CTRI/2025/02/080893 [Registered on: 19/02/2025] Trial Registered Prospectively
Last Modified On:
17/02/2025
Post Graduate Thesis
Yes
Type of Trial
Interventional
Type of Study
Surgical/Anesthesia Diagnostic Preventive
Study Design
Randomized, Parallel Group Trial
Public Title of Study
Donor-Derived Cell-Free DNA: A Breakthrough Biomarker for Early Rejection Detection and Immunosuppression Management in Liver Transplant Patients
Scientific Title of Study
DONOR-DERIVED CIRCULATING CELL-FREE DNA AS A REJECTION BIOMARKER AND IMMUNOSUPPRESSION MODULATION TOOL IN LIVER TRANSPLANTATION
Trial Acronym
NIL
Secondary IDs if Any
Secondary ID
Identifier
NIL
NIL
Details of Principal Investigator or overall Trial Coordinator (multi-center study)
Name
Dr Dinesh Balakrishnan
Designation
Clinical Professor- Department of Gastrointestinal surgery
Affiliation
Amrita Institute of Medical Sciences and Research Centre: Amrita Institute of Medical Sciences
Address
Department of Gastrointestinal Surgery and Solid Organ Transplantation, G-block:2nd floor, Amrita institute of Medical Sciences and Research, Ponekkara P.O, Ernakulam, Pin-682041
Ernakulam KERALA 682041 India
Phone
9946861474
Fax
Email
dineshb@aims.amrita.edu
Details of Contact Person Scientific Query
Name
Dr.Sudhindran S
Designation
Chief Transplant Surgeon-Department of Gastroinetstinal Surgery
Affiliation
Amrita Institute of Medical Sciences and Research Centre: Amrita Institute of Medical Sciences
Address
G-block,2nd floor, Room No:10 Amrita Institute of Medical Sciences and Research, Ponekkara P.O, Ernakulam, Pin-682041
Ernakulam KERALA 682041 India
Phone
7306716242
Fax
Email
sudhisalini@icloud.com
Details of Contact Person Public Query
Name
Dr. Lakshmi.V.U
Designation
Research scholar- Department of Gastrointestinal surgery
Affiliation
Amrita Institute of Medical Sciences and Research Centre: Amrita Institute of Medical Sciences
Address
Department of Gastrointestinal surgery-block, 2nd floor Amrita Institute of Medical Sciences and Research, Ponekkara P.O, Ernakulam, Pin-682041
Ernakulam KERALA 682041 India
Phone
8304023632
Fax
Email
laxmisadhi123@gmail.com
Source of Monetary or Material Support
Amrita Centre for NanoSciences and Molecular Medicine, B-block, 1st floor, Amrita Institute of Medical Sciences and Research, Ponekkara P.O, Ernakulam, Pin-682041
The Liver Transplant Society-India, Room No:1246,2nd floor Indraprastha,Apollo Hospotal,Sarita Vihar, New Delhi, Pin-110076
Primary Sponsor
Name
Liver Transplant Society-India
Address
Room number 1246, 2nd Floor Indraprastha Apollo Hospital Sarita Vihar New Delhi 110076 India
Type of Sponsor
Research institution and hospital
Details of Secondary Sponsor
Name
Address
Amrita institute of medical sciences and research
Amrita institute of medical sciences, Ponekkara P.O, Ernakulam, Pin-682041
Countries of Recruitment
India
Sites of Study
No of Sites = 1
Name of Principal
Investigator
Name of Site
Site Address
Phone/Fax/Email
Dr Dinesh Balakrishnan
Amrita Institute of Medical Sciences Ethics Committee
Department of Gastrointestinal Surgery and Solid Organ Transplantation, G-block,2nd floor, Room No:10 Amrita institute of medical sciences, Ponekkara P.O, Ernakulam, Pin-682041 Ernakulam KERALA
8304023632
dineshb@aims.amrita.edu
Details of Ethics Committee
No of Ethics Committees= 1
Name of Committee
Approval Status
Amrita Institute of Medical Sciences Ethics Committee
Approved
Regulatory Clearance Status from DCGI
Status
Not Applicable
Health Condition / Problems Studied
Health Type
Condition
Patients
(1) ICD-10 Condition: O||Medical and Surgical,
Intervention / Comparator Agent
Type
Name
Details
Intervention
donor-derived cell-free DNA and liver function tests
Blood samples (9 ml) were collected on postoperative days 3, 7, 14, 30, and 90, and dd-cfDNA was quantified. If dd-cfDNA levels were found to be elevated, suggesting a possible early rejection, immunosuppression was adjusted as follows:
1.The primary goal of immunosuppression modulation was to maintain Tacrolimus (TAC) blood levels at 8 ng/ml. If TAC levels were already at this target, immunosuppressive therapy was intensified by either increasing the dosage or adding alternative immunosuppressants such as steroids or Mycophenolate Mofetil (MMF).
2.The steroid dose was increased by additional 10 mg of prednisolone, or Mycophenolate Mofetil was increased to 500 mg thrice daily. Adjustments were made based on platelet and total WBC counts.
3. If dd-cfDNA levels were elevated after postoperative day 30 and the recipient was already on adequate triple immunosuppression, an mTOR inhibitor (Everolimus) was added. This was avoided within the initial 30 days post-transplant due to the risk of impaired wound healing.
4.Granzyme-B levels were to be done in patients who underwent a rejection to differentiate antibody-mediated rejection, so that each type of rejection could be treated accordingly.
Simultaneously, if liver function tests (LFTs) showed significant abnormalities, particularly a more than two-fold rise in LFTs without evident causes such as sepsis or graft-related issues (e.g., biliovascular problems), a liver biopsy was performed to confirm rejection. The biopsy results were used to calculate the Rejection Activity Index (RAI) to quantify rejection severity. Biopsy-proven rejection was treated according to the units protocol with Methylprednisolone pulse doses of 10 mg/kg for 3-5 days.
If dd-cfDNA levels were normal, patients continued with standard immunosuppressive therapy and were followed up regularly to ensure stable transplant function. dd-cfDNA analysis included assessment of 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 normal profiles. This facilitated precise identification of the presence and type of rejection in each patient. This intervention is to be carried out in each subject over a period of 3 months and then the morphology of allografts in both groups will be compared with CT/MRI or Fibro Scan. The total duration of the study will be 2 years.
Comparator Agent
Liver function tests alone
Patients were monitored using standard liver function tests alone, according to standard protocol. This included liver function tests daily for the initial 2 weeks post-transplantation. Then these tests were done twice a week, then once a week, then once in 2 weeks and later on monthly basis for a year, unless any complication was suspected, and additional testing is required in that case. Biopsies were performed if there was a two-fold rise in LFTs without evident causes such as sepsis or graft-related issues (e.g., biliovascular problems). This intervention is to be carried out in each subject over a period of 3 months and then the morphology of allografts in both groups will be compared with CT/MRI or Fibro Scan. The total duration of the study will be 2 years.
Inclusion Criteria
Age From
18.00 Year(s)
Age To
80.00 Year(s)
Gender
Both
Details
All adults admitted to undergo liver transplantation
ExclusionCriteria
Details
Multi-organ transplantation, identical twin donor and recipient, ABO-incompatible transplantation
Method of Generating Random Sequence
Computer generated randomization
Method of Concealment
Sequentially numbered, sealed, opaque envelopes
Blinding/Masking
Not Applicable
Primary Outcome
Outcome
TimePoints
Primary Outcome: Efficacy of dd-cfDNA in indicating adequate immunosuppression levels
Post-operative day 3, 7, 14,30 and 90
Secondary Outcome
Outcome
TimePoints
a) Early detection of allograft rejection compared to liver function tests.
Post-operative day3,7,14,30 and 90
b) Correlation of Granzyme-B levels and antibody mediated rejection
Post-operative day 3,7,14,30 and 90
c)Comparison of graft morphology 1-year post-transplant in both groups
1-year post-transplantation
Target Sample Size
Total Sample Size="136" Sample Size from India="136" Final Enrollment numbers achieved (Total)= "Applicable only for Completed/Terminated trials" Final Enrollment numbers achieved (India)="Applicable only for Completed/Terminated trials"
Phase of Trial
Phase 4
Date of First Enrollment (India)
28/02/2025
Date of Study Completion (India)
Applicable only for Completed/Terminated trials
Date of First Enrollment (Global)
Date Missing
Date of Study Completion (Global)
Applicable only for Completed/Terminated trials
Estimated Duration of Trial
Years="2" Months="0" Days="0"
Recruitment Status of Trial (Global)
Open to Recruitment
Recruitment Status of Trial (India)
Open to Recruitment
Publication Details
N/A
Individual Participant Data (IPD) Sharing Statement
Will individual participant data (IPD) be shared publicly (including data dictionaries)?
Response - NO
Brief Summary
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 rejection in 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.