Contrast-enhanced Ultrasound for Complex Kidney Lesion Diagnosis in Patients With CKD Extension (CEUS CKDx)
Phase: PHASE2
Conditions: Chronic Kidney Diseases, Cystic Kidney Disease
Sponsor: University of North Carolina, Chapel Hill

 
Contrast-enhanced Ultrasound for Complex Kidney Lesion Diagnosis in 
Patients with CKD extension (CEUS CKDx) 
 
 
NCT number NCT03196076 
Document Date April 2, 2018 
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
1 
Contrast-enhanced Ultrasound for Complex Kidney Lesion Diagnosis in 
Patients with CKD extension (CEUS CKDx) 
 
Principal Investigators  
Emily H. Chang, MD 
UNC Kidney Center, CB #7155 
919-445-2621 (Phone) 
919-966-4251 (Fax) 
919-216-0932 (Pager) 
Email: emily_chang@med.unc.edu 
 
Lauren Burke, MD  
Radiology, CB# 7510  
Email: lauren_burke@med.unc.edu  
 
Biostatistician  
Dr. Jason Fine, ScD 
Department of Biostatistics  
3103 Mcgavran-Greenberg, CB 7420  
919-966-7259 (Phone) 
Email: jfine@email.unc.edu 
  
Funding Sponsor: Lantheus Medical Imaging 
331 Treble Cove Road 
North Billerica, MA  01862 
800-362-2668 (phone) 
 
Study Product: Perflutren Lipid Microsphere Injectable Suspension (Definity) 
 
Protocol Number: 17-1130 
 
IND Number: 127535 
 
Initial Version: June 6, 2017 
Version Date: April 2, 2018  
 
 
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
2 
Signature Page  
The signature below constitutes the approval of this protocol and the attachments, 
and provides the necessary assurances that this trial will be conducted according to 
all stipulations of the protocol, including all statements regarding confidentiality, 
and according to local legal and regulatory requirements and applicable U.S. federal 
regulations and ICH guidelines.  
 
Principal Investigator (PI) Name:_______________________  
 
PI Signature: ___________________  
 
Date:____________________  
 
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
3 
TABLE OF CONTENTS  
1.0 BACKGROUND AND RATIONALE .................................................................... 5  
1.1 Study Synopsis ...................................................................................................... 5  
1.2 Background ............................................................................................................ 5  
1.3 Perflutren Lipid Background and Associated Known Toxicities..... 7  
1.4 Rationale.................................................................................................................. 10  
 
2.0 STUDY OBJECTIVES............................................................................................. 10  
2.1 Primary Objective ................................................................................................10  
2.2 Secondary Objectives.......................................................................................... 10 
 
3.0 PATIENT ELIGIBILITY ....................................................................................... 10  
3.1 Inclusion Criteria.................................................................................................. 10  
3.2 Exclusion Criteria................................................................................................. 11  
 
4.0 STUDY PLAN ......................................................................................................... 12  
4.1 Schema ..................................................................................................................... 12 
4.2 Study Design .......................................................................................................... 13  
4.3 Follow-up ……………............................................................................................. 15  
4.4 Duration of Study Intervention ..................................................................... 15  
4.5 Duration of Follow Up ....................................................................................... 16  
4.6 Removal of Patients from Protocol .............................................................. 16  
 
5.0 DRUG INFORMATION ......................................................................................... 16  
5.1 Perflutren Lipid Microspheres (Lantheus Medical Imaging)............ 16  
5.2 Supplier/How Supplied .................................................................................... 16  
5.3 Handling and Dispensing of Perflutren.......................................................16  
5.4 Storage Requirements/Stability ................................................................... 17  
5.5 Preparation ............................................................................................................ 17  
5.6 Dosage and Administration ............................................................................ 17  
5.7 Clinical Safety Summary ................................................................................... 17  
5.8 Return and Retention of Study Drug............................................................ 18  
 
6.0 EVALUATIONS AND ASSESSMENTS................................................................ 18  
6.1 Pre-Study Assessments ..................................................................................... 18  
6.2 Treatment Assessments ................................................................................... 19  
6.3 Follow-up Assessments..................................................................................... 19  
6.4 Assessment of Safety ......................................................................................... 19  
6.5 Tissue Collection and Optional Storage..................................................... 19  
 
7.0 ADVERSE EVENTS ............................................................................................... 19  
7.1 Definitions .............................................................................................................. 19  
7.2 Documentation of non-serious AEs or SARs............................................ 21  
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
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7.3 SAEs or Serious SARs ......................................................................................... 21  
7.4 Data and Safety Monitoring Plan .................................................................. 22  
 
8.0 STATISTICAL CONSIDERATIONS .................................................................... 24  
8.1 Study Design/ Study Endpoints .................................................................... 24  
8.2 Sample Size and Accrual.................................................................................... 24  
8.3 Data Analysis Plans.............................................................................................. 24  
 
9.0 STUDY MANAGEMENT ....................................................................................... 25  
9.1 Institutional Review Board (IRB) Approval and Consent................... 25  
9.2 Required Documentation.................................................................................. 26  
9.3 Data Management and Monitoring/Auditing .......................................... 26  
9.4 Adherence to the Protocol ............................................................................... 27  
9.5 Amendments to the Protocol ......................................................................... 28  
9.6 Record Retention ................................................................................................. 28  
9.7 Obligations of Investigators ............................................................................ 29  
 
10.0 REFERENCES....................................................................................................... 29  
 
11.0 APPENDICES ....................................................................................................... 32  
 
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
5 
1.0  BACKGROUND AND RATIONALE   
1.1  Study Synopsis   
This is an investigator-initiated, prospective study designed to evaluate the accuracy 
of contrast-enhanced ultrasound (CEUS) with microbubble contrast agent 
(perflutren lipid; Definity®) to identify malignancy in patients with kidney disease, 
a known risk factor for kidney malignancy, in whom a conventional ultrasound (US) 
or other prior imaging shows an indeterminate or suspicious kidney lesion.   
The primary objective of the study is to estimate the sensitivity of CEUS in 
diagnosing kidney malignancy in patients with a risk factor for kidney malignancy 
and compare it to the current truth standard test in this patient population. 
Secondary analysis will include more optimal contrast-enhanced imaging with 
computed tomography (CT) or magnetic resonance imaging (MRI) in a subset of 
patients who can receive these studies. The study outcome will lead to immediate 
clinical application in patients with chronic kidney disease. Given the cost-
effectiveness and adverse event profile, it has excellent potential to become 
established as first line diagnostics in the general patient population as well. 
1.2  Background   
Introduction  
According to the US Renal Data System 2014 report, there are over 600,000 people 
in the US living with end stage kidney disease (ESKD) with almost 450,000 receiving 
dialysis. In a large collaborative international study, kidney cancers have a 
standardized incidence ratio of 3.6 in patients receiving dialysis1 meaning that for 
the nearly 500,000 patients receiving dialysis, there are an additional 50,000 cases 
of kidney cancer that occur above that of the general population. More recent 
findings show that patients with chronic kidney disease (CKD) are also at increased 
risk of kidney cancer, with hazard ratios increasing with CKD stage2. For this reason, 
patients on the waiting list for renal transplant and those receiving dialysis have 
been advised to have annual screenings for kidney malignancy.  
 
Currently, contrast-enhanced CT and MRI are the truth standard imaging studies for 
kidney lesion characterization. However iodinated contrast agents are nephrotoxic 
in patients with renal insufficiency3 and therefore contraindicated in patients with 
moderate to severe CKD (stages IV and V) as they can lead to permanent 
deterioration of kidney function. Until recently, contrast MRI was a suitable 
alternative. However, Nephrogenic Systemic Fibrosis (NSF) is now regarded as a 
serious, potentially lethal complication of the gadolinium contrast agents used in 
MRI if administered to patients with impaired renal function. Inadequate 
preoperative imaging can lead to unnecessary nephrectomy, which would further 
limit already compromised renal function4. There is therefore a need for an imaging 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
6 
method for evaluating renal masses that does not use iodinated or gadolinium 
contrast agents. CEUS represents one potential alternative.  
Contrast-Enhanced US Diagnostic Imaging of the Kidney  
For CKD patients screened annually for kidney malignancy, US imaging of the kidney 
is typically performed in the United States without contrast as no US contrast agent 
is approved for this indication. Without contrast, malignant tumors cannot be 
differentiated from benign tumors or pseudotumors. Typically, following the 
identification of a kidney lesion via US without contrast, contrast-CT or MRI may 
then be performed to stage and grade the lesion, depending on a risk/benefit 
analysis performed by the patient’s physician. If a contrast-CT or MRI is deemed too 
risky, the patient may proceed directly to surgery, or more likely, will be followed 
closely with repeated non-contrast imaging, typically anywhere from 1-5 years.  
US contrast agents are gas-filled microbubbles which are intravenously 
administered in very small volume boluses or slow infusions. These microbubbles 
typically have mean diameters between 1 to 6 μm5, remain intravascular for several 
minutes, and do not diffuse into the interstitium (pure blood-pool agents)5.  
Advancements in imaging technique combined with the nonlinear properties of 
microbubbles have spurred the development of contrast-specific US techniques such 
as CPS (Contrast Pulse Sequencing). Such imaging techniques provide high-
resolution images of tissue vasculature and allow the assessment of the 
microcirculation patterns in real-time6-12. Outside of the United States, or in the 
context of clinical trials, US contrast agents have been used in kidney imaging8-11, 13-
20. Levovist® (lipid and galactose microparticle suspension) and Sonovue® 
(lyophilized sulfur hexafluoride microbubbles), two non-FDA approved similar US 
contrast agents approved for use in countries outside of the United States, have 
been used in the general population to identify and diagnose renal pseudotumors10, 
18, 21, cystic renal lesions10, 14, 16, 18, 22 and solid renal lesions10-13, 15, 17-19. See a 
descriptive table of these studies below. Two recent studies have included patients 
with CKD and shown similar accuracy in this population.23, 24 
Author 
(year) 
Contrast 
Agent 
Imaging 
Method 
# 
lesions  
Lesion 
Type 
Results 
Kim 
(1999)14 
Levovist
®  
Power 
Doppler  
13  
Cystic  
PPV: 100% NPV: 57% DxA: 77%  
Ascenti 
(2001)13 
Levovist
®  
Power 
Doppler  
41  
Solid  
DxA: 78%  
Ascenti 
(2004)15 
SonoVue
®  2
nd 
Harmonic  40  
Solid  
Se: 85.7%  
Park 
(2005)17 
Levovist
®  
2
nd 
Harmonic  30  
Solid  
Se: 97%  
Sp: 93% DxA: 95%  

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
7 
Tamai 
(2005)19 
Levovist
®  
SieFlow  
29  
Solid  
Se: 94.4%  
Sp: 45.4% PPV: 100%  
Wink 
(2007)8 
SonoVue
®  CPS  
20  
Solid & 
Cystic  
Se: 95%  
Quaia 
(2008)9 
SonoVue
®  CPS  
40  
Cystic  
Se: 89% Sp: 73.6% PPV: 79.3% 
NPV: 86% DxA: 82%  
Fan 
(2008)12 
SonoVue
®  CPS  
72  
Solid 
Sp: 96.4% Se: 77.3%  
Meloni 
(2008)11  
SonoVue
®  CPS  
30  
Solid  
Se: 96.6% Sp: 100% PPV: 100% 
NPV: 95.8% DxA: 98.1%  
Roy 
(2008)18 
SonoVue
®  CPS  
86  
Solid & 
Cystic  
Sp: 92.9% - Papillary carcinoma 
57.1% - Clear Cell carcinoma 100% - 
Oncocytoma 100% - Benign  
Abbreviations: PPV – Positive Predictive Value, NPV – Negative Predictive Value, DxA – 
Diagnostic Accuracy, Se – Sensitivity, Sp – Specificity  
 
In the United States, where there has been significantly less research on using US 
contrast in humans, the US contrast agent, Definity®, has recently been used in one 
study to measure renal blood flow in a healthy population20, effectively reproducing 
previous animal studies in the human population. Three US contrast agents are FDA 
approved for human use: perflutren lipid microspheres (Definity®), sulfur 
hexafluoride lipid microspheres (Lumason®) and perflutren protein-type A 
microspheres (Optison®). All are FDA indicated for use in cardiac studies, and 
Lumason® is also FDA approved for liver and urinary tract imaging.  
1.3 Perflutren Lipid Background and Associated Known Toxicities  
See http://www.accessdata.fda.gov/scripts/cder/drugsatfda/ for full prescribing 
information on perflutren when used according to its FDA-approved indication. Also 
see section 5.7.  
1.3.1 Background  
Definity® (perflutren lipid) is an FDA-approved US contrast agent indicated to 
opacify the left ventricular chamber and to improve the delineation of the left 
ventricular endocardial border in patients with sub-optimal echocardiograms. It is 
activated by mechanical agitation with a Vialmix® which produces a milky white 
injectable suspension of perflutren lipid microspheres composed of 
octafluoropropane. Activated perflutren may be injected by either an intravenous 
bolus or infusion. See http://www.accessdata.fda.gov/scripts/cder/drugsatfda/ for 
full prescribing information when used in this indication. When used in this setting, 
the maximum dose of perflutren is administered as either two bolus doses or one 
single intravenous infusion.  
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
8 
1.3.2 Associated Toxicities  
In pre-market clinical trials, 1716 subjects were evaluated with activated perflutren 
lipid. Of the 1716 subjects, 144 (8.4%) had at least one treatment-related adverse 
reaction. There were 26 serious adverse events and 15 (0.9%) subjects discontinued 
because of an adverse event. Nineteen subjects (1.1%) suffered serious 
cardiopulmonary adverse events including 8 deaths. The deaths occurred several 
days after activated perflutren lipid administration and appear to be related to the 
course of underlying disease. Of the 11 other serious adverse events, which 
appeared within 2-15 days of the drug administration, all appeared to be a 
progression of underlying cardiac and non-cardiac disease. However, a role for 
perflutren lipid in the initiation or course of these adverse events cannot be ruled 
out.  
There were 15 discontinuations reported. Nine of these patients were discontinued 
after the first injection. One patient experienced a hypersensitivity reaction with 
urticaria and pruritus and all the other patients experienced dizziness, chest pain, 
dyspnea or back pain. Adverse events (AEs) appeared within 1 – 15 minutes of the 
drug administration and were of moderate intensity resolving usually without 
treatment within minutes or hours after onset.  
For all AEs, there were no differences in the overall incidence based on age, gender, 
or route of administration. The most common events were (% of patients 
experiencing): headache (2.3%), back and renal pain (1.2%), flushing (1.1%) and 
nausea (1.0%).  
Cardiopulmonary Reactions  
In 2007, in response to post-marketing reports of 4 deaths and 190 serious 
cardiopulmonary reactions, the FDA issued a black box warning for both Definity® 
and Optison® adding disease state contraindications and a mandatory 30 minute 
monitoring period following administration in all patients. Following this there have 
been several large-scale safety studies looking into the records of a total of more 
than 200,000 patients who received one of these contrast agents. In all those studies 
a composite rate of serious adverse events was calculated to be 1 – 3 in 10,000,25 
compared to gadolinium-based MRI contrast which has an incidence of NSF of 2 – 5 
in 100 patients with chronic kidney disease.26  
 
Following a meeting of the FDA Cardio-renal Advisory Committee in 2008, the black 
box warning was revised. The revisions shortened the contraindications to include 
cardiac shunts and hypersensitivity to perflutren and mandated the 30-minute 
monitoring period be limited to patients with pulmonary hypertension or unstable 
cardiopulmonary conditions. The black box warning was further revised in 2011, 
removing the mandatory 30-minute monitoring period, but stating that most 
serious cardiopulmonary reactions occur within 30 minutes of administration. For 
this reason, the label states that cardiopulmonary resuscitation personnel and 
equipment be readily available prior to perflutren administration, and that all 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
9 
patients be monitored for acute reactions.  
 
Patients with a history of cardiac shunts, pulmonary hypertension or unstable 
cardiopulmonary conditions will be excluded from our study. In addition, all 
patients will be monitored for 30-minute post-perflutren administration by the 
research or clinical nurse or research physician.  
Hypersensitivity Reactions  
The real risk of perflutren in our study is to the small number of potential patients 
with an undiagnosed allergy to perflutren. Post-marketing reports have included 
anaphylactoid events and other serious but non-fatal adverse reactions, typically 
within 30 minutes of drug administration (see the package insert, and section 5.7 
for additional information. In order to avoid a potentially fatal event, EpiPen® 
(epinephrine) injections will be kept near the US machine for all patients.  
High Ultrasound Mechanical Index (MI)  
High ultrasound MI values may cause microsphere cavitation or rupture and lead to 
ventricular arrhythmias. In addition, end-systolic triggering with high MI has been 
reported to cause ventricular arrhythmias. While the safety of perflutren at MIs >0.8 
has not been established by the manufacturer, real-time measurement of renal 
blood flow in 19 healthy subjects using perflutren was recently reported, using a 
flash replenishment high-MI (MI of 1.0) US technique. The contrast agent was well 
tolerated with no serious adverse events. One patient each had a 20 mmHg increase 
in systolic blood pressure, a very brief and mild episode of flushing, and mild back 
pain, but no cardiac arrhythmias were recorded during the study period.20   
 
A second study using the Sonovue® contrast agent with flash-replenishment high-
MI to detect changes in kidney perfusion via US in 10 normal volunteers reported 
the agent was well tolerated, and not associated with any adverse events.27 The 
safety of using a flash-replenishment high MI echocardiography with Sonovue®28 
has been established in patients with chest pain of undetermined origin as well as in 
patients undergoing stress testing for detection of ischemia.29 
 
Use in Renal Insufficiency  
US contrast agents including perflutren should carry no additional risks in patients 
with renal insufficiency, as peflutren is cleared by the lungs. The phospholipid 
component of perflutren lipid microspheres is thought to be metabolized to free 
fatty acids, while the octafluoropropane (OFP), as a stable gas, is not metabolized. In 
a small (n=8) pharmacokinetic study in healthy subjects, OFP was undetectable after 
10 minutes in most subjects either in the blood or expired air, with a mean half-life 
of 1.3 minutes (Definity® Prescribing Information).  
 
The UNC Division of Cardiology routinely uses perflutren as an echocardiographic 
contrast agent, including in patients with renal insufficiency.  

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
10
1.4 Rationale  
At this time, contrast-enhanced CT and MRI are the standard imaging studies for 
evaluation of suspected renal tumors. However, there are serious adverse effects 
from iodinated CT contrast and gadolinium MRI contrast in patients with renal 
insufficiency. Currently, to reduce exposure of potentially nephrotoxic contrast 
agents in patients with renal insufficiency, use of iodinated CT and gadolinium MR is 
limited to those patients in whom US without contrast indicates a suspicious or 
indeterminate lesion, and for whom the risk/benefit ratio warrants this risk.  
 
Our primary objective is to compare the accuracy of perflutren enhanced US to the 
current clinical truth standard of follow-up non-contrasted imaging in patients with 
CKD. The accuracy of perflutren-enhanced US will be determined by comparing 
results of the perflutren-enhanced US with the Truth Standard (tissue diagnosis or 
change in 1-year follow-up non-contrast US imaging). In patients where contrasted 
imaging (CT or MRI) is available, these results will be incorporated into the Truth 
Standard in a secondary analysis. 
Unlike CT and MRI contrast agents, US contrast agents are not associated with 
complications in patients with impaired kidney function. We hope to show that 
contrast-enhanced US may provide a viable alternative to contrast-enhanced CT and 
MRI for identifying malignancy in kidney lesions in patients with impaired kidney 
function in view of the serious adverse effects associated with CT and MR contrast 
agents in this patient population.  
2.0  STUDY OBJECTIVES   
2.1  Primary Objective   
To estimate the sensitivity and specificity of qualitative interpretations of CEUS in 
diagnosing kidney malignancy in patients with CKD and a suspicious or 
indeterminate lesion on non-contrasted imaging compared to the truth standard 
described in 1.4 and 4.2.6.  
   
2.2  Secondary Objectives   
To compare sensitivity and specificity of qualitative interpretations to contrast-CT 
or MR in a subset of patients. 
 
To compare sensitivity and specificity of two different scoring/classification 
systems (Bosniak and Barr 2014) for use in CEUS of kidney lesions in patients with 
CKD.  
 
 
 
3.0  PATIENT ELIGIBILITY   
3.1  Inclusion Criteria   

CEUS CKDx 
PI: Emily Chang 
 
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UNIVERSITY OF NORTH CAROLINA 
 
 
11
To be eligible for the present study, patients must meet the following criteria:   
 
3.1.1  Able to provide written informed consent   
 
3.1.2  Willing to comply with protocol requirements   
 
3.1.3  At least 18 years of age   
 
3.1.4  Have kidney disease, defined as either CKD II-V, determined by estimated 
glomerular filtration rate (GFR) of <90 and derived from serum creatinine 
measurements, or albuminuria/proteinuria, determined by albumin to creatinine 
ratio or protein to creatinine ratio of >30mg/gm within 3 months of recruitment, or 
on dialysis or having received a kidney transplant or have biopsy proven kidney 
disease. In these latter cases, blood and urine tests are not necessary. 
  
3.1.5  Have at least one kidney lesion identified but incompletely characterized on a 
non-contrasted US, CT, or MR exam for which the patient’s provider recommends 
follow-up studies or further evaluation with additional imaging test(s). 
 
3.2 Exclusion Criteria  
Patients who meet any of the following criteria will be excluded for enrollment:  
 
3.2.1  Critically ill or medically unstable and whose critical course during the 
observation period would be unpredictable (e.g., chronic obstructive pulmonary 
disease (COPD) requiring oxygen)   
 
3.2.2  Known hypersensitivity to sulfur hexafluoride or to any component of 
perflutren lipid (Definity®)   
 
3.2.3  Right to left shunt, severe pulmonary hypertension (Pulmonary artery 
pressure >90mmHg), or adult respiratory distress syndrome   
 
3.2.4  Active cardiac disease including any of the following:  
• Severe congestive heart failure (class IV in accordance with the 
 classification of the New York Heart Association)   
• Unstable angina.   
• Severe arrhythmia (i.e. ventricular tachycardia, flutter fibrillation; 
 ventricular premature complexes occurring close to the preceding T- 
 wave, multifocal complexes).   
• Myocardial infarction within 14 days prior to the date of proposed Definity® 
administration.   
• Uncontrolled systemic hypertension (systolic blood pressure (BP)>180 mm 
Hg and/or diastolic BP>100 mm Hg despite optimal medical management)   
 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
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UNIVERSITY OF NORTH CAROLINA 
 
 
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3.2.5  Is in an intensive care setting   
 
3.2.6  Has an unstable neurological disease (e.g cerebrovascular accident (including 
transient ischemic attacks (TIAs) within the 3 months before signing of informed 
consent  
 
3.2.7  Has undergone an invasive procedure on kidney lesion (e.g. tissue biopsy, 
surgery, nonsurgical cytoreductive procedure) since identification of lesion via US 
without contrast  
 
3.2.8  Has any other medical condition or other circumstances that would 
significantly decrease the chances of obtaining reliable data or of achieving the 
study objectives such as:   
• Mental illness  
• Drug abuse   
 
3.2.9  Female patient who is pregnant or lactating (the possibility of pregnancy has 
to be excluded by negative POC, serum or urine Β-HCG results, obtained within 24 
hours before the perflutren lipid administration, or on the basis of patient history, 
e.g.: tubal ligation, hysterectomy or a minimum of 1 year without menses)  
 
3.2.10 Obesity that limits obtainment of acceptable images 
 
4.0  STUDY PLAN   
 
4.1  Schema  
 
 
Patients who meet study inclusion and exclusion criteria (see Sections 3.1 and 3.2) 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
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and provide informed consent will be subjected to a baseline conventional, non-
contrasted lesion-focused ultrasound on Day 1 and CEUS. One year after initial 
study, a follow-up non-contrasted ultrasound will be obtained. The results of any 
other additional studies obtained clinically, including histology, contrasted or non-
contrasted CT/MR, will be collected. Once all CEUS studies are completed, blinded 
radiologists will grade each lesion based on Bosniak criteria. Once all follow-up non-
contrasted ultrasounds are obtained, radiologists will grade each lesion as stable, 
progressed, or regressed. 
 
Please see the Time and Events Table in Appendix 2 for a full listing of evaluations 
and assessments throughout the course of the study.  
 
4.2  Study Design  
 
4.2.1 Patient Identification and Recruitment 
Patients will be identified through UNC’s nephrology, transplant and dialysis clinics. 
Additionally, community collaborators will be alerted to the study and asked for 
referrals. Patients who participated in a prior pilot study (IRB 12-2314) with similar 
inclusion and exclusion criteria will be screened to determine if they still meet 
criteria. The PI will communicate with the patient’s referring clinician and the 
patient to determine whether they fit the inclusion/exclusion criteria and would like 
to participate in the study. The study coordinator will then be notified of these 
patients and approach the patient to further explain the study and obtain their 
signature on the informed consent form if they wish to participate. If patient 
consents, they will undergo perflutren-enhanced US. We will provide $50 
compensation and a parking voucher for completion of initial CEUS study. If two 
lesions are identified and imaged, either the day of the initial study visit or return 
visit, they will receive $50.00 for each lesion imaged .  Individuals will also be 
compensated an additional $50.00 if a lesion needs to be reimaged on a return visit.  
We will offer an additional $50 for completion of all follow-up studies (1-year 
urinalysis and serum creatinine and 1-year follow-up conventional ultrasound or 
other imaging study) and $75 if they are not cared for at UNC and need to make an 
additional trip to UNC to obtain the studies. We plan on enrolling a total of 20 
patients that fit the above criteria over a 6-month period.  
 
Healthy volunteers will also be recruited at study initiation to optimize the imaging 
technique. These volunteers will receive equivalent compensation. In the case of any 
abnormalities found during the study, the subject will be informed of the finding, 
and the finding will be communicated to the subject’s primary provider by study PI 
for further management.  Imaging optimization is needed to optimize the method of 
the administration of Definity.  A total of 5 health volunteers will be included in the 
imaging optimization phase. 
 
Patients that decline enrollment will be asked if they would be willing to participate 

CEUS CKDx 
PI: Emily Chang 
 
CONFIDENTIAL 
April 2, 2018 
 
UNIVERSITY OF NORTH CAROLINA 
 
 
14
as controls. This would involve no CEUS study and only collection of serum 
creatinine levels over a 1-year follow-up period from the patient’s primary provider.  
These patients will be consented by the study coordinator to allow patients to be 
followed. 
 
4.2.2 Multicenter sites.  
This will be a single institution study. 
 
4.2.3  Sonographic Evaluation  
Study subjects will be asked to lie on an examination table in the UNC Radiology 
Department of US. Once the patient is positioned, US gel will be applied to the 
patient’s back or flank. The transducer will be placed on top of the gel. Once the 
kidney lesion is located, the distance from the transducer to the nearest edge of the 
target lesion will be recorded. Pre-contrast Doppler and B-mode investigations of 
the target lesion will be performed at baseline using commercially available US 
equipment and standard technique. Subsequent to this, either color or power 
Doppler imaging should be used to study the anatomy of the target lesion and 
surrounding vessels. Images (baseline and perflutren-enhanced) will be digitally 
recorded on a cine loop.  
 
4.2.4  Perflutren Administration   
See http://www.definityimaging.com/pdf/VIALMIX%20Users%20Guide.pdf, and 
the package insert, for instructions on perflutren lipid preparation and activation. 
Peflutren lipid is intended for intravenous (IV) administration only after activation 
in the Vialmix® apparatus.  
 
Resuscitative carts and trained personnel will be immediately available during 
Perflutren administration and imaging. 
 
Perflutren will be administered in a bolus or continuous infusion using the dosing 
range and administration type within the perflutren prescribing information (see 
http://www.definityimaging.com/how-administration.html). If a subject has a 
central port 20 gauge or greater and does not have a filter then perflutren will be 
administered through the central port. If a subject does not have a central port then 
perflutren will be administered through the antecubital vein, preferably on their 
dominant hand. 
 
Once perflutren lipid has been administered, the transducer is maintained in a 
constant position over the area of interest to show the target lesion in order to 
assess the enhancement pattern during the early, mid and late vascular phases. 
Either bolus or infusion imaging with flash replenishment imaging approaches will 
be used with MI not to exceed 1.1. Images will also be taken of kidney parenchyma 
in a suitable longitudinal plane. Imaging with perflutren lipid will be performed with 
software (Siemens Medical Solutions USA, Inc) using the flash-replenishment high-

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MI imaging technique for parenchymal imaging.  
 
The contrast-enhanced US examination will consist of the complete dynamic real- 
time assessment of the contrast enhancement profile of the lesion in comparison to 
the surrounding renal parenchyma. If there are multiple lesions in one subject 
requiring a second dose, the subject will have the option to undergo a 2nd contrast-
enhanced study 30-minutes after the initial contrast dose, per dosing instructions in 
the package insert.  Individuals may also be asked to return on a different study date 
to reimage a lesion or to image additional lesions. 
 
Upon completion of imaging study, the patient will be brought back to the radiology 
holding area and monitored by nursing staff for 30 minutes after study completion. 
Blood pressure and heart rate will be obtained 15 and 30 minutes post procedure 
and recorded on the flowsheet. 
 
4.2.5 Assessment of Imaged Lesions  
At least two blinded radiologists will interpret the US images independently. Each 
interpreter will be asked to assess multiple lesion characteristics including 
echogenicity, size, septations, vascularity, and patterns of enhancement which will 
be used to classify the lesion. A diagnostic conclusion (e.g. benign, malignant or 
indeterminate) will be derived based on the Bosniak category if the lesion is cystic. 
Bosniak categories I, II and IIF will be classified as benign, and categories III and IV 
as malignant. The Bosniak classification system can be found in Appendix I. Readers 
will also be asked to score lesions based on a classification scheme presented by 
Barr in 2014.30  
 
Results of the perflutren-enhanced scans will not be used to dictate treatment and 
will not be shared with patients.  
 
4.2.6 Truth Standard  
Because the majority of patients will not undergo surgery/biopsy or receive a 
contrasted CT or MRI, the truth standard will be the change from baseline non-
contrasted US at one year follow-up non-contrasted US, the most typical clinical 
approach to these lesions. Follow-up US will be categorized as either stable, 
regression or progression. Lesions that are stable or have regressed will be deemed 
a negative test. Lesions that have progressed will be positive. When clinically 
indicated, if tissue pathology/biopsy or other imaging studies including contrasted 
CT or MRI are available, these will be collected and used in secondary analyses.  
 
4.3  Follow-up  
For all patients, the treatment path will be decided by the subject’s primary 
physician. We will review the medical records from each patient for a minimum of 
12 months post CEUS and document results of 1-year urinalysis and serum 
creatinine and follow-up non-contrasted US 12 months after CEUS. Additionally, any 

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contrast-enhanced or non-contrast CT and/or MRI findings or surgical procedures 
performed as part of their routine standard of care will be collected and reviewed.  
  
4.4  Duration of Study Intervention   
The study intervention is complete once the patient receives perflutren-enhanced 
US (see Study Schema). We anticipate that CEUS imaging will last approximately 30 
minutes for each subject, including set-up, pre-scan, and image capture. The 
patient may be withdrawn from the study prior to this point if any of the following 
apply:  
• Inter-current illness prevents completion of perflutren-enhanced US   
• Unacceptable adverse event(s) prevents completion of perflutren-enhanced 
US   
• Patient decides to withdraw from the study, OR   
• General or specific changes in the patient’s condition render the  patient 
unacceptable for completion of perflutren-enhanced US in the judgment of 
the investigator.  
 
4.5  Duration of Follow Up   
Patients will be followed up via review of their medical records and through receipt 
of 1-year urinalysis and serum creatinine and a 1-year follow-up non-contrast US. 
Additional imaging studies and histopathological diagnoses will also be obtained for 
any scheduled biopsy and/or surgery as dictated by standard of care. Patients will 
also be followed indefinitely after the initial year for lesion changes/histologic 
diagnosis or changes in kidney function. 
  
4.6 Removal of Patients from Protocol  
Patients will be removed from study when any of the criteria listed in Section 4.4 
apply. The Principal Investigator will be notified, and the reason for study removal 
and the date the patient was removed documented in the study documentation 
sheets. The patient should be followed-up per protocol.  
 
5.0  DRUG INFORMATION   
5.1  Perflutren Lipid Microspheres (Lantheus Medical Imaging)   
The Definity® vial contains components that upon activation yield perflutren lipid 
microspheres composed of octafluoropropane. Perflutren is a diagnostic drug that is 
intended to be used for contrast enhancement. The vial contains a clear, colorless, 
sterile, non-pyrogenic, hypertonic solution which is activated by mechanical 
agitation with Vialmix®.   
 
Vialmix® is the activation device used in the preparation of US contrast imaging 
agents, including Definity®. Prior to activation, each Definity® vial contains 6.52 
mg/mL octafluoropropane in the headspace and 0.75 mg lipid blend (0.045 mg 
DPPA, 0.401 mg DPPC, and 0.304 mg MPEG5000 DPPE), 103.5 mg propylene glycol, 
126.2 mg glycerin, 2.34 mg sodium phosphate monobasic monohydrate, 2.16 mg 

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sodium phosphate dibasic heptahydrate and 4.87 mg sodium chloride in water in 
the clear liquid. Upon activation, each mL of the milky white suspension contains a 
maximum of 1.2 x 1010 perflutren lipid microspheres with approximately 150 
μL/mL octafluoropropane.   
 
5.2  Supplier/How Supplied   
Perflutren (Definity®) will be provided to study subjects at no cost. Perflutren is 
supplied as a single use 2mL clear glass vial containing clear liquid. Each package 
contains 4 single-use vials. 
 
5.3  Handling and Dispensing of Perflutren   
Perflutren lipid must be dispensed only from official study sites by authorized 
personnel according to local regulations. Perflutren should be stored in a secure 
area according to local regulations. It is the responsibility of the Investigator to 
ensure that study drug is only dispensed to study patients.  
 
5.4  Storage Requirements/Stability   
The drug product should be stored in a secure location with limited access under 
controlled temperature conditions of 2-8° C (36° -46° F) in a refrigerator.   
 
5.5  Preparation   
See http://www.definityimaging.com/pdf/VIALMIX%20Users%20Guide.pdf, and 
the package insert for instructions on the use of Vialmix®.   
 
5.6  Dosage and Administration   
See Section 4.2.4.   
 
5.7  Clinical Safety Summary   
See prescribing information on perflutren when used according to its FDA 
indication (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/ and highlights 
for perflutren prescribing at 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021064s011lbl.pdf), 
 
In addition, the following warnings and precautions are noted in the October 2011 
labeling:   
 
Serious Cardiopulmonary Reactions 
Serious cardiopulmonary reactions including fatalities have occurred uncommonly 
during or shortly following perflutren-containing microsphere administration, 
typically within 30 minutes of administration. The risk for these reactions may be 
increased among patients with unstable cardiopulmonary conditions (acute 
myocardial infarction, acute coronary artery syndromes, worsening or unstable 
congestive heart failure, or serious ventricular arrhythmias). Always have 
cardiopulmonary resuscitation personnel and equipment readily available prior to 

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perflutren administration and monitor all patients for acute reactions.  The 
reported reactions include: fatal cardiac or respiratory arrest, shock, syncope, 
symptomatic arrhythmias (atrial fibrillation, tachycardia, bradycardia, 
supraventricular tachycardia, ventricular fibrillation, ventricular tachycardia), 
hypertension, hypotension, dyspnea, hypoxia, chest pain, respiratory distress, 
stridor, wheezing, loss of consciousness, and convulsions.   
 
Anaphylactoid Reactions  
In postmarketing use, uncommon but serious anaphylactoid reactions were 
observed during or shortly following perflutren-containing microsphere 
administration including: shock, hypersensitivity, bronchospasm, throat tightness, 
angioedema, edema (pharyngeal, palatal, mouth, peripheral, localized), swelling 
(face, eye, lip, tongue, upper airway), facial hypoesthesia, rash, urticaria, pruritus, 
flushing, and erythema. These have occurred in patients with no prior exposure to 
perflutren-containing microsphere products.   
 
Systemic Embolization of Perflutren in Patients with Cardiac Shunts  
In patients with right-to-left, bi-directional, or transient right-to-left cardiac shunts 
phospholipid-encapsulated microspheres can bypass the pulmonary particle-
filtering mechanisms and directly enter the arterial circulation resulting in 
microvascular occlusion and ischemia. In an animal study utilizing intra-arterial 
administration of activated perflutren, microsphere trapping was seen in small 
arterioles <15 μm, especially at branch points and in capillaries at all doses tested, 
including doses directly applicable to those used in humans. An animal study 
utilizing intravenous administration did not result in arterial microvascular 
obstruction presumably because of filtering by the lungs. Do not administer 
perflutren by intra-arterial injection.  
 
High Ultrasound Mechanical Index  
High ultrasound mechanical index values may cause microsphere cavitation or 
rupture and lead to ventricular arrhythmias. Additionally, end-systolic triggering 
with high mechanical indices has been reported to cause ventricular arrhythmias. 
The safety of activated perflutren at mechanical indices greater than 0.8 has not 
been evaluated.  
 
QTc Prolongation  
ECG parameters for doses up to10 μL/kg were monitored in 221 subjects at multiple 
time points from 1 hour to 72 hours after the first bolus injection. In the 221 
subjects, QTc prolongations of >30 msec were noted in 64 (29%) subjects. Forty-six 
out of 64 subjects with QTc prolongations were further evaluated and 39% (18/46) 
showed associated cardiac rhythm changes. The effects of concomitant drugs were 
not studied.  
 
5.8 Return and Retention of Study Drug  

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Remaining reagent at the completion of the study or expired reagent will be 
destroyed.  
 
6.0  EVALUATIONS AND ASSESSMENTS 
   
6.1  Pre-Study Assessments   
Written informed consent will be obtained from the subjects prior to the 
implementation of any study procedures. The subject will receive a signed and 
dated informed consent form. A complete medical history will be taken at 
enrollment and reviewed on presentation for the study ultrasound. A serum or 
urine β-HCG test will be performed within 24 hours prior to perflutren 
administration if clinically appropriate.  All medications (prescription and over-
the-counter) taken within 24 hours prior to perflutren lipid administration will be 
recorded. 
   
6.2  Treatment Assessments   
Subjects will be monitored for any untoward medical occurrences or toxicities from 
the time of signed informed consent through 30 minutes after perflutren lipid 
administration.   
 
6.3  Follow-up Assessments   
All post-CEUS clinical diagnoses based on 1-year follow-up non-contrasted US will 
be reviewed. Any post-operative histopathological diagnoses for patients who 
undergo biopsy/surgery within the 12-month follow-up period post perflutren lipid 
administration will be reviewed. Any additional non-contrasted or contrasted 
imaging (CT or MRI) within the 12-month follow-up period post perflutren lipid 
administration will be reviewed.  
 
6.4  Assessment of Safety   
Any patient in whom perflutren administration is initiated will be evaluable for 
toxicity.  
 
Baseline creatinine (within 12 weeks prior to study) and urinalysis (on day of study, 
pre and post-CEUS) will be collected from each patient prior to CEUS. At and 1-year 
post-CEUS, serum creatinine levels and urinalysis will be obtained from primary 
providers. Serum creatinine from a comparison group drawn from patients who 
meet study inclusion and exclusion criteria but decline to participate in the study 
will be followed over the study period to detect potential adverse effects the study 
intervention may have on kidney function of the study subjects. Their serum 
creatinine values will be collected at 1-year post initial recruitment with a minimum 
of 1 value at 1-year timepoint. 
 
6.5  Tissue Collection and Optional Storage   
If a patient undergoes nephrectomy, all nephrectomy specimens will be collected by 

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the tissue procurement facility. Final diagnoses will be obtained by chart review.   
 
7.0  ADVERSE EVENTS  
 
7.1  Definitions   
 
7.1.1  Adverse Event (AE)   
An adverse event (AE) is any untoward medical occurrence (e.g., an abnormal 
laboratory finding, symptom, or disease temporally associated with the use of a 
drug) in a patient or clinical investigation subject administered a pharmaceutical 
product and which does not necessarily have a causal relationship with this 
treatment. An AE can therefore be any unfavorable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease temporally associated with 
the use of a medicinal product, whether or not related to the medicinal product. 
Each AE will be graded on a mild-moderate-severe scale. 
 
Hospitalization for elective surgery or routine clinical procedures that are not the 
result of an AE (e.g., surgical insertion of central line) need not be considered AEs 
and should not be recorded as an AE. Disease progression should not be recorded as 
an AE, unless it is attributable by the investigator to the study therapy.  
 
7.1.2  Suspected Adverse Reaction (SAR)   
A suspected adverse reaction (SAR) is any AE for which there is a reasonable 
possibility that the drug is the cause. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the drug and the AE. A suspected 
adverse reaction implies a lesser degree of certainty about causality than adverse 
reaction, which means any adverse event caused by a drug. Each SAR will be graded 
on a mild-moderate-severe scale. 
 
Causality assessment to a study drug is a medical judgment made in consideration of 
the following factors: temporal relationship of the AE to study drug exposure, 
known mechanism of action or side effect profile of study treatment, other recent or 
concomitant drug exposures, normal clinical course of the disease under 
investigation, and any other underlying or concurrent medical conditions. Other 
factors to consider in considering drug as the cause of the AE:  
• Single occurrence of an uncommon event known to be strongly associated 
with drug exposure (e.g., angioedema, hepatic injury, Stevens-Johnson 
Syndrome)   
• One or more occurrences of an event not commonly associated with drug 
exposure, but otherwise uncommon in the population (e.g., tendon rupture); 
often more than one occurrence from one or multiple studies would be 
needed before the sponsor could determine that there is reasonable 
possibility that the drug caused the event.   
• An aggregate analysis of specific events observed in a clinical trial that 

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indicates the events occur more frequently in the drug treatment group than 
in a concurrent or historical control group 
 
7.1.3  Unexpected AE or SAR   
An AE or SAR is considered unexpected if the specificity or severity of it is not 
consistent with the applicable product information (e.g., Investigator’s Brochure 
(IB) for an unapproved investigational product or package insert/summary of 
product characteristics for an approved product). Unexpected also refers to AEs or 
SARs that are mentioned in the IB as occurring with a class of drugs or as 
anticipated from the pharmacological properties of the drug, but are not specifically 
mentioned as occurring with the particular drug under investigation.  
 
7.1.4  Serious AE or SAR   
An AE or SAR is considered serious if, in the view of either the investigator or 
sponsor, it results in any of the following outcomes:   
• Death 
• Is life-threatening (places the subject at immediate risk of death from the 
event as it occurred) 
• Requires inpatient hospitalization (>24 hours) or prolongation of existing 
hospitalization 
• Results in congenital anomaly/birth defect 
• Results in a persistent or significant incapacity or substantial disruption of 
the ability to conduct normal life functions 
• Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered a serious adverse drug experience 
when, based upon appropriate medical judgment, they may jeopardize the 
patient or subject and may require medical or surgical intervention to 
prevent one of the outcomes listed in the definition. For reporting purposes, 
also consider the occurrences of pregnancy as an event which must be 
reported as an important medical event.  
 
*Hospitalization for anticipated or protocol specified procedures such as 
administration of chemotherapy, central line insertion, metastasis interventional 
therapy, resection of primary tumor, or elective surgery, will not be considered 
serious adverse events.  
 
Pregnancy that occurs during the study must also be reported as an SAE.  
 
7.2  Documentation of non-serious AEs or SARs   
For non-serious AEs or SARs, documentation must begin from day 1 of study 
treatment and continue through 24 hours after treatment is discontinued. 
 Collected information should be recorded in the Case Report Forms (CRF) for that 
patient. A description of the event, its severity or toxicity grade, onset and resolved 
dates (if applicable), and the relationship to the study drug will be documented.  

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7.3  SAEs or Serious SARs  
 
7.3.1  Timing   
For any experience or condition that meets the definition of an SAE or a serious SAR, 
recording of the event must begin from day 1 of study treatment and continue 
through the 30-day follow-up period after treatment is discontinued.  
 
7.3.2  Documentation and Notification   
These events (SAEs or Serious SARs) must be recorded within 24 hours of learning 
of its occurrence.  
 
7.3.3  Reporting  
IRB Reporting Requirements:  
• UNC will submit an aggregated list of all SAEs to the UNC IRB annually at the 
time of study renewal according to the UNC IRB policies and procedures.   
• The UNC-IRB will be notified of all SAEs that qualify as an Unanticipated 
Problem as per the UNC IRB Policies using the IRB’s web-based reporting 
system (see section 9.4.3) within 7 days of the Investigator becoming aware 
of the problem.  
 
Pregnancy  
Pregnancies and suspected pregnancies (including a positive pregnancy test 
regardless of age or disease state) of a female subject occurring while the subject is 
on study should be recorded as SAEs. The patient is to be discontinued immediately 
from the study. The female subject should be referred to an obstetrician-
gynecologist, preferably one experienced in reproductive toxicity for further 
evaluation and counseling.  
 
The Investigator will follow the female subject until completion of the pregnancy, 
and must document the outcome of the pregnancy (either normal or abnormal 
outcome). If the outcome of the pregnancy was abnormal (e.g., spontaneous or 
therapeutic abortion), the Investigator should report the abnormal outcome as an 
AE. If the abnormal outcome meets any of the serious criteria, it must be reported as 
an SAE.  
 
FDA Expedited Reporting requirements for studies conducted under an IND:  
If an investigator deems that an event is both a serious SAR AND unexpected, it must 
also be recorded on the MedWatch Form 3500A as per 21 CFR 312.32. The 
MedWatch 3500a form can be accessed at: 
http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.ht
m. (Please be sure and access form 3500a, and not form 3500). For Affiliate 
Investigators, the MedWatch form should be faxed to the UNC Study Coordinator at 
919-966-0817 along with supporting documentation defining the event and 

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causality. UNC, as the Sponsor of the study, will make the final determination 
regarding FDA submission. 
  
Once the UNC Principal Investigator determines an event is a serious SAR AND 
unexpected, the MedWatch 3500A form will be submitted to the FDA.  
 
The UNC Study Coordinator will also be responsible for informing each Affiliate site 
of all serious and unexpected SARs reported to the FDA via fax as soon as possible.   
 
7.4 Data and Safety Monitoring Plan  
The Principal Investigator will provide continuous monitoring of patient safety in 
this trial with periodic reporting to the Data and Safety Monitoring Committee 
(DSMC).  
 
Meetings/teleconferences will be held at a frequency dependent on study accrual, 
and in consultation with the study Biostatistician.  These meetings will include the 
investigators as well as protocol nurses, clinical research associates, regulatory 
associates, data managers, biostatisticians, and any other relevant personnel the 
principal investigators may deem appropriate.  At these meetings, the research team 
will discuss all issues relevant to study progress, including enrollment, safety, 
regulatory, data collection, etc. 
 
The team will produce summaries or minutes of these meetings. These summaries 
will be available for inspection when requested by any of the regulatory bodies 
charged with the safety of human subjects and the integrity of data including, but 
not limited to, the oversight (Office of Human Research Ethics (OHRE) Biomedical 
IRB, the Oncology Protocol Review Committee (PRC) or the North Carolina TraCS 
Institute Data and Safety Monitoring Board (DSMB).   
 
The UNC LCCC Data and Safety Monitoring Committee (DSMC) will review the study 
on a regular (quarterly to annually) basis, with the frequency of review based on 
risk and complexity as determined by the UNC Protocol Review Committee.  The 
UNC PI will be responsible for submitting the following information for review: 1) 
safety and accrual data including the number of patients treated; 2) significant 
developments reported in the literature that may affect the safety of participants or 
the ethics of the study; 3) preliminary response data; and 4) summaries of team 
meetings that have occurred since the last report.  Findings of the DSMC review will 
be disseminated by memo to the UNC PI, PRC, and the UNC IRB and DSMB.   
 
7.4.1 Monitoring Safety of Participants 
Blood and urine test results within 12 weeks of study recruitment will be obtained 
from the subject’s clinical provider at the time of enrollment, as a part of the 
standard of care disease management and for determination of inclusion (unless the 
patient has a transplant or is on dialysis). In particular, serum creatinine and any 

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evidence of hematuria will be noted. Urinalyses will be obtained just prior and post-
CEUS to detect any evidence of hematuria. Serum creatinine measurements and 
urinalyses will be followed during the duration of study follow-up with intervals at 
the discretion of the primary provider. This information will be compiled by study 
coordinators by review of the medical chart and when necessary, obtaining records 
directly from the clinical provider.  
 
7.4.2 Individual Subject Stopping 
Patient safety will be considered during the study intervention with individual 
patient withdrawal for criteria outlined in 4.4 and managed as described in 4.6. 
There are no subsequent interventions after the initial contrast-enhanced 
ultrasound.  
 
7.4.3 Study-wide Stopping 
If there is a severe SAR or a severe AE suspected to be related to the study 
intervention, CEUS, the study will be put on hold and re-evaluated as this would not 
be an expected event. 
 
7.4.4 Ensuring data accuracy and protocol compliance 
The PI will monitor the accruing data and make decisions about the ongoing safety 
of the trial. A Medical Monitor outside the study will be asked to provide an 
independent review of the study and data. The PI will be responsible for providing 
the following information for such a review: 1) safety and accrual data including the 
number of patients treated; 2) significant developments reported in the literature 
that may affect the safety of participants or the ethics of the study; 3) preliminary 
diagnostic data; and 4) summaries of team meetings that have occurred since the 
last report.  
 
8.0  STATISTICAL CONSIDERATIONS   
 
8.1  Study Design/Study Endpoints   
This is an investigator-initiated, prospective pilot study evaluating the sensitivity of 
contrast enhanced ultrasound using microbubble contrast agent (perflutren lipid; 
Definity®) in diagnosing renal malignancy in patients with kidney disease and an 
indeterminate or possibly malignant kidney mass on a screening CT/MR/US. The 
study is designed to estimate the sensitivity of CEUS as a surrogate for conventional 
imaging.  
 
8.2  Sample Size and Accrual   
This study will recruit to a target goal of 20 patients total which will be combined 
with 75 patients from a previous study (15-1866) under a similar protocol. Both 
studies use the same inclusion/exclusion criteria and patient population.  However, 
this study has slightly modified imaging techniques based on the information 
learned from the 15-1866.   

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We will also include 5 healthy volunteers for image optimization, as described on 
page 13. 
 
We expect to complete the enrollment in 6 months. Based on available literature 
regarding incidence of malignancy in indeterminate (Bosniak IIF and III) lesions31, 32 
and accuracy of CEUS for cystic kidney lesion characterization in a general, non-CKD 
population9, 16, 22, 30, 33 and our experience from a pilot study including 27 patients 
with kidney disease and an indeterminate lesion, we approximate a true positive 
rate of 10%, false positive rate of 8%, true negative rate of 80% and false negative 
rate of 2%. Given the sample size of 75 (from previous study) and these 
approximated rates, we project an achievable sensitivity of 83%, specificity of 91%, 
PPV of 56% and NPV of 98%. The 95% confidence interval for this sensitivity 
estimate is 74.5-91.5% with a width of 17%. With the additional 20 lesions, we can 
improve confidence in our sensitivity and specificity measures. 
 
8.3 Data Analysis Plans  
Primary analyses will include 1) estimating the sensitivity of CEUS qualitative 
interpretations using the truth standard of findings on 1-yr follow-up non-
contrasted US compared to baseline non-contrasted US and 2) comparing the 
sensitivity and specificity of two different classification schemes for classifying 
lesions on CEUS images compared to the truth standard of findings on 1-yr follow-
up non-contrasted US compared to baseline non-contrasted US. 
 
Descriptive summary statistics such as mean, median and standard deviation of 
tumor measurement will be provided to quantify and characterize kidney lesions.  
Similarly, other patient clinical and demographic characteristics will be 
summarized, using frequencies in the case of categorical variables.  
 
For the primary objective, sensitivity and specificity and their 95% confidence 
intervals will be computed for CEUS qualitative interpretations compared to change 
in baseline non-contrasted US. Based on the truth standard diagnosis of lesion 
progression at one year follow-up, a 95% confidence interval for the Mantel-
Haenszel odds ratio will be constructed to quantify the odds that a subject with a 
diagnosis of a malignant lesion by non-contrasted US will be diagnosed with a 
malignant lesion by CEUS relative to the odds for a subject without a malignant 
lesion diagnosis by non-contrasted US.  A similar odds ratio and 95% confidence 
interval will be constructed for subjects with a truth standard diagnosis of stable or 
lesion regression.  Sensitivities, specificities are based on diagnoses made using 
CEUS-based risk classification (class I, II, and IIF considered negative, class III and IV 
as positive, using modified Bosniak criteria).  Estimation of sensitivity and 
specificity will be carried out for each reader.  Agreement between readers will be 
based on kappa statistics for the dichotomous diagnosis and using weighted kappas 
based on the ordinal Bosniak scale.  

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As part of a the secondary object, a  sensitivity analysis, the sensitivity of CEUS 
(qualitative interpretation metrics), will be compared to the sensitivity when other 
standards are incorporated when available, including pathology and additional 
imaging with contrasted or non-contrasted CT or MR. 
   
For the secondary objective, the prediction performance of the two different 
classification schemes will be compared by generating sensitivity, specificity and 
their 95% confidence intervals compared to the truth standard. 
 
9.0  STUDY MANAGEMENT 
 
9.1  Institutional Review Board (IRB) Approval and Consent   
It is expected that the IRB will have the proper representation and function in 
accordance with federally mandated regulations. The IRB should approve the 
consent form and protocol. In obtaining and documenting informed consent, the 
investigator should comply with the applicable regulatory requirement(s), and 
should adhere to Good Clinical Practice (GCP) Good Laboratory Practice (GLP) and 
to ethical principles that have their origin in the Declaration of Helsinki. Before 
recruitment and enrollment into this study, the patient will be given a full 
explanation of the study and will be given the opportunity to review the consent 
form. Each consent form must include all the relevant elements currently required 
by the FDA Regulations and local or state regulations. Once this essential 
information has been provided to the patient and the investigator is assured that the 
patient understands the implications of participating in the study, the patient will be 
asked to give consent to participate in the study by signing an IRB-approved consent 
form.  Prior to a patient’s participation in the trial, the written informed consent 
form should be signed and personally dated by the patient and by the person who 
conducted the informed consent discussion.  
 
9.2  Required Documentation   
Before the study can be initiated at any site, the following documentation must be 
provided to the Study Sponsor, Lantheus.  
• A copy of the official IRB approval letter for the protocol and informed 
consent   
• CVs and medical licensure for the principal investigator and any co-
investigators who will be involved in the study 
• Form FDA 1572 appropriately filled out and signed with appropriate 
 documentation (NOTE: this is required if UNC holds the IND. Otherwise, 
the Investigator’s signature documenting understanding of the protocol and 
providing commitment that this trial will be conducted according to all 
stipulations of the protocol is sufficient to ensure compliance)   
• Executed clinical research contract  
  

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9.3  Data Management and Monitoring/Auditing   
The Radiology Research Department will serve as the coordinating center for this 
trial. Data will be collected on paper charts, translated into Excel flowsheets and 
housed in the Radiology Research Department. Radiology Research Department will 
coordinate and manage data for quality control assurance and integrity. There will 
be three components of data collection: 1) CEUS data, stored in the Dayton lab under 
the unique identifier; 2) general patient data, stored in the radiology study 
coordinator database; 3) study specific patient clinical information including blood 
and urine tests and “truth standard” conventional US results and any additional 
imaging tests or histology, stored in the study coordinator and PI’s office on a 
password-protected, secured network drive.  
 
All data will be collected and stored by Study Coordinators from the Radiology 
Research Department. All electronic data will be stored on a password-protected, 
secured computer. The study files will be password-protected. Paper records will be 
housed in a secured lockbox in a locked office. The investigators will allow monitors 
to review all source documents supporting stored data.   
 
The data management plan includes assigning a unique identifier to each patient. 
For example, 101, 102, 103, etc. If a patient has multiple lesions, these lesions will be 
designated A, B, etc. The numbering will begin where IRB study 15-1866 left off. 
These will be the unique de-identified codes for each lesion with the master code 
held in the radiology study coordinator’s office in a separate, password-protected 
database. Prior to statistical analysis, a general quality control will be performed to 
look for invalid entries/out of range findings/outliers. Only principal investigators, 
research coordinators and members of Dr. Dayton’s lab performing image and data 
analysis will have access to study data. The study biostatistician will only have 
access to de-identified data (with the possible exception of dates that are clinically 
meaningful, e.g., study visit dates). 
  
9.4  Adherence to the Protocol   
Except for an emergency situation in which proper care for the protection, safety, 
and well-being of the study patient requires alternative treatment, the study shall be 
conducted exactly as described in the approved protocol.  
 
9.4.1  Emergency Modifications   
Investigators may implement a deviation from, or a change of, the protocol to 
eliminate an immediate hazard(s) to trial subjects without prior IRB/IEC 
approval/favorable opinion. For any such emergency modification implemented, a 
UNC IRB modification form must be completed by Research Personnel within five 
(5) business days of making the change.  
 
9.4.2  Single Patient/Subject Exceptions   
Any request to enroll a single subject who does not meet all the eligibility criteria of 

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this study requires the approval of the Principal Investigator and the IRB.  
 
9.4.3  Other Protocol Deviations/Violations   
According to UNC’s IRB, a protocol deviation is any unplanned variance from an IRB 
approved protocol that:   
• Is generally noted or recognized after it occurs  
• Has no substantive effect on the risks to research participants   
• Has no substantive effect on the scientific integrity of the research plan or the 
value of the data collected   
• Did not result from willful or knowing misconduct on the part of the 
investigator(s).  
 
An unplanned protocol variance is considered a violation if the variance meets any 
of the following criteria:   
• Has harmed or increased the risk of harm to one or more research 
participants   
• Has damaged the scientific integrity of the data collected for the study 
• Results from willful or knowing misconduct on the part of the 
 investigator(s)  
• Demonstrates serious or continuing noncompliance with federal regulations, 
State laws, or University policies.  
 
If a deviation or violation occurs please follow the guidelines below:   
 
Protocol Deviations: UNC Research personnel will report the deviation to any 
sponsor or data and safety monitoring committee in accordance with their policies. 
Deviations should be summarized and reported to the IRB at the time of continuing 
review.   
 
Protocol Violations: Violations should be reported within one (1) week of the 
investigator becoming aware of the event using the same IRB online mechanism 
used to report Unanticipated Problems.   
 
Unanticipated Problems:  Any events that meet the criteria for “Unanticipated 
Problems” as defined by the IRB must be reported by the Study Coordinator using 
the IRB’s web- based reporting system.  
 
9.5  Amendments to the Protocol   
Should amendments to the protocol be required, the amendments will be originated 
and documented by the Principal Investigator. It should also be noted that when an 
amendment to the protocol substantially alters the study design or the potential risk 
to the patient, a revised consent form might be required. The written amendment, 
and if required the amended consent form, must be sent to the IRB for approval 
prior to implementation.  

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9.6  Record Retention   
Study documentation includes all CRFs, data correction forms or queries, source 
documents, Sponsor-Investigator correspondence, monitoring logs/letters, and 
regulatory documents (e.g., protocol and amendments, IRB correspondence and 
approval, signed patient consent forms).  
 
Source documents include all recordings of observations or notations of clinical 
activities and all reports and records necessary for the evaluation and 
reconstruction of the clinical research study.  
 
Government agency regulations and directives require that all study documentation 
pertaining to the conduct of a clinical trial must be retained by the study 
investigator. In the case of a study with a drug seeking regulatory approval and 
marketing, these documents shall be retained for at least two years after the last 
approval of marketing application in an International Conference on Harmonization 
(ICH) region. In all other cases, study documents should be kept on file until three 
years after the completion and final study report of this investigational study.  
 
9.7 Obligations of Investigators  
The Principal Investigator is responsible for the conduct of the clinical trial at the 
site in accordance with Title 21 of the Code of Federal Regulations and/or the 
Declaration of Helsinki. The Principal Investigator is responsible for personally 
overseeing the treatment of all study patients. The Principal Investigator must 
assure that all study site personnel, including sub-investigators and other study staff 
members, adhere to the study protocol and all FDA/GCP/GLP/NCI regulations and 
guidelines regarding clinical trials both during and after study completion.  
 
The Principal Investigator will be responsible for assuring that all the required data 
will be collected and entered into the eCRFs. Periodically, monitoring visits will be 
conducted and the Principal Investigator will provide access to his/her original 
records to permit verification of proper entry of data. At the completion of the study, 
all eCRFs will be reviewed by the Principal Investigator and will require his/her 
final signature to verify the accuracy of the data.  
 
10.0 REFERENCES  
 
1. 
Maisonneuve, P., Agodoa, L., Gellert, R. et al.: Cancer in patients on dialysis for 
end-stage renal disease: an international collaborative study. Lancet, 354: 
93, 1999 
2. 
Lowrance, W. T., Ordonez, J., Udaltsova, N. et al.: CKD and the Risk of Incident 
Cancer. J Am Soc Nephrol, 2014 

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3. 
Lind Ramskov, K., Thomsen, H. S.: Nephrogenic systemic fibrosis and contrast 
medium-induced nephropathy: a choice between the devil and the deep blue 
sea for patients with reduced renal function? Acta Radiol, 50: 965, 2009 
4. 
Huang, W. C., Levey, A. S., Serio, A. M. et al.: Chronic kidney disease after 
nephrectomy in patients with renal cortical tumours: a retrospective cohort 
study. Lancet Oncol, 7: 735, 2006 
5. 
Lindner, J. R.: Microbubbles in medical imaging: current applications and 
future directions. Nat Rev Drug Discov, 3: 527, 2004 
6. 
von Herbay, A., Vogt, C., Willers, R. et al.: Real-time imaging with the 
sonographic contrast agent SonoVue: differentiation between benign and 
malignant hepatic lesions. J Ultrasound Med, 23: 1557, 2004 
7. 
Celli, N., Gaiani, S., Piscaglia, F. et al.: Characterization of liver lesions by real-
time contrast-enhanced ultrasonography. Eur J Gastroenterol Hepatol, 19: 3, 
2007 
8. 
Wink, M. H., de la Rosette, J. J., Laguna, P. et al.: Ultrasonography of renal 
masses using contrast pulse sequence imaging: a pilot study. J Endourol, 21: 
466, 2007 
9. 
Quaia, E., Bertolotto, M., Cioffi, V. et al.: Comparison of contrast-enhanced 
sonography with unenhanced sonography and contrast-enhanced CT in the 
diagnosis of malignancy in complex cystic renal masses. AJR Am J Roentgenol, 
191: 1239, 2008 
10. 
Setola, S. V., Catalano, O., Sandomenico, F. et al.: Contrast-enhanced 
sonography of the kidney. Abdom Imaging, 32: 21, 2007 
11. 
Meloni, M. F., Bertolotto, M., Alberzoni, C. et al.: Follow-up after percutaneous 
radiofrequency ablation of renal cell carcinoma: contrast-enhanced 
sonography versus contrast-enhanced CT or MRI. AJR Am J Roentgenol, 191: 
1233, 2008 
12. 
Fan, L., Lianfang, D., Jinfang, X. et al.: Diagnostic efficacy of contrast-enhanced 
ultrasonography in solid renal parenchymal lesions with maximum 
diameters of 5 cm. J Ultrasound Med, 27: 875, 2008 
13. 
Ascenti, G., Zimbaro, G., Mazziotti, S. et al.: Usefulness of power Doppler and 
contrast-enhanced sonography in the differentiation of hyperechoic renal 
masses. Abdom Imaging, 26: 654, 2001 
14. 
Kim, A. Y., Kim, S. H., Kim, Y. J. et al.: Contrast-enhanced power Doppler 
sonography for the differentiation of cystic renal lesions: preliminary study. J 
Ultrasound Med, 18: 581, 1999 
15. 
Ascenti, G., Gaeta, M., Magno, C. et al.: Contrast-enhanced second-harmonic 
sonography in the detection of pseudocapsule in renal cell carcinoma. AJR 
Am J Roentgenol, 182: 1525, 2004 
16. 
Ascenti, G., Mazziotti, S., Zimbaro, G. et al.: Complex cystic renal masses: 
characterization with contrast-enhanced US. Radiology, 243: 158, 2007 
17. 
Park, B. K., Kim, S. H., Choi, H. J.: Characterization of renal cell carcinoma 
using agent detection imaging: comparison with gray-scale US. Korean J 
Radiol, 6: 173, 2005 

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18. 
Roy, C., Gengler, L., Sauer, B. et al.: [Role of contrast enhanced US in the 
evaluation of renal tumors]. J Radiol, 89: 1735, 2008 
19. 
Tamai, H., Takiguchi, Y., Oka, M. et al.: Contrast-enhanced ultrasonography in 
the diagnosis of solid renal tumors. J Ultrasound Med, 24: 1635, 2005 
20. 
Kalantarinia, K., Belcik, J. T., Patrie, J. T. et al.: Real-time measurement of renal 
blood flow in healthy subjects using contrast-enhanced ultrasound. Am J 
Physiol Renal Physiol, 297: F1129, 2009 
21. 
Ascenti, G., Zimbaro, G., Mazziotti, S. et al.: Contrast-enhanced power Doppler 
US in the diagnosis of renal pseudotumors. Eur Radiol, 11: 2496, 2001 
22. 
Clevert, D. A., Minaifar, N., Weckbach, S. et al.: Multislice computed 
tomography versus contrast-enhanced ultrasound in evaluation of complex 
cystic renal masses using the Bosniak classification system. Clin Hemorheol 
Microcirc, 39: 171, 2008 
23. 
Sawhney, S., Wilson, S. R.: Can Ultrasound With Contrast Enhancement 
Replace Nonenhanced Computed Tomography Scans in Patients With 
Contraindication to Computed Tomography Contrast Agents? Ultrasound Q, 
2017 
24. 
Girometti, R., Stocca, T., Serena, E. et al.: Impact of contrast-enhanced 
ultrasound in patients with renal function impairment. World J Radiol, 9: 10, 
2017 
25. 
Main, M. L., Goldman, J. H., Grayburn, P. A.: Ultrasound contrast agents: 
balancing safety versus efficacy. Expert Opin Drug Saf, 8: 49, 2009 
26. 
Chrysochou, C., Buckley, D. L., Dark, P. et al.: Gadolinium-enhanced magnetic 
resonance imaging for renovascular disease and nephrogenic systemic 
fibrosis: critical review of the literature and UK experience. J Magn Reson 
Imaging, 29: 887, 2009 
27. 
Schneider, A. G., Hofmann, L., Wuerzner, G. et al.: Renal perfusion evaluation 
with contrast-enhanced ultrasonography. Nephrol Dial Transplant, 27: 674, 
2012 
28. 
Gaibazzi, N., Squeri, A., Ardissino, D. et al.: Safety of contrast flash-
replenishment stress echocardiography in 500 patients with a chest pain 
episode of undetermined origin within the last 5 days. Eur J Echocardiogr, 
10: 726, 2009 
29. 
Aggeli, C., Giannopoulos, G., Roussakis, G. et al.: Safety of myocardial flash-
contrast echocardiography in combination with dobutamine stress testing 
for the detection of ischaemia in 5250 studies. Heart, 94: 1571, 2008 
30. 
Barr, R. G., Peterson, C., Hindi, A.: Evaluation of indeterminate renal masses 
with contrast-enhanced US: a diagnostic performance study. Radiology, 271: 
133, 2014 
31. 
Hindman, N. M., Hecht, E. M., Bosniak, M. A.: Follow-up for Bosniak category 
2F cystic renal lesions. Radiology, 272: 757, 2014 
32. 
Smith, A. D., Remer, E. M., Cox, K. L. et al.: Bosniak category IIF and III cystic 
renal lesions: outcomes and associations. Radiology, 262: 152, 2012 

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33. 
Chen, Y., Wu, N., Xue, T. et al.: Comparison of contrast-enhanced sonography 
with MRI in the diagnosis of complex cystic renal masses. J Clin Ultrasound, 
2014 
34. 
Israel, G. M., Bosniak, M. A.: An update of the Bosniak renal cyst classification 
system. Urology, 66: 484, 2005 
 
 
 

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11.0 APPENDICES  
 
Appendix I  
The Bosniak classification system is as follows34:  
 
 
Appendix 2 
Time and Events Table 
 
Screening 
Pre-CEUS 
Visit 
CEUS 
study 
Mo12 
(Wk 48) 
Early  
Termination1 
Inclusion/ 
Exclusion Criteria 
X 
 
 
 
 
Comprehensive Medical 
History 
X2 
X2 
 
 
 
Informed Consent 
X3 
X3 
 
 
 
Education of potential 
side effects 
 
X 
 
 
 
Pregnancy test (urine), if 
applicable 
 
X4 
 
 
 
Serum creatinine 
X 
 
 
X5 
X5 
Urinalysis 
 
X6 
 
X6 
 
Contrast-Enhanced US 
(CEUS) 
 
 
X7 
 
 
AE Assessment 
 
 
X8 
 
 
Request tissue diagnosis 
 
 
 
X9 
 
Obtain follow-up 
conventional US and any 
other imaging studies 
 
 
 
X 
X 

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obtained during the 
follow-up period 
 
Patients declining CEUS 
but allowing creatinine 
follow-up10 
 
 
 
 
 
Serum creatinine 
X 
 
 
X 
X 
 
1For any patients that do not complete the CEUS study, an ongoing adverse 
events/serious adverse events will be followed to the resolution of the event. For 
any patient that is deceased before the 1-year follow-up most recent creatinine 
levels and imaging studies will be collected and autopsy results documented, if 
available.  
 
2Comprehensive medical history obtained at time of enrollment; thereafter history 
focused on symptoms and assessments. 
 
3Consent can be obtained and signed at either screening visit or during the pre-
study visit. It will always be reviewed at pre-study visit, even if signed during 
screening visit. 
 
4Women of childbearing potential must have negative urine or serum pregnancy 
test on the day of CEUS study, prior to the study. 
 
5Serum creatinine levels will be obtained clinically at the discretion of the patient’s 
primary provider. We will obtain results of serum creatinine levels drawn by the 
primary provider. 
 
6Urinalysis will be obtained prior to and post CEUS study. This will be obtained by 
Radiology Nursing staff as per agreement. Results will be obtained and recorded by 
Study Coordinator. At approximate 6 and 12-month intervals, urinalysis will also be 
obtained. This can be done locally with results sent to UNC. 
 
7During the CEUS study, Study Coordinator will record time of injection. 
 
8Adverse event assessment will happen during the CEUS study and the 30-minute 
post-study monitoring period, where they will be monitored by the Radiology 
nursing staff with vital signs (blood pressure and heart rate) recorded in the 
hospital’s electronic monitoring system. These results will be collected by the Study 
Coordinator and documented in the Monitoring Logs/Flowsheets housed in the 
Radiology Research Coordinator Office. 
 
9For patients who undergo biopsy or nephrectomy, tissue diagnosis will be 
requested after the procedure. 
 

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10For patients who decline study enrollment with CEUS but meet all study 
inclusion/exclusion criteria but agree to allow us to collect creatinine values from 
their primary providers over the study period, serum creatinine will be collected, if 
available at the designated time periods with a minimum value at 1-year.