High Frame Rate 3-D Super Resolution Ultrasound Microvascular Imaging
Phase: PHASE2
Conditions: Breast Cancer, Thyroid Cancer
Sponsor: UNC Lineberger Comprehensive Cancer Center

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LCCC1915: High Frame Rate 3-D Super Resolution Ultrasound Microvascular 
Imaging  
 
Principal Investigator 
Yueh Lee, MD, PhD 
University of North Carolina 
Department of Radiology  
 
yueh_lee@med.unc.edu 
 
Principal Co-Investigator  
Cherie Kuzmiak, DO 
University of North Carolina 
Department of Radiology 
 
cherie_kuzmiak@med.unc.edu   
 
Lauren M.B Burke, MD 
University of North Carolina 
Department of Radiology 
 
lauren burke@med.unc.edu  
 
 
Biostatistician  
Thad Benefield, MS 
University of North Carolina 
Department of Radiology 
thad benefield@med.unc.edu 
 
 
Sponsor:  Lineberger Comprehensive Cancer Center 
 
Funding Source: NIH (National Cancer Institute) 
 
Date: August 19, 2022 

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LCCC1915: High Frame Rate 3-D Super Resolution Ultrasound Microvascular 
Imaging 
 
Principal Investigator 
Yueh Lee, MD, PhD 
University of North Carolina 
Department of Radiology  
919-537-3730 phone 
yueh lee@med.unc.edu  
 
 
 
 
 
 
 
 
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:____________________ 
 
Date of Protocol: August 19, 2022 
 
 
 
 

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TABLE OF CONTENTS 
1.0 ............................................................................ BACKGROUND AND RATIONALE
..............................................................................................................................................4 
1.1 
Study Synopsis ...................................................................................................4 
1.2 
Disease Background ..........................................................................................4 
1.3 
Ultra-fast and Super-Resolution Imaging .......................................................5 
1.4 
Software-based Ultrasound Systems ..............................................................7 
1.5 
Impact of ultrasound as a diagnostic tool .......................................................7 
1.6 
Ultrasound-localization microscopy/Contrast Enhanced Super Resolution 
(CESR) Imaging .............................................................................................................7 
1.7 
Investigational super-resolution imaging methods ........................................8 
1.8 
Perflutren Lipid Background and Known Toxicities .......................................8 
1.9 
Rationale ............................................................................................................11 
2.0 .................................................................................................... STUDY OBJECTIVES
............................................................................................................................................11 
2.1 
Primary Objectives ...........................................................................................12 
2.2 
Secondary Objectives ......................................................................................12 
2.3 
Endpoints ...........................................................................................................12 
3.0 ................................................................................................... PATIENT ELIGIBILITY
............................................................................................................................................12 
3.1 
Arm 1: Healthy Volunteers ..............................................................................12 
3.2 
Arm 2: Breast Imaging Patients .....................................................................13 
3.3 
Arm 3: Thyroid Imaging Patients ....................................................................15 
4.0 ................................................................................................................... STUDY PLAN
............................................................................................................................................16 
4.1 
STUDY SCHEMA .............................................................................................16 

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4.2 
Study Procedures .............................................................................................16 
5.0 ........................................................................................ INVESTIGATIONAL DEVICE
............................................................................................................................................18 
5.1 
Investigational Device Description .................................................................19 
6.0 ............................ Perflutren Lipid Microspheres (Lantheus Medical Imaging)
............................................................................................................................................20 
6.1 
Investigational Contrast Agent Description and Management ..................21 
6.2 
Return and Retention of Study Contrast Agent ...........................................22 
7.0 ............................................................. UNANTICIPATED CONCERNS (DEVICES)
............................................................................................................................................22 
8.0 .......................................... ADVERSE EVENTS (DRUGS- CONTRAST AGENTS)
............................................................................................................................................24 
8.1 
Definitions ..........................................................................................................24 
8.2 
Documentation of non-serious AEs or SARs ...............................................26 
8.3 
SAEs or Serious SARs ....................................................................................26 
9.0 ............................................................................ STATISTICAL CONSIDERATIONS
............................................................................................................................................27 
9.1 
Study Design/Study Endpoints .......................................................................28 
9.2 
Data Analysis Plans .........................................................................................28 
9.3 
Sample Size Considerations in ROC Analysis ............................................28 
9.4 
Secondary Analyses ........................................................................................29 
10.0 .............................................................................................. STUDY MANAGEMENT
............................................................................................................................................29 
10.1 
Institutional Review Board (IRB) Approval and Consent ...........................29 
10.2 
Registration Procedures ..................................................................................30 
10.3 
Data Management and Monitoring/Auditing .................................................30 

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10.4 
Adherence to the Protocol ...............................................................................30 
10.5 
Amendments to the Protocol ..........................................................................31 
10.6 
Record Retention ..............................................................................................31 
10.7 
Obligations of Investigators .............................................................................32 
11.0 ............................................................................................................... REFERENCES
............................................................................................................................................32 
12.0 ................................................................................................................. APPENDICES
............................................................................................................................................34 
12.1 
Appendix A: Reader Study Data Collection Form .......................................34 
12.2 
Appendix B: Previous Clinical Investigation Use of Verasonics System .38 
 
 
 

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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 or thyroid cancer could provide substantial clinical 
benefit by improving diagnosis, preventing over-treatment, and reducing 
healthcare costs. Contrast enhanced super-resolution (CESR) imaging is 
a new type of contrast enhanced ultrasound imaging which is specifically 
sensitive to microvascular structure and density.  It evaluates tumor micro-
vasculature on the order of 10 – 100 microns in diameter and may provide 
a powerful prognostic tool for the diagnosis of breast cancer, and 
eventually for treatment evaluation.   
 
This is a 3-arm single center study of 45 patients.  These cohorts will 
include 15 breast patients scheduled to undergo a biopsy, and 15 thyroid 
patients scheduled to undergo fine needle aspiration, biopsy, or 
thyroidectomy that consent to undergo CESR imaging in conjunction with 
b-mode ultrasound prior to their scheduled biopsy.  Prior to imaging 
clinical patients, the third arm will include 15 healthy volunteers that will be 
imaged to optimize imaging parameters. Despite the similarity to our 
previous study, LCCC 1748, optimization is required because we will 
employ a new imaging technique using a different US system. The 
optimization includes adjusting the frame rate, power, depth of imaging, 
and the linear translation rate.  The primary objectives of this study are to 
evaluate the sensitivity and specificity of CESR imaging in the analysis of 
known breast and thyroid lesions by comparing image analyses to the 
pathological results for these lesions. The secondary objectives of this 
study are to compare the sensitivity and specificity of CESR imaging with 
traditional b-mode ultrasound in the distinction of malignant versus benign 
breast and thyroid lesions.   
1.2 
Disease Background  
1.2.1 Breast Cancer 
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.[1, 2] 
Mammography is an effective tool for the early detection of breast cancer 
in the majority of women.[3] 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.[4] For young women with heritable mutations 
who wish to begin screening at a younger age, these limitations are 
especially problematic.[5] 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). 

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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%), 
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.  
 
1.2.2 Thyroid Cancer 
The most common endocrine cancer is thyroid cancer (approximately 
1.0%–1.5% of all new cancers diagnosed each year in the USA). In the 
United States, 53,000 people are diagnosed with thyroid cancer each 
year.[6] In many countries in the developed world, thyroid cancer 
incidence has increased dramatically over the past three decades.[7] 
Increased frequency in use of sensitive diagnostic procedures, including 
ultrasound, Doppler examination and imaging techniques like CT, MRI, or 
PET scanning, has amplified the detection of thyroid cancer among other 
cancer types.[8] However, thyroid cancer incidence and mortality 
tendencies have been recognized as being consistent with 
overdiagnosis.[9] The financial impact of overdiagnosis in a thyroid cancer 
patient can range from hundreds to thousands dollars. It may also cause 
prospective harm in standings of avoidable distress and conceivable 
adverse consequences of avoidable treatment. [10] 
 
1.3 
Ultra-fast and Super-Resolution Imaging 
Recent developments in ultrasound hardware and software have enabled 
a substantial leap forward in ultrasound imaging technology. New 
programmable ultrasound systems can utilize software beamformers, 
parallel and distributed computing architectures, and large onboard 
memory to perform ultra-fast imaging, on the order of thousands of frames 
per second, compared to ultrasound systems still utilized in the clinic 
which are limited to only slightly past 30 frames per second. This ‘quantum 
leap’ in system performance has enabled several revolutionary 
advancements that will ensure that ultrasound in the clinic will soon offer 
substantial capabilities beyond what is currently available. One such novel 
technology is ‘ultrasound localization microscopy’, also referred to in this 
proposal as contrast enhanced super-resolution (CESR) imaging.[11] This 
imaging approach has been developed by researchers in the medical 
ultrasound domain who have made an effort to mimic the revolutionary 
new technology of optical localization microscopy. Optical localization 

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microscopy exploits the stochastic blinking of specific fluorescent sources 
and super-localizes the center of each source by virtue of its separability. 
[12, 13] By accumulating these center positions over thousands of 
acquisitions, the resulting image achieves a ten-fold resolution 
improvement and enables imaging cell membrane and small organelles 
with a resolution beyond the diffraction limit. In the medical ultrasound 
domain, different techniques have been investigated to achieve an 
ultrasound super resolution image, using the blinking of contrast agent 
microbubbles as an acoustic equivalent of the fluorescent sources. Some 
groups used highly diluted contrast agents to meet the key requisite of 
separable microbubble detection.[14, 15] Although these groups obtained 
super-resolved images using conventional ultrasound scanners, the long 
acquisition time necessary to perform diluted microbubble super resolution 
imaging is likely to impinge upon its practicality. Tanter’s group first 
demonstrated the application of ultrafast acquisition and spatiotemporal 
filtering to separate microbubbles even at a clinically-relevant 
concentration, by exploiting the decorrelation of microbubbles from a stack 
of images.[14, 16] This technique is a direct analog to fluorescence 
photoactivation localization microscopy (FPALM) in optics and the 
acquisition time is more reasonable for clinical translation. By localizing 
the centers of separable scattering microbubbles, this ultrasound 
localization microscopy technique allows imaging of microvessels at 
resolutions as small as ten micrometers, over an order of magnitude 
smaller than the ultrasound diffraction limit. Their team has recently 
published the use of this novel approach in Nature, demonstrating super-
resolved vasculature maps of rat brain slices using this super resolution 
contrast ultrasound technique (Fig. 2.).[11] The Dayton Lab at UNC, 
following work from Tanter’s group, has taken this approach a step further, 
applying it with a mechanically scanned system to obtain 3-D images of 
tumor-associated angiogenesis.[17] Our results illustrate that we can 
observe the same microvascular abnormalities we observe with Acoustic 
Angiography in tumors – supporting our hypothesis that we will be able to 
use super-resolution imaging to image cancer biomarkers in humans. It is 
of particular note that CESR can be performed effectively at clinical 
frequencies (our data to date has been acquired at 4.5 MHz with a clinical 
ATL probe), and depths up to 10 cm are theoretically achievable while still 
retaining resolution better than 100 microns, as long as microbubbles are 
detectable in original B mode imaging!  
 
Another notable advantage of CESR is that it can be performed at low 
mechanical indices, less than 0.2, which means that it is a non-destructive 
imaging technique, and can readily be performed within guidelines for 
contrast in humans. 

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1.4 
Software-based Ultrasound Systems 
Within the last few years, computational power has finally achieved the 
performance required to design almost all components of an ultrasound 
system with dedicated programmable integrated circuits. Consequently, 
instead of large analog componentry with fixed capabilities, modern 
ultrasound systems can fit into the case of a simple PC, and still be highly 
programmable. Commercial vendors such as Verasonics, Cephasonics, 
and others, are now making programmable ultrasound systems widely 
available. The high-performance hardware means that these systems can 
transmit and receive data at rates up to the pulse repetition frequency limit 
based on speed of sound (thousands of frames per second, depending on 
tissue depth). It is this advancement which has enabled super-resolution 
imaging as described above. 
1.5 
Impact of ultrasound as a diagnostic tool 
While there are clearly clinical applications for MRI, PET, SPECT, and CT 
that will never be supplanted by acoustics, there are clear clinical 
applications where ultrasound is well positioned to improve clinical 
outcome. For example, breast ultrasound has poor specificity and a high 
false positive rate, and hence it is not used as a screening tool for breast 
cancer. Furthermore, ultrasound is challenged to detect breast lesions 
smaller than a few millimeters. The same can be said about ultrasound’s 
sensitivity to malignant thyroid and prostate cancer. Improvements in 
specificity in these fields would have a significant clinical impact. 
Furthermore, due to the low-cost and portability of the next generation of 
laptop or handheld-sized ultrasound systems, this modality is uniquely 
poised to expand diagnostic capability to rural and underserved locations 
and populations worldwide. Other clinical applications, such as assessing 
vasa vasorum in atherosclerotic plaques, or assessing angiogenesis in 
wound healing, may also benefit from high-resolution microvascular 
ultrasound imaging. Furthermore, the applications of high resolution 
microvascular imaging in pre-clinical cancer research are readily apparent. 
1.6 
Ultrasound-localization microscopy/Contrast Enhanced Super 
Resolution (CESR) Imaging 
The methods described in this protocol are based on the novel imaging 
technique of Ultrasound Localization Microscopy, also called Contrast 
Enhanced Super-Resolution Imaging (CESR). This technology is only 
about five years old – with the seminal paper being published in Nature in 
2015.[11] Its growth has been explosive because of the enormous 
potential– with at least 8 talks in this area at the 2016 International 
Ultrasonics Symposium. Further innovation from this technique arises 
from our 3-D CESR implementation, which has been published in 
November 2016.[17] In this project, we will advance this technology to the 
next step – fast 3-D CESR with a matrix transducer, combined with unique 
approaches such as multi-focus adaptive beamforming to increase 

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sensitivity, pushing its innovation to the next level with the goal of clinical 
translation. 
1.7 
Investigational super-resolution imaging methods 
We will perform contrast enhanced super-resolution (CESR) imaging of 
tumors using a Verasonics Vantage system (Verasonics Inc., Redmond, 
WA, USA) with one of three different ultrasound probes, using plane-wave 
imaging at a pulse repetition frequency of 500 Hz. Two of the possible 
probes are 32x32 element matrix arrays with center frequencies of 1.5 and 
3 MHz. The third probe is a hybrid dual-frequency array that transmits at 
1.7 MHz with two single elements and receives at 20 MHz on a linear 
array. The transmitted pulses will be 1 cycle sinusoids at 1.5, 1.7, or 3 
MHz with mechanical index less than 0.3. This low mechanical index was 
chosen to minimize bubble destruction under high frame rate 
insonification.  
 
For data acquired with the matrix probes, a high-pass spatiotemporal 
singular value decomposition (SVD) filter is applied to detect the 
decorrelation of bubbles, yielding individual sources on the filtered images. 
This spatiotemporal filter can discriminate bubble signals whose spatial 
coherence is low from tissue signals whose spatial coherence is high 
because their temporal variations affect many neighboring pixels the same 
way.[18] For data acquired with the dual-frequency probe, bubbles are 
detected based on their nonlinear response relative to tissue. Hysteresis 
thresholding is used to localize the bubbles on the filtered images. Bubble 
centers are detected and center positions from all the frames are 
accumulated to get a super-resolution image, with a pixel size of 10 μm × 
10 μm, for each scan slice.  
1.8 
Perflutren Lipid Background and Known Toxicities 
See https://definityimaging.com/pdf/definity-pi.pdf for full prescribing 
information on perflutren when used according to its FDA-approved 
indication.   
1.8.1 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 
microvascular imaging in the breast or for thyroid imaging. 
 

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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.  We will request an investigational new drug 
(IND) exemption at the time of the IRB application because the 
investigators feel this study meets the criteria of an IND exempt study. 
1.8.2 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 
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 

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10,000,[19] compared to gadolinium-based MRI contrast which has an 
incidence of NSF of 2 – 5 in 100 patients with chronic kidney disease.[20]  
 
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 15-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 for additional information. In order to avoid a potentially 
fatal event, epinephrine will be readily accessible in the code cart in the 
mammography clinic.  
 
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).  

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1.9 
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 CESR imaging, 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 and thyroid 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. 
 
LCCC1915 is designed to evaluate the sensitivity and specificity of 
contrast enhanced super-resolution ultrasound imaging in the analysis of 
known breast and thyroid lesions in patients with pathologically confirmed 
diagnoses of breast or thyroid cancer comparted with 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. For the breast cohort, this study is limited to women with a score 
of 4-5 based on pre-study imaging.  We selected this group of patients 
because there is more likely to be a cancer diagnosis than BIRADS 1-3.   
 
For the thyroid cohort, we will include patients scheduled for a core needle 
or surgical thyroid biopsy, fine needle aspiration, or thyroidectomy of at 
least one sonographically visible thyroid lesion that is 3 cm in depth from 
the skin surface.  We will recruit patients with high TIRADS risk scores 
(4c, moderately suspicious and 5, high risk), that have an anticipated risk 
range of 50% to in excess of 85%, which will provide an estimated relative 
risk of 75% to be congruent with the breast population. 
2.0 
STUDY OBJECTIVES 

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2.1 
Primary Objectives 
2.1.1 To evaluate the sensitivity and specificity of contrast enhanced super-
resolution imaging in the analysis of known breast lesions by comparing 
image analyses to the pathological results for these lesions. 
2.1.2 To evaluate the sensitivity and specificity of contrast enhanced super-
resolution imaging in the analysis of known thyroid lesions by comparing 
image analyses to the pathological results for these lesions. 
2.2 
Secondary Objectives  
2.2.1 To compare (using a reader study) the sensitivity and specificity of 
contrast enhanced super-resolution imaging to the sensitivity and 
specificity of conventional b-mode ultrasound in evaluation of known 
breast lesions for predicting malignancy. 
2.2.2 To compare (using a reader study) the sensitivity and specificity of 
contrast enhanced super-resolution imaging to the sensitivity and 
specificity of conventional b-mode ultrasound in evaluation of known 
thyroid lesions for predicting malignancy. 
2.2.3 To compare the area under the curve (AUC) of contrast enhanced super-
resolution imaging to the AUC of the b-mode ultrasound. 
2.2.4 To compare radiologist preference of contrast enhanced super-resolution 
imaging to conventional b-mode ultrasound for each lesion characteristic 
(shape, margins, and vascularity). 
2.3 
Endpoints 
We will utilize pathological status as derived from patient biopsy pathology 
reports.  
3.0 
PATIENT ELIGIBILITY 
3.1 
Arm 1: Healthy Volunteers 
3.1.1 Inclusion Criteria 
Subject must meet all of the inclusion criteria to participate in this study:  

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3.1.1.1 Adults ≥18 years old 
3.1.1.2 Able to provide informed consent 
3.1.1.3 Negative urine pregnancy test in women of child-bearing potential 
3.1.2 Exclusion Criteria 
All subjects meeting any of the exclusion criteria at baseline will be 
excluded from study participation 
3.1.2.1 Institutionalized subject (prisoner or nursing home patient) 
3.1.2.2 Critically ill or medically unstable and whose critical course during the 
observation period would be unpredictable (e.g., chronic obstructive 
pulmonary disease (COPD)  
3.1.2.3 Known hypersensitivity to sulfur hexafluoride or to any component of 
perflutren lipid (Definity®)  
3.1.2.4 Active cardiac disease including any of the following:  
• Severe congestive heart failure (class IV in accordance with the 
classification of the New York Heart Association)  
• Unstable angina.  
• Severe arrhythmia (i.e. ventricular tachycardia, flutter fibrillation; 
ventricular premature complexes occurring close to the preceding 
T-wave, multifocal complexes).  
• Myocardial infarction within 14 days prior to the date of proposed 
Definity® administration.  
• Pulmonary hypertension 
• Cardiac shunts  
3.1.2.5 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.) 
3.2 
Arm 2: Breast Imaging Patients 
3.2.1 Inclusion Criteria 
Subject must meet all of the inclusion criteria to participate in this study:  
3.2.1.1 Women ≥18 years old 
3.2.1.2 Patient had a diagnostic breast ultrasound study performed at UNC 

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3.2.1.3 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 
3.2.1.4  Lesion visualized on ultrasound 
3.2.1.5 Able to provide informed consent 
3.2.1.6 Negative urine pregnancy test in women of child-bearing potential 
3.2.1.7 BIRADS score of 4 or 5. 
3.2.2 Exclusion Criteria 
All subjects meeting any of the exclusion criteria at baseline will be 
excluded from study participation 
3.2.2.1 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) 
3.2.2.2 Institutionalized subject (prisoner or nursing home patient) 
3.2.2.3 Critically ill or medically unstable and whose critical course during the 
observation period would be unpredictable (e.g., chronic obstructive 
pulmonary disease (COPD)  
3.2.2.4 Sonographically visible breast lesion larger than 2cm or greater than 
3cm in depth from the skin surface 
3.2.2.5 Known hypersensitivity to sulfur hexafluoride or to any component of 
perflutren lipid (Definity®)  
3.2.2.6 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.  
• Pulmonary hypertension 
• Cardiac shunts 
3.2.2.7 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 

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before the perflutren lipid administration, or on the basis of patient 
history, as defined by the UNC IRB SOP 4801.) 
3.3 
Arm 3: Thyroid Imaging Patients 
3.3.1 Inclusion Criteria 
Subject must meet all of the inclusion criteria to participate in this study:  
3.3.1.1 Adults ≥18 years old 
3.3.1.2 Patient had a diagnostic thyroid ultrasound study performed at UNC  
3.3.1.3 TIRADS risk score of 4c or 5 
3.3.1.4 Scheduled for a core needle or surgical thyroid biopsy, fine needle 
aspiration, or thyroidectomy of at least one sonographically visible 
thyroid lesion that is 3 cm in depth from the skin surface 
3.3.1.5 Lesion visualized on ultrasound 
3.3.1.6 Able to provide informed consent 
3.3.1.7 Negative urine pregnancy test in women of child-bearing potential 
3.3.2 Exclusion Criteria 
All subjects meeting any of the exclusion criteria at baseline will be 
excluded from study participation 
3.3.2.1 Institutionalized subject (prisoner or nursing home patient) 
3.3.2.2 Critically ill or medically unstable and whose critical course during the 
observation period would be unpredictable (e.g., chronic obstructive 
pulmonary disease (COPD)  
3.3.2.3 Known hypersensitivity to sulfur hexafluoride or to any component of 
perflutren lipid (Definity®)  
3.3.2.4 Active cardiac disease including any of the following:  
• Severe congestive heart failure (class IV in accordance with the 
classification of the New York Heart Association)  
• Unstable angina.  
• Severe arrhythmia (i.e. ventricular tachycardia, flutter fibrillation; 
ventricular premature complexes occurring close to the preceding 
T-wave, multifocal complexes).  
• Myocardial infarction within 14 days prior to the date of proposed 
Definity® administration.  
• Pulmonary hypertension 
• Cardiac shunts 

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3.3.2.5 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 
STUDY SCHEMA 
This is a 3-arm single center study of 45 patients scheduled to undergo a 
breast or thyroid biopsy that consent to undergo a CESR scan in 
conjunction with b-mode ultrasound prior to their scheduled biopsy.  
 
 
 
Figure 1. Study scheme.  
4.2 
Study Procedures 
Study participants will be enrolled for 1 research imaging visit.  In addition, 
their medical charts will be followed for 6 months. 
4.2.1 Enrollment/Recruitment 
A total of 45 adults will be enrolled to this study. The study subjects will be 
consecutively recruited from adults 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.   
Once a patient has been referred, the patient will be approached by a 
coordinator from Radiology to assess interest in participation.  
 
All eligible subjects 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.   
 

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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 childbearing 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. 
4.2.2 Research Imaging 
4.2.2.1 Contrast Administration (if applicable) 
At the time of imaging, the contrast agent perflutren lipid will be 
administered. See https://definityimaging.com/using-definity 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.  
 
Monitoring will include taking vitals (O2 sat, HR, RR, BP).  Vitals will first 
be collected at arrival to the clinic.  All patients will be visually monitored 
for signs or symptoms of a contrast administration reaction once the drug 
is administered for a 15-minute period.  Once 15 minutes have passed, 
vitals will be taken again. This study will be conducted in Mammography of 
the UNC Cancer Hospital, so trained medical personnel will be available 
as needed. 
 
4.2.2.2 Imaging Procedures 
CESR imaging involves a research ultrasound scanner as well as 
conventional b-mode ultrasound to guide the location of the imaging. The 
conventional ultrasound will be conducted just prior to CESR imaging 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). 
 
CESR imaging will be performed by 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.  The 
field of view of the research images will be limited to clinically identified 

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lesions. Incidental findings are not anticipated due to the sensitivity 
limitations of this imaging modality. Information regarding additional 
lesions or other incidental findings discovered during research imaging 
procedures will not be recorded or communicated to the patient or their 
provider because this imaging modality is not standard of care. 
4.2.3 Standard of Care Biopsy (if applicable) 
The patient will then undergo his or her scheduled biopsy procedure 
based on the pre-study diagnostic imaging. The research CESR imaging 
will NOT be interpreted prior to the biopsy and therefore will not influence 
any clinical decision concerning the biopsy.  
4.2.4 Medical Record Abstraction (if applicable) 
The primary objective of this study is to determine the sensitivity and 
specificity of CESR 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.   
4.2.5 Reader Study  
A reader study will be performed after the completion of patient accrual to 
study lesion characteristics under CESR as compared to conventional b-
mode ultrasound. In these pilot observational studies, the primary aim will 
be to evaluate the imaging approach for application in these two organs. 
We will also estimate the receiver operating characteristic (ROC) curve for 
the CESR system. For scientific rigor, a total of five readers who are not 
investigators on this study (radiologists trained in breast imaging or 
ultrasound imaging) will be recruited to participate for each reader study. 
The readers will be asked to assign a probability score (1 to 5) and 
confidence for each lesion for each modality (0 to 100%). ROC analysis 
will be performed as the primary analysis for the first aim. To compare the 
results from the two imaging modalities, we will adopt the mixed effect 
ANOVA based on the Dorfman-Berbaum-Metz method. The outcome 
variable is the Tukey’s jackknife pseudovalues of the AUCs from each 
reader and each patient under either modality, and separately for each 
anatomic region. The fixed effect in the independent list will correspond to 
the difference between the two modalities, and the random effects will be 
used to account for within-patient and within-reader’s correlations. 
Possible interactions between the modalities and the readers and the 
patients will also be included and tested for statistical significance. To test 
the main hypothesis, F-test statistic from the model parameter estimates 
will be used to compare the mean AUCs between the b-mode ultrasound 
images and the CESR ultrasound images only.  
5.0 
INVESTIGATIONAL DEVICE 

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similar
 
 
   
 
Figure 3. Vermon 32 x 32 matrix array.  
 
Hybrid Dual-frequency Ultrasound Array 
 
 
 
 
 
 
 
 
 
 
 This dual-frequency 
imaging technique enables the detection of perflutren microbubbles 
without the need for spatiotemporal filtering.[22] Imaging with the dual-
frequency probe will be performed with one Verasonics Vantage 256 
system from the Vantage 1024 described above. For 3D acquisition, the 
transducer will be translated with a linear motion stage, as used in our 
previous study, LCCC 1748.  
6.0 
Perflutren Lipid Microspheres (Lantheus Medical Imaging) 
 
 

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.  
6.1 
Investigational Contrast Agent Description and Management 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
 
.com/pdf/vialmix-user-guide.pdf and the 
package insert for instructions on the use of Vialmix®.  
6.1.1 Known and Associated Risks 
For indication see https://www.definityimaging.com/pdf/definity-pi.pdf and 
see section 1.8.2 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 

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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, 
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.  
 
Hypersensitivity Reactions:  
In postmarketing use, serious hypersensitivity reactions were observed 
during or shortly following perﬂutren-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, ﬂushing, 
and erythema have occurred in patients with no prior exposure to perﬂutren-
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 ﬁltering 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. 
6.2 
Return and Retention of Study Contrast Agent 
Incomplete vials of perflutren lipid remaining at the completion of the 
study, or expired perflutren lipid will be destroyed by UNC IDS. 
7.0 
UNANTICIPATED CONCERNS (DEVICES) 
7.1.1 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 

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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)). 
7.1.2 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. 
7.1.3 Reporting 
7.1.4 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  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. 
7.1.5 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.   
 

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Any unanticipated problem that occurs during the conduct of this study 
and that meets at least the first two criteria listed in section 7.1.2 must be 
reported to the UNC IRB using the IRB’s web-based reporting system.   
8.0 
ADVERSE EVENTS (DRUGS- CONTRAST AGENTS) 
8.1 
Definitions  
8.1.1 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. 
8.1.2 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 

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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 
8.1.3 Unexpected AE or SAR 
An AE or SAR is considered unexpected if the specificity or severity of it is 
not consistent with the applicable product information (e.g., Investigator’s 
Brochure (IB) for an unapproved investigational product or package 
insert/summary of product characteristics for an approved product).  
Unexpected also refers to AEs or SARs that are mentioned in the IB as 
occurring with a class of drugs or as anticipated from the pharmacological 
properties of the drug but are not specifically mentioned as occurring with 
the particular drug under investigation.   
8.1.4 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. 

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8.2 
Documentation of non-serious AEs or SARs 
For non-serious AEs or SARs, documentation must begin from day 1 of 
study treatment and continue through the 30-day follow-up period after 
treatment is discontinued.   
 
Collected information should be recorded in the Case Report Forms (CRF) 
for that patient.  Please include a description of the event, its severity or 
toxicity grade, onset and resolved dates (if applicable), and the 
relationship to the study drug.    Documentation should occur at least 
monthly.  
8.3 
SAEs or Serious SARs  
8.3.1 Timing 
After informed consent but prior to initiation of study medications, only 
SAEs caused by a protocol-mandated intervention will be collected (e.g. 
SAEs related to invasive procedures such as biopsies, medication 
washout.  
 
For any other experience or condition that meets the definition of an SAE 
or a serious SAR, recording of the event must begin from day 1 of study 
treatment and continue through the 30-day follow-up period after 
treatment is discontinued.   
8.3.2 Documentation and Notification 
These events (SAEs or Serious SARs) must be recorded in the SAE 
console within Oncore™ for that patient within 24 hours of learning of its 
occurrence.  
8.3.3 Reporting  
IRB Reporting Requirements: 
• The UNC-IRB will be notified of all SAEs that qualify as an 
Unanticipated Problem as per the UNC IRB Policies using the IRB’s 
web-based reporting system within 7 days of the Investigator 
becoming aware of the problem.   
Pregnancy 
Pregnancies and suspected pregnancies (including a positive pregnancy 
test regardless of age or disease state) of a female subject occurring while 
the subject is on study should be recorded as SAEs.  The patient is to be 
discontinued immediately from the study. The female subject should be 
referred to an obstetrician-gynecologist, preferably one experienced in 
reproductive toxicity for further evaluation and counseling. 
 
The Investigator will follow the female subject until completion of the 
pregnancy and must document the outcome of the pregnancy (either 
normal or abnormal outcome). If the outcome of the pregnancy was 

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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.   
 
8.3.4 Data and Safety Monitoring Plan 
The Principal Investigator will provide continuous monitoring of patient 
safety in this trial with periodic reporting to the Data and Safety Monitoring 
Committee (DSMC).  
 
Meetings/teleconferences will be held at a frequency dependent on study 
accrual, and in consultation with the study Biostatistician.  These meetings 
will include the investigators as well as protocol nurses, clinical research 
associates, regulatory associates, data managers, biostatisticians, and 
any other relevant personnel the principal investigators may deem 
appropriate.  At these meetings, the research team will discuss all issues 
relevant to study progress, including enrollment, safety, regulatory, data 
collection, etc. 
 
The team will produce summaries or minutes of these meetings. These 
summaries will be available for inspection when requested by any of the 
regulatory bodies charged with the safety of human subjects and the 
integrity of data including, but not limited to, the oversight (Office of 
Human Research Ethics (OHRE) Biomedical IRB, the Oncology Protocol 
Review Committee (PRC) or the North Carolina TraCS Institute Data and 
Safety Monitoring Board (DSMB).   
 
The UNC LCCC Data and Safety Monitoring Committee (DSMC) will 
review the study on a regular (quarterly to annually) basis, with the 
frequency of review based on risk and complexity as determined by the 
UNC Protocol Review Committee.  The UNC PI will be responsible for 
submitting the following information for review: 1) safety and accrual data 
including the number of study participants treated; 2) significant 
developments reported in the literature that may affect the safety of 
participants or the ethics of the study; 3) preliminary response data; and 4) 
summaries of team meetings that have occurred since the last report.  
Findings of the DSMC review will be disseminated by memo to the UNC 
PI, PRC, and the UNC IRB and DSMB.   
9.0 
STATISTICAL CONSIDERATIONS 
This is a nonrandomized, single-center study.  The primary purpose and 
endpoint of this study is to evaluate the sensitivity and specificity of CESR 
imaging in the analysis of known breast and thyroid lesions by comparing 
image analyses to the pathological results for these lesions. 

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9.1 
Study Design/Study Endpoints 
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 super-resolution imaging device in 
the analysis of known breast and thyroid lesions as compared to 
pathological results of these lesions. . 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 CESR imaging 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 
9.2 
Data Analysis Plans 
ROC analysis will be performed as the primary analysis for the first aim. 
To compare the results from the two imaging modalities, we will adopt the 
mixed effect ANOVA based on the Dorfman-Berbaum-Metz method. The 
outcome variable is the Tukey’s jackknife pseudovalues of the AUCs from 
each reader and each patient under either modality, and separately for 
each anatomic region. The fixed effect in the independent list will 
correspond to the difference between the two modalities and the random 
effects will be used to account for within-patient and within- reader’s 
correlations. 
 
Possible interactions between the modalities and the readers and the 
patients will also be included and tested for statistical significance. To test 
the main hypothesis, F-test statistic from the model parameter estimates 
will be used to compare the mean AUCs between the b- mode ultrasound 
images and the CESR ultrasound images only.  
9.3 
Sample Size Considerations in ROC Analysis 
For each lesion type, we assume 8 malignant and 7 benign and the AUC 
for conventional imaging to be 0.7 based on our previous breast studies. 
We will recruit patients with high TIRADS risk scores (4c, moderately 

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suspicious and 5, high risk), that have an anticipated risk range of a 50% 
to in excess of 85%, which will provide an estimated relative risk of 75% to 
be congruent with the breast population. The power to detect 0.2 AUC 
difference is about 30%. However, our primary goal is to evaluate the 
technical ability of the CESR approach to examine lesions in each of these 
superficial anatomic regions. We will also be evaluating the number of 
vessels visualized, the effects of respiratory motion and radiologist 
confidence in interpreting the CESR microvascular images. These studies 
will guide further development of the imaging approach for a prospective 
trial. 
9.4 
Secondary Analyses 
Radiologist Preference 
The secondary analysis will be to evaluate reader preference for specific 
lesion characteristics. 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. 
10.0 STUDY MANAGEMENT 
10.1 
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. 
10.2 
Registration Procedures 
Study participants will be registered into OnCore®, a web based clinical 
research platform by one of the Study Coordinators. 
10.3 
Data Management and Monitoring/Auditing 
The 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 as appropriate. 
10.4 
Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, 
safety, and well-being of the study patient requires alternative treatment, 
the study shall be conducted exactly as described in the approved 
protocol.   
10.4.1 Emergency Modifications 
UNC investigators may implement a deviation from, or a change of, the 
protocol to eliminate an immediate hazard(s) to trial subjects without prior 
UNC’s IRB/IEC approval/favorable opinion.   
 
For any such emergency modification implemented, an IRB modification 
form must be completed by UNC Research Personnel within five (5) 
business days of making the change.   
10.4.2 Single Subject Exceptions 
10.4.3 Eligibility single subject exceptions are not permitted for Lineberger 
Comprehensive Cancer Center Investigator Initiated Trials under any 
circumstances. Other types of single subject exceptions may be 
allowed if proper regulatory review has been completed in 
accordance with Lineberger Comprehensive Cancer Center’s Single 
Subject Exceptions Policy. Other Protocol Deviations/Violations 
According to UNC’s IRB, a protocol deviation is any unplanned variance 
from an IRB approved protocol that:  
• Is generally noted or recognized after it occurs 
• Has no substantive effect on the risks to research participants 

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• Has no substantive effect on the scientific integrity of the 
research plan or the value of the data collected  
• Did not result from willful or knowing misconduct on the part of 
the investigator(s).  
 
An unplanned protocol variance is considered a violation if the variance 
meets any of the following criteria:  
• Has harmed or increased the risk of harm to one or more 
research participants. 
• Has damaged the scientific integrity of the data collected for the 
study. 
• Results from willful or knowing misconduct on the part of the 
investigator(s). 
• Demonstrates serious or continuing noncompliance with federal 
regulations, State laws, or University policies. 
 
If a deviation or violation occurs, please follow the guidelines below: 
 
Protocol Deviations: UNC personnel will record the deviation in 
OnCore®, and report to any sponsor or data and safety monitoring 
committee in accordance with their policies.  Deviations should be 
summarized and reported to the IRB at the time of continuing review. 
 
Protocol Violations: Violations should be reported by UNC personnel 
within one (1) week of the investigator becoming aware of the event using 
the same IRB online mechanism used to report Unanticipated Problems.   
 
Unanticipated Problems: 
Any events that meet the criteria for “Unanticipated Problems” as defined 
by UNC’s IRB must be reported by the study team using the IRB’s web-
based reporting system.   
10.5 
Amendments to the Protocol 
Should amendments to the protocol be required, the amendments will be 
originated and documented by the Principal Investigator at UNC.  It should 
also be noted that when an amendment to the protocol substantially alters 
the study design or the potential risk to the patient, a revised consent form 
might be required.   
 
The written amendment, and if required the amended consent form, must 
be sent to UNC’s IRB for approval prior to implementation.  
10.6 
Record Retention 
Study documentation includes all eCRFs, data correction forms or queries, 
source documents, Sponsor-Investigator correspondence, monitoring 

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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. 
10.7 
Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial 
at the site in accordance with Title 21 of the Code of Federal Regulations 
and/or the Declaration of Helsinki.  The Principal Investigator is 
responsible for personally overseeing the treatment of all study 
participants.  The Principal Investigator must assure that all study site 
personnel, including sub-investigators and other study staff members, 
adhere to the study protocol and all FDA/GCP/NCI regulations and 
guidelines regarding clinical trials both during and after study completion. 
11.0 REFERENCES 
[1] C. Demene, T. Deffieux, M. Pernot, B. F. Osmanski, V. Biran, J. L. Gennisson, et al., 
"Spatiotemporal Clutter Filtering of Ultrafast Ultrasound Data Highly Increases Doppler 
and fUltrasound Sensitivity," IEEE Trans Med Imaging, vol. 34, pp. 2271-85, Nov 2015. 
 
[2] S. E. Shelton, B. D. Lindsey, J. K. Tsuruta, F. S. Foster, and P. A. Dayton, "Molecular 
Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound 
Molecular Imaging," Ultrasound Med Biol, vol. 42, pp. 769-81, Mar 2016. 
 
[3] J. E. Streeter, R. Gessner, I. Miles, and P. A. Dayton, "Improving sensitivity in 
ultrasound molecular imaging by tailoring contrast agent size distribution: in vivo 
studies," Mol Imaging, vol. 9, pp. 87-95, Apr 2010. 
 
[4] G. F. Pinton, G. E. Trahey, and J. J. Dahl, "Sources of image degradation in 
fundamental and harmonic ultrasound imaging using nonlinear, full-wave simulations," 
IEEE Trans Ultrason Ferroelectr Freq Control, vol. 58, pp. 754-65, Apr 2011. 
 
[5] M. Tanter, J. Bercoff, L. Sandrin, and M. Fink, "Ultrafast compound imaging for 2-D 
motion vector estimation: application to transient elastography," IEEE Trans Ultrason 
Ferroelectr Freq Control, vol. 49, pp. 1363-74, Oct 2002. 
 

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[6] M. P. Curado, B. Edwards, H. R. Shin et al., Cancer Incidence in Five Continents, vol. 
9 of IARC Scientific Publications, No. 160, IARC, Lyon, France, 2007. 
 
[7] Pellegriti G, Frasca F, Regalbuto C, Squatrito S, Vigneri R. Worldwide increasing 
incidence of thyroid cancer: update on epidemiology and risk factors. J Cancer 
Epidemiol2013;2013:965212. doi:10.1155/2013/965212 pmid:23737785. 
 
[8] E. L. Mazzaferri, “Management of a solitary thyroid nodule,” The New England 
Journal of Medicine, vol. 328, no. 8, pp. 553–559, 1993. 
 
[9] The thyroid cancer epidemic, 2017 perspective. Roman BR1, Morris LG, Davies L. 
Curr Opin Endocrinol Diabetes Obes. 2017 Oct;24(5):332-336. doi: 
10.1097/MED.0000000000000359. 
 
[10] H. G. Welch and P. C. Albertsen, “Prostate cancer diagnosis and treatment after the 
introduction of prostate-specific antigen screening: 1986–2005,” Journal of the National 
Cancer Institute, vol. 101, no. 19, pp. 1325–1329, 2009.  
 
[11] C. Errico, J. Pierre, S. Pezet, Y. Desailly, Z. Lenkei, O. Couture, et al., "Ultrafast 
ultrasound localization microscopy for deep super-resolution vascular imaging," 
Nature, vol. 527, pp. 499-502, Nov 26 2015. 
 
[12] E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. 
Bonifacino, et al., "Imaging intracellular fluorescent proteins at nanometer resolution," 
Science, vol. 313, pp. 1642-5, Sep 15 2006. 
 
[13] M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic 
optical reconstruction microscopy (STORM)," Nat Methods, vol. 3, pp. 793-5, Oct 
2006. 
 
[14] K. Christensen-Jeffries, R. J. Browning, M. X. Tang, C. Dunsby, and R. J. Eckersley, 
"In vivo acoustic super-resolution and super-resolved velocity mapping using 
microbubbles," IEEE Trans Med Imaging, vol. 34, pp. 433-40, Feb 2015. 
 
[15] M. A. O'Reilly and K. Hynynen, "A super-resolution ultrasound method for brain 
vascular mapping," Med Phys, vol. 40, p. 110701, Nov 2013. 
 
[16] Y. Desailly, O. Couture, M. Fink, and M. Tanter, "Sono-activated ultrasound 
localization microscopy," Applied Physics Letters, vol. 103, Oct 21 2013. 
 
[17] F. Lin, S. E. Shelton, D. Espindola, J. D. Rojas, G. Pinton, and P. A. Dayton, "3-D 
Ultrasound Localization Microscopy for Identifying Microvascular Morphology 
Features of Tumor Angiogenesis at a Resolution Beyond the Diffraction Limit of 
Conventional Ultrasound " Theranostics, vol. 7, pp. 196-204, 2016. 
 
[18] S. E. Shelton, Y. Z. Lee, M. Lee, E. Cherin, F. S. Foster, S. R. Aylward, and P.A. 
Dayton, "Quantification of Microvascular Tortuosity during Tumor Evolution Using 
Acoustic Angiography," Ultrasound Med Biol, vol. 41, no. 7, pp. 1896-1904, 2015.  
 
[19] S. L. Mulvagh, "Safety of Ultrasound Contrast Studies," in Diagnostic Ultrasound 
Annual Conference, 2010.J. Holash, P. C. Maisonpierre, D. Compton, P. Boland, 

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C. R. Alexander, D. Zagzag, et al., "Vessel cooption, regression, and growth in 
tumors mediated by angiopoietins and VEGF," Science, vol. 284, pp.1994-8, Jun 
18 1999. 
 
[20] J. Holash, S. J. Wiegand, and G. D. Yancopoulos, "New model of tumor 
angiogenesis: dynamic balance between vessel regression and growth mediated 
by angiopoietins and VEGF," Oncogene, vol. 18, pp. 5356-62, Sep 20 1999. 
 
[21] E. Cherin, J. Yin, A. Forbrich, C. White, P.A. Dayton, F.S. Foster, and C.E.M. 
Demore, “In vitro superharmonic contrast imaging using a hybrid dual-frequency 
probe,” Ultrasound Med Biol, vol. 45, no. 9, pp. 2525-2539, 2019.  
 
[22] T.M. Kierski, D. Espindola, I.G. Newsome, E. Cherin, J. Yin, F.S. Foster, C.E.M. 
Demore, G.F. Pinton, and P.A. Dayton, “Super harmonic ultrasound for motion-
independent localization microscopy: applications to microvascular imaging from 
low to high flow rates,” IEEE Trans Ultrason Ferroelectr Freq Control, in press, 
2019.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12.0 APPENDICES 
12.1 
Appendix A: Reader Study Data Collection Form 
 
Specimen:   
 
 
 
 
 
 
Reader: 
 
 

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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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12.2 
Appendix B: Previous Clinical Investigation Use of Verasonics 
System 
 
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: 

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10.1016/j.ultrasmedbio.2017.01.001. This study compared a 
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.