Academic-Industrial Partnership for Translation of Acoustic Angiography
Phase: PHASE4
Conditions: Signs and Symptoms
Sponsor: UNC Lineberger Comprehensive Cancer Center

LCCC  1748:  Academic-Industrial Partnership for Translation of Acoustic 
Angiography 
 
 
NCT Number: NCT03544268 
Document Date: 2/8/18 
 

LINEBERGER COMPREHENSIVE CANCER CENTER 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
 
LCCC  1748:  Academic-Industrial Partnership for Translation of Acoustic 
Angiography 
 
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: NIH (National Cancer Institute) 1R01CA189479 - 01 
Version Date: February 8, 2018 

LINEBERGER COMPREHENSIVE CANCER CENTER 
CLINICAL ONCOLOGY RESEARCH PROGRAM 
UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL 
 
LCCC  1748:  Academic-Industrial Partnership for Translation of Acoustic 
Angiography 
  
 
 
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 1748 
 
CONFIDENTIAL 
PI: Lee 
 
UNIVERSITY OF NORTH CAROLINA 
February 8, 2018 
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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 ................................................................................................6 
4.0 
STUDY PLAN .........................................................................................................7 
4.1 
Schema ................................................................................................................7 
4.2 
Enrollment/Recruitment .....................................................................................8 
4.3 
Acoustic Angiography ........................................................................................8 
4.4 
Standard of Care Biopsy ...................................................................................9 
4.5 
Medical Record Abstraction ..............................................................................9 
4.6 
Reader Study ....................................................................................................10 
5.0 
Research Ultrasound Scanner: FUJIFILM VisualSonics Vevo 2100 ....10 
5.1 
Expected Risks .................................................................................................12 

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5.2 
Perflutren lipid (Definity) ..................................................................................12 
5.3 
Duration of Study ..............................................................................................12 
6.0 
Drug Information ................................................................................................12 
6.1 
Perflutren Lipid Microspheres (Lantheus Medical Imaging) ......................12 
7.0 
ADVERSE EXPERIENCES-DRUGS ................................................................14 
7.1 
Definitions ..........................................................................................................14 
7.2 
Documentation of non-serious AEs or SARs ...............................................16 
7.3 
SAEs or Serious SARs ....................................................................................16 
8.0 
UNANTICIPATED CONCERNS ........................................................................17 
8.1 
Unanticipated Adverse Device Effect (UADE) .............................................17 
8.2 
Unanticipated Problems (UP) .........................................................................18 
8.3 
Reporting ...........................................................................................................18 
9.0 
Data and Safety Monitoring Plan ...................................................................18 
10.0 STATISTICAL CONSIDERATIONS .................................................................19 
10.1 
Sample Size and Accrual.............................................................................19 
10.2 
Data Analysis Plans ......................................................................................20 
11.0 STUDY MANAGEMENT .....................................................................................21 
11.1 
Institutional Review Board (IRB) Approval and Consent ........................21 
11.2 
Required Documentation .............................................................................22 
11.3 
Registration Procedures ..............................................................................22 
11.4 
Data Management and Monitoring/Auditing .............................................22 
11.5 
Adherence to the Protocol ...........................................................................22 
11.6 
Amendments to the Protocol .......................................................................24 
11.7 
Record Retention ..........................................................................................24 

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11.8 
Obligations of Investigators .........................................................................24 
12.0 References ...........................................................................................................26 
13.0 APPENDICES.......................................................................................................31 
13.1 
Appendix A: Reader Study Data Collection Form ...................................31 
13.1 
Appendix B: Previous Clinical Investigation Use of Device: ..................33 

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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, and comparison of 
radiologist preference for the two imaging techniques for each of 3 lesion 
characteristics.  
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 
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%), 

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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, 
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 

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patient monitoring 30 minutes after ultrasound contrast examination, 
based on these and other recent safety data.  The ultrasound contrast 
agent to be used in LCCC1748 is perflutren lipid.  An IND exemption will 
be requested from the FDA for the usage of perflutren prior to usage in 
this study. 
1.4 
Perflutren Lipid Background and Known Toxicities 
See http://www.definityimaging.com/pdf/DEFINITY_US_PI_515987-
0117.pdf 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. 
Adverse events (AEs) appeared within 1 – 15 minutes of the drug 

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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. 
 
LCCC1748 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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UNIVERSITY OF NORTH CAROLINA 
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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 known breast lesions for predicting 
malignancy. 
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) 
2.3 
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 Patient had a diagnostic breast ultrasound study performed at UNC 
3.1.3 Scheduled for a core needle or surgical breast biopsy of at least one 
breast lesion that is 2 cm or less in size and 3 cm in depth from the skin 
surface 
3.1.4  Lesion visualized on ultrasound 
3.1.5 Able to provide informed consent 
3.1.6 Negative urine pregnancy test in women of child-bearing potential 
3.2 
Exclusion Criteria 
All subjects meeting any of the exclusion criteria at baseline will be 
excluded from study participation 

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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 Sonographically visible breast lesion larger than 2cm or greater than 3cm 
in depth from the skin surface 
3.2.5 Known hypersensitivity to sulfur hexafluoride or to any component of 
perflutren lipid (Definity®)  
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 14 days 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, 
as defined by the UNC IRB SOP 4801.) 
4.0 
STUDY PLAN 
4.1 
Schema 
This is a one arm single center study of 60 patients scheduled to be 
biopsied from the UNC Breast Clinic that consent to undergo an acoustic 
angiography in conjunction with b-mode ultrasound prior to their 
scheduled biopsy.   
 
 

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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 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. 
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 

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perflutren administration, and all patients will be monitored for acute 
reactions.  
 
Perflutren will be administered in split doses using the dosing range and 
administration type IV bolus) within the perflutren prescribing information 
(see http://www.definityimaging.com/how-administration.html). When 
administered as a bolus, the package insert recommends 10 uL/kg patient 
weight administered within 30-60 seconds, followed by a 10mL saline 
flush with a second dose of 10 uL/kg patient weight 30 minutes following 
the first dose, if needed.  All patients will be monitored for 15-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 15 min.  The blood pressure will be monitored at 15 minutes.  This 
study will be conducted in Mammography of the UNC Cancer Hospital, so 
trained medical personnel will be available as needed. 
 
We will infuse via hand bolus injection for a "precision controlled bolus".   
4.3.2 Acoustic Angiography Imaging Procedures 
Acoustic angiography imaging involves a research ultrasound scanner 
(Verasonics Vantage 256; see section 5.0) as well as conventional b-
mode ultrasound to guide the location of the imaging. The conventional 
ultrasound will be conducted just prior to the acoustic angiography for 
localization. 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. Total imaging time 
is estimated to be less than 15 minutes.  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 
The primary objective of this study is to determine the sensitivity and 
specificity of acoustic angiography imaging. In order to meet this objective, 
we will review each patient’s clinical records, including their pathology 

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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 
LCCC1748.    
 
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 order that the modalities will be presented to the 
radiologists are randomized.  The first modality presented for the 
specimen will be considered the primary and the second modality 
presented will be considered the secondary for the survey instrument. 
After a washout period of four weeks, the modalities will be presented in 
reverse.  
 
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. 
5.0 
Research Ultrasound Scanner: Verasonics Vantage 256 
The device used for this study will be a programmable research 
ultrasound scanner (Vantage 256, Verasonics, Inc., Kirkland, WA, USA) 
and a unique probe developed in conjunction with the research lab run by 
Dr. Stuart Foster (Sunnybrook Research Institute, Toronto, ON, Canada) 
and VisualSonics (FUJIFILM VisualSonics, Inc., Toronto, ON, Canada).    
The ultrasound probe developed is a modification of a preclinical high 
frequency transducer, modified to house a dual-frequency linear array.   
 
The Vantage 256 is a programmable research ultrasound system.  It is 
marketed commercially by Verasonics, Inc.  For this study, two Vantage 
256 platforms will be used.  Each system will be programmed for 

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diagnostic ultrasound imaging at acoustic exposures similar to those 
approved for clinical use.  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 when acoustic output is maintained 
at or below FDA-approved levels (see section 13.1, Appendix B). 
 
 
 
 
Figure 1. Device Image 
 
Dual-Frequency Transducers: 
The  probes we plan to use in this study are modified VisualSonics 
probes.  They are dual-frequency linear transducer arrays.  We plan to run 
the transducers in 2 modes during the study: b-mode and dual-frequency 
contrast mode.  In b-mode, only the high frequency elements are 
transmitting and receiving ultrasound.  B-mode imaging will be performed 
using a designated b-mode program on one Vantage 256 scanner.  In 
dual-frequency contrast mode, the low frequency elements transmit 
ultrasound and the high frequency elements receive the signal.  To do so, 
one Vantage 256 controls the low frequency elements, and a second 
Vantage 256 controls the high frequency elements.  Operation of the 
systems is synchronized and controlled by one designated dual-frequency 
program.  It should be noted that only one Vantage system will be 
transmitting at any given time. The center frequency is 2 MHz for the low 
frequency elements and 20 MHz for the high frequency elements.   

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5.1 
Expected Risks  
5.1.1 Verasonics Vantage 256 
This research protocol presents minimal risk to participants, investigators 
and study personnel. Our preliminary experimental and theoretical data 
show that the proposed stack design does effectively preserve excellent 
independent transmission of the two beams from the same aperture. 
Additional FEM modeling will be conducted to look at minimizing HF pulse 
distortion by varying the interlayer thicknesses and acoustic properties. The 
LF device will be designed to produce pulses with a mechanical index below 
the FDA limit of 1.9. Materials and processes will be adjusted to ensure that 
the probe temperature does not increase more than 6°C, as stated by the 
FDA.52 Thermal effects will also be modeled using finite element methods 
(PZFlex). Analysis of the low frequency beam will be performed to optimize 
the number and lateral extent of low frequency elements. The current 
transducer design contains 32 LF elements, which we believe should be 
enough in the low frequency array. Based on simulations and experimental 
findings, however, future transducer iterations may extend this to 64 
elements for improved imaging performance. Although the high frequency 
array by itself has been previously optimized with respect to its use in 
preclinical VisualSonics systems, the dual-frequency variant and effects of 
the various polymer and metal layers within flex circuits required for element 
connectivity will need to be included within the acoustic modeling of the 
transducer stack. 
5.2 
Perflutren lipid (Definity) 
See sections 1.4 and 6.1.5. 
5.3 
Duration of Study 
It is anticipated that the total clinical study duration encompassing 
recruitment, enrollment, and data analysis will take approximately 2 years 
and will begin in year 4 of the grant.  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 

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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.  
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.definityimaging.com/pdf/DEFINITY_US_PI_515987-
0117.pdf), 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 
January 2017 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 DEFINITY 
administration and monitor all patients for acute reactions. The reported 
reactions include: fatal cardiac or respiratory arrest, shock, syncope, 

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symptomatic arrhythmias (atrial ﬁ brillation, tachycardia, bradycardia, 
supraventricular 
tachycardia, 
ventricular 
ﬁ 
brillation, 
ventricular 
tachycardia), hypertension, hypotension, dyspnea, hypoxia, chest pain, 
respiratory distress, stridor, wheezing, loss of consciousness, and 
convulsions. Hypersensitivity Reactions:  
In postmarketing use, serious hypersensitivity reactions were observed 
during or shortly following perﬂ utren-containing microsphere administration 
including: Shock, bronchospasm, throat tightness, angioedema, edema 
(pharyngeal, palatal, mouth, peripheral, localized), swelling (face, eye, lip, 
tongue, upper airway), facial hypoesthesia, rash, urticaria, pruritus, ﬂ 
ushing, and erythema have occurred in patients with no prior exposure to 
perﬂ utren-containing microsphere products.  Always have cardiopulmonary 
resuscitation personnel and equipment readily available prior to DEFINITY 
administration and monitor all patients for hypersensitivity reactions.  
Systemic Embolization of Perflutren in Patients with Cardiac Shunts:  
When administering DEFINITY to patients with a cardiac shunt, the 
microspheres can bypass ﬁ ltering by the lung and enter the arterial 
circulation.  Assess patients with shunts for embolic phenomena following 
DEFINITY administration.   DEFINITY is only for intravenous administration; 
do not administer DEFINITY by intra-arterial injection.  
Ventricular Arrhythmia Related to High 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.  DEFINITY is not recommended for use at 
mechanical indices greater than 0.8.. 
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 
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.   
 

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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 
treatment group than in a concurrent or historical control group 
7.1.3 Unexpected AE or SAR 
An AE or SAR is considered unexpected if the 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 
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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, 
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).  
 

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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.  
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 
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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)).  
 
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 

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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.   
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 
10.1.1 Primary analysis 
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% 

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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 88% 
(the variance of the estimated specificity is calculated to be less than 
0.6*0.25/30) at the significance level 0.05 using one-side test.  Although 
we will study both sensitivity and specificity, our power calculation is 
based on specificity. 
10.1.2 Secondary Analysis 
Sixty (60) women scheduled to undergo breast biopsy based on an 
abnormal ultrasound finding will be enrolled. We assume the AUC for the 
b-mode ultrasound images is 60% with range 60%~80%, and AUC 
difference of 10%. Assuming that the prevalence of noncancerous lesions 
in the sample population will be approximately 50%, the study will be 
powered at least at 80% with 56 patients and 5 readers.  
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 
assumption.  
 
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 
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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. 
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 
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 accordance with 
Lineberger Comprehensive Cancer Center’s Single Subject Exceptions 
Policy. 
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 
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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 
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, 

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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 
1. Nagy, J.A., et al., Heterogeneity of the tumor vasculature. Semin Thromb 
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14. Aylward, S., et al. Intra-operative 3D ultrasound augmentation in IEEE 
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with acoustic angiography yields quantifiable differences between healthy and 
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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 
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31. Willett, C.G., et al., Direct evidence that the VEGF-specific antibody 
bevacizumab has antivascular effects in human rectal cancer. Nat Med, 2004. 
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32. Yuan, F., et al., Time-dependent vascular regression and permeability 
changes in established human tumor xenografts induced by an anti-vascular 
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33. Kopans, D.B., Breast Imaging. 2nd ed. 1997, New York: Lippincott Williams 
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34. Highnam, R. and M. Brady, Mammographic image analysis. 1999: Kluwer 
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35. Bassett, L.W., Diagnosis of Diseases of the Breast. 1997, Philadelphia: WB 
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36. Wu, T., et al., A comparison of reconstruction algorithms for breast 
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38. Checka, C.M., et al., The relationship of mammographic density and age: 
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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 
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and peritumoral lymphatic vessel invasion are relevant prognostic markers in 
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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.  
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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 
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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: 
Primary 
 
Overall specimen malignancy score (-2 to +2):   
 
 
Malignancy confidence (0-100)   
% 
 
Secondary 
 
Overall specimen malignancy score (-2 to +2):   
 
 
Malignancy confidence (0-100) 
 
% 
 
 
 
1.  Primary versus Secondary Shape/Morphology 
+3 
I am significantly more confident in the Primary representation of the 
lesion shape/morphology I described as compared to Secondary 
representation of the same lesion 
+2 
I am more confident in the Primary representation of the lesion 
shape/morphology I described as compared to Secondary 
representation of the same lesion 
+1 
I am slightly more confident in the Primary representation of the lesion 
shape/morphology I described as compared to the Secondary 
representation of the same lesion. 
0 
I have the same confidence in the Primary representation of the lesion 
shape/morphology I described as I do in the Secondary representation 
of the same lesion 
-1 
I am slightly less confident in the Primary representation of the lesion 
shape/morphology I described as compared to the Secondary 
representation of the same lesion. 
-2 
I am less confident in the Primary representation of the lesion 
shape/morphology I described as compared to the Secondary 
representation of the same lesion. 
-3 
I am significantly less confident in the Primary representation of the 
lesion shape/morphology I described as compared to the Secondary 
representation of the same lesion. 
 
 
 

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

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13.1 Appendix B: Previous Clinical Investigation Use of Device: 
 
The Verasonics Vantage investigational device and its predecessors 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 
• Duke University, Durham, NC: Improved Visualization of 
Endocardial Borders with Short-Lag Spatial Coherence Imaging 
of Fundamental and Harmonic Ultrasound Data. DOI: 
10.1109/ULTSYM.2012.0531. This study used a Verasonics 
scanner to implement a new imaging scheme to improve 
endocardial delineation. 
• University of Washington, Seattle, WA: Ultrasonic propulsion of 
kidney stones: preliminary results of human feasibility study. 
DOI: 10.1109/ULTSYM.2014.0126. This study utilized a 
Verasonics system to perform ultrasonic propulsion of kidney 
stones as an alternative to surgery.  
• Mayo Clinic College of Medicine, Rochester, MN: Pediatric 
Cardiac Shear Wave Elastography for Quantitative Assessment 
of Myocardial Stiffness: A Pilot Study in Healthy Controls. DOI: 
10.1016/j.ultrasmedbio.2016.03.009. This study implemented 
shear wave elastography on a Verasonics Vantage system to 
evaluate myocardial stiffness in children.  
• Mayo Clinic College of Medicine, Rochester, MN: Comb-Push 
Ultrasound Shear Elastography (CUSE) for Evaluation of 
Thyroid Nodules: Preliminary In Vivo Results. DOI: 
10.1109/TMI.2014.2346498. This study used a Verasonics 
platform to measure tissue stiffness of benign and malignant 
thyroid nodules with elastography.  
• Mayo Clinic College of Medicine, Rochester, MN: Probe 
Oscillation Shear Wave Elastography: Initial In Vivo Results in 
Liver. DOI: 10.1109/TMI.2017.278085. This study employed a 
Verasonics system to perform a novel elastography technique in 
the liver.  
• Mayo Clinic College of Medicine, Rochester, MN: Effect of 
Calcifications on Breast Ultrasound Shear Wave Elastography: 
An Investigational Study. DOI: 10.1371/journal.pone.01378. 
This study used a Verasonics scanner to evaluate the results of 
shear wave elastography when breast calcifications are present.  
• Columbia University, New York, NY: Evaluation of Coronary 
Artery Disease Using Myocardial Elastography with Diverging 
Wave Imaging: Validation Against Myocardial Perfusion Imaging 
and Coronary Angiography. DOI: 
10.1016/j.ultrasmedbio.2017.01.001. This study compared a 

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novel elastography technique on a Verasonics system to non-
ultrasound standard of care imaging methods used for the 
assessment of coronary artery disease.  
 
CANADA 
• University of Montreal Hospital, Montreal, QC: High-Frame-Rate 
Echocardiography Using Coherent Compounding with Doppler-
Based Motion-Compensation. DOI: 10.1109/TMI.2016.2523346. 
This study utilized a Verasonics scanner to implement motion-
corrected echocardiography at high frame rates.  
• University of Montreal Hospital, Montreal, QC: High-Frame-Rate 
Speckle-Tracking Echocardiography. DOI: 
10.1109/TUFFC.2018.2809553. This study used a Verasonics 
system to assess a novel image processing technique on 
echocardiography images.   
 
EUROPE 
• Imperial College, London, UK: High Frame-Rate Contrast 
Echocardiography: In-Human Demonstration. DOI: 
10.1016/j.jcmg.2017.09.011. This study used a Verasonics 
scanner to demonstrate an improvement in contrast 
echocardiography by using high frame rate imaging.