Clinical trial NCT03571672
Conditions: Cardiac Disease
Keywords: Left Ventricular Ejection Fraction DEFINITY Enhanced Echocardiogram Cardiac Magnetic Resonance
Sponsor: Lantheus Medical Imaging
Start date: 2018-10-24; Completion date: 2019-08-30
Enrollment: 153 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is a Phase 3, prospective, open-label, multicenter study to evaluate Left Ventricular Ejection Fraction (LVEF) measurement accuracy and reproducibility of DEFINITY® contrast-enhanced and unenhanced echocardiography as compared with non-contrast cardiac magnetic resonance imaging (CMR) used as the truth standard.

Detailed description:

Arms: arm 1: Each patient will undergo an unenhanced ultrasound examination and a DEFINITY contrast-enhanced ultrasound
Interventions: intervention 1: DEFINITY
Intervention descriptions: intervention 1: All subjects will receive a single dose of DEFINITY®

Protocol documents:
Prot_000.pdf: 
 
 
Clinical Study Protocol:  DEF-314 
Study Title: 
A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection 
Fraction, End-Diastolic and End-Systolic Volumes, by Unenhanced 
and DEFINITY®-enhanced 2D-Echo and Magnetic Resonance 
Imaging 
Study Number: 
DEF-314 
Study Phase: 
Phase 3 
Product Name: 
DEFINITY® 
IND Number: 
048626 
Indication: 
DEFINITY® is indicated to improve the accuracy of left ventricular 
ejection fraction measurements with echocardiography. 
Investigators: 
Multicenter 
Sponsor: 
Lantheus Medical Imaging, Inc. 
Sponsor 
Contact: 
331 Treble Cove Road 
North Billerica, MA 01862 
+1 (978)-667-9531 
Medical 
Monitor: 
Cesare Orlandi, MD 
 
 
 
Date 
Original Protocol 
07 February 2018  
Amendment 1 
02 May 2018 
Amendment 2 
12 June 2018 
Amendment 3 
15 August 2018 
 
Confidentiality Statement 
This document is the confidential property of Lantheus Medical Imaging (LMI). No part of it 
may be transmitted, reproduced, published, or used by other persons without the permission 
of LMI. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 2 of 49 
SYNOPSIS 
Sponsor:  Lantheus Medical Imaging, Inc. 
Study Title: 
A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection Fraction, End-Diastolic 
and End-Systolic Volumes, by Unenhanced and DEFINITY®-enhanced 2D-Echo and 
Magnetic Resonance Imaging 
Study Number:  DEF-314 
Study Phase:  Phase 3 
Primary Objective(s):  
The primary objective of this study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography.  
Secondary Objective(s): 
The secondary objectives of this study are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography in 
subjects with suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 3 of 49 
Study Design: 
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF 
measurement accuracy and reproducibility of DEFINITY® contrast-enhanced and 
unenhanced echocardiography as compared with non-contrast cardiac magnetic resonance 
imaging (CMR) used as the truth standard.   
Subjects will be enrolled over approximately 10 months at approximately 10 centers 
located in the United States.  Subjects will be screened based on LVEF measurements 
obtained via 2D echo with or without contrast or other methods (e.g. CMR, MUGA scan) 
obtained within 6 months of enrollment (Day 0) to achieve an approximately even 
distribution within four pre-defined LVEF groups (>50, 41-50, 30–40, <30%).  Subjects 
with optimal and sub-optimal echocardiograms will be enrolled.  
Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® 
contrast-enhanced examination on the same day at Day 0.  Subjects will remain at the 
clinical site for at least 30 minutes of observation after DEFINITY® administration.  The 
site investigator will determine whether an echocardiogram is optimal or sub-optimal 
based on the unenchanced echocardiogram performed on Day 0.   
A non-contrast CMR will be used as the truth standard.  CMR studies will be performed 
within ±30 days of the echocardiographic assessments at Day 0. Final assignment of each 
subject to an LVEF group will be determined by CMR. 
In addition, a safety follow-up phone call will be conducted at 72±24 hours after 
DEFINITY® administration.   
All echocardiography images will be interpreted by 3 experienced independent blinded 
readers at a central imaging core laboratory.  A single, independent, blinded 
echocardiologist will read each subject’s unenhanced echocardiogram as either optimal or 
suboptimal.  This will be independent of the site investigator determination.  All CMR 
images will be read centrally by a single reader.  Echocardiographic imaging data will be 
compiled, analyzed, and stored without knowledge of the CMR findings. 
 
Study Population:  
Approximately one hundred and fifty (150) evaluable subjects will be enrolled at 
approximately 10 US centers.  Subjects who have had a 2D echo with or without contrast 
or other methods (e.g. CMR, MUGA scan) to evaluate screening LVEF within 6 months 
prior to enrollment (Day 0) may be enrolled.  The study population will consist of males 
and non-pregnant and non-lactating females 18 years-of-age or older. 
Duration of Treatment:  
Subjects will receive DEFINITY® treatment once as a part of a single day imaging session.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 4 of 49 
Study Treatment:  
DEFINITY® will be administered as a diluted bolus injection.  1.3 mL of activated 
DEFINITY® will be diluted with 8.7 mL of preservative-free saline to evenly distribute 
microspheres.  An initial injection of up to 3 mL of diluted DEFINITY® will be 
administered with subsequent injections of 1 to 2 mL, as needed. 
Efficacy Assessments: 
The primary endpoint is to demonstrate an improvement in LVEF accuracy from 
unenhanced imaging to imaging with DEFINITY® contrast enhancement using CMR as 
the truth standard.   
The secondary endpoints are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography 
in subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
Safety Assessments: 
Enrolled subjects will be followed for adverse events (AEs), serious adverse events (SAEs) 
and changes in concomitant medications from the time the Informed Consent (IC) is 
signed through 72±24 hours after completion of DEFINITY® administration.  All AEs will 
be recorded on the electronic case report form (eCRF).  The National Cancer Institute 
Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 will be 
utilized for Adverse Event (AE) reporting of severity. 
Statistical Methods for Primary Objective:  
The primary objective of the study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced 
over unenhanced echocardiography, using CMR as the truth standard.   
The following analyses will be carried out for each of the three blinded readers.  For each 
subject, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF 
will be calculated.  Similarly, the absolute value of the difference of unenhanced imaging 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 5 of 49 
LVEF minus CMR LVEF will be calculated for each subject.  The primary analysis is to 
assess the significance of the difference between DEFINITY® and unenhanced echo with 
respect to the mean “absolute value of the difference vs. CMR”.  These means will be 
compared with a paired t-test at a two-sided 0.05 level of significance.  Specifically, the 
null and alternative hypotheses are: 
H0: µD = µU vs. H1: µD ≠ µU 
Where µD is the mean of the absolute value of the differences between the DEFINITY®-
enhanced echo LVEF and the CMR LVEF. Similarly, µU is the mean of the absolute value 
of the differences between the unenhanced echo LVEF and the CMR LVEF.  A sample of 
size of 150 enrolled subjects has 90% power to reject the null hypothesis in favor of the 
alternative if the true difference µD - µU is at least 2.75 with a standard deviation of 10 or 
less and allows for approximately 5% premature withdrawal.  If the null hypothesis is 
rejected in favor of DEFINITY® for at least two of three blinded readers, the primary 
analysis will be considered a success. 
The following are additional analyses to be carried out separately for each blinded reader 
to achieve the primary objective: 
Bias is the mean of the actual (not absolute value) per-subject differences between the 
imaged LVEF and CMR LVEF.  The bias, the two-sided 95% confidence interval of the 
bias, the precision (standard deviation of the per-subject differences) and the root mean 
square error (RMSE) will be calculated for DEFINITY®.  The RMSE is the square root of 
the bias-squared + precision-squared and is considered a measure of overall accuracy.  
These analyses will be repeated for unenhanced echocardiography vs. CMR.   It is 
anticipated that the RMSE will be smaller for DEFINITY®-enhanced LVEF than for 
unenhanced LVEF. 
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for 
measurement accuracy against the reference CMR using Bland-Altman analysis and 
Deming regression analysis.  For the Bland-Altman analysis, a plot of per-subject actual 
difference between DEFINITY®-enhanced echocardiography and CMR LVEF will be 
plotted vs. the per-subject sum of the two measurements. Limits of agreement (defined as 
the mean of the DEFINITY® vs. CMR difference) +/- 2 SD will be shown on the plot.  
Similar analyses will be carried out for the unenhanced echocardiography LVEF again 
using CMR as the reference standard. 
Deming regression plots of LVEF vs. CMR LVEF will be generated for each of 
DEFINITY®-enhanced and unenhanced echocardiography.  Unweighted Deming 
regression will be employed to estimate the regression slope and intercept with two-sided 
95% confidence intervals of each, assuming the measurement error is the same for CMR 
and each of the echocardiography error techniques. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 6 of 49 
The intra-class correlation coefficient (ICC) and its two-sided 95% confidence interval will 
be calculated for DEFINITY®-enhanced echo vs. CMR and for unenhanced echo vs. 
CMR.    
Statistical Methods for Secondary Objectives: 
A secondary objective of this study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography in subjects with suboptimal echocardiograms.  For this, the 
above analyses for the primary objective will be repeated on subjects with suboptimal 
echocardiograms. 
An additional secondary objective is to demonstrate a reduction in inter-reader variability 
for the assessment of LVEF and End-Diastolic/Systolic volumes using DEFINITY® 
contrast-enhanced versus unenhanced echocardiography.  The inter-observer variability 
among each pair of readers within each imaging modality will be estimated using an intra-
class correlation coefficient (ICC); descriptive comparisons of the ICC between 
DEFINITY® and unenhanced echocardiography will be carried out.  Similar analyses will 
be conducted for the final secondary objective of assessing inter-reader variability within 
subjects with suboptimal echocardiograms.   
 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 7 of 49 
TABLE OF CONTENTS 
SYNOPSIS .................................................................................................................................2 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..........................................11 
1 
INTRODUCTION ...........................................................................................................14 
1.1 
Background ............................................................................................................14 
2 
STUDY OBJECTIVES ....................................................................................................15 
2.1 
Primary Objective ..................................................................................................15 
2.2 
Secondary Objectives.............................................................................................15 
3 
INVESTIGATIONAL PLAN ..........................................................................................16 
3.1 
Overall Study Design and Plan ..............................................................................16 
3.2 
Rationale for Study Design ....................................................................................17 
3.3 
Study Duration .......................................................................................................18 
4 
STUDY POPULATION ..................................................................................................19 
4.1 
Inclusion Criteria ...................................................................................................19 
4.2 
Exclusion Criteria ..................................................................................................19 
5 
STUDY TREATMENT(S) ..............................................................................................21 
5.1 
Description of Treatment(s) ...................................................................................21 
5.1.1 
Investigational Product ................................................................................21 
5.2 
Treatments Administered .......................................................................................21 
5.2.1 
Administering Study Drug ...........................................................................21 
5.3 
Selection and Timing of Dose for Each Patient .....................................................21 
5.4 
Method of Assigning Subjects to Treatment Groups .............................................21 
5.5 
Blinding..................................................................................................................22 
5.6 
Concomitant Therapy.............................................................................................22 
5.7 
Treatment Compliance ...........................................................................................22 
5.8 
Packaging and Labeling .........................................................................................22 
5.9 
Storage and Accountability ....................................................................................22 
5.10 
Investigational Product Retention at Study Site ....................................................22 
6 
STUDY PROCEDURES .................................................................................................23 
6.1 
Screening................................................................................................................23 
6.2 
Informed Consent...................................................................................................23 
6.3 
Medical History .....................................................................................................23 
6.4 
Physical Examination.............................................................................................24 
6.5 
Vital Signs ..............................................................................................................24 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 8 of 49 
6.6 
Pregnancy Test .......................................................................................................24 
6.7 
Cardiac Magnetic Resonance Imaging ..................................................................24 
6.7.1 
Image Acquisition ........................................................................................24 
6.7.2 
Imaging Core Laboratory .............................................................................25 
6.8 
Echocardiography Imaging Procedures .................................................................25 
6.8.1 
Image Acquisition ........................................................................................25 
6.8.2 
Imaging Core Laboratory .............................................................................26 
6.9 
Efficacy Assessments.............................................................................................26 
6.9.1 
Primary Efficacy Assessments .....................................................................26 
6.9.2 
Secondary Efficacy Assessments .................................................................26 
6.10 
Adverse Event Assessments ..................................................................................26 
6.10.1 
Performing Adverse Event Assessments .....................................................26 
6.10.2 
Timing ..........................................................................................................28 
6.10.3 
Severity ........................................................................................................28 
6.10.4 
Relationship .................................................................................................29 
6.10.5 
Clinical Significance ....................................................................................29 
6.10.6 
Serious Adverse Events ...............................................................................29 
6.10.6.1 
Definition ...........................................................................................29 
6.10.6.2 
Reporting Serious Adverse Events ....................................................30 
6.11 
Removal of Subjects from the Trial or Investigational Product ............................31 
7 
QUALITY CONTROL AND ASSURANCE .................................................................32 
8 
PLANNED STATISTICAL METHODS ........................................................................33 
8.1 
Statistical Analyses ................................................................................................33 
8.1.1 
General Considerations ................................................................................33 
8.1.2 
Analysis Populations ....................................................................................33 
8.1.2.1 
Safety Population ...............................................................................33 
8.1.2.2 
Intent-to-Treat ....................................................................................33 
8.1.2.3 
Modified Intent-to-Treat Population ..................................................33 
8.1.2.4 
Per-Protocol Population .....................................................................33 
8.2 
Efficacy Endpoint(s) ..............................................................................................34 
8.2.1 
Primary Efficacy Endpoint ..........................................................................34 
8.2.2 
Other Primary Endpoint(s) ...........................................................................36 
8.3 
Secondary Endpoint(s) ...........................................................................................36 
8.3.1 
Secondary Efficacy Endpoint(s) ..................................................................36 
8.3.1.1 
Improvement in accuracy in left ventricular ejection fraction 
(LVEF) assessment using DEFINITY® contrast-enhanced over 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 9 of 49 
unenhanced echocardiography in subjects with suboptimal 
echocardiograms ................................................................................36 
8.3.1.2 
Reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography ...............................................................................36 
8.3.1.3 
Reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography ................................................37 
8.3.1.4 
Reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms ......37 
8.3.1.5 
Reduction in inter-reader variability for the assessment of End-
Diastolic/Systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms ................................................................................37 
8.3.2 
Other Secondary Endpoint(s) .......................................................................37 
8.4 
Safety Endpoint(s) .................................................................................................37 
8.4.1 
Primary Safety Endpoint(s)..........................................................................37 
8.4.2 
Secondary Safety Endpoint(s) ......................................................................37 
8.5 
Demographics and Baseline Characteristics ..........................................................38 
8.6 
Interim Analysis .....................................................................................................38 
8.7 
Determination of Sample Size ...............................................................................40 
9 
ADMINISTRATIVE CONSIDERATIONS ....................................................................41 
9.1 
Investigators and Study Administrative Structure .................................................41 
9.2 
IRB or IEC Approval .............................................................................................41 
9.3 
Ethical Conduct of the Study .................................................................................41 
9.4 
Patient Information and Consent ...........................................................................42 
9.5 
Patient Confidentiality ...........................................................................................42 
9.6 
Study Monitoring ...................................................................................................43 
9.7 
Case Report Forms and Study Records .................................................................43 
9.8 
Data Monitoring Committee ..................................................................................44 
9.9 
Protocol Violations/Deviations ..............................................................................44 
9.10 
Access to Source Documentation ..........................................................................44 
9.11 
Data Generation and Analysis ...............................................................................45 
9.12 
Retention of Data ...................................................................................................45 
9.13 
Financial Disclosure...............................................................................................45 
9.14 
Publication and Disclosure Policy .........................................................................46 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 10 of 49 
10 REFERENCES ................................................................................................................47 
 
LIST OF IN-TEXT FIGURES 
Figure 3-1 
Study Event Overview ..................................................................................17 
 
LIST OF APPENDICES 
Appendix 1 Schedule of Procedures .................................................................................48 
Appendix 2 Dose Preparation and Administration Guide ................................................49 
 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 11 of 49 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
A2C 
apical 2-chamber 
A3C 
apical 3-chamber 
A4C 
apical 4-chamber 
AE 
adverse event 
BMI 
body mass index 
CABG 
coronary artery bypass graft 
CAD 
coronary artery disease 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
cm 
centimeter 
CMR 
cardiac magnetic resonance 
CRO 
Contract Research Organization 
DMC 
Data Monitoring Committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
ED 
effective dose 
Echo 
Echocardiogram 
EEG 
electroencephalography 
EF 
ejection fraction 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GSM 
Group sequential method 
HEENT 
head, ears, eyes, nose and throat  
IB 
Investigator’s Brochure 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 12 of 49 
Abbreviation 
Definition 
IC 
informed consent 
ICC 
intra-class correlation coefficient 
ICF 
informed consent form 
ICH 
International Conference on Harmonization 
IEC 
Institutional Ethics Committee 
IRB 
Institutional Review Board 
ITT 
intent-to-treat 
LC 
liquid chromatography 
LCX 
left circumflex 
LMI 
Lantheus Medical Imaging 
LV 
left ventricular 
LVEF 
left ventricular ejection fraction 
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
myocardial infarction 
mITT 
modified intent-to-treat 
MUGA 
Multiple-gated acquisition 
g 
microgram 
mg 
milligram 
MI 
mechanical index 
min 
minute 
mL 
milliliter 
mmHg 
millimeters of mercury 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse 
Events 
O2 Sat 
oxygen saturation 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 13 of 49 
Abbreviation 
Definition 
pH 
potential of hydrogen 
PFP 
phospholipids-encapsulated perfluoropropane 
PT 
preferred term 
QC 
quality control 
QT [interval] 
interval on the ECG from the onset of the QRS complex to the end of the 
T-wave 
QTcB 
Bazett-Corrected QT interval 
RCA 
right coronary artery  
RMSE 
root mean square error 
RR 
respiratory rate 
SAE 
serious adverse event 
SAF 
Safety Evaluable Population 
SAS® 
Statistical Analysis System 
sec 
second(s) 
SOC 
system organ class 
SP 
safety population 
TE 
echo time 
TEAE 
treatment emergent adverse event 
TIA 
transient ischemic attack 
TR 
repetition time 
US 
United States 
WOCBP 
women of childbearing potential 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 14 of 49 
1 
INTRODUCTION 
1.1 
Background 
Left ventricular (LV) volumes and ejection fraction (EF) are important clinical parameters 
for the diagnosis, management, and prognosis of subjects with cardiac diseases. Important 
treatment decisions and the evaluation of therapeutic effects are based on these parameters.  
Several techniques have been used for the analysis of LV volumes and EF, among them cine 
ventriculography, echocardiography, cardiac magnetic resonance (CMR) and computed 
tomography (Foley 2012).1 CMR has evolved into the preferred reference technique because 
of its high spatial resolution and ability to obtain complete volumetric data sets, allowing 
very accurate determinations of regional and global LV function.  Although the most 
frequently used modality in clinical practice, echocardiography has been hampered by its 
moderate reproducibility and accuracy to define LVEF. Poor acoustic windows and 
inadequate discrimination of the endocardial border are the main reasons for the poor 
reproducibility and accuracy of the test, besides geometric assumptions resulting from the 
two-dimensional approach (Foley 2012).1 In a few studies, contrast echocardiography has 
been shown to allow improved assessment of LV volumes and EF, especially in subjects with 
difficult imaging conditions (Hoffman 2005, 2014,).2,3  Recent innovations in contrast-
specific ultrasound techniques have further enabled improvements in visualization of the LV 
endocardial border above the level already shown in previous trials with the use of contrast-
enhanced ultrasound imaging. 
DEFINITY® (perflutren injectable suspension) is a sterile, non-pyrogenic suspension of 
phospholipids-encapsulated perfluoropropane microbubbles that is activated by shaking with 
the aid of the Vialmix™.  In subjects with suboptimal echocardiograms, the addition of 
DEFINITY® improves the assessments of cardiac structure (ventricular chambers and 
endocardial borders) and function (regional wall motion). 
The objective of this multicenter study is to determine the accuracy and inter-reader 
agreement of DEFINITY® contrast-enhanced and unenhanced 2D echocardiography for the 
assessment of LV volumes and EF in comparison with non-contrast CMR. 
This trial will be conducted strictly according to the protocol, Good Clinical Practice (GCP), 
the sites’ Independent Ethics Committees (IECs)/Institutional Review Boards (IRBs), and 
other related laws and regulations as applicable. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 15 of 49 
2 
STUDY OBJECTIVES 
2.1 
Primary Objective  
The primary objective of the study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography. 
2.2 
Secondary Objectives 
The secondary objectives of the study are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography in 
subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 16 of 49 
3 
INVESTIGATIONAL PLAN 
3.1 
Overall Study Design and Plan 
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF measurement 
accuracy and reproducibility of DEFINITY® contrast-enhanced and unenhanced 
echocardiography as compared with non-contrast cardiac magnetic resonance imaging 
(CMR) used as the truth standard.  Approximately one-hundred fifty (150) evaluable subjects 
will be enrolled and will undergo unenhanced and DEFINITY®-enhanced echocardiograms 
and CMR.   
Subjects will be screened based on LVEF measurements obtained via 2D echo with or 
without contrast or other methods (e.g. CMR, MUGA scan) obtained within 6 months prior 
to enrollment.    Subjects will be stratified to achieve an approximately even distribution 
within four pre-defined LVEF groups (>50, 41-50, 30–40, <30%) as measured by CMR.  
Subjects with optimal and suboptimal echocardiograms will be enrolled.   
Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® 
contrast-enhanced examination on the same day.  A minimum of 360 seconds of images will 
be collected during both the unenhanced and the DEFINITY® contrast-enhanced 
examinations.   
The site investigator will determine whether an echocardiogram is optimal or sub-optimal 
based on the unenchanced echocardiogram performed on Day 0.  An echocardiogram is 
considered suboptimal if 2 or more segments of the ventricular border are classified as not 
adequately visualized in any one of the 3 apical views.   
Subjects will remain at the clinical site for at least 30 minutes of observation after the end of 
the administration of DEFINITY®.  A safety follow-up telephone call will be conducted for 
all subjects at approximately 72±24 hours after completion of the imaging sessions.   
Enrolled subjects will be followed for AEs, SAEs, and changes in concomitant medications 
from the time the Informed Consent is signed through the safety follow-up telephone call.   
Unenhanced and DEFINITY®-enhanced echocardiograms will be performed with standard 
apical 2 chamber, apical 3 chamber, and apical 4 chamber, (A2C, A3C, and A4C, 
respectively) views.  DEFINITY®-enhanced echocardiograms will be performed using low or 
very low mechanical index (MI) imaging.  Images will be recorded in standard digital format, 
masked to patient identifiers, and sent to a central echocardiography facility for analysis.  
There, 3 experienced independent blinded readers will interpret the results according to the 
Image Review Charter; additional analyses will also be performed as described in the Image 
Review Charter.  Each reader’s independent interpretation will be recorded in a database. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 17 of 49 
CMR images will also be collected and independently read at a central laboratory by a single 
reader. 
The schedule of events is provided in Appendix 1. 
Figure 3-1 displays an overview of study events. 
Figure 3-1 
Study Event Overview 
 
 
3.2 
Rationale for Study Design 
The study is intended to demonstrate the utility of DEFINITY® contrast-enhanced 
echocardiography in providing accurate and reproducible measurements of LVEF and LV 
volumes as compared with unenhanced 2D echocardiography using non-contrast CMR as the 
truth standard. 
LVEF measurement obtained via 2D Echo or other 
methods (e.g. CMR, MUGA scan) within 6 months prior 
to enrollment Day 0  
Screening 
Days -7 to Day 0 
Echocardiograms & DEFINITY® 
administration  
Day 0 
 
Safety telephone 
follow-up 
72 ± 24 hours 
CMR within ±30 days of Day 0 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 18 of 49 
A broad-based population with known or suspected cardiac disease with either optimal or 
suboptimal 2D echocardiograms or other imaging methods from which LVEF can be derived 
(e.g. CMR, MUGA scan) will be included.   
The primary endpoint of improved accuracy in LVEF measurement will be assessed from the 
results of an independent assessment and subsequent analysis conducted by the central 
imaging laboratory.  Secondary endpoints will also be assessed, including accuracy in the 
suboptimal echocardiogram subset and inter-reader variability in the overall population and 
the suboptimal echocardiogram subset. 
Interpretation of images will be conducted with a blinded read by three experienced 
physicians at a central imaging laboratory who are not associated with patient recruitment.  
In addition, a single, independent, blinded echocardiologist will read each subject’s 
unenhanced echocardiogram as either optimal or suboptimal.  This will be independent of the 
site investigator determination performed at Day 0.  The independent readers will employ a 
prospectively defined and standardized methodology described in the Image Review Charter, 
which will reduce the potential for variation in the criteria used for image reads. 
CMR images will also be collected and read independently at a central imaging laboratory, 
using a methodology that will also be described in the Image Review Charter. 
3.3 
Study Duration 
All screening assessments will occur within 7 days prior to enrollment/DEFINITY® 
administration (Day 0).  Non-contrast CMR studies will occur within ± 30 days of 
DEFINITY® administration.  Safety monitoring will begin with the signing of the informed 
consent and continue up to 72±24 hours post-DEFINITY® administration.  The expected 
duration of subject participation is not more than 41 days.  Subjects are expected be enrolled 
over a 10 month period.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 19 of 49 
4 
STUDY POPULATION 
Approximately one hundred and fifty (150) evaluable subjects who have previously 
undergone a 2D echocardiogram with or without contrast or other assessment (CMR, MUGA 
scan) will be enrolled in this trial.  The study population will consist of male and female 
subjects 18 years of age or older.   
Enrollment will be stratified according to CMR LVEF to achieve an approximately even 
distribution across four subgroups (LVEF >50, 41-50, 30–40, <30%).  LVEF for screening 
purposes will be determined by the investigator and based on measurements with 2D echo or 
other methods (e.g. CMR, MUGA scan) obtained within 6 months prior to enrollment (Day 
0). Subjects with optimal and sub-optimal echocardiograms will be enrolled.  Suboptimal 
echocardiograms will be based on the site investigator’s evaluation of the unenhanced 
echocardiography images obtained on Day 0.  An echocardiogram is considered suboptimal 
if 2 or more segments of the ventricular border are classified as not adequately visualized in 
any one of the 3 apical views. 
4.1 
 Inclusion Criteria 
Potential study subjects must meet the following inclusion criteria to be enrolled in this 
study. 
1. Men and women ≥ 18 years of age in sinus rhythm 
2. Able to communicate effectively with trial personnel 
3. LVEF measurements obtained via 2D Echo with or without contrast or other methods 
(e.g. CMR, MUGA scan) obtained within 6 months prior to enrollment (Day 0) 
4. Has provided signed informed consent after receiving a verbal and written explanation of 
this clinical trial 
 
4.2 
Exclusion Criteria 
Potential study subjects who meet any of the following criteria will be excluded from the 
study: 
1. Female subjects who are pregnant or lactating.  All women of child bearing potential 
[WOCBP] must have a negative urine pregnancy test at screening regardless of 
contraceptive use history. 
2. Women of child-bearing potential are excluded unless they: 
a. are post-menopausal defined as amenorrhea ≥ 12 consecutive months, OR 
b. have undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation 
or bilateral oophorectomy), OR  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 20 of 49 
c. have been using an adequate and medically approved method of contraception to 
avoid pregnancy for at least 1 month prior to DEFINITY® dose administration and be 
willing to continue using the same method for the duration of the study. 
3. Current illness or pathology that would prevent undergoing investigational product 
administration due to a significant safety risk to the patient.  
4. Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or 
diastolic blood pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood 
pressure ≤ 90 mmHg). 
5. Unstable cardiovascular status defined as: 
a. myocardial infarction or unstable angina pectoris within 6 months prior to 
enrollment/DEFINITY® dose administration day  
b. transient ischemic attack or stroke within 3 months prior to DEFINITY® dose 
administration  
c. symptomatic valvular heart disease or moderate to severe stenotic valvular heart 
disease  
d. clinically significant congenital heart defects 
e. current uncontrolled cardiac arrhythmias causing symptoms or hemodynamic 
compromise  
f. acute pulmonary embolus or pulmonary infarction  
g. acute myocarditis or pericarditis  
h. acute aortic dissection   
i. atrial fibrillation 
6. any major surgery within 4 weeks prior to screening 
7. known contraindications to undergoing CMR (e.g. implanted pacemakers, cardioverter, 
defibrillators) or claustrophobia 
8. participation in any investigational drug, device, or placebo study within 30 days prior to 
screening  
9. known hypersensitivity to perflutren, or any of the excipients in DEFINITY® 
10. prisoners or those who are subject to compulsory detention or involuntary incarceration 
for treatment of either a psychiatric or physical illness (e.g., infectious disease) 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 21 of 49 
5 
STUDY TREATMENT(S) 
5.1 
Description of Treatment(s) 
5.1.1 
Investigational Product 
DEFINITY® is an ultrasound contrast agent consisting of phospholipids-encapsulated 
perfluoropropane (PFP), also known as perflutren, microspheres, which is designed to 
enhance echocardiographic ultrasound images.  DEFINITY® (perflutren injectable 
suspension) will be provided in a 2-mL clear vial containing a 1.5 mL fill volume. 
DEFINITY® contains no preservative.  Bacterial contamination with the risk of 
post-administration septicemia can occur following the puncture of the elastomeric septum.  
It is essential to follow directions for preparation of DEFINITY® carefully and to adhere to 
strict aseptic procedures during preparation. 
The chemical, and pharmacologic properties and formulation of DEFINITY® are described in 
detail in the Investigator’s Brochure (IB).   
5.2 
Treatments Administered 
All treatments will be administered as described in the Dose Preparation and Administration 
Guide.  Appendix 2. 
5.2.1 
Administering Study Drug  
DEFINITY® will be administered as a diluted bolus injection.  1.3 mL of activated 
DEFINITY® will be diluted with 8.7 mL of preservative-free saline to evenly distribute 
microspheres.  An initial injection of up to 3 mL of diluted DEFINITY® will be administered 
with subsequent injections of 1 to 2 mL, as needed.  Full details are available in the Dose 
Preparation and Administration Guide – Appendix 2. 
5.3 
Selection and Timing of Dose for Each Patient 
All subjects will receive administration of DEFINITY® as described above and in the Dose 
Preparation and Administration Guide – Appendix 2. 
5.4 
Method of Assigning Subjects to Treatment Groups 
There is only one treatment group in this study. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 22 of 49 
5.5 
Blinding 
This is an open-label study; therefore, no blinding procedures are required for investigators 
and subjects.   
5.6 
Concomitant Therapy 
Concomitant medications will be collected from the screening visit to the 72±24 hour safety 
follow-up phone call, and will be recorded on the eCRF.   
5.7 
Treatment Compliance 
The investigator will comply with the protocol and all applicable regulatory requirements in 
assuring that the study drug is dispensed to subjects by authorized personnel only. 
5.8 
Packaging and Labeling 
The labeling of the study drug will meet local regulatory requirements, but at a minimum will 
provide the investigational product name, expiry, lot number, manufacturer name and 
address, storage conditions, and caution warnings for investigational product use.   
5.9 
Storage and Accountability 
The investigator is responsible for ensuring that deliveries of study drug are safely and 
properly received, recorded, handled, and stored in accordance with the national and/or local 
laws and are used in accordance with this protocol. 
DEFINITY® should be stored in a refrigerator (2-8° C) upon receipt of the vials.  Prior to 
activation, the vial should be warmed to room temperature.  Following activation, 
DEFINITY® can be stored at room temperature (do not store above 30°C) and should be used 
within 12 hours of preparation.  Refer to DEFINITY® Dose Preparation and Administration 
Guide (Appendix 2) for full storage details. 
5.10 Investigational Product Retention at Study Site 
The site will document the administration of the investigational product for each subject. All 
investigational product, used and unused, will be accounted for by a study monitor.   
All unused or residual study drug must be properly destroyed at the study site in accordance 
with national and/or local laws and institutional policies and properly documented. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 23 of 49 
6 
STUDY PROCEDURES 
6.1 
Screening 
The timing of study procedures is provided in Appendix 1.  All Screening/Baseline 
assessments will occur within 7 days prior to administration of study drug (Day 0). 
6.2 
Informed Consent 
Informed consent (IC) must be obtained from each patient prior to the initiation of any study 
related procedures unless those procedures are considered standard of care for the diagnosis 
and/or treatment of the underlying disease process.  The IC must meet the requirements as 
defined by International Conference on Harmonization (ICH) - Good Clinical Practice (GCP) 
guidelines, US Federal Regulations and/or conform to national or regional regulatory agency 
requirements, or local laws, whichever provides the greatest level of protection.  Each patient 
must be provided, in an understandable manner, a written informed consent form (ICF) that 
describes the nature and duration of the study.  Additionally, the patient must be allowed 
adequate time to consider the potential risks and benefits associated with his/her participation 
in the study.  The IC must also contain language that allows the Sponsor (or designated 
representative), regulatory authorities, and the Institutional Review Board (IRB)/ Institutional 
Ethics Committee (IEC) direct access to the patient’s medical records for the purpose of 
review. 
In situations where study subjects are not legally competent to provide consent (i.e., 
cognitively incapacitated patient), written consent must be obtained from an authorized 
guardian or representative.  In these situations, the ICF must be signed and dated by a witness 
and, if possible, assent must be obtained from the patient.  The ICF must also be signed and 
dated by the individual obtaining the IC. 
The Investigator, as part of the study documentation, must retain the original signed and 
dated ICF and must provide a signed copy to each patient or authorized representative.  The 
Investigator will not undertake any measures specifically required only for the clinical study until 
valid consent has been obtained. 
6.3 
Medical History 
A general medical history for each patient will be obtained by interview and through a 
review of available medical records.  Data to be collected and recorded on the eCRF will 
include demographics (e.g. age, gender, race/ethnicity) and an overview of significant past or 
present illnesses, including previous administration of DEFINITY®.  Known drug allergies, 
other allergies, or history of hypersensitivity reactions will also be recorded on the eCRF.  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 24 of 49 
Information obtained via interview should be supplemented with data from hospital records 
or clinic charts as available. 
All concomitant medications will be recorded on the eCRF.  This information will be 
obtained by interview and through a review of available medical records. 
6.4 
Physical Examination 
A physical examination will be performed at screening and will include height, weight, 
general appearance, and assessment of appearance (head, ears, eyes, nose and throat 
[HEENT]); neck; cardiovascular; lungs; abdomen; lymph nodes; extremities; neurological; 
skin; musculoskeletal; and other).  All physical examination abnormalities will be recorded 
on the eCRF. 
6.5 
Vital Signs 
All vital sign measurements should be obtained with the patient lying supine, having rested 
quietly for at least 2 minutes, as feasible.  Vital signs (including heart rate, respiratory rate, 
and systolic and diastolic blood pressure) will be collected at the Screening visit only. 
6.6 
Pregnancy Test 
A urine sample for pregnancy testing will be obtained for all women of childbearing potential 
at screening and within 24 hours prior to dosing the study drug. 
6.7 
Cardiac Magnetic Resonance Imaging 
6.7.1 
Image Acquisition 
Subjects will receive a non-contrast enhanced CMR examination. MRI examinations will be 
performed by a qualified technologist supervised by a physician experienced with cardiac 
MRI. Steady-state free precession (SSFP) MRI will be performed using a 1.5 T (or above) 
imaging system and a standardized and harmonized pre-specified method in accordance with 
practice guidelines for images that are to be used for LVEF evaluation. Cardiac slices relative 
to single apical 2- and 4-chamber views as well as multi-slice short-axis images running from 
the apex to the base will be obtained for each patient. The apical 2-chamber view should be 
parallel to the interventricular septum and intersect the cardiac apex and mid-mitral valve; 
whereas, the apical 4-chamber view should be perpendicular to the interventricular septum, 
maximizing ventricular size, and should intersect the apex and mid-mitral valve. The left 
ventricular outflow tract and aorta must not be visualized in this view. Parasternal short-axis 
images of the left ventricle will be acquired, perpendicular to the long axis of the ventricle 
spanning apex to base.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 25 of 49 
Consecutive breath held short-axis cine images will be obtained with full ventricular 
coverage.   
All images will then be transferred to an Imaging Core Laboratory for further analysis. No 
site analysis will be performed. Instructions for MRI data transfer and storage at the central 
imaging laboratory will be provided in the study Imaging Manual.   
6.7.2 
Imaging Core Laboratory 
To identify the LV end-diastolic and end-systolic images, the full stack of short axis images 
will be evaluated.  The LV end-diastolic image will be chosen as the phase with the largest 
LV blood volume.  The LV end-systolic image will be chosen as the phase with the smallest 
LV blood volume.   The Simpson’s method of summation of discs will then then applied 
(sum over all slices of cross sectional area multiplied by the sum of the slice thickness and 
the inter-slice gap) to determine LV end-diastolic (LVEDV) and LV end-systolic (LVESV) 
volumes for entire LV. Quantification of LVEF will then be calculated based on volumes 
using the formula: LVEF = (LVEDV − LVESV)/LVEDV.4,5 
A single central MR reader will verify and correct if necessary the identification of the LV 
end-systolic and diastolic images provided by a trained and blinded technologist to determine 
the LV end diastolic and end-systolic volumes. Similarly a trained and blinded technologist 
will provide initial ventricular contours which will be verified and corrected if necessary by 
the central MR reader prior to final calculation of the volumes. 
6.8 
Echocardiography Imaging Procedures 
6.8.1 
Image Acquisition 
A bedside resting transthoracic echocardiogram will be performed by personnel trained in 
study procedures according to standardized instructions provided here and in the study 
Imaging Manual.  Unenhanced echocardiography will be performed in each of the standard 
A2C, A3C, and A4C views after optimization of these views.  At least 3 consecutive cardiac 
cycles should be recorded in standard digital format in each view.  After unenhanced imaging 
has been conducted, study drug will be administered as described above and in the Dose 
Preparation and Administration Guide.  DEFINITY®-enhanced images will be obtained with 
low or very low MI imaging using a standardized and harmonized method consistent with 
practice guidelines.  After study drug administration and subsequent image optimization, the 
same A2C, A3C, and A4C views should be obtained as with unenhanced imaging over at 
least 3 consecutive cardiac cycles. 
Subjects will remain at the site for observation until at least 30 minutes after the end of the 
administration of study drug.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 26 of 49 
All images will then be transferred to an Imaging Core Laboratory for further analysis. No 
site analysis will be performed. Instructions for echo data transfer and storage at the central 
imaging laboratory will be provided in the study Imaging Manual.   
6.8.2 
Imaging Core Laboratory 
Left ventricular end-diastolic and end-systolic volumes will be measured using the modified 
biplane Simpson's method in the apical 4 chamber and apical 2 chamber views. End-diastole 
will be identified as the frame in which the left ventricular cavity size is visually 
largest.  End-systole will be identified as the frame in which the left ventricular cavity size is 
visually smallest, just before mitral valve opening. The inner contour of the LV cavity 
(interface between left ventricular cavity and compacted myocardium) will be traced 
according to the recommendations of the American Society of Echocardiography (ASE), 
leaving the papillary muscles and trabeculations within the cavity.  Left ventricular ejection 
fraction will be calculated from the biplane volumes using the formula: 
LVEF = (LVEDV − LVESV)/LVEDV.6 
A blinded echocardiography technologist at the Imaging Core Laboratory will provide an 
initial contour of the LV cavity for the blinded read.  The blinded echocardiography readers 
will adjust contours based on their judgement and will provide independent LV 
measurements.  The same method for LVEF calculation will be used for all echocardiograms, 
including suboptimal and non-suboptimal and both enhanced and non-enhanced.   
6.9 
Efficacy Assessments 
Three blinded independent readers will perform all of the efficacy assessments following the 
methodology described in the Imaging Review Charter. 
6.9.1 
Primary Efficacy Assessments 
The primary efficacy assessment will be the measurement of LVEF. 
6.9.2 
Secondary Efficacy Assessments 
Secondary efficacy assessments will be the end-systolic and end-diastolic LV volumes. 
6.10 Adverse Event Assessments 
6.10.1 
Performing Adverse Event Assessments 
In this clinical trial, an AE is defined as any new untoward medical occurrence or worsening 
in severity or frequency of a pre-existing medical condition in a study patient, which does not 
necessarily have a causal relationship with investigational product.  AEs occurring from the 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 27 of 49 
time the informed consent is signed until the 72±24 hour telephone follow-up will be 
reported.  Treatment-emergent adverse events (TEAEs) are a safety endpoint and are 
discussed in Section 8.4.1.  The NCI-CTCAE version 4.0 will be utilized for Adverse Event 
(AE) reporting of severity by grade. 
An AE can, therefore, be any unfavorable and unintended sign (including an abnormal 
laboratory finding, for example), symptom or disease temporally associated with the use of a 
medicinal (investigational or marketed) product, whether or not considered related to the 
medicinal (investigational or marketed) product. 
Note:  For this clinical trial, observations and findings (previously unknown) discovered 
during the procedures and medically assessed as most-likely pre-existing (e.g., myocardial 
wall hypokinesis, thrombus, cardiac valve deficits, and so on) will not be considered AEs and 
will not be reported as such in the eCRF.  However, these observations and any 
hospitalizations/treatments required to treat the new findings will be reported elsewhere in 
the eCRF.  Any complications resulting from the study procedures (e.g., intravenous 
extravasations) will be recorded as an AE.   
A medication error is defined as any event related to the administration of a medicinal or 
investigational product that may cause or lead to inappropriate use or patient harm.  
Medication errors can be actual or potential errors and may include misuse, administration 
error, overdose, under-dose, or abuse.  For this clinical trial, any medication errors (e.g., 
administration error, overdose, etc., that occur at the time of study product administration) 
must be recorded as an AE.  
All identified AEs must be recorded and described on the appropriate AE section of the 
eCRF by the investigator (or designee). If known, a diagnosis should be recorded, rather than 
multiple individual symptoms.  In addition to the event term, the following information 
should be captured for all AEs: 
• Date (and time) of onset and resolution  
• Severity of the event (see definitions below)  
• Investigator’s opinion of the causal relationship to study drug or comparator, procedure, 
or underlying disease (see definitions below)  
• Treatment action taken  
• Information regarding resolution/outcome 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 28 of 49 
6.10.2 
Timing 
Timely and complete reporting of safety information helps to identify any untoward medical 
occurrence, thereby allowing: 
• Protection of safety of study subjects, 
• Greater understanding of the overall safety profile of the investigational product, 
• Appropriate modification of study protocols, and 
• Adherence to worldwide regulatory requirements. 
 
Adverse event monitoring will be initiated at the time the informed consent is signed and will 
continue until completion of all echo assessments and patient discharge.  All subjects will be 
followed up by telephone assessments for AEs at 72±24 hours following DEFINITY® 
administration. 
6.10.3 
Severity 
The Investigator (or designee) will assess the severity of the AE using the National Cancer 
Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 will 
be utilized for Adverse Event (AE) reporting.  The NCI-CTCAE definitions of severity are 
below.   
Grade 
Degree of Severity 
1 
Mild, with mild or no symptoms; no interventions required 
2 
Moderate; minimal intervention indicated; some limitation of activities 
3 
Severe but not life threatening; hospitalization required; limitation of patient's ability 
to care for him/herself 
4 
Life threatening; urgent intervention required 
5 
Death related to adverse event 
 
This assessment is subjective and the Investigator should use good clinical judgment to 
compare the reported AE to similar type experiences observed in clinical practice. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 29 of 49 
6.10.4 
Relationship 
The Investigator will assess the relationship of the AE to DEFINITY®. The following 
categories and definitions of causal relationship should be used for this study: 
• Not related: indicates that there is little or no chance that the study treatment or 
procedure caused the reported AE; other conditions, including concomitant illnesses, 
progression or expression of the disease state, or a reaction to a concomitant medication, 
appear to explain the reported AE.  For the purposes of determining expedited 
reportability, AEs categorized as unlikely related to investigational product will not be 
considered associated with the investigational product. 
• Possibly related: indicates that the association of the AE with the study treatment or 
procedure is unknown; however, the AE is not reasonably attributed to any other 
condition 
• Related: indicates that a reasonable temporal association exists between the AE and 
study treatment or procedure and, based upon the investigator’s clinical experience, the 
association of the event with the study treatment seems likely 
 
6.10.5 
Clinical Significance 
Any abnormality in physical examinations or vital signs not present at Baseline should be 
judged for clinical significance by the Investigator. 
6.10.6 
Serious Adverse Events 
6.10.6.1 
Definition 
A serious adverse event (SAE) is defined by regulation as any AE occurring at any dose that 
results in any of the following outcomes:  
• Death 
• Life-threatening AE 
• In-patient hospitalization or prolongation of existing hospitalization 
• Persistent or significant disability/incapacity, or  
• Congenital anomaly/birth defect. 
 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, 
they may jeopardize the patient and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition.  Examples of such medical events include 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 30 of 49 
allergic bronchospasm requiring intensive treatment in an emergency room or at home, and 
blood dyscrasias or convulsions that do not result in inpatient hospitalization. 
Note: Hospitalization for scheduled elective procedures or hospitalization from clinically 
indicated patient management decisions based on pre-study conditions should not be 
classified as SAEs. Examples of this would include a scheduled coronary intervention or 
CABG procedure within the SAE reporting period.  Additionally, a treatment procedure 
scheduled due to diagnosis during the investigational procedure would not be classified as an 
SAE.  An example of this would be angioplasty to treat a patient’s underlying CAD.  Events 
resulting from the investigational procedure or a worsening of a pre-study condition which 
satisfy the definition of a SAE must be reported.  
6.10.6.2 
Reporting Serious Adverse Events 
All SAEs, whether related or unrelated to the investigational product, must be reported 
within 1 calendar day to LMI Drug Safety by confirmed facsimile transmission, email or 
phone.  Overnight express mail may also be used in lieu of a facsimile transmission.  If only 
limited information is initially available, follow-up reports are required.  The original SAE 
form must be kept on file at the study site. 
Collection of complete information concerning SAEs is extremely important.  Thus, 
follow-up information that becomes available as the SAE evolves, as well as supporting 
documentation (e.g., hospital discharge summaries and autopsy reports), should be collected 
subsequently, if not available at the time of the initial report, and immediately sent using the 
same procedure as the initial SAE report. 
The contact information for reporting SAEs and follow-up SAE information is: 
Contact 
Telephone Number 
Fax Number 
Email Address 
LMI Safety 
1-800-343-7851 
+ 978-677-9531 
FAX: 1-978-436-7296 
lantheussafety@lantheus.com 
 
 
If the investigator needs to discuss an SAE or any other aspect of the protocol with the 
Medical Monitor, s/he can be reached at the following numbers:  
LMI Medical Monitor 
24-Hour phone number:  +1-240-481-8818 
Fax:  +1-978-436-7296  
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 31 of 49 
In the event of AEs that are serious, unexpected, and possibly related or related to 
DEFINITY® administration, LMI will contact the Investigator and will provide a MedWatch 
and/or Council for International Organizations of Medical Sciences (CIOMS) narrative of the 
case, which will include an Analysis of Similar Events.  The Investigator is responsible for 
notifying the appropriate IRB in accordance with institutional requirements.  The Investigator 
and IRB will determine if the IC requires revision.  The Investigator should also comply with 
the IRB procedures for reporting any other safety information. 
6.11 Removal of Subjects from the Trial or Investigational Product 
The investigator may withdraw a patient from the study for any of the following reasons: 
• Patient withdraws consent 
• Patient is lost to follow up 
• Patient has an AE that, in the opinion of the Investigator, requires the patient’s 
discontinuation 
• Discretion of the Investigator  
• The Sponsor or Investigator terminates the study  
 
All events that result in discontinuation of study treatment will be appropriately recorded and 
reported.  In addition, for all subjects who discontinue prematurely, an evaluation which 
reflects the status of the patient at premature termination, along with a final assessment and 
the reasons for termination, will be recorded on the eCRF.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 32 of 49 
7 
QUALITY CONTROL AND ASSURANCE 
The sponsor has implemented and maintains Quality Assurance and Quality Control systems 
with written procedural documents (e.g. SOPs, work instructions, monitoring plans, etc.) to 
ensure that the study is conducted, and data are generated, recorded, and reported in 
compliance with the protocol, ICH-GCP, and all applicable regulatory requirements. 
These quality systems include monitoring (see Section 9.6); data collection, review and entry 
(see Section 9.11); training; and audit by the sponsor’s Quality group or representative.  
Audits are independent of routine monitoring and quality control functions, and are 
conducted to evaluate trial conduct and compliance with the protocol, procedural documents, 
ICH-GCP and applicable regulatory requirements. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 33 of 49 
8 
PLANNED STATISTICAL METHODS 
8.1 
Statistical Analyses 
8.1.1 
General Considerations 
The contract research organization (CRO) will be responsible for data management and 
statistical analysis in this trial.  All statistical analyses will be performed using SAS (SAS 
Institute, Inc., Cary, NC) version 9.4 or higher.  Patient data listings and tabular presentations 
of results will be provided.  Presentation of summary statistics for continuous variables will 
include N, mean, median, and standard deviation, as well as the minimum and maximum 
values.  For categorical variables, the number and percent of each category within a 
parameter for non-missing data will be calculated.  All statistical tests will be two-sided 
employing a significance level of 5% unless otherwise specified.  Further details of the 
criteria and conduct of the statistical analyses are below and will be included in the Statistical 
Analysis Plan for this study. 
8.1.2 
Analysis Populations 
8.1.2.1 
Safety Population 
The Safety Population (SP) will include all subjects who have signed informed consent and 
who have received any amount of DEFINITY® in the study. This is the primary analysis 
population for the safety analysis.  
8.1.2.2 
Intent-to-Treat 
The Intent-to-Treat (ITT) population will include all subjects who have signed informed 
consent. 
8.1.2.3 
Modified Intent-to-Treat Population 
The Modified Intent-to-Treat (mITT) population will include all ITT subjects who complete 
unenhanced imaging, DEFINITY® -enhanced imaging, and cardiac MR studies.  This is the 
primary analysis population for the efficacy endpoints. 
8.1.2.4 
Per-Protocol Population 
The Per-Protocol (PP) population will include all subjects in the mITT population who a) did 
not violate inclusion and exclusion criteria that would likely have an effect on the primary 
outcome, b) do not have major protocol violations; c) have LVEF data on both unenhanced 
and DEFINITY®- enhanced for at least one reader; (d) have CMR LVEF.  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 34 of 49 
Efficacy analyses will be conducted for both mITT and PP population sets.  Differences in 
results using the two populations will be carefully examined and the results of that evaluation 
presented in the study report. 
8.2 
Efficacy Endpoint(s) 
8.2.1 
Primary Efficacy Endpoint  
The primary analysis is to demonstrate an improvement in LVEF accuracy from unenhanced 
imaging to imaging with DEFINITY® contrast enhancement using CMR as the truth 
standard.  For each patient, the absolute value of the difference of DEFINITY® LVEF minus 
CMR LVEF will be calculated.  Similarly, the absolute value of the difference of unenhanced 
imaging LVEF minus CMR LVEF will be calculated for each patient.  The primary analysis 
is to assess the significance of the difference between DEFINITY® and unenhanced echo 
with respect to the mean “absolute value of the difference vs. CMR”.  These means will be 
compared with a paired t-test at a two-sided 0.05 level of significance.  Specifically, the null 
and alternative hypotheses are: 
H0: µD = µU vs. H1: µD ≠ µU 
where µD and µU are the mean of the DEFINITY® and unenhanced echo absolute value of 
the difference vs. CMRs, respectively.  A sample of size 150 enrolled subjects has 90% 
power to reject the null hypothesis in favor of the alternative if the true difference µD - µU is 
at least 2.75 (in favor of DEFINITY®) with a standard deviation of 10 or less and allows for 
approximately 5% premature withdrawal. 
The analyses will be conducted separately for each of the three blinded readers of the 
DEFINITY® enhanced and unenhanced images; the criterion for success is that the null 
hypothesis is rejected in favor of DEFINITY® for at least 2 of the 3 blinded readers.  CMR 
LVEF is interpreted by a single reader; this is the CMR LVEF that will be used as the 
comparator for each of the three blinded readers. 
The primary analysis will be performed on mITT subjects with non-missing LVEF for 
DEFINITY®, unenhanced echocardiography, and the reference standard.  A supportive 
analysis will be run where a missing echocardiography or CMR LVEF is multiply imputed 
using the fully conditional specification (FCS) multiple regression, as outlined below and as 
will be further detailed in the Statistical Analysis Plan. 
The following are additional analyses to be carried out separately for each blinded reader to 
achieve the primary objective: 
Bias is the mean of the actual (not absolute value) per-subject differences between the 
imaged LVEF and CMR LVEF.  The bias, the two-sided 95% confidence interval of the bias, 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 35 of 49 
the precision (standard deviation of the per-subject differences) and the root mean square 
error (RMSE) will be calculated for DEFINITY®.  The RMSE is the square root of the bias-
squared + precision-squared and is considered a measure of overall accuracy.  These analyses 
will be repeated for unenhanced echocardiography vs. CMR.   It is anticipated that the RMSE 
will be smaller for DEFINITY®-enhanced LVEF than for unenhanced LVEF. 
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for 
measurement accuracy against the reference CMR using Bland-Altman analysis and Deming 
regression analysis.  For the Bland-Altman analysis, a plot of the per-subject difference 
between DEFINITY®-enhanced echocardiography and CMR LVEF will be plotted vs. the 
per-subject sum of the two measurements. Limits of agreement (defined as the mean of the 
DEFINITY® vs. CMR difference) +/- 2 SD will be shown on the plot.  The same analyses 
will be performed for unenhanced echocardiography using CMR as the reference standard. 
Deming regression plots of LVEF vs. CMR LVEF will be generated for each of 
DEFINITY®-enhanced and unenhanced echocardiography.  Unweighted Deming regression 
will be employed to estimate the regression slope and intercept with two-sided 95% 
confidence intervals of each assuming the measurement error is the same for CMR and each 
of the echocardiography error techniques. 
The intra-class correlation coefficient (ICC) and its two-sided 95% confidence interval will 
be calculated for DEFINITY®-enhanced echo vs. CMR and for unenhanced echo vs. CMR.  
The ICCs and their two-sided confidence intervals will be calculated using between and 
within mean squares from an ANOVA model with method (echocardiography, CMR) and 
subject as the main effects and LVEF as the dependent variable.  The ICC will be calculated 
using the SAS macro developed by Hamer (1990).7 
Assessment Across Study Centers:  The mean “absolute value of the difference vs. CMR” 
for each DEFINITY® and unenhanced echocardiography, as well as the difference between 
the two treatments with respect to this mean, will be presented for each study center and 
blinded reader.  Within each blinded reader, an assessment of study center effect on the mean 
treatment difference will be carried out using one-way analysis of variance. For each blinded 
reader, a site difference that is not significant at the 0.15 level of significance, or a site 
difference that is significant but where for every site the mean treatment difference is more 
favorable for DEFINITY® than enhanced echocardiography, will support pooling subjects 
across sites for the primary analysis for that reader. 
Missing Data:  Within each blinded reader, the primary analysis will be performed on 
subjects with non-missing LVEF for DEFINITY®, unenhanced echocardiography, and the 
reference standard.  A supportive analysis will be run where, within each blinded reader, 
missing LVEF is multiply imputed using the fully conditional specification (FCS) multiple 
regression.  The covariates in the imputation model will be fully specified in the formal 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 36 of 49 
statistical analysis plan and will include baseline and demographic variables and will also 
include imaging method (DEFINITY®, unenhanced, or CMR reference standard).  A total of 
50 imputations will be generated; the paired t-test comparing mean bias between 
DEFINITY® and unenhanced will be performed separately on each of the 50 imputed 
datasets, and the t-test results will be combined across datasets using the usual multiple 
imputation techniques to create one overall paired t-test results on imputed data. 
8.2.2 
Other Primary Endpoint(s) 
None 
8.3 
Secondary Endpoint(s) 
8.3.1 
Secondary Efficacy Endpoint(s) 
8.3.1.1 
Improvement in accuracy in left ventricular ejection fraction (LVEF) assessment 
using DEFINITY® contrast-enhanced over unenhanced echocardiography in 
subjects with suboptimal echocardiograms  
The above primary analyses will be repeated here, but on the subgroup of subjects with 
suboptimal echocardiograms.  There will be no imputation of missing data for this analysis. 
8.3.1.2 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography 
The inter-observer variability among each pair of readers within each imaging modality will 
be estimated using an intra-class correlation coefficient (ICC).  The ICC assesses rating 
reliability by comparing the variability of different ratings of the same subject with the total 
variation across all ratings and all subjects. The inter-observer variability in the assessment of 
LVEF between two readers will be determined by percentage of error.  The percentage of 
error will be calculated using the formula: 
Percentage of error = SD between 2 measurements/ mean of the 2 measurements x 100 
The mean percentage of error and its 95% confidence interval will be calculated for each pair 
of readers within each imaging modality.  
There will be no imputation of missing data for this analysis. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 37 of 49 
8.3.1.3 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on 
end-diastolic/systolic volumes.  There will be no imputation of missing data for this analysis. 
8.3.1.4 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in 
subjects with suboptimal echocardiograms 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on 
the subgroup of subjects with suboptimal echocardiograms.  There will be no imputation of 
missing data for this analysis. 
8.3.1.5 
Reduction in inter-reader variability for the assessment of End-Diastolic/Systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms 
The above analyses used to assess inter-reader variability on LVEF in subjects with 
suboptimal echocardiograms will be carried out, but on end-diastolic/systolic volumes.  
There will be no imputation of missing data for this analysis. 
8.3.2 
Other Secondary Endpoint(s) 
None. 
8.4 
Safety Endpoint(s) 
8.4.1 
Primary Safety Endpoint(s) 
Safety analyses will include tabulations of the incidence (number and percent of subjects) of 
treatment-emergent adverse events (TEAEs) and procedure-related AEs by MedDRA system 
organ class (SOC) and preferred term (PT).  This will be repeated for serious adverse events, 
for adverse events leading to study withdrawal and for adverse events at least possibly related 
to study procedures.  This will be performed on the safety population. 
8.4.2 
Secondary Safety Endpoint(s) 
None. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 38 of 49 
8.5 
Demographics and Baseline Characteristics 
Demographic profiles including age, gender, race, ethnicity, height, weight and BMI will be 
summarized for all Safety evaluable subjects.  Descriptive statistics (n, mean, median, 
standard deviation, minimum and maximum) will be provided for each quantitative variable, 
and frequencies and percentages will be provided for each categorical variable. 
8.6 
Interim Analysis 
This study will utilize an adaptive design that allows one interim sample size re-calculation 
after a minimum of 75 subjects have been enrolled and followed. The pre-specified 
maximum allowable adjusted sample size following re-estimation will be 300 enrolled, or 
two times the initial planned sample size of 150 enrolled. The re-estimation of sample size 
will be conducted by an independent biostatistician following a pre-specified plan using the 
method of by Mehta and Pocock (2011).1 Specifically, at the interim stage the conditional 
power (CP) for obtaining a significant beneficial effect of DEFINITY® over unenhanced 
imaging with respect to the primary endpoint will be calculated for each blinded reader, 
using the protocol-specified planned sample size of 150 enrolled subjects (approximately 143 
evaluable subjects assuming 5% premature withdrawal). This conditional power will be 
calculated under the assumption that the interim estimate of DEFINITY® -unenhanced 
difference is the true population difference. Specifically, the calculation of conditional power 
will be as follows: 
Let µD be the true mean of the absolute value of the LVEF differences between the 
DEFINITY®-enhanced echo LVEF and the CMR LVEF, and let µU be the true mean of the 
absolute value of the LVEF differences between the unenhanced echo LVEF and the CMR 
LVEF.  After 75 patients are enrolled and followed (which should lead to approximately 71 
evaluable patients), an unblinded interim analysis will be conducted to determine whether 
sample size should be increased to maintain adequate conditional power of up to 90% for 
each reader. Conditional Power (CP) for rejecting the null hypothesis in favor of 
DEFINITY® by the planned final sample size of 143 evaluable patients is calculated as 
follows for each reader: 
CP = 𝑃(𝑍>
𝐶2√𝑖2−𝑡1√𝐼1−(𝐼2−𝐼1)∆
√𝐼2−𝐼1
) 
where  
a. Z is a random standard random variate 
b. c2 is the t-critical value to be used in the final analysis = 1.97681 (one-sided 0.025 level 
of significance; , with 142 df assuming the final evaluable sample size is 143, which is 
the number of evaluable subjects expected for the final analysis with 150 enrolled 
subjects with 5% premature withdrawal) 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 39 of 49 
c. Δ is the assumption of the true difference “µU minus µD” where µD is the mean of the 
absolute value of the differences between the DEFINITY®-enhanced echo LVEF and the 
CMR LVEF, and µU is the mean of the absolute value of the differences between the 
unenhanced echo LVEF and the CMR LVEF; Δ will be set to the value observed at the 
interim (the calculation of Δ is described below; it is not correct to calculate it as the 
interim point estimate of µU minus the interim point estimate of µD). 
d. I1 is the observed “information” at the interim analysis based on the interim observed 
standard deviation of µD minus µE; its calculation is described below. 
e. I2 is the anticipated “information” at the final analysis; its calculation is described below. 
f. t1 is the interim paired t-statistic testing the null hypothesis, calculated from the data 
observed at the interim 
 
Further information on the above parameters is as follows: 
Assuming the Definity-CMR raw LVEF difference (not absolute value) is normally 
distributed with mean μ and sd σ, then its absolute value, D, follows the folded normal 
distribution with true mean and sd as follows: 
𝜇𝐷= √2
𝜋𝜎𝑒−𝜇2
𝜎2 + 𝜇[1 −2Φ (−𝜇
𝜎)]         𝜎𝐷= √𝜇2 + 𝜎2 −𝜇𝐷
2 
where ϕ is the standard normal cumulative distribution function. A similar algorithm can be 
applied to the unenhanced-CMR LVEF raw difference and its absolute value U to determine 
µU  and σU.  In the above CP formula, Δ will be set to the estimate of µD - µU, where the 
estimates of µU and µD  are each calculated from the above equation using point estimates of 
the relevant μ and σ.  The standard deviation of the difference between the absolute value of 
D minus the absolute value of U is  
√𝜎𝐷
2 + 𝜎𝑈
2 −2 ∗𝐶𝑜𝑣(𝐷, 𝑈) where 2 ∗𝐶𝑜𝑣(𝐷, 𝑈) = 𝜌∗ 𝜎𝐷∗𝜎𝑈  
 
where ρ is the correlation between D and U. This standard will be used in the calculation of 
the “information”, as further described below.  
So, in the CP formula for this example, 
a. Δ will be set to the estimate of µD - µU 
b. I1  = 
1
𝑠2/𝑛1 where s = the interim estimate of the above standard deviation between the 
absolute values D and U 
c. I2  = 
1
𝑠2/𝑛2 where s - the interim estimate of the above standard deviation between the 
absolute values D and U and n2 is the planned final sample size = 143.   


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 40 of 49 
•  
The study will not be stopped for overwhelming efficacy or futility at this interim stage. 
Instead, based on Table 1 of Mehta and Pocock (2011),1 the following algorithm will be 
carried out:  (1) if the CP ≤ 36% for at least two of the three blinded readers based on the 
final sample size, the study will continue to its original planned sample size of 150 enrolled 
subjects; otherwise (2) if the CP ≥ 90% for at least two of the three blinded readers based on 
the final sample size, the study will continue to its original planned sample size of 150 
enrolled subjects; (3) if 36% < CP <90% (the Mehta and Pocock “promising zone”) for at 
least two of the three readers based on the final sample size, then the sample size will be 
increased so that the CP is at least 90% for every reader whose CP fell in the promising zone.  
However, if there is a reader who does not fall in the promising zone, then that reader’s 
primary results will be based only on the patients enrolled under the original protocol-
specified sample size.  Further, for every reader for whom the sample size is increased, that 
reader’s results will only be based on the first X images, where X is the number of images 
that yielded CP of 90%.  The sample size may be increased  up to 300 enrolled subjects 
(twice the protocol-planned sample size) for a given reader in order to obtain 90% CP.  If for 
a given reader that falls into the promising zone, the maximum increase to 300 enrolled 
subjects still does not yield 90% CP, the sample size may still be increased to 300 for this 
reader to maximize conditional power, at the discretion of the independent statistician).  If 
(1), (2), and (3) above are not satisfied, the study will continue to its original planned sample 
size of 150 enrolled subjects. This approach to sample size increase does not inflate the 
overall Type I error for a given reader. 
The sponsor and investigators will remain blinded to the interim results for the duration of 
the ongoing study.  After the interim analysis is carried out, the recommendation made to the 
sponsor will only be to keep sample size as is or to increase enrolled sample size to a given 
value; the reason for the recommendation will not be given to the sponsor. 
8.7 
Determination of Sample Size 
See Section 8.2.1 on the primary efficacy endpoint for the determination of sample size. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 41 of 49 
9 
ADMINISTRATIVE CONSIDERATIONS 
9.1 
Investigators and Study Administrative Structure 
Each Investigator is responsible for conducting the study in accordance with the protocol, all 
applicable laws, regulations and ICH-GCP.   
Lantheus Medical Imaging is the Sponsor of this study and is responsible for overseeing its 
conduct.  Study-specific information regarding the imaging procedures will be described in 
the Imaging Manual. 
9.2 
IRB or IEC Approval 
It is the Investigator’s responsibility to ensure that all aspects of the ethics review are 
conducted in accordance with the current ICH and GCP guidelines, and/or in accordance 
with local laws, whichever provides the greatest level of protection for the study subjects.  
The protocol and any information supplied to the patient to obtain informed consent, 
including written ICF(s), patient recruitment procedures (e.g., advertisements) and written 
information (e.g., information leaflets), must be reviewed and approved by a qualified 
IRB/IEC prior to enrolling subjects in the study.  Before study initiation, the Sponsor must 
receive documentation of the IRB/IEC approval, which specifically identifies the 
study/protocol, and the assurance number or a list of the committee members. 
Amendments to the protocol and revisions to the IC must also be submitted to and approved 
by the IRB/IEC. 
At intervals required by the IRB/IEC, but not less than annually, the Investigator must submit 
to the IRB/IEC a progress report with a request for re-evaluation and re-approval of the 
study.  A copy of the progress report and re-approval of the study must be sent to the 
Sponsor. 
When the Sponsor provides the Investigator with a Safety Report, the Investigator must 
promptly forward a copy to the IRB/IEC. 
After completion or termination of the study, the Investigator must submit a final report to 
the IRB/IEC and forward a copy to the Sponsor.  As part of the record retention requirements 
for the study, the Investigator must maintain documentation of all submissions, 
correspondence and approvals to and from the IRB/IEC. 
9.3 
Ethical Conduct of the Study 
This study will be conducted in conformance with all applicable country or local 
requirements regarding ethical committee review and informed consent using the guidelines 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 42 of 49 
for the protection of the rights and welfare of subjects participating in biomedical research, as 
described in the following documents: 
• US Code of Federal Regulations (CFR) dealing with clinical studies (21 CFR including 
parts 50 and 56 concerning informed consent and IRB US regulations) 
• Good Clinical Practice:  Consolidated Guidance (ICH-E6 R2) 
 
By signing the protocol, the investigator(s) will agree to adhere to the instructions and 
procedures described in it and thereby to adhere to the principles of GCPs to which it 
conforms. 
9.4 
Patient Information and Consent 
Informed consent must be obtained from each patient prior to entering the study, and must 
meet the requirements as defined by the ICH GCP guideline, US Federal Regulations and/or 
conform to local laws, whichever provides the greatest level of protection.  Each patient must 
be provided, in an understandable manner, written and verbal information that describes the 
nature and duration of the study.  Additionally, the patient must be allowed adequate time to 
consider the potential risks and benefits associated with his/her participation in the study.  
The IC must also contain language that allows the Sponsor (or designated representative), 
regulatory authorities, and the IRB/IEC direct access to the patient’s medical records for the 
purpose of review and copying. 
The ICF must be signed and dated by the study patient.  In situations where a study patient is 
not legally competent to provide consent (i.e., a mentally incapacitated patient), written 
consent must be obtained from an authorized guardian or representative.  In these situations, 
the consent form must be signed and dated by a witness and, if possible, assent must be 
obtained from the patient.  The ICF must also be signed and dated by the individual obtaining 
the IC. 
The Investigator, as part of the study documentation, must retain the original signed and 
dated ICF and must provide a signed copy to each patient or authorized representative.  The 
Investigator will not undertake any measures specifically required only for the clinical study until 
valid consent has been obtained. 
9.5 
Patient Confidentiality 
The Sponsor affirms and upholds the principle of the patient’s right to protection against 
invasion of privacy.  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 43 of 49 
9.6 
Study Monitoring 
Each study will be monitored by qualified representatives of the Sponsor.  Monitoring visits 
provide the Sponsor with the opportunity to: evaluate the progress of the study; verify the 
accuracy and completeness of eCRFs; assure that all protocol requirements, applicable laws 
and/or regulations and Investigator obligations are being fulfilled; and resolve any 
inconsistencies in the study records.  The Investigator must allow the Sponsor’s 
representatives direct access to periodically review, at mutually convenient times during the 
study and after the study has been completed, all eCRFs and office, hospital, and laboratory 
records supporting the participation of each patient in the study.  In the case where medical 
records are stored electronically, direct access to the relevant records relating to the clinical 
trial including past medical history must be provided to the study monitor, auditors or other 
representatives of the sponsor.  eCRFs and supporting documentation of the study conduct 
must be kept up-to-date and available for each monitoring visit. 
Deviations from the protocol with regard to patient enrollment, study conduct or in any other 
category will be recorded.  The Investigator is responsible for abiding by the IRB/EC’s rules 
and regulations for reporting protocol deviations.  The Sponsor may adjust the monitoring 
schedule for more frequent visits to allow more prompt collection of the data; provide 
continued training to the study center personnel on an as needed basis; and/or to follow up on 
any issues identified at the study center. 
Details of the monitoring activities will be described in the Clinical Monitoring Plan.   
9.7 
Case Report Forms and Study Records 
Data reflecting the patient’s participation in the study and experiences with the study 
treatment must be reported by the Investigator to the Sponsor.  These data must be recorded 
on eCRFs or other media approved by the Sponsor.  For rules regarding completion and 
correction of eCRFs, see the eCRF completion guidelines that accompany the eCRFs.  
eCRFs must be reviewed, electronically signed and dated by the Investigator or Sub-
investigator and must be submitted in a timely manner to the Sponsor.  Significant delays in 
submission of study data to the sponsor may result in a site being placed on enrollment hold. 
Study documentation includes all eCRFs; Safety Reports received from the Sponsor; SAE 
reports sent to the Sponsor; data correction forms; source documents; monitoring logs; 
Sponsor-Investigator correspondence; protocols and amendments; clinical supplies receipts; 
dispensing and final disposition records; IRB/IEC correspondence and approvals; signed 
consent forms; and Statement of Investigator forms. 
Source documents include all original records of observations, results and activities 
necessary to reconstruct and evaluate the study.  Source documents include, but are not 
limited to, laboratory reports, electrocardiogram tracings, X-ray films, ultrasound 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 44 of 49 
photographs, patient diaries, patient progress notes, hospital charts, appointment books, 
radiology reports or pharmacy records, and any other records or reports of procedures 
performed during the study.  Source documents also may include copies of the eCRF or 
Sponsor supplied worksheets when original information is recorded directly onto these 
forms. 
Whenever possible, an original recording of an observation must be retained as the source 
document.  However, a photocopy of a record is acceptable provided it is legible and is a 
verified copy of the original document. 
9.8 
Data Monitoring Committee 
No DMC will be formed for this study. 
9.9 
Protocol Violations/Deviations 
The Investigator will not intentionally deviate from the protocol without prior written 
agreement from the Sponsor and the IRB/IEC except when necessary to eliminate an 
immediate hazard(s) to study subjects.  The investigator must take all possible steps to avoid 
unintentional deviations.  
All deviations, whether intentional or unintentional, must be documented and submitted to 
the appropriate parties (Sponsor, IRB/IEC, etc.) as required by applicable regulation or local 
practice.  
9.10 Access to Source Documentation 
In compliance with international and federal (US) regulations relative to monitoring clinical 
studies and the fulfillment of its obligation to verify compliance with this protocol, the 
Sponsor will require the investigator to permit the Sponsor’s representatives and, when 
necessary, representatives of the FDA and other regulatory authorities, to review and/or copy 
any or all portions of the patient medical records relevant to this study.  The potential for 
disclosure of patient’s confidential medical information will be included in the informed 
consent form signed by all subjects in the study. 
At some point during the study, individuals from the Sponsor’s Quality group or their 
authorized representative may visit the Investigator’s site to conduct an audit of the study.  
The purpose of this visit will be to determine the Investigator’s adherence to the protocol, 
applicable regulations and the Sponsor’s procedures, in addition to assessing the accuracy of 
the study data.  Before initiating this audit, the Investigator will be contacted by the Sponsor 
to arrange a convenient time for this visit.  The Investigator and staff are expected to 
cooperate with the auditors and allow access to all patient records supporting the eCRFs and 
other study-related documents. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 45 of 49 
9.11 Data Generation and Analysis 
All database and data management activities will be handled by a CRO contracted by LMI 
using qualified and validated database systems.  At scheduled monitoring visits, eCRFs will 
be verified against source documentation and submitted as final data.  Any subsequent 
changes to the eCRFs must be performed in accordance with the CRO’s standard operating 
procedures for editing and clarifying eCRFs.  Data entry will be performed by the sites using 
an electronic data capture (EDC) system.  Comment fields on the eCRFs will be used as a 
means of clarification and communication between the investigator and the sponsor; 
however, comments entered in these fields will not be edited or clarified. 
9.12 Retention of Data 
The Investigator must ensure that all records pertaining to the conduct of the clinical study, 
including signed eCRFs, informed consent forms, drug accountability records, source 
documents and other study documentation are adequately stored for the required time period 
to allow for review and reconstruction of the study.  This documentation must be retained 
for: 
• 2 years following approval of the last marketing application that the data was used to 
support; or 
• 2 years after formal discontinuation of the clinical development program; or 
• a record retention period mandated by any national and/or local laws, regulations, and 
customs. 
 
The Investigator must not destroy any records associated with the study without receiving 
written approval from the Sponsor.  The Investigator must notify the Sponsor in the event of 
accidental loss or destruction of any study records.  If the Investigator leaves the institution 
where the study was conducted, the Sponsor must be contacted to arrange alternative record 
storage options. 
9.13 Financial Disclosure 
All Clinical Investigators participating in clinical studies subject to FDA Regulation 21 CFR 
Part 54 – Financial Disclosure by Clinical Investigators are required before study initiation to 
submit a completed Clinical Investigator Financial Certification/Disclosure Request Form 
that sufficiently details any financial interests and arrangements that apply.  For the purpose 
of this regulation, Clinical Investigator is defined as any Investigator or Sub-investigator who 
is directly involved in the treatment or evaluation of research subjects, including the spouse 
and any dependent child of each Investigator and Sub-investigator.  These requirements 
apply to both US and foreign Clinical Investigators conducting covered clinical studies. 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 46 of 49 
The Clinical Investigators will also be reminded that they must report any changes in their 
financial information regarding significant equity interests and significant payments for the 
duration of the trial, and for a period of 1 year after completion of their participation in the 
covered clinical study.  At the conclusion of the study, the Clinical Investigators may, again, 
be required to submit a completed Clinical Investigator Financial Certification/Disclosure 
Request Form to document any changes in financial interests. 
9.14 Publication and Disclosure Policy 
All information collected during this study will be regarded as confidential.  Investigators 
must obtain written permission from the Sponsor prior to disclosure or publication of data 
obtained during the conduct of this study.  Manuscripts prepared for publication will be 
submitted to the Sponsor for review and comment prior to submission to a publisher, in 
accordance with policies set forth in the Clinical Trail Agreement signed by the Principal 
Investigator.  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 47 of 49 
10 REFERENCES 
1. Foley TA, Mankad SV, Anavekar NS, Bonnichsen CR, Morris MF, Miller TD, et al.  
Measuring left ventricular ejection fraction – techniques and potential pitfalls.  Eur 
Cardiol. 2012;8(2):108114. 
2. Hoffman R, von Bardeleben S, ten Cate F, Borges AC, Kasprzak J, Firschke C, et al.  
Assessment of systolic left ventricular function: A multi-centre comparison of 
cineventriculography, cardiac magnetic resonance imaging, unenhanced and contrast-
enhanced echocardiography.  Eur Heart J. 2005;26:607-616. 
3. Hoffman R, Barletta G, von Bardeleben S, Vanoverschelde JL, Kasprzak J, Greis C, et al.  
Analysis of left ventricular volumes and function:  A multicenter comparison of cardiac 
magnetic resonance imaging, cine ventriculography, and unenhanced and contrast-
enhanced two-dimensional and three-dimensional echocardiography.  J Am Soc 
Echocardiogr. 2014;27:292-301. 
4. ACCF/ACR/AHA/NASCI/SCMR 2010 Expert Consensus Document on CMR. 
Circulation. 2010;121(22):2461-2508. 
5. Schulz-Menger J, Bluemke DA, Bremerich J, Flamm SD, Fogel MA, Friedrich MG, et al.  
Standardized image interpretation and post processing in cardiovascular magnetic 
resonance: Society for Cardiovascular Magnetic Resonance (SCMR) board of trustees 
task force on standardized post processing.  J Cardiovasc Magn Reson. 2013 May 
1;15:35.  
6. Lang RM, Badano LP, Mor-Avi V, Afilalo J, Armstrong A, Ernande L, et al. 
Recommendations for cardiac chamber quantification by echocardiography in adults: an 
update from the American Society of Echocardiography and the European Association of 
Cardiovascular Imaging. J Am Soc Echocardiogr. 2015;28(1):1-39.e14. 
7. Hamer RM.  INTRACC.SAS Macro to Calculate Reliabilities for Intraclass Correlations 
[Internet].  SAS Institute;1990 [cited 15 January 2018].  Available from  
https://github.com/friendly/SAS-macros/blob/master/intracc.sas. 
8. Mehta CR and Pocock SJ.  Adaptive increase in sample size when interim results are 
promising:  A practical guide with examples.  Statist Med. 2011;30:3267-3284. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 48 of 49 
Appendix 1 
Schedule of Procedures 
Study Procedures 
Screening/Baseline1 
Echo Imaging Session 
Day 0 
Telephone Follow-up 
CMR Imaging Session 
Informed consent 
X 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
Medical history 
X 
 
 
 
Physical exam 
X 
 
 
 
Body weight and height 
X 
 
 
 
Vital Signs 
X 
 
 
 
Urine pregnancy test2 
X 
X 
 
 
Resting unenhanced echocardiogram 
 
X 
 
 
DEFINITY® administration and resting 
echocardiogram3 
 
X 
72 ± 24 hours after imaging 
session completion 
Within ±30 days of imaging 
session 
Concomitant medications 
X 
X 
X 
 
Safety assessments (AEs, SAEs)4 
 
 
1Procedures may be conducted up to 7 days prior to administration of study drug (Day 0). 
2A urine pregnancy test will be performed at screening and within 24 hours prior to dosing study drug. 
3Patient will remain at the clinical site for observation until at least 30 minutes after the end of study drug administration. 
4AEs will be recorded from at the time the ICF is signed until the 72 ± 24 hour telephone follow-up. 
 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-314 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 49 of 49 
Appendix 2 
Dose Preparation and Administration Guide 
 

SAP_001.pdf: 
 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 1 of 35 
06 October 2018 
 
 
 
Statistical Analysis Plan 
Lantheus Medical Imaging, Inc.  
 
Protocol: DEF-314  
 
Investigational Product: DEFINITY®  
 
 
A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection Fraction, 
End-Diastolic and End-Systolic Volumes, by Unenhanced and DEFINITY®-
enhanced 2D-Echo and Magnetic Resonance Imaging  
 
 
 
Author(s): 
 Joseph Massaro, Ph.D. 
 
Version Dates: 
Version 1.0 Draft 1: 07 February 2018 
Version 1.0 Draft 2: 12 June 2018 
Version 1.0 Draft 3: 15 August 2018 
Version 1.0 Final:  06 October 2018 
 
Pages: 
35  
 
 
 
 
 
 Confidentiality Statement 
This document is the confidential property of Lantheus Medical Imaging (LMI). No part of it 
may be transmitted, reproduced, published, or used by other persons without the permission 
of LMI. 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 2 of 35 
06 October 2018 
 
 
Contents 
ABBREVIATIONS ....................................................................................................................... 4 
1 
INTRODUCTION................................................................................................................. 5 
2 
STUDY OBJECTIVES......................................................................................................... 6 
2.1 
PRIMARY ENDPOINT AND ANALYSES ................................................................................ 6 
2.2 
SECONDARY ENDPOINTS AND ANALYSES ......................................................................... 6 
2.3 
SAFETY ENDPOINTS .......................................................................................................... 7 
3 
STUDY DESIGN................................................................................................................... 8 
3.1 
DISCUSSION OF STUDY DESIGN........................................................................................ 8 
3.2 
SCHEDULE OF PROCEDURES ........................................................................................... 10 
3.2.1 Screening .................................................................................................................. 11 
3.2.2 Echocardiography Imaging Session ....................................................................... 11 
3.2.3 Telephone Assessment ............................................................................................. 12 
3.2.4 Cardiac Magnetic Resonance Imaging ................................................................... 12 
3.3 
EFFICACY EVALUATIONS ............................................................................................... 12 
3.4 
ADVERSE EVENTS .......................................................................................................... 13 
3.5 
CONCOMITANT MEDICATION ......................................................................................... 13 
3.6 
STUDY ANALYSIS POPULATIONS.................................................................................... 13 
3.6.1 Safety Population ..................................................................................................... 13 
3.6.2 Intent-to-Treat Population....................................................................................... 13 
3.6.3 Modified Intent-to-Treat Population ....................................................................... 13 
3.6.4 Per-Protocol Population .......................................................................................... 14 
3.6.5 Other Population Defined for Tables and Listings................................................. 14 
3.7 
WITHDRAWN SUBJECTS ................................................................................................. 14 
3.8 
RANDOMIZATION ........................................................................................................... 14 
3.9 
BLINDING ....................................................................................................................... 14 
3.10 
SAMPLE SIZE.................................................................................................................. 14 
4 
STATISTICAL METHODOLOGY.................................................................................. 16 
4.1 
PLANNED ANALYSES ..................................................................................................... 16 
4.2 
INTERIM ANALYSIS ........................................................................................................ 16 
4.3 
DISPOSITION OF SUBJECTS ............................................................................................. 16 
4.4 
BASELINE AND DEMOGRAPHIC CHARACTERISTICS ........................................................ 17 
4.5 
EXPOSURE...................................................................................................................... 17 
4.6 
CONCOMITANT MEDICATION ......................................................................................... 17 
4.7 
EFFICACY / PRIMARY AND SECONDARY ANALYSIS ........................................................ 18 
4.7.1 Primary endpoint...................................................................................................... 18 
4.7.2 Primary analysis for the primary endpoint ............................................................. 18 
4.7.3 Other analyses for the primary endpoint ................................................................ 20 
4.7.4 Secondary endpoints ................................................................................................ 21 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 3 of 35 
06 October 2018 
 
 
4.7.5 Methods of analysis for secondary outcomes.......................................................... 22 
4.7.5.1 
Demonstrate improvement in accuracy in LVEF assessment using 
DEFINITY® contrast-enhanced over unenhanced echocardiography in subjects with 
suboptimal echocardiograms............................................................................................. 22 
4.7.5.2 
Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography. ............... 22 
4.7.5.3 
Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. ............................................................................................................. 23 
4.7.5.4 
Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects 
with suboptimal echocardiograms. ................................................................................... 23 
4.7.5.5 
Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms..................................... 23 
4.8 
SAFETY ANALYSIS ......................................................................................................... 23 
4.8.1 Adverse events .......................................................................................................... 23 
4.8.2 Vital Signs ................................................................................................................ 25 
4.8.3 Physical exam........................................................................................................... 26 
4.8.4 Study Center effects.................................................................................................. 26 
4.9 
PROTOCOL VIOLATIONS ................................................................................................. 26 
4.9.1 Violation criteria ...................................................................................................... 26 
4.9.2 Protocol violations.................................................................................................... 26 
4.10 
MISSING VALUES AND MISSING VISIT DATES................................................................ 27 
4.11 
DEVIATIONS FROM SAP ................................................................................................. 28 
4.12 
CHANGES IN CONDUCT OR PLANNED ANALYSES FROM THE PROTOCOL ........................ 28 
4.13 
ALGORITHMS/SAS CODES ............................................................................................. 29 
5 
TABLES AND LISTINGS ................................................................................................. 30 
5.1 
TABLE FORMAT ............................................................................................................. 30 
5.2 
CONVENTIONS................................................................................................................ 30 
5.3 
TABLES .......................................................................................................................... 31 
5.3.1 Section 14.1: Demographic and baseline ................................................................ 31 
5.3.2 Section 14.2: Primary .............................................................................................. 31 
5.3.3 Section 14.2: Secondary........................................................................................... 31 
5.3.4 Section 14.3: Safety .................................................................................................. 32 
5.3.4.1 
Adverse events .................................................................................................. 32 
5.3.4.2 
Vital signs ......................................................................................................... 32 
5.4 
FIGURES ......................................................................................................................... 33 
5.4 
LISTINGS ........................................................................................................................ 34 
6 
REFERENCE LIST ............................................................................................................ 35 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 4 of 35 
06 October 2018 
 
 
Abbreviations 
 
A2C 
Apical 2-chamber 
A3C 
Apical 3-chamber 
A4C 
Apical 4-chamber 
AE 
Adverse Event 
BMI 
Body Mass Index 
CI 
Confidence Interval 
cm 
Centimeters 
CP 
Conditional Power 
CMR 
Cardiac Magnetic Resonance Imaging 
CRF 
Case Report Form 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
HEENT 
Head, Eyes, Ears, Nose, and Throat 
ITT 
Intent-to-Treat 
Kg 
Kilogram 
LVEF 
Left Ventricular Ejection Fraction 
MedDRA  
Medical Dictionary for Regulatory Activities  
m 
Meter 
MI 
Medical Imaging 
mITT 
Modified Intent-to-Treat 
mL 
Millilitre 
MRI 
Magnetic Resonance Imaging 
MUGA scan 
Multiple-gated acquisition scan 
PP 
Per-Protocol 
PT 
Preferred Term 
RMSE 
Root Mean Square Error 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SP 
Safety Population 
SD 
Standard Deviation 
SOC 
System Organ Class 
TEAE 
Treatment Emergent Adverse Event 
WHO 
World Health Organization 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 5 of 35 
06 October 2018 
 
 
1 Introduction 
This document presents the statistical analysis plan (SAP) for Lantheus Medical Imaging (LMI) 
Protocol No. DEF-314:  A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection 
Fraction, End-Diastolic and End-Systolic Volumes, by Unenhanced and DEFINITY®-enhanced 
2D-Echo and Magnetic Resonance Imaging.  
This analysis plan is based on the protocol dated 15 August 2018.  
The purpose of the SAP for this study is to provide a framework in which answers to the 
protocols’ objectives may be achieved in a statistically rigorous fashion, without bias or 
analytical deficiencies.  Specifically, this SAP has the following purpose: To prospectively (a 
priori) outline the types of analyses and presentations of data that will form the basis for 
conclusions to be reached that will answer the studies’ objectives outlined in the protocol, and to 
explain in detail how the data will be handled and analyzed, adhering to commonly accepted 
standards and practices of biostatistical analysis in the clinical trial industry. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 6 of 35 
06 October 2018 
 
 
2 Study Objectives 
The objectives of this study are: 
Primary objective: 
• Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography. 
Secondary objectives: 
• Demonstrate improvement in accuracy in LVEF assessment using DEFINITY® contrast-
enhanced over unenhanced echocardiography in subjects with suboptimal 
echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
2.1 Primary endpoint and analyses 
The primary endpoint of this study is left ventricular ejection fraction (LVEF) accuracy.  The 
primary analysis is to compare LVEF accuracy from unenhanced imaging to imaging with 
DEFINITY® contrast enhancement using cardiac magnetic resonance imaging (CMR) as the 
truth standard. 
 
2.2 Secondary endpoints and analyses 
The secondary endpoints and analyses of this study are: 
• Demonstrate improvement in accuracy in LVEF assessment using DEFINITY® contrast-
enhanced over unenhanced echocardiography in subjects with suboptimal 
echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 7 of 35 
06 October 2018 
 
 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
2.3 Safety endpoints 
Enrolled subjects will be followed for adverse events (AEs), serious adverse events (SAEs) and 
changes in concomitant medications from the time the Informed Consent (IC) is signed through 
72±24 hours after completion of DEFINITY® administration. 
 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 8 of 35 
06 October 2018 
 
 
3 Study Design 
 
3.1 Discussion of Study Design 
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF measurement 
accuracy and reproducibility of DEFINITY® contrast-enhanced and unenhanced 
echocardiography as compared with non-contrast cardiac magnetic resonance imaging (CMR) 
used as the truth standard.  Approximately one-hundred fifty (150) subjects will be enrolled over 
approximately 10 months at approximately 10 centers located in the United States.  Subjects will 
undergo unenhanced and DEFINITY®-enhanced echocardiograms and CMR.  The study 
population will consist of male and female subjects 18 years of age or older.   
Subjects will be screened for enrollment if they have undergone a 2D echocardiogram with or 
without contrast or other methods (e.g. CMR, MUGA scan) within 6 months prior to enrollment 
(Day 0).  Subjects will be stratified to achieve an even distribution within four pre-defined LVEF 
groups (>50, 41-50, 30–40, <30%).  Subjects with optimal and sub-optimal echocardiograms 
(based on the investigator opinion) will be enrolled.  An echocardiogram is considered sub-
optimal if 2 or more segments of the ventricular border are classified as not adequately 
visualized. 
Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® contrast-
enhanced examination on the same day.  A minimum of 360 seconds of images will be collected 
during both the unenhanced and the DEFINITY® contrast-enhanced examinations.   
Subjects will remain at the clinical site for at least 30 minutes of observation after the end of the 
administration of DEFINITY®.  A safety follow-up telephone call will be conducted for all 
subjects at approximately 72±24 hours after completion of the imaging sessions.   
Enrolled subjects will be followed for adverse events (AEs), serious adverse events (SAEs), and 
changes in concomitant medications from the time the Informed Consent is signed through the 
safety follow-up telephone call.   
Unenhanced and DEFINITY®-enhanced echocardiograms will be performed with standard 
apical 2 chamber, apical 3 chamber, and apical 4 chamber (A2C, A3C, and A4C, respectively) 
views using harmonic imaging.  Images will be recorded in standard digital format, masked to 
subject identifiers, and sent to a central imaging core laboratory for analysis.  There, 3 
experienced independent blinded readers will interpret the results according to the Image Review 
Charter. Additional analyses will also be performed as described in the Image Review Charter.  
Each reader’s independent interpretation will be recorded in a database. 
CMR images will also be collected and independently read by 3 experienced blinded readers at a 
central imaging core laboratory. 
The figure below displays an overview of study events.  The table that follows is a schedule of 
events: 
 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 9 of 35 
06 October 2018 
 
 
All screening assessments will occur within 7 days prior to enrollment/DEFINITY® 
administration (Day 0).  CMR studies will occur within ± 30 days of DEFINITY® 
administration.  Safety monitoring will continue up to 72±24 hours post-DEFINITY® 
administration.  The expected duration of subject participation is not more than 41 days.  
Subjects will be enrolled over a 10-month period. 
 
Figure: Schedule of Events 
 
 
 
LVEF measurement obtained via 2D Echo or other methods (e.g. 
CMR, MUGA scan) within 6 months prior to enrollment Day 0  
Screening 
Days -7 to Day 0 
Echocardiograms & DEFINITY® 
administration  
Day 0 
 
Safety telephone follow-
up 
72 ± 24 hours 
CMR within ±30 days of Day 0 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 10 of 35 
06 October 2018 
 
 
3.2 Schedule of Procedures 
 
Study Procedures 
Screening/Baseline1 
Echo Imaging Session 
Day 0 
Telephone Follow-up 
CMR Imaging Session 
Informed consent 
X 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
Medical history 
X 
 
 
 
Physical exam 
X 
 
 
 
Body weight and height 
X 
 
 
 
Vital Signs 
X 
 
 
 
Urine pregnancy test2 
X 
X 
 
 
Resting unenhanced echocardiogram 
 
X 
 
 
DEFINITY® administration and 
resting echocardiogram3 
 
X 
72 ± 24 hours after 
imaging session 
completion 
Within ±30 days of 
imaging session 
Concomitant medications 
X 
X 
X 
 
Safety assessments (AEs, SAEs)4 
 
 
1Procedures may be conducted up to 7 days prior to administration of study drug (Day 0). 
2A urine pregnancy test will be performed at screening and within 24 hours prior to dosing study drug. 
3Patient will remain at the clinical site for observation until at least 30 minutes after the end of study drug administration. 
4AEs will be recorded from at the time the ICF is signed until the 72 ± 24 hour telephone follow-up. 
 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 11 of 35 
06 October 2018 
 
 
3.2.1 Screening 
All Screening/Baseline assessments will occur within 7 days prior to administration of study 
drug (Day 0); 
• Determine eligibility according to the inclusion/exclusion criteria; 
• Informed consent has been obtained; 
• General medical history has been obtained;   
• Concomitant medications collected; 
• Physical examination: A physical examination will be performed at screening and will 
include height (cm), weight (kg), general appearance, and normal/abnormal assessment 
and description of abnormalities for body systems (head, ears, eyes, nose and throat 
[HEENT]); neck; cardiovascular; lungs; abdomen; lymph nodes; extremities; 
neurological; skin; musculoskeletal; and other).  All physical examination abnormalities 
will be recorded on the electronic case report form (eCRF); 
• Vitals signs including heart rate, respiratory rate and systolic and diastolic blood pressure 
will be collected at the Screening visit only; 
•  Urine pregnancy test:  A urine sample for pregnancy testing will be obtained for all 
women of childbearing potential; 
• Adverse event collection. 
 
3.2.2 Echocardiography Imaging Session 
Subjects providing informed consent and meeting the inclusion criteria and none of the exclusion 
criteria will next undergo the following assessments during the Echocardiography Imaging 
Session: 
•  Urine pregnancy test:  A urine sample for pregnancy testing will be obtained for all 
women of childbearing potential within 24 hours prior to dosing the study drug. 
• A bedside resting transthoracic echocardiogram will be performed according to 
standardized instructions provided in the protocol and in the Study Imaging Manual.   
• After unenhanced imaging has been conducted, the study drug (DEFINITY®) will be 
administered as described in the Dose Preparation and Administration Guide.   
• After study drug administration and subsequent image optimization, the same A2C, A3C, 
and A4C views will be obtained as instructed in the Study Imaging Manual. 
• Subjects will remain at the site for observation until at least 30 minutes after the end of 
the administration of study drug.   
• Concomitant Medications collected. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 12 of 35 
06 October 2018 
 
 
• Adverse event collection. 
 
3.2.3 Telephone Assessment 
• A safety follow-up telephone call will be conducted for all subjects at approximately 
72±24 hours after completion of the imaging sessions.  Concomitant medications and 
adverse events will be solicited on this call. 
 
3.2.4 Cardiac Magnetic Resonance Imaging 
• Within +/- 30 days of imaging session, subjects will receive a non-contrast enhanced 
CMR examination as described in the Study Imaging Manual.  
Instructions for MRI data transfer and storage at the central imaging core laboratory is 
provided in the Study Imaging Manual. 
 
3.3 Efficacy Evaluations 
Three blinded independent readers will perform all efficacy assessments following the 
methodology described in the Imaging Review Charter.  Analyses discussed below will be 
conducted separately for each reader. 
The primary endpoint of this study is left ventricular ejection fraction (LVEF) accuracy.  The 
primary analysis is to compare LVEF accuracy from unenhanced imaging to imaging with 
DEFINITY® contrast enhancement using cardiac magnetic resonance imaging (CMR) as the 
truth standard. 
The secondary endpoints and analyses of this study are: 
• Demonstrate improvement in accuracy in LVEF assessment using DEFINITY® contrast-
enhanced over unenhanced echocardiography in subjects with suboptimal 
echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 13 of 35 
06 October 2018 
 
 
3.4 Adverse Events 
An AE is defined as any new untoward medical occurrence or worsening in severity or 
frequency of a pre-existing medical condition in a study patient, which does not necessarily have 
a causal relationship with investigational product.  AEs occurring from the time the informed 
consent is signed until the 72±24 hour telephone follow-up will be reported.  Included in the AE 
data collection are start date and time, severity (Mild/ Moderate/ Severe), relationship (Not 
Related/ Related to DEFINITY®/ Related to Study Procedure), outcome (Resolved/ Resolved 
with Sequelae/ Ongoing/ Death/ Unknown), end date and time, treatment action taken (Yes/No), 
serious (Yes/ No), resulted in death and date of death if applicable, is life threatening, requires 
hospitalization or prolonged hospitalization, results in persistent or significant 
disability/incapacity, is a congenital anomaly/birth defect, other significant medical event). 
 
3.5 Concomitant Medication 
All medications (over the counter or prescription only medication) are permitted during this 
study at the discretion of the Investigator and will be captured in the database.  Enrolled subjects 
will be followed for changes in concomitant medication use from the time the Informed Consent 
is signed through 72±24 hours after completion of DEFINITY® administration.  Included in data 
collection are the indication, dose, dose units, frequency, route, start date, stop date or indicator 
of ongoing at end of study. 
 
3.6 Study Analysis Populations 
The following analysis populations are defined: 
 
3.6.1 Safety Population 
The Safety Population (SP) will include all subjects who have signed informed consent and who 
have received any amount of DEFINITY® in the study. This is the primary analysis population 
for the safety analysis. 
 
3.6.2 Intent-to-Treat Population 
The Intent-to-Treat (ITT) will include all subjects who have signed informed consent. 
 
3.6.3 Modified Intent-to-Treat Population 
The Modified Intent-to-Treat (mITT) population will include all ITT subjects who complete 
unenhanced imaging and DEFINITY® enhanced imaging.  This is the primary analysis 
population for the efficacy endpoints. 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 14 of 35 
06 October 2018 
 
 
3.6.4 Per-Protocol Population 
The Per-Protocol (PP) population will include modified ITT subjects who a) did not violate 
inclusion and exclusion criteria that would likely have an effect on the primary outcome, b) do 
not have major protocol violations; c) have LVEF data on both unenhanced and DEFINITY®- 
enhanced images for at least one reader; (d) have CMR LVEF.  
Efficacy analyses will be conducted for both modified ITT and PP population sets, differences in 
results using the two populations will be carefully examined descriptively.  
 
3.6.5 Other Population Defined for Tables and Listings 
For the purposes of tables and listings population includes: 
• All screened subjects. 
 
3.7 Withdrawn Subjects 
The investigator may withdraw a subject from the study for any of the following reasons: 
• Subject withdraws consent. 
• Subject is lost to follow up. 
• Subject has an AE that, in the opinion of the Investigator, requires the subject’s 
discontinuation. 
• Discretion of the Investigator. 
• The Sponsor or Investigator terminates the study.  
 
All events that result in discontinuation of study treatment will be appropriately recorded and 
reported.  In addition, for all subjects who discontinue prematurely, an evaluation which reflects 
the status of the patient at premature termination, along with a final assessment and the reasons 
for termination, will be recorded on the eCRF. 
 
3.8 Randomization 
There is no randomization for this trial as all subjects will be receiving DEFINITY®. 
 
3.9 Blinding 
Blinding to treatment is not applicable for this single arm open-label trial.    
 
3.10 Sample Size 
The primary analysis is to demonstrate an improvement in LVEF accuracy from unenhanced 
imaging to imaging with DEFINITY® contrast enhancement using CMR as the truth standard.   


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 15 of 35 
06 October 2018 
 
 
For each patient, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF 
will be calculated.  Similarly, the absolute value of the difference of unenhanced imaging LVEF 
minus CMR LVEF will be calculated for each patient.  The primary analysis is to assess the 
significance of the difference between DEFINITY® and unenhanced echo with respect to the 
mean “absolute value of the difference vs. CMR”.  These means will be compared with a paired 
t-test at a two-sided 0.05 level of significance.  Specifically, the null and alternative hypotheses 
are: 
 
H0: µD = µU vs. H1: µD ≠ µU 
where µD and µU are the mean of the DEFINITY® unenhanced echo absolute value of the 
difference vs.  CMRs, retrospectively.  A sample size of 150 enrolled subjects has 90% power to 
reject the null hypothesis in favor of the alternative if the true difference µD - µU is at least 2.75 
(in favor of DEFINITY®) with a standard deviation of 10 or less and allows for approximately 
5% premature withdrawal.  Power was calculated using the PASS 15 software. 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 16 of 35 
06 October 2018 
 
 
4 Statistical Methodology 
 
4.1 Planned Analyses 
A contract research organization (CRO) will be responsible for data management and statistical 
analysis.  All statistical analyses will be performed using SAS (SAS Institute, Inc., Cary, NC) 
version 9.4 or higher.   Patient data listings and tabular presentations of results will be provided.  
Presentation of summary statistics for continuous variables will include N, mean, median, and 
standard deviation, as well as the minimum and maximum values.  For categorical variables, the 
number and percent of subjects in each category will be calculated; the number of subjects with 
missing data will be presented under a “Missing” category. Unless otherwise stated, subjects 
with missing values will be included in the denominator count when computing percentages.  
Results will be presented to 1 decimal place when applicable.  All statistical tests will be two-
sided employing a significance level of 5% unless otherwise specified.  Further details of the 
criteria and conduct of the statistical analyses are below. 
Demographic and efficacy analyses will be carried out using the modified intent-to-treat (mITT) 
population as the main analysis population; it will also be carried out on the per-protocol (PP) 
population.  The safety analysis will be carried out on the Safety Population (SP). 
The primary analysis will be performed on subjects with non-missing LVEF for DEFINITY®-
enhanced echocardiography imaging, unenhanced imaging, and the truth standard, cardiac 
magnetic resonance.  A supportive analysis will be run where missing LVEF is multiply imputed 
using the fully conditional specification (FCS) multiple regression, as detailed below.  
Otherwise, there will be no imputation of missing data.   
 
4.2 Interim Analysis 
This study will utilize an adaptive design that allows one interim sample size re-estimation after 
a minimum of 75 subjects have been enrolled and imaged. Further details are described below in 
the section describing the primary analysis. 
 
4.3 Disposition of Subjects 
Subject disposition data will include the number and percentage of ITT subjects who were 
enrolled in the trial, who received DEFINITY®, who completed the trial, and who discontinued 
the trial will be presented.  The number and percentage of ITT subjects who discontinued the 
trial will also be presented by reason of discontinuation.  The number and percentage of subjects 
in each analysis population will be presented.  All percentages will be based on the total number 
of ITT subjects.   
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 17 of 35 
06 October 2018 
 
 
4.4 Baseline and Demographic Characteristics 
All baseline and demographic characteristics will be summarized overall. This will include age at 
Screening, age group at Screening, gender, ethnicity, race, height, weight, body mass index 
(BMI), and active and past medical history. Descriptive statistics will be provided for each 
quantitative (continuous) variable; frequencies and percentages of subjects will be provided for 
each qualitative variable.  
Notes:  
• Age will be calculated as (year of screening date – year of birth) and presented to 1 
decimal place. No rounding will be carried out prior to summarising age. 
• Age group will be categorized as < 65 years and ≥ 65 years.  
• BMI will be calculated as weight (kg)/height2(m). 
• Medical history data will be collected by body system for all enrolled subjects.  The 
details of history by body system are collected as open text. Medical history will be 
coded according to Medical Dictionary for Regulatory Activities (MedDRA) version 
20.0; the number and percentage of subjects within each system organ class (SOC) and 
preferred term (PT) will be presented; a patient experiencing a medical history within 
more than one SOC or PT will be counted only once within that SOC and PT, 
respectively. 
• Active medical history is defined as histories marked as ongoing at time of screening. 
• Past medical histories are defined as histories marked as resolved at time of screening 
(ongoing not selected). 
 
4.5 Exposure 
DEFINITY® will be administered as a diluted bolus injection.  1.3 mL of activated DEFINITY® 
will be diluted with 8.7 mL of preservative-free saline to evenly distribute microspheres.  An 
initial injection of up to 3 mL of diluted DEFINITY® will be administered with subsequent 
injections of 1 to 2 mL, as needed.  Information about the administration of DEFINITY® is 
collected in the eCRF.   Exposure to DEFINITY® study drug will be summarized as described 
below: 
The number and percentage of ITT subjects who are administered DEFINITY® will be 
presented.  Summary statistics of the dose administered (mL) will be summarized for the safety 
population. 
 
4.6 Concomitant Medication 
Medications are coded using World Health Organization (WHO) Drug dictionary, version Q3-
2016.  The number and percentage of subjects with prior and concomitant medication will be 
presented overall by, WHO therapeutic area and WHO preferred drug name.  Prior medications 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 18 of 35 
06 October 2018 
 
 
are those that started and stopped before exposure to contrast agents; concomitant medications 
are all medications taken during the study period, including those started before but on going at 
first administration. 
Medications for each subject will be assigned as prior or concomitant by comparing the start and 
stop date of medication and the date of each dose.   Where a medication start date is partially or 
fully missing, and it is unclear as to whether the medication is prior or concomitant, it will be 
assumed that it is concomitant.   
 
4.7 Efficacy / Primary and Secondary Analysis 
All efficacy parameters will be summarized and presented in tables based on the Modified 
Intention-to-Treat population and reanalysed using the Per Protocol population. 
 
4.7.1 Primary endpoint 
The primary endpoint of this study is LVEF accuracy.  The primary analysis is to compare LVEF 
accuracy from unenhanced imaging to that derived from imaging with DEFINITY® contrast 
enhancement using cardiac magnetic resonance imaging (CMR) as the truth standard. 
 
4.7.2 Primary analysis for the primary endpoint 
The primary analysis is to demonstrate an improvement in LVEF accuracy from unenhanced 
imaging to that derived from imaging with DEFINITY® contrast enhancement using CMR as 
the truth standard.   
For each patient, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF 
will be calculated.  Similarly, the absolute value of the difference of unenhanced imaging LVEF 
minus CMR LVEF will be calculated for each patient.  The primary analysis is to assess the 
significance of the difference between DEFINITY® and unenhanced echo with respect to the 
mean “absolute value of the difference vs. CMR”.  Specifically, the null and alternative 
hypotheses are: 
 H0: µD = µU vs. H1: µD ≠ µU 
or i.e.,  
H0: µU - µD =0 vs. H1: µU - µD ≠ 0 
where µD and the µU are the mean of the DEFINITY® and unenhanced echo absolute value of 
the difference vs. CMR, retrospectively.   The null hypothesis will be tested at a two-sided 0.05 
level of significance using a paired t-test derived from the sample point estimates of the mean 
and standard deviation of the difference between unenhanced echo’s and DEFINITY’s® 
“absolute value of the difference vs. CMR”.    


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 19 of 35 
06 October 2018 
 
 
The analyses will be conducted separately for each of the three blinded readers of the 
DEFINITY® enhanced and unenhanced images; the criterion for success is that the null 
hypothesis is rejected in favor of DEFINITY® for at least 2 of the 3 blinded readers for all 
subjects.  CMR LVEF is interpreted by a single reader; this is the CMR LVEF that will be used 
as the comparator for each of the three blinded readers.  
The primary analysis will be performed on mITT subjects with non-missing LVEF for 
DEFINITY®, unenhanced echocardiography, and the truth standard.  A supportive analysis will 
be run where a missing echocardiography or CMR LVEF is multiply imputed using the fully 
conditional specification (FCS) multiple regression.  
Interim Analysis: This study will utilize an adaptive design that allows one interim sample size 
re-calculation after a minimum of 75 subjects have been enrolled and followed. The pre-
specified maximum allowable adjusted sample size following re-estimation will be 300 enrolled, 
or two times the initial planned sample size of 150 enrolled. The re-estimation of sample size 
will be conducted by an independent biostatistician following a pre-specified plan using the 
method of by Mehta and Pocock (2011).1 Specifically, at the interim stage, the conditional power 
(CP) for obtaining a significant beneficial effect of DEFINITY® over unenhanced imaging with 
respect to the primary endpoint will be calculated for each blinded reader, using the protocol-
specified planned sample size of 150 enrolled subjects (approximately 143 evaluable subjects 
assuming 5% premature withdrawal). This conditional power will be calculated under the 
assumption that the interim estimate of the mean unenhanced- DEFINITY® difference in LVEF 
accuracy is the true population mean difference. Specifically, the calculation of conditional 
power and sample size increase will be as follows: 
Let µD be the true mean of the absolute value of the differences between the DEFINITY®-
enhanced echo LVEF and the CMR LVEF, and let µU be the true mean of the absolute value of 
the differences between the unenhanced echo LVEF and the CMR LVEF.   After 75 patients are 
enrolled and followed (which should lead to approximately 71 evaluable patients), an unblinded 
interim analysis will be conducted to determine whether sample size should be increased to 
maintain adequate conditional power of up to 90% for each reader. Conditional Power (CP) for 
rejecting the null hypothesis in favor of DEFINITY® by the planned final sample size of 143 
evaluable patients is calculated as follows for each reader: 
CP = 𝑃𝑃൬𝑍𝑍>
𝐶𝐶2ඥ𝑖𝑖2−𝑡𝑡1ඥ𝐼𝐼1−(𝐼𝐼2−𝐼𝐼1)∆
ඥ𝐼𝐼2−𝐼𝐼1
൰ 
where  
a. Z is a random standard random variate 
b. c2 is the t-critical value to be used in the final analysis = 1.97681 (one-sided 0.025 level of 
significance; with 142 df assuming the final evaluable sample size is 143, which is the 
number of evaluable subjects expected for the final analysis with 150 enrolled subjects 
with 5% premature withdrawal) 
c. Δ is the assumption of the true difference “µU minus µD” where µD is the mean of the 
absolute value of the differences between the DEFINITY®-enhanced echo LVEF and the 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 20 of 35 
06 October 2018 
 
 
CMR LVEF, and µU is the mean of the absolute value of the differences between the 
unenhanced echo LVEF and the CMR LVEF; Δ will be set to the interim sample’s point 
estimate of µU minus µD (i.e., Δ will be set to 𝑋𝑋ത𝑈𝑈−𝑋𝑋ത𝐷𝐷, where 𝑋𝑋ത𝑈𝑈 and 𝑋𝑋ത𝐷𝐷 are the sample 
mean point estimates of µU and µD, respectively).  
d. I1 is the observed “information” at the interim analysis; specifically, I1  = 
1
𝑠𝑠2/𝑛𝑛1 where s = 
the interim sample point estimate of the standard deviation of the difference between the 
unenhanced and DEFINITY® echo’s “absolute value of the difference vs. CMR”  and n1 
is the interim sample size. 
e. I2 is the anticipated “information” at the final analysis; specifically, I2  = 
1
𝑠𝑠2/𝑛𝑛2 where s is as 
defined in item d directly above and n2 is the planned final evaluable sample size = 143.   
f. t1 is the interim paired t-statistic testing the null hypothesis, derived from the interim 
sample point estimates of the mean and standard deviation of the difference between the 
unenhanced and DEFINITY® echo’s “absolute value of the difference vs. CMR”.  
The study will not be stopped for overwhelming efficacy or futility at this interim stage. Instead, 
based on Table 1 of Mehta and Pocock (2011),1 the following algorithm will be carried out:  
• If 36% < CP < 90% for all three readers: Let n be the total protocol-specified enrolled sample 
size.  Assume na is the total enrolled sample size for reader 1 to reach 90% CP, nb is the total 
enrolled sample size for reader 2 to reach 90% CP, and nc is the total enrolled sample size for 
reader 3 to reach 90% CP. The final total number of enrolled subjects is min(max(na, nb, nc), 
2n). Each reader’s final analysis will be based on min(max(na, nb, nc), 2n) enrolled subjects. 
• If 36% < CP < 90% for exactly two out of three readers, for example reader 1 and reader 2 
where and reader 3’s CP is not in the promising zone. The final total number of enrolled 
subjects will be the sample size with which both readers have more than 90% CP but not 
exceeding 2n, i.e. min(max(na, nb), 2n). Each reader’s final analysis will be based on this 
min(max(na, nb), 2n) enrolled subjects. 
The number of enrolled subjects is not changed from the final protocol-specified sample size in 
any other cases. That is, in all such other cases, each reader’s final analysis will be based on the n 
enrolled subjects. 
The sponsor and investigators will remain blinded to the interim results for the duration of the 
ongoing study.  After the interim analysis is carried out, the recommendation made to the 
sponsor will only be to keep sample size as is or to increase enrolled sample size to a given 
value; the reason for the recommendation will not be given to the sponsor. 
 
4.7.3 Other analyses for the primary endpoint 
The following will be carried out separately for each of the three blinded readers without 
imputation of missing LVEF. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 21 of 35 
06 October 2018 
 
 
Bias is the mean of the actual (not absolute value) per-subject differences between the imaged 
LVEF and CMR LVEF.  The bias, the two-sided 95% confidence interval of the bias, the 
precision (standard deviation of the per-subject differences) and the root mean square error 
(RMSE) will be calculated for the LVEF derived from the DEFINITY®-enhanced images.  The 
RMSE is the square root of the bias-squared + precision-squared and is considered a measure of 
overall accuracy.  These analyses will be repeated for LVEF values derived from the unenhanced 
echocardiography vs. those from CMR.   It is anticipated that the RMSE will be smaller for 
DEFINITY®-enhanced LVEF than for unenhanced LVEF.   
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for 
measurement accuracy against the reference CMR using Bland-Altman analysis and Deming 
regression analysis.  For the Bland-Altman analysis, a plot of the per-subject actual difference 
between DEFINITY®-enhanced echocardiography and CMR LVEF will be plotted vs. the per-
subject sum of the two measurements. Limits of agreement (defined as the mean of the 
DEFINITY® vs. CMR difference) +/- 2 standard deviations (SDs) will be shown on the plot.  
The same analyses will be performed for unenhanced echocardiography using CMR as the 
reference standard.   
Deming regression plots of LVEF vs. CMR LVEF will be generated for each of DEFINITY®-
enhanced and unenhanced echocardiography.  Unweighted Deming regression will be employed 
to estimate the regression slope and intercept with two-sided 95% confidence intervals of each 
assuming the measurement error is the same for CMR and each of the echocardiography error 
techniques. 
The intra-class correlation coefficient (ICC) and its two-sided 95% confidence interval (CI) will 
be calculated for DEFINITY®-enhanced echo vs. CMR and for unenhanced echo vs. CMR.  The 
ICCs and their two-sided confidence intervals will be calculated using between and within mean 
squares from an ANOVA model with method (echocardiography, CMR) and subject as the main 
effects and LVEF as the dependent variable.  The ICC will be calculated using the SAS macro 
developed by Hamer (1990).2  
 
4.7.4 Secondary endpoints 
The secondary endpoints and analyses of this study are: 
• Demonstrate improvement in accuracy in LVEF assessment using DEFINITY® contrast-
enhanced over unenhanced echocardiography in subjects with suboptimal 
echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 22 of 35 
06 October 2018 
 
 
• Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
• Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
4.7.5 Methods of analysis for secondary outcomes 
 
4.7.5.1 Demonstrate improvement in accuracy in LVEF assessment using 
DEFINITY® contrast-enhanced over unenhanced echocardiography in 
subjects with suboptimal echocardiograms 
The above primary endpoint analyses will be repeated in the subset of subjects with suboptimal 
echocardiograms.  The study is not powered to reject the primary endpoint null hypothesis in 
Section 4.7.2, so the focus is more on the estimate of µD - µU, on Bland-Altman plots and on the 
Deming regression for each blinded reader.   There will be no imputation of missing LVEF data 
for this analysis. 
 
4.7.5.2 Demonstrate a reduction in inter-reader variability for the assessment of 
LVEF 
using 
DEFINITY® 
contrast-enhanced 
versus 
unenhanced 
echocardiography. 
 
The inter-reader variability among each pair of readers within each imaging modality will be 
estimated using an intra-class correlation coefficient (ICC) and its two sided 95% CI.  The ICC 
assesses rating reliability by comparing the variability of different ratings of the same subject with 
the total variation across all ratings and all subjects. The inter-observer variability in the 
assessment of LVEF between two readers will be determined by percentage of error.  The 
percentage of error will be calculated using the formula: 
Percentage of error = SD between 2 measurements/mean of the 2 measurements x 100 
The mean percentage of error and its 95% confidence interval will be calculated for each pair of 
readers within each imaging modality.  
There will be no imputation of missing data for this analysis.   The pairwise ICCs and percentages 
of error differences will be compared descriptively between contrast-enhanced and unenhanced 
echocardiography. 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 23 of 35 
06 October 2018 
 
 
4.7.5.3 Demonstrate a reduction in inter-reader variability for the assessment of 
end-diastolic/systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography. 
 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on end-
diastolic/systolic volumes.  There will be no imputation of missing data for this analysis.  
 
4.7.5.4 Demonstrate a reduction in inter-reader variability for the assessment of 
LVEF using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on the 
subgroup of subjects with suboptimal echocardiograms.  There will be no imputation of missing 
data for this analysis. 
 
4.7.5.5 Demonstrate a reduction in inter-reader variability for the assessment of 
end-diastolic/systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms. 
 
The above analyses used to assess inter-reader variability on LVEF in subjects with suboptimal 
echocardiograms will be carried out, but on end-diastolic/systolic volumes.  There will be no 
imputation of missing data for this analysis. 
 
4.8 Safety Analysis 
The safety analyses that are described below will be carried out on the Safety Population. 
 
4.8.1 Adverse events 
A treatment emergent adverse event (TEAE) is an adverse event that started or worsened in 
severity following the start of administration of DEFINITY®. A table of adverse events will be 
summarized including: 
• Number of subjects having experienced at least one TEAE, 
• Number of subjects having experienced at least one TEAE related to the DEFINITY®, 
• Number of subjects having experienced at least one TEAE related to a  study procedure, 
• Number of subjects having experienced at least one severe TEAE, 
• Number of subjects definitively removed from the contrast agent due to a TEAE, 
• Number of subjects definitively removed from the contrast due to a TEAE related to the 
study contrast agent, 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 24 of 35 
06 October 2018 
 
 
• Number of subjects definitively removed from the contrast due to a TEAE related to the 
study procedure, 
• Number of subjects having experienced at least one serious TEAE, 
• Number of subjects having experienced at least one serious TEAE related to the study 
contrast agent, 
• Number of subjects having experienced at least one serious TEAE related to a study 
procedure, 
• Number of subjects having experienced at least one severe serious TEAE, 
• Number of subjects definitively removed from the contrast agent due to a serious TEAE, 
• Number of subjects definitively removed from the contrast agent due to a serious TEAE 
related to the study contrast agent, 
• Number of subjects definitively removed from the contrast agent due to a serious TEAE 
related to the study procedure, 
• Number of subjects having experienced a fatal serious TEAE, 
• Number of subjects having experienced a fatal serious TEAE related to the study contrast 
agent, 
• Number of subjects having experienced a fatal serious TEAE related to a study procedure. 
A summary table of TEAEs (number and % of subjects who experienced an adverse event and 
number of events) grouped by primary SOC and PT will be presented for the following categories 
of events: 
• All TEAEs,  
• All TEAEs related to the study contrast agent, 
• All TEAEs related to a study procedure, 
• All serious TEAEs, 
• All serious TEAEs related to the study contrast agent, 
• All serious TEAEs related to a study procedure, 
• All severe TEAEs, 
• All TEAEs leading to study discontinuation. 
A summary table of TEAEs by SOC and PT will be also presented for each severity.   
A subject with more than one occurrence of the same adverse event in a particular SOC and PT 
will be counted only once in the total of subjects experiencing adverse events in that particular 
SOC and PT, respectively. If a subject experiences the same adverse event at more than one 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 25 of 35 
06 October 2018 
 
 
severity, or with more than one relationship category, the most severe rating or the stronger causal 
relationship will be given precedence.  
Any missing severity or relationship of an AE should be replaced by the worst case as follows: 
• If severity is missing, then the AE will be included in “severe” category.  
• If relationship is missing, then the AE will be included as “related to the study contrast 
agent”. 
Time to onset and duration of events in days will also be listed where: 
• Time to onset is defined as (AE start date – date of DEFINITY®). 
• Duration of event is defined as (AE stop date - AE start date + 1). 
Note: AEs with missing start dates will be included in the count of events, but a time will not be 
calculated.  
AEs will be coded using MedDRA version 19.0. 
 
4.8.2 Vital Signs  
The vital sign measurements are only obtained at screening/baseline visit. The vital sign 
measurements (systolic and diastolic blood pressure, heart rate, and respiratory rate) at baseline 
will be summarized using descriptive statistics.  
The number and percentage of subjects with potentially clinically significant vital sign values at 
baseline will be tabulated. Potentially clinically significant vital signs values are detailed below: 
 
 
 
 
Parameter 
Unit 
Normal 
range 
Potentially Clinically 
Significant 
vital sign 
 
Heart Rate 
Beats 
per 
minute 
60-100 
1) 
<50 bpm and >/=25% 
decrease from baseline 
2) 
>100 bpm and >/=25% 
increase from baseline 
Systolic 
Blood 
Pressure 
mmHg 
90~139 
1) 
＞190 mmHg (entry is < 
160 mmHg)  
2) 
Systolic <80 mm Hg 
Decrease from baseline >30 
mmHg 
Diastolic 
Blood 
Pressure 
mmHg 
60~89 
1) 
110 mmHg (entry is < 
90 mmHg)  
2) 
<50 mm Hg  
3) 
Change from baseline of 
20mmHg (increase or 
decrease) 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 26 of 35 
06 October 2018 
 
 
4.8.3 Physical exam 
Physical examination body system inspection results will be listed only.  The physical exam is 
only conducted at Screening. 
 
4.8.4 Study Center effects 
The mean “absolute value of the difference vs. CMR” for each of DEFINITY® and unenhanced 
echocardiography, as well as the difference between the two treatments with respect to this 
mean, will be presented for each study center and each blinded reader.  Within each blinded 
reader, and assessment of study center effect on the mean treatment difference will be assessed 
using one-way analysis of variance. For each blinded reader, a site difference that is not 
significant at the 0.15 level of significance, or a site difference that is significant but where for 
every site the mean treatment difference is more favorable for DEFINITY® than enhanced 
echocardiography, will support pooling subjects across sites for the primary analysis for that 
reader. 
 
4.9 Protocol Violations 
The finalization of protocol violations and excluded data will be made prior to the database lock. 
 
4.9.1 Violation criteria 
Subjects who meet any of the following criteria will be listed and presented in the study report: 
• Non-compliance with inclusion criteria. 
• Non-compliance with exclusion criteria. 
• Non-compliance with study contrast agent. 
• Non-compliance with study procedures. 
• Other.  
Other reasons for violation may be added to this list and will be done so prior to database lock of 
the study. 
 
4.9.2 Protocol violations 
Deviations from the protocol, as defined in the protocol, will be documented and monitored on 
an ongoing basis by the Sponsor, study monitors and project manager throughout the study 
period.  
At the time of database lock, while the protocol violations are being reviewed, the Sponsor will 
forward all relevant documentation highlighting protocol violations to the study statistician. 
These violations will be included in the protocol violation document for agreement and will be 
listed with the protocol violations in the clinical study report. 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 27 of 35 
06 October 2018 
 
 
4.10 
Missing Values and Missing Visit Dates 
Within each blinded reader, the primary hypothesis test on the primary endpoint will be 
performed on subjects with non-missing LVEF for DEFINITY®, unenhanced echocardiography, 
and the reference standard.  A supportive analysis will be run where, within each blinded reader, 
missing LVEF is multiply imputed using the fully conditional specification (FCS) multiple 
regression prior to carrying out the primary endpoint analysis.  The covariates in the imputation 
model will be study center, age, gender, body weight (kg), race and ethnicity; this will be done 
separately for each imaging method (DEFINITY® enhanced, unenhanced, and CMR reference 
standard).  A total of 50 imputations will be generated; the paired t-test comparing mean bias 
between DEFINITY® and unenhanced (see primary endpoint null and alternative hypotheses 
above) will be performed separately on each of the 50 imputed datasets, and the t-test results will 
be combined across datasets using the usual multiple imputation techniques to create one overall 
paired t-test results on imputed data. 
There is no intention to implement any procedure for replacing missing data for any analyses 
except the primary endpoint paired t-test.  For all other analyses, the number of subjects with 
missing data will be presented under a “Missing” category. Unless otherwise stated, subjects 
with missing values will be included in the denominator count when computing percentages. 
When continuous data are being summarized, only the non-missing values will be evaluated for 
computing summary statistics. 
In case of missing date, replacement will be applied in order to be in a worst case: 
• Concomitant medication start date:  
o in case of completely missing date, it will be estimated as the DEFINITY® 
administration date; 
o if the day and the month are missing: 
 if the year is the same as the year of the  DEFINITY® administration date, 
it will be estimated by the  DEFINITY® administration date; 
 if the year is different to the year of the   administration date, it will be 
estimated as 1st January of that year; 
o if day only is missing: 
 if the month/year are the same as the month/year of the DEFINITY® 
administration date, it will be estimated by the DEFINITY® 
administration date; 
 if the month/year are different from the month/year of the DEFINITY® 
administration date, it will be estimated by the first day of the month; 
• Concomitant medication end date (if ongoing is not ticked at the end of the study):  
o in case of missing date, it will be estimated by the End of Study date; 
o if the day and the month are missing: 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 28 of 35 
06 October 2018 
 
 
 if the year is the same as the year of the End of Study date, it will be 
estimated by End of Study date; 
 if the year is prior to the year of End of Study date, it will be estimated by 
as 31 December of that year; 
o if day only is missing: 
 if the month/year are the same as the month/year of End of Study date, it 
will be estimated by the End of Study date; 
 if the year or month is different to the year or month of End of Study date, 
it will be estimated by the last day of the month. 
• Start date of an adverse event:  
o In case of completely missing date, it will be estimated by the DEFINITY® 
administration date. 
 
4.11 
Deviations from SAP 
Any deviations from the original statistical plan will be described and justified in the final 
clinical study report, whether written post interim or final analysis. 
 
4.12 
Changes in Conduct or Planned Analyses from the Protocol 
There have been no changes in analyses from those defined in the protocol.  
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 29 of 35 
06 October 2018 
 
 
4.13 
Algorithms/SAS Codes 
• Tables that need descriptive statistics – continuous variables: 
PROC UNIVARIATE DATA=dset NOPRINT; 
    VAR var1 var2 var3 …varn; 
   BY byvar; (optional) 
   OUTPUT OUT=outname 
   N=n MEAN=mean MIN=min MAX=max MEDIAN=median STD=std; 
RUN; 
• Tables that need frequency counts: 
PROC FREQ DATA=dset NOPRINT; 
  
BY byvar; (optional) 
   TABLES var1*var2; 
  
OUTPUT OUT=outname; 
RUN; 
• Tables that need 95% CIs within group for continuous variables: 
DATA outdata;  
 
SET outname; 
   LCL=mean-(TINV(0.975,n-1)*(std/SQRT(n))); 
   UCL=mean+(TINV(0.975,n-1)*(std/SQRT(n))); 
RUN; 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 30 of 35 
06 October 2018 
 
 
5 Tables and Listings 
5.1 Table Format 
All output will be produced using SAS version 9.4 or a later version. 
In the top left portion of each table/listing, a table/listing number followed by the title of the 
table/listing will be presented. After the title line, optional sub-title or population information 
can be presented. Horizontal lines will appear before and after the column heading of the 
table/listing. Footnotes will be put under the main body of text at the bottom of the page.  
The sponsor name, protocol number, programmers User ID, status of the table/listing (i.e. draft 
or final) and SAS program name will appear bottom left in a string and the page number will 
appear on the bottom right corner of each table/listing. The date and time of creation of 
table/listing will appear bottom left under the sponsor name. The source listing number will 
appear bottom left.  
A landscape layout is proposed for both table and listing presentations. 
The left and right margins of all tables and listings will be a minimum of 2.1 cm from the left 
and 1.9cm from the right. The top and bottom margins will be a minimum 2.92cm. Header and 
footer will be both 1.27 cm. 
There is no special requirement of font type and size, but an 8-point font size for tables and 7or 8-
point for listings is proposed using Courier New font. A maximum SAS line size=141 and page 
size=44 for 8-point font size, and line size=161 and page size=50 for 7-point will be used so as to 
fit on both UK and US paper sizes. 
In a listing, in the case that a subject’s record has been continued to the next page, an appropriate 
identification (e.g., the subject ID number) must be presented at the beginning of that page. 
 
5.2 Conventions 
Unless otherwise specified, in summary tables of continuous variables, the minimum and 
maximum values will be displayed to the same number of decimal places as the raw data, the 
mean and median will be presented to one extra decimal place compared to the raw data, and the 
standard deviation will be displayed to two extra decimal places compared to the raw data. 
Wherever possible data will be decimal aligned. 
Unless otherwise specified frequency tabulations will be presented by number and percentage, 
where the percentage is presented in brackets to 1 decimal place. 
P-values, if applicable, will be presented to 3 decimal places. If a p-value is less than 0.05 but is 
greater than or equal to 0.01, then an asterisk (*) will be added next to this value. If a p-value is 
less than 0.01 but is greater than or equal to 0.001, then two asterisks (**) will be added next to 
this value. Finally, if the p-value is less than 0.001 then three asterisks (***) will be added next 
to this value and it will be presented as <0.001. If the rounded result is a value of 1.000, it will be 
displayed as >0.999. Any date information in the listing will use the date9. format, for example, 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 31 of 35 
06 October 2018 
 
 
07MAY2002. In the listing, a unit associated with a variable will be presented only once within 
parentheses either below or next to that variable in the heading portion. If a parameter has 
multiple units, each unit will be displayed only once, as applicable. 
All tables will have their source listing referenced in a footnote. Listings should be sorted by 
Sequence, and subject and have the source data received by data management referenced in a 
footnote. All tables and listings will be converted into Microsoft Word documents and collated 
into two complete documents.  
 
5.3 Tables 
 
5.3.1 Section 14.1: Demographic and baseline 
Table 14.1.1 
Subject Disposition (All screened population) 
Table 14.1.2 
Demographics (Modified ITT  populations) 
Table 14.1.3.1 
Active Medical History (Modified ITT populations)  
Table 14.1.3.2 
Past medical history (Modified ITT populations)  
Table 14.1.4 
Exposure (Safety population)  
Table 14.1.5.1 
Prior medications (Safety population)  
Table 14.1.5.2 
Concomitant medications (Safety population)  
 
 
5.3.2 Section 14.2: Primary 
Table 14.2.1.1.1 
LVEF – Subjects with Available Data (Modified ITT population)  
Table 14.2.1.1.2 
LVEF by Study Center – Subjects with Available Data (Modified ITT 
population)  
Table 14.2.1.2.1 
LVEF – All Subjects - Multiple Imputation (Modified ITT population) 
Table 14.2.1.2.2 
LVEF by Study Center – All Subjects - Multiple Imputation (Modified ITT 
population) 
Table 14.2.1.3.1 
LVEF – All Subjects (Per-Protocol population) 
Table 14.2.1.3.2 
LVEF by Study Center – All Subjects (Per-Protocol population) 
 
5.3.3 Section 14.2: Secondary 
Table 14.2.2.1 
LVEF for Suboptimal Echocardiograms – Subjects with Available Data 
(Modified ITT population)  
Table 14.2.2.2 
LVEF for Suboptimal Echocardiograms – All Subjects (Modified PP 
population)  
Table 14.2.3.1 
Inter-Reader Variability - LVEF – Subjects with Available Data 
(Modified ITT population) 
Table 14.2.3.2 
Inter-Reader Variability - LVEF – All Subjects (Modified PP 
population) 
Table 14.2.4.1 
Inter-Reader Variability – End-Diastolic/Systolic Volumes – Subjects 
with Available Data (Modified ITT population) 
Table 14.2.4.2 
Inter-Reader Variability – End Diastolic/Systolic Volumes – All 
Subjects (Modified PP population) 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 32 of 35 
06 October 2018 
 
 
Table 14.2.5.1 
Inter-Reader Variability - LVEF for Suboptimal Echocardiograms – 
Subjects with Available Data (Modified ITT population) 
Table 14.2.5.2 
Inter-Reader Variability - LVEF for Suboptimal Echocardiograms - All 
Subjects (Modified PP population) 
Table 14.2.6.1 
Inter-Reader Variability – End-Diastolic/Systolic Volumes for 
Suboptimal Echocardiograms – Subjects with Available Data (Modified 
ITT population) 
Table 14.2.6.2 
Inter-Reader Variability – End Diastolic/Systolic Volumes for 
Suboptimal Echocardiograms – All Subjects (Modified PP population) 
 
 
5.3.4 Section 14.3: Safety 
5.3.4.1 Adverse events  
Table 14.3.1.1 
Summary of Treatment Emergent Adverse Events 
Table 14.3.1.2.1 
Treatment Emergent Adverse Events by System Organ Class and Preferred 
Term 
Table 14.3.1.2.2 
Study Contrast Agent Related Treatment Emergent Adverse Events by 
System Organ Class and Preferred Term 
Table 14.3.1.2.3 
Procedure Related Treatment Emergent Adverse Events by System Organ 
Class and Preferred Term 
Table 14.3.1.3.1 
Serious Treatment Emergent Adverse Events by System Organ Class and 
Preferred Term 
Table 14.3.1.2.2 
Study Contrast Agent Related Serious Treatment Emergent Adverse Events 
by System Organ Class and Preferred Term 
Table 14.3.1.2.3 
Procedure Related Serious Treatment Emergent Adverse Events by System 
Organ Class and Preferred Term 
Table 14.3.1.4 
Severe Treatment Emergent Adverse Events by System Organ Class and 
Preferred Term 
Table 14.3.1.5 
Treatment Emergent Adverse Events Leading to Study Withdrawal by 
System Organ Class and Preferred Term 
Table 14.3.1.6 
Treatment Emergent Adverse Events by System Organ Class and Preferred 
Term Classified According to Severity 
 
5.3.4.2 Vital signs  
Table 14.3.3.1 
Vital signs  
Table 14.3.3.2 
Number (Percentage) of Subjects with Potentially Clinically Significant 
(PCS) Values in Vital Signs 
 
All safety tables will be produced using the Safety Population. 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 33 of 35 
06 October 2018 
 
 
5.4 Figures 
Figure 14.2.1.1.1 
Bland-Altman 
Plot of DEFINITY®-enhanced 
LVEF(%) 
vs. CMR 
LVEF(%) - Patients with Available Data (Modified ITT population) 
Figure 14.2.1.1.2 
Bland-Altman 
Plot of DEFINITY®-enhanced 
LVEF(%) 
vs. CMR 
LVEF(%) – All Patients (PP population) 
Figure 14.2.1.2.1 
Bland-Altman Plot of Unenhanced LVEF(%) vs. CMR LVEF(%) - Patients 
with Available Data (Modified ITT population) 
Figure 14.2.1.2.2 
Bland-Altman Plot of Unenhanced LVEF(%) vs. CMR LVEF(%) - All 
Patients (PP population) 
Figure 14.2.2.1.1 
Deming Regression Plot of DEFINITY®-enhanced LVEF(%) vs. CMR 
LVEF(%) - Patients with Available Data (Modified ITT population) 
Figure 14.2.2.1.2 
Deming Regression Plot of DEFINITY®-enhanced LVEF(%) vs. CMR 
LVEF(%) – All Patients (PP population) 
Figure 14.2.2.2.1 
Deming Regression Plot of Unenhanced LVEF(%) vs. CMR LVEF(%) - 
Patients with Available Data (Modified ITT population) 
Figure 14.2.2.2.2 
Deming Regression Plot of Unenhanced LVEF(%) vs. CMR LVEF(%) - All 
Patients (PP population) 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 34 of 35 
06 October 2018 
 
 
5.4 Listings 
 
Listing 16.2.1.1 Eligibility 
Listing 16.2.1.2  Subject disposition 
Listing 16.2.2.1 Inclusion/Exclusion descriptions 
Listing 16.2.2.2 Inclusion/Exclusion criteria 
Listing 16.2.2.3 Protocol violations 
Listing 16.2.4.1  Subject demography 
Listing 16.2.4.2  Medical history, including reported and coded terms 
Listing 16.2.4.3 Prior medications, including reported and coded terms 
Listing 16.2.4.4 Concomitant medications, including reported and coded terms 
Listing 16.2.5.1 DEFINITY® Exposure 
Listing 16.2.5.2 Visit Dates 
Listing 16.2.6.1 LVEF 
Listing 16.2.6.2 End Diastolic and Systolic Volumes 
Listing 16.2.7.1 Adverse events, including reported and coded terms 
Listing 16.2.7.2 Related adverse events 
Listing 16.2.7.3 Adverse events leading to withdrawal 
Listing 16.2.7.4 Serious adverse events 
Listing 16.2.7.5 Deaths 
Listing 16.2.8.1 Vital signs 
Listing 16.2.8.2 Physical examination 
 
 


 
 
Sponsor: 
Lantheus Medical Imaging 
Protocol Number: 
DEF-314  
 
 
CONFIDENTIAL 
 
SAP Version 1.0, Final                                                Page 35 of 35 
06 October 2018 
 
 
6 Reference List 
 
1.  Mehta CR and Pocock SJ.  Adaptive increase in sample size when interim results are promising:  A practical 
guide with examples.  Statist Med 2011;30:3267-3284. 
 
2.  Hamer RM.  INTRACC.SAS Macro to Calculate Reliabilities for Intraclass Correlations [Internet].  SAS 
institute;1990 [cited 15 January 2018].  Available from https://github.com/friendly/SAS-
macros/blob/master/intracc.sas.