Comparison of the Sensitivity and Specificity of Acoustic Angiography (Micro-tumor Detection by Quantifying Tumor-induced Vascular Abnormalities) to the Sensitivity and Specificity of Conventional Ultrasound
Phase: NA
Conditions: Signs and Symptoms
Sponsor: UNC Lineberger Comprehensive Cancer Center

 
LCCC 1405: Comparison of the sensitivity and specificity of acoustic 
angiography (Micro-tumor detection by quantifying tumor-induced 
vascular abnormalities) to the sensitivity and specificity of 
conventional ultrasound 
 
NCT number NCT02175628 
Document Date July 17, 2017 
 

LINEBERGER COMPREHENSIVE CANCER CENTER 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
 
LCCC  1405:  Comparison of the sensitivity and specificity of acoustic 
angiography (Micro-tumor detection by quantifying tumor-induced vascular 
abnormalities) to the sensitivity and specificity of conventional ultrasound 
 
Principal Investigator 
Yueh Lee, MD, PhD 
University of North Carolina 
Department of Radiology  
919-537-3730 phone 
yueh_lee@med.unc.edu 
 
Co-Investigators  
 
Cherie Kuzmiak, DO 
University of North Carolina 
Department of Radiology 
919-966-6392 phone 
cherie_kuzmiak@med.unc.edu 
 
Sheila Lee, MD 
University of North Carolina 
Department of Radiology 
sheila_lee@med.unc.edu 
 
Biostatistician  
Donglin Zeng, PhD 
Department of Biostatistics 
University of North Carolina 
919-966-7273 – phone 
dzeng@bios.unc.edu 
 
Sponsor:  Lineberger Comprehensive Cancer Center 
Funding Source: National Institutes of Health 3R01CA170665-03S1  
Version Date: July 17, 2017 

LINEBERGER COMPREHENSIVE CANCER CENTER 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
 
LCCC 1405:  Comparison of the sensitivity and specificity of acoustic 
angiography (Micro-tumor detection by quantifying tumor-induced vascular 
abnormalities) to the sensitivity and specificity of conventional ultrasound 
  
 
 
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:_______________ 
 
Version Date: _______________

LCCC 1405 
 
CONFIDENTIAL 
PI: Lee 
 
UNIVERSITY OF NORTH CAROLINA 
September 26, 2014 
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TABLE OF CONTENTS 
1.0 
BACKGROUND AND RATIONALE ...................................................................1 
1.1 
Study Synopsis ...................................................................................................1 
1.2 
Traditional Breast Cancer Screening and Diagnosis ....................................1 
1.3 
Angiogenesis .......................................................................................................2 
1.4 
Perflutren Lipid Background and Known Toxicities .......................................3 
1.5 
Rationale ..............................................................................................................5 
2.0 
STUDY OBJECTIVES AND ENDPOINTS ........................................................6 
2.1 
Primary Objective ...............................................................................................6 
2.2 
Secondary Objectives ........................................................................................6 
2.3 
Primary Endpoint ................................................................................................6 
3.0 
ELIGIBILITY ...........................................................................................................6 
3.1 
Inclusion Criteria .................................................................................................6 
3.2 
Exclusion Criteria ................................................................................................7 
4.0 
STUDY PLAN .........................................................................................................7 
4.1 
Schema ................................................................................................................8 
4.2 
Enrollment/Recruitment .....................................................................................8 
4.3 
Acoustic Angiography ........................................................................................9 
4.4 
Standard of Care Biopsy .................................................................................10 
4.5 
Medical Record Abstraction ............................................................................10 
4.6 
Reader Study ....................................................................................................10 
5.0 
Research Ultrasound Scanner: FUJIFILM VisualSonics Vevo 770 ......11 
5.1 
Expected Risks .................................................................................................12 

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5.2 
Duration of Study ..............................................................................................13 
6.0 
Drug Information ................................................................................................13 
6.1 
Perflutren Lipid Microspheres (Lantheus Medical Imaging) ......................13 
7.0 
ADVERSE EXPERIENCES-DRUGS ................................................................15 
7.1 
Definitions ..........................................................................................................16 
7.2 
Documentation of non-serious AEs or SARs ...............................................17 
7.3 
SAEs or Serious SARs ....................................................................................18 
8.0 
UNANTICIPATED CONCERNS ........................................................................19 
8.1 
Unanticipated Adverse Device Effect (UADE) .............................................19 
8.2 
Unanticipated Problems (UP) .........................................................................19 
8.3 
Reporting ...........................................................................................................19 
9.0 
Data and Safety Monitoring Plan ...................................................................20 
10.0 STATISTICAL CONSIDERATIONS .................................................................21 
10.1 
Sample Size and Accrual ................................................................................21 
10.2 
Data Analysis Plans .........................................................................................21 
11.0 STUDY MANAGEMENT .....................................................................................23 
11.1 
Institutional Review Board (IRB) Approval and Consent ...........................23 
11.2 
Required Documentation .................................................................................24 
11.3 
Registration Procedures ..................................................................................24 
11.4 
Data Management and Monitoring/Auditing .................................................24 
11.5 
Adherence to the Protocol ...............................................................................24 
11.6 
Amendments to the Protocol ..........................................................................25 
11.7 
Record Retention ..............................................................................................26 
11.8 
Obligations of Investigators .............................................................................26 

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12.0 References ...........................................................................................................27 
13.0 APPENDICES.......................................................................................................32 
13.1 
Appendix A: Reader Study Data Collection Form .......................................32 
13.1 
Appendix B: Previous Clinical Investigation Use of Device: ......................34 

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1.0 
BACKGROUND AND RATIONALE 
1.1 
Study Synopsis 
Increasing the sensitivity and specificity of diagnostic imaging in patients 
at high risk for breast cancer could provide substantial clinical benefit by 
improving diagnosis, preventing over-treatment, and reducing healthcare 
costs. Acoustic angiography is a new type of contrast enhanced 
ultrasound imaging which is specifically sensitive to microvascular 
structure and density.  It evaluates tumor micro-vasculature and may 
provide a powerful prognostic tool for the diagnosis of breast cancer, and 
eventually for treatment evaluation.   
 
Sixty patients who are to have a clinical surgical breast biopsy based on 
results from pre-study standard of care (SOC) imaging will be recruited 
from the UNC Breast Clinic for participation in the study. The primary 
objective of this single arm study is to compare the sensitivity and 
specificity of acoustic angiography with traditional b-mode ultrasound in 
the distinction of malignant versus benign breast lesions.  Secondary 
objectives include a comparison of area under the curve (AUC) for 
acoustic angiography versus b-mode ultrasound, comparison of radiologist 
preference for the two imaging techniques for each of 3 lesion 
characteristics, and quantification of vessel tortuosity based on acoustic 
angiography imaging results.  These metrics will be used to develop a 
predictive model of malignancy which will subsequently be compared to 
results from radiology review.   
1.2 
Traditional Breast Cancer Screening and Diagnosis 
Breast cancer is the most common cancer type among women. 
Approximately 10% of women in the United States develop breast cancer 
during their lifetime and 30% to 40% of these patients will die from it.[33, 
34] Mammography is an effective tool for the early detection of breast 
cancer in the majority of women.[35] However, for women with dense 
breast tissue (considered an independent risk factor for breast cancer) 
and younger women, mammography performs poorly due to lower 
sensitivity and specificity in these groups.[37] For young women with 
heritable mutations who wish to begin screening at a younger age, these 
limitations are especially problematic.[38] Additionally, mammography is 
less sensitive in women who have undergone breast augmentation. Given 
these limitations, most of these women may undergo additional imaging 
with breast ultrasound or Magnetic Resonance Imaging (MRI). 
 
MRI is time consuming, extraordinarily costly, and has limited availability, 
especially among rural and underserved populations. Another screening 
option, breast ultrasound (without contrast), is widely used when additional 
imaging beyond mammogram is required due to its real-time imaging 

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capability (>30 images per second), portability, safety (does not involve 
radiation), and relatively low cost compared to breast MRI. Unfortunately, 
while breast ultrasound is highly sensitive (96%), it is less specific (70%), 
resulting in a high false positive rate.  This results in unnecessary biopsies 
with associated complications, additional follow-up and negative 
psychosocial impacts on patients, e.g., significant anxiety. A significant 
clinical need exists to improve breast ultrasound sensitivity and specificity.  
1.3 
Angiogenesis 
Angiogenesis is the development of new microvasculature, and is well 
recognized to be involved in the growth of solid tumors as well as tumor 
invasion and metastasis. Recent studies have reported an association 
between microvessel density and poorer recurrence-free, cancer-specific 
and overall survival.[39-46]. Furthermore, both microvessel density and 
microvessel morphology have been reported to be associated with the 
clinical response to chemotherapy.[16, 47-49] However, no current clinical 
imaging modality can directly evaluate the microvasculature associated 
with suspected breast tumors.[50] Histological techniques based on core 
needle biopsy or surgical biopsy may evaluate only a small portion of a 
lesion, and require additional invasive procedures. Thus, direct non-
invasive evaluation of the tumor micro-vasculature may provide a powerful 
prognostic tool for the diagnosis of breast cancer, and also provide a 
potential tool for treatment evaluation. 
1.3.1 Screening Based on Angiogenesis-Acoustic Angiography 
Acoustic angiography allows for the viewing of vessel shape and 
delineation of ‘tortuosity,’ which can indicate the presence and progression 
of cancer. This ability to image the microvasculature depends on the use 
of a multi (high) frequency ultrasound scanner in addition to a traditional 
single-frequency pulse-echo ultrasound scanner (b-mode ultrasound), the 
latter to ensure accurate anatomical location of the lesion.  Because of this 
dependence on pulse-echo, the technique is referred to as “acoustic” 
angiography [62].   
 
Acoustic angiography also depends on use of an ultrasound contrast 
agent. Contrast enhanced ultrasound imaging has been used for nearly 
two decades for clinical cardiology in the United States (and is currently 
routinely used in UNC Hospital Cardiology Clinics), and is much more 
widely used in Europe and Asia for visualization of blood perfusion in 
organs, tissues, and tumors.[51] Early concerns about the safety of 
ultrasound contrast (specifically with the contrast agent (perflutren lipid; 
Definity®)  due to events in a clinical trial have been resolved; the 
overwhelming amount of more recent evidence from large clinical studies 
has shown that contrast ultrasound is very safe.[52-54] In fact ,it is much 
safer than other commonly used techniques, such as coronary 
angiography, exercise ECG, or myocardial scintigraphy.[55] Furthermore, 

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it does not have the risks of nephrogenic systemic fibrosis associated with 
gadolinium based MRI contrast agents when used in renally compromised 
patients. On October 27, 2011, the FDA eliminated the requirement for 
patient monitoring 30 minutes after ultrasound contrast examination, 
based on these and other recent safety data.  The ultrasound contrast 
agent to be used in LCCC1405 is perflutren lipid.   
1.4 
Perflutren Lipid Background and 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 6.1.5. 
1.4.1 Background and Current Indications 
Definity® (perflutren lipid) is an FDA-approved lipid-shell microbubble 
ultrasound (US) contrast agent that may be administered by an 
intravenous (IV) bolus or infusion. Currently, this contrast agent is 
approved for use in patients with suboptimal echocardiograms to opacify 
the left ventricular chamber and to improve delineation of the left 
ventricular endocardial border. It is not approved as a contrast agent for 
acoustic angiography in the breast. 
 
When used according to its approved indication, the maximum dose of 
perflutren is administered as either two bolus doses or one single 
intravenous infusion.  
For our study, perflutren lipid will be administered intravenously by a nurse 
or trained medical personnel (see section 4.3). 
1.4.2 Associated Toxicities 
In pre-market clinical trials 1716 subjects were evaluated with activated 
perflutren lipid. Of the 1716 subjects, 144 subjects (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 eight 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. 

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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,[22] compared to gadolinium-based MRI contrast which has an 
incidence of NSF of 2 – 5 in 100 patients with chronic kidney disease.[23]  
 
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 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 nurse or research physician.  
 
Hypersensitivity Reactions  
The real risk of perflutren in our study is to the small number of potential 
patients with 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 6.1.5 for additional information. In order to 
avoid a potentially fatal event, EpiPen® (epinephrine) injections will be 
kept near the US machine for all patients.  

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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 mechanical indices has been reported to cause ventricular 
arrhythmias.  
 
Use in Patients with Known Breast Lesions  
US contrast agents including perflutren should carry no additional risks in 
patients with a known breast lesions, as they are cleared by the lungs. 
The phospholipid component of perflutren lipid microspheres are 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) .  
1.5 
Rationale 
One main limitation to the widespread use of contrast enhanced 
ultrasound clinically has been the lack of availability of state-of-the art 
contrast imaging approaches available to clinicians. However, contrast 
ultrasound will likely become far more widespread as new imaging 
techniques, such as acoustic angiography, demonstrate their usefulness 
and become available on commercial ultrasound systems. We propose to 
evaluate a novel ultrasound method that could potentially improve the 
sensitivity and specificity of traditional breast ultrasound.   Increasing the 
accuracy of diagnostic imaging in high risk patients could provide 
substantial clinical benefit by improving diagnosis, preventing over-
treatment, and reducing healthcare costs. 
 
LCCC1405 is designed to compare the sensitivity and specificity of 
contrast enhanced ultrasound (acoustic angiography) to the sensitivity and 
specificity of conventional ultrasound in women scheduled to undergo a 
biopsy based on pre-study imaging results.  The gold standard for 
sensitivity and specificity, then, will be based on pathological results. The 
Breast Imaging Reporting and Data System (BIRADS) is used by 
radiologists who read mammograms, ultrasounds and MRIs to indicate 
their level of suspicion of the possibility of breast cancer. Scores range 
from 0 to 6, with scores of 4-5 indicating suspicious results, while 6 
indicates an existing diagnosis of breast cancer. This study is limited to 
women with a score of 4-5 based on pre-study imaging. 
 
 
 
 
 

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2.0 
STUDY OBJECTIVES AND ENDPOINTS  
2.1 
Primary Objective 
To compare (using a reader study) the sensitivity and specificity of 
acoustic angiography to the sensitivity and specificity of conventional b-
mode ultrasound in evaluation of breast lesions 
2.2 
Secondary Objectives 
2.2.1 To compare the area under the curve (AUC) of acoustic angiography to 
the AUC of the b-mode ultrasound  
2.2.2 To compare radiologist preference of acoustic angiography to 
conventional b-mode ultrasound for each lesion characteristic (shape, 
margins and vascularity) 
2.2.3 To quantify vessel tortuosity metrics for the acoustic angiograph images, 
and to use these metrics to develop a model for predicting malignancy (a 
model-based malignancy score) 
2.2.4 To compare the model-based malignancy score to the acoustic 
angiography reader study  
2.3 
Primary Endpoint 
Sensitivity and specificity for our study is defined as the ability of readers 
(radiologists) to use the acoustic angiography or b-mode ultrasound to 
distinguish between malignant and non-malignant breast lesions known to 
exist based on pathological results (the gold standard). 
3.0 
ELIGIBILITY 
3.1 
Inclusion Criteria 
Subject must meet all of the inclusion criteria to participate in this study:  
3.1.1 Women ≥18 years old 
3.1.2 Scheduled for breast core needle or surgical biopsy of at least one breast 
lesion based on suspicious breast lesion (BIRADS score of 4 or 5) from 
pre-study SOC imaging studies 
3.1.3 Center of suspicious lesion is not deeper than 1.5 cm. 
3.1.4 Able to provide informed consent 
3.1.5 Negative urine pregnancy test in women of child-bearing potential 
 

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3.2 
Exclusion Criteria 
All subjects meeting any of the exclusion criteria at baseline will be 
excluded from study participation 
3.2.1 Male (it is uncommon for men to present for imaging and the 
overwhelming majority of findings are non-cancerous and do not lead to 
biopsy; male breast cancer represents <1% of newly diagnosed breast 
cancer) 
3.2.2 Institutionalized subject (prisoner or nursing home patient) 
3.2.3 Critically ill or medically unstable and whose critical course during the 
observation period would be unpredictable (e.g., chronic obstructive 
pulmonary disease (COPD)  
3.2.4 Known hypersensitivity to any component of perflutren lipid (Definity®)  
3.2.5 Right to left shunt, severe pulmonary hypertension (pulmonary artery 
pressure >90mmHg), or adult respiratory distress syndrome  
3.2.6 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 1 year prior to the date of proposed 
Definity® administration.  
• Uncontrolled systemic hypertension (systolic blood pressure (BP) 
>150 mm Hg and/or diastolic BP >90 mm Hg despite optimal 
medical management  
3.2.7 Any woman who is pregnant or has reason to believe she is pregnant or 
any woman who is lactating (the possibility of pregnancy has to be 
excluded by negative 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) 
 
 
 
 
 
 
 
4.0 
STUDY PLAN 

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4.1 
Schema 
This is a one arm single center study of 60 patients with suspicious breast 
lesions (BIRADS 4 or 5) from the UNC Breast Clinic that consent to 
undergo an acoustic angiography in conjunction with b-mode ultrasound 
prior to their scheduled biopsy.   
 
4.2 
Enrollment/Recruitment 
A total of 60 women will be enrolled to this study. The 60 study subjects 
will be consecutively recruited from women who are scheduled to undergo 
core needle or surgical biopsy (BIRADS 4 or 5) to have pathological 
confirmation of malignancy status.  Eligible patients will be identified by 
research staff review in coordination with the UNC Breast Clinic.   
Once a patient has been referred, the patient will be approached by a 
coordinator from Radiology to assess interest in participation.  
 
All eligible women who agree to participate in the study will be asked to 
come to their scheduled biopsy appointment thirty minutes early to 
complete the informed consent process.   
 
Review of the consent will take place in the privacy of an exam room, or 
when possible, a sample consent form will be sent to the patient via email 
prior to the patient’s visit to allow for ample review.  Once the patient has 
consented, women of child bearing potential (WCBP) will be given a urine 
pregnancy test in order to ensure that they are not pregnant.  If a urine 
pregnancy test shows a result positive for pregnancy, the patient will be 
excluded from the study per the exclusion criteria because the 
investigators cannot, in good conscience, expose a fetus to the contrast 
agent used.  Women who consent for the study and are eligible will be 
escorted by the research coordinator to a dressing room, where the 
subject will change into a gown. 

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4.3 
Acoustic Angiography 
4.3.1 Perflutren Administration 
At the time of imaging, the contrast agent perflutren lipid (see section 4.3) 
will be administered. See 
http://www.definityimaging.com/pdf/VIALMIX_Users_Guide.pdf, and the 
package insert, for instructions on perflutren lipid preparation and 
activation. Perflutren lipid is intended for intravenous (IV) administration 
only after activation in the Vialmix® apparatus. Cardiopulmonary 
resuscitation personnel and equipment will be readily available prior to 
perflutren administration, and all patients will be monitored for acute 
reactions.  
 
Perflutren will be administered in a single dose using a bolus 
administration.  (see http://www.definityimaging.com/how-
administration.html). Although the prescribing information allows for two 
10 uL/kg doses separated by 30 minutes, we will be administering double 
the package recommended dose (administering 20 uL/kg) administered 
within 30-60 seconds, followed by a 10mL saline flush. No second dose 
will be administered.   All patients will be monitored for 30-minute post-
perflutren administration by the research nurse or research physician.  
Monitoring will include taking vitals (O2 sat, HR, RR, BP).  The oxygen 
saturation, heart rate, and respiration rate will be monitored continuously 
for 30 min.  The blood pressure will be monitored every 15 minutes.  This 
study will be conducted in Mammography of the UNC Cancer Hospital, so 
trained medical personnel will be available as needed. 
 
The initial concentration will be diluted, although the final dilution amount 
is not finalized (most likely to range from 1:1 (full dose) to - 1:5 dilution in 
saline). This dilution technique is still to be determined based on 
performance because this is a new type of imaging. We will infuse via 
hand bolus injection.  The infusion time will be approximately 30 - 60 
seconds for the dose. 
4.3.2 Acoustic Angiography Imaging Procedures 
Acoustic angiography imaging involves a research high frequency 
ultrasound scanner (VisualSonics Vevo 770; see section 5.0) in contrast 
imaging mode.  In addition, we may use a standard FDA-approved clinical 
ultrasound system to obtain a second conventional B-mode ultrasound 
image as a gold standard.  Imaging will be performed within the package 
insert guidelines for ultrasound system mechanical index (a measurement 
of output power) when imaging perflutren contrast agent (less than 0.8). 
 
Acoustic angiography imaging will be performed by a trained medical 
personnel using mild compression to eliminate motion.  A linear translation 
stage will be attached to the transducer to mechanically translate the 

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transducer the imaging distance (up to approximately 3 cm).Total imaging 
time per pass is estimated to be 60 seconds, similar to a standard 
ultrasonography exam   All image data will be de-identified and transferred 
for off-line analysis based on a study ID. The research images will NOT be 
interpreted or analyzed for clinical decisions related to the patient.  See 
section 5.0 for additional information on the acoustic angiography device. 
4.4 
Standard of Care Biopsy 
The patient will then undergo her scheduled breast biopsy procedure 
based on the pre-study diagnostic imaging. The research acoustic 
angiography imaging will NOT be interpreted prior to the breast biopsy 
and therefore will not influence any clinical decision concerning the biopsy. 
4.5 
Medical Record Abstraction 
A secondary objective of this study is to quantify the vessel tortuosity and 
develop a “model-based malignancy score” to predict malignancy of a 
lesion based on quantitative tortuosity metrics from the acoustic 
angiography imaging. In order to meet this objective, we will review each 
patient’s clinical records, including their pathology report from biopsy.  The 
malignancy will be determined as indicated by the pathology report.   
4.6 
Reader Study    
A total of five readers (radiologists trained in breast imaging) will be 
recruited to participate in evaluation of all imaging performed under 
LCCC1405.    
 
In the reader preference study, each reader will be asked to compare the 
acoustic angiography case to the conventional b-mode ultrasound case to 
evaluate the imaging characteristics based on the BIRADS ultrasound 
lexicon 
(http://www.acr.org/~/media/ACR/Documents/PDF/QualitySafety/Resourc
es/BIRADS/USLexiconClass.pdf). Specifically the relative ability to 
evaluate shape, margins and vascularity will be evaluated using a seven-
point scale (-3 to +3) for the paired modality comparisons for each of the 
60 cases.  
 
The data collection form for the reader study portion is given in Appendix 
A, section 13.1.  
 
The readers will be asked to assign a subjective malignancy score (-2 
(highly not malignant) to +2 (highly malignant)) and their confidence for 
each lesion for each modality (0 to 100%). These will also be documented 
on the form in Appendix A.  These scores will be used combined for a 
binary analysis.  Malignancy scores of +1 and +2 will be considered 
malignant.  Scores of -2, -1, and 0 will be considered not malignant. 
The confidence of malignancy will be used independently in the analysis. 

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5.0 
Research Ultrasound Scanner: FUJIFILM VisualSonics Vevo 770 
The device used for this study will be a research ultrasound scanner 
(FUJIFILM VisualSonics Vevo 770) and unique probes developed in 
conjunction with the research lab run by the founder of VisualSonics, Dr. 
Stuart Foster.  The ultrasound probes are modifications of Vevo 770 
probes (RMV716 probes), modified to house 2 single-element transducers 
instead of 1.   
 
The Vevo 770 is a research ultrasound system designed for pre-clinical, 
high resolution imaging of small animals.  It is marketed commercially by 
VisualSonics, Inc., a division of SonoSite, Inc., under the parent company 
FUJIFILM.  This ultrasound system and similar models have been 
designated to pose no significant risk in other clinical trials by the 
respective IRBs or other similar regulatory agencies responsible for 
compliance oversight of these studies. (See section 13.1, Appendix B). 
 
 
 
Figure 1. Device Image 
 
3 Dual-Frequency Transducers: 
The probes we plan to use in this study are modified VisualSonics 
RMV716 probes.  They have been modified to house a high frequency 
inner element and a low frequency annular ring element, instead of a 
single transducer element.  We plan to run the transducers in dual-
frequency contrast mode during the study.  In dual-frequency contrast 
mode, the low frequency (outer) element transmits ultrasound and the 

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high frequency (outer) element ring receives the signal.  In this mode, the 
low frequency element is run by an external waveform generator and 
amplifier, and the high frequency element is run by the Vevo 770 system.   
 
 
Figure 2. Device Transducers 
 
5.1 
Expected Risks  
5.1.1 VisualSonics Vevo 770 
This research protocol presents minimal risk to participants, investigators 
and study personnel. The ultrasound imaging equipment, though 
specifically designed for preclinical use and not FDA approved, is 
calibrated and will be used according to FDA limits on diagnostic imaging 
ultrasound exposure to patients (Mechanical Index of less than 1.9 and 
spatial-peak temporal average intensity of less than 720 mW/cm2).  For 

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the imaging studies proposed here, the maximum mechanical index and 
intensity will not exceed 0.8 and 300 mW/cm2, respectively, less than half 
of the FDA limits, and within the range of standard output parameters 
regularly used in diagnostic ultrasound imaging.  Furthermore, this is 
within the mechanical index range listed on the prescribing information for 
use of perflutren contrast agent. The 1.5 cycle pulse length and 2-4 MHz 
center frequency of the ultrasound that is produced by our transducers are 
within the range of common parameters regularly used by other FDA 
approved diagnostic ultrasound imaging systems.  The unique aspect of 
this imaging system is that we receive data at a higher frequency (15-45 
MHz) than typical diagnostic ultrasound imaging systems used for 
transcutaneous imaging.   The transmitted ultrasound parameters are 
within standard diagnostic imaging ranges for frequency and acoustic 
output.   
5.1.2  Perflutren lipid (Definity) 
See sections 1.4 and 6.1.5. 
5.2 
Duration of Study 
It is anticipated that the total study duration encompassing recruitment, 
enrollment, and data analysis will take approximately 2 years.  Active 
patient participation will last approximately 1 visit (consent and 15 minutes 
imaging). 
6.0 
Drug Information 
6.1 
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 for use 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.  

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6.1.1 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.  
6.1.2 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.  
6.1.3 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. 
6.1.4 Preparation  
See http://www.definityimaging.com/pdf/VIALMIX_Users_Guide.pdf, and 
the package insert for instructions on the use of Vialmix®.  
6.1.5 Clinical Safety Summary  
See prescribing information on perflutren when used according to its FDA 
indication (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/), and 
see section 1.3 for a summary of toxicities reported in clinical trials. 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 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:  

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In post-marketing 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 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 15 
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 microL/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.  
6.1.6 Return and Retention of Study Drug 
Incomplete vials of perflutren lipid remaining at the completion of the study, 
or expired perflutren lipid will be destroyed by UNC IDS. 
 
7.0 
ADVERSE EXPERIENCES-DRUGS 

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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.   
 
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.   
 
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 
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7.1.3 Unexpected AE or SAR 
An AE or SAR is considered unexpected if the sensitivity and 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 the 30 day follow-up period after 
treatment is discontinued.   
 
Collected information should be recorded in the electronic Case Report 
Forms (e-CRF) for that patient.  Please include a description of the event, 

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its severity or toxicity grade, onset and resolved dates (if applicable), and 
the relationship to the study drug.    Documentation should occur at least 
monthly.  
7.3 
SAEs or Serious SARs  
7.3.1 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, or no treatment run-in).  
 
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.  
7.3.2 Documentation and Notification 
These events (SAEs or Serious SARs) must be recorded for that patient 
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 8.2 ) 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.  
 

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8.0 
UNANTICIPATED CONCERNS 
8.1 
Unanticipated Adverse Device Effect (UADE) 
The investigational device exemption (IDE) regulations define an 
unanticipated adverse device effect (UADE) as “any serious adverse 
effect on health or safety or any life-threatening problem or death caused 
by, or associated with, a device, if that effect, problem, or death was not 
previously identified in nature, severity, or degree of incidence in the 
investigational plan or application (including a supplementary plan or 
application), or any other unanticipated serious problem associated with a 
device that relates to the rights, safety, or welfare of subjects” (21 CFR 
812.3(s)). 
8.2 
Unanticipated Problems (UP) 
As defined by UNC’s IRB, unanticipated problems involving risks to study 
subjects refers to any incident, experience, or outcome that: 
• Is unexpected (in terms of nature, severity, or frequency) given (a) 
the research procedures that are described in the protocol-related 
documents, such as the IRB-approved research protocol and 
informed consent document; and (b) the characteristics of the 
subject population being studied; 
• Is related or possibly related to a subject’s participation in the 
research; and  
• Suggests that the research places subjects or others at a greater 
risk of harm (including physical, psychological, economic, or social 
harm) related to the research than was previously known or 
recognized. 
8.3 
Reporting 
8.3.1 UADEs 
UADEs must be reported by the clinical investigator to the sponsor and 
the reviewing IRB, as described below:  
 
For this device study, investigators are required to submit a report of a 
UADE to the FDA, the manufacturer of the device and the UNC IRB as 
soon as possible, but in no event later than 10 working days after the 
investigator first learns of the event (§ 812.150(a)(1)), using the 
MedWatch Form 3500A.   Sponsors (LCCC) must immediately conduct an 
evaluation of a UADE and must report the results of the evaluation to 
FDA, the UNC IRB, and participating investigators within 10 working days 
after the sponsor first receives notice of the effect (§§ 812.46(b), 
812.150(b)(1)).  
 

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For this device study, we will submit a report of a UADE to the 
manufacturer and the IRB as soon as possible, but no later than 10 
working days after the investigators first learn of the event. 
8.3.2 UP 
Any events that meet the criteria for “Unanticipated Problems” as defined 
by UNC’s IRB must be reported by the Study Coordinator using the IRB’s 
web-based reporting system.   
 
Any unanticipated problem that occurs during the conduct of this study 
and that meets at least the first two criteria listed in section 8.2 must be 
reported to the UNC IRB using the IRB’s web-based reporting system.   
9.0 
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.   
 

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10.0 STATISTICAL CONSIDERATIONS 
This is a nonrandomized, single-center study.  The primary purpose and 
endpoint of this study is to compare, in a radiologist reader study, the 
sensitivity and specificity of the acoustic angiography system to the b-
mode ultrasound with pathology as the reference standard.   
10.1 
Sample Size and Accrual 
 
 
Per the revision in increased Perflutren dose, we have updated our 
sample size statistics as follows.  
  
For power calculation, the null hypothesis is that the specificity of acoustic 
angiography system is the same as the standard of care (b-mode 
ultrasound), which is assumed to be 70%. (We will determine the 
specificity of b-mode ultrasound during the trial to confirm that the 70% 
represents the specificity in the hands of UNC radiologists)  Under the 
alternative, we expect that the specificity of the new device is at least 
90%. The specificities are measured relative to pathological diagnosis as 
the reference standard. With 60 lesions and 5 readers, where we 
anticipate that roughly half will be malignant, assuming the correlation 
from the same patient to be 0.5 and the readers to read different patients 
independently, the power to see specificity at least larger than 90% is 98% 
at the significance level 0.05 using one-side test. With 50 lesions (half are 
benign), the power to see specificity of 90% is >96%; with 25 lesions 
(about 13 are benign), this power becomes 72%.  The power to reject the 
null hypothesis is 98%.  Although we will study both sensitivity and 
specificity, our power calculation is based on specificity, which is the 
primary interest. 
10.2 
Data Analysis Plans 
10.2.1 Primary Analysis 
This is a prospective study to assess the diagnostic performance of the 
newly developed device. The primary outcome of interest is the 
sensitivity and specificity of the acoustic angiography device as 
compared to b-mode ultrasound. The sensitivity and specificity can be 
estimated non-parametrically as the proportion of the lesions which are 
distinguished as malignant versus non-malignant compared to the 
reference. This estimate will be calculated for each reader then averaged 
over all the readers. The standard error of the average sensitivity and 
specificity estimate will be calculated using the bootstrap method, where 
each patient is treated as independent unit with 5 ratings. The confidence 
interval of the final estimate will be provided using the normality 
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Furthermore, we will estimate the sensitivity and specificity of the b-mode 
ultrasound in this study and compare it with the sensitivity and specificity 
estimate of the acoustic angiography device using the bootstrap approach 
and the confidence interval of their differences will be provided using the 
normality assumption.   
 
Malignancy scores of +1 and +2 will be considered malignant.  Scores of -
2, -1, and 0 will be considered not malignant.  The confidence of 
malignancy will be used independently 
10.2.2 Secondary Analyses 
Radiologist Preference 
The secondary analysis will be to estimate the receiver operating 
characteristic (sensitivity and specificity) curve for the acoustic 
angiography system, with an additional aim of evaluating reader 
preference for specific breast lesion characteristics.  Specifically, to 
compare the diagnostic performance, we will non-parametrically calculate 
the area under the ROC curve for each reader and each modality, where 
the ROC curve is derived using the different cut-off of the probability 
scores across 60 patients. We then fit a mixed effect model, where the 
outcomes are the estimated area under the ROC curves and the fixed 
effect is the dummy variable of the acoustic angiography system vs. 
conventional b-mode ultrasound.  The readers will be treated as random 
effect. F-test from model fitting will be used to test whether the acoustic 
angiograph has a significant larger AUC than the conventional b-mode 
ultrasound (with significance level 0.05).  
 
To assess the reader preference for modality for each characteristic 
including shape, margins and vascularity, we will fit a random effect model 
with only intercept and random terms for patients and readers while the 
outcomes are the confidence scores (-3 to +3) . By testing the intercept 
significantly larger than zero, we will conclude that the new modality 
provides more confidence for readers than the conventional one. 
 
Image Segmentation and Tortuosity Quantification 
In order to derive quantitative vessel tortuosity metrics for the acoustic 
angiography images, we will perform an image processing workflow. The 
de-identified DICOM images will be converted to a 3-D volume and 
linearly up-sampled using MATLAB to create isotropic 50 x 50 x 50 micron 
voxels. Then, the lesion will be manually segmented to identify a region of 
interest (ROI) for vessel analysis. Using a series of algorithms [59, 60], 
each vessel in the ROI will be segmented and a series of tortuosity 
metrics will be calculated. The vessels are segmented using semi-
automated multi-scale ridge traversal and stored as a series of center-line 
point coordinates (x,y,z) forming a spline. The radius of the vessel is 
estimated at each point and saved as a fourth parameter. [14, 61] Each 

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tortuosity metric will be used individually to contrast benign and malignant 
lesions using a two-sided t-test, incorporating a Bonferroni correction to 
account for multiple hypothesis testing.  
 
Predicting Malignancy 
In order to predict malignancy based on the quantitative vessel tortuosity 
metrics, the set of images will be randomly divided into two halves to form 
a training data set and a test data set. The malignant lesions and the 
benign lesions will be randomly assigned to the training or test group 
separately to ensure that both groups contain both types of images. 
Multivariate binary logistic regression will be used to predict malignancy 
based on the full set of tortuosity metrics for the training data set of 30 
images. We will examine the model for multicollinearity using pairwise 
correlations and perform linear discriminant analysis to choose a smaller 
subset of metrics if over-fitting due to multicollinearity appears to be a 
problem. The model will then be used to predict the probability of 
malignancy of the test set of 30 images (via development of a model-
based malignancy score). We will construct a receiver operator curve 
(ROC) by determining the sensitivity and specificity of the model when 
varying the threshold of probability of malignancy. We can then compare 
the performance of the quantitative tortuosity model to the reader study by 
comparing the area under the curve for both ROCs. 
11.0 STUDY MANAGEMENT 
11.1 
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 participating in the study, the patient will be asked to give 
consent to participate in the study by signing an IRB-approved consent 
form. 
 

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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. 
11.2 
Required Documentation 
Before the study can be initiated at any site, the following documentation 
must be provided to the Study Sponsor.  
 
• A copy of the official IRB approval letter for the protocol and 
informed consent 
• IRB membership list 
• CVs and medical licensure for the principal investigator and any 
associate investigators who will be involved in the study 
• A copy of the IRB-approved consent form 
• Executed clinical research contract (if applicable)  
 
The above documentation will be provided to our Study Sponsor (LCCC). 
11.3 
Registration Procedures 
Patients will be registered into OnCore®, a web based clinical research 
platform by one of the Study Coordinators.  The spread sheet contains 
each subject enrolled in the study identified by the patient first and last 
initial, study id, date of enrollment into study, race and ethnicity.  
11.4 
Data Management and Monitoring/Auditing 
The breast images of all eligible enrolled subjects that are obtained and 
contribute to the ultimate diagnosis leading to biopsy will be de-identified for 
inclusion in the reader study. Copies of the clinical report forms as well as 
the de-identified images described in the preceding will be submitted for 
each case to the Study Coordinators for maintaining the study record and 
entering the data into a spreadsheet in preparation for the reader study. 
  
As an investigator initiated study, this trial may also be audited by the 
Lineberger Cancer Center audit committee every twelve months. 
11.5 
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.   
11.5.1 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 IRB approval.  
 

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For any such emergency modification implemented, a UNC IRB 
modification form must be completed by UNC Research Personnel within 
five (5) business days of making the change.   
11.5.2 Single Patient/Subject Exceptions 
Any request to enroll a single subject who does not meet all the eligibility 
criteria of this study requires the approval of the UNC Principal 
Investigator and the UNC IRB.  
11.5.3 Other Protocol Deviations/Violations 
All other planned deviations from the protocol must have prior approval by 
the Principal Investigator and the UNC IRB.  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: 
• 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 without prior approval from the Principal 
Investigator, please follow the guidelines below: 
 
Protocol Deviations: UNC personnel will keep a log of any protocol 
deviations and report them to the study 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.   
11.6 
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 

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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 UNC’s IRB for approval prior to implementation.   
11.7 
Record Retention 
Study documentation includes all Case Report Forms, 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. 
11.8 
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/NCI regulations and guidelines regarding 
clinical trials both during and after study completion. 
 
The Principal Investigator at each institution or site will be responsible for 
assuring that all the required data will be collected and entered onto the 
Case Report Forms. 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 case report forms will be reviewed by the Principal Investigator and will 
require his/her final signature to verify the accuracy of the data. 
 
 

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12.0 References 
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10. Folkman, J., Angiogenesis. Annu Rev Med, 2006. 57: p. 1-18.  
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2005. 23(8): p. 1782-90.  

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14. Aylward, S., et al. Intra-operative 3D ultrasound augmentation in IEEE 
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3683-8.  
19. Bullitt, E., et al., Malignancy-associated vessel tortuosity: a computer-
assisted, MR angiographic study of choroid plexus carcinoma in genetically 
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20. Gessner, R., et al. Ultrasonic assessment of disease using a quantitative 
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25. Gessner, R.C., S.R. Aylward, and P.A. Dayton, Mapping microvasculature 
with acoustic angiography yields quantifiable differences between healthy and 
tumor-bearing tissue volumes in a rodent model. Radiology, 2012. 264(3): p. 
733-40.  

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26. Bullitt, E., et al., Computerized assessment of vessel morphological changes 
during treatment of glioblastoma multiforme: report of a case imaged serially by 
MRA over four years. Neuroimage, 2009. 47 Suppl 2: p. T143-51.  
27. Bullitt, E., et al., Tumor therapeutic response and vessel tortuosity: 
preliminary report in metastatic breast cancer. Med Image Comput Comput 
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28. Bullitt, E., et al., Blood vessel morphologic changes depicted with MR 
angiography during treatment of brain metastases: a feasibility study. Radiology, 
2007. 245(3): p. 824-30.  
29. Bullitt, E., et al., Vessel tortuosity and brain tumor malignancy: a blinded 
study. Acad Radiol, 2005. 12(10): p. 1232-40.  
30. Jain, R.K., Normalization of tumor vasculature: an emerging concept in 
antiangiogenic therapy. Science, 2005. 307(5706): p. 58-62.  
31. Willett, C.G., et al., Direct evidence that the VEGF-specific antibody 
bevacizumab has antivascular effects in human rectal cancer. Nat Med, 2004. 
10(2): p. 145-7.  
32. Yuan, F., et al., Time-dependent vascular regression and permeability 
changes in established human tumor xenografts induced by an anti-vascular 
endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad 
Sci U S A, 1996. 93(25): p. 14765-70.  
33. Kopans, D.B., Breast Imaging. 2nd ed. 1997, New York: Lippincott Williams 
and Wilkins.  
34. Highnam, R. and M. Brady, Mammographic image analysis. 1999: Kluwer 
Academic Pub.  
35. Bassett, L.W., Diagnosis of Diseases of the Breast. 1997, Philadelphia: WB 
Saunders  
36. Wu, T., et al., A comparison of reconstruction algorithms for breast 
tomosynthesis. Med Phys, 2004. 31(9): p. 2636-47.  
37. Pisano, E.D., et al., Diagnostic accuracy of digital versus film mammography: 
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38. Checka, C.M., et al., The relationship of mammographic density and age: 
implications for breast cancer screening. AJR Am J Roentgenol, 2012. 198(3): p. 
W292-5.  

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39. Bevilacqua, P., et al., Prognostic value of intratumoral microvessel density, a 
measure of tumor angiogenesis, in node-negative breast carcinoma--results of a 
multiparametric study. Breast Cancer Res Treat, 1995. 36(2): p. 205-17.  
40. Bosari, S., et al., Microvessel quantitation and prognosis in invasive breast 
carcinoma. Hum Pathol, 1992. 23(7): p. 755-61.  
41. Gasparini, G., et al., Tumor microvessel density, p53 expression, tumor size, 
and peritumoral lymphatic vessel invasion are relevant prognostic markers in 
node-negative breast carcinoma. J Clin Oncol, 1994. 12(3): p. 454-66.  
42. Ogawa, Y., et al., Microvessel quantitation in invasive breast cancer by 
staining for factor VIII-related antigen. Br J Cancer, 1995. 71(6): p. 1297-301.  
43. Toi, M., J. Kashitani, and T. Tominaga, Tumor angiogenesis is an 
independent prognostic indicator in primary breast carcinoma. Int J Cancer, 
1993. 55(3): p. 371-4.  
44. Weidner, N., et al., Tumor angiogenesis: a new significant and independent 
prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst, 1992. 
84(24): p. 1875-87.  
45. Murri, A.M., et al., The relationship between the systemic inflammatory 
response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, 
microvessel density and survival in patients with primary operable breast cancer. 
Br J Cancer, 2008. 99(7): p. 1013-9.  
46. Martin, L., et al., Examining the technique of angiogenesis assessment in 
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47. Protopapa, E., G.S. Delides, and L. Revesz, Vascular density and the 
response of breast carcinomas to mastectomy and adjuvant chemotherapy. Eur J 
Cancer, 1993. 29A(10): p. 1391-3.  
48. Foekens, J.A., et al., High tumor levels of vascular endothelial growth factor 
predict poor response to systemic therapy in advanced breast cancer. Cancer 
Res, 2001. 61(14): p. 5407-14.  
49. Manders, P., et al., Vascular endothelial growth factor is associated with the 
efficacy of endocrine therapy in patients with advanced breast carcinoma. 
Cancer, 2003. 98(10): p. 2125-32.  
50. Heimann, R., et al., Assessment of intratumoral vascularization 
(angiogenesis) in breast cancer prognosis. Breast Cancer Res Treat, 1998. 52(1-
3): p. 147-58.  

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51. Goldberg, B.B., J.S. Raichlen, and F. Forsberg, Ultrasound Contrast Agents: 
Basic principals and clinical applications. 2001, London: Martin Dunitz.  
52. Abdelmoneim, S.S., et al., Safety of contrast agent use during stress 
echocardiography: a 4-year experience from a single-center cohort study of 
26,774 patients. JACC Cardiovasc Imaging, 2009. 2(9): p. 1048-56.  
53. Abdelmoneim, S.S., et al., Safety of contrast agent use during stress 
echocardiography in patients with elevated right ventricular systolic pressure: a 
cohort study. Circ Cardiovasc Imaging, 2010. 3(3): p. 240-8.  
54. Dolan, M.S., et al., Safety and efficacy of commercially available ultrasound 
contrast agents for rest and stress echocardiography a multicenter experience. J 
Am Coll Cardiol, 2009. 53(1): p. 32-8.  
55. Mulvagh, S.L. Safety of Ultrasound Contrast Studies. in Diagnostic 
Ultrasound Annual Conference. 2010.  
56. Huang, S.F., et al., Analysis of tumor vascularity using three-dimensional 
power Doppler ultrasound images. IEEE Trans Med Imaging, 2008. 27(3): p. 
320-30.  
57. Molinari, F., et al., Characterization of single thyroid nodules by contrast-
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58. Hanley, J.A. and B.J. McNeil, A method of comparing the areas under 
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60. Bullitt, E., et al. (2004) Determining Malignancy of Brain Tumors by Analysis 
of Vessel Shape. Medical Image Computing and Computer-Assisted Intervention 
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61. Bullitt, E., et al., Measuring tortuosity of the intracerebral vasculature from 
MRA images. IEEE Trans Med Imaging, 2003. 22(9): p. 1163-71. 
62. Gessner, R.C., et al., An In Vivo Validation of the Application of Acoustic 
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D Ultrasound. Ultrasound Med Biol, 2012. 38(4) p. :651-60. 
 
 

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13.0 APPENDICES 
13.1 
Appendix A: Reader Study Data Collection Form 
 
Specimen:   
 
 
 
 
 
 
Reader: 
 
 
 
 
Date:  
 
 
 
Overall Assessment: 
Acoustic Angiography 
 
Overall specimen malignancy score (-2 to +2):   
 
 
Malignancy confidence (0-100)   
% 
 
ULTRASOUND 
 
Overall specimen malignancy score (-2 to +2):   
 
 
Malignancy confidence (0-100) 
 
% 
 
 
 
1.  Acoustic Angiography versus ULTRASOUND Shape/Morphology 
+3 
I am significantly more confident in the Acoustic Angiography 
representation of the lesion shape/morphology I described as compared 
to ULTRASOUND representation of the same lesion 
+2 
I am more confident in the Acoustic Angiography representation of the 
lesion shape/morphology I described as compared to ULTRASOUND 
representation of the same lesion 
+1 
I am slightly more confident in the Acoustic Angiography 
representation of the lesion shape/morphology I described as compared 
to the ULTRASOUND representation of the same lesion. 
0 
I have the same confidence in the Acoustic Angiography 
representation of the lesion shape/morphology I described as I do in the 
ULTRASOUND representation of the same lesion 
-1 
I am slightly less confident in the Acoustic Angiography representation 
of the lesion shape/morphology I described as compared to the 
ULTRASOUND representation of the same lesion. 
-2 
I am less confident in the Acoustic Angiography representation of the 
lesion shape/morphology I described as compared to the ULTRASOUND 
representation of the same lesion. 
-3 
I am significantly less confident in the Acoustic Angiography 
representation of the lesion shape/morphology I described as compared 
to the ULTRASOUND representation of the same lesion. 
 
 
 

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2.  Acoustic Angiography versus ULTRASOUND: Vascularity 
 
+3 
I am significantly more confident in the Acoustic Angiography 
representation of the lesion vascularity I described as compared to the 
ULTRASOUND representation of the same lesion. 
+2 
I am more confident in the Acoustic Angiography representation of the 
lesion vascularity I described as compared to ULTRASOUND 
representation of the same lesion 
+1 
I am slightly more confident in the Acoustic Angiography 
representation of the lesion vascularity I described as compared to the 
ULTRASOUND representation of the same lesion. 
0 
I have the same confidence in the Acoustic Angiography 
representation of the lesion vascularity I described as I do in the 
ULTRASOUND representation of the same lesion. 
-1 
I am slightly less confident in the Acoustic Angiography representation 
of the lesion vascularity I described as compared to the ULTRASOUND 
representation of the same lesion. 
-2 
I am less confident in the Acoustic Angiography representation of the 
lesion vascularity I described as compared to the ULTRASOUND 
representation of the same lesion. 
-3 
I am significantly less confident in the Acoustic Angiography 
representation of the lesion vascularity I described as compared to the 
ULTRASOUND representation of the same lesion. 
 
3.  Acoustic Angiography versus ULTRASOUND Margins /Distribution 
+3 
I am significantly more confident in the Acoustic Angiography 
representation of the lesion margins/distribution I described as compared 
to ULTRASOUND representation of the same lesion 
+2 
I am more confident in the Acoustic Angiography representation of the 
lesion margins/distribution I described as compared to ULTRASOUND 
representation of the same lesion 
+1 
I am slightly more confident in the Acoustic Angiography 
representation of the lesion margins/distribution I described as compared 
to the ULTRASOUND representation of the same lesion. 
0 
I have the same confidence in the Acoustic Angiography 
representation of the lesion margins/distribution I described as I do in the 
ULTRASOUND representation of the same lesion 
-1 
I am slightly less confident in the Acoustic Angiography representation 
of the lesion ULTRASOUND representation of the same lesion. 
-2 
I am less confident in the Acoustic Angiography representation of the 
lesion margins/distribution I described as compared to the 
ULTRASOUND representation of the same lesion. 
-3 
I am significantly less confident in the Acoustic Angiography 
representation of the lesion margins/distribution I described as compared 
to the ULTRASOUND representation of the same lesion. 

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13.1 
Appendix B: Previous Clinical Investigation Use of Device: 
 
The Vevo 770 investigational device and its successor the Vevo 2100 
have been utilized in a number of clinical research studies deemed non-
significant risk by the respective IRB or other regulatory agency 
responsible for human subject safety.  
 
UNITED STATES 
• University of Louisville, Louisville: Novel Pediatric Applications 
of High Resolution Ultrasound Imaging. This study evaluates the 
Vevo 2100 in comparison with standard ultrasound in the 
imaging of head, heart, and kidneys in premature infants. 
• Jewish Hospital and St. Mary's Healthcare, Louisville: High 
Resolution Ultrasound in the Study of Arteriopathy in Composite 
Tissue Allotransplantation of the Hand. This study utilizes the 
Vevo 2100 to monitor blood flow, vessels, arteries and artery 
intima thickness, for signs of potential allograft rejection. 
• Hospital of the University of Pennsylvania, Philadelphia: High-
Frequency Ultrasound for Evaluation of Intimal Thickness. This 
study utilized a Vevo 770 to measure the intima separately from 
the media in the brachial, radial, and anterior tibial arteries. 
• University of Pittsburgh Medical Center, Pittsburgh: Non-
invasive High Resolution Ultrasound Evaluation. This study 
utilizes a Vevo 2100 to upper extremity arterial vasculature. 
 
CANADA 
• St. Michael's Hospital, Toronto (ITA 123218): Maturation of the 
Arterio-venous Fistula for Hemodialysis. This study utilizes the 
Vevo 770 to track the AVF maturation process by monitoring of the 
vascular remodeling of the blood vessels, to better predict AVF 
readiness for cannulation. 
• Hospital for Sick Kids, Toronto (ITA 137454): Very High-Resolution 
Vascular Imaging in Children with Normal and Abnormal Vascular 
Development and Maturation. This study utilizes the Vevo 770 to 
determine the vascular function of peripheral arteries in children 
with or without abnormal pre- and post-natal hemodynamics. 
• Mt. Sinai Hospital, Toronto (ITA 163547): High-Resolution Micro 
Ultrasound in the Diagnosis and Treatment of Male Infertility. This 
study utilizes the Vevo 2100 to establish normal parameters for the 
human testes using high resolution ultrasound to demonstrate its 
diagnostic advantage over standard ultrasound. 
 
 
 
 
 

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NETHERLANDS 
• Erasmus Medical Center, Rotterdam: High-Resolution 
Ultrasonography of the Cutaneous Nerve Branches in the Hand 
and Wrist. This study utilized the Vevo 770 to image the median, 
radial, ulnar, and digital nerves to evaluate the Vevo in the 
diagnosis of various neuropathies, including nerve injury. 
• Erasmus Medical Center, Rotterdam: Radial Access Research: 
Echo Based Radial Artery Evaluation for Diagnostic or Therapeutic 
Coronary Intervention. This study utilizes the Vevo 2100 to assess 
arterial wall healing following radial artery cannulations. 
 
SWEDEN 
• University Hospital, Uppsala: Thicker Carotid Intima Layer and 
Thinner Media Layer in Subjects with Cardiovascular Diseases. 
This study utilizes the Vevo 21 00 to evaluate vascular risk in 
connection with systemic lupus erythematosus {SLE) and hormone 
replacement therapy. 
• Sahlgrenska University Hospital, Goteborg: High-Resolution 
Ultrasound Showing Increased Intima and Media Thickness of the 
Radial Artery in Patients with End-Stage Renal Disease. This study 
utilized the Vevo 770 to measure intima and media thickness 
separately in patients with end-stage renal disease in order to 
investigate the radial arterial wall layers in patients with chronic 
renal failure. 
• Sahlgrenska University Hospital, Goteborg: Increased Intima 
Thickness of the Radial Artery in Patients with Coronary Heart 
Disease. This study utilized the Vevo 770 to assess intima 
thickness separately from intima-media thickness as a potential tool 
for non-invasive early detection of atherosclerosis. 
• Sahlgrenska University Hospital, Goteborg: Increasing Peripheral 
Artery Intima Thickness from Childhood to Seniority. This study 
utilized the Vevo 770 in carotid, radial and anterior tibial arteries, 
measuring intima thickness and intima-media thickness to establish 
that intima thickness-to-lumen diameter ratio increases with age.