DONOR-DERIVED CIRCULATING CELL-FREE DNA AS A REJECTION BIOMARKER AND IMMUNOSUPPRESSION MODULATION TOOL IN LIVER TRANSPLANTATION

Following liver transplantation, acute cellular rejection remains a significant challenge, affecting 10-40% of recipients despite advancements in immunosuppressive therapies. While liver biopsy is the gold standard for diagnosing rejection, it is invasive, costly, and associated with potential complications. Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising noninvasive biomarker, providing real-time insights into graft health through simple blood sampling. This randomized controlled trial aims to evaluate the efficacy of dd-cfDNA-modulated immunosuppression in reducing acute T-cell mediated rejection. Participants undergoing live donor liver transplantation will be randomized into two groups. Immediately after liver transplantation, both groups of patients will initially be started on immunosuppression as follows :

• Tacrolimus will be started at 2mg/day in a divided or single dose depending on the formulation to aim for a trough level of 8ng/ml. The dosage will be altered depending on the renal function.
• Methylprednisolone will be started at 250 mg IV on Day 1, 125mg IV on Day 2,  80mg IV on Day 3, and 40 mg IV on Day 4 . Subsequently, oral prednisolone is started at 30mg daily and tapered weekly by 5mg and stopped at 6 weeks.
• Mycophenolate Mofetil (MMF) will be started at 500 mg twice daily from day 1, provided the platelet count is more than 25,000/mm3. It will be stopped at 4-6 months. Due to the complexity of the interaction between immunosuppressants and other drugs and hepatic and renal function, some degree of immunosuppression tweaking will be left to the physician’s discretion.

On postoperative day 3, the participants will be randomly assigned into two groups using a computerized randomization technique. The assigned group will be enclosed in sealed, opaque envelopes opened only by the study coordinator.

**Group 1 (Test group):**Blood samples (9ml) will be collected at the post-operative days 3,7,14,30 & 90 and the dd-cfDNA will be quantified. At any point during the analysis, if dd-cfDNA levels are found to be elevated, this possibly suggests an early rejection, and immunosuppression will be adjusted as follows:

• Immunosuppression modulation aims to maintain Tacrolimus (TAC) blood levels at 8 ng/ml. If TAC levels are already at this target level, immunosuppressive therapy will be intensified by either increasing the dosage or adding alternative immunosuppressants such as steroids or Mycophenolate mofetil (MMF) depending on clinical parameters
• Steroid dose will be increased by 10mg of prednisolone or Mycophenolate mofetil will be increased to 500mg thrice a day.
• This will be based on the platelet count and total WBC count. If the dd-cfDNA levels are elevated on postoperative day 30 or 90, and the recipient is already on adequate triple immunosuppression, an mTOR inhibitor (Everolimus) will be added. This is a relative contraindication within the initial 30 days of transplant due to poor wound healing.

Simultaneously, if liver function tests (LFTs) show significant abnormalities, particularly a more than two-fold rise in LFTs without evident causes like sepsis or graft-related issues (such as biliovascular problems), a liver biopsy will be performed to confirm a rejection. The biopsy results will be used to calculate the Rejection Activity Index (RAI) to quantify the severity of the rejection. Biopsy-proven rejection will be treated according to the unit protocol: methylprednisolone pulsed doses at 10 mg/kg for 3-5 days. If dd-cfDNA levels are found to be normal, the patient will continue with standard immunosuppressive therapy, with regular follow-up to ensure stable transplant function.

dd-cfDNA analysis will involve assessing parameters like total cfDNA, dd-cfDNA percentage, integrity ratio, T-cell markers, and Granzyme-B to differentiate between infection, T-cell-mediated rejection, antibody-mediated rejection, and a normal profile. This will help precisely identify the presence and type of rejection in each patient.

Group 2(Standard group): Patients are monitored using standard liver function tests alone. The 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 graft-related issues (such as biliovascular problems) or sepsis (based on elevated WBC, CRP or procalcitonin, and culture reports) Biopsy-proven rejection will be treated as elaborated in Group 1.

Steroid-resistant rejection is defined as a form of acute rejection in which the liver transplant recipient does not respond adequately to three aliquots of pulsed steroids This will be confirmed by liver biopsy and treated usually by Antithymocyte globulin (ATG). The measurement of the study outcome will be the number of biopsy-proven rejections in the test group & standard group.

We have completed a pilot study as per our previous protocol with CTRI registration.l The study included a total of 50 patients, with 25 allocated to the standard care group and 25 to the test group. In the test group, 8 patients experienced allograft rejection, of whom 4 cases were confirmed via liver biopsy (T-cell-mediated rejection and antibody-mediated rejection). Biopsy could not be performed in the remaining 4 patients due to complications such as sepsis and thrombocytopenia. In the standard group, 6 patients experienced allograft rejection, with 4 cases confirmed by liver biopsy (all T-cell-mediated rejection). Among the 8 patients who experienced rejectionin the test group, dd-cfDNA levels increased before abnormalities in liver function tests (LFTs) in 3 patients**, with elevations occurring 5 days, 6 days, and 20 days before LFT changes were detected.**Exclusions were necessary for 3 patients from the standard group and 1 from the test group due to mortality within 75 days post-transplantation. Importantly, there were no false-positive or false-negative results in rejection detection using dd-cfDNA.Among patients who did not experience rejection, dd-cfDNA levels steadily declined from approximately 10% on postoperative day 7 to less than 1% by day 180. In contrast, a marked elevation in dd-cfDNA levels was observed in patients undergoing rejection. Additionally, Granzyme-B levels were significantly elevated during antibody-mediated rejection, rising from the normal range of 20–40 pg/mL to 140 pg/mL.