Clinical trial NCT00320606
Conditions: Liver Transplant Liver Transplantation
Keywords: immunosuppression withdrawal (IS) anti-rejection drugs tolerance allograft function
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2006-06-05; Completion date: 2017-03-13
Enrollment: 20 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Antirejection medicines, also known as immunosuppressive drugs, are prescribed to organ transplant recipients to prevent their bodies from rejecting the new organ. Long-term use of these drugs places transplant recipients at higher risk of serious infections and certain types of cancer. The purpose of this study is to determine whether immunosuppressive drugs can be safely withdrawn over a minimum of 9 months from children who received liver transplants at least 4 years ago.

Detailed description:
In order to prevent the rejection of transplanted organs, transplant recipients are prescribed a strict, lifelong regimen of immunosuppressive drugs. While these drugs help prevent the body from rejecting the transplant, they carry numerous complications, including increased risk of serious infections and certain types of cancer. However, there is mounting evidence that a significant percentage of liver transplant recipients can maintain a healthy, functioning transplant without ongoing immunosuppression. This study will determine whether gradual withdrawal and eventual discontinuation of all immunosuppressive medication can be safely accomplished in children who received a liver transplant from a parent. Twenty eligible participants who were under 18 years old at the time of transplant, whose donor was a parent, and who received the transplant at least four years ago will be enrolled in the study.

Liver recipients will have an initial screening assessment consisting of a medical history, liver biopsy, and urine and blood collection. Eligible recipients will be placed on a modified medication schedule to gradually decrease their immunosuppression medication slowly over a 9- to 12-month period, during which time they will be closely monitored by study staff. Immunosuppressive drugs will not be provided by this study. For a minimum of 3 and up to a maximum of 7 years, monthly telephone consultations and quarterly study visits will occur. Visits will include physical exams and blood collection to monitor the children's health during the withdrawal phase. The exact schedule of immunosuppressant withdrawal will be determined by study physicians based on participant's health and immune function test results. Donor and nondonor parents will be asked to each provide one blood sample during the initial study visits for immunologic and genetic testing.

\*\*\* IMPORTANT NOTICE: \*\*\* The National Institute of Allergy and Infectious Diseases and the Immune Tolerance Network do not recommend the discontinuation of immunosuppressive therapy for recipients of cell, organ, or tissue transplants outside of physician-directed, controlled clinical studies. Discontinuation of prescribed immunosuppressive therapy can result in serious health consequences and should only be performed in certain rare circumstances, upon the recommendation and with the guidance of your health care provider.

Arms: arm 1: Recipients of parental living donor liver transplants 4 or more years prior to trial enrollment, who also had stable allograft function during the preceding 6 months while taking a single immunosuppressive drug were permitted to undergo withdrawal of immunosuppression therapy. With high dose, daily dose reduction by 25% for 8 weeks. With low dose, daily dose reduction by 25% for 4 weeks.

Participants are carefully evaluated/monitored throughout the study by assessments including but not limited to liver biopsy, liver tests and clinic visits, alloantibodies, autoantibodies and quantitative immunoglobulin G test results.
Interventions: intervention 1: Immunosuppression Withdrawal
Intervention descriptions: intervention 1: Gradual withdrawal of immunosuppressive medication. With high dose, daily dose reduction by 25% for 8 weeks. With low dose, daily dose reduction by 25% for 4 weeks.

Protocol documents:
Prot_000.pdf: 
 
Immune Tolerance Network
Protocol ITN029ST 
Immunosuppression Withdrawal for Pediatric Living-donor  
Liver Transplant Recipients 
Short Title: Immunosuppression Withdrawal for Pediatric Liver Recipients 
Version  5.0  (April 19, 2011) 
This clinical study is supported and conducted by the Immune Tolerance Network, which is sponsored by the 
National Institute of Allergy and Infectious Diseases. 
Protocol Chair 
 
 
 
 
 
 
 
ITN Clinical Trial Physician 
 
 
 
 
 
NIAID Medical Monitor 
 
 
 
  
 
 
ITN Manager of Clinical Operations 
 
 
 
 
 
NIAID Project Manager 
 
 
  
 
 
 
 
 
Confidentiality Statement 
This document is confidential.  It is provided for review only to investigators, potential investigators, consultants, study staff, 
and applicable independent ethics committees or institutional review boards.  It is understood that the contents of this 
document will not be disclosed to others without written authorization from ITN and NIAID unless it is necessary to obtain 
informed consent from potential study participants. 


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Protocol Approval 
Protocol: ITN029ST 
Version 5.0          Date: April 19, 2011 
IND: Not Applicable 
Protocol Chair: Sandy Feng, MD, PhD  
Short Title:   Immunosuppression Withdrawal for Pediatric Liver Recipients 
I have read protocol ITN029ST and I approve it.  As the principal investigator, I agree to conduct this 
protocol using good clinical practices, as delineated in ICH Guidance for Industry: E6 Good Clinical 
Practice: Consolidated Guidance (April 1996), and according to the criteria specified in the protocol. 
 
_______________________________ 
Principal Investigator (Print) 
 
_______________________________                                 _______________ 
Principal Investigator (Signature)                                                      Date 
 


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Synopsis 
Title 
Immunosuppression Withdrawal for Pediatric Living-donor Liver Transplant 
Recipients 
Short Title 
Immunosuppression Withdrawal for Pediatric Liver Recipients 
Sponsor 
National Institute of Allergy and Infectious Diseases 
Conducted by 
Immune Tolerance Network 
Protocol Chair 
 
Accrual Objective 
20 participants 
Study Design 
This is a prospective multicenter, open-label, single-arm trial in which 20 pediatric 
recipients of parental living-donor liver allografts will undergo gradual withdrawal 
of immunosuppression with the goal of complete withdrawal.  Patients on stable 
immunosuppression regimens with good organ function and no evidence of acute or 
chronic rejection or other forms of allograft dysfunction will be enrolled.  
Participants will undergo gradual withdrawal of immunosuppression and will be 
followed for a minimum of 8 years after completion of immunosuppression 
withdrawal.  Immunologic and genetic profiles will be collected at multiple time 
points and compared between tolerant and nontolerant participants.   
Pace of Enrollment 
and Study Duration 
Enrollment is defined as starting on the day that immunosuppression withdrawal is 
initiated.  Enrollment is planned for 2 years and will be limited to a maximum of 
two participants every 4 weeks.  Maximum patient participation is up to 11 years; 
therefore, the total study duration is projected to be a maximum of 13 years. 
 
Primary Endpoint 
The proportion of participants who are successfully withdrawn from 
immunosuppression, which is defined as those who remain off immunosuppression 
for at least 1 year. 
 
Inclusion Criteria 
1. Living-donor liver transplantation from a parental donor. 
2. Age less than 18 years at the time of transplantation. 
3. At least 4 years since transplantation. 
4. Availability and willingness of parental liver donor to participate in the trial. 
5. Liver biopsy at screening demonstrating no evidence of acute or chronic 
rejection and a less than stage 2 fibrosis on the Ishak scale. 
6. Negative urine pregnancy test at entry and agreement to use birth control 
during the study for women of childbearing potential. 
7. Negative purified protein derivative (PPD) test results or history of appropriate 
treatment. 
Exclusion Criteria 
1. Indication for transplantation liver failure due to autoimmune disease, such as 
autoimmune hepatitis, primary sclerosing cholangitis, or primary biliary 
cirrhosis. 
 


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2. Hepatitis B infection, as defined by the presence of HBSAg or active treatment 
for hepatitis B. 
3. Hepatitis C infection, as defined by the presence of antibody against hepatitis 
C. 
4. Serologic evidence of autoimmunity, defined as abnormal antinuclear, anti-
smooth-muscle, antimitochondrial, or anti-liver-kidney microsomal antibody 
titers greater than or equal to 1:160. 
5. Transplantation of a second organ before, simultaneous with, or after liver 
transplantation; or liver retransplantation. 
6. Aspartate or alanine aminotransferase (AST or ALT) of greater than 2 times 
the upper limit of normal. 
7. Total bilirubin and direct bilirubin, and either alkaline phosphatase or gamma-
glutamyl transferase (GGT) greater than 2 times the upper limit of normal. 
8. Clinically significant change in hepatic function in the past 26 weeks. 
9. GFR less than 40 mL/min/1.73 m2. 
10. Immunosuppression with 
a. 50% dose increase in a current agent within 26 weeks of screening, or  
b. more than one agent within 52 weeks of screening. 
11. Any systemic illness requiring, or likely to require, immunosuppressive drug 
use. 
12. Human immunodeficiency virus (HIV) infection. 
13. Pregnancy or breast-feeding. 
14. Unwillingness or inability to comply with study requirements and procedures.   


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Table of Contents 
1. 
BACKGROUND ............................................................................................................11 
1.1 
Summary .................................................................................................................11 
1.2 
Clinical Rationale ...................................................................................................11 
1.2.1  Pediatric Liver Transplant Recipients ....................................................................11 
1.2.2  Complications of Long-term Immunosuppression ................................................12 
1.2.3  Clinical Reports of Immunosuppression Withdrawal ............................................14 
1.2.4  Late Allograft Dysfunction in Uncontrolled and Controlled Settings ...................17 
1.3 
Scientific Rationale .................................................................................................21 
1.3.1  Rationale for Immune Studies ...............................................................................21 
1.3.2  Unique Immunobiology of the Liver .....................................................................21 
1.3.3  Planned Immunologic and Genetic Assessments ..................................................22 
1.4 
Potential Benefits and Risks to Human Subjects ....................................................23 
1.4.1  Potential Benefits ...................................................................................................23 
1.4.2  Risks .......................................................................................................................23 
2. 
OBJECTIVES ...............................................................................................................23 
2.1 
Primary Objective ...................................................................................................23 
2.2 
Secondary Objectives .............................................................................................23 
2.2.1  Safety of Immunosuppression Withdrawal ............................................................23 
2.2.2  Duration .................................................................................................................23 
2.2.3  Tolerance Predictive Profiles .................................................................................23 
2.2.4  Rejection Profiles ...................................................................................................23 
3. 
STUDY DESIGN ...........................................................................................................23 
3.1 
Description ..............................................................................................................23 
3.2 
Assent and Consent for Participation ......................................................................24 
3.3 
Study Endpoints ......................................................................................................24 
3.3.1  Primary Endpoint ...................................................................................................24 
3.3.2  Secondary Endpoints .............................................................................................24 
3.4 
Rationale for Selection of Study Population ...........................................................25 
3.5 
Stopping Rules ........................................................................................................25 
3.5.1  Ongoing Review ....................................................................................................25 
3.5.2  Specific Adverse Events Independent of Participant Enrollment ..........................25 
3.5.3  Specific Adverse Events as a Proportion of Enrolled Participants ........................26 
3.6 
Pace of enrollment and Study Duration ..................................................................27 
4. 
ELIGIBILITY ...............................................................................................................27 
4.1 
Study Population .....................................................................................................27 
4.2 
Inclusion Criteria ....................................................................................................27 
4.3 
Exclusion Criteria ...................................................................................................27 


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4.4 
Premature Termination ...........................................................................................28 
4.4.1  Premature Termination of Immunosuppression Withdrawal .................................28 
4.4.2  Premature Termination from the Trial ...................................................................28 
5. 
STUDY INTERVENTION ...............................................................................................28 
5.1 
Immunosuppression Withdrawal ............................................................................28 
5.2 
Assessment of Compliance with Study Intervention ..............................................30 
5.3 
Interruption or Discontinuation of Study Intervention ...........................................30 
5.3.1  Interruption of Study Intervention .........................................................................30 
5.3.2  Discontinuation of Study Intervention ...................................................................30 
5.4 
Concomitant Medications .......................................................................................30 
5.5 
Assessment of Allograft Dysfunction and Treatment of Rejection ........................31 
5.5.1  Definition of Allograft Dysfunction and Indication for an Allograft Biopsy ........31 
5.5.2  Diagnosis, Grading, and Monitoring of Rejection .................................................31 
5.5.3  Treatment of Acute Rejection ................................................................................31 
5.5.4  Treatment of Chronic Rejection .............................................................................32 
5.6 
Prophylactic Medications .......................................................................................33 
5.6.1  For Participants Receiving Corticosteroids ...........................................................33 
5.6.2  For Participants Receiving Antibody Therapy ......................................................33 
6. 
STUDY PROCEDURES ..................................................................................................35 
6.1 
Visit Windows ........................................................................................................35 
6.2 
General Assessments ..............................................................................................35 
6.3 
Study Site and Local Laboratory Assessments .......................................................36 
6.4 
Central Laboratory Assessments ............................................................................36 
6.5 
Liver Biopsies .........................................................................................................36 
6.5.1  Biopsy Technique ..................................................................................................37 
6.5.2  Tissue Handling and Disposition During Rejection Episodes ...............................37 
6.6 
Follow-up Assessments ..........................................................................................37 
7. 
TOLERANCE ASSAYS ..................................................................................................40 
7.1 
Cell-Based Assays ..................................................................................................40 
7.1.1  Whole Blood–Flow Cytometry Panel Staining ......................................................40 
7.1.2  Frozen PBMC–T-cell Assays ................................................................................40 
7.1.3  Liver Biopsy–Histology .........................................................................................40 
7.2 
Whole Blood DNA–HLA genotypes ......................................................................41 
7.3 
Gene Expression Profiling ......................................................................................41 
7.3.1  Whole Blood–Gene Expression Profiling ..............................................................41 
7.3.2  Liver Biopsy RNA–Gene Expression Profiling .....................................................41 
7.4 
Serum Assays ..........................................................................................................41 
7.4.1  Serum–Secreted Cytokines ....................................................................................41 
7.4.2  Serum–HLA Alloantibodies ..................................................................................42 


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8. 
ADVERSE EVENTS ......................................................................................................42 
8.1 
Overview .................................................................................................................42 
8.2 
Definitions ..............................................................................................................42 
8.2.1  Adverse Event ........................................................................................................42 
8.2.2  Serious Adverse Event ...........................................................................................43 
8.2.3  Unexpected Adverse Events ..................................................................................43 
8.3 
Collecting Adverse Events ......................................................................................43 
8.3.1  Methods of Collection............................................................................................43 
8.3.2  Collecting Serious Adverse Events ........................................................................43 
8.3.3  Recording Adverse Events .....................................................................................44 
8.3.4  Recording Serious Adverse Events ........................................................................44 
8.4 
Grading and Attribution of Adverse Events ...........................................................44 
8.4.1  Grading Criteria .....................................................................................................44 
8.4.2  Attribution Definitions ...........................................................................................44 
8.5 
Reporting Serious Adverse Events .........................................................................45 
8.5.1  Reporting Timeline ................................................................................................45 
8.5.2  Options for Reporting Serious Adverse Events .....................................................45 
8.5.3  Reporting Serious Adverse Events to the Data Safety Monitoring Board .............45 
8.5.4  Reporting Pregnancy ..............................................................................................45 
9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ........................................46 
9.1 
Analysis Samples ....................................................................................................46 
9.2 
Analysis Plan ..........................................................................................................46 
9.2.1  Analysis of Primary Endpoint ................................................................................46 
9.2.2  Analysis of Secondary Endpoints ..........................................................................46 
9.3 
Sample Size .............................................................................................................47 
9.4 
Participant and Demographic Data .........................................................................47 
9.4.1  Study Completion ..................................................................................................47 
9.4.2  Description of Baseline Characteristics and Demographics ..................................47 
9.4.3  Medical History .....................................................................................................48 
9.4.4  Use of Medications ................................................................................................48 
9.4.5  Safety .....................................................................................................................48 
9.5 
Randomization, Stratification, and Blinding ..........................................................48 
9.6 
Planned Interim Analyses .......................................................................................48 
9.7 
Reporting Deviations from the Original Statistical Plan ........................................48 
10.  ACCESS TO SOURCE DATA OR DOCUMENTS .............................................................48 
11.  QUALITY CONTROL AND QUALITY ASSURANCE .......................................................49 
12.  ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE 49 
12.1 
Statement of Compliance ........................................................................................49 
12.2 
Informed Consent ...................................................................................................49 


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12.3 
Privacy and Confidentiality ....................................................................................50 
13.  PUBLICATION POLICY ................................................................................................50 
14.  REFERENCES ..............................................................................................................50 
 
List of Appendices 
Appendix 1.  Schedule of Events:  Gradual to Complete Withdrawal Plus 3 Months of High-intensity 
Follow-up ....................................................................................................................................... 54 
Appendix 2.  Schedule of Events: Medium-intensity Follow-up ................................................... 57 
Appendix 3.  Schedule of Events: Low-intensity Follow-up ......................................................... 59 
Appendix 4. Schedule of Events: Extended Participant Follow-up ............................................... 60 
 
List of Tables 
Table 1.  Overall outcomes in the University of Pittsburgh study*................................................ 15 
Table 2.  Outcomes of children vs. adults in the University of Pittsburgh study* ......................... 16 
Table 3.  Outcomes in the Kyoto University study ........................................................................ 16 
Table 4.  Comparison of immunosuppression withdrawal trial designs ......................................... 20 
Table 5.  Qualifying event thresholds triggering suspension of enrollment and immunosuppression 
withdrawal based on the number of evaluable participants ............................................................ 26 
Table 6.  Attribution of adverse events .......................................................................................... 44 
 
List of Figures 
Figure 1.  Immunosuppression withdrawal.  (*See section 5.1 for definition of high dose and low 
dose.) .............................................................................................................................................. 29 
Figure 2.  Management of acute rejection.  (*See section 5.5.3.3 for definitions.) ........................ 34 
Figure 3.  Participant follow-up ...................................................................................................... 39 
 


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Glossary of Abbreviations 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
AMA 
antimitochondrial antibody 
ANA 
antinuclear antibody 
ALKMA 
anti-liver-kidney microsome antibody 
ASMA 
anti-smooth-muscle antibody 
CFR 
Code of Federal Regulations 
CMV 
cytomegalovirus 
CRF 
case report form 
CsA 
cyclosporine 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAIT 
Division of Allergy, Immunology, and Transplantation 
D5W 
5% dextrose and water 
DSMB 
Data Safety and Monitoring Board 
EBV 
Epstein-Barr virus 
EDTA 
ethylenedinitrilo tetra-acetic acid 
ELISPOT 
enzyme-linked immunospot 
GCP 
good clinical practice 
GFR 
glomerular filtration rate 
GGT 
gamma-glutamyl transferase 
HCC 
hepatocellular carcinoma 
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
IRB 
institutional review board 
IS 
immunosuppression 
INR 
international normalized ratio 
ICH 
International Conference on Harmonization 
IND 
investigational new drug 
IRB 
Institutional review board 
ITN 
Immune Tolerance Network 
ITT 
intent-to-treat 
IV 
intravenous 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMF 
mycophenolate mofetil 


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NCI 
National Cancer Institute 
NCI-CTCAE 
National Cancer Institute Common Toxicity Criteria for Adverse Events 
NIAID 
National Institute of Allergy and Infectious Diseases 
NK cells 
natural killer cells 
PBC 
primary biliary cirrhosis 
PBMC 
peripheral blood mononuclear cells 
PBS 
phosphate buffered saline 
PCP 
Pneumocystis carinii pneumonia 
PCR 
polymerase chain reaction 
PI 
principal investigator 
PP 
per protocol 
PSS 
Product Safety Scientist 
RHO 
Rho Federal Systems Division, Inc. 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SMT 
study management team 
TMP 
trimethoprim 
TSH 
thyroid-stimulating hormone 
ULN 
upper limit of normal 
WHO 
World Health Organization 
 


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1. 
BACKGROUND 
1.1 
SUMMARY 
Currently, transplantation of any solid organ incurs a lifelong burden of immunosuppression for the 
recipient.  In spite of many advances, including the development of new agents, the basic premises of 
immunosuppression strategies remain unchanged and, as such, substantial metabolic, infectious, and 
neoplastic complications continue to threaten the recipient’s life and well-being.  Several reports, 
however, have shown that a significant proportion of liver recipients (19%–42%) can maintain 
normal allograft function without immunosuppression—the definition of “functional tolerance.”  
Although drug weaning precipitates rejection in some recipients, most episodes are mild or moderate, 
are easily reversed, and do not result in long-term consequences. 
These reports have motivated us to propose gradual and complete immunosuppression withdrawal in 
a highly selected subgroup of liver transplant recipients:  those who underwent living-donor liver 
transplantation as a child (<18 years of age) 4 or more years ago for diseases other than viral hepatitis 
and autoimmune liver disorders, who continue to have excellent graft function, and who are on a 
stable single-agent immunosuppression regimen.  Recipients will be closely monitored during 
tapering to ensure expeditious recognition, diagnosis, and, if necessary, treatment of liver 
dysfunction. 
The main clinical endpoints measure the outcome of immunosuppression withdrawal.  They target the 
success rate of withdrawal; the duration for which recipients remain off of immunosuppression; and 
the overall incidence, severity, and timing of rejection.  The current trial also encompasses a 
complementary scientific effort to identify, quantify, and characterize donor-specific immune 
responses, immunologic interactions, and genetic characteristics that may predict or correlate with 
functional tolerance.  
1.2 
CLINICAL RATIONALE 
1.2.1 Pediatric Liver Transplant Recipients 
Historically, the pediatric patient has been instrumental in the initial establishment of, and subsequent 
innovation in, the field of liver transplantation.  Both the first attempted liver transplant in 1963 and 
the first successful liver transplant in 1967 involved young children.  Later, during the late eighties 
and early nineties, special challenges facing pediatric liver transplantation motivated the development 
and refinement of innovative surgical strategies (i.e., reduced-size, split, and, finally, living-donor 
liver transplantation techniques) that have permeated and changed not only pediatric but also adult 
liver transplantation. 
While these contributions have been predominantly surgical and technical in nature, pediatric 
transplant recipients may now present a unique opportunity to explore tolerance.  Specifically, there 
now exists a significant number of stable living-donor recipients who underwent transplantation as 
children, typically from parent donors.  The current trial proposes to identify a cohort of stable, 
pediatric living-donor recipients 4 or more years after transplantation who are willing to undergo 
gradual immunosuppression withdrawal under close and prolonged supervision with an aim to 
identify and quantify those who are tolerant—defined as those able to maintain normal allograft 
function in the absence of immunosuppression.  Previous reports regarding prospective withdrawal 


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trials would suggest that a significant proportion of such patients can be successfully and safely 
withdrawn.  Releasing young patients from the lifelong burden of immunosuppression has obvious 
benefit and appeal, particularly considering the reality that complications and toxicities of 
immunosuppression (e.g., renal dysfunction, infection, malignancy, metabolic abnormalities, and 
growth retardation) represent the major threats to long-term quality of life and survival in this 
posttransplant population.1 
1.2.2 Complications of Long-term Immunosuppression 
The general risk of long-term renal dysfunction is illustrated by a review of 69,321 solid organ 
transplant recipients (median age 45 years) who were followed for a median of 36 months.2 Chronic 
renal failure was reported in 16.5%; 29% of these recipients required dialysis.  Among 2261 liver 
transplant recipients followed for 10 years, the cumulative incidence of chronic renal failure was 
26%.  Among pediatric non-renal-transplant recipients, the rate of chronic renal failure, defined as a 
serum creatinine of greater than 2.5 mg/dL or chronic dialysis, appears to be much lower.  Of all heart 
and liver recipients undergoing transplantation between 1990 and 1999 who survived more than 1 
year, 5.6% developed chronic renal failure; the actuarial risk at 5 years was 3%.3  Among pediatric 
lung transplant recipients, a large, single center study of 125 one-year survivors reported that serum 
creatinine nearly doubled from pretransplant to 1-year post transplant and tripled by 7 years post 
transplant.  Mean glomerular filtration rate (GFR), as estimated by the Schwartz formula, dropped 
from a baseline of 163 ± 5.9 mL/min to 69 ± 9.0 by 10 years after transplant.  Five years after 
transplant, 38% of patients had GFR <60mL/min and seven patients developed end-stage kidney 
disease.4 
More recently, detailed data has emerged from three single-center studies specifically regarding the 
renal function of pediatric liver transplant recipients 10 years after transplantation.  All three reported 
that the calculated GFR by the Schwartz formula significantly overestimated actual GFR as measured 
by nuclear medicine renal isotopic clearance studies.  Alonso reported on the 10-year calculated GFR 
for 32 pediatric liver transplant recipients from the University of Chicago.5  Two patients had 
moderate renal insufficiency (calculated GFR < 60mL/min), whereas three had mild renal 
insufficiency (calculated GFR of 60–80mL/min).5  The data from the two groups that measured GFR 
were quite different.  The group from Lille, France reported on seven recipients.6  Four of the seven 
had mild renal insufficiency (measured GFR 60–80mL/min) and two of the seven had moderate renal 
insufficiency (measured GFR of 20-60mL/min).  Similarly, the group from Toronto reported on 26 of 
their 32 10-year survivors who underwent GFR measurements.7 At 10 years post transplant, 20 of the 
26 (77%) had abnormal renal function.  Renal insufficiency was mild (measured GFR 75–
100mL/min) for 4 (15%), moderate (measured GFR 50–75mL/min) for 15 (58%), and severe 
(measured GFR <50mL/min) for 1 (4%).  Moreover, two additional patients did not undergo GFR 
measurement at their 10-year anniversary; one was hemodialysis dependent and the second had 
undergone combined liver and kidney transplantation 8 years after initial liver transplantation.   
Therefore, while overall rates of renal failure are low, available data suggest that the prevalence of 
renal insufficiency is high.  The concern is that children who have a longer potential life span after 
liver transplantation will “evolve from asymptomatic decreased glomerular filtration rate to severe 
renal impairment”.1  In children, progressive renal insufficiency leads to many well-characterized 
complications including poor growth, anemia, hypertension, secondary 
hyperparathyroidism/metabolic bone disease, and electrolyte abnormalities.8  Although these 
morbidities become clinically apparent typically at GFR <30 mL/min, corresponding to chronic 


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kidney disease stages 4 or 5, children may be most sensitive to these metabolic derangements in 
chronic kidney disease stage 3.  As pediatric transplant recipients mature into adulthood, concern 
focuses on the cardiovascular morbidity incurred by suboptimal renal function.  It is clear that poor 
quality renal function is associated with cardiovascular morbidity independent of other known 
cardiovascular risk factors such as hypertension and dyslipidemia.9, 10  
In addition to predisposing pediatric transplant recipients to metabolic abnormalities indirectly 
through renal dysfunction, immunosuppression predisposes directly to metabolic conditions such as 
hypertension, diabetes, and hyperlipidemia.  In general, their impact on pediatric liver transplant 
recipients appears less than on their adult counterparts.  However, at 10 years post transplant, Avitzur 
and associates reported that 8 of 32 patients (25%) required antihypertensive treatment, a rate 
consistent with several previous reports.7, 11, 12  Moreover, a recent publication suggests that 
hypertension in pediatric liver transplant recipients may be substantially underdiagnosed.  Del 
Compare and associates compared office blood pressure measurements (mean of three measurements 
1 minute apart) with 24-hour ambulatory blood pressure monitoring (APBM) in stable pediatric liver 
transplant recipients 1.1–11.5 years (median 5.1 years) after liver transplantation who were not being 
treated with an antihypertensive medication.13  Of 61 eligible patients, 32 were excluded: 18 (30%) 
because of chronic renal insufficiency (calculated GFR <80mL/min), 12 (20%) because of recent 
changes in immunosuppression, and 2 because of declined consent.  Of the 29 stable patients with 
relatively normal renal function who were studied, 8 (28%) met criteria for hypertension by APBM; 
only 1 of the 8 was hypertensive based on office blood pressure measurements.13 Overall, the 
incidence of insulin-dependent diabetes mellitus in 10-year liver transplant survivors has been 
reported at 6%.7  Although 6 (26%) of 10-year pediatric liver transplant recipients had elevated 
fasting cholesterol and 10 (45%) had elevated triglycerides, respectively, none were prescribed lipid-
lowering agents.7  Again, although these metabolic abnormalities were most often mild and affected a 
modest proportion of patients, the potential for cumulative morbidity particularly over the anticipated 
long life of a pediatric transplant recipient span merits concern.  
Infection has been reported as a major cause of late pediatric liver allograft loss and late pediatric 
liver recipient mortality.14, 15 In particular, five different papers have reported on a total of 58 late 
deaths (>1 year after transplantation) that occurred in a total of 856 pediatric liver transplant 
recipients.  Overwhelming infection was the most common cause, accounting for 35 (60%) of all 
deaths  secondary to overwhelming infection.14, 16-19  Although infectious complications do diminish 
over time after transplantation, presumably since immunosuppression decreases, they continue to 
cause morbidity between 5 and 10 years after transplantation.  In a report of a single center’s 32 
pediatric liver transplant recipients who survived more than 10 years after liver transplantation, the 
majority developed infections requiring hospitalization or medical intervention between 5 and 10 
years after transplantation.7  Sixteen were diagnosed with viral infections (herpes zoster = 6;  
chickenpox = 4;  CMV colitis/hepatitis = 2; EBV hepatitis = 1; parvovirus = 1; HCV = 1; and vulvar 
condyloma = 1), two with bacterial infections (mycoplasma = 1;  periorbital cellulitis = 1), and one 
with a fungal infection (trichophyton).  There were three additional infections in long-term recipients 
for whom no bacteriologic diagnosis could be made (pneumonia = 2; epididymitis = 1). 
As for malignancy after transplantation, the most notable malignancies considered as directly related 
to immunosuppression are posttransplant lymphoproliferative disorder (PTLD) and skin cancer.  In a 
single-center study, 19 of 335 pediatric  transplant recipients (ages 0–17 years) developed PTLD, 
which was typically associated with Epstein-Barr virus (EBV).20  Thirty-one developed EBV 
infection or reactivation without PTLD.  Of those with PTLD, 32% died; of those with EBV without 


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PTLD, 6% died.  Antibody-based treatment for rejection was common among those whose developed 
EBV-related disease.  This report confirms the high mortality rate associated with EBV-related 
disease in patients receiving chronic immunosuppression after liver transplantation.  With regard to 
skin cancer, the risks of squamous cell carcinoma and melanoma are increased by 65 and  3.4 times 
higher, respectively, in renal and cardiac transplant recipients than in the general population.21  A 
retrospective single-center analysis of liver transplant recipients demonstrated a 4.5% incidence of 
malignancy with a 1.6% mortality rate.22  Specific information on pediatric transplant recipients 
suggest that skin cancers typically do not occur during childhood.23  Rather, in the largest series, skin 
cancers developed during the second decade after transplantation (range 5.5–292 months), at an 
average age of 26–28 years.  As in adults, but even more so, there was reversal of the squamous cell 
to basal cell carcinoma ratio compared with the general population.  Spread to lymph nodes was also 
more common in pediatric recipients than in adult recipients (9% vs. 6%).  Finally, the third most 
frequent malignancy in pediatric kidney transplant recipients was anogenital cancer, accounting for 
4% of tumors of pediatric transplant recipients.  Similar to skin cancers, the tumors occurred on 
average 12 years (range 3.5–22 years) after transplantation, at a mean age of 27 years (range 20–39 
years) childhood.23  
1.2.3 Clinical Reports of Immunosuppression Withdrawal 
1.2.3.1 
Summary of Clinical Results 
Reports from four transplant centers suggest that 19%–42% of liver transplant recipients are 
functionally tolerant.24-29  These series included pediatric and adult recipients who underwent 
deceased- or living-donor liver transplantation for wide-ranging etiologies, including autoimmune 
disorders and viral hepatitis.  Recipients were electively, incidentally (i.e., secondary to 
noncompliance), or obligatorily (i.e., secondary to development of a major contraindication to 
ongoing immunosuppression) weaned from immunosuppression.  Episodes of subsequent rejection 
were easily reversed in nearly all cases; only one of the 54 reported cases of patients who experienced 
rejection required antilymphocyte therapy.  In light of the major differences in the study populations, 
it is worthwhile to consider each of these experiences individually to clarify important differences 
between these historical cohorts and the one proposed in the current trial.  
1.2.3.2 
Deceased Donors 
Three studies focus on recipients of deceased-donor liver transplants.  The Kings College group 
reported on a cohort of 18 adult liver transplant recipients prospectively withdrawn from 
immunosuppression.24 After 3 years, five recipients (28%) remained completely off 
immunosuppression.  Of the remaining 13 recipients, 4 (22%) experienced allograft rejection, of 
which 1 was severe in grade; 8 developed a hepatitis-like picture, which for the majority of cases 
appeared to represent an exacerbation of findings identified in the prewithdrawal biopsies; and 1 
recipient developed acute hepatic necrosis.  Similar to the Pittsburgh approach, all treatment 
comprised corticosteroid pulses and /or resumption or escalation of baseline immunosuppression, 
including conversion to alternative agents without need for antilymphocyte therapy.  The authors 
reported that transplantation for nonimmunologic liver disease, fewer HLA mismatches, and lower 
incidence of early rejection were associated with successful withdrawal.   
A small study involving nine prospectively weaned adult recipients has been reported recently from 
Spain.27 Three recipients (33%) were successfully weaned; acute rejection was diagnosed in two; and 


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the remaining four developed nondiagnostic portal inflammation.  Again, there was no need for 
antilymphocyte therapy.     
The largest study comes from the University of Pittsburgh group who reported on 95 prospectively 
withdrawn recipients; at study entry, 31 were 20 years old or younger and the remaining were 21–68 
years old.25, 28  At last publication, 19% were completely off immunosuppression, 39% were still 
being tapered, 29% had experienced rejection, and 13% had withdrawn from the study.  The pediatric 
cohort had better outcomes than the adult cohort did; it had a higher weaning success rate and a lower 
rejection rate.  Of the 28 patients with rejection, 18 had biopsy-proven episodes, 7 had clinically 
suspected episodes, and 3 were withdrawn from the study because biopsy findings were suggestive of 
incipient chronic rejection although not diagnostic of chronic rejection.  Treatment of recipients 
considered to have acute or chronic rejection comprised corticosteroid pulses and/or resumption or 
escalation of baseline immunosuppression, including conversion to alternative agents, typically from 
cyclosporine-based to tacrolimus-based regimens.  Again, no recipient received antilymphocyte 
antibody therapy, and there were no graft losses related to allograft dysfunction. 
Updated information regarding the Pittsburgh cohort was presented at the recent American Transplant 
Congress (Seattle, May 2005; Tables 1 and 2).  Their withdrawal experience now encompasses 120 
liver transplant recipients: 70 adults and 50 children.  Overall, 33 (28%) were successfully weaned, 
30 (25%) were still weaning, 47 (39%) had failed, and an additional 10 (8%) were on “hold”: these 10 
were no longer weaning but had not achieved any failure endpoints of acute or chronic rejection.  
Outcomes were distinctly better in the pediatric subgroup (n=50): 20 (40%) were successfully 
weaned, 23 (46%) were still weaning, and the remaining 7 (14%) had acute rejection and thus failed 
weaning (Table 2). 
 
Table 1.  Overall outcomes in the University of Pittsburgh study* 
 
Status/Outcome 
Off drugs 
Withdrawing 
Failure 
Withdrawal  
on hold 
Patients, N (%) 
33 (28) 
30 (25) 
47 (39) 
10 (8) 
Age at transplantation, yr 
15.3 
8.2 
29.6 
36.5 
Transplantation to 
withdrawal start, yr 
6.8 
7.4 
9.0 
8.4 
Withdrawal to outcome, yr 
1.5 
N/A 
1.5 
2.8 
Current follow-up, yr 
9.1 (2.5–12.6) 
7.6 (2.0–11.0) 
8.7 (2.7–9.3) 
7.6 (4.0–6.8) 
*Updated May 2005. 
 
 


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 Table 2.  Outcomes of children vs. adults in the University of Pittsburgh study* 
Status/Outcome 
Children 
N = 50 
Adults 
N = 70 
Off drugs 
20 (40%) 
13 (19%) 
Withdrawing 
23 (46%) 
7 (10%) 
Acute cellular rejection 
7 (14%) 
30 (43%) 
Chronic rejection 
0 
1 (1%) 
Duct injury 
0 
5 (7%) 
Recurrent primary biliary cirrhosis 
0 
4 (6%) 
On hold 
0 
10 (14%) 
*Updated May 2005. 
1.2.3.3 
Living Donors 
Perhaps the experience closest to the currently proposed trial was that from Kyoto University.  This 
patient group, like the one we are proposing, was limited to pediatric recipients of living-donor liver 
transplants.26, 29  An overall success rate for complete immunosuppression withdrawal of 42.6% (49 of 
115 recipients) was observed.  It should be noted, however, that the success rate was 69% (33 of 48) 
for those obligatorily or incidentally (nonelectively) weaned, and 24% (16 of 67) for those electively 
weaned.  Overall, 20 patients (17%) experienced rejection: 12 (25%) in the nonelective and 8 (12%) 
in the elective weaning group.  All rejection episodes were easily reversed except one occurring in a 
nonelectively weaned recipient who required OKT3 therapy.  The authors could not identify any 
clinical parameters associated with successful weaning. 
Similarly, the Kyoto group provided updated information regarding their withdrawal cohort at the 
American Transplant Congress (Table 3).  As of June 2004, 55 patients were completely off of 
immunosuppression and 39 patients were continuing to wean.  One patient developed chronic 
rejection which was successfully treated with return to a triple immunosuppression regimen.  
Unfortunately, the Kyoto group did not provide us with the number of patients for whom weaning 
was attempted to enable calculation of percentages. 
 
Table 3.  Outcomes in the Kyoto University study 
 
Transplantation 2001 
N = 63 
Update as of June 2004*  
N = Unknown† 
Off drugs, n: 
24 (38%) 
55  
     Drug free time, mo 
     Median (range) 
23.5  
(3–69) 
Scheduled:  41 (10-81) 
EBV:           50  (9–124) 
Others:        57  (22–93) 
Withdrawing,  n: 
23 (37%) § 
39  


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Transplantation 2001 
N = 63 
Update as of June 2004*  
N = Unknown† 
Acute cellular rejection, n: 
16 (25%) 
All episodes easily treated with 
tacrolimus  corticosteroids 
Unknown  
      
     Time of rejection, mo 
     Median (range) 
 
9.5  
(1–63) 
 
Unknown 
Chronic rejection, n: 
0% 
1 
Successfully treated with 
prednisone/tacrolimus/MMF 
*Presented at the American Transplant Congress (Seattle, May 2005). 
† Number of patients in whom withdrawal was unattempted is unknown. 
§Six of these 23 withdrawal patients are taking less than once weekly tacrolimus. 
1.2.3.4 
Summary of Clinical Experience With Withdrawal  
In summary, these four studies suggest that, 19%–43% of all liver transplant recipients and 40-43% of 
pediatric liver transplant candidates can be prospectively and successfully gradually withdrawn from 
immunosuppression and identified as functionally tolerant.  Conversely, 14%–43% developed acute 
rejection.  The remaining recipients failed withdrawal after developing nonspecific abnormalities that 
were not attributed to rejection, had withdrawal held without either succeeding or failing, or were still 
undergoing withdrawal.25, 26, 28, 29
  The four studies provide very consistent data regarding the severity 
and treatability of the rejection episodes: the majority of episodes were graded as mild to moderate 
and reversed without the use of antilymphocyte therapy.  Finally, there were no instances of graft loss 
related to immunosuppression withdrawal. 
1.2.4 Late Allograft Dysfunction in Uncontrolled and Controlled Settings 
A fundamental premise of the current trial is that the acute rejection that occurs during controlled, 
closely supervised immunosuppression withdrawal as planned in the current trial is unlikely to have 
severe or lasting negative consequences.  This is supported by reports of the several trials of gradual 
immunosuppression withdrawal presented above in which rejection was consistently observed to be 
mild to moderate and easily reversed.24-29  In contrast to these experiences, late allograft dysfunction 
that occurs outside controlled immunosuppression withdrawal trials has been associated with variable 
results and more complications.30-33 
1.2.4.1 
Uncontrolled Settings 
A major confounding factor in interpreting reports of allograft dysfunction after liver transplantation 
is the widely varying definition of “late,” which has ranged from greater than 30 days to greater than 
365 days after transplantation.  Other factors include the variable etiologies (e.g., acute rejection vs. 
de novo autoimmune hepatitis) and additional factors (e.g., noncompliance or viral infection) that 
have frequently precipitated and/or complicated late allograft dysfunction.   
With regard to “late” acute rejection, the literature on adult recipients is conflicting.  Some reports 
suggest that late episodes of rejection are more refractory than early episodes to standard therapy30-32 
and that they predispose to the development of chronic rejection,30, 31, 33 whereas other reports indicate 


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that their response to therapy is similar to that of early episodes33, 34 and they do not predispose to 
chronic rejection.32, 34   
Two reports focus on late rejection in pediatric recipients.  The University of Pennsylvania group 
found that 32% (18 of 57) of recipients had rejection diagnosed more than 100 days after 
transplantation.35  Seventeen patients (94%) responded well to therapy, with only one requiring 
OKT3.  One recipient who had two episodes of early rejection and two episodes of late rejection did 
develop chronic rejection.  Function was stabilized by conversion to tacrolimus-based 
immunosuppression.  
The King’s College group, however, reported a different experience.  Twenty children were 
diagnosed with “late cellular” rejection greater than 6 months after transplantation.36 At the time of 
diagnosis, 5 were considered to be on “adequate” immunosuppression while 15 were considered to be 
on “inadequate” immunosuppression.  Ten of the 20 (50%) returned quickly to normal allograft 
function with treatment; only 1 of these required high-dose corticosteroids (“adequate” 
immunosuppression group) for reversal; 9 (all from the “inadequate” immunosuppression group) 
were treated simply with increased baseline immunosuppression or conversion from cyclosporine to 
tacrolimus. 
The 10 other children, however, did not respond readily to treatment, including 3 who had recurrent 
or persistent CMV infection, of which 1 also had hepatic artery thrombosis; 1 who progressed to 
chronic rejection and underwent retransplantation; and 5 who progressed to de novo autoimmune 
hepatitis.  These five were treated with immunosuppression designed for autoimmune hepatitis.  All 
responded, although one has had several relapses of autoimmune hepatitis attributed to 
noncompliance.  In retrospect, when late acute cellular rejection was diagnosed, three of these five 
already had histologic features suggestive of de novo autoimmune hepatitis (plasma cell infiltrates 
with perivenular dropout or bridging collapse) and two of five had detectable autoantibodies. 
Therefore, the King’s College experience suggests that uncomplicated late acute rejection responds 
readily to treatment.  However, concurrent processes such as CMV infection or hepatic artery 
thrombosis make reversal of dysfunction more difficult.  Moreover, serologic and/or histologic 
features of autoimmunity necessitate a different treatment approach (see section 1.2.3.1). 
Posttransplant de novo autoimmune hepatitis was first described in 1995.  Currently, the diagnosis 
can be made when a transplant recipient without previous diagnosis of autoimmune disease develops 
serologic evidence of autoimmunity (high immunoglobulin G titers with positive titers of antinuclear 
(ANA), smooth muscle (ASMA), mitochondrial (AMA) or liver-kidney microsomal (ALKMA) 
antibodies in conjunction with an allograft biopsy that shows histopathologic features of chronic 
hepatitis (portal and periportal hepatitis with lymphocytes and plasma cells, bridging collapse, and 
perivenular cell necrosis).37 Although this does occur in adult recipients,38, 39 it has been reported 
more often in pediatric recipients,37, 40-42 with an estimated incidence of approximately 5% (2%–11%).   
De novo autoimmune hepatitis in pediatric liver recipients typically occurs several years after 
transplantation (often 2–4 years but as late as 13 years) when immunosuppression doses are 
commonly low.  The overall incidence and time of development of de novo AIH is quite similar in the 
living-donor and deceased-donor liver transplant groups.  The etiology is not fully clarified and may 
in fact be heterogeneous.  Some have speculated that it can result from an immune response to a 
foreign antigen rather than a self-antigen.  De novo hepatitis has been reported as an immune response 
directed against glutathione S-transferase T1 expressed in a donor liver by a transplant recipient 


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bearing the glutathione S-transferase T1-null genotype.43  Viral and other infections have also been 
suggested as precipitating events as they may expose self-antigens and thereby disrupt peripheral 
tolerance.  Moreover, some have speculated that calcineurin inhibitors might influence the 
development of AIH by activating autoreactive T-cell clones.37, 39, 44  The literature recommends 
treatment with corticosteroids and azathioprine, at doses used to treat autoimmune hepatitis, rather 
than the immunosuppression protocols typically used to treat acute cellular rejection.  The short-term 
response rates to such treatment have been reported to be high with biochemical and/or histologic 
improvement.36, 37, 41  However, the long-term, natural history of de novo autoimmune hepatitis may 
be less favorable.  Histologic progression to cirrhosis or the necessity for retransplantation has been 
reported.42, 45  The expanded range of immunosuppression today may, however, decrease the 
likelihood of progressive de novo hepatitis.  Kerkar and colleagues from Mt. Sinai have just reported 
their experience using rapamycin as rescue therapy for pediatric recipients with posttransplant (de 
novo and recurrent) autoimmune hepatitis that was unresponsive to corticosteroids and azathioprine.40  
Five nonresponders to standard therapy (two de novo and three recurrent) all responded to rapamycin, 
with normalization of liver function.   
1.2.4.2 
Controlled Settings 
Several reports indicate that acute rejection that occurs during controlled immunosuppression 
withdrawal is most frequently mild to moderate and easily reversed.24-29 Nearly all reported episodes 
have been successfully treated, typically with return to baseline immunosuppressants, with or without 
bolus corticosteroid therapy.  To date, there has been no reported graft loss or patient death as a direct 
result of elective immunosuppression withdrawal.  Presumably, the close surveillance regimen 
dictated by withdrawal protocols has ensured the expeditious detection, diagnosis, and treatment of 
allograft dysfunction that has resulted in resolution.  The literature supports the intuitive concept that 
delay in diagnosis and treatment plays a substantial role in the more varied outcomes of allograft 
dysfunction that occur outside of close surveillance.46  
As for chronic rejection, one study reported that 3 of 95 prospectively weaned patients were 
terminated from the withdrawal protocol because of “suspicion of incipient chronic rejection” 
without, however, the diagnosis of chronic rejection on liver biopsy.25 All of these patients were 
adults.  If there is histologic or serologic evidence of incipient chronic rejection or de novo hepatitis, 
the currently available armamentarium of immunosuppressants, many of which have been reported to 
be efficacious rescue therapies, provides some semblance of a safety net.40, 47-49 
1.2.4.3 
The Current Trial 
There are several features of our trial design that should minimize the likelihood of an unfavorable 
outcome (Table 4).  First of all, unlike previous controlled withdrawal studies, we are excluding all 
recipients who underwent transplantation for liver diseases that might recur.   
Second, we require thorough biochemical, serologic, and histologic assessments aimed to detect 
recipients with subclinical allograft dysfunction to exclude them from participation.  Only the King’s 
College group performed prewithdrawal biopsies; those with mild, nonspecific abnormalities were 
allowed to withdraw.  A significant number of their patients who could not be successfully withdrawn 
developed exacerbations of the abnormalities evident on their initial biopsies rather than acute 
rejection.   
Third, our protocol incorporates serologic screening for markers of autoimmunity before enrollment 
and periodically during the withdrawal phase with an aim to identify those who may be evolving 


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toward de novo autoimmune hepatitis.  Although  de novo autoimmune hepatitis clearly occurs 
spontaneously, outside of controlled immunosuppression withdrawal, we are certainly concerned that 
this condition would be exacerbated by gradual withdrawal of immunosuppression.   
Fourth, our study enrolls only pediatric recipients of parental living-donor grafts.  Although there is 
doubt that HLA matching and donor-recipient relatedness result in a significant immunologic benefit, 
there is nevertheless a distinct thread of literature that suggests limited immunologic advantage to the 
parental living-donor setting.  Some centers have reported that, while the incidence of acute rejection 
is comparable between living- and deceased-donor transplantation, that the acute rejection episodes 
that occur after living-donor transplantation are less severe and that living-donor liver transplantation 
is associated with substantially lower risk of chronic rejection than deceased-donor transplantation.42, 
50, 51 
Fifth, only parental living-donor recipients who are 4 or more years post transplant (compared with 2 
years in the Kyoto report) are eligible for our withdrawal trial.  Our patients will likely start on lower 
relative immunosuppression doses since they are further out from transplantation and have undergone 
more “natural weaning” by continued growth.   
A sixth and important consideration is our requirement for evidence of good medical compliance for 
continued trial participation.  A safety feature of supervised immunosuppression withdrawal is the 
close monitoring of liver function tests which is entirely dependent upon compliance.   
Finally, we have set low thresholds to mandate liver biopsy to ensure swift detection of rejection, if 
and when it occurs.  We believe that prompt treatment provides the best opportunity for a good 
response.  Our protocol also specifies ongoing assessment of the severity and treatability of acute 
rejection episodes with concomitant stopping rules if unfavorable events occur with undue frequency.  
Table 4.  Comparison of immunosuppression withdrawal trial designs 
 
Adult or 
pediatric 
Deceased 
or living 
donor 
Inclusion of 
HCV/HBV/
AIH/PBC/ 
PSC? 
Screening 
for de 
novo 
AIH? 
Time 
after tx 
Prewith-
drawal 
biopsy? 
Postwith-
drawal 
biopsies? 
King’s 
College 
Adult 
Deceased 
Yes 
No 
> 5yr 
Yes 
Yes 
University 
of 
Pittsburgh 
Adult and 
Pediatric 
Deceased 
Yes 
No 
 5yr for 
elective 
weaning 
No 
No 
Kyoto 
University 
Pediatric 
Living 
No 
No 
> 2 yr 
for 
elective 
weaning 
No 
No 
ITN trial 
Pediatric 
Living 
No 
Yes 
 4 yr 
Yes 
Yes 


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Overall, we anticipate that our rate of successful withdrawal will be at least 20% but possibly higher, 
based on published literature of previous elective withdrawal trials,  and in particular, the experience 
with pediatric liver transplant recipients.24-29, 37, 40  Features of the transplant recipients in the current 
trial, as outlined above, may however, favor a higher success rate and a higher safety rate.     
1.3 
SCIENTIFIC RATIONALE 
1.3.1 Rationale for Immune Studies 
While the clinical reports regarding immunosuppression withdrawal do provide a context of clinical 
expectations for our proposed study, they, unfortunately, have yielded little information regarding the 
mechanism(s) of functional tolerance or biomarkers that may predict, characterize, or identify 
functional tolerance.  Three of the four experiences involved recipients of livers from deceased 
donors and, as such, were severely limited in the performance of tolerance studies by the lack of 
extant donor tissue.  The Kyoto experience, which comprised solely pediatric recipients of living-
donor liver transplants, did begin to explore potential mechanisms of functional tolerance with very 
limited findings.  They suggested that downregulation of interferon-gamma secretion may be a 
mechanism responsible for the observed donor-specific hyporeactivity in mixed lymphocyte 
reactions.52  Clearly, much more can and must be done to elucidate mechanisms and biomarkers of 
operational tolerance, as we are currently proposing. 
If biomarkers of clinical tolerance can be identified, our serial evaluations of peripheral blood and 
liver tissue before, during, and after withdrawal should enable us to delineate the time course of their 
presence.  It is hoped that an emerging signature of tolerance will yield clues about the operational 
mechanism(s) of tolerance.  We plan to enroll patients who will undergo drug withdrawal without any 
peritransplant “tolerizing” immunosuppression protocol; therefore, our study does not induce 
tolerance because it is simply designed to uncover preexisting, but unidentified, tolerance. 
An integral part of the success of this study is the identification of immune cell or molecular markers 
that can segregate patients who have successfully achieved tolerance following withdrawal of 
immunosuppression vs. those patients who failed.  Through the discovery of such markers it is hoped 
that predictive markers can be extrapolated, such that parameters measured before withdrawing 
immunosuppression would help guide whether or not drug withdrawal should be attempted.  Thus, 
the use of surrogate markers could create a powerful means for stratification of patients into those 
“potentially tolerizable” or those who will likely never achieve tolerance. 
Using many of the latest cellular immunological techniques combined with molecular profiling, the 
measurements of peripheral blood and liver biopsy material will be done in patients before, during, 
and after immunosuppression withdrawal.  Importantly, assays will also be performed at time of 
rejection so that comparisons can be made between withdrawal successes and failures.  
1.3.2 Unique Immunobiology of the Liver 
Of all solid organs that are transplanted, the liver has been regarded historically and consistently as 
being immunologically privileged—the most resistant to immunologic attack and damage.  
Presentation of antigens via the portal venous system has long been recognized as more likely to 
result in a tolerizing response than presentation via the systemic venous system.  In several allogeneic 
models ranging from rodent to canine to swine, minimal or even no exposure to immunosuppression 
has resulted in successful and durable graft function after transplantation.53-56 


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In the human arena, the liver is unique among transplanted solid organs in several ways.  First, while 
episodes of acute rejection commonly connote deleterious outcomes for nearly all other solid organs 
that we transplant, acute rejection after liver transplantation has had no such long-term 
connotations.57, 58  Second, it is well known that some episodes of acute rejection may resolve 
spontaneously without treatment.59-61  Third, although chronic rejection in varied manifestations 
represents a major threat to the longevity of other transplanted solid organs, its incidence and 
therefore, its importance, is substantially less in the post-liver-transplant setting, particularly in the 
age of modern immunosuppression.62  Furthermore, it has been suggested that the advent of 
tacrolimus immunosuppression has nearly eliminated the threat of chronic rejection for pediatric liver 
transplant recipients,63, 64 and that living-donor liver transplants may be particularly immune to 
chronic rejection.42  Finally, while the importance of humoral mechanisms of acute and chronic 
allograft has recently emerged for other solid organs, there is little literature to substantiate their 
relevance and importance in the setting of liver transplantation.  
At present, the biologic basis for these observed differences between the liver and other solid organs 
remains imprecisely defined.  While the liver’s unique regenerative capacity enabling repair of injury 
may be important to buffer the impact of immunologic attack, most believe that the primary reason 
for the liver’s privileged position of relative resistance against immunologic attack is predominantly 
immunologic in nature and results from the unique anatomy and function of the liver.65, 66  The liver 
has two blood supplies—the portal venous and the hepatic arterial systems—which mixes in the 
hepatic sinusoids, specialized blood channels in the liver which are lined by fenestrated endothelial 
cells and lack a discrete basement membrane.  This unique architectural arrangement and the 
sluggishness of sinusoidal flow facilitates the entry of circulating antigens and immune cells into 
these blood spaces where they can interact with endothelial cells, hepatocytes, Kupffer cells, and 
other resident cells.  
This design is likely integral to the liver’s unique immunobiology, which has been delineated only 
recently.66 Compared with other organs and peripheral blood, the liver has a unique complement of 
lymphocytes.  Natural killer (NK) and NK-like T cells strikingly account for approximately 60% of 
resident lymphocytes compared with approximately 15% in the peripheral blood compartment.  The 
liver is also enriched for CD8+ over CD4+ T cells.67 When challenged by pathogens, the liver has the 
demonstrable capability to generate a protective immune response.  However, since portal venous 
flow constitutively exposes the liver to nonpathogenic foreign antigens (e.g., food derivatives, 
environmental toxins, and bacterial products), the liver must also possess potent mechanisms that 
suppress immune activation.  One of the scientific goals of this proposal is to define the immunologic 
interactions that critically determine the nature and scope of the immune response. 
1.3.3 Planned Immunologic and Genetic Assessments 
A variety of immunologic and genetic assessments will be performed at several time points to study 
changes in immune responses during and after immunosuppression withdrawal.  These will include 
measures of cellular reactivity, antibody response, and patterns of gene expression.  Assessments will 
be performed on peripheral blood samples and on liver tissue.  The results of immune and genetic 
assessments will be compared between tolerant and nontolerant individuals; that is, between subjects 
who are successfully withdrawn from immunosuppression and those who experience rejection or 
some other complication during tapering. 


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1.4 
POTENTIAL BENEFITS AND RISKS TO HUMAN SUBJECTS 
1.4.1 Potential Benefits 
In the current trial, participants undergo gradual withdrawal of immunosuppression.  Previous clinical 
experience, described in section 1.2, demonstrates that a proportion of these participants will likely be 
able to be withdrawn completely from immunosuppression.  The long-term risks of 
immunosuppression are detailed in section 1.2.1.  This approach provides study participants the 
possibility of graft survival in the absence of immunosuppression.  The thorough clinical and 
laboratory monitoring of potential adverse events during the gradual withdrawal process allow the 
risks of withdrawal to be closely managed. 
1.4.2 Risks 
Standard care in liver transplantation currently includes lifelong administration of 
immunosuppression, typically a calcineurin inhibitor with or without corticosteroids or other 
immunosuppressants.  In the current trial the gradual withdrawal of immunosuppression among 
eligible participants is likely to increase the risk of acute allograft rejection.  In this case additional 
diagnostic procedures and the reinstitution of immunosuppression may be required.  This could lead 
to an increased risk of opportunistic infection. 
2. 
OBJECTIVES 
2.1 
PRIMARY OBJECTIVE 
To evaluate allograft tolerance in pediatric recipients of livers from parental living related donors. 
2.2 
SECONDARY OBJECTIVES 
2.2.1 Safety of Immunosuppression Withdrawal  
To assess the safety of immunosuppression withdrawal. 
2.2.2 Duration 
To assess the durability of allograft tolerance. 
2.2.3 Tolerance Predictive Profiles 
To define profiles of immunologic and genetic features present before or during gradual withdrawal 
of immunosuppression that distinguish tolerant and nontolerant allograft recipients. 
2.2.4 Rejection Profiles 
To define profiles of immunologic and genetic features associated with allograft rejection. 
3. 
STUDY DESIGN 
3.1 
DESCRIPTION 
This is a prospective multicenter, open-label, single-arm trial in which 20 pediatric recipients of 
parental living-donor liver allografts will be gradually withdrawn from immunosuppression with the 
goal of complete withdrawal.  Patients on stable immunosuppression regimens with good organ 


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function and no evidence of acute or chronic rejection or other forms of allograft dysfunction will be 
enrolled.  Participants will undergo gradual withdrawal of immunosuppression and will be followed 
for a minimum of 8 years after completion of withdrawal.  Immunologic and genetic profiles will be 
collected at multiple time points and compared between tolerant and nontolerant individuals.   
3.2 
ASSENT AND CONSENT FOR PARTICIPATION 
Consent must be obtained from individuals eligible for screening assessments before any screening 
assessments are performed to determine their eligibility for trial participation.  Participants who meet 
the eligibility criteria (defined in sections 4.2 and 4.3) and have continued willingness to participate 
will then be enrolled in the study. 
Requirements for the assent and informed consent process will depend on the participant’s age:   
4–6 years  A parent or legal guardian will sign the consent form for the participant. 
7–12 years  The participant will sign the assent form and a parent or legal guardian will sign the 
consent form.  
13–17 years  The participant and a parent or legal guardian will both sign the consent form.   
>18 years  Only the participant will be required to sign the consent form.  
Informed consent for blood drawing must also be obtained from the parent who was the liver donor 
and will be requested from the parent who is not the donor.  
3.3 
STUDY ENDPOINTS 
3.3.1 Primary Endpoint 
The proportion of participants who are successfully withdrawn from immunosuppression, which is 
defined as those who remain off immunosuppression for at least 1 year. 
3.3.2 Secondary Endpoints 
3.3.2.1 
Safety of Immunosuppression Withdrawal 
1. 
The proportion of participants who have graft loss or who die after initiation of 
immunosuppression withdrawal. 
2. 
The time from the start of immunosuppression withdrawal to  
a. the first episode of acute rejection requiring treatment,  
b. the second episode of acute rejection not requiring treatment, or  
c. the diagnosis of chronic rejection. 
3. 
The distribution of histologic severity among rejection episodes. 
4. 
The incidence of adverse events. 
5. 
Changes in renal function, blood pressure, cholesterol level, and glucose control. 
3.3.2.2 
Duration 
Immunosuppression-free duration, defined as the time from discontinuation of immunosuppression to 
end of trial participation or to time of restarting immunosuppression.  


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3.3.2.3 
Tolerance Predictive Profiles 
Results of mechanistic studies and clinical assessments at various time points that allow for the 
definition of profiles associated with tolerance.  
3.3.2.4 
Rejection Profiles 
Results of mechanistic studies and clinical assessments that allow for definition of profiles associated 
with liver allograft rejection.  
3.3.2.5 
Assessment of Fibrosis Over Time for Participants Undergoing Extended Follow 
Up 
The change in periportal and pervenular fibrosis on liver biopsy over time will be analyzed only for 
participants who remain off immunosuppression and enter Extended Participant Follow Up (see 
Appendix 4). 
3.4 RATIONALE FOR SELECTION OF STUDY POPULATION 
The study population is defined in section 4.1.  The proposed study aims to prospectively identify a 
cohort of stable patients who underwent parental living-donor liver transplantation when they were 
less than 18 years of age.  This choice stems from the following: 
 The concerning incidence of renal dysfunction, hypertension, and metabolic abnormalities 
related to long-term use of immunosuppressive medications in long-term (5–10 years) 
pediatric survivors of liver transplantation. 
 The substantial potential benefit (physical, psychosocial, and financial) resulting from 
immunosuppressive withdrawal that participants can enjoy over a long lifetime.  
 The availability of donor blood and recipient blood and tissue for mechanistic testing. 
 The minimal maintenance immunosuppression currently utilized in these patients (a single 
agent) may increase the likelihood of successful withdrawal. 
Patients for whom the indication for transplantation was hepatitis B, hepatitis C, autoimmune 
hepatitis, or primary sclerosing cholangitis are excluded because these diseases may recur after 
transplantation and their posttransplant course may be affected by withdrawal or intensification of 
immunosuppression as necessitated by a precipitated episode of rejection. 
3.5 STOPPING RULES 
3.5.1 Ongoing Review 
The protocol chair, the ITN clinical trial physician, the NIAID medical monitor, and the NIAID 
Transplant Data and Safety Monitoring Board (DSMB) will periodically review safety data.  
Enrollment of participants in the trial and withdrawal of immunosuppression in current trial 
participants will be suspended at any time if any of these reviews concludes that there are significant 
safety concerns.   
3.5.2 Specific Adverse Events Independent of Participant Enrollment 
Enrollment and immunosuppression withdrawal in trial participants will be suspended if any of the 
following occurs: 
 Any death 
 Any graft loss 


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 Two participants requiring antibody therapy for graft rejection 
 Two participants diagnosed with chronic rejection 
3.5.3 Specific Adverse Events as a Proportion of Enrolled Participants 
An undue incidence of the following qualifying events will be grounds for suspending enrollment and 
immunosuppression withdrawal in trial participants: 
 Severe graft rejection by Banff criteria. 
 Rejection that requires more than one course of bolus corticosteroids. 
Table 5 illustrates the thresholds for the number of qualifying events that would trigger suspension of 
enrollment and further withdrawal of immunosuppression in participants depending on the number of 
evaluable participants.  The incidence rate is the number of participants with a qualifying event 
observed at any time after enrollment divided by the number of evaluable participants enrolled.  An 
evaluable participant is defined as a participant who has been enrolled for at least 4 weeks or a 
participant who has had a qualifying event.  These thresholds reflect the assumption that qualifying 
events should not occur in significantly more than 15% of participants in the trial.  The basis for the 
stopping rule is that if there is reasonable evidence that the true event rate exceeds that value, 
suspension should occur.  Each time a qualifying event occurs, the current number of evaluable 
participants can be compared with the number in Table 5 for the corresponding threshold.  The trial 
will continue if the current number of evaluable participants for the threshold is greater than the 
number in Table 5 and will be suspended otherwise.  
An 80% confidence interval is chosen, instead of a larger one, to reduce the chance of not suspending 
soon enough.  Assessments begin with the second event. 
Table 5.  Qualifying event thresholds triggering suspension of enrollment and 
immunosuppression withdrawal based on the number of evaluable participants 
Threshold for 
Number of Qualifying 
Events 
Evaluable 
Participant 
Number  
Observed 
Event Rate 
80% Confidence Interval 
Lower Bound 
Upper Bound 
2 
3 
0.67 
0.20 
0.97 
3 
7 
0.43 
0.17 
0.72 
4 
11 
0.36 
0.17 
0.60 
5 
16 
0.31 
0.16 
0.50 
6 
20 
0.30 
0.17 
0.47 


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3.6 PACE OF ENROLLMENT AND STUDY DURATION 
Enrollment is defined as starting on the day that immunosuppression withdrawal is initiated.  
Enrollment is planned for 2 years and will be limited to a maximum of two participants every 4 
weeks.  Maximum patient participation is up to 11 years; therefore, the total study duration is 
projected to be a maximum of 13 years. 
4. 
ELIGIBILITY 
4.1 
STUDY POPULATION 
Pediatric recipients of parental living related donor hepatic allografts with adequate and stable graft 
function and no evidence of rejection or significant allograft dysfunction. 
4.2 
INCLUSION CRITERIA 
1. 
Living-donor liver transplantation from a parental donor. 
2. 
Age less than 18 years at the time of transplantation. 
3. 
At least 4 years since transplantation. 
4. 
Availability and willingness of parental liver donor to participate in the trial. 
5. 
Liver biopsy at screening demonstrating no evidence of acute or chronic rejection and a 
less than stage 2 fibrosis on the Ishak scale. 
6. 
Negative urine pregnancy test at entry and agreement to use a medically acceptable form 
of birth control during the study for women of childbearing potential. 
7. 
Negative purified protein derivative (PPD) test results or history of appropriate treatment. 
4.3 
EXCLUSION CRITERIA 
1. 
Indication for transplantation liver failure due to autoimmune disease, such as 
autoimmune hepatitis, primary sclerosing cholangitis, or primary biliary cirrhosis. 
2. 
Hepatitis B infection as defined by the presence of HBSAg or active treatment for 
hepatitis B. 
3. 
Hepatitis C infection as defined by the presence of antibody against hepatitis C. 
4. 
Serologic evidence of autoimmunity defined as abnormal antinuclear, anti-smooth-muscle, 
antimitochondrial, or anti-liver-kidney microsomal antibody titers greater than or equal to 
1:160. 
5. 
Transplantation of a second organ before, simultaneously with, or after liver 
transplantation; or liver retransplantation. 
6. 
Aspartate or alanine aminotransferase (AST or ALT) greater than 2 times the upper limit 
of normal. 
7. 
Total bilirubin and direct bilirubin, and either alkaline phosphatase or gamma-glutamyl 
transferase (GGT) greater than 2 times the upper limit of normal. 
8. 
Clinically significant change in hepatic function in the past 26 weeks. 
9. 
GFR less than 40 mL/min/1.73 m2. 
10. Immunosuppression with 
a. a 50% dose increase in a current agent within 26 weeks of screening, or 
b. more than one agent within 52 weeks of screening. 
11. Any systemic illness requiring or likely to require immunosuppressive drug use. 


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12. Human immunodeficiency virus (HIV) infection. 
13. Pregnancy or breastfeeding. 
14. Unwillingness or inability to comply with study requirements and procedures.  
4.4 
PREMATURE TERMINATION 
4.4.1 Premature Termination of Immunosuppression Withdrawal 
Section 5.3 outlines parameters for discontinuation of immunosuppression withdrawal.  All 
participants who fail to withdraw will be followed as specified. 
4.4.2 Premature Termination from the Trial 
Withdrawal of consent.  Participants who withdraw consent will undergo all mechanistic 
assessments scheduled for visit 12 (see Appendices 1–3) at the time that consent is withdrawn.  
Such participants will be followed but will not be replaced. 
Failure to return.  Participants who do not return for visits and do not respond to repeated 
attempts by the site staff to attend study visits will be considered lost to follow-up.  Such 
participants will not be replaced.  
5. 
STUDY INTERVENTION 
5.1 
IMMUNOSUPPRESSION WITHDRAWAL  
The algorithm for immunosuppression withdrawal is shown in Figure 1.  For tacrolimus, high dose is 
defined as ≥ 0.08 mg/kg/day; low dose is < 0.08 mg/kg/day.  For cyclosporine, high dose is defined as 
≥ 3 mg/kg/day; low dose is < 3 mg/kg/day. 
During the withdrawal process (see Figure 1), additional monitoring at the current 
immunosuppression dose level may be done before continuing on to the next scheduled dose 
reduction.  In such instances, gradual withdrawal must resume, or a biopsy must be performed, within 
4 weeks of the previously scheduled dose reduction. 


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Figure 1.  Immunosuppression withdrawal.  (*See section 5.1 for definition of high dose and 
low dose.) 


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5.2 
ASSESSMENT OF COMPLIANCE WITH STUDY INTERVENTION 
Compliance will be assessed at each scheduled visit or telephone contact in accordance with the 
schedule of events (Appendices 1–4). 
5.3 
INTERRUPTION OR DISCONTINUATION OF STUDY INTERVENTION 
5.3.1 Interruption of Study Intervention 
Immunosuppression withdrawal may be temporarily interrupted for up to 4 weeks for the following 
reasons: 
As clinically indicated.   
To accommodate the post-immunosuppression-withdrawal liver biopsy visit window.  The 
duration of the lowest taper level before completing immunosuppression withdrawal may be 
extended for up to 4 weeks in order for the liver biopsy to be performed within the allowed visit 
window (see section 6.1). 
During this time the participant will be followed per the withdrawal schedule (see Appendix 1). 
Immunosuppression withdrawal may be resumed at any time during the 4-week period as indicated.  
Participants whose immunosuppression withdrawal has been suspended for more than 4 weeks and 
for whom a biopsy is not indicated will be considered to have failed immunosuppression withdrawal. 
Participants who fail immunosuppression withdrawal will move into the medium-intensity follow-up 
schedule (see Appendix 2) for 52 weeks and then will be discharged from the study. 
5.3.2 Discontinuation of Study Intervention 
Immunosuppression withdrawal will be discontinued for a participant if he or she experiences any of 
the following: 
 A second episode of mild rejection not requiring treatment. 
 Any episode of rejection (mild, moderate, or severe) requiring treatment. 
 Chronic rejection. 
 Abnormal LFTs in the absence of biopsy-proven rejection that do not substantially improve 
(see section 5.5.2) within the maximum interval specified in section 5.3.1, Interruption of 
Study Intervention. 
Such participants will be considered to have failed immunosuppression withdrawal. 
Participants who fail immunosuppression withdrawal will move into the medium-intensity follow-up 
schedule (see Appendix 2) for 52 weeks after their LFTs have improved (see section 5.5.2) and then 
will be discharged from the study. 
5.4 
CONCOMITANT MEDICATIONS 
Participants may receive all required concomitant medications as clinically indicated.  


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5.5 
ASSESSMENT OF ALLOGRAFT DYSFUNCTION AND TREATMENT OF 
REJECTION 
5.5.1 Definition of Allograft Dysfunction and Indication for an Allograft 
Biopsy 
Allograft dysfunction occurs when liver function test (LFT) values are elevated compared with 
baseline, as defined below.  LFTs comprise ALT, alkaline phosphatase, and GGT assessments.  
Baseline LFTs are the mean of the screening value, the prebiopsy value, and—if available—the most 
recent home laboratory value.  Allograft dysfunction occurs when either ALT or both alkaline 
phosphatase and GGT are elevated compared with baseline, as defined below:  
If normal or below normal at baseline: Allograft dysfunction occurs when the value reaches 
twice the upper limit of normal.   
If above normal at baseline: Allograft dysfunction occurs when the value reaches twice the 
baseline value. 
If allograft dysfunction is unexplained, a liver biopsy must be performed.  Liver tests can be repeated 
once for confirmation of allograft dysfunction before biopsy. 
5.5.2 Diagnosis, Grading, and Monitoring of Rejection 
Allograft biopsy will be used to diagnose and grade rejection.  Biopsies will be read locally for 
clinical decision-making, according to the Banff global assessment criteria.68  The liver biopsy will 
then be reviewed centrally at the ITN Liver Pathology Core Laboratory at which time protocol 
grading will be determined.  If the biopsy does not demonstrate rejection, an evaluation for other 
causes of liver dysfunction will be performed.  A diagnosis of rejection will necessitate monitoring of 
LFTs according to clinical standards.  Changes in LFTs will be evaluated as follows: 
 If normal or below normal at baseline: 
o Substantially improved when the value is less than or equal to the upper limit of normal. 
o Improved when the value is less than 1.5 times the upper limit of normal. 
o Not improved when the value is greater than or equal to 1.5 times the upper limit of 
normal. 
 If above normal at baseline: 
o Substantially improved when the value is less than or equal to 1.2 times baseline. 
o Improved when the value is less than 1.5 times baseline. 
o Not improved when the value is greater than or equal to 1.5 times baseline. 
An episode of rejection is deemed resolved when LFTs are substantially improved per definitions 
provided.  
5.5.3 Treatment of Acute Rejection  
5.5.3.1 
Additional Assessments Performed During a Rejection Episode 
During a period of diagnosed rejection, additional assessments will be performed according to clinical 
standard of care: 
LFTs. LFTs will be performed upon diagnosis of allograft dysfunction or rejection and at the 
resolution of rejection. 


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CMV and EBV.  During rejection, collection will continue as scheduled in the SOE (see 
Appendices 1–3).   
Quantitative IgGs and autoantibodies.  Blood samples will be drawn upon diagnosis of 
rejection unless these have been drawn within the previous 4 weeks. 
5.5.3.2 
Mild Rejection Not Requiring Treatment 
Mild rejection will be treated as clinically indicated at the discretion of the investigator; management 
will depend on the results of additional follow-up as, outlined in Figure 2.  
If this is the first episode of mild rejection and it resolves without treatment, the participant may 
resume immunosuppression withdrawal at the same dose and level at which withdrawal was 
interrupted. 
If this is the second episode of mild rejection not requiring treatment, the participant will not be 
permitted to resume tapering and will be considered to have failed immunosuppression withdrawal 
(see section 5.3). 
5.5.3.3 
Any Rejection Requiring Treatment 
If a participant experiences a single episode of mild rejection requiring treatment, or moderate or 
severe rejection, immunosuppression withdrawal will be discontinued.  These participants will be 
considered to have failed immunosuppression withdrawal (see section 5.3). The algorithm for 
treatment of rejection is outlined in Figure 2.  These definitions apply to the use of 
immunosuppressive agents for treatment of rejection: 
Reinstitution.  Returning to the regimen used before beginning immunosuppression withdrawal.  
Intensification.  Increasing the dose of immunosuppressant compared with that used before 
beginning immunosuppression withdrawal.  
Addition.  Initiating another nonsteroid immunosuppressant. 
Conversion.  Changing from one immunosuppressant to another. 
Steroid boluses.  Use of high-dose corticosteroids for a defined course, which may or may not be 
followed by an addition of corticosteroids to the maintenance immunosuppression regimen. 
5.5.3.4 
Rejection after Successful Completion of Immunosuppression Withdrawal 
Participants who experience any episode of rejection after successful completion of 
immunosuppression withdrawal will not be eligible for further attempts at withdrawal. These 
participants will undergo reinstitution of maintenance immunosuppression after resolution of  the 
rejection episode as defined in section 5.5.2.  They will undergo safety follow up as described in 
section 6.6.  
 
5.5.4 Treatment of Chronic Rejection 
A diagnosis of chronic rejection is indicated when a biopsy fulfills the Banff criteria and total and 
direct bilirubin are above normal.  Chronic rejection will be treated per the standard of care.  
Participants who experience chronic rejection will be considered to have failed immunosuppression 
withdrawal (see section 5.3). 


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5.6 
PROPHYLACTIC MEDICATIONS 
5.6.1 For Participants Receiving Corticosteroids 
Participants who receive corticosteroid bolus for treatment of allograft dysfunction will receive 
nystatin to prevent fungal infection and trimethoprim/sulfamethoxazole ([TMP/SMX] Bactrim™ or 
Septra®), pentamidine inhalation therapy, or dapsone to prevent Pneumocystis carinii pneumonia 
(PCP).  
5.6.2 For Participants Receiving Antibody Therapy 
Participants who receive antibody therapy for the treatment of allograft dysfunction will receive 
prophylaxis for a minimum of 12 weeks with nystatin to prevent fungal infections; with trimethoprim/ 
sulfamethoxazole ([TMP/SMX] Bactrim™ or Septra®), pentamidine inhalation therapy, or dapsone 
to prevent PCP; and with valganciclovir to prevent cytomegalovirus (CMV).   


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LFTs?
LFTs within 
2 wk?
Banff grade?
If withdrawing, no further 
withdrawal
Steroid boluses
8-wk observation
Biopsy
Reinstitution, 
intensification,
conversion, or addition of 
another agent plus steroid 
boluses *
Continue 
immunosuppression
per investigator
Consider rejection 
refractory, 
antilymphocyte 
antibody, or additional 
immunosuppressants
Moderate
Not improved
Not improved
Improved
Worse
Yes
Banff
stable?
Rx
Not
improved
Improved or
substantially
improved
Improved or
substantially improved
Banff grade?
Mild
Severe
LFTs?
Biopsy
Banff grade?
Improved
Same or
worse
Improved or
substantially
improved
Not improved
Requires
treatment?
Recurrent
episode?
Banff
improved?
LFTs?
4-wk observation
8-wk observation
LFTs?
Biopsy
If withdrawing, 
resume tapering
Improved
Improved or
substantially improved
No
Bx
No
Not
improved
Substantially
improved
Yes
No
Yes
No
If withdrawing, no 
further withdrawal
Biopsy-proven rejection during 
or after withdrawal
If withdrawing, no further 
withdrawal
Yes
Reinstitution, intensification,
conversion, or addition of 
another agent plus steroid 
boluses *
Biopsy or 
treat
Rx
Medium-intensity 
f/u  for 52 wk
Treat
Biopsy or 
treat
8-wk observation
 
Figure 2.  Management of acute rejection.  (*See section 5.5.3.3 for definitions.) 


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6. 
STUDY PROCEDURES 
6.1 
VISIT WINDOWS 
All visits, except as noted below, should be completed within ±2 weeks of the scheduled time points 
in the SOE (see Appendices 1–4): 
 Screening 
o Interval between visits −2 and −1 will not exceed 9 weeks. 
o Interval between visits −1 and 0 will not exceed 2 weeks. 
 Initiation or interruption of gradual withdrawal of immunosuppression 
o Immunosuppression withdrawal (visit 0) may begin on the same day as the liver 
biopsy (visit −1) or up to 2 weeks afterwards. 
o Additional monitoring at the current immunosuppression dose level may be indicated 
before continuing with the gradual withdrawal of immunosuppression.  If so, the 
maximum interruption period may not exceed 4 weeks (see section 5.1). 
 Liver biopsies 
o A liver biopsy to determine eligibility at screening (visit −1) may be performed no 
later than 9 weeks after the initial screening visit (visit −2). 
o If a for-cause biopsy is performed within 6 weeks of a scheduled study visit that 
includes a protocol biopsy, then all the tests (including ITN Core Laboratory blood 
draws, clinical assessments, etc.) for the scheduled visit can be conducted at the time 
as the for-cause biopsy. 
o For participants who have successfully withdrawn from immunosuppression, a 
biopsy will be performed within 4 to 8 weeks after the last dose of 
immunosuppressant was taken.  In order for the biopsy to be performed within this 
time period, the window for the visit on which this biopsy occurs will be extended to 
±6 weeks. 
6.2 
GENERAL ASSESSMENTS 
These general assessments will be performed (see Appendices 1– 4): 
 Informed consent.  Written informed consent and subject assent (if applicable) will be 
obtained before performing any study assessments or procedures. 
 Demographic history.  A history will be taken to obtain the participant’s date of birth, sex, 
race, and ethnicity. 
 General medical history.  A history will be taken to document any present or past diseases, 
any past or planned medical/surgical procedures, and information on the condition under 
study.  Additionally, the most recent results of any previous testing of ALT or alkaline 
phosphatase and GGT will be documented. 
 Liver transplant specific medical history.  Rejection episodes, history of any cancer, CMV, 
infectious episodes, medication change histories, indication for liver transplantation, and 
results from any liver biopsy performed less than 1 year before screening.  (If results for 
that period are not available, results of the most recent liver biopsy before screening will be 
collected). 


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 Physical examination. 
 Vital signs.  Temperature, blood pressure, pulse, respiration, weight, and height will be 
recorded. 
 Review of inclusion and exclusion criteria. 
 PPD skin test.  A purified protein derivative (PPD) test to assess infection with tuberculosis 
will be performed at screening.  Results of a prior test performed within 1 year prior to 
screening will be accepted in lieu of the test at screening and will be recorded on the case 
report forms (CRFs) 
 Telephone consultation. Telephone consultations will be performed as scheduled to assess 
adverse events, changes in concomitant medications, and compliance with study therapy 
and laboratory assessments. 
 Adverse events.  Participants will be assessed for adverse events at all site visits.  All 
adverse events will be recorded on the CRFs. 
 Concomitant medications.  All reported concomitant medications will be recorded on the 
CRFs.  
6.3 
STUDY SITE AND LOCAL LABORATORY ASSESSMENTS 
These laboratory assessments will be performed at study sites or at local laboratories where each 
participant resides: 
 Hematology includes CBC with differential and platelets, INR, and PTT. 
 Comprehensive chemistry includes Na, K, Cl, CO2, Mg, Ca, PO4, BUN, Cr, glucose AST, 
ALT, GGT, alkaline phosphatase, LDH, albumin, total protein, total bilirubin, direct 
bilirubin, uric acid, and fasting cholesterol panel (total cholesterol, HDL, LDL, and 
triglycerides). 
 Basic chemistry includes Na, K, Cl, CO2, BUN, Cr, and glucose. 
 Liver panel includes AST, ALT, GGT, alkaline phosphatase, total bilirubin, and direct 
bilirubin. 
 Autoantibodies include AMA, ASMA, ANA, and ALKMA. 
 Quantitative immunoglobulin (IgG). 
 Viral serology includes evaluation of antibodies to CMV, EBV, hepatitis A, hepatitis B, 
hepatitis C, and HIV. 
 Urine hCG. 
 GFR (i.e., measurement of serum creatinine and child’s height). 
 Hemoglobin A1C. 
 Tacrolimus or cyclosporine serum levels. 
6.4 
CENTRAL LABORATORY ASSESSMENTS 
Whole blood–quantitative PCR for CMV and EBV reactivation will be performed for all participants. 
6.5 
LIVER BIOPSIES 
Liver biopsies will be performed as follows: 
1. 
At screening. 
2. 
For those participants who successfully withdraw from immunosuppression:  


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a. Upon successful completion of immunosuppression withdrawal.  
b. Upon completion of 104 weeks of medium-intensity follow-up, if still 
immunosuppression free. 
c. Upon completion of 104 weeks of low-intensity follow-up, if still immunosuppression 
free. 
d. Upon completion of 156 weeks of Extended Participant follow-up, if still 
immunosuppression free. 
3. 
For any unexplained allograft dysfunction (see section 5.5.1). 
6.5.1 Biopsy Technique 
A consent specific for this procedure will be obtained according to guidelines at the investigative 
sites.  Liver biopsies will be performed with an 18-gauge or larger needle using a percutaneous 
technique.  Investigators may use ultrasound guidance at their discretion.  A minimum of 4 cm of core 
tissue will be obtained: 
 1 cm in RPMI with fetal calf serum will be sent to the protocol chair for intrahepatic 
lymphocyte studies; however, if this laboratory is not receiving samples, than the tissue will 
be divided equally among the three core labs below.  
 1 cm will be formalin fixed and paraffin imbedded at screening (visit −1).  Local laboratory 
will keep two slides for local diagnosis and the rest of the block will be sent to the ITN 
Pathology Core Laboratory for routine histology and immunohistochemical analysis.  For 
all other visits the full 1 cm in formalin will be sent to the ITN Pathology Core Laboratory 
for routine histology and immunohistochemical analysis.  
 1 cm placed in RNAlater® will be sent on dry ice to the ITN RT-PCR Core Laboratory for 
intrahepatic gene expression profiling. 
 1 cm embedded in OCT media.  Cryomold will be frozen and sent to the ITN Core 
Laboratory for immunohistochemical analysis. 
6.5.2 Tissue Handling and Disposition During Rejection Episodes 
Biopsies to rule out rejection will be handled similarly, except that 1 cm in saline will be sent in 
formalin to the local pathology laboratory for analysis instead of to the protocol chair.  Any extra 
tissue beyond 4 cm, however, will be placed in RPMI with 20% fetal calf serum and sent to the 
protocol chair for intrahepatic lymphocyte studies. 
6.6 
FOLLOW-UP ASSESSMENTS 
Frequency of visits and the number of assessments performed will depend on whether the patient is 
undergoing high-, medium-, or low-intensity follow-up (see Figure 3 and Appendices 1–3). 
All participants who successfully withdraw from immunosuppression must complete 12 weeks of 
high-intensity follow-up, 104 weeks of medium-intensity follow-up, and 104 weeks of low-intensity 
follow-up (see Appendices 1–3).  The 12 weeks of high-intensity follow-up will begin immediately 
after the last dose of immunosuppressant medication is taken. 
All participants who remain off immunosuppression after successful completion of low- intensity 
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Participants who experience any episode of rejection after successful complete of immunosuppression 
withdrawal will move into the medium-intensity follow-up schedule (see Appendix 2) for 52 weeks 
after their LFTs have improved (see section 5.5.2).  They will then be discharged from the study. 
 
Participants who fail immunosuppression withdrawal will move into the medium-intensity follow-up 
schedule (see Appendix 2) for 52 weeks and then will be discharged from the study. 
 


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Figure 3.  Participant follow-up 


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7. 
TOLERANCE ASSAYS 
7.1 
 CELL-BASED ASSAYS 
7.1.1 Whole Blood–Flow Cytometry Panel Staining   
Process and achievement of tolerance may be closely related to the types and relative frequencies of 
different immune cell populations.  Five-color flow cytometry for multiparameter cell visualization 
will be used to monitor the dynamic changes in T-cell subsets, naïve vs. memory T cells, regulatory T 
cells, activated cytotoxic T cells, and NK cells.  We will test the hypothesis that changes in the 
proportion of plasmacytoid, as compared with myeloid dendritic, cells have an effect on, and/or 
reflect, tolerance.  In addition, the presence and frequency of regulatory T cells will be monitored to 
see if there are differences between tolerant and nontolerant participants.  Functionality of such 
populations will be tested in donor-specific T-cell assays as described in section 7.1.2. 
7.1.2 Frozen PBMC–T-cell Assays 
There are two pathways that contribute to MHC allorecognition: the direct and indirect pathways.  
The direct pathway of T-cell activation is mediated by recognition of intact donor MHC alloantigens 
presented on the surface of donor cells, whereas the indirect pathway involves presentation of 
allopeptides by MHC molecules on recipient cells.  Monitoring antidonor responses mounted by 
recipient cells is critical since high frequency of donor-specific IFN-producing cells appear to 
correlate with the risk of renal allograft rejection. 
Relative contributions of direct and indirect pathways of antigen presentation to allograft rejection 
can be measured by the ELISPOT assay.  The number of recipient T cells stimulated by whole donor 
PBMC to produce IFN can be used to assess direct T-cell allorecognition.  Similarly, frequencies of 
IFN-producing recipient T cells can also be measured by stimulated self-APC pulsed with sonicated 
donor cells or donor class I or class II peptides.  With the use of these assays, it should be possible to 
determine the relative number of donor-specific recipient T cells primed via the direct or indirect 
pathways.   
In this study, we will focus on the direct pathway measures by looking at changes in the nature of T-
cell response to alloantigen.  In particular, we will look for a shift in the profile of cytokines produced 
in response to alloantigen from TH1 to TH2 type cytokines.  The ELISPOT assay can be used in this 
context to measure frequencies of alloreactive T cells that produce IL-4 or IL-5 vs. IFN-.  This 
immune response transition of cytokine production will be measured by ELISPOT assay or 
intracellular staining.  
Whole blood will be collected from parental living donors.  The donor blood cells obtained will be 
cryopreserved and used in the flow cytometry lymphocytic cross match and donor-specific cell-based 
assays. 
7.1.3 Liver Biopsy–Histology 
Tissue obtained by liver biopsy will be stained by hematoxylin-and-eosin and will be assessed for 
histologic evidence of graft rejection.  Immunohistochemistry will be performed to identify the types 
of recipient cells that infiltrate the allograft and may contribute to rejection.  Conversely, tolerant 
grafts may contain cell infiltrates that are protective, another parameter that can be monitored.  These 


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studies may reveal distinctions in immune cell populations in the liver of individuals with successful 
immunosuppression withdrawal, as compared with nontolerant individuals.  
7.2 
WHOLE BLOOD DNA–HLA GENOTYPES 
Since the development of tolerance may depend on the degree of HLA-matching between donor and 
recipient, DNA collected from participants (transplant recipients and living donors) will be used to 
perform sequence-based HLA typing.  A complete class I and class II haplotype will be completed, 
including fine typing of the DQB and DRB regions.  The degree of matching between donor and 
recipient haplotypes will be correlated to phenotypic tolerance.  
Genotyping for single nucleotide polymorphisms (SNPs) in select immune response genes will also 
be performed.  These analyses will be used to correlate clinical tolerance with genotypes of candidate 
immune response genes. 
7.3 
GENE EXPRESSION PROFILING 
7.3.1 Whole Blood–Gene Expression Profiling 
Previous gene profiling studies in biopsy material of transplant recipients indicate that changes in 
expression of a panel of genes, as measured by quantitative real-time PCR (RT-PCR) or DNA 
microarrays can be predictive of acute allograft rejection.  These genes include perforin, 
cyclooxygenase, and IL-7, among others.  Interestingly, some studies indicate that these changes are 
also measurable by RT-PCR of RNA made from peripheral blood.   
Additionally, peripheral blood RNA samples taken just before withdrawal of immunosuppression, 
during withdrawal, and post withdrawal will be measured on microarray and RT-PCR to see if we can 
discover new patterns of genes that indicate tolerance-like state or a predisposition to tolerance vs. 
rejection before drug withdrawal.  Both microarray and high-throughput quantitative RT-PCR will be 
used to define global changes in gene expression.  Monitoring during withdrawal of 
immunosuppression will allow the detection of changes in gene expression patterns predictive of, or 
correlated with, rejection.  Peripheral blood expression of acute rejection–associated genes will be 
complemented by profiles of these same genes in protocol-directed or clinically indicated liver 
biopsies. 
7.3.2 Liver Biopsy RNA–Gene Expression Profiling 
Transcriptional profiling will be performed at time points described in the schedule of events (see 
Appendices 1–3).  Total RNA will be isolated and analyzed for expression of selected immune 
response genes related to rejection and immunoregulatory functions, including granzyme B, 
cyclooxygenase, IFN, TGF-β, IL-4, IL-7, IL-10, and IL-15.  In addition, we will study genes, such as 
FoxP3, which may indicate immune regulation in the liver.  The success or failure of 
immunosuppression withdrawal will be correlated with the expression of both rejection-associated 
genes and immune regulation genes.  The analysis will be performed using TaqMan® RT-PCR at the 
ITN Core Laboratory.  
7.4 
SERUM ASSAYS 
7.4.1 Serum–Secreted Cytokines  
Levels of various immune cytokines will be measured in participant’s serum to determine if cytokine 
levels are linked to induction of clinical tolerance or are indicative of allograft rejection.  Serum will 


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be collected at regular intervals during the study as well as at the time of clinically directed biopsies.  
The cytokines analyzed will include IL-2, IL-5, IL-10, TNF-α, and IFN-γ.  Higher levels of IL-2, 
TNF-α, and IFN-γ are indicative of a TH1-mediated immune response.  Higher levels of IL-5 and IL-
10 are indicative of a TH2-mediated immune response. 
By using a multianalytical profiling system, the quality, sensitivity, and throughput of cytokine and 
chemokine assays can be improved. 
7.4.2 Serum–HLA Alloantibodies  
Donor-specific alloantibodies have been shown to be less of a risk factor for graft rejection in liver 
transplant recipients than in recipients of other organ allografts.  Their presence, however, may 
preclude development or signal the absence of functional tolerance.  Serum samples collected from 
participants, therefore, will be evaluated for the presence or absence of donor-specific alloantibodies. 
Alloantibody assessments will be performed before immunosuppression withdrawal (baseline) and at 
selected time points during and after withdrawal.  Assessments will include donor-specific cross 
matching with cryopreserved cells, testing against a panel of cryopreserved lymphocytes or HLA 
antigen-coated microparticles. 
8. 
ADVERSE EVENTS 
8.1 
OVERVIEW 
Safety data will be recorded on a case report form (CRF) specifically designed for this purpose.  All 
the SAE information will be recorded on the source document and transcribed onto an SAE report; 
pertinent data will be collected on the appropriate CRF.  All data will be reviewed periodically by the 
Data and Safety Monitoring Board (DSMB).  The DSMB has the authority to withdraw any 
participants and/or terminate the study because of safety concerns. 
Adverse events that are classified as serious according to the definition of health authorities must be 
reported promptly and appropriately to the NIAID, ITN, principal investigators in the trial, IRBs, and 
health authorities.  This section defines the types of adverse events and outlines the procedures for 
appropriately collecting, grading, recording, and reporting them.  Information in this section complies 
with ICH Guideline E2A: Clinical Safety Data Management: Definitions and Standards for Expedited 
Reporting, ICH Guideline E-6: Guideline for Good Clinical Practice, and applies the standards set 
forth in the National Cancer Institute (NCI), Common Terminology Criteria for Adverse Events, 
Version 3.0 (June 10, 2003). 
8.2 
DEFINITIONS 
8.2.1 Adverse Event 
An adverse event is any occurrence or worsening of an undesirable or unintended sign, symptom, 
laboratory finding, or disease that occurs during participation in the trial.  An adverse event will be 
followed until it resolves or until 30 days after a participant terminates from the study, whichever 
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8.2.2 Serious Adverse Event 
A serious adverse event (SAE) or reaction is defined as “any adverse event that suggests a significant 
hazard, contraindication, side effect, or precaution.”  This includes, but is not limited to, any of the 
following events: 
 Death:  A death that occurs during the study or that comes to the attention of the 
investigator during the protocol-defined follow-up period must be reported whether it is 
considered treatment related or not. 
 A life-threatening event:  Any adverse experience that, in the view of the investigator, 
places the participant at immediate risk of death. 
 Inpatient hospitalization or prolongation of existing hospitalization. 
 Persistent or significant disability. 
 An event that requires intervention to prevent permanent impairment or damage.  An 
important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based on appropriate medical judgment, it 
may jeopardize the participant and may require medical or surgical intervention to prevent 
one of the outcomes listed above. 
 Congenital anomaly or birth defect. 
Regardless of the relation of the adverse event to study participation, the event must be reported as an 
SAE if it meets any of the above definitions. 
8.2.3 Unexpected Adverse Events 
An adverse event is considered “unexpected” when its nature (specificity) or severity is not consistent 
with the usual clinical course of individuals who would qualify for this trial but who were not 
enrolled.  
8.3 
COLLECTING ADVERSE EVENTS 
8.3.1 Methods of Collection 
Adverse events will be collected from the time consent is obtained from the participant  until the time 
the event resolves or until he/she completes study participation, whichever comes first. 
Adverse events may be discovered by any of these methods: 
 Observing the participant. 
 Questioning the participant in an objective manner. 
 Receiving an unsolicited complaint from the participant. 
An abnormal value or result from a clinical or laboratory evaluation (e.g., a radiograph, an ultrasound, 
or an electrocardiogram) can also indicate an adverse event if it is determined by the investigator to 
be clinically significant.  If this is the case, it must be recorded in the source document and as an 
adverse event on the appropriate adverse event form(s).  The evaluation that produced the value or 
result should be repeated until that value or result returns to normal or can be explained and the 
participant’s safety is not at risk. 
8.3.2 Collecting Serious Adverse Events 
Serious adverse events will be collected from the time the participant begins study participation until 
30 days after he/she completes or prematurely withdraws from the study. 


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8.3.3 Recording Adverse Events 
Throughout the study, the investigator will record all adverse events on the appropriate adverse event 
case report form (CRF) regardless of their severity or relation to study participation.  The investigator 
will treat participants experiencing adverse events appropriately and observe them at suitable 
intervals until their symptoms resolve or their status stabilizes. 
8.3.4 Recording Serious Adverse Events 
Serious adverse events will be recorded on the adverse event CRF and on the SAE form, and health 
authorities will be notified as outlined in section 8.5. 
8.4 
GRADING AND ATTRIBUTION OF ADVERSE EVENTS 
8.4.1 Grading Criteria 
The study site will grade the severity of adverse events experienced by study participants according to 
the criteria set forth in the National Cancer Institute’s Common Toxicity Criteria for Adverse Events 
Version 3.0 (published June 10, 2003).  This document (referred to herein as the NCI-CTCAE 
manual) provides a common language to describe levels of severity, to analyze and interpret data, and 
to articulate the clinical significance of all adverse events. 
Adverse events will be graded on a scale from 1 to 5 according to the following standards in the NCI-
CTCAE manual: 
Grade 1 = mild adverse event. 
Grade 2 = moderate adverse event. 
Grade 3 = severe and undesirable adverse event.  
Grade 4 = life-threatening or disabling adverse event. 
Grade 5 = death. 
All adverse events will be reported and graded whether they are or are not related to disease 
progression or study participation. 
8.4.2 Attribution Definitions 
The relation, or attribution, of an adverse event to study participation will be determined by the site 
investigator.  The site investigator will also record the determination of attribution on the appropriate 
CRF and/or SAE reporting form.  The relation of an adverse event to study participation will be 
determined using the descriptors and definitions provided in Table 6. 
For additional information and a printable version of the NCI-CTCAE manual, consult the NCI-
CTCAE web site:   http://ctep.cancer.gov/reporting/ctc.html. 
Table 6.  Attribution of adverse events 
Code 
Descriptor 
Definition 
Unrelated Category  
1 
Unrelated 
The adverse event is clearly not related to study participation. 


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8.5 
REPORTING SERIOUS ADVERSE EVENTS 
8.5.1 Reporting Timeline 
When an investigator identifies a serious adverse event (as defined in section 8.2.2), he or she must 
notify the Rho Federal Systems Division, Inc. (Rho Fed) Safety Reporting Center within 24 hours 
of discovering the event using the Rho Fed  24-Hour SAE Reporting Hotline (1-888-746-3293).  In 
addition to being reported by telephone, these events will be entered on the serious adverse event 
form and the adverse event CRF.  Both forms will be faxed to the Rho Fed Safety Reporting Center 
(1-888-746-7231) or E-MAIL ( rho_productsafety@rhoworld.com ). within 24 hours. Rho Fed is 
responsible for notifying the sponsor within 48 hours of receipt of the event.   
8.5.2 Options for Reporting Serious Adverse Events 
After the SAE has been assessed by the investigator, there are three options for reporting an event to 
the appropriate health authorities: 
 
 
No requirement to report.  This option applies if the adverse event is deemed not serious by 
 
the Rho Fed Safety PSS, the ITN clinical physician and the NIAID  
medical monitor. 
 Standard reporting is required.  This option applies if the adverse event is classified as one 
of the following: (a) serious, expected, and study related; (b) serious, expected, and not 
study related; or (c) serious, unexpected and not study related. 
 Expedited reporting is required.  This option applies if the adverse event is considered 
serious, unexpected, and study related.  These events must be reported by the sponsor to the 
appropriate health authorities within 15 days; fatal or life-threatening events must be 
reported within 7 days.  For expedited SAEs, all sites must attach the notification letter and 
MedWatch/CIOMS to the current investigator’s brochure. 
All investigators must report serious adverse events to their respective IRBs as mandated by them. 
 
8.5.3 Reporting Serious Adverse Events to the Data Safety Monitoring 
Board 
The DSMB will be provided listings of all SAEs on an ongoing basis.  Furthermore, the DSMB will 
be informed of expedited SAEs by the Regulatory CRO at the same time as health authorities. 
8.5.4 Reporting Pregnancy 
Any pregnancy that occurs during a clinical study will be reported on an SAE form for tracking 
purposes only.  The investigator should be informed immediately of any pregnancy and should report 
all pregnancies within 24 hours (as described in section 8.5.1) using the SAE form.  The investigator 
should counsel the participant and discuss the risks of continuing with the pregnancy and the possible 
Related Categories  
2 
Unlikely 
The adverse event is doubtfully related to study participation. 
3 
Possible 
The adverse event may be related to study participation. 
4 
Probable 
The adverse event is likely related to study participation. 
5 
Definite 
The adverse event is clearly related to study participation. 


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effects on the fetus.  Monitoring of the participant should continue until the conclusion of the 
pregnancy, and a follow-up SAE reporting form detailing the outcome of the pregnancy should be 
submitted. 
9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 
9.1 
ANALYSIS SAMPLES 
The following groups of participants will define samples for endpoint analysis: 
 Intent-to-treat (ITT) sample:  Subjects who have signed informed consent and are enrolled. 
 Per-protocol (PP) sample:  All participants in whom immunosuppression withdrawal is 
attempted. 
9.2 
ANALYSIS PLAN 
9.2.1 Analysis of Primary Endpoint 
The proportion of participants who are successfully withdrawn from immunosuppression, defined as 
those who remain off immunosuppression for at least 1 year, will be analyzed using the PP sample 
and descriptively summarized with a two-sided, 95% CI.  Participants with acute rejection occurring 
after 1 year following complete immunosuppression withdrawal will be considered successfully 
withdrawn for the purpose of the primary endpoint.  This endpoint will also be analyzed using the 
ITT sample. 
9.2.2 Analysis of Secondary Endpoints 
9.2.2.1 
Safety of Immunosuppression Withdrawal 
The following endpoints will be analyzed using the PP and ITT samples: 
 The proportion of participants who have graft loss or who die after initiation of 
immunosuppression withdrawal will be descriptively summarized with a two-sided, 95% 
CI. 
 Median time from initiation of immunosuppression withdrawal to first episode of acute 
rejection or to first diagnosis of chronic rejection, with a corresponding two-sided 95% CI, 
will be estimated using the Kaplan-Meier method. 
 Distribution of severity among rejection episodes and the incidence of adverse events will 
be descriptively summarized using frequency tables with enumerations and percentages. 
 Descriptive statistics (mean, standard deviation, mean change from baseline) for renal 
function, blood pressure, cholesterol level, and glucose control over time will be presented. 
9.2.2.2 
Duration 
Median immunosuppression-free duration with a corresponding two-sided 95% CI will be estimated 
with the Kaplan-Meier method using the PP and ITT samples. 
9.2.2.3 
Tolerance Predictive Profiles 
Immune and genetic profiles will be described and compared between tolerant and nontolerant 
individuals.  Mechanistic endpoints will be analyzed using the PP sample.  An objective of the study 
is to define a predictive profile that differentiates liver transplant recipients who are successfully 
withdrawn from immunosuppression from those recipients who are not successfully withdrawn, that 


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is, to see if any one mechanistic assay or combination of mechanistic assays can serve as surrogate 
markers of tolerance by identifying a subpopulation of liver transplant recipients who subsequently 
are found to be functionally tolerant.  The mechanistic data analysis will involve determining whether 
each mechanistic assay taken alone or taken in combination with other mechanistic assays can 
discriminate significantly between tolerant individuals and those at risk of developing alloimmune-
mediated graft injury. 
9.2.2.4 
Rejection Profiles 
Rejection profiles will be analyzed in a manner similar to that described above for tolerance 
predictive profiles. 
9.2.2.5 
Assessment of Fibrosis Over Time 
The change in periportal and perivenular fibrosis will be analyzed using a mixed model repeated 
measures ANOVA. This endpoint will be analyzed using all subjects who enter Extended Participant 
Follow up (Appendix 4).  
9.3 
SAMPLE SIZE 
There are no published data available with respect to the success of immunosuppression withdrawal 
exclusively in living-donor pediatric liver transplant patients 4 years or more post transplant.  As 
described in section 1.2.2, the single source of immunosuppression withdrawal data available in 
living-donor pediatric liver transplant recipients is from the Kyoto study, which differs from the 
present study in terms of patient eligibility with respect to time since transplantation (2 years in the 
Kyoto study vs. 4 years in the present study).29 The present study will therefore likely include a larger 
percentage of patients entering on lower relative immunosuppression.  The sample size of 20 is thus 
based on clinical experience and judgment in order to provide a broad, initial pilot estimate of the 
proportion of patients meeting the primary endpoint in this patient population for which no prior data 
exist.  For example, if 9 of 20 participants are successfully withdrawn from immunosuppression, the 
point estimate of the success proportion is 45% (95% CI:  23.1%, 68.5%, based on the exact binomial 
method).  If 4 of 20 are successfully tapered, the point estimate is 20% (95% CI: 5.7%, 43.7%).  This 
broad, initial estimate can subsequently serve in the design of future immunosuppression withdrawal 
studies in this patient population. 
9.4 
PARTICIPANT AND DEMOGRAPHIC DATA 
9.4.1 Study Completion 
The percentage of participants who complete the study, loss to follow-up, time to loss of follow-up, 
and reasons for discontinuation (adverse events, etc.) will be presented.   
9.4.2 Description of Baseline Characteristics and Demographics 
Summary descriptive statistics for demographic, baseline, and transplant-related clinical history 
characteristics will be provided for all enrolled participants.  Demographic characteristics will include 
age, race, sex, body weight, and height.  Continuous data (e.g., age, body weight, and height) will be 
summarized descriptively by mean, standard deviation, median, and range.  Enumerations and 
percentages will be presented for categorical data (e.g., sex and race).   


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9.4.3 Medical History 
Medical history, including the existence of current signs and symptoms and clinical significance, will 
be collected for each body system. 
9.4.4 Use of Medications 
All medications used will be coded using the WHO drug dictionary.  The number and percentage of 
participants receiving concomitant medications/therapies will be presented.   
9.4.5 Safety 
All adverse events, including infections, malignancies, morbidity, and side effects associated with 
immunosuppression withdrawal and/or study participation, will be classified by body system and 
preferred term according to the Medical Dictionary for Regulatory Activities (MedDRA). 
Frequency tables by category of event (e.g., serious, related to study participation, causing the 
discontinuation from the study) and by NCI-CTCAE grade will be presented.  Laboratory values and 
vital signs will also be summarized by mean, standard deviation, and change from baseline. 
9.5 
RANDOMIZATION, STRATIFICATION, AND BLINDING 
Individuals who are deemed qualified for the study will be enrolled and assigned a unique participant 
number within each site.  All clinical activities performed in conjunction with this study will be 
performed in an unblinded manner.  However, mechanistic assays will be performed in a blinded 
fashion because group assignments of tolerant or nontolerant individuals will be unknown until the 
completion or failure of immunosuppressive withdrawal.  Given that this is a one-arm study, there 
will be no randomization procedure performed.   
9.6 
PLANNED INTERIM ANALYSES 
No interim analyses are planned. 
9.7 
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL PLAN 
The principal features of the study design and of the plan for statistical analysis of the data are 
outlined in this protocol and in the subsequent SAP.  Any changes in these principal features will 
require a protocol or an SAP amendment, which will be subject to review by the independent DSMB, 
the study sponsor(s), and the regulatory agencies.  These changes will be described in the final report 
as appropriate.  
10. ACCESS TO SOURCE DATA OR DOCUMENTS 
The investigational sites participating in this study will maintain the highest degree of confidentiality 
permitted for the clinical and research information obtained from the participants and donors in this 
clinical trial.  Medical and research records should be maintained at each site in the strictest 
confidence.  However, as a part of the quality assurance and legal responsibilities of an investigation, 
the investigational sites must permit authorized representatives of the sponsor(s) and regulatory 
agencies to examine (and when required by applicable law, to copy) clinical records for the purpose 
of quality assurance reviews, audits, and evaluations of the study safety and progress.  Unless 
required by the laws that permit copying of records, only the coded identity associated with 
documents or with other participant data may be copied (and all personally identifying information 
must be obscured).  Authorized representatives as noted above are bound to maintain the strict 


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confidentiality of medical and research information that is linked to identified individuals.  The 
investigational sites will normally be notified before auditing visits occur. 
11. QUALITY CONTROL AND QUALITY ASSURANCE 
The investigator is required to keep accurate records to ensure that the conduct of the study is fully 
documented.  The investigator is required to ensure that all case report forms are legibly completed 
for every participant entered in the trial. 
The sponsor is responsible for regularly reviewing the conduct of the trial, for verifying adherence to 
the protocol, and for confirming the completeness, consistency, and accuracy of all documented data. 
To ensure the reliability of the data recorded in the database, double-data entry will be used for all 
fields on the CRF.  The data will be verified by a series of computerized edit checks, and all relevant 
data queries will be resolved regularly.  When the CRFs are complete, they will be reviewed and 
signed by the investigator and returned to the sponsor or the CRO.  All data from the original signed 
CRF will be entered in the database, and a comparison program will be run again.  All discrepancies 
will be reviewed, and any resulting queries will be resolved with the investigator and amended in the 
database.  All elements of data entry (i.e., time, date, verbatim text, and the name of the person 
performing the data entry) will be recorded in an electronic audit trail to allow all data changes in the 
database to be monitored and maintained in accordance with federal regulations. 
12. ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD 
CLINICAL PRACTICE 
12.1 
STATEMENT OF COMPLIANCE 
This trial will be conducted in compliance with the protocol, with current good clinical practices 
(GCP), the principles of the Declaration of Helsinki, and with all applicable regulatory requirements. 
Before study initiation, the protocol and the informed consent documents will be reviewed and 
approved by an appropriate ethics review committee or institutional review board.  Any amendments 
to the protocol or to the consent materials must also be approved before they are implemented. 
12.2 
INFORMED CONSENT 
The informed consent form is a means of providing information about the trial to a prospective 
participant and allows for an informed decision about participation in the study.  All participants (or 
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study or undergoing any study-specific procedures.  The consent process must be recorded in the 
source document.  Consent materials for participants who do not speak or read English must be 
translated into the participant’s appropriate language. 
The informed consent form must be revised whenever important new safety information is available, 
whenever the protocol is amended, and/or whenever any new information becomes available that may 
affect participation in the trial. 
A copy of the informed consent will be given to a prospective participant for review.  The attending 
physician, in the presence of a witness, will review the consent and answer questions.  The 
prospective participant will be told that being in the trial is voluntary and that he or she may withdraw 
from the study at any time and for any reason. 
12.3 
PRIVACY AND CONFIDENTIALITY 
A participant’s privacy and confidentiality will be respected throughout the study.  Each participant 
will be assigned a sequential identification number, and these numbers rather than names will be used 
to collect, store, and report participant information. 
13. PUBLICATION POLICY 
The ITN policy on the publication of study results will apply to this study.  Authorized participants 
can find details of the policy statement on the ITN internet website at 
http://www.immunetolerance.org. 
14. REFERENCES 
1. 
Bucuvalas JC, Ryckman FC. Long-term outcome after liver transplantation in children. 
Pediatr.Transplant. 2002;6(1):30-36. 
2. 
Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl 
J Med. 2003;349(10):931-940. 
3. 
Lee CK, Christensen LL, Magee JC, Ojo AO, Bridges ND. Chronic renal failure in pediatric heart and 
liver recipients: risk factors and impact on survival. [Abstract]. Am J Transplant. 2005;5(5). 
4. 
Hmiel SP, Beck AM, de la Morena MT, Sweet S. Progressive chronic kidney disease after pediatric 
lung transplantation. Am J Transplant. 2005;5(7):1739-1747. 
5. 
Alonso EM. Long-term renal function in pediatric liver and heart recipients. Pediatr Transplant. 
2004;8(4):381-385. 
6. 
Mention K, Lahoche-Manucci A, Bonnevalle M, et al. Renal function outcome in pediatric liver 
transplant recipients. Pediatr Transplant. 2005;9(2):201-207. 
7. 
Avitzur Y, De Luca E, Cantos M, et al. Health status ten years after pediatric liver transplantation--
looking beyond the graft. Transplantation. 2004;78(4):566-573. 
8. 
Hogg RJ, Furth S, Lemley KV, et al. National Kidney Foundation's Kidney Disease Outcomes Quality 
Initiative clinical practice guidelines for chronic kidney disease in children and adolescents: 
evaluation, classification, and stratification. Pediatrics. 2003;111(6 Pt 1):1416-1421. 
9. 
Henry RM, Kostense PJ, Bos G, et al. Mild renal insufficiency is associated with increased 
cardiovascular mortality: The Hoorn Study. Kidney Int. 2002;62(4):1402-1407. 
10. 
Manjunath G, Tighiouart H, Coresh J, et al. Level of kidney function as a risk factor for cardiovascular 
outcomes in the elderly. Kidney Int. 2003;63(3):1121-1129. 
11. 
Bartosh SM, Alonso EM, Whitington PF. Renal outcomes in pediatric liver transplantation. Clin 
Transplant. 1997;11(5 Pt 1):354-360. 
12. 
Berg UB, Ericzon BG, Nemeth A. Renal function before and long after liver transplantation in 
children. Transplantation. 2001;72(4):631-637. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
13. 
Del Compare ME, D'Agostino D, Ferraris JR, Boldrini G, Waisman G, Krmar RT. Twenty-four-hour 
ambulatory blood pressure profiles in liver transplant recipients. Pediatr Transplant. 2004;8(5):496-
501. 
14. 
Sudan DL, Shaw BW, Jr., Langnas AN. Causes of late mortality in pediatric liver transplant recipients. 
Ann Surg. 1998;227(2):289-295. 
15. 
Wallot MA, Mathot M, Janssen M, et al. Long-term survival and late graft loss in pediatric liver 
transplant recipients--a 15-year single-center experience. Liver Transpl. 2002;8(7):615-622. 
16. 
Asfar S, Metrakos P, Fryer J, et al. An analysis of late deaths after liver transplantation. 
Transplantation. 1996;61(9):1377-1381. 
17. 
Fridell JA, Jain A, Reyes J, et al. Causes of mortality beyond 1 year after primary pediatric liver 
transplant under tacrolimus. Transplantation. 2002;74(12):1721-1724. 
18. 
Migliazza L, Lopez SM, Murcia J, et al. Long-term survival expectancy after liver transplantation in 
children. J Pediatr Surg. 2000;35(1):5-7. 
19. 
Ryckman FC, Alonso MH, Bucuvalas JC, Balistreri WF. Long-term survival after liver transplantation. 
J Pediatr Surg. 1999;34(5):845-849. 
20. 
Hurwitz M, Desai DM, Cox KL, Berquist WE, Esquivel CO, Millan MT. Complete 
immunosuppressive withdrawal as a uniform approach to post-transplant lymphoproliferative disease 
in pediatric liver transplantation. Pediatr Transplantation. 2004;8:267-272. 
21. 
Otley CC, Pittelkow MR. Skin cancer in liver transplant recipients. Liver Transpl. 2000;6(3):253-262. 
22. 
Levy M, Backman L, Husberg B, et al. De novo malignancy following liver transplantation: a single-
center study. Transplant Proc. 1993;25(1 Pt 2):1397-1399. 
23. 
Euvrard S, Kanitakis J, Cochat P, Claudy A. Skin cancers following pediatric organ transplantation. 
Dermatol Surg. 2004;30(4 Pt 2):616-621. 
24. 
Devlin J, Doherty D, Thomson L, et al. Defining the outcome of immunosuppression withdrawal after 
liver transplantation. Hepatology. 1998;27(4):926-933. 
25. 
Mazariegos GV, Reyes J, Marino IR, et al. Weaning of immunosuppression in liver transplant 
recipients. Transplantation. 1997;63(2):243-249. 
26. 
Oike F, Yokoi A, Nishimura E, et al. Complete withdrawal of immunosuppression in living donor liver 
transplantation. Transplant Proc. 2002;34(5):1521. 
27. 
Pons JA, Yelamos J, Ramirez P, et al. Endothelial cell chimerism does not influence allograft tolerance 
in liver transplant patients after withdrawal of immunosuppression. Transplantation. 2003;75(7):1045-
1047. 
28. 
Ramos HC, Reyes J, Abu-Elmagd K, et al. Weaning of immunosuppression in long-term liver 
transplant recipients. Transplantation. 1995;59(2):212-217. 
29. 
Takatsuki M, Uemoto S, Inomata Y, et al. Weaning of immunosuppression in living donor liver 
transplant recipients. Transplantation. 2001;72(3):449-454. 
30. 
Anand AC, Hubscher SG, Gunson BK, McMaster P, Neuberger JM. Timing, significance, and 
prognosis of late acute liver allograft rejection. Transplantation. 1995;60(10):1098-1103. 
31. 
Florman S, Schiano T, Kim L, et al. The incidence and significance of late acute cellular rejection 
(>1000 days) after liver transplantation. Clin Transplant. 2004;18(2):152-155. 
32. 
Mor E, Gonwa TA, Husberg BS, Goldstein RM, Klintmalm GB. Late-onset acute rejection in 
orthotopic liver transplantation--associated risk factors and outcome. Transplantation. 1992;54(5):821-
824. 
33. 
Ramji A, Yoshida EM, Bain VG, et al. Late acute rejection after liver transplantation: the Western 
Canada experience. Liver Transpl. 2002;8(10):945-951. 
34. 
Yoshida EM, Shackleton CR, Erb SR, et al. Late acute rejection occurring in liver allograft recipients. 
Can.J Gastroenterol. 1996;10(6):376-380. 
35. 
Sellers M, Singer A, Maller E, Olthoff K, Jacobowski D, Shaked A. Incidence of late acute rejection 
and progression to chronic rejection in pediatric liver recipients. Transplant Proc. 1997;29(1-2):428-
429. 
36. 
D'Antiga L, Dhawan A, Portmann B, et al. Late cellular rejection in paediatric liver transplantation: 
aetiology and outcome. Transplantation. 2002;73(1):80-84. 
37. 
Kerkar N, Hadzic N, Davies ET, et al. De-novo autoimmune hepatitis after liver transplantation. 
Lancet. 1998;351(9100):409-413. 
38. 
Heneghan MA, Portmann BC, Norris SM, et al. Graft dysfunction mimicking autoimmune hepatitis 
following liver transplantation in adults. Hepatology. 2001;34(3):464-470. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
39. 
Salcedo M, Vaquero J, Banares R, et al. Response to steroids in de novo autoimmune hepatitis after 
liver transplantation. Hepatology. 2002;35(2):349-356. 
40. 
Kerkar N, Dugan C, Rumbo A, et al. Rapamycin successfully treats post-transplant autoimmune 
hepatitis. Am J Transplant. Online Early article 15 February 2005. 
41. 
Andries S, Casamayou L, Sempoux C, et al. Posttransplant immune hepatitis in pediatric liver 
transplant recipients: incidence and maintenance therapy with azathioprine. Transplantation. 
2001;72(2):267-272. 
42. 
Gupta P, Hart J, Cronin D, Kelly S, Millis JM, Brady L. Risk factors for chronic rejection after 
pediatric liver transplantation. Transplantation. 2001;72(6):1098-1102. 
43. 
Aguilera I, Wichmann I, Sousa JM, et al. Antibodies against glutathione S-transferase T1 (GSTT1) in 
patients with de novo immune hepatitis following liver transplantation. Clin Exp.Immunol. 
2001;126(3):535-539. 
44. 
Mieli-Vergani G, Vergani D. De novo autoimmune hepatitis after liver transplantation. J Hepatol. 
2004;40(1):3-7. 
45. 
Minagawa R, Okano S, Tomita Y, et al. The critical role of Fas-Fas ligand interaction in donor-specific 
transfusion-induced tolerance to H-Y antigen. Transplantation. 2004;78(6):799-806. 
46. 
Neil DA, Hubscher SG. Delay in diagnosis: a factor in the poor outcome of late acute rejection of liver 
allografts. Transplant Proc. 2001;33(1-2):1525-1526. 
47. 
Nishida S, Pinna A, Verzaro R, et al. Sirolimus (rapamycin)-based rescue treatment following chronic 
rejection after liver transplantation. Transplant Proc. 2001;33(1-2):1495. 
48. 
Reyes J, Jain A, Mazariegos G, et al. Long-term results after conversion from cyclosporine to 
tacrolimus in pediatric liver transplantation for acute and chronic rejection. Transplantation. 
2000;69(12):2573-2580. 
49. 
Sher LS, Cosenza CA, Michel J, et al. Efficacy of tacrolimus as rescue therapy for chronic rejection in 
orthotopic liver transplantation: a report of the U.S. Multicenter Liver Study Group. Transplantation. 
1997;64(2):258-263. 
50. 
Alonso EM, Piper JB, Echols G, Thistlethwaite JR, Whitington PF. Allograft rejection in pediatric 
recipients of living related liver transplants. Hepatology. 1996;23(1):40-43. 
51. 
Toyoki Y, Renz JF, Mudge C, Ascher NL, Roberts JP, Rosenthal P. Allograft rejection in pediatric 
liver transplantation: Comparison between cadaveric and living related donors. Pediatr Transplant. 
2002;6(4):301-307. 
52. 
Takatsuki M, Uemoto S, Inomata Y, et al. Analysis of alloreactivity and intragraft cytokine profiles in 
living donor liver transplant recipients with graft acceptance. Transpl Immunol. 2001;8(4):279-286. 
53. 
Calne RY, Sells RA, Pena JR, et al. Induction of immunological tolerance by porcine liver allografts. 
Nature. 1969;223(205):472-476. 
54. 
Kamada N, Davies HS, Roser B. Reversal of transplantation immunity by liver grafting. Nature. 
1981;292(5826):840-842. 
55. 
Qian S, Demetris AJ, Murase N, Rao AS, Fung JJ, Starzl TE. Murine liver allograft transplantation: 
tolerance and donor cell chimerism. Hepatology. 1994;19(4):916-924. 
56. 
Starzl TE, Demetris AJ, Trucco M, et al. Cell migration and chimerism after whole-organ 
transplantation: the basis of graft acceptance. Hepatology. 1993;17(6):1127-1152. 
57. 
Neuberger J, Adams DH. What is the significance of acute liver allograft rejection? J Hepatol. 
1998;29(1):143-150. 
58. 
Wiesner RH, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection: incidence, risk factors, 
and impact on outcome. Hepatology. 1998;28(3):638-645. 
59. 
Dousset B, Hubscher SG, Padbury RT, et al. Acute liver allograft rejection--is treatment always 
necessary? Transplantation. 1993;55(3):529-534. 
60. 
Klompmaker LJ, Gouw AS, Haagsma EB, Ten Vergert EM, Verwer R, Slooff MJ. Selective treatment 
of early acute rejection after liver transplantation: effects on liver, infection rate, and outcome. Transpl 
Int. 1997;10(1):40-44. 
61. 
Schlitt HJ, Nashan B, Krick P, et al. Intragraft immune events after human liver transplantation. 
Correlation with clinical signs of acute rejection and influence of immunosuppression. 
Transplantation. 1992;54(2):273-278. 
62. 
Libby P, Pober JS. Chronic rejection. Immunity. 2001;14(4):387-397. 
63. 
Cao S, Cox KL, Berquist W, et al. Long-term outcomes in pediatric liver recipients: comparison 
between cyclosporin A and tacrolimus. Pediatr.Transplant. 1999;3(1):22-26. 


Immune Tolerance Network 
CONFIDENTIAL 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
64. 
Jain A, Mazariegos G, Pokharna R, et al. Almost total absence of chronic rejection in primary pediatric 
liver transplantation under tacrolimus. Transplant Proc. 2002;34(5):1968-1969. 
65. 
Bertolino P, McCaughan GW, Bowen DG. Role of primary intrahepatic T-cell activation in the 'liver 
tolerance effect'. Immunol Cell Biol. 2002;80(1):84-92. 
66. 
Crispe IN. Hepatic T cells and liver tolerance. Nat.Rev.Immunol. 2003;3(1):51-62. 
67. 
Norris S, Collins C, Doherty DG, et al. Resident human hepatic lymphocytes are phenotypically 
different from circulating lymphocytes. J Hepatol. 1998;28(1):84-90. 
68. 
Banff schema for grading liver allograft rejection: an international consensus document. Hepatology. 
1997;25(3):658-663. 
 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
Appendix 1.  Schedule of Events:  Gradual to Complete Withdrawal Plus 
3 Months of High-intensity Follow-up 
 
Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
General Assessments 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Liver transplant: specific 
medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Vital signs 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Inclusion/exclusion 
criteria 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
PPD skin test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Telephone consultation 
 
 
X 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant  
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study Site and Local Laboratory Assessments 
Hematology 
X1 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Comprehensive 
chemistry 
X1 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Basic chemistry 
 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Liver panel 
 
X 
Every 2 weeks starting at visit 02 
Autoantibodies 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Quantitative 
immunoglobulins 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Viral serology3 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                          
1    Can be performed within 9 weeks before the liver biopsy visit. 
2  LFTs must be performed upon diagnosis of allograft dysfunction or rejection and at the resolution of rejection 
(see sections 5.5.2 and 5.5.3.1).  Additional LFTs may be performed during rejection at the investigator’s 
discretion.    
3 See section 6.3 for a description of the viral tests to be performed. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
 
Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
Urine hCG  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Glomerular filtration rate 
(creatinine and height) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hemoglobin A1C 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tacrolimus or 
cyclosporine serum 
levels 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Central Laboratory Assessments 
Whole blood–
quantitative PCR for 
CMV reactivation 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Whole blood–
quantitative PCR for 
EBV reactivation 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver Biopsies 
Liver biopsies4 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Tolerance Assessments7 
Whole blood–flow 
cytometry panel staining X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Frozen PBMC–T-cell 
assays 9 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver biopsy–histology  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Whole-blood–gene 
expression profiling 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver biopsy RNA–gene 
expression profiling 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Serum–secreted 
cytokines 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
                                                          
4 Additional biopsies will be done to rule out rejection if necessary. 
5 Will be performed within 4 to 8 weeks after the last dose of immunosuppressant is taken. 
6 Will be performed only at visit 12 and not at visit 24. 
7 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits 12 
and 24 (except for whole blood–flow cytometry panel staining) will be collected at this time. 
8 Please try to collect blood at assigned visit -2; if this is not possible,  collect at visit −1.  If not able to collect at 
visit −1,  please collect at visit 0. 
9 To be collected from living donor and nondonor parents after they have signed the informed consent; blood 
draw can be done at any visit during the recipient’s trial participation. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
 
Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
Serum–HLA 
alloantibodies 
X8 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood DNA–HLA 
genotypes9,  10 
X8 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                          
10 Collection from the participant may be deferred to any visit during the trial depending on participant’s weight 
and blood volume status. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
Appendix 2.  Schedule of Events: Medium-intensity Follow-up 
 
Medium-intensity Follow-up 1 
Monthly visit 
M1 
M2 
M3 
M4 
M5 
M6 
M7 
M8 
M9 
M10 
M11 
M12 
M13 M14 
M15 
M16 
M17 
M18 
M19 
M20 
M21 
M22 
M23 
M24 
General Assessments 
Physical examination 
 
 
 
 
 
X 
 
 
 
 
 
X 
Vital signs 
 
 
 
 
 
X 
 
 
 
 
 
X 
Telephone consultation 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study Site and Local Laboratory Assessments 
Hematology 
 
 
 
 
 
X 
 
 
 
 
 
X 
Comprehensive 
chemistry 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver panel 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
 
Autoantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 
Quantitative IgG 
 
 
 
 
 
X 
 
 
 
 
 
X 
Glomerular filtration rate 
(creatinine and height) 
X2 
 
 
 
 
 
 
 
 
 
 
X 
Hemoglobin A1C 
X2 
 
 
 
 
 
 
 
 
 
 
X 
Central Laboratory Assessments 
Whole blood–
quantitative PCR for 
CMV reactivation2 
X3 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood–
quantitative PCR for 
EBV reactivation 2 
X3 
 
 
 
 
X 
 
 
 
 
 
X 
Liver Biopsies 
Liver biopsies4, 5 
 
 
 
 
 
 
 
 
 
 
 
X 
Tolerance Assessments6,7 
Whole blood–flow 
cytometry panel staining 
 
 
 
 
 
X 
 
 
 
 
 
X 
Frozen PBMC–T-cell 
assays 
 
 
 
 
 
X 
 
 
 
 
 
X 
                                                          
1 Participants enter medium-intensity follow-up after completing 3 months of high-intensity follow-up or after 
failing immunosuppression withdrawal (see section 5.3).  Participants who have successfully completed 
immunosuppression withdrawal will remain in medium-intensity follow up for 24 months.  Participants who 
fail immunosuppression withdrawal will remain in medium-intensity follow-up for 12 months and then be 
discharged from the study. 
2 Will be performed only at visit M1 and not at visit M13. 
3 Do not collect if last sample was collected less than 6 weeks before visit M1. 
4 Additional biopsies will be performed if necessary to rule out rejection. 
5 Will be performed only at the end of the second year (i.e., month 24) of medium-intensity follow-up for those 
who have successfully withdrawn.  Will not be done for those who experienced rejection and have completed 
1 year of medium-intensity follow-up. 
6 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits 
M12 and M24 (except for whole blood–flow cytometry panel staining) will be collected at this time. 
7 Do not collect for participants who have failed immunosuppression withdrawal.  


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
 
Medium-intensity Follow-up 1 
Monthly visit 
M1 
M2 
M3 
M4 
M5 
M6 
M7 
M8 
M9 
M10 
M11 
M12 
M13 M14 
M15 
M16 
M17 
M18 
M19 
M20 
M21 
M22 
M23 
M24 
Liver biopsy–histology 4 
 
 
 
 
 
 
 
 
 
 
 
X 
Whole-blood–gene 
expression profiling 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver biopsy RNA–gene 
expression profiling4 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–secreted 
cytokines 
 
 
 
 
 
X 
 
 
 
 
 
X 
Serum–HLA 
alloantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
Appendix 3.  Schedule of Events: Low-intensity Follow-up 
 
Low-intensity Follow-up1 
Monthly visit 
L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L15 
L16 
L17 
L18 
L19 
L20 
L21 
L22 
L23 
L24 
General Assessments 
Physical examination 
 
 
 
 
 
X 
 
 
 
 
 
X 
Vital signs 
 
 
 
 
 
X 
 
 
 
 
 
X 
Telephone consultation 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
Adverse events 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Concomitant medications 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Study Site and Local Laboratory Assessments 
Hematology 
 
 
 
 
 
X 
 
 
 
 
 
X 
Comprehensive chemistry 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver panel 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
Autoantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 
Quantitative IgG 
 
 
 
 
 
X 
 
 
 
 
 
X 
Glomerular filtration rate 
(creatinine and height) 
 
 
 
 
 
 
 
 
 
 
 
X 
Hemoglobin A1C 
 
 
 
 
 
 
 
 
 
 
 
X 
Central Laboratory Assessments 
Whole blood–quantitative 
PCR for CMV reactivation 
 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood–quantitative 
PCR for EBV reactivation 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver Biopsies 
Liver biopsies2, 3 
 
 
 
 
 
 
 
 
 
 
 
X 
Tolerance Assessments4 
Whole blood–flow 
cytometry panel staining 
 
 
 
 
 
 
 
 
 
 
 
X 
Frozen PBMC–T-cell 
assays 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver biopsy–histology 2 
 
 
 
 
 
 
 
 
 
 
 
X 
Whole blood–gene 
expression profiling 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver biopsy RNA–gene 
expression profiling 2 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–secreted cytokines 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–HLA alloantibodies 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
                                                          
1 Participants enter low-intensity follow-up after completing medium-intensity follow-up. 
2 Additional biopsies will be performed if necessary to rule out rejection. 
3 Will be performed at the end of the second year (i.e., monthly visit L24) of low-intensity follow-up. 
4 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits 
L12 and L24 (except for whole blood–flow cytometry panel staining) will be collected at this time. 


Immune Tolerance Network 
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Protocol ITN029ST 
Immunosuppression Withdrawal                        
 
 Version 5.0 (April 19, 2011) 
for Pediatric Liver Recipients 
Appendix 4. Schedule of Events: Extended Participant Follow-up 
 
Extended Participant Follow-up 1 
Extended Follow-Up Week: 
8 
17 
26 
35 
44 
52 
Visit Number: Year 1  
E2 
E4 
E6 
E8 
E10 
E12 
Visit Number: Year 2  
E14 
E16 
E18 
E20 
E22 
E24 
Visit Number: Year 3  
E26 
E28 
E30 
E32 
E34 
E36 
Visit Number: Year 4  
E38 
E40 
E42 
E44 
E46 
E48 
General Assessments 
Physical examination 
 
 
 
 
X 
Vital signs 
 
 
 
 
X 
Telephone consultation 
X 
X 
X 
X 
X 
X 
Adverse events 
X 
X 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
Study Site and Local Laboratory Assessments 
Hematology 
 
 
 
 
X 
Comprehensive chemistry 
 
 
 
 
X 
Liver panel 
X 
X 
X 
X 
X 
 
Autoantibodies 
 
 
 
 
X 
Quantitative IgG 
 
 
 
 
X 
Glomerular filtration rate (creatinine and height) 
 
 
 
 
 
X 
Hemoglobin A1C 
 
 
 
 
 
X 
Liver Biopsies 
Liver biopsies2  
 
 
 
 
 
X 
Tolerance Assessments3 
Whole blood–flow cytometry panel staining 
 
 
 
 
 
X 
Frozen PBMC–T-cell assays 
 
 
 
 
 
X 
Liver biopsy–histology 2 
 
 
 
 
 
X 
Whole blood–gene expression profiling 
 
 
 
 
 
X 
Liver biopsy RNA–gene expression profiling 2 
 
 
 
 
 
X 
Serum–secreted cytokines 
 
 
 
 
 
X 
Serum–HLA alloantibodies 
 
 
 
 
 
X 
 
                                                          
1 Participants enter extended follow-up after completing low-intensity follow-up. 
2 Liver biopsy will be performed as part of the E36 visit. Additional biopsies will be performed according to 
protocol guidelines or at the discretion of the site investigator. 
3 If a rejection episode occurs, samples for all tolerance assessments that are scheduled for E36 will be collected 
at this time – including liver biopsy related collections. 

SAP_001.pdf: 
1 
 
 
FINAL STATISTICAL ANALYSIS PLAN 
Version 1.0 
 
 
 
 
Immunosuppression Withdrawal for Pediatric Living-donor Liver 
Transplant Recipients  
 
 
 
PROTOCOL NUMBER ITN029ST 
 
 
 
SPONSOR 
 
This clinical study is supported and conducted by the Immune Tolerance Network, which is 
sponsored by the National Institute of Allergy and Infectious Diseases. 
 
Immune Tolerance Network 
 
 
 
National Institute of Allergy and Infectious Diseases 
 
 
 
 
 
PREPARED BY 
 
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Statistical Analysis Plan 
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DOCUMENT VERSION CONTROL 
Version Number 
Date 
Comments/Changes 
0.1 
06 October 2009 
First Draft 
1.0    
23 April 2010 
Final 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPROVALS 
Approved: 
 
Date: 
 
 
 
  
Protocol Chair 
 
 
 
 
Date: 
 
 
 
NIAID Medical Monitor 
 
 
 
 
Date: 
 
 
 
ITN Clinical Trial Physician 
 
 
 
 
Date: 
 
 
 
Senior Statistical Scientist 
 
 
 
 
Date: 
 
 
 
Biostatistician 
 
 


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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS .............................................................................................. 5 
LIST OF IN-TEXT TABLES AND FIGURES .................................................................... 7 
1. 
INTRODUCTION ....................................................................................................... 7 
2. 
CLINICAL BACKGROUND ....................................................................................... 7 
3. 
PROTOCOL SUMMARY ........................................................................................... 8 
3.1 
Study Objectives ............................................................................................ 8 
3.1.1 
Primary Objective ................................................................................. 8 
3.1.2 
Secondary Objectives ........................................................................... 8 
3.2 
Study Endpoints ............................................................................................. 8 
3.2.1 
Primary Endpoints ................................................................................ 8 
3.2.2 
Secondary Endpoints ............................................................................ 9 
3.3 
Stratification Variables.................................................................................... 9 
3.4 
Overall Study Design and Plan ...................................................................... 9 
3.5 
Study Population ............................................................................................ 9 
3.5.1 
Inclusion and Exclusion Criteria .......................................................... 10 
3.6 
Treatment Regimens .................................................................................... 11 
3.7 
Sample Size Determination .......................................................................... 12 
4. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS .................................. 12 
5. 
ANALYSIS POPULATIONS .................................................................................... 14 
6. 
STUDY SUBJECTS ................................................................................................. 14 
6.1 
Disposition of Subjects ................................................................................. 14 
6.2 
Protocol Deviations ...................................................................................... 15 
7. 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ......................... 15 
7.1 
Recipient and Donor Demographics ............................................................ 15 
8. 
MEASUREMENTS OF TREATMENT COMPLIANCE ............................................ 16 
9. 
EFFICACY EVALUATION ....................................................................................... 16 
9.1 
Overview of Efficacy Analysis Issues ........................................................... 16 
9.1.1 
Handling of Dropouts or Missing Data ................................................ 16 
9.1.2 
Multicenter Studies ............................................................................. 16 
9.1.3 
Assessment Time Windows ................................................................ 16 
9.2 
Efficacy Variables ......................................................................................... 17 
9.2.1 
Primary Efficacy Variable .................................................................... 17 
9.2.2 
Secondary Efficacy Variables ............................................................. 17 
9.3 
Analysis Methods ......................................................................................... 18 
9.3.1 
Primary Efficacy Analyses .................................................................. 18 
9.3.2 
Secondary Efficacy Analyses.............................................................. 19 
9.4 
Examination of Subgroups ........................................................................... 19 
10.  SAFETY EVALUATION .......................................................................................... 19 
10.1 
Overview of Safety Analysis Methods .......................................................... 19 
10.2 
Extent of Exposure ....................................................................................... 19 
10.3 
Adverse Events ............................................................................................ 19 
10.4 
Deaths, Serious Adverse Events, and Other Significant Adverse Events .... 20 


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10.4.1 
Deaths and Serious Adverse Events .................................................. 20 
10.4.2 
Histologic Severity of Rejection Episodes ........................................... 20 
10.4.3 
Graft Loss ........................................................................................... 21 
10.4.4 
Allograft Dysfunction Episodes ........................................................... 21 
10.4.5 
Change in Renal Function, Blood Pressure, Cholesterol, and Glucose 
Control ................................................................................................ 21 
10.4.6 
Infections ............................................................................................ 21 
10.5 
Clinical Laboratory Evaluation ...................................................................... 21 
10.6 
Vital Signs, Physical Findings, and Other Observations Related to Safety .. 22 
10.6.1 
Vital Signs ........................................................................................... 22 
10.6.2 
Physical Examinations ........................................................................ 22 
11.  EXPLORATORY ANALYSES ................................................................................. 23 
12.  OTHER ANALYSES ................................................................................................ 23 
12.1 
Use of Medications ....................................................................................... 23 
12.2 
Immunosuppression Medication History ...................................................... 23 
12.3 
Medical History ............................................................................................. 23 
12.3.1 
Liver Transplant Specific Medical History ........................................... 23 
12.3.2 
Rejection History ................................................................................. 24 
12.3.3 
Prescreening Biopsies ........................................................................ 24 
12.3.4 
Blood Typing ....................................................................................... 24 
13.  INTERIM ANALYSES AND DATA MONITORING .................................................. 24 
14.  CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL ........................ 25 
15.  REFERENCES ........................................................................................................ 25 
16.  APPENDICES .......................................................................................................... 25 
16.1 
Schedule of Events ...................................................................................... 25 
 
LIST OF ABBREVIATIONS 
ALT   
alanine aminotransferase 
AST   
aspartate aminotransferase 
AMA   
antimitochondrial antibody 
ANA   
antinuclear antibody 
ALKMA  
anti-liver-kidney microsome antibody 
ASMA  
anti-smooth-muscle antibody 
CFR   
Code of Federal Regulations 
CMV   
cytomegalovirus 
CRF 
 
case report form 
CsA   
cyclosporine 
CTCAE  
Common Terminology Criteria for Adverse Events 
DAIT   
Division of Allergy, Immunology, and Transplantation 
 


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D5W   
5% dextrose and water 
DSMB  
Data Safety and Monitoring Board 
EBV   
Epstein-Barr virus 
EDTA  
ethylenedinitrilo tetra-acetic acid 
ELISPOT  
enzyme-linked immunospot 
GCP   
good clinical practice 
GFR   
glomerular filtration rate 
GGT   
gamma-glutamyl transferase 
HCC   
hepatocellular carcinoma 
HIV   
human immunodeficiency virus 
HLA   
human leukocyte antigen 
IRB  
 
institutional review board 
IS  
 
immunosuppression 
INR   
international normalized ratio 
ICH   
International Conference on Harmonization 
IND   
investigational new drug 
IRB  
 
Institutional review board 
ITN  
 
Immune Tolerance Network 
ITT  
 
intent-to-treat 
IV  
 
intravenous 
MedDRA  
Medical Dictionary for Regulatory Activities 
MMF   
mycophenolate mofetil 
NCI   
National Cancer Institute 
NCI-CTCAE  National Cancer Institute Common Toxicity Criteria for 
Adverse Events 
NIAID  
National Institute of Allergy and Infectious Diseases 
NK cells  
natural killer cells 
PBC   
primary biliary cirrhosis 
PBMC  
peripheral blood mononuclear cells 
PBS   
phosphate buffered saline 
PCP   
Pneumocystis carinii pneumonia 
PCR   
polymerase chain reaction 
 


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PI  
 
principal investigator 
PP  
 
per protocol 
SAE   
serious adverse event 
SAEC   
safety adverse event coordinator 
SAP   
statistical analysis plan 
SMT   
study management team 
TMP   
trimethoprim 
TSH   
thyroid-stimulating hormone 
ULN   
upper limit of normal 
WHO   
World Health Organization 
 
 
 
 
 
 
LIST OF IN-TEXT TABLES AND FIGURES 
Table 9-1 
Table of Efficacy Variables and Analysis Methods ........................ 18 
 
 
1. 
INTRODUCTION 
This statistical analysis plan only includes analyses related to the clinical endpoints.  
Mechanistic analyses will be performed at the ITN core facilities and a separate analysis plan 
will be created to detail the planned analyses.  Relevant clinical data from the study will be 
submitted to the core facility to augment the mechanistic analyses. 
2. 
CLINICAL BACKGROUND 
Currently, transplantation of any solid organ incurs a lifelong burden of immunosuppression 
for the recipient.  In spite of many advances, including the development of new agents, the 
basic premises of immunosuppression strategies remain unchanged and, as such, substantial 
metabolic, infectious, and neoplastic complications continue to threaten the recipient’s life 
and well-being.  Several reports, however, have shown that a significant proportion of liver 
recipients (19%–42%) can maintain normal allograft function without immunosuppression—
the definition of “functional tolerance.”  Although drug weaning precipitates rejection in 
some recipients, most episodes are mild or moderate, are easily reversed, and do not result in 
long-term consequences. 
 
These reports have motivated us to propose gradual and complete immunosuppression 
withdrawal in a highly selected subgroup of liver transplant recipients:  those who underwent 


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living-donor liver transplantation as a child (<18 years of age) 4 or more years ago for 
diseases other than viral hepatitis and autoimmune liver disorders, who continue to have 
excellent graft function, and who are on a stable single-agent immunosuppression regimen.  
Recipients will be closely monitored during tapering to ensure expeditious recognition, 
diagnosis, and, if necessary, treatment of liver dysfunction. 
 
The main clinical endpoints measure the outcome of immunosuppression withdrawal.  They 
target the success rate of withdrawal; the duration for which recipients remain off of 
immunosuppression; and the overall incidence, severity, and timing of rejection.  The current 
trial also encompasses a complementary scientific effort to identify, quantify, and 
characterize donor-specific immune responses, immunologic interactions, and genetic 
characteristics that may predict or correlate with functional tolerance.  
 
3.  PROTOCOL SUMMARY 
 
3.1 
Study Objectives 
3.1.1 
Primary Objective 
 
The primary objective is to evaluate allograft tolerance in pediatric recipients of livers from 
parental living related donors.   
 
3.1.2 
Secondary Objectives 
The secondary objectives of this study are 
• To assess the safety of immunosuppression withdrawal in pediatric recipients of 
livers from parental living related donors;  
• To assess the durability of allograft tolerance; 
• To define profiles of immunologic and genetic features present before or during 
gradual withdrawal of immunosuppression that distinguishes tolerant and nontolerant 
allograft recipients. 
• To define profiles of immunologic and genetic features associated with allograft 
rejection. 
 
3.2 
Study Endpoints 
3.2.1 
Primary Endpoints 
The primary endpoint is the proportion of subjects who are successfully withdrawn from 
immunosuppression, which is defined as those who remain off immunosuppression for at 
least 1 year. 
 


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3.2.2 
Secondary Endpoints 
The following secondary endpoints are related to the safety of immunosuppression 
withdrawal. 
o The proportion of subjects who have graft loss or who die after initiation of 
immunosuppression withdrawal. 
o The time from the start of immunosuppression withdrawal to 
o the first episode of acute rejection requiring treatment, 
o the second episode of acute rejection not requiring treatment, or 
o the diagnosis of chronic rejection. 
o The distribution of histologic severity among rejection episodes. 
o The incidence of adverse events. 
o Changes in renal function, blood pressure, cholesterol level, and glucose control. 
 
Other secondary endpoints include 
o Immunosuppression-free duration, defined as the time from discontinuation of 
immunosuppression to end of trial participation or to time of restarting 
immunosuppression; 
o Results of mechanistic studies and clinical assessments at various time points that 
allow for the definition of profiles associated with tolerance; 
o Results of mechanistic studies and clinical assessments that allow for definition of 
profiles associated with liver allograft rejection. 
 
3.3 
Stratification Variables 
Selected analyses will present data grouped by clinical phenotypes of tolerant and not-
tolerant.  A subject is defined to be tolerant if s/he is successfully withdrawn from 
immunosuppression for at least one year.  The definition of tolerant corresponds to the 
primary endpoint. 
3.4 
Overall Study Design and Plan 
This is a prospective multicenter, open-label, single-arm trial in which 20 pediatric recipients 
of parental living-donor liver allografts will undergo gradual withdrawal of 
immunosuppression with the goal of complete withdrawal. Subjects on stable 
immunosuppression regimens with good organ function and no evidence of acute or chronic 
rejection or other forms of allograft dysfunction will be enrolled. Subjects will undergo 
gradual withdrawal of immunosuppression and will be followed for a minimum of 4 years 
after completion of immunosuppression withdrawal. Immunologic and genetic profiles will 
be collected at multiple time points and compared between tolerant and nontolerant subjects. 
 
3.5 
Study Population 
Subjects will be selected from those pediatric recipients of parental living related donor 
hepatic allografts with adequate and stable graft function and no evidence of rejection or 


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significant allograft dysfunction. Subjects must meet all of the inclusion criteria and none of 
the exclusion criteria outlined in the following section. 
 
3.5.1 
Inclusion and Exclusion Criteria 
 
Inclusion Criteria 
 
o Living-donor liver transplantation from a parental donor. 
o Age less than 18 years at the time of transplantation. 
o At least 4 years since transplantation. 
o Availability and willingness of parental liver donor to participate in the trial. 
o Liver biopsy at screening demonstrating no evidence of acute or chronic rejection and 
a less than stage 2 fibrosis on the Ishak scale. 
o Negative urine pregnancy test at entry and agreement to use a medically acceptable 
form of birth control during the study for women of childbearing potential. 
o Negative purified protein derivative (PPD) test results or history of appropriate 
treatment. 
 
Exclusion Criteria 
 
o Indication for transplantation liver failure due to autoimmune disease, such as 
autoimmune hepatitis, primary sclerosing cholangitis, or primary biliary cirrhosis. 
o Hepatitis B infection as defined by the presence of HBSAg or active treatment for 
hepatitis B. 
o Hepatitis C infection as defined by the presence of antibody against hepatitis C. 
o Serologic evidence of autoimmunity defined as abnormal antinuclear, anti-smooth-
muscle, antimitochondrial, or anti-liver-kidney microsomal antibody titers greater 
than or equal to 1:160. 
o Transplantation of a second organ before, simultaneously, or after liver 
transplantation; or liver retransplantation. 
o Aspartate or alanine aminotransferase (AST or ALT) greater than 2 times the upper 
limit of normal. 
o Total bilirubin and direct bilirubin, and either alkaline phosphatase or gamma-
glutamyl transferase (GGT) greater than 2 times the upper limit of normal. 
o Clinically significant change in hepatic function in the past 26 weeks. 
o Glomerular filtration rate (GFR) less than 40 mL/min/1.73 m2. 
o Immunosuppression with  
o 50% dose increase in a current agent within 26 weeks of screening, or  
o more than one agent within 52 weeks of screening. 
o Any systemic illness requiring or likely to require immunosuppressive drug use. 
o Human immunodeficiency virus (HIV) infection. 
o Pregnancy or breastfeeding. 
o Unwillingness or inability to comply with study requirements and procedures.  
 
 


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3.6 
Treatment Regimens 
The subjects in this study will be gradually withdrawn from immunosuppression with the 
goal of complete withdrawal. The algorithm for immunosuppression withdrawal is shown in 
Figure 1. For tacrolimus, high dose is defined as ≥0.08 mg/kg/day; low dose is <0.08 
mg/kg/day. For cyclosporine, high dose is defined as ≥3 mg/kg/day; low dose is <3 
mg/kg/day. 
 
During the withdrawal process (see Figure 1), additional monitoring at the current 
immunosuppression dose level may be done before continuing on to the next scheduled dose 
reduction. In such instances, gradual withdrawal must resume, or a biopsy must be 
performed, within 4 weeks of the previously scheduled dose reduction. 
 


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Figure 1. Immuosuppression withdrawal. (*See protocol section 5.1 for definition of high 
dose and low dose.) 
 
3.7 
Sample Size Determination 
A total of 20 subjects will be enrolled in the study. There are no published data available with 
respect to the success of immunosuppression withdrawal exclusively in living-donor 
pediatric liver transplant subjects 4 years or more post transplant. The sample size of 20 is 
thus based on clinical experience and judgment in order to provide a broad, initial pilot 
estimate of the proportion of subjects meeting the primary endpoint in this subject population 
for which no prior data exist. For example, if 9 of 20 subjects are successfully withdrawn 
from immunosuppression, the point estimate of the success proportion is 45% (95% CI: 
23.1%, 68.5%, based on the exact binomial method). If 4 of 20 are successfully tapered, the 
point estimate is 20% (95% CI: 5.7%, 43.7%). This broad, initial estimate can subsequently 
serve in the design of future immunosuppression withdrawal studies in this subject 
population. 
 
 
4. GENERAL ANALYSIS AND REPORTING CONVENTIONS 
The following analyses and reporting conventions will be used: 
• Categorical variables will be summarized using counts (n) and percentages (%) and 
will be presented in the form n (%).  Percentages will be rounded to 1 decimal place. 
• In general, in frequency tables of variables with different categories (e.g., for 
discontinuation reason, race): if no subjects belong to a certain category, including 
Missing, across all treatment groups, then the printing of this category should be 
suppressed.  Otherwise, if the number of subjects in a category for a treatment group 
is zero, then a zero should be displayed for the number, and the percentage should be 
left blank. 
• Numeric variables will be summarized using n, mean, standard deviation (SD), 
median, min, max.  The min/max will be reported at same level of significance as 
original data.  The mean and median will be reported at 1 more significant digit than 
the precision of the data and SD will be reported at 2 more significant digits than the 
precision of the data.  Descriptive statistics will be displayed in the order: n, mean, 
SD, median, min, max 


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• Following SAS default rules, the median will be reported as the average of the two 
middle numbers if the dataset contains even numbers. 
• Test statistics including t and z test statistics will be reported to two decimal places.  
• P-values will be reported to 3 decimal places if greater than 0.001.  If less than 0.001 
than report ‘<0.001’.  A p-value can be reported as 1.000 only if it is exactly 1.000 
without rounding.  A p-value can be reported as 0.000 only if it is exactly 0.000 
without rounding. 
• All statistical calculations will be performed using the SAS System version 9.1 (or 
higher). 
• Tables and listings will be generated as RTF files with Courier New font, size 8.  
Figures will be generated as CGM files.  Separate stacked files will be created for the 
tables, listings, and figures and converted to PDF prior to delivery. 
• The program name, creation date, and data extraction date will be included in the 
footer of all displays (For example, Program: Program Name 
 Creation Date: 
HH:MM/DDMMMYYYY  Data Extraction Date: DDMMMYYYY) 
• In general, columns with character values will have the header and column values left 
aligned. Numeric columns will be centered around their decimal place with headers 
also centered. 
• In the first column, if text wraps onto another line indent 1 additional space. For 
subgroups, indent 2 spaces. 
• Units of measurement - International units SI will be used for clinical laboratory data 
as a standard presentation.  The metric system will be used whenever possible.  Thus, 
weight will be in kilograms and height in centimeters.  Temperature will be presented 
in Celsius degrees. 
• For general footnotes, ‘Note:’ will come before any bracketed footnotes. 
• All listings will be sorted in order of treatment, subject, and time of assessment (e.g., 
visit, time, and/or event). 
• Episodes of rejection will be determined by the diagnoses from the central 
pathologist.  All references to rejection episodes in this Statistical Analysis Plan refer 
to central pathology reads.   
If departures from these general conventions are present in the specific evaluations section of 
this SAP then those conventions will take precedence over these general conventions.  
 


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5. ANALYSIS POPULATIONS 
Intent to treat (ITT) sample will be defined as all subjects who have signed informed 
consent and are enrolled.  For the purpose of these analyses enrollment is defined as 
beginning the screening process for participation in the study. 
 
 
Per protocol (PP) samples will be defined as all subjects in whom immunosuppression 
withdrawal is attempted. 
• Per protocol 1 (PP1) sample will be defined as all subjects in whom 
immunosuppression withdrawal is attempted. 
• Per protocol 2 (PP2) sample will be defined as all subjects without 
inclusion/exclusion criteria protocol deviations in whom immunosuppression 
withdrawal is attempted. 
 
 
6. STUDY SUBJECTS 
Subject Profiles will be created for a subset of the data collected.  Profiles are similar to data 
listings however instead of listing clinical data by the type of data, profiles will list the data 
by subject in order to provide a comprehensive overview of the clinical status of each 
subject. 
6.1 
Disposition of Subjects 
The disposition of enrolled subjects throughout the study will be listed and summarized in 
tables.  
The numbers and percentages of subjects enrolled, completed, discontinued from 
immunosuppression withdrawal, terminated from protocol, in each analysis population, as 
well as reasons for terminating the protocol early will be presented.  For subjects 
discontinuing immunosuppression withdrawal early, the reasons for discontinuing 
withdrawal early will also be presented.   Analysis populations will be derived according to 
the definitions in Section 5. 
 
Reasons for terminating the study early include: 
• ‘Voluntary Withdrawal’ 
• ‘Death’ 
• ‘Lost to Follow Up’ 
• ‘Adverse Event’ 
• ‘Protocol Deviation’ 


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• ‘Other’ 
 
Reasons for discontinuing study medication/treatment early include: 
•  ‘Protocol Deviation’ 
• ‘Voluntary Discontinuation from Weaning’ 
• ‘Terminated from Study’ 
• ‘Second episode of mild rejection not requiring treatment’ 
• ‘Any episode of Rejection (mild, moderate, or severe) requiring treatment’ 
• ‘Chronic rejection’ 
• ‘Requirement for use of corticosteroid bolus therapy’ 
• ‘Abnormal LFTs in the absence of biopsy-proven rejection that do not substantially 
improve within maximum interval’ 
• ‘Other’ 
 
6.2 
Protocol Deviations 
Protocol deviations will be listed by site and subject with information such as deviation date 
and who identified it, deviation type and details, steps taken to resolve the deviation, and an 
indicator for whether the subject will continue in the trial. Protocol deviations will also be 
summarized in tabular format.  The summary will be reported by clinical phenotype and will 
include the total number of protocol deviations, the number of subjects with at least one 
deviation, and indicators for whether the deviation was an inclusion/exclusion criteria 
deviation and whether the subject will continue in the trial. 
 
 
7. DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS 
7.1 
Recipient and Donor Demographics 
Summary descriptive statistics for demographic characteristics will be reported for the ITT 
sample, the PP1, and the PP2 sample by clinical phenotype.  
Demographic data will include age at transplant, age at enrollment, sex, race (primary and 
secondary), ethnicity, weight at enrollment, and height; these data will be presented 
separately for recipients and donors in the following manner: 
• Continuous data (e.g., age, body weight, and height) will be summarized descriptively 
by mean, standard deviation, median, and range as well as categorized into groups 
(e.g., Age of donors (years): 18-33, 34-49, 50-65) 
• Categorical data (e.g., sex, race, and ethnicity) will be presented as frequencies and 
percentages. 
Recipient demographic data will also be presented in a data listing by site and subject and 
will include birth date, age at enrollment, sex, primary and secondary race, ethnicity, height, 


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weight and BMI.  Donor demographic data will be presented in a data listing by site and 
subject and will include birth date, age at enrollment, sex, primary and secondary race, and 
ethnicity. 
 
8. MEASUREMENTS OF TREATMENT COMPLIANCE 
Compliance to immunosuppression withdrawal schedules will be presented in a data listing 
by subject and dose interval and will include dose, frequency, start date, stop date, duration, 
expected duration per protocol, and tapering status for dose interval.  Expected duration per 
protocol for each dose interval is described above in Figure 1 (Section 3.6).  
 
 
9. EFFICACY EVALUATION 
9.1 
Overview of Efficacy Analysis Issues 
9.1.1 
Handling of Dropouts or Missing Data 
All efforts will be made via the querying and monitoring phases to avoid missing data. In 
general, missing data will not be imputed. 
Missing endpoint data for Kaplan-Meier analyses (time off immunosuppression) will be 
accounted for by censoring the data after the last time point where the subject’s relevant 
endpoints are known. This approach allows all subjects to contribute data at all points where 
subject’s treatment response are known, excluding them only when their data is unknown. 
Endpoint data that is missing due to the subject being lost to follow-up will be imputed as 
failures in the primary endpoint analysis. 
9.1.2 
Multicenter Studies 
Study subjects will be recruited from 3 study sites.  Due to the small number of subjects in 
the study, study data will be analyzed as a whole and no formal accommodation for site-to-
site variation will be made. If any one site is a large enroller, basic descriptive analyses of 
baseline demographics, physical examination findings, medical history, and key study 
endpoints will be repeated for each site individually in order to allow qualitative exploration 
of site-to-site variability.  
9.1.3 
Assessment Time Windows 
All visits, except as noted below, should be completed within ±2 weeks of the scheduled time 
points in the Schedule of Events. 


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-Screening 
• Interval between visits −2 and −1 will not exceed 9 weeks. 
• Interval between visits −1 and 0 will not exceed 2 weeks. 
-Initiation or interruption of gradual withdrawal of immunosuppression 
• Immunosuppression withdrawal (visit 0) may begin on the same day as the liver 
biopsy 
(visit −1) or up to 2 weeks afterwards. 
• Additional monitoring at the current immunosuppression dose level may be indicated 
before continuing with the gradual withdrawal of immunosuppression. If so, the 
maximum interruption period may not exceed 4 weeks (see section 5.1). 
- Liver biopsies 
• A liver biopsy to determine eligibility at screening (visit −1) may be performed no 
later than 9 weeks after the initial screening visit (visit −2). 
• If a for-cause biopsy is performed within 6 weeks of a scheduled study visit that 
includes a protocol biopsy, then all the tests (including ITN Core Laboratory blood 
draws, clinical assessments, etc.) for the scheduled visit can be conducted at the time 
as the for-cause biopsy. 
• For subjects who have successfully withdrawn from immunosuppression, a biopsy 
will be performed within 4 to 8 weeks after the last dose of immunosuppressant was 
taken. In order for the biopsy to be performed within this time period, the window for 
the visit on which this biopsy occurs will be extended to ±6 weeks. 
Unscheduled visits may also occur throughout the study.   
Measurements from unscheduled visits, scheduled visits obtained outside the scheduled 
assessment time windows, and windowed measurements not closest to the scheduled target 
day will be included in listings only and not in by-visit tabular displays. 
9.2 
Efficacy Variables 
9.2.1 
Primary Efficacy Variable 
The proportion of subjects who are successfully withdrawn from immunosuppression, 
defined as those who remain off immunosuppression for at least 1 year.  Subjects with acute 
rejection occurring after 1 year following complete immunosuppression withdrawal will be 
considered successfully withdrawn for the purpose of the primary endpoint.  
 
9.2.2 
Secondary Efficacy Variables 
1)  Immunosuppression-free duration: a continuous variable measured in days and is defined 
as: date of end of trial participation or date of restarting immunosuppression – date of 
completing withdrawal.  All subjects completing withdrawal at any time will be included. 
2) Time to Rejection: time from initiation of immunosuppression withdrawal to first episode 
of acute rejection or first diagnosis of chronic rejection measured in days and defined as date 
of first episode of acute rejection or date of first diagnosis of chronic rejection – date of 


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initiation of immunosuppression withdrawal.  All subjects initiating withdrawal (PP1 sample) 
will be included. 
3) Graft Loss or Death: 
Proportion of subjects who have graft loss or who die after initiation of immunosuppression 
withdrawal: a continuous variable taking on values between 0 and 1, the numerator is the 
total number of subjects who experience either graft loss or death after initiation of 
immunosuppression withdrawal and the denominator is the total number of subjects initiating 
immunosuppression withdrawal (PP1 sample).  This analysis will also be done on the PP2 
sample.  
 
 
 
9.3 
Analysis Methods 
Analysis methods for clinical end points, described in detail below, are summarized in Table 
9-1.  
Table 9-1 
Table of Efficacy Variables and Analysis Methods 
Endpoint (section reference) 
Analysis Technique 
(section reference) 
Analysis 
Sample 
Primary 
 
 
Proportion of subjects who are 
successfully withdrawn from 
immunosuppression ‡ 
Proportion with exact 
95% confidence 
interval   
PP1, PP2 
 
 
 
Secondary 
 
 
1)  Immunosuppression-free 
duration 
Median Time and 
95% CI 
All 
subjects 
completing 
withdrawal
2) Time to Rejection 
Median Time and 
95% CI 
PP1 
3)  Graft Loss or Death  
Proportion with exact 
95% confidence 
interval   
PP1, PP2  
‡ Defined as those who remain off immunosuppression for at least 1 year. 
 
9.3.1 
Primary Efficacy Analyses 
The proportion of subjects in the PP1 and PP2 samples who are successfully withdrawn will 
be descriptively summarized with 95% confidence intervals using an exact binomial method.   


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
23April2010 
 
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9.3.2 
Secondary Efficacy Analyses 
1)  Median immunosuppression-free duration will be estimated for all subjects completing 
withdrawal.  Duration will be displayed graphically with a Kaplan-Meier curve and the 
corresponding median and two-sided 95% confidence interval.  
2) Median time from initiation of immunosuppression withdrawal to first episode of acute 
rejection or to first diagnosis of chronic rejection will be estimated for the PP1 sample.  
Duration will be displayed graphically with a Kaplan-Meier curve and the corresponding 
median and two-sided 95% confidence interval. 
3)  The proportion of subjects experiencing graft loss or death after initiation of 
immunosuppression withdrawal will be descriptively summarized for the PP1 and PP2 
samples as well as the ITT sample with 95% confidence intervals using an exact binomial 
method. 
9.4 
Examination of Subgroups 
No subgroup analyses are planned. 
10. SAFETY EVALUATION 
 
10.1 
Overview of Safety Analysis Methods 
All safety analysis will be carried out using the PP1 sample and the PP2 sample defined in 
Section 5 unless otherwise noted.  Missing safety information will not be imputed.  These 
analyses will not be stratified by site.  Safety will be analyzed through the reporting of 
adverse events, vital signs, physical examinations, and changes in routine laboratory values. 
Listings will be prepared for all safety measurements.  All listings will be sorted in order of 
subject ID and time of assessment (e.g., visit, time, and/or event). 
 
10.2 
Extent of Exposure 
The displays that are used to describe the total burden of immunosuppression and treatment 
compliance (detailed in section 8 and 9.2.2) will also be used to assess treatment exposure.  
10.3 
Adverse Events 
All adverse events will be coded using the MedDRA dictionary, including classification by 
system organ class (SOC) and preferred term.  The severity of adverse events (AE) will be 
classified using the National Cancer Institute’s Common Toxicity Criteria for Adverse 
Events toxicity scale.  


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
23April2010 
 
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An overall summary table will be developed to report the number of events and the number 
and percentage of subjects having at least one event in the following categories:  
• Adverse events (AEs) 
• Serious Adverse Events (SAEs) 
• Deaths 
• AEs reported as definitely, probably, or possibly related to immunosuppression 
withdrawal 
• AEs reported by severity 
The tabulations described above will not include classifications by SOC and preferred term. 
In addition, a summary table of adverse events classified by MedDRA SOC and preferred 
term will be provided for each of the following: 
• AEs 
• AEs by maximum severity  
• AEs by relationship to immunosuppression withdrawal. 
The summary tables will present the total number of events as well as the number and 
percentage of subjects experiencing the events by clinical phenotype.  When reporting the 
number of AEs, if the same AE occurs for a subject on multiple occasions the event will be 
counted once for each occurrence.  When reporting the number of subjects experiencing the 
events, a subject will only be counted once if they ever experience an event within the 
particular system organ class or preferred term.  Percentages will be based on the number of 
subjects categorized in each clinical phenotype. 
Separate data listings will be provided for all AE and AEs leading to treatment 
discontinuation/study termination ordered by subject id. 
10.4 
Deaths, Serious Adverse Events, and Other Significant Adverse 
Events 
10.4.1 
Deaths and Serious Adverse Events 
SAEs will be listed and summarized in the same manner described in section 10.3.  Separate 
displays listing and summarizing death, including time to death and cause of death will also 
be created.  
10.4.2 
Histologic Severity of Rejection Episodes 
The Banff global assessment grade indicating histologic severity of rejection episodes will be 
summarized for the PP1, PP2 and ITT samples by clinical phenotype.  Counts and 
percentages will be based on the number of rejection episodes. 


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
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10.4.3 
Graft Loss 
Data listings will be provided for graft loss, including the date of graft loss, study day, and 
primary cause of graft loss. 
10.4.4 
Allograft Dysfunction Episodes 
 Data listings will be provided for allograft dysfunction episodes, including the date, final 
clinical diagnosis, whether follow-up biopsies were performed, date of biopsies, and whether 
treatment was provided for rejection. 
10.4.5 
Change in Renal Function, Blood Pressure, Cholesterol, and 
Glucose Control 
Renal function is measured by creatinine and GFR.  The GFR for each subject will be 
estimated by the Schwartz Formula: 
 
GFR (mL/min/1.73 m2) = creatinine
height
k
)
(
; 
where k = 0.33 in premature infants, k = 0.45 in full term infants to 1 year old, k = 
0.55 in children 1 year old up to 13 years old, k = 0.65 in adolescent males greater 
than 13 years old (k remains 0.55 for adolescent females greater than 13 years old); 
height is measured in cm; serum creatinine is measured in mg/dL. 
 
 
Renal function, blood pressure, cholesterol, and glucose results and their respective changes 
from baseline will be summarized by visit for subjects in the PP1, PP2 and the ITT samples 
by clinical phenotype.   
 
Spaghetti plots for post-screening visits will be created for renal function measurements, 
blood pressure, cholesterol, and glucose. 
 
10.4.6 
Infections 
Infection site, start date, stop date, culture site, result, and organism will be listed for all 
infections for all subjects. 
 
 
10.5 
Clinical Laboratory Evaluation 
Clinical laboratory measurements include serum chemistry, urinalysis, and hematology.  
Results will be standardized to the international system of units (SI), where possible.   All 
clinical laboratory evaluations were analyzed at study sites or at local laboratories where 
each subject resides.  Laboratory results and the change from baseline will be summarized by 
visit for subjects in the PP1 sample.  Results for chemistry and hematology labs will be listed 
in separate listings.    
 


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN029ST 
 
23April2010 
 
22 
 
All laboratory tests will be listed, but if there are duplicate laboratory tests for one study 
period then the earliest observation will be used in the table summaries and graphic displays.  
They will be sorted by subject ID, laboratory parameter and time of assessment.  Laboratory 
normal ranges will be included and out-of-range flags (H or L) will be used to denote values 
that are above (H) or below (L) the relevant reference range.  Reference ranges will be 
obtained from The Harriet Lane Handbook1.   
 
Spaghetti plots for baseline and post-screening visits will be created for liver function tests 
including: AST, ALT, alkaline phosphatase, GGT, total bilirubin, and direct bilirubin.   
 
Autoantibodies and quantitative immunoglobulins results and the change from baseline will 
be summarized using descriptive statistics by visit for subjects in the PP1 and PP2 samples as 
well as listed in separate listings. 
 
Subject serology will be summarized by number and percentages for subjects in the PP1 and 
PP2 samples.  CMV and EBV Reactivation will be summarized by descriptive statistics by 
visit for subjects in the PP1 and PP2 samples.  Serology and CMV and EBV Reactivation 
variables will also be presented in separate listings. 
 
10.6 
Vital Signs, Physical Findings, and Other Observations Related to 
Safety 
 
10.6.1 
Vital Signs 
Descriptive statistics of vital signs results and change from baseline of vital signs will be 
summarized for the PP1 and PP2 samples.  Data listings will be provided for vital signs 
measurements.  They will be sorted by subject, vital sign parameter and time of assessment. 
Data reported in English units will be converted to corresponding metric units (e.g., cm, kg, 
degrees C). 
 
10.6.2 
Physical Examinations 
A listing of the physical examination data will be created.  Physical exam status at baseline 
and change in physical examination will be summarized at each post-baseline visit by body 
system.  The categories of change include no change, improved, and worsened.    
 
 


Immune Tolerance Network 
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Protocol No. ITN029ST 
 
23April2010 
 
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11. EXPLORATORY ANALYSES 
All analyses performed on the PP2 sample will also be performed on the PP2 sample 
excluding subjects with selected liver pathologies as exploratory analyses. 
12. OTHER ANALYSES 
12.1 
Use of Medications 
Medications reported on the CRF will be categorized into prior, concomitant or after for 
analysis.  Prior, concomitant or after medications will be identified by comparing the 
medication start and stop dates with the date of initiation of immunosuppression withdrawal.  
Prior medications will have both the medication start and stop date prior to the first dose of 
study medication date.  After medications will have both the medication start and stop date 
after the last dose of study medication date.  All other medications will be classified as 
concomitant, indicating that use of the medication overlapped with use of the study 
medication by at least one day. 
The number and percentage of subjects receiving prior, concomitant and after medications 
will be presented overall and by medication class.  When reporting the number of subjects 
receiving the medication, a subject will only be counted once if they ever received the 
medication within the medication class.  Percentages will be based on the number of subjects 
in the PP1 sample.  Separate data listings will be provided for prior, concomitant and after 
medications. 
 
12.2 
Immunosuppression Medication History 
Number and percentage for types of medication given during induction, given post-
discharge, and used within the 12 months prior to enrollment will be summarized for all 
subjects by visit in the ITT sample.  Separate data listings will be provided for induction, 
post-discharge, and detailed 12 month prior to enrollment to current immunosuppresion.  
 
12.3 
Medical History 
The number and percentage of subjects with clinically significant diseases including allergies 
and current or past medical procedures other than the disease under study, by body system 
will be listed and summarized for the ITT sample.  
12.3.1 
Liver Transplant Specific Medical History 
The liver transplant specific medical history includes indication for liver transplant, whether 
subject has experienced infections associated with liver transplant and the infection type, and 
whether subject has experienced malignancy since the liver transplant.  A data listing will be 
created for liver transplant specific medical history data for the ITT sample. 


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
23April2010 
 
24 
 
 
 
12.3.2 
Rejection History 
Rejection episode date, histological grading and treatment for rejection will be listed for all 
rejection history for the ITT sample.  
 
12.3.3 
Prescreening Biopsies 
Reason for biopsy, BANFF global assessment grade, other findings and Ishak fibrosis score 
will be listed for all pre-screening biopsies for the ITT sample. 
 
12.3.4 
Blood Typing 
The number and percents for blood type and Rh type for all subjects and their donors will be 
summarized.  Blood typing data for all subjects in the ITT sample and their donors will also 
be listed. 
 
 
 
 
13. INTERIM ANALYSES AND DATA MONITORING 
Per protocol version 4.0, there are not any planned interim analyses for this study. 
 
The protocol chair, the ITN clinical trial physician, the NIAID medical monitor, and the 
NIAID Transplant Data and Safety Monitoring Board (DSMB) will periodically review 
safety data. Enrollment of subjects in the trial and withdrawal of immunosuppression in 
current trial subjects will be suspended at any time if any of these reviews concludes that 
there are significant safety concerns. Stopping rules are described in Section 3.5 of the 
protocol. 
 
The progress of the study will be monitored by the NIAID Data and Safety Monitoring Board 
(NIAID DSMB). The NIAID transplant DSMB will be chartered to review safety data and to 
make recommendations regarding continuation, termination, or modification of the study. 
The DSMB will formally review the safety data at least yearly. The discontinuation of study 
treatment will also be periodically reported to the DSMB.  
 
In addition, safety data will be reviewed by the DSMB when an event occurs that is of 
sufficient concern to the NIAID medical monitor or protocol co-chairs to warrant review, or 
when an event occurs that could contribute to a pre-defined stopping rule specified in the 
Protocol.  
 


Immune Tolerance Network 
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Findings will be reported to IRBs and health authorities. 
 
 
14. CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
The principal features of the plan for statistical analysis of the data are outlined in this SAP. 
Any changes in these principal features will require a SAP amendment, which will be subject 
to review by the independent DSMB, the study sponsor(s), and the regulatory agencies. 
These changes will be described in the final report as appropriate. 
 
15. REFERENCES 
 
1. The Harriet Lane Handbook, Fifteenth edition, Editors George K. Siberry MD, MPH; 
Robert Iannone, MD; pg 119-121. (Publisher - Mosby INC).   
  
16. APPENDICES 
16.1 
Schedule of Events 
 
 
Appendix 1.  Schedule of Events:  Gradual to Complete Withdrawal Plus 
3 Months of High-intensity Follow-up 
 
Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
General Assessments 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Liver transplant: specific 
medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN029ST 
 
23April2010 
 
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Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
Vital signs 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Inclusion/exclusion 
criteria 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
PPD skin test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Telephone consultation 
 
 
X 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant  
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study Site and Local Laboratory Assessments 
Hematology 
X1 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Comprehensive 
chemistry 
X1 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Basic chemistry 
 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Liver panel 
 
X 
Every 2 weeks starting at visit 02 
Autoantibodies 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Quantitative 
immunoglobulins 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Viral serology3 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine hCG  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Glomerular filtration rate 
(creatinine and height) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hemoglobin A1C 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tacrolimus or 
cyclosporine serum 
levels 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Central Laboratory Assessments 
                                                 
1    Can be performed within 9 weeks before the liver biopsy visit. 
2  LFTs must be performed upon diagnosis of allograft dysfunction or rejection and at the resolution of rejection 
(see sections 5.5.2 and 5.5.3.1).  Additional LFTs may be performed during rejection at the investigator’s 
discretion.    
3 See section 6.3 for a description of the viral tests to be performed. 


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
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Withdrawal Plus 3-Month High-intensity Follow-up 
Monthly visits 
–2 
–1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
 
 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
Whole blood–
quantitative PCR for 
CMV reactivation 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Whole blood–
quantitative PCR for 
EBV reactivation 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver Biopsies 
Liver biopsies4 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Tolerance Assessments7 
Whole blood–flow 
cytometry panel staining X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Frozen PBMC–T-cell 
assays 9 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver biopsy–histology  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Whole-blood–gene 
expression profiling 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Liver biopsy RNA–gene 
expression profiling 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X5,6 
Serum–secreted 
cytokines 
X8 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Serum–HLA 
alloantibodies 
X8 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood DNA–HLA 
genotypes9,  10 
X8 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                 
4 Additional biopsies will be done to rule out rejection if necessary. 
5 Will be performed within 4 to 8 weeks after the last dose of immunosuppressant is taken. 
6 Will be performed only at visit 12 and not at visit 24. 
7 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits 12 
and 24 (except for whole blood–flow cytometry panel staining) will be collected at this time. 
8 Please try to collect blood at assigned visit -2; if this is not possible,  collect at visit −1.  If not able to collect at 
visit −1,  please collect at visit 0. 
9 To be collected from living donor and nondonor parents after they have signed the informed consent; blood 
draw can be done at any visit during the recipient’s trial participation. 
10 Collection from the subject may be deferred to any visit during the trial depending on subject’s weight and 
blood volume status. 


Immune Tolerance Network 
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Statistical Analysis Plan 
Protocol No. ITN029ST 
 
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Appendix 2.  Schedule of Events: Medium-intensity Follow-up 
 
Medium-intensity Follow-up 11 
Monthly visit 
M1 
M2 
M3 
M4 
M5 
M6 
M7 
M8 
M9 
M10 
M11 
M12 
M13 M14 
M15 
M16 
M17 
M18 
M19 
M20 
M21 
M22 
M23 
M24 
General Assessments 
Physical examination 
 
 
 
 
 
X 
 
 
 
 
 
X 
Vital signs 
 
 
 
 
 
X 
 
 
 
 
 
X 
Telephone consultation 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study Site and Local Laboratory Assessments 
Hematology 
 
 
 
 
 
X 
 
 
 
 
 
X 
Comprehensive 
chemistry 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver panel 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
 
Autoantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 
Quantitative IgG 
 
 
 
 
 
X 
 
 
 
 
 
X 
Glomerular filtration rate 
(creatinine and height) 
X2 
 
 
 
 
 
 
 
 
 
 
X 
Hemoglobin A1C 
X2 
 
 
 
 
 
 
 
 
 
 
X 
Central Laboratory Assessments 
Whole blood–
quantitative PCR for 
CMV reactivation12 
X13 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood–
quantitative PCR for 
EBV reactivation 2 
X3 
 
 
 
 
X 
 
 
 
 
 
X 
Liver Biopsies 
Liver biopsies14, 15 
 
 
 
 
 
 
 
 
 
 
 
X 
Tolerance Assessments16,17 
Whole blood–flow 
cytometry panel staining 
 
 
 
 
 
X 
 
 
 
 
 
X 
                                                 
11 Subjects enter medium-intensity follow-up after completing 3 months of high-intensity follow-up or after failing 
immunosuppression withdrawal (see section 5.3).  Subjects who have successfully completed 
immunosuppression withdrawal will remain in medium-intensity follow up for 24 months.  Subjects who fail 
immunosuppression withdrawal will remain in medium-intensity follow-up for 12 months and then be 
discharged from the study. 
12 Will be performed only at visit M1 and not at visit M13. 
13 Do not collect if last sample was collected less than 6 weeks before visit M1. 
14 Additional biopsies will be performed if necessary to rule out rejection. 
15 Will be performed only at the end of the second year (i.e., month 24) of medium-intensity follow-up for those who 
have successfully withdrawn.  Will not be done for those who experienced rejection and have completed 1 year 
of medium-intensity follow-up. 
16 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits M12 and 
M24 (except for whole blood–flow cytometry panel staining) will be collected at this time. 
17 Do not collect for subjects who have failed immunosuppression withdrawal.  


Immune Tolerance Network 
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Medium-intensity Follow-up 11 
Monthly visit 
M1 
M2 
M3 
M4 
M5 
M6 
M7 
M8 
M9 
M10 
M11 
M12 
M13 M14 
M15 
M16 
M17 
M18 
M19 
M20 
M21 
M22 
M23 
M24 
Frozen PBMC–T-cell 
assays 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver biopsy–histology 4 
 
 
 
 
 
 
 
 
 
 
 
X 
Whole-blood–gene 
expression profiling 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver biopsy RNA–gene 
expression profiling4 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–secreted 
cytokines 
 
 
 
 
 
X 
 
 
 
 
 
X 
Serum–HLA 
alloantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Immune Tolerance Network 
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Appendix 3.  Schedule of Events: Low-intensity Follow-up 
 
Low-intensity Follow-up1 
Monthly visit 
L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13
L14 
L15 
L16 
L17 
L18 
L19 
L20 
L21 
L22 
L23 
L24 
General Assessments 
Physical examination 
 
 
 
 
 
X 
 
 
 
 
 
X 
Vital signs 
 
 
 
 
 
X 
 
 
 
 
 
X 
Telephone consultation 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
Adverse events 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Concomitant medications 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Study Site and Local Laboratory Assessments 
Hematology 
 
 
 
 
 
X 
 
 
 
 
 
X 
Comprehensive chemistry 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver panel 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
Autoantibodies 
 
 
 
 
 
X 
 
 
 
 
 
X 
Quantitative IgG 
 
 
 
 
 
X 
 
 
 
 
 
X 
Glomerular filtration rate 
(creatinine and height) 
 
 
 
 
 
 
 
 
 
 
 
X 
Hemoglobin A1C 
 
 
 
 
 
 
 
 
 
 
 
X 
Central Laboratory Assessments 
Whole blood–quantitative 
PCR for CMV reactivation 
 
 
 
 
 
X 
 
 
 
 
 
X 
Whole blood–quantitative 
PCR for EBV reactivation 
 
 
 
 
 
X 
 
 
 
 
 
X 
Liver Biopsies 
Liver biopsies2, 3 
 
 
 
 
 
 
 
 
 
 
 
X 
Tolerance Assessments4
Whole blood–flow 
cytometry panel staining 
 
 
 
 
 
 
 
 
 
 
 
X 
Frozen PBMC–T-cell 
assays 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver biopsy–histology 2 
 
 
 
 
 
 
 
 
 
 
 
X 
Whole blood–gene 
expression profiling 
 
 
 
 
 
 
 
 
 
 
 
X 
Liver biopsy RNA–gene 
expression profiling 2 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–secreted cytokines 
 
 
 
 
 
 
 
 
 
 
 
X 
Serum–HLA alloantibodies 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
1 Subjects enter low-intensity follow-up after completing medium-intensity follow-up. 
2 Additional biopsies will be performed if necessary to rule out rejection. 
3 Will be performed at the end of the second year (i.e., monthly visit L24) of low-intensity follow-up. 
4 If a rejection episode occurs, samples for all mechanistic assessments that are scheduled for monthly visits 
L12 and L24 (except for whole blood–flow cytometry panel staining) will be collected at this time.