Imaging and Biomarker Evaluation of Hepatic Stiffness in Children Enrolled in the Fontan Udenafil Exercise Longitudinal Study
Phase: PHASE3
Conditions: Single Ventricle Heart Disease
Sponsor: Mezzion Pharma Co. Ltd

 
Pediatric Heart Network/Mezzion Pharma Co. Ltd. 
Imaging and Biomarker Evaluation of Hepatic Stiffness in Children Enrolled in the Fontan Udenafil Exercise 
Longitudinal Study (FALD) 
Page 1 of 36 
 
 
Clinical Study Protocol 
PROTOCOL NUMBER: PHN-Udenafil-04 
  
Title 
IMAGING AND BIOMARKER EVALUATION OF HEPATIC 
STIFFNESS IN CHILDREN ENROLLED IN THE FONTAN 
UDENAFIL EXERCISE LONGITUDINAL STUDY 
Phase 
Open Label Extension of parent study 
IND Number 
121648 
Investigational 
Study Products 
Udenafil 
Protocol Version 
Version 3.0 
Final Date 
December 11, 2017 
Sponsor: 
Mezzion Pharma Co. Ltd. in Partnership with Pediatric Heart 
Network (PHN) 
Sponsor Contact: 
 
Mezzion Pharma Co. Ltd. James L. Yeager, RPh, PhD, Tel: 847-283-
9176 
Pediatric Heart Network (PHN) Gail D. Pearson, MD, ScD, Tel: 301-
435-0524 
Sponsor’s 
Responsible 
Medical 
Monitor: 
 
Brian Feingold, MD 
Contract 
Research 
Organization 
(CRO) Contact: 
 
New England Research Institute, Inc. 
Julie Miller, MPH, PMP, Tel: 617-972-3197 
CONFIDENTIAL 

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PROTOCOL REVISION HISTORY 
 
Version History 
Version Number 
Version Date 
Comment(s) 
Version 1.0 
November 1, 2017 
Original Protocol 
Version 2.0 
November 17, 
2017 
Administrative clarification as it relates to 
consent/assent timing. 
Version 3.0 
December 11, 2017 
Change of planned sample size from 100-150 
subjects to 50-150 subjects. 
Removal of Certificate of Confidentiality 
language. 
 
 
 
 
 

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SIGNATURE PAGE
IMAGING AND BIOMARKER EVALUATION OF HEPATIC STIFFNESS IN CHILDREN 
ENROLLED IN THE FONTAN UDENAFIL EXERCISE LONGITUDINAL STUDY
Protocol Number: PHN-Udenafil-04
Sponsor Approvals:
Signature:
___________________________________
Date:
_______________
James L. Yeager, RPh, PhD
Signature:
___________________________________
Date:
_______________
Gail D. Pearson, MD, ScD
James L. 
Yeager
Digitally signed by 
James L. Yeager 
Date: 2017.12.18 
15:00:52 -06'00'
Gail D. 
Pearson -S
Digitally signed by 
Gail D. Pearson -S 
Date: 2017.12.19 
16:08:33 -05'00'

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INVESTIGATOR AGREEMENT PAGE 
 
I have read the protocol and agree to conduct the study as outlined herein.  
I will use the informed consent form approved by the NHLBI and will fulfill all responsibilities 
for submitting pertinent information to the Institutional Review Board or Ethics Committee 
responsible for this study. 
I further agree that the NHLBI and/or its designee has access to any source documents from 
which case report form information may have been generated. 
I also agree to handle all clinical supplies (including drugs, biologics, and/or devices) provided 
by Mezzion® and collect and handle all clinical specimens in accordance with the protocol. 
 
Signature: 
  
 
Date:   
 
Name: 
(print): 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
SIGNATURE PAGE .................................................................................................................................... 3 
INVESTIGATOR AGREEMENT PAGE .................................................................................................... 4 
TABLE OF CONTENTS .............................................................................................................................. 5 
LIST OF FIGURES ...................................................................................................................................... 8 
1.0 
PROTOCOL SYNOPSIS .................................................................................................................. 9 
2.0 
LIST OF ABBREVIATIONS ......................................................................................................... 11 
3.0 
BACKGROUND INFORMATION AND RATIONALE .............................................................. 13 
3.1 
Discussion of Hepatic fibrosis and FUEL Trial Extension Study ............................................... 13 
3.1.1 
FALD .................................................................................................................................. 13 
3.1.2 
Characterization of FALD .................................................................................................. 14 
3.1.3 
FALD and FUEL Extension Study ..................................................................................... 14 
3.2 
Rationale for the Study and Study Outcomes ............................................................................. 15 
3.2.1 
Study Rationale ................................................................................................................... 15 
3.2.2 
Rationale for Study Outcomes ............................................................................................ 15 
3.2.2.1 
The use of ultrasound SWE to evaluate liver fibrosis ..................................................... 15 
3.2.2.2 
The use of Magnetic Resonance Elastrography to evaluate liver fibrosis ...................... 17 
3.2.2.3 
Biomarkers and liver stiffness ......................................................................................... 18 
4.0 
CLINCIAL TRIAL OBJECTIVES, AIMS, AND HYPOTHESES ................................................ 19 
4.1 
Clinical Trial Objective ............................................................................................................... 19 
4.2 
Primary Aim and Hypothesis ...................................................................................................... 19 
4.3 
Secondary Aims and Hypotheses ................................................................................................ 19 
5.0 
STUDY DESIGN ............................................................................................................................ 20 
5.1 
Study Duration ............................................................................................................................ 20 
6.0 
SELECTION AND DISPOSITION OF SUBJECTS ..................................................................... 21 
6.1 
Number of Subjects ..................................................................................................................... 21 
6.2 
Clinical Trial Population Characteristics .................................................................................... 21 
6.3 
Subject Inclusion Criteria............................................................................................................ 21 
6.4 
Subject Exclusion Criteria .......................................................................................................... 21 
6.5 
Subject Withdrawal Criteria........................................................................................................ 21 
7.0 
TREATMENT DESCRIPTION ..................................................................................................... 22 

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7.1 
Description of Treatment ............................................................................................................ 22 
7.2 
Subject Identification Number .................................................................................................... 22 
7.3 
Method of Treatment Assignment and Blinding ......................................................................... 22 
7.4 
Sample Management ................................................................................................................... 22 
8.0 
STUDY ASSESSMENTS/MEASUREMENTS AND PROCEDURES ........................................ 24 
8.1 
Primary Assessments .................................................................................................................. 24 
8.2 
Primary Assessment Measurement ............................................................................................. 24 
8.3 
Additional Measurements ........................................................................................................... 25 
9.0 
PLANNED STATISTICAL ANALYSES AND STATISTICAL CONSIDERATIONS .............. 25 
9.1 
Statistical Analysis Plan .............................................................................................................. 25 
9.2 
Sample Size Determination ......................................................................................................... 25 
9.3 
Analysis Populations ................................................................................................................... 25 
9.4 
Statistical Analysis ...................................................................................................................... 26 
9.4.1 
General Methods ................................................................................................................. 26 
9.4.2 
Primary Outcome ................................................................................................................ 26 
9.4.3 
Secondary Outcomes........................................................................................................... 26 
10.0 
ETHICS AND GENERAL CLINCIAL TRIAL CONDUCT CONSIDERATIONS ..................... 27 
10.1 
Institutional Review Board or Independent Ethics Committee ................................................... 27 
10.2 
Ethical Conduct of the Clinical Trial, Confidentiality, and Potential Risks ............................... 27 
10.3 
Potential Risks ............................................................................................................................ 27 
10.4 
Protection against Risks .............................................................................................................. 28 
10.5 
Potential Benefits ........................................................................................................................ 28 
10.6 
Risk/Benefit Ratio and Importance of Information to be Obtained ............................................ 29 
10.7 
Study Limitations ........................................................................................................................ 29 
11.0 
REGULATORY/ADMINISTRATIVE PROCEDURES AND DOCUMENTATION .................. 30 
11.1 
Data Collection ........................................................................................................................... 30 
11.2 
Quality Assurance ....................................................................................................................... 31 
11.3 
Data Management ....................................................................................................................... 32 
11.3.1 
Data Entry ........................................................................................................................... 32 
11.3.2 
Data Validation and Monitoring ......................................................................................... 33 
11.3.3 
Data Security and Integrity ................................................................................................. 33 
11.3.4 
Biospecimen Tracking ........................................................................................................ 33 

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12.0 
REFERENCES ............................................................................................................................... 34 
 
 
 

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LIST OF FIGURES 
 
Figure 1: Description of Fontan Associated Liver Disease ........................................................................ 13 
Figure 2: Box plot showing increasing liver shear wave speed measurements with increasing parenchymal 
fibrosis. ....................................................................................................................................................... 16 
Figure 3: Individual profile plots from subjects undergoing stage 3 Fontan palliation .............................. 17 
Figure 4: 13 year-old patient status post Fontan palliation due to Single Ventricle Congenital Heart 
Disease. ....................................................................................................................................................... 18 
 
 

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1.0 
PROTOCOL SYNOPSIS 
 
TITLE OF STUDY 
AN IMAGING AND BIOMARKER EVALUATION OF 
HEPATIC STIFFNESS IN CHILDREN ENROLLED IN THE 
FONTAN UDENAFIL EXERCISE LONGITUDINAL STUDY 
PHASE 
Open Label Extension 
STUDY 
OBJECTIVE 
Determine scope of hepatic stiffness in Fontan patients by 
ultrasound and magnetic resonance imaging elastography and 
evaluate the efficacy of udenafil in reducing liver stiffness. 
SIGNIFICANCE 
Study will obtain necessary and broad information regarding hepatic 
stiffness in Fontan patients and will be the first to evaluate a potentially 
efficacious treatment to reduce liver stiffness. 
STUDY DESIGN 
Prospective cohort study integrated with the Fontan Udenafil Exercise 
Longitudinal (FUEL) clinical trial 
PLANNED SAMPLE 
SIZE  
50-150 Subjects 
KEY SUBJECT 
SELECTION 
CRITERIA 
Inclusion Criteria: 
1. Enrollment in FUEL Extension Trial 
2. Informed assent from subject, informed consent from 
parent/legal guardian as appropriate 
 
Exclusion Criteria: 
1. Non-enrollment in the FUEL Extension Trial 
2. Subjects with contra-indications for MRI (these subjects will 
be excluded from the MRI component of this study) 
3. Other exclusionary criteria will match those used for the 
FUEL Extension Trial  
TREATMENT 
One year of udenafil treatment (87.5 mg twice daily) 
PRIMARY AIM 
Define the range of liver stiffness in those who have had the surgical 
creation of a total cavopulmonary connection. 

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SECONDARY 
AIMS: 
• Determine the impact of udenafil treatment on liver stiffness in 
a large cross-section of adolescents who have undergone total 
cavopulmonary connection. 
• Determine the relationship between clinical outcomes and liver 
stiffness. 
• Assess the association between biomarkers of both heart failure 
and liver fibrosis and liver stiffness. 
EFFICACY 
PARAMETERS 
Reduction in liver stiffness after initiating udenafil therapy adolescents 
with Fontan physiology.   
EFFICACY 
ASSESSMENTS 
Efficacy will be assessed at FUEL-OLE initiation and conclusion. 
EFFICACY 
ENDPOINTS 
Absolute liver stiffness as measured by ultrasound (and MR) and 
change in liver stiffness.   
SAFETY 
PARAMETERS 
As this study has no active intervention, there are no specific safety 
measures beyond FUEL-OLE protocols. 
SAFETY 
ASSESSMENTS 
As this study has no active intervention, there are no specific safety 
assessments beyond FUEL-OLE protocols. 
STASTICAL 
ANALYSES 
 Analysis Populations: All individuals enrolling in FUEL-OLE will be 
recruited for enrollment in the FALD study.  At completion of FUEL-
OLE, once FUEL assignments are unblended (udenafil or placebo), the 
cohort will be stratified into udenafil naïve vs. udenafil continuation 
groups.  Analysis will be done both on an intent-to-treat and a per-
protocol basis. 
 
 General Statistical Methods: This is prospective observational study 
of liver stiffness in Fontan patients.  Absolute liver stiffness and 
association of degree of stiffness liver biomarkers will be assessed for 
the entire cohort.  Change in liver stiffness over the 12-month FUEL-
OLE duration will be assessed both for the overall cohort and after 
stratification into udenafil naïve vs. udenafil continuation groups.   
FINAL DATE 
December 11, 2017 
 
 

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2.0 
LIST OF ABBREVIATIONS 
 
Abbreviation or Term 
Definition/Explanation 
ARFI 
Acoustic Radiation Force Impulses 
BNP 
Brain natriuretic peptide 
CAE 
Common adverse event 
CRF 
Case report form 
CHD 
Congenital Heart Disease 
DCC 
Data coordinating center 
DMS 
Data Management System 
EDC 
Electronic data capture 
ELF 
Enhanced Liver Fibrosis 
ELISA 
Enzyme linked immunosorbent assay  
FALD 
Fontan-associated Liver Disease 
FDA 
Food and drug administration 
FUEL 
Fontan Udenafil Exercise Longitudinal Trial 
HA 
Hyaluronic Acid 
ID 
Identification 
IRB 
Institutional Review Board 
MRI 
Magnetic Resonance Imaging 
NHLBI 
National Heart, Lung, and Blood Institute 
PAT 
Pulse amplitude tonometry 
PDE5i 
Phosphodiesterase type 5 inhibitor 
PHN 
Pediatric Heart Network 
PI 
Principal investigator 
PIIINP 
Amino-terminal propeptide of type III collagen 
RCT 
Randomized control trial 
ROC 
Receiver operating characteristic 
RT 
Reverse Transcription 
SAP 
Statistical Analysis Plan 
SWE 
Shear Wave Elastography 
SAE 
Serious adverse event 

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TCPC 
Total Cavo-pulmonary Connection 
TIMP-1 
Tissue inhibitor of metalloproteinase-1 
UMPL 
University of Michigan Pathology Laboratory 
US 
Ultrasound 
 
 
 

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3.0 
BACKGROUND INFORMATION AND RATIONALE 
 
3.1 
Discussion of Hepatic fibrosis and FUEL Trial Extension Study 
3.1.1 FALD 
Fontan-associated liver disease (FALD) is defined as abnormalities in liver structure and 
function resulting from the abnormal circulation created by the total cavopulmonary connection 
(TCPC) and not related to any other process (1). FALD is increasingly recognized as one of the 
most common complications of the TCPC (2, 3) and one which is universally present in patients 
with TCPC physiology (8-11). After the Fontan operation, blood from the inferior and superior 
vena cava are routed directly to the pulmonary arteries bypassing the heart (Figure 1). In this 
circulation, blood is not “pumped” through the lungs but is “pushed” through by chronic 
elevation of the central venous pressure. It is postulated that the Fontan’s elevated, non-phasic 
systemic venous pressure results in obligate chronic hepatic venous hypertension and congestion 
that is likely an important driver of hepatic fibrosis. As part of the injury response, activated 
myofibroblasts deposit excess extracellular matrix in the perisinusoidal space, a process which, if 
it persists, can lead to increased hepatocellular injury, progressive fibrosis, organ dysfunction, 
and, ultimately, to cirrhosis and hepatic failure. The congestion and fibrosis result in a wide 
spectrum of liver disease, including synthetic dysfunction, ascites, portal hypertension, cirrhosis, 
and hepatocellular carcinoma, and may culminate in fulminant liver failure. The severity of liver 
fibrosis is known to increase with time after TCPC and occurs in the absence of any other 
identifiable etiology of chronic liver disease. 
Figure 1: Description of Fontan Associated Liver Disease  
 
 
 
 
 
 
 
 
 

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3.1.2 Characterization of FALD 
A roadblock to understanding and treating FALD is the lack of an established, noninvasive 
means of detecting it. Basic laboratory testing (platelet count, AST, ALT, bilirubin, GGT, INR) 
and standard imaging (conventional ultrasound (US), computed tomography (CT) or magnetic 
resonance imaging (MRI)) are insensitive to liver congestion and early liver fibrosis in these 
patients (8). Percutaneous core needle liver biopsy, while the current standard of care, is not an 
ideal surveillance tool as the core sample may falsely under- or over-estimate the degree of 
fibrosis given the significant heterogeneity that often exists. Liver biopsy is also invasive and 
carries a small but real risk of bleeding, particularly in patients who require anti-coagulation. 
Ultrasound shear wave elastography (SWE), based on the measurement of tissue shear wave 
speed, provides a non-invasive measure of liver stiffness and is an extremely promising 
technology to monitor the congestion and early fibrosis that precedes overt FALD. The utility of 
ultrasound SWE in assessing liver fibrosis has been well-documented (9, 10) and image-guided 
ultrasound SWE has been used in TCPC patients to demonstrate that liver stiffness correlates 
with hepatic congestion (as determined by elevated central venous pressures) and hepatic fibrosis 
(as determined by liver biopsy) (11-13). MRI-elastography may be of even greater utility as it 
allows sampling over a much larger region of the liver (thus, minimizing sampling error) and 
may potentially allow differentiation of stiffness due to fibrosis versus congestion, a feature 
which may be particularly useful in the evaluation of Fontan patients and in determining the best 
approach to targeted therapy in this population (14). 
3.1.3 FALD and FUEL Extension Study 
The FUEL Extension trial is an NHLBI-sponsored study that will assess the efficacy of safety 
and efficacy of udenafil, a phosphodiesterase-5 inhibitor (PDE-5i) that causes relaxation and 
decongestion of the pulmonary and systemic venous vasculature, to improve Fontan cardiac 
function, peripheral endothelial function, and exercise performance in adolescents who have 
undergone TCPC. By partnering with the FUEL Extension trial, we will have the unique 
opportunity to 1) measure liver stiffness across a large cross-section of children and adolescents 
after TCPC; 2) assess how liver stiffness responds to vasodilator therapy determine; 3) determine 
if liver stiffness correlates with important functional outcomes; and 4) determine the relationship 
between biomarkers of heart failure and liver fibrosis and liver stiffness. Currently, PDE-5i 
therapy after the Fontan is reserved for those patients who have clinically symptomatic heart 
failure. However, a significant and sustained reduction in liver stiffness with PDE-5i therapy 
would suggest that earlier treatment might reduce the incidence, severity and rate of progression 
of FALD. This has important implications for a population in whom the rate of liver fibrosis is a 
critical determinant of overall long-term health. 
 

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3.2 
Rationale for the Study and Study Outcomes 
3.2.1 Study Rationale 
While there have been many small studies that have used SWE to evaluate the degree of liver 
stiffness in patients who have undergone TCPC, there has not been a large-scale evaluation that 
defines “normal” in this population or seeks to correlate liver stiffness with functional outcome.  
Similarly, the impact of treatment with pulmonary vasodilators, medications that should lower 
systemic venous pressure, has not been evaluated.  The FUEL Extension trial presents a unique 
opportunity to measure liver stiffness in those with TCPC, to evaluate the impact of udenafil on 
liver stiffness, to correlate liver stiffness with functional outcomes, and to gain valuable insight 
into the relationship between liver stiffness and biomarkers of heart failure and fibrosis. 
3.2.2 Rationale for Study Outcomes 
3.2.2.1 The use of ultrasound SWE to evaluate liver fibrosis 
Ultrasound SWE uses one or more acoustic radiation force impulses (ARFI) or “push pulses” to 
generate shear waves in the tissue of interest. These shear waves can be tracked and their speed 
measured, with increasing tissue stiffness associated with increasing tissue shear wave speed. 
Shear wave speed can be mathematically converted to both shear and Young’s moduli (in kPa), 
mechanical properties related to tissue rigidity and elasticity. There are two major forms of 
ultrasound SWE – “point” and “2D”.  Point ultrasound SWE provides a very focal estimate of 
liver stiffness using a small region-of-interest, while 2D ultrasound SWE provides a regional 
color parameteric map (elastogram) of image stiffness and allows measurement over a much 
larger area of the liver.  
We have demonstrated excellent inter- and intra-observer reproducibility of ultrasound SWE 
measurements in soft and hard tissue-mimicking elasticity phantoms using two different US 
systems (Supersonic Imagine and Siemens Medical Solutions USA) and five different US 
transducers.  Coefficients of variation between various combinations of US systems, transducers, 
phantoms, and phantom depths were low, ranging from 0.5-6.8%, and inter-operator agreement 
(four operators) was near-perfect (ICCs ≥ 0.99) (15). We also have explored the in vivo 
relationship between liver shear wave speed and histologic fibrosis and observed significant 
positive correlations between liver shear wave speed and histologic fibrosis using point and 2D 
ultrasound SWE (Siemens Acuson S3000) (r = 0.68 and r = 0.73; p-values <0.0001) (Figure 2). 
 
 
 

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Figure 2: Box plot showing increasing liver shear wave speed measurements with 
increasing parenchymal fibrosis. 
 
 
Receiver operating characteristic (ROC) areas under the curve for discriminating Ishak fibrosis 
scores “0-2” (no/mild fibrosis) from “3-6” (moderate/severe fibrosis) were 0.84 and 0.86 for 
point and 2D ultrasound SWE, respectively. In a recent prospective longitudinal observation 
study, we have shown that children with single ventricle congenital heart disease experience 
marked liver stiffening immediately after the TCPC. Five subjects returned at a mean of 185 ± 
28 days, and mean liver stiffness remained elevated above baseline but with a variable response 
between patients when comparing the change in stiffness from early (2.5 and 7.5 days post-op) to 
late (6 month post-op) time points, which may reflect individual differences in cardiovascular 
performance or functional reserve (Figure 3). 
 
 
 
 
 
 
 
 
 

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Figure 3: Individual profile plots from subjects undergoing stage 3 Fontan palliation  
 
 
 
 
 
 
 
 
 
These plots show changes in liver shear wave speed (stiffness) over time (follow #1 = 2.5 days 
post-op, follow-up #2 = 7.5 days post-op, and follow-up #3 = 185 days post-Fontan). 
3.2.2.2 The use of Magnetic Resonance Elastrography to evaluate liver fibrosis 
MR elastography uses a vibrating passive driver or “paddle” placed on the abdominal wall over 
the liver in order to generate in vivo shear waves. These shear waves can be tracked using an 
MRI modified phase contrast pulse sequence. Shear wave data is then used to generate color 
parameteric maps (elastograms) of liver stiffness that allow quantitative assessment (Figure 4). 
We have demonstrated excellent agreement when using intra-class correlation coefficients to 
assess reproducibility of MR elastography measurements across MRI scanners (Philips and GE 
Healthcare), field strengths (1.5T and 3T), and pulse sequences in 24 adult healthy volunteers. 
Pairwise ICCs ranged from 0.67-0.82 when agreement was assessed between individual 
sequences across manufacturers (GE vs. Philips); the greatest agreement while fixing the pulse 
sequence was at 1.5T using 2D GRE sequences (ICC=0.82, r=0.85) (16). 
A study by Wallihan et al (11) showed that MRI-derived liver stiffness was universally elevated 
in a cohort of children and young adults that had undergone the Fontan procedure. They also 
found a statistically significant inverse correlation between liver stiffness and cardiac index 
(p=0.02) and ejection fraction (p=0.002). Patients with long Fontan duration (>20 years post-op) 
had greater liver stiffness compared with those having a shorter duration (p=0.02). The study 
provided evidence that elevated liver stiffness in Fontan patients is likely due to both fibrosis and 
congestion. 
 

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Figure 4: 13 year-old patient status post Fontan palliation due to Single Ventricle 
Congenital Heart Disease.  
 
 
 
 
 
 
A, MR elastography color wave image shows mechanically-induced shear waves in the liver. B, 
Color parametric map (elastogram) shows the distribution of stiffness within the upper abdomen, 
including the liver. The liver is very stiff and heterogeneous. 
3.2.2.3 Biomarkers and liver stiffness 
As noted previously, increased liver stiffness can be secondary to hepatic congestion and/or 
hepatic fibrosis. Circulating biomarkers previously demonstrated to be associated with 
congestive heart failure or tissue fibrosis may help to determine the primary driver of liver 
stiffness in a particular patient and help to monitor the response to targeted therapy. For instance, 
we would anticipate that patients with increased liver stiffness due to hepatic congestion would 
have increased biomarkers of heart failure (e.g., BNP, NT-proBNP). Similarly, patients in whom 
fibrosis significantly contributes to increased liver stiffness may have elevation in biomarkers 
associated with liver fibrosis (e.g., ELF panel, miR-138, miR-143, or galectin-3). The relative 
contribution of congestion or fibrosis (as determined by biomarker profiles) to the observed level 
of liver stiffness may correlate with which therapeutic strategies are likely to be successful in a 
particular patient. Therefore, in this aim, we will determine which circulating biomarkers 
correlate with measures of liver stiffness and which correlate with a change in liver stiffness with 
udenafil treatment. We will use both a directed approach, with an analysis of candidate 
biomarkers validated for other disease processes, and a non-directed approach, which will 
identify novel biomarkers potentially associated with FALD. 
 
 

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4.0 
CLINCIAL TRIAL OBJECTIVES, AIMS, AND HYPOTHESES 
 
4.1 
Clinical Trial Objective 
Determine scope of hepatic stiffness in Fontan patients by ultrasound and magnetic resonance 
imaging elastography and evaluate the efficacy of udenafil in reducing liver stiffness. 
4.2 
Primary Aim and Hypothesis 
Primary Aim: Define the range of liver stiffness in those who have had the surgical creation of a 
total cavopulmonary connection. 
Hypothesis: Following total cavopulmonary connection, children with single ventricle congenital 
heart disease will have elevated measures of liver stiffness. 
4.3 
Secondary Aims and Hypotheses 
Secondary Aim 1: Determine the impact of udenafil treatment on liver stiffness in a large cross-
section of adolescents who have undergone total cavopulmonary connection. 
Hypothesis: Udenafil treatment will reduce liver stiffness as measured by non-invasive 
elastography by decreasing venous congestion.   
 
Secondary Aim 2: Determine the relationship between clinical outcomes and liver stiffness. 
Hypothesis: Liver stiffness will be associated with measures of ventricular diastolic function, 
exercise performance, and endothelial function. 
 
Secondary Aim 3: Assess the association between biomarkers of both heart failure and liver 
fibrosis and liver stiffness. 
Hypothesis: Biomarkers of heart failure and liver fibrosis will directly correlate with liver 
stiffness as measured by ultrasound and magnetic resonance elastography. 
 
 

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5.0 
STUDY DESIGN 
 
This is an open-label extension study that integrates with the FUEL Extension Trial. FUEL-
enrolled patients will have been randomized and double-blinded to either placebo or udenafil, 
and all patients entering the FUEL Extension trial will be placed on udenafil therapy.  Therefore, 
approximately 50% of patients eligible to be enrolled in our study will be udenafil-naïve (U-) 
and 50% will have already completed 6-months of therapy (U+).  As a part of FUEL-OLE, each 
participant will receive one year of udenafil treatment (87.5 mg twice daily). Both the U- cohort 
and the U+ cohort will have liver stiffness measured by ultrasound and MR elastography (where 
available), as described above. Thus, the U- subjects will have elastography measurements pre-
udenafil and after 1 year of treatment. This will determine the impact of udenafil therapy on liver 
stiffness. Additionally, the U+ cohort from FUEL will allow us to determine if potential benefits 
of udenafil therapy are sustained over time by comparing measurements after 6 (baseline entry 
into our study) and 18 months of therapy (after 1-year of our study). The study visits are as 
follows: 
1. Baseline visit: during the baseline visit, subjects will undergo ultrasound SWE and, if 
available at their center, magnetic resonance elastography.  These will be coordinated 
with FUEL extension testing to ensure that the primary study is not interrupted.  The 
additional blood work for this protocol will be obtained at the same time as the blood 
work for the FUEL extension study. 
2. Follow-up visit: during the follow-up visit, subjects will undergo ultrasound SWE and, if 
available at their center, magnetic resonance elastography.  These will be coordinated 
with FUEL extension testing to ensure that the primary study is not interrupted.  The 
additional blood work for this protocol will be obtained at the same time as the blood 
work for the FUEL extension study. 
5.1 
Study Duration 
The planned clinical trial duration from the start of subject screening and enrollment to last 
subject out is anticipated to be from 12 to 24 months concurrent with FUEL-OLE.  The final 
evaluation within the current study will be concurrent with the 12-month FUEL-OLE completion 
visit.  Thus, each individual subject will be enrolled in the current study for only 12 months.  
 
 

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6.0 
SELECTION AND DISPOSITION OF SUBJECTS 
 
6.1 
Number of Subjects 
Subjects for this protocol will be recruited from the pool of patients in the FUEL Extension trial, 
from all participating centers that have the capacity to perform either ultrasound SWE or 
magnetic resonance elastography. We anticipate that we will have access to 100-200 subjects, 
although this will vary based on the start date for this study.  If we achieve between a 50-75% 
consent rate, it would provide a sample size of 50-150 subjects. 
6.2 
Clinical Trial Population Characteristics 
In order to be eligible for the clinical trial, subjects must fulfill the following criteria. Male and 
female subjects 12-19 years of age who meet all the Inclusion criteria and none of the Exclusion 
criteria will be enrolled.  
6.3 
Subject Inclusion Criteria 
1. Enrollment in FUEL Extension Trial 
2. Informed assent from subject, informed consent from parent/legal guardian as appropriate 
6.4 
Subject Exclusion Criteria 
1. Non-enrollment in the FUEL Extension Trial 
2. Subjects with contra-indications for MRI including presence of any indwelling, 
significant, ferromagnetic objects (including pacemakers and implantable cardiac 
defibrillators, or those unable to lie in the scanner without sedation (these subjects will be 
excluded from only the MRI components of this study) 
3. Other exclusionary criteria will match those used for the FUEL Extension Trial 
6.5 
Subject Withdrawal Criteria 
Subjects may voluntarily withdraw from the study at any time.  In addition, the site principal 
investigators are free to withdraw a subject at any time for reasons of medical prudence.  Any 
subject withdrawn from the FUEL Extension study will also be withdrawn from this ancillary 
study. 
 
 

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7.0 
TREATMENT DESCRIPTION 
 
7.1 
Description of Treatment 
All subjects participating in the present protocol will be participants in the FUEL-OLE protocol.  
Subjects in FUEL-OLE will be treated with udenafil for one year’s time (87.5 mg twice daily).  
No additional treatment will occur in the present study. 
7.2 
Subject Identification Number 
Each subject is assigned a subject identification number (SID).  All interview and clinical 
research data are stripped of identifiers and labeled with the study number. The enrollment log 
with participant identifiers will be maintained at each site in a secured, locked location available 
only to the study staff. The informed consent form states that study data will be made available 
to the Data Coordinating Center (DCC) and NIH/NHLBI to ensure study safety and quality 
control. The subject’s name and any other identifying information will not appear in any 
presentation or publication resulting from this study. 
7.3 
Method of Treatment Assignment and Blinding 
Not applicable. This study is open label.  
7.4 
Sample Management 
Sample collection and brain natriuretic peptide (BNP) testing will be performed during the 
collection of samples for the FUEL-OLE study. A plasma sample for BNP level will be obtained 
at the time of entry into the study and at the end of the 1-year open label extension trial. The 
samples will be collected and processed on-site and the plasma will be shipped on dry ice to the 
FUEL-OLE Central Laboratory located at the University of Michigan. The Central Laboratory 
will aliquot a portion of the sample for BNP testing in the University of Michigan Pathology 
Laboratory (UMPL) which performs this test for all patients seen in the University of Michigan 
Health System. In addition to this sample, FALD specific labs will be obtained at the same 
venipuncture.  The samples that will be collected for those patients enrolled in the FUEL-OLE 
and the ancillary study are as follows: 
Blood Samples 
• 3 mL in a purple top (K2EDTA) PPT tube for BNP measurement (FUEL-OLE sample) 
o Invert the tube gently five times. Further inversion may cause alterations in 
sample integrity. 
o Centrifuge 10 minutes at 1600 x g using a refrigerated centrifuge (4°C) 

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o For each sample, the separated plasma will be transferred into two labeled 
polypropylene tubes (approximately 750 μL each), which will be stored at 
approximately -70°C within 2 hours of collection. 
• 3.5 mL in a yellow top (SST) tube for serum isolation (Liver study sample) 
o Invert the tube gently five times. Further inversion may cause alterations in 
sample integrity. 
o Allow to clot for 30 min at room temperature 
o Centrifuge 15 minutes at 1600 x g using a refrigerated centrifuge (4°C) 
o For each sample, the separated serum will be transferred into two labeled 
polypropylene tubes (approximately 750 μL each), which will be stored at 
approximately -70°C within 2 hours of collection. 
Biomarker Testing 
• BNP testing will be performed by the UMPL  
• NT-proBNP levels measurements will be performed on the residual plasma sample using 
a commercially available enzyme linked immunosorbent assay (ELISA) that will be run 
in the FUEL-OLE Central Laboratory.  
• MicroRNA measures will be performed on the residual plasma sample in the FUEL-OLE 
Central Laboratory on total RNA isolated using miRNeasy total RNA preps (QIAGEN, 
Valencia, CA). miRNA qRT-PCR analysis will be performed using Taqman miRNA 
assays (Applied Biosystems, Foster City, CA). 5 ng total RNA will be used as an input 
into each reverse transcription reaction (RT) for each miRNA. Four replicates will be 
done for each miRNA, consisting of two replicate PCR reactions from each of the two 
replicate RT reactions, and the results were averaged. PCR reactions will be run on a 
7500 Real Time PCR machine (Applied Biosystems) and analyzed using 7500 System 
SDS software (v1.4). For the candidate biomarkers, an equivalent number of anonymous, 
age-matched, healthy controls will be used for comparison. The control cohort will be 
derived from healthy individuals who present to the University of Michigan for elective 
surgical procedures. 
• Enhanced Liver Fibrosis (ELF) score which is based on the serum levels of hyaluronic 
acid (HA), amino-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor 
of metalloproteinase-1 (TIMP-1). Testing will be performed by the FUEL-OLE Central 
Laboratory using the serum sample. In accordance with prior studies, we will assign 
those patients with scores of <7.7 to the no or mild fibrosis group, 7-7-9.8 to the 
moderate fibrosis group, and >9.8 to the severe fibrosis group. 
BNP testing will be performed by the UMPL, as described above. NT-proBNP levels 
measurements will be performed using a commercially available enzyme linked immunosorbent 
assay (ELISA) that will be run in the Russell laboratory. MicroRNA measures will be performed 

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in the Russell laboratory on total RNA isolated from plasma using miRNeasy total RNA preps 
(QIAGEN, Valencia, CA). miRNA qRT-PCR analysis will be performed using Taqman miRNA 
assays (Applied Biosystems, Foster City, CA). 5 ng total RNA will be used as an input into each 
reverse transcription reaction (RT) for each miRNA. Four replicates will be done for each 
miRNA, consisting of two replicate PCR reactions from each of the two replicate RT reactions, 
and the results were averaged. PCR reactions will be run on a 7500 Real Time PCR machine 
(Applied Biosystems) and analyzed using 7500 System SDS software (v1.4). For the candidate 
biomarkers, an equivalent number of anonymous, age-matched, healthy controls will be used for 
comparison. The control cohort will be derived from healthy individuals who present to the 
University of Michigan for elective surgical procedures. 
Proteomic and transcriptomic analysis will take place using a multiplex Tandem Mass Tag 
(TMT) system allowing concurrent analysis of multiple samples. Plasma samples will be 
depleted of 14 high-abundant proteins using MARS14 (Agilent) or Seppro IgY14 (Sigma) 
columns.  Depleted plasma samples (~50ug) will be alkylated and digested overnight. Samples 
will be labeled with the TMT Reagents per manufacturer’s recommended protocol and mixed 
before sample fractionation (12 fractions). Each fraction will be analyzed by high-resolution, 
nano-LC-MS/MS using Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher, Waltham, 
MA) to identify peptides/proteins and quantify reporter ion relative abundance. Data will be 
collected using the recently developed multinotch-MS3 method which improves the quantitation 
accuracy by minimizing the reporter ion ratio distortion resulting from fragmentation of co-
isolated peptides (17).  The data analysis will be performed using both commercial (Proteome 
Discoverer v 2.1) and publicly available software (e.g., MaxQuant). The performance and 
analysis of the proteomic profiling will be done in the Proteomics Resource Facility at the 
Department of Pathology, University of Michigan. 
 
8.0 
STUDY ASSESSMENTS/MEASUREMENTS AND PROCEDURES 
 
8.1 
Primary Assessments 
• 
Range of liver stiffness in subjects with a surgically created total cavopulmonary 
connection 
8.2 
Primary Assessment Measurement 
• Liver stiffness as determined by ultrasound shear wave elastography 
• Liver stiffness as determined by magnetic resonance elastrography 

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8.3 
Additional Measurements  
• Evaluate serum biomarkers of heart failure, brain type natriuretic peptide (BNP) and n-
terminal BNP (NT-BNP) 
• Evaluate biomarkers of liver fibrosis  
• Proteomic and transcriptomic analyses on subcohorts of patients with the following: 
o Low levels of liver stiffness 
o Very high levels of liver stiffness 
o Decrease in liver stiffness with udenafil treatment 
 
9.0 
PLANNED STATISTICAL ANALYSES AND STATISTICAL 
CONSIDERATIONS 
 
9.1 
Statistical Analysis Plan 
A Statistical Analysis Plan (SAP) will be developed and finalized prior to database lock. The 
SAP will contain detailed descriptions of data conventions and statistical procedures. Any 
deviation(s) from the SAP will be described and justified in the clinical study report.  
9.2 
Sample Size Determination 
Given the primary outcome is descriptive, sample size estimate is provided for secondary 
outcome 1 – evaluating the change in liver stiffness before and after udenafil treatment compared 
to a patient receiving on-going udenafil treatment.  Elastography indices measured by US or 
MRI elastography from baseline to 12 months will be examined in both udenafil treatment 
groups (U+ and U-), using paired t-test or Wilcoxon signed- rank test, as appropriate. DiPaola et 
al. (17) demonstrated that liver stiffness measured by US-SWE significantly increased from 
baseline to 6-month post-TCPC (mean 1.18 ± SD 0.29 m/s to 2.08 ± 0.24 m/s, p<.0001). 
Assuming a mean liver sheer wave speed (SWS) of 2.08 with known SD 0.24 as our baseline 
liver stiffness, a total sample size of 24 subjects (12 per group) is required to achieve 85% power 
with alpha=0.05 to detect 10% reduction of liver stiffness (deemed clinically meaningful in an 
individual) at 12 months from baseline in the U- group. Assuming a maximal enrollment of 100 
subjects, (approximately 50% of whom will be from the FUEL U- cohort), we would be powered 
to detect a difference of 3.75% reduction in liver stiffness. 
9.3 
Analysis Populations 
All individuals enrolling in FUEL-OLE will be recruited for enrollment in the FALD study.  At 
completion of FUEL-OLE, once FUEL assignments are unblinded (udenafil or placebo), the 

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cohort will be stratified into udenafil naïve vs. udenafil continuation groups.  Analysis will be 
done both on an intent-to-treat and a per-protocol basis. 
9.4 
Statistical Analysis 
9.4.1 General Methods 
This is a prospective, observational, cohort study.  
9.4.2 Primary Outcome 
The absolute stiffness by SWE and by MR elastography will be reported using descriptive 
statistics as appropriate. 
9.4.3 Secondary Outcomes 
Secondary outcome 1 
Baseline liver stiffness measured by ultrasound or MRI elastography will be compared between 
the two groups (U+ vs. U-) using appropriate statistics. Next we will assess change in liver 
stiffness from pre- to post-1 year udenafil treatment in the U-group as well as from 6 to 18 
months of therapy in the U+ group. 
Secondary outcome 2  
Changes in liver stiffness, diastolic cardiac function (including E/E’ and MPI), PAT 
measurement, and exercise measurements (including peak VO2 and submaximal exercise 
measures) from baseline to 12 months will be quantified. Correlation between change in liver 
stiffness and changes in diastolic cardiac function and exercise measurements in the entire cohort 
and in the U- group and U+ group separately.  
Secondary outcome 3  
Bivariate analyses will examine 1) the correlation between each biomarker and liver stiffness 
measured by ultrasound and MRI elastography and 2) the correlation between change in each 
biomarker and change in liver stiffness from baseline to 12 months in both the U+ and U- 
cohorts. 
The proteomic and transcriptomic data will be presented primarily as a descriptive report.  
Pairwise differences between groups may be sought using appropriate 2-group statistics, but we 
expect the limited sample size in this exploratory outcome to limit analysis to descriptive 
statistics. 
 
 

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10.0 
ETHICS AND GENERAL CLINCIAL TRIAL CONDUCT CONSIDERATIONS 
 
10.1 
Institutional Review Board or Independent Ethics Committee 
10.2 
Ethical Conduct of the Clinical Trial, Confidentiality, and Potential Risks 
The clinical studies in this proposal meet the criteria in the NIH Supplemental Instructions for 
Preparing the Human Subjects Section of the Research Plan for Scenario B: Non-exempt human 
subject research, non-clinical trial. Although this will be a study ancillary to a clinical trial, this 
study is not a clinical trial; it collects additional data and measurements on all patients regardless 
of their status within FUEL-OLE. 
10.3 
Potential Risks 
Human Subjects Involvement, Characteristics, and Design 
Young adults and pediatric patients with single ventricle congenital heart disease will be 
managed as per the parent FUEL-OLE trial and otherwise per the standard of care; laboratory, 
radiologic, and echocardiographic testing will be performed within the framework of the FUEL-
OLE trial and at interim time points per the standard of care. The research team will not be 
involved in clinical decisions.  Patient demographics and available clinical and laboratory data 
will be recorded for the purposes of research.  
Biospecimen Collection 
No additional venipuncture beyond that already being obtained in FUEL-OLE will occur. Minor 
temporary discomfort may be associated with the removal of blood by venipuncture.  There is a 
risk of bruising and a very small amount of bleeding associated with the blood drawing.  There is 
also a very small risk of infection at the site. Whenever possible, blood samples will be gathered 
when the participant is scheduled for routine blood testing or procedures. Serum collection will 
occur via venipuncture simultaneously with FUEL-OLE collection.  The total amount of serum 
obtained for both studies will be 6mL (3mL for FUEL-OLE and 3mL for this study), which is 
within safe range for adolescent patients within FUEL-OLE’s height and weight criteria.  No 
DNA samples specific to FALD beyond what is already collected in FUEL-OLE will be 
obtained. 
MRI 
Risks related to MRI include subject distress/claustrophobia, projectile injury, and scanning with 
ferromagnetic implanted material (e.g., pacemaker or aneurysm clip) in the subject. Subjects 
experiencing distress/claustrophobia during MRI will be allowed to opt out of the MRI portion of 
the study; their US results will still be included in our results. Additionally, individuals with any 

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significant ferromagnetic objects, specifically implantable cardiac defibrillators or pacemakers, 
should not undergo MRI and will have US results included in the study. MRI scanners will be 
operated within U.S. Food and Drug Administration guidance to minimize energy imparted into 
the subject and tissue heating.   
Ultrasound 
There are no known risks to ultrasound SWE. FDA-approved US systems will be employed that 
operate within U.S. Food and Drug Administration guidance with respect to energy imparted into 
the patient (thermal index and mechanical index). 
10.4 
Protection against Risks 
Investigators will take all reasonable measures to protect the confidentiality of subjects and their 
families, including the following: 
Reporting of Test Findings 
The results of future tests on biological specimens will not be released to the subject/family.  
There is a reasonable possibility that no findings will result from this research effort.   If findings 
are detected, it may be years before any utility of these findings is realized.  Further, if samples 
are “anonymized” prior to release to other investigators for research, it may not be possible to 
trace the results back to the subject. 
If an incidental finding is found on a study clinical test such, the PI or other qualified member of 
the research team will take full responsibility for disclosing the findings to the patients/parents, 
communicating with their primary cardiologist with permission, or making appropriate 
cardiology referrals as indicated.   The subject may choose to seek a second opinion and/or 
appropriate clinical care.  This might change the subject's insurability and employability as it 
relates to the clinical finding only.  The presumption is that detection of a potentially clinically 
significant finding will prove to be beneficial. 
10.5 
Potential Benefits 
Study Findings 
At the end of the study study results will be provided to treating/referring physicians 
(cardiologist, etc).  If udenafil is shown to reduce liver stiffness, it is possible that this can 
modify progression of FALD and could directly benefit the enrollee.  This may directly benefit 
an individual by attenuating the disease.  It is possible that the liver scans obtained for the study 
will demonstrate significant abnormalities in liver stiffness, and they may also find areas of 
significant fibrosis or cirrhosis.  Although the clinical significance of such findings remains 

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unclear to the field, appreciation of these individual findings is likely to be beneficial for an 
individual’s care.   
Biospecimens 
Identification of optimal serum biomarkers of liver stiffness may directly benefit the individual 
by allowing serial monitoring of FALD.  In addition, we hope that the proteomic and 
transciptomic portion will help investigators to learn more about the relationship between genetic 
factors and pathophysiology of FALD.  This information may help physicians provide better 
answers to families’ questions regarding causes and risk.  It may also provide clues to future 
interventions and/or treatments. 
Indirect Benefit 
There might be an indirect benefit from the awareness that study results may help to improve the 
care of children with similar problems in the future.  Families may derive a sense of altruism, 
accomplishment, and contribution to furthering understanding of the problem through their 
participation. 
10.6 Risk/Benefit Ratio and Importance of Information to be Obtained 
The risk/benefit ratio is favorable for this study and adverse events are not anticipated. The 
baseline risk is minimal because there are no therapeutic interventions specifically for the FALD 
study. There are direct potential benefits as described above.  In addition, although an individual 
subject may not benefit from participation, the results of this study will make important 
contributions to the improvement of knowledge of the causes of congenital heart disease (CHD), 
in the development of new diagnostic tests, and ultimately in the improvement of treatment and 
prognosis. 
10.7 
Study Limitations 
While integrating with FUEL-OLE is a clear advantage and makes this FALD study feasible, the 
study will not have a placebo control arm.  However, an active control (U+) should still allow 
assessment of how udenafil affects liver stiffness in a naïve patient. Each center may have access 
to only US SWE, MR elastography, or both. We expect most, if not all, centers to have access to 
US SWE, as it is now widely available, commonly employed in practice, and straightforward to 
use.  Our (CCHMC) radiology core (including MRI physicist Suraj Serai, PhD, and imaging 
post-processing laboratory) will be able to facilitate MR elastography capabilities at participating 
centers, ensuring the use of a standardized protocol across scanners.  Experience in processing 
and analyzing/interpretation of this relatively new measurement technique should be mitigated 
by use of a radiology core. MR elastography is more difficult to add for currently non-capable 
centers, thus, MR elastography measurements will be limited to centers that have capabilities.   

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11.0 
REGULATORY/ADMINISTRATIVE PROCEDURES AND DOCUMENTATION 
 
11.1 
Data Collection 
Data for each SWE and magnetic resonance elastography will be generated from each site and 
recorded on a case report form.  This form will serve as the source document for each test and 
will be used to enter data into an electronic data capture (EDC) system (eCOS).  US SWE data 
will be recorded from each site. MREL data will need to be sent anonymized to CCHMC for 
central processing/analysis. 
The biomarker core lab will generate results for each blood specimen and will transmit that data 
electronically into the EDC system (eCOS). Subject identification will follow the same system as 
for the FUEL Extension trial.  Each imaging study will be associated with the FUEL Extension 
identification number and each blood sample will be labeled with that same number. 
FUEL-OLE enrollment takes place over a 2 day period.  For clarity, often the initial FUEL-OLE 
day (called hereafter OLE-D1) and the final day of FUEL are the same visit.  This visit consists 
of informed consent and assent, blood sample collection, EndoPAT testing, an echocardiogram, 
and exercise testing.  Based on experience with the Udenafil study, this day takes 5-6 hours in 
total.  Subjects will then return the next day (OLE-D2) for drug administration and 6-minute 
walk.  Subjects will return at 12 months of FUEL-OLE for a final day repeating the above tests.  
FALD will ideally be integrated into this schedule with keen attention to preserving the integrity 
of the FUEL-OLE parent trial and preventing study fatigue as detailed below for each step.  This 
plan was vetted and approved by the FUEL study subcommittee.   
Subject recruitment:  FALD will only be recruited by either the site PI or a study coordinator 
after a commitment is given to participate in FUEL-OLE.  Recruiting should be done at the same 
time point as OLE to prevent unnecessarily repetitive patient contact. 
Consent/Assent:   Consent and assent for FALD will be obtained immediately following but only 
after consent/assent for FUEL-OLE has been obtained.    
Serum testing:  Serum for the ancillary study will be obtained during the same venipuncture as 
used in FUEL-OLE.  The serum sample will be processed, stored and shipped in parallel with the 
plasma sample that is being collected for the FUEL-OLE Trial. The processed serum (and the 
processed plasma for the FUEL-OLE protocol) will be shipped to the FALD core lab (University 
of Michigan) for testing.  The ancillary study will require an additional 3 ml sample of blood (for 
a total of 6 ml). 
Imaging:  We have identified the morning of OLE-D2 as the optimal time for hepatic imaging. 

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• US SWE:  A complete US SWE exam lasts 5-10 minutes only.  It is a portable test, and 
thus may be performed in either a radiology suite or any other clinical cardiology location 
used by the study.  Additional limited anatomic imaging will also be obtained that takes 
approximately 1 minute. 
• MRE:  A complete MRE exam lasts approximately 15-20 minutes. Additional limited 
anatomic imaging will also be obtained that takes approximately 1 minute. Including 
patient travel to and from the MR scanner we anticipate an additional 20-40 minutes, 
making the total duration of MR testing 35-60 minutes. 
All liver imaging should be able to be completed in 60-90 minutes.   
• For initial FALD imaging, by starting OLE-D2 immediately with hepatic imaging at 7:30 
or 8:00 AM, we would anticipate FUEL-OLE can begin its formal day’s schedule with 
study drug administration by 9-9:30 AM.  In total, we would expect OLE-D2 including 
hepatic imaging to still conclude by midday as anticipated by the FUEL-OLE protocol.   
• For the OLE and FALD 12-month follow-up visit, we would again anticipate beginning 
the day for with hepatic imaging.  To make this feasible, we can pay for hotel costs for 
those individuals enrolled in FALD at the 12-month visit so that the testing day may start 
early. Alternatively, imaging may be obtained at the end of the day following FUEL-OLE 
testing, although this is less ideal because exercise testing may influence FALD 
measures.  Integration of same day testing should be feasible based on projected length of 
the testing day.   
Imaging identifiers for these research studies will include only the site and subject study-specific 
ID. Following image obtainment at both time points, images from both US SWE and MRE will 
be sent to the imaging core.  Images transferred will contain the site and subject identifiers and 
no protected health information. 
 
 
11.2 
Quality Assurance 

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The DCC has primary responsibility for QC/QA activities of the phenotypic data. The DCC also 
requires that the sites complete certain QC activities, most of which are monitored by the DCC.   
The key QC/QA activities include the following: 
• Development of a Study Manual; 
• Clearly formatted and carefully constructed Data Forms with clear, up-to-date manuals of 
instruction; 
• Sign-Off Procedures for all CRFs; 
• Central protocol training and certification of all site data collection staff with the use of 
standardized checklists; 
• Data management training and certification of site personnel completing data entry 
and/or data management; 
• Verification of patient eligibility;  
• On-going monitoring of all protocols/data collection activities; 
• Completion of reliability and/or pilot studies for key measurements as appropriate;  
• Inclusion of repeat measurements, as feasible, in the course of the study; and 
• Monitoring visits to sites as required with pre-specified goals and/or remote monitoring 
activities. 
The DCC may conduct site visits to the Core Laboratories and/or Biorepository to review QA 
and QC procedures and data transfer to the DCC.  Review of central laboratory-related reports 
will be conducted at least monthly to identify overall or site-specific problems in data or 
specimen acquisition and reporting of results.    
11.3 
Data Management 
An EDC system will be used for the study that is designed to support reliable and secure data 
entry for clinical research purposes. The system also provides seamless integration of eCRFs and 
paper-based CRFs within a single protocol if desired; implementation of protocol amendments; 
and SAS and XML study data exports. 
11.3.1 Data Entry 
Data can be entered directly from multiple study sites via a fully validated and 21 CFR Part 11 
compliant, secure Web application and stored centrally.  A configurable sample-based double 
data entry system is available. Data are entered by subject study identification number; names 
will not be linked with subject data in the database. Study sites will maintain records in secure 
areas linking the subject name with the identification number assigned for the study. Study sites 
will have full access to their own data and be able to view these data remotely. Study staff will 
not be able to view subject data associated with other sites. 

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11.3.2 Data Validation and Monitoring 
Integrated into the data entry system are real time validations, including both inter- and intra-
instrument data checks.  Inconsistent or questionable values are flagged during entry, and an edit 
report is automatically generated to the data entry client. These edit reports provide the 
information necessary to investigate any data entry errors or resolved questions regarding out-of-
range or questionable values.  Second level query tracking allows monitors and data managers 
real time access to unresolved queries as well as the date and time of query generation and 
resolution. 
11.3.3 Data Security and Integrity 
All data changes are written to an audit trail. The audit trail identifies the data item by table, 
column and key field. The entry includes the user, date and time, as well as the old value and 
new value. Both patient related data as well as trial configuration data are written to the audit 
trail. Data are saved at regular intervals during data entry to prevent loss of information in the 
event of a disruption of the Internet connection. In the unlikely event of a major disruption, a 
backup connection allows full access to the DMS.  
Several levels of security are employed to ensure privacy and integrity of the study data, 
including the following: Study access requires use of assigned user names and passwords. 
Individual roles and access levels are assigned by the study data manager. Passwords are 
changed regularly. Web-based entry uses secure socket layer data encryption.  Data will not be 
stored on laptop computers. 
11.3.4 Biospecimen Tracking 
Specimen tracking is started from the time of receipt at the site, through shipment to the central 
biorepository.  Each specimen will be labeled with a bar-coded label identified by a unique 
specimen number that is different from the subject’s unique study ID number.  The master list 
linking the barcode numbers to the subject study ID numbers will be maintained under password 
protection in the data management system at the DCC.  This blinding code system will maintain 
the confidentiality of the specimens yet allowing linkage of the specimens with clinical study 
data for analyses. 
 
 

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12.0 
REFERENCES 
 
1) Greenway SC, Crossland DS, Hudson M, Martin SR, Myers RP, Prieur T, Hasan A, Kirk R. 
Fontan-associated liver disease: Implications for heart transplantation. J Heart Lung 
Transplant2016 Jan;35(1):26-33. 
2) Rychik J, Veldtman G, Rand E, Russo P, Rome JJ, Krok K, Goldberg DJ, Cahill AM, Wells 
RG. The precarious state of the liver after a Fontan operation: summary of a 
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