from necrotic and apoptotic donor cells, increase in the bloodstream after transplantation and stabilize within a week. During rejection episodes, dd-cfDNA levels can rise to fivefold, allowing for early detection approximately 1 to 2 weeks before clinical symptoms or biochemical abnormalities manifest.³ With a short half-life of 1.5 hours, dd-cfDNA provides real-time insights into graft health and requires only a blood sample, with a turnaround time of one day.^4,5 Serial monitoring of dd-cfDNA can assess treatment efficacy, as persistent elevation indicates ongoing graft injury. Elevated dd-cfDNA on day 7 post-transplant is particularly useful for predicting rejection risk and differentiating rejection from other confounding conditions, thereby improving post-transplant prognostication and management.⁶

Previous research on donor-derived cell-free DNA (dd-cfDNA) in liver transplantation reveals promise and limitations. Kanamori et al.'s study across eight transplant centers in Japan (involving 19 patients) found that dd-cfDNA levels correlated with rejection activity but had limited efficacy in detecting sub-clinical rejection.⁷ Schütz et al.'s multicenter study in Germany (with 88 patients), demonstrated that dd-cfDNA had higher sensitivity and specificity for detecting graft injury compared to liver function tests (LFTs); however, it lacked time-matched samples of LFT and dd-cf DNA levels, and protocolized patient care.⁸ Baumann et al.'s larger cohort study (with 108 patients) at Hannover Medical School showed increased dd-cfDNA levels with graft rejection, but its accuracy in predicting sub-clinical damage was still limited, and no correlation with donor-specific antibodies was observed.⁹ These findings underscore the need to validate the diagnostic reliability of dd-cfDNA and its clinical utility in guiding immunosuppression modulation to prevent post-transplant rejection^.

This study is particularly relevant for various reasons. Firstly, it is the first randomized controlled trial that explores the potential of dd-cfDNA levels to preemptively modulate immunosuppression levels, an area that has not been investigated in the past. This study will thus analyze whether dd-cfDNA-directed immunosuppression can prevent rejection. Secondly, there will be a comparative assessment of liver integrity and morphology at one-year post-transplant, using Fibroscan, or CT-MRI, to assess whether dd-cfDNA modulated immunosuppression may have long-term beneficial effects. Thirdly, this study will analyze the integrity ratio of short and long DNA fragments, percentage of dd-cfDNA specific to allograft, and total dd-cfDNA to differentiate from other potential causes of raised dd-cfDNA levels as well as to correlate with types of rejection. Finally, it aims to establish optimal thresholds and ideal sampling frequencies for cfDNA, which remain poorly defined in the current literature.

Harm & Benefits: There is no harm at all, as the study involves only blood samples which will anyway be performed during the routine follow-up as part of standard protocol. The benefits will include avoidance of invasive liver biopsy if the study proves the benefit of dd-cfDNA in the diagnosis of rejection. Furthermore, early detection may aid in the prevention of graft cellular damage by preemptive immunosuppression modulation.

B. Explanation of choice of comparators

The study compares a test group and a standard group in liver transplant patients. The standard group follows protocolized care, adjusting immunosuppression based on trough levels, and clinical, and biochemical parameters. The test group undergoes additional monitoring with dd-cfDNA, allowing earlier immunosuppression adjustments. In both groups, liver biopsies for rejection diagnosis are based on LFTs. This approach in the test group may enable earlier intervention, potentially reducing rejection rates.

Interventions

A. Interventions for each group

Post liver transplantation, both groups of patients will initially be started on immunosuppression as follows:

Group 2 (Standard group): Patients are monitored using standard liver function tests alone. A biopsy will be performed if there is a two-fold rise in liver function tests (aminotransferases, alkaline phosphatase, gamma-glutamyl transferases) without evident causes like sepsis or graft-related issues (such as biliovascular problems). Biopsy-proven rejection will be treated as elaborated in Group 1.

Criteria for discontinuation- There are no criteria for discontinuation, as the study involves only collecting blood samples for dd-cfDNA analysis along with those required for follow-up lab investigations and immunosuppression modulation will be followed according to the protocol. However, there may be a requirement to change or even cease immunosuppression in case of adverse effects which will be captured in the data sheet for analysis.

B. Strategies to improve adherence to protocol- Regular follow-ups and tests, educating participants on the protocol's importance, and using reminders (SMS, calls) for appointments and medication.

D. Relevant concomitant care and interventions prohibited/permitted during the trial

Outcomes

a. Efficacy of dd-cfDNA in indicating adequate immunosuppression levels.

b. Detection of biochemical rejection.

c. Graft morphology at 1 year.

Participant timeline

The inclusion and exclusion criteria are outlined in Figure 1, while the detailed methodology of the study is presented in Figure 2. Additionally, the SPIRIT figure, which illustrates the timeline for eligibility screening, patient enrollment, and assessment, is provided in Table 1.

Figure 1. Inclusion and exclusion criteria.

Figure 2. Methodology of the study.

Sample size & its determination

Based on the proportion of rejection in the treatment group (33%) and in the control group (13%) in liver transplant patients, was observed in a pilot study conducted with 30 samples (15 in each group) and with 80% power and 95% confidence, the sample size comes to 136 with 68 in each group.

Recruitment

All patients undergoing live donor liver transplantation in the Department of Gastrointestinal Surgery, Amrita Institute of Medical Sciences and Research will be evaluated.

Allocation

Blinding

Data collection methods

A. Collection & Assessment of Outcomes

Blood samples will be collected on designated postoperative days and during follow-up visits, with demographic and clinical data recorded on a predefined form and in Excel. To ensure data quality, the study will implement standardized protocols, conduct audits every three months, utilize dual entry methods, maintain thorough documentation, and involve a biostatistician for data analysis.

B. Plans to promote participant retention & follow-up

To ensure participants fully understand the study's purpose, procedures, and benefits, detailed information will be provided upfront. Regular follow-up visits and check-ins, via phone or messages, will help maintain engagement and monitor participants' well-being. Flexible appointment times will be offered to accommodate participants' schedules, reducing barriers to attendance, while appointment reminders will be sent via phone calls to ensure follow-up compliance. Direct contact will also be made available for participants to discuss any concerns or issues they encounter during the study.

Outcome data to be collected if the patient deviates or discontinues

For participants who discontinue or deviate from the trial, the specific reasons for withdrawal, such as adverse events or personal reasons, will be documented. The last available dd-cfDNA levels, LFT results, and Tacrolimus (TAC) levels will be collected, alongside any clinical assessments or adverse events experienced before discontinuation. Follow-up information on the participant's health status post-discontinuation will also be gathered. Updated demographic data will be maintained to assess any potential biases or trends among participants who deviate from the study.

Plans for data entry, and security & including any related processes to promote data quality

Standardized data entry forms will ensure uniform data collection across participants. A double data entry process will be used, with researchers entering data both on paper and in Excel for validation and error-checking. Automated checks will detect implausible values, and regular audits will correct errors promptly. Data entry personnel will receive training on procedures, with a feedback system in place for ongoing improvement.

Data security will be ensured through restricted access for authorized personnel only, with encryption protecting data during storage and transmission. Secure servers with firewalls and antivirus software will guard against unauthorized access, and regular backups will enable recovery from system failures. Allocation of unique ID numbers will be done to safeguard participant confidentiality. Physical data copies will be stored in locked locations, and long-term storage plans will be developed for future research and data sharing.

Statistical methods

To test the statistical significance of the difference in the proportion of rejection between the two groups, the Chi-square test will be used.

For secondary outcomes, appropriate models will be applied:

Subgroup analyses will be conducted to explore potential variations in treatment effects across different groups (e.g., age, sex, disease severity). Stratified analysis may also be performed to compare subgroups independently. The adjusted analysis will account for potential confounding variables using multivariable regression models to adjust for pre-specified covariates (such as age, baseline disease status, graft parameters, and comorbidities). Sensitivity analysis may be performed to explore the robustness of the findings under different modeling assumptions.

The primary analysis will follow the intention-to-treat (ITT) principle, analyzing participants based on their original randomization group, regardless of intervention adherence. A per-protocol analysis may also be performed as a secondary analysis for those who adhered to the protocol. Multiple imputation will be used to handle missing data, assuming it is missing at random (MAR), by generating and analyzing multiple complete datasets and then pooling the results. Sensitivity analyses may assess the impact of different assumptions about missing data, such as missing not at random (MNAR).

Data monitoring

Interim analyses will occur every 6 months to assess safety and early efficacy. Only the DMC will access these results, keeping researchers, sponsors, and participants blinded to maintain study integrity. The DMC can recommend study modifications or early termination if interim data show harm, overwhelming efficacy, or futility. The final decision to terminate will be made jointly by the DMC, principal investigators, and ethics board based on the interim findings.

Plans to collect, assess, report & manage adverse events and other unintended effects of trial

Adverse events are not expected in this trial, as the study involves only the modulation of immunosuppression based on cfDNA analysis, which is validated with LFTs and liver biopsies. Since no new drugs or invasive interventions are introduced, the risk of unintended side effects is minimal. However, any serious adverse events (SAEs) related to routine clinical care will still be recorded and reported per ethics committee and regulatory guidelines to ensure patient safety.

Frequency and procedures of auditing trial conduct, and whether it will be independent of investigators and sponsors

Auditing procedures, conducted every 6 months will include a review of the trial protocol, regulatory submissions, and guidelines, along with document checks to ensure compliance with protocols, CRFs, and informed consent forms. Data will be verified to match source documents, and participants' rights and consent will be checked for adherence. Safety monitoring will involve reviewing the management of adverse events (AEs) and serious adverse events (SAEs), while data management systems will be inspected for confidentiality and integrity. Findings will be reported with recommendations, and corrective actions will be taken for non-compliance, followed by additional audits if necessary. Independent auditors from the institutional ethics committee will oversee these audits to ensure objectivity.

Plans to seek IEC approval: The study was approved by the Institutional Ethics Committee (IEC) of Amrita Institute of Medical Sciences [IEC-AIMS-2024-PHARMA-304] on 26.11.2024

Institutional Ethics Committee statement:

The Ethics Committee reviewed the documents pertaining to the study protocol titled ‘Donor-derived circulating cell-free DNA as a rejection biomarker and modulator of immunosuppression following liver transplantation: A randomized controlled trial’ presented by Dr.Lakshmi.V.U as investigator with Dr. Dinesh Balakrishnan, Dept of GI surgery and Dr.Narmadha.M.P, School of Pharmacy as the guides.

After reviewing the protocol and hearing the Investigator, the Committee finds that the study is a randomized controlled study with the primary objective to assess the efficacy of donor-derived cell-free DNA (dd-cfDNA) modulated immunosuppression in reducing acute T-cell mediated rejection following liver transplantation. Additionally, the study aims to evaluate and compare the effectiveness of dd-cfDNA as an early biomarker for detecting allograft rejection in liver transplant recipients, in contrast to conventional liver function tests.

PI submits that dd-cfDNA analysis has not been studied in the Indian population with a significant sample from a high-volume transplant center in a tertiary care setting, especially in live donor liver transplant. Internal Scientific Review Report says that the study benefits early detection of allograft rejection, prompt identification of steroid reftactory rejection and tailoring of immunosuppression. The study is an academic study in public interest. No ethical issues are noted. Approval granted. PI to ensure that the data collected are anonymized and confidential. Data privacy of the subjects shall be protected. It is mandatory that PI shall submit interim report at 6-months intervals about the progress of the study and a detailed final report upon closure of the study to the Ethics Committee.

Plans to communicate important protocol modifications (Ex: Changes in eligibility centers, outcome analysis) to relevant parties

Any important protocol modifications, such as changes in eligibility criteria, study centers, or outcome analysis, will be promptly communicated to all relevant parties. This includes notifying investigators, the Research Ethics Committee (REC)/Institutional Review Board (IRB), trial registries, regulatory authorities, and journals. Updated protocols will be submitted for approval, and participants will be re-consented if necessary. All changes will be documented and publicly reported as required by trial registries and journals.