Contrast-Enhanced Ultrasound for Kidney Cancer Subtyping and Staging
Phase: PHASE2
Conditions: Kidney Cancer
Sponsor: UNC Lineberger Comprehensive Cancer Center

Study Title: Contrast-Enhanced Ultrasound for Kidney Cancer 
Subtyping and Staging 
 
NCT Number: NCT04021238 
 
Study Protocol 
 
Date of Document: January 4, 2022 
 
 

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LCCC  1922: Contrast-Enhanced Ultrasound for Kidney Cancer Subtyping 
and Staging 
 
 
Principal Investigator 
Emily Chang, MD 
Department of Medicine, Division of Nephrology 
7024 Burnett Womack, CB 7155 
(919) 445-2621 
 
Principal Co-Investigator  
Lauren Burke, MD 
 
 
Biostatistician  
Thad Benefield, MS 
 
 
Sponsor:  Lineberger Comprehensive Cancer Center 
 
Funding Source: Lantheus Medical Imaging 
 
Version Date: January 4, 2022 
 

LINEBERGER COMPREHENSIVE CANCER CENTER 
Imaging Protocol Template 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
 
LCCC  1922: Contrast-Enhanced Ultrasound for Kidney Cancer Subtyping 
and Staging 
 
Principal Investigator 
Emily Chang, MD 
Department of Medicine, Division of Nephrology 
7024 Burnett Womack, CB 7155 
(919) 445-2621 
 
 
 
 
 
 
 
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:__Emily Chang________ 
 
PI Signature: _____
______________ 
 
Date:____________________ 
 
Date of Protocol: January 4, 2022 
 
 
 
 

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TABLE OF CONTENTS 
1.0 
BACKGROUND AND RATIONALE ...................................................................4 
1.1 
Study Synopsis ...................................................................................................4 
1.2 
Kidney Cancer .....................................................................................................4 
1.3 
Kidney Cancer Histologic Subtypes ................................................................5 
1.4 
Tumor staging .....................................................................................................5 
1.5 
Chronic Kidney Disease ....................................................................................5 
1.6 
Contrast Enhanced Ultrasound ........................................................................6 
1.7 
Contrast-Enhanced Ultrasound Diagnostic Imaging of the Kidney ............7 
1.8 
Contrast Agents ..................................................................................................7 
1.9 
Associated Toxicities .........................................................................................8 
1.10 
Cardiopulmonary Reactions ..........................................................................8 
1.11 
Hypersensitivity Reactions ............................................................................9 
1.12 
High Ultrasound Mechanical Index (MI) ......................................................9 
1.13 
Preclinical Data ...............................................................................................9 
1.14 
Clinical Data to Date .....................................................................................10 
1.15 
Dose Rationale and Risk/Benefits ..............................................................10 
2.0 
STUDY OBJECTIVES.........................................................................................11 
2.1 
Primary Objectives ...........................................................................................11 
2.2 
Secondary Objectives ......................................................................................11 
2.3 
Endpoints ...........................................................................................................11 
3.0 
PATIENT ELIGIBILITY .......................................................................................11 
3.1 
Inclusion Criteria ...............................................................................................11 
3.2 
Exclusion Criteria ..............................................................................................11 

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4.0 
STUDY PLAN .......................................................................................................12 
4.1 
STUDY SCHEMA .............................................................................................12 
4.2 
Study Procedures .............................................................................................13 
4.3 
Time and Events Table ....................................................................................14 
5.0 
INVESTIGATIONAL CONTRAST AGENT ......................................................14 
5.1 
Description .........................................................................................................14 
5.2 
IND Exemption ..................................................................................................15 
5.3 
Treatment Regimen .........................................................................................15 
5.4 
Preparation and Administration of Study Drug ............................................15 
5.5 
Subject Compliance Monitoring .....................................................................15 
5.6 
Prior and Concomitant Therapy .....................................................................15 
5.7 
Packaging ..........................................................................................................15 
5.8 
Receiving, Storage, Dispensing and Return ................................................16 
6.0 
ADVERSE EVENTS ............................................................................................16 
6.1 
Definitions ..........................................................................................................16 
6.2 
Documentation of non-serious AEs or SARs ...............................................18 
6.3 
SAEs or Serious SARs ....................................................................................18 
6.4 
Data and Safety Monitoring Plan ...................................................................19 
7.0 
STATISTICAL CONSIDERATIONS .................................................................20 
7.1 
Sample Size and Accrual ................................................................................20 
7.2 
Data Analysis Plans .........................................................................................21 
8.0 
STUDY MANAGEMENT .....................................................................................23 
8.1 
Institutional Review Board (IRB) Approval and Consent ...........................23 
8.2 
Registration Procedures ..................................................................................24 

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8.3 
Data Management and Monitoring/Auditing .................................................24 
8.4 
Adherence to the Protocol ...............................................................................24 
8.5 
Amendments to the Protocol ..........................................................................25 
8.6 
Record Retention ..............................................................................................26 
8.7 
Obligations of Investigators .............................................................................26 
9.0 
REFERENCES .....................................................................................................26 
 
 
 

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1.0 
BACKGROUND AND RATIONALE 
1.1 
Study Synopsis 
This is a single arm, non-randomized study of 25 adult subjects with 
suspected kidney cancer and planned surgical nephrectomy that will 
undergo a contrast enhanced ultrasound (CEUS) of the kidneys to predict 
kidney mass histologic diagnosis, subtype and stage. 
1.2 
Kidney Cancer 
Kidney cancer incidence has more than doubled over the past 2 decades 
with nearly 70,000 new cases projected for 2019[1, 2]. This exponential 
growth is attributable largely to increased detection of incidental small 
kidney masses[3, 4]. Most of these small masses are resected; however, 
only 20% harbor aggressive disease. The remaining 80% consist of benign 
tumors or non-aggressive cancers[5-7] that may not need resection. 
Unfortunately, current diagnostic techniques do not reliably differentiate 
aggressive from non-aggressive subtypes. Identifying non-aggressive 
subtypes pre-resection would reduce unnecessary surgeries and 
associated risks including kidney function loss, a risk particularly high 
among individuals with chronic kidney disease (CKD).  
 
Contrast-enhanced computed tomography (CT) or magnetic resonance 
imaging (MRI) are the standard tests to diagnose kidney cancer and guide 
management. However, CT and MRI have key disadvantages that limit their 
ability to precisely diagnose and stage kidney cancer resulting in costly 
surgeries and their associated morbidities in patients that may not have 
needed resection. First, both modalities capture image snapshots over time, 
and important enhancement characteristics may be missed if the snapshot 
is not timed correctly. Second, contrast extravasation leads to decreased 
sensitivity to vascular enhancement, limiting their staging ability. Third, CT 
and MRI are contraindicated among individuals with moderate and severe 
CKD, contrast allergies and/or metal implants, relegating these patients to 
substandard diagnostics. Ultrasound (US) is a safe, inexpensive and 
accessible imaging tool often used in patients with CKD, but it suffers from 
a lack of sensitivity. Given the rising incidence of kidney cancer and the 
increasing number of individuals with CKD who cannot undergo CT/MRI, 
there is an urgent and unmet need to identify imaging that can safely and 
accurately differentiate kidney masses needing resection from those that do 
not. Moreover, among all individuals with kidney masses, including those 
without CT/MRI contraindications, there is a clear and unmet need to 
identify imaging that can accurately diagnose subtype and characterize 
vascular involvement, a key factor in cancer staging.  
 
We propose to investigate an advanced contrast-enhanced ultrasound 
(CEUS) technique as a novel imaging tool to address these critical gaps in 
kidney mass diagnosis and staging. We will accomplish the following aims: 

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1) to predict histologic subtype and stage using 3D CEUS-generated 
metrics in humans and 2) to compare the predictive capabilities of 2 different 
CEUS imaging techniques (one using bolus and one using infusion 
injection) in humans. 
1.3 
Kidney Cancer Histologic Subtypes 
The majority of kidney cancers are renal cell carcinomas (RCC) of which 
there are three main subtypes: clear cell, papillary and chromophobe. The 
subtypes differ in incidence, prognosis[8, 9] and vascularity[10]. Accurate 
subtyping is critical to clinical decision-making as less aggressive subtypes 
do not require immediate resection. Differentiation of clear cell (a higher risk 
subtype requiring resection) from other subtypes or benign kidney masses 
has the potential to reduce unnecessary surgeries[6]. CT/MRI, the 
predominant imaging modalities used to diagnose RCC, do not reliably 
predict cancer subtype or grade. Other than surgical resection, biopsy is the 
only reliable way to preoperatively determine histologic subtype and tumor 
grade. However, biopsy is not performed at all centers[11] and is subject to 
sampling error, with as many as 20% of biopsies being non-diagnostic[12].  
 
Differences in tissue vascularity cause differing tissue enhancement[13]. 
CEUS is highly sensitive for tissue enhancement which can be leveraged 
to differentiate kidney mass subtype based on enhancement properties. 
Previous studies have reported accurate diagnosis[14] and subtyping[15] of 
kidney masses using single plane CEUS with qualitative interpretations. 3D 
CEUS-generated metrics will capture enhancement in multiple planes and 
thus produce a more complete picture of the entire mass. Specifically, we 
will use 3D CEUS-generated quantitative metrics to characterize mass 
vascularity. 
1.4 
Tumor staging 
For malignant masses requiring resection, treatment decisions are often 
based on CT/MRI-derived staging. Tumors confined to the kidney are stage 
1 or 2. Vascular involvement classifies a tumor as stage 3 or higher. 
However, CT/MRI often miss subtle vascular involvement, leaving such 
involvement undetected until pathologic inspection post-resection, making 
preoperative prognosis estimation inaccurate. Stages 1, 2 and 3 have very 
different survival rates (81%, 74% and 53%[1], respectively), rendering 
accurate preoperative staging essential for surgical planning and patient 
counseling. 3D CEUS’ vascular enhancement sensitivity and real-time 
imaging may better characterize small vessel involvement, aiding surgical 
planning and patient counseling. In addition, 3D volume rendering of the 
vessels may be useful for vasculature-based mass staging.  
1.5 
Chronic Kidney Disease 
Individuals with advanced CKD and a kidney mass offer a unique challenge. 
These patients have an increased risk of kidney cancer, and risk rises with 

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CKD severity[16]. As CKD severity worsens, contrast CT/MRI risks also 
increase. If such patients undergo contrast CT, they risk kidney function 
loss and potential progression to end-stage kidney disease. If they undergo 
contrast MRI, they risk development of a rare but devastating disease, 
nephrogenic sclerosing fibrosis. However, without such imaging, they either 
risk missed detection of a mass needing resection or undergo preemptive 
resection, potentially of a mass not needing resection and resulting in 
further loss of kidney function. Identification of a contrast imaging tool that 
does not pose increased risks to patients with CKD would eliminate these 
unnecessary risks. 
The advanced CEUS techniques of 3D imaging provides a powerful 
opportunity to fill existing gaps in kidney mass care, including subtype 
prediction and staging. Additionally, for those with advanced CKD and other 
contraindications to CT/MRI, CEUS allows for contrast imaging not possible 
with existing imaging options. 
1.6 
Contrast Enhanced Ultrasound 
CEUS has several features that distinguish it from contrast CT/MRI. Unlike 
contrast CT/MRI, CEUS captures real-time images, enabling dynamic 
visualization of contrast over time. In addition, the CEUS contrast agent, 
microbubbles, remains intravascular, and is thus sensitive to lesion 
enhancement and small vessel involvement, giving CEUS the potential to 
inform kidney mass staging and surgical planning, capabilities not currently 
possessed by CT/MRI.  
 
In addition, CEUS microbubbles differ from CT/MRI contrast agents in that 
they are cleared by the lungs within minutes of administration and have no 
known nephrotoxicity, making CEUS safe for patients with advanced CKD. 
For these individuals, CEUS availability would mean access to contrast-
enhanced imaging, which would significantly improve the ability to provide 
a definitive mass diagnosis and increase the chance of avoiding 
unnecessary surgery. CEUS maintains the advantages of conventional US 
(lower cost in comparison to CT/MRI and safety).  
 
To-date, most CEUS research has utilized a 2D approach. By adapting 
routine imaging techniques, we can generate three-dimensional (3D) CEUS 
images. Our preliminary work suggests that expanding 2D CEUS to a 3D 
technique will provide deeper data about mass enhancement, facilitating 
subtype differentiation. Moreover, our group has developed image analysis 
techniques that can generate 3D renderings of the vasculature (described 
below).  
 
In 2D or 3D CEUS, microbubbles can be administered by bolus or 
continuous infusion dosing which correspond with low mechanical index 
(MI) and flash-replenishment imaging (F-R), respectively. The two 
approaches yield different information. The optimal approach is unknown. 

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With bolus administration of undiluted microbubbles, CEUS quantifies 
measurements like peak intensity, time to peak intensity, and wash-
in/wash-out rates from the time intensity curve (TIC), each of which may be 
used to predict mass subtype. Alternatively, infusion of diluted microbubbles 
facilitates longer imaging times and uses faster frame rates. Longer imaging 
time allows for imaging in multiple planes which can capture multiple 
masses or different planes in a single mass, providing richer information. 
Faster frame rates can capture rapid wash-in rates with greater granularity 
than bolus dosing. However, the infusion method TIC does not quantify a 
wash-out rate, eliminating one metric that may contribute to subtype 
classification accuracy. Existing data suggest that bolus dosing 2D CEUS 
has comparable accuracy for malignancy detection compared to contrast-
enhanced CT and MRI[17]. The accuracy of infusion dosing is unknown. 
We will adapt both approaches to generate 3D CEUS images. 
1.7 
Contrast-Enhanced Ultrasound Diagnostic Imaging of the Kidney 
Ultrasound (US) imaging of the kidney is typically performed in the United 
States without contrast as no US contrast agent is currently approved for 
this indication. Without contrast, malignant tumors often cannot be 
definitively differentiated from benign tumors or pseudotumors. For this 
reason, following the identification of a kidney lesion via US without 
contrast, contrast-enhanced CT or MRI is then performed to stage and 
grade the lesion. 
 
CEUS of the kidney for mass or lesion characterization is currently being 
performed in only certain centers in the United States and is more 
widespread outside the United States. 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 μm, remain intravascular for several minutes, and 
do not diffuse into the interstitium (pure blood-pool agents)[18]. Three US 
contrast agents are Food and Drug Administration (FDA) approved for 
human use: perflutren lipid microspheres (Definity®), sulfur hexafluoride 
lipid microspheres and perflutren protein-type A microspheres (Optison®). 
All are FDA indicated for use in cardiac studies. The intent of this study is 
not to change the labeling of either FDA-approved agent. 
1.8 
Contrast Agents 
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 

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http://www.accessdata.fda.gov/scripts/cder/drugsatfda/ for full prescribing 
information. When used in this setting, the maximum dose of perflutren is 
administered as either bolus doses or one single intravenous infusion.  
 
1.9 
Associated Toxicities 
Most safety studies have been performed in cardiac patients as this was the 
only FDA approved indication. Thus far, microbubble agents have been 
shown to be quite safe[19, 20]. 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, 1 due to a hypersensitivity reaction and the rest due to 
dizziness, chest pain, dyspnea or back pain. The events appeared within 1 
– 15 minutes of the drug administration and were of moderate intensity 
resolving usually without treatment within minutes or hours after onset. 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. 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.  
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%).  
Initial post-marketing experience, which included over 1 million patients with 
5 years surveillance, showed the only medically significant risk was rare 
allergic events, occurring at a rate of 1 in 10,000[20].  
1.10 Cardiopulmonary Reactions 
Cardiopulmonary 
contraindications 
include 
cardiac 
shunts 
and 
hypersensitivity to perflutren. The initial mandated 30-minute monitoring 
period was limited to patients with pulmonary hypertension or unstable 
cardiopulmonary conditions. The mandatory 30-minute monitoring period 
was removed in 2011, although a statement remained 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 patients be monitored for acute reactions.  
 
Although recent studies have shown no increased risk in patients with 
pulmonary hypertension[21], patients with a history of known cardiac 
shunts, pulmonary hypertension or unstable cardiopulmonary conditions 

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will be excluded from our study. In addition, any patient with a suspected 
reaction during imaging will be monitored 30-minutes post-contrast agent 
administration by clinical or research nurse.  
1.11 Hypersensitivity Reactions 
Anaphylaxis to perflutren is extremely rare, however, in case of a severe 
reaction, EpiPen® (epinephrine) injections will be kept near the US machine 
for all patients.  
1.12 High Ultrasound Mechanical Index (MI) 
While high ultrasound MI of the heart may cause microbubble cavitation and 
subsequent ventricular arrhythmias, this was seen only at MI>1.6, higher 
than the typical MI used for microbubble disruption, with an agent no longer 
in clinical use.[22] No premature ventricular contractions were seen with 
MI≤1.1. Large clinical trials using MI≤1.0 for expanded cardiac indications 
have shown no significant concerns for arrhythmias[9, 20, 23]. 
Nevertheless, the safety of perflutren at MIs >0.8 has not yet been 
established by the manufacturer. However, real-time measurement of renal 
blood flow in 19 healthy subjects using perflutren was 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 
20mmHg 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[24]. A second study using 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[25].  
Small pre-clinical studies performed in rodents and pigs[26-29] showed 
conflicting results. One group showed glomerular capillary hemorrhage in 
exposed animals while other groups showed no sign of damage. Recently 
published work in a rat model using clinically relevant parameters for CEUS 
high MI flash-replenishment imaging showed no long-term changes in 
histology or clinical chemistry measurements[30]. The clinical relevance of 
these findings has not yet been investigated and is believed to be minimal, 
if any. 
1.13 Preclinical Data 
Numerous pre-clinical studies of CEUS have been performed, primarily in 
rodent kidneys but also larger animals like rabbit and pig, that investigate 
overall perfusion of diseased kidneys[31-33] or kidneys exposed to various 
medications[34, 35]. In the field of oncology, many pre-clinical studies have 
been performed to determine the sensitivity of CEUS for detection of 
malignancy[36-38]. The use of targeted microbubbles (with antibodies 
attached to the microbubble surface) has been investigated in this field as 
well[39, 40]. 

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1.14 Clinical Data to Date 
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-time[41-47]. Outside 
of the United States, or in the context of clinical trials, other US contrast 
agents have been used in kidney imaging[43, 44, 47-51]. 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 also 
been used in the general population to identify and diagnose kidney 
pseudotumors[52], cystic kidney lesions[44, 48, 50, 53], and solid kidney 
lesions[47, 51]. 
In the United States, where there has been significantly less research on 
using US contrast in humans, the US contrast agent, Definity®, was used 
in one study to measure renal blood flow in a healthy population[24], 
effectively reproducing previous animal studies in the human population. 
Multiple contrast agents were used in a large retrospective study of CEUS 
in patients with kidney lesions[54] and showed excellent sensitivity (100%) 
and specificity (99%). Since that study, several smaller studies in the United 
States have looked at CEUS in the kidney and consistently shown excellent 
sensitivity[55, 56]. Studies outside the United States show similar 
sensitivities and specificities[17, 57]. 
1.15 Dose Rationale and Risk/Benefits 
Dosages for Definity are based on package inserts, expert opinion from 
radiologists who routinely use CEUS for kidney imaging, and our previous 
experience from clinical trials (IRB 12-2314, 15-1866, 17-1130). Because 
the kidney is a very vascular organ, use of 0.2mL Definity has been 
adequate for visualization of lesion enhancement. Our experience has been 
with both bolus doses of 0.2mL and infusion at set rates based on BMI. The 
infusion rate or dose can be increased or decreased if visualization is not 
adequate. Use of either small boluses or infusion allows for multiple 
injections for patients with multiple lesions. Patients with greater abdominal 
girth and multiple lesions may require a 2nd vial as their bolus doses or rate 
of infusion will be higher. All microbubble contrast agents are injected 
intravenously. 
The greatest risk is of an unknown hypersensitivity reaction to the 
microbubbles as many patients will not have previous exposure to these 
contrast agents. However, the rate of true allergic reactions is low and 
similar to that for gadolinium-based contrast agents. Other risks are 
transient and resolve when the contrast agent is cleared. Overall the risk is 
minimal. Benefits include improved visualization of kidney lesions 
compared to non-contrasted ultrasound and the potential reduction of total 

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lifetime load of gadolinium, exposure to radiation from CT and improvement 
in cost-effectiveness. 
2.0 
STUDY OBJECTIVES 
2.1 
Primary Objectives 
2.1.1 To predict kidney mass diagnosis using 3D CEUS generated metrics (see 
Section 7.2.1). 
2.2 
Secondary Objectives  
2.2.1 To compare diagnostic accuracy of F-R infusion imaging technique to low 
MI bolus imaging technique. 
2.2.2 To predict kidney mass stage using 3D CEUS-generated metrics 
2.3 
Endpoints 
The primary study endpoint will be 3D CEUS generated metric(s) that best 
predict kidney mass diagnosis based on pathologic examination. 
3.0 
PATIENT ELIGIBILITY 
3.1 
Inclusion Criteria 
3.1.1 A suspected diagnosis of kidney cancer with a solid or partially solid lesion 
and planned surgical nephrectomy within 3 months before surgery 
3.1.2 Able to provide written informed consent  
3.1.3 Willing to comply with protocol requirements 
3.1.4 At least 18 years of age 
3.2 
Exclusion Criteria 
3.2.1 Critically ill or medically unstable or in an intensive care setting and whose 
critical course during a potential observation period would be 
unpredictable  
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 

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3.2.4 Has any other medical condition or other circumstances that would 
significantly decrease the chances of obtaining reliable data or of 
achieving the study objectives 
3.2.5 Unstable cardiopulmonary 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 
• 
Symptomatic arrhythmia (i.e. tachycardia, bradycardia, 
supraventricular tachycardia, ventricular fibrillation, ventricular 
tachycardia, atrial flutter or fibrillation) 
• 
Myocardial infarction within 14 days prior to the date of proposed 
microbubble administration.  
3.2.6 Any woman who is pregnant or has reason to believe she is pregnant (the 
possibility of pregnancy has to be excluded by negative urine β-HCG 
results, obtained the same day as the CEUS, or on the basis of patient 
history, e.g.: tubal ligation, hysterectomy or a minimum of 1 year without 
menses) 
3.2.7 Obesity that limits obtainment of acceptable images 
4.0 
STUDY PLAN 
4.1 
STUDY SCHEMA 
 
 
Clinical Scan
Patient Receives clinical 
scan
Contrast Enhanced 
Ultrasound
Patient Receives 
investigational scan at 
research visit
Clinical Nephrectomy
Patient receives standard of 
care treatment
Chart Review
Patient charts are reviewed 
to obtain pathology results
CEUS Quantitative Analysis
Metrics will be used to 
generate a score which will 
be used to predict 
pathologic diagnosis

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4.2 
Study Procedures 
4.2.1 Enrollment/Recruitment 
We will recruit subjects from UNC urology and urologic oncology clinics. 
Patients ≥18 years of age who have at least one suspected kidney cancer 
with a solid or partially solid lesion and planned surgical resection will be 
eligible. Pregnant patients and patients with known adverse microbubble 
reactions will be excluded. We will approach patients during clinic visits or 
by telephone and schedule consenting individuals for 3D CEUS within 3 
months before planned surgery. If surgery is delayed, lesions will be 
reimaged prior to surgery to meet the 3-month window. The goal is to 
minimize changes that may occur in tumor pathology between the time of 
imaging and time of resection. Subjects may also be asked to return for an 
additional imaging visit if they are determined to have more than one eligible 
lesion.  
4.2.2 Imaging Procedures 
4.2.2.1 Low MI imaging with sweeps 
Upon subject arrival, a urine pregnancy test will be conducted for subjects 
with child bearing potential.  Once confirmed that a subject is eligible, a 
nurse will place an intravenous (IV) catheter, and a sonographer will 
determine mass location and approach with a baseline B-mode US. The 
nurse will prepare the CEUS contrast agent, Definity®, to be administered 
via infusion pump according to package insert instructions. We will use a 
Siemens ACUSON Sequoia S512 with a 4C1 curved array transducer 
equipped with cadence pulse sequencing technology for contrast-enhanced 
imaging.  
 
Each subject will be injected with a bolus of 0.2-0.4 mL undiluted 
microbubbles placed on an infusion pump for consistent injection rate and 
followed by 5mL sterile saline. The sonographer will capture sweeps 
through the kidney in 2-3 different planes (i.e. x-y, y-z, or x-z) of imaging 
during contrast infusion. Each imaging plane will require a new bolus. 
During imaging, the subject will be instructed to take shallow breaths to 
minimize movement artifact.We will capture a B-mode image perpendicular 
to each sweep direction to determine the total length of each sweep. The 
subject will be instructed to take shallow breaths to minimize movement 
artifact. Imaging time will be 15-20 minutes.  
4.2.2.2 Flash-replenishment infusion imaging 
Following low MI bolus imaging, we will use the F-R infusion protocol and 
infuse diluted microbubbles with a calibrated syringe pump. We will 
translate the transducer across the mass in 0.5-1 cm steps to image the 
entire mass, executing a F-R pulse at each step. Imaging time will be 3-15 
minutes depending on mass size. We will monitor the subject for adverse 

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reactions during the procedure. Images will be coded and stored for later 
analysis. 
4.2.3 Medical Record Abstraction 
We will perform chart review to obtain histologic diagnosis of subtype as 
determined by standard of care pathologic examination. Routine formalin 
fixed specimens will be examined with standard light microscopy. An 
experienced UNC pathologist will document size, gross description, and 
tumor subtype, grade and stage per standard-of-care clinical pathology 
processes using the American Joint Committee on Cancer TNM staging 
system and the World Health Organization/International Society of Urologic 
Pathologists grading system. All pathological studies will be conducted per 
institutional standards. 
4.3 
Time and Events Table 
 
 
Screening 
Pre-
CEUS 
CEUS 
study 
Early  
Termination1 
Post-CEUS 
study 
Inclusion/ 
Exclusion Criteria 
X 
 
 
 
 
Comprehensive Medical 
History 
X 
X2 
 
 
 
Informed Consent 
X1 
X1 
 
 
 
Education of potential side 
effects 
 
X 
 
 
 
Pregnancy test (urine), if 
applicable 
 
X3 
 
 
 
Contrast-Enhanced US 
(CEUS) 
 
 
X 
 
 
AE Assessment 
 
 
X4 
X 
 
Review medical record for 
pathology results 
 
 
 
X 
X 
1Consent 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. 
2Comprehensive medical history obtained at time of enrollment; thereafter history focused on symptoms and assessments. 
3Women of childbearing potential must have negative urine pregnancy test on the day of CEUS study, prior to the study. 
4Adverse event assessment will happen during the CEUS study when they will be monitored by nursing staff with vital signs 
(blood pressure and heart rate) recorded in the hospital’s electronic monitoring system. Adverse events will be recorded on 
an adverse events CRF. 
5.0 
INVESTIGATIONAL CONTRAST AGENT 
5.1 Description 
5.1.1 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®.  
 

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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 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 IND Exemption 
The investigators feel that this study meets the regulatory definition of a 
study that is exempt from obtaining an IND. 
5.3 Treatment Regimen 
The contrast agent will be dosed undiluted (0.2-0.4 mL Definity) for up to 7 
intravenous injections, depending on the number of lesions (2-3 bolus 
injections per lesion and one infusion injection). The activated undiluted 
dose will be placed in the tubing and attached to an infusion pump loaded 
with a syringe of sterile saline. The pump will be programmed to a standard 
rate for all patients and set to administer a total of 5 mL saline flush after 
the contrast agent is fully injected. Infusion dosing will be used after 
completion of bolus dosing. 
5.4 Preparation and Administration of Study Drug 
The contrast agent, Definity will be dispensed from the UNC Investigational 
Drug Service (IDS). It will be dispensed in inactivated form to a study team 
member. The contrast agent will be activated just prior to administration 
(ideally to be used within 5 minutes of activation). Contrast agent will be 
activated by nursing staff trained in the activation of Definity and according 
to package insert instructions, including use of VialMix®, the device used to 
activate Definity. Administration will occur IV in coordination with 
sonography staff trained specifically in contrast ultrasound imaging. 
5.5 Subject Compliance Monitoring 
Since the study only involves a single visit for most subjects, there will be 
no issues with compliance and no role for compliance monitoring. 
5.6 Prior and Concomitant Therapy 
Comparison imaging studies (ultrasound and MRI) completed prior, 
concomitantly or post-CEUS will also be collected. 
5.7 Packaging 
• Definity comes in single use 2-mL clear glass vials. 

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5.8 Receiving, Storage, Dispensing and Return 
5.8.1 Receipt of Drug Supplies 
Upon receipt of the of the study treatment supplies at the Investigational 
Drug Service (IDS), an inventory must be performed and a drug receipt log 
filled out and signed by the person accepting the shipment.  It is important 
that the designated study staff counts and verifies that the shipment 
contains all the items noted in the shipment inventory.  Any damaged or 
unusable study drug in a given shipment (active drug or comparator) will be 
documented in the study files. The investigator must notify study sponsor of 
any damaged or unusable study treatments that were supplied to the 
investigator’s site. 
5.8.2 Storage 
• Definity is stored between 2-8°C. 
5.8.3 Dispensing of Study Drug 
Study drug (contrast agent) is not dispensed to the subject. It is used one-
time, at the study visit and administered by study members. 
5.8.4 Return or Destruction of Study Drug 
At the completion of the study, there will be a final reconciliation of drug 
shipped, drug consumed, and drug remaining.  This reconciliation will be 
logged on the drug reconciliation form, signed and dated. Any discrepancies 
noted will be investigated, resolved, and documented prior to return or 
destruction of unused study drug. Drug destroyed on site will be 
documented in the study files. 
6.0 
ADVERSE EVENTS  
6.1 
Definitions  
6.1.1 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.   
 
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 

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should not be recorded as an AE, unless it is attributable by the investigator 
to the study therapy. 
6.1.2 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.   
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 once 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 indicates the events occur more frequently in the drug 
treatment group than in a concurrent or historical control group 
6.1.3 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.   
6.1.4 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; 

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• 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. 
6.2 
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 the 30-day follow-up period after 
treatment is discontinued.   
 
Collected information should be recorded in the Case Report Forms (CRF) 
for that patient. Please include a description of the event, its severity or 
toxicity grade, onset and resolved dates (if applicable), and the relationship 
to the study drug.  Documentation should occur at least monthly.  
6.3 
SAEs or Serious SARs  
6.3.1 Timing 
After informed consent but prior to initiation of study medications, only SAEs 
caused by a protocol-mandated intervention will be collected (e.g. SAEs 
related to invasive procedures such as biopsies, medication washout).  
 
For any other 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.   

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6.3.2 Documentation and Notification 
These events (SAEs or Serious SARs) must be recorded in the SAE 
console within Oncore™ for that patient within 24 hours of learning of its 
occurrence.  
6.3.3 Reporting  
IRB Reporting Requirements: 
• 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 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.   
6.4 
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 

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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 study participants 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.   
7.0 
STATISTICAL CONSIDERATIONS 
7.1 
Sample Size and Accrual 
We will enroll 25 adult subjects with suspected with suspected kidney 
cancer and planned surgical nephrectomy.  We have deep recruitment 
experience for kidney CEUS imaging. Approximately 150 nephrectomies for 
localized disease are performed at UNC yearly. Based on our previous 
recruitment rate of ~75% and a strong partnership with our urology 
colleagues, we anticipate no problems meeting our recruitment goal of 25 
subjects. 
7.1.1 Primary Objective 
7.1.1.1 Predicting kidney mass diagnosis using 3D CEUS generated 
metrics. 
 
The proposed sample of 25, assuming 60% clear cell legions, achieves 63.2% 
power to detect a difference of 0.1000 between a diagnostic test with an area 
under the ROC curve (AUC) of 0.8000 and another diagnostic test with an AUC 
of 0.9000 using a two-sided z-test at a significance level of 0.050.  The 
correlation between the two diagnostic tests is assumed to be 0.9 for the positive 
group and 0.9 for the negative group.  
 
The proposed sample of 25, assuming 60% clear cell legions, achieves 88.7% 
power to detect a difference of 0.1500 between a diagnostic test with an area 
under the ROC curve (AUC) of 0.8000 and another diagnostic test with an AUC 
of 0.9500 using a two-sided z-test at a significance level of 0.050.  The 
correlation between the two diagnostic tests is assumed to be 0.9 for the positive 
group and 0.9 for the negative group.  

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7.1.2 Secondary Objectives 
7.1.2.1 To compare diagnostic accuracy of F-R infusion imaging technique 
to low MI bolus imaging technique. 
The proposed tests with sample size 25 have 63.2% power to detect a 
difference of 0.1 in the AUCs (.8 vs .9) and 88.7% power to detect a 
difference of 0.15 (0.8 vs 0.95). 
7.1.2.2 To predict kidney mass stage using 3D CEUS-generated metrics 
Based on our clinical experience, we estimate 15 patients will have a T2a 
category or lower lesion and 25% of those will have a change from clinical 
to pathological staging. A sample size of 15 patients will not have sufficient 
power to detect areasonable effect size but computation of parameter 
estimates and standard errors will inform larger future studies. 
7.2 
Data Analysis Plans 
7.2.1 Predicting kidney mass diagnosis using 3D CEUS generated metrics. 
Image Analysis (Low MI TIC analysis): We will use Matlab (Mathworks, 
Natick, MA) for time intensity curve (TIC) analysis and 3D volume rendering. 
For each imaging plane, we will create a region of interest (ROI) by 
delineating the entire mass in the image slice. We will produce a TIC by 
taking the mean signal intensity in the ROI at each time point and calculate 
the metrics of wash-in rate (WI), wash-out rate (WO), mean transit time 
(MTT), time to peak (TTP) and peak enhancement relative to the 
surrounding parenchyma (PE) for each x-sweep position. A weighted 
average based on the ROI area at each position will be used to produce 
single values for the 3D volume for each TIC metric (WI, WO, MTT, TTP 
and PE). We will repeat this process for each plane of imaging.  
3D volume rendering: We will also create 3D renderings of the tumor 
vasculature from the CEUS images. We will sort the image data collected 
over the 3-minute period after a bolus injection by the x- (or y-) sweep 
position, then by capture time in relation to the start of bolus injection 
through the wash-out period. We will produce a cumulative contrast image 
(CCI) at each position with the time-dependent image data by summing all 
time-dependent image data at each position. The images show where the 
microbubbles have travelled throughout the acquisition period, and since 
microbubbles are confined to the vascular spaces, the images provide 
excellent delineation of the vasculature. We will stack the CCIs together to 
create a 3D rendering.  
Multiparametric score and statistical analysis: We will combine the 
quantitative data derived from 
CEUS image analyses into a 
single score. For the primary 
analysis 
using 
the 
gold 

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standard of histologic diagnosis to differentiate clear cell from non-clear cell 
tumors, we will use logistic regression to build predictive models based on 
multiple parameters from CEUS (WI, WO, MTT, TTP, PE and tumor 
volume), B-mode US images (length, echogenicity, shape), as well as 
patient factors (age, sex, race) (Table 1). We will assess the effects of 
individual parameters using hypothesis tests based on parameter estimates 
from the fitted logistic regression model. This approach facilitates 
assessment of the added value of the CEUS measurements over other 
patient factors. We will calculate nonparametric estimates and 95% 
confidence intervals for area under the curve (AUC) using the fitted logistic 
regression models, both with and without non-CEUS patient factors. We will 
use model selection procedures, including AUC, to identify a final model, 
with a one-sided test of AUC ≤.8 vs AUC ≥.8 at alpha=.05 for the final 
model. We will use model selection procedures based on optimizing AUC 
to identify a final model, with a one-sided test of AUC<=0.8 vs AUC>=.8 at 
alpha=.05 for the final model.  
Gold Standard Comparator: Histologic diagnosis from pathologic tissue. 
7.2.2 Comparing diagnostic accuracy of F-R infusion imaging technique to 
low MI bolus imaging technique. 
Image Analysis (Flash-Replenishment TIC analysis): For each 0.5-1 cm 
step, we will produce a TIC by delineating an ROI of the entire mass and 
measuring the mean contrast signal intensity over the time course of the F-
R. We will model TIC using a mono-exponential equation, UI=A(1 - e-βt), 
where UI is ultrasound intensity and β, the slope of the tangent to the curve, 
represents the rate of perfusion. The height of the curve, A, represents the 
perfusion volume. To produce a single value for β for the entire tumor 
volume, we will calculate a weighted average based on the ROI area of the 
mass at each step. This value will serve as the WI rate.  
Gold Standard Comparator: Histologic diagnosis from pathologic tissue. 
Statistical Analysis: We will follow the model building and evaluation 
strategy used for the primary objective. This will be done separately for 
bolus and infusion imaging modalities, as well as combining the two 
multiparametric scores. We will compare AUCs from final logistic regression 
models obtained for bolus and infusion using a Z-test at alpha=0.05, with 
standard errors obtained by bootstrapping correctly adjusting for the fact 
that the two AUCs are calculated using the same data and hence correlated. 
We will conduct similar tests comparing the AUC based on the combined 
model to those based on bolus and infusion multiparametric scores 
separately. The proposed tests with sample size 25 have 80% power to 
detect a difference of .1 in the AUCs (0.8 vs 0.9) and 90% power to detect 
a difference of 0.15 (0.8 vs 0.95). 

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7.2.3 Predicting kidney mass stage using 3D CEUS-generated metrics 
Image Eligibility: CEUS mass images from individuals with clinical stage 
T2a or lower tumors will be eligible for this sub-analysis. We estimate that 
15 tumors will meet these criteria since over 50% of kidney cancer 
resections at UNC are for T2a or lower clinical stage tumors.  
Imaging Procedure and Analysis: We will use Matlab (Mathworks, Natick, 
MA) to measure WI and WO rates from TIC analyses. We will apply the 3D 
volume rendering exploratory analyses described in the primary outcome to 
this outcome measure. Qualitative interpretation of 3D volume renderings 
will be assessed and compared to the clinical outcome. 
Gold Standard Comparator: We will compare the gold standard of post-
surgical pathologic stage to WI/WO rates generated from CEUS TICs. 
Statistical Analysis: We will use the continuous variables of WI and WO to 
predict the binary outcome measure of pathologic stage T3 disease or 
greater. We will calculate means (standard deviations) and 95% confidence 
intervals for WI and WO for those ≥ stage T3 vs. < stage T3, and compare 
via t-tests at alpha=.05. We will use logistic regression to assess ability of 
WI and WO to predict stage T3 disease or greater, both separately and 
together. We will obtain nonparametric estimates of AUC along with 95% 
confidence intervals for WI alone, WO alone, and WI and WO combined 
using the fitted logistic regression models. We will also add patient specific 
factors to the logistic regression models, to assess the added value of the 
WI and WO measurements, with analyses to include tests of the effect 
parameters from the fitted logistic regression models and estimates and 
confidence intervals for AUCs from those models. 
8.0 
STUDY MANAGEMENT 
8.1 
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) and to ethical principles that have their origin 
in the Declaration of Helsinki. 
 
 
 
Before recruitment and enrollment onto 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 

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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. 
8.2 
Registration Procedures 
Study participants will be registered into OnCore®, a web based clinical 
research platform by one of the Study Coordinators. 
8.3 
Data Management and Monitoring/Auditing 
Copies of completed CRFs with subject IDs will stored within subjects’ 
binders and data will be entered in the secure REDCap database.  Study 
personnel will have access to enter data in the study database.  Data should 
be entered in the study database within 5 business days to ensure timely 
entry. 
The coordinator will complete the first CRF together with the PI to verify that 
it is completed correctly.  We will verify a randomly selected 25% of all 
source documents.  The randomization for this verification will be generated 
using a random number generator in Excel.  Source document verification 
and monitoring will be documented and stored with the regulatory files. 
8.4 
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.   
8.4.1 Emergency Modifications 
UNC investigators may implement a deviation from, or a change of, the 
protocol to eliminate an immediate hazard(s) to trial subjects without prior 
UNC’s IRB/IEC approval/favorable opinion.   
 
For any such emergency modification implemented, an IRB modification 
form must be completed by UNC Research Personnel within five (5) 
business days of making the change.   
8.4.2 Single Subject Exceptions 
8.4.3 Eligibility single subject exceptions are not permitted for Lineberger 
Comprehensive Cancer Center Investigator Initiated Trials under any 
circumstances. Other types of single subject exceptions may be 
allowed if proper regulatory review has been completed in 

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accordance with Lineberger Comprehensive Cancer Center’s Single 
Subject Exceptions Policy. 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 personnel will record the deviation in OnCore®, 
and report 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 by UNC personnel 
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 UNC’s IRB must be reported by the study team using the IRB’s web-
based reporting system.   
8.5 
Amendments to the Protocol 
Should amendments to the protocol be required, the amendments will be 
originated and documented by the Principal Investigator at UNC.  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.   

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CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
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The written amendment, and if required the amended consent form, must 
be sent to UNC’s IRB for approval prior to implementation.  
8.6 
Record Retention 
Study documentation includes all eCRFs, 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. 
8.7 
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 participants.  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/NCI regulations and guidelines regarding clinical 
trials both during and after study completion. 
9.0 
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LINEBERGER COMPREHENSIVE CANCER CENTER 
Imaging Protocol Template 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
27 
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LINEBERGER COMPREHENSIVE CANCER CENTER 
Imaging Protocol Template 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
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LINEBERGER COMPREHENSIVE CANCER CENTER 
Imaging Protocol Template 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
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29 
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LINEBERGER COMPREHENSIVE CANCER CENTER 
Imaging Protocol Template 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
 
 February 2017 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
30 
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