Interstitial Fluid Pressure Estimation in Breast Cancer Using 3D Subharmonic Signals From Contrast-Enhanced Ultrasound
Phase: EARLY_PHASE1
Conditions: Breast Carcinoma
Sponsor: Thomas Jefferson University

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THOMAS JEFFERSON UNIVERSITY 
Sidney Kimmel Cancer Center 
 
Interstitial Fluid Pressure Estimation in Breast Cancer using 3D Subharmonic 
Signals from Contrast-enhanced Ultrasound 
 
Principal Investigator: 
Kibo Nam, PhD 
Department of Radiology 
132 South 10th Street, Philadelphia, PA 19107 
(215) 955-6261 
Co-Investigator(s): 
Flemming Forsberg, PhD 
Department of Radiology 
132 South 10th Street, Philadelphia, PA 19107 
(215) 955-4870 
 
John Eisenbrey, PhD 
Department of Radiology 
132 South 10th Street, Philadelphia, PA 19107 
(215) 503-5188 
 
Andrej Lyshchik, MD, PhD 
Department of Radiology 
132 South 10th Street, Philadelphia, PA 19107 
(215) 955-8558 
 
Lydia Liao, MD 
Department of Radiology 
1100 Walnut Street, Philadelphia, PA 19107 
(215) 955-9545 
 
Jason Shames, MD 
Department of Radiology 
1100 Walnut Street, Philadelphia, PA 19107  
(215) 503-3724 
 
Maysa Abu-Khalaf, MD 
Department of Medical Oncology 
1025 Walnut Street, Suite 700, Philadelphia, PA 19107 
(215) 955-8874 
 
Christopher Sebastiano, MD 
Department of Pathology 
132 South 10th Street, Philadelphia, PA 19107 
(215) 955-1197 
Statistician: 
Scott Keith, PhD 
Pharmacology and Experimental Therapeutics 
1015 Chestnut Street, Philadelphia, PA 19107 

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(215) 503-9876 
Funding Sponsor: 
R37 CA234428 
National Cancer Institute 
National Institutes of Health (NIH) 
9000 Rockville Pike, Bethesda, MD 20892 
(301) 402-9612 
 
Additional Support: Lantheus Medical Imaging 
IND Holder: 
Flemming Forsberg 
IND Number: 
IND no. 112,241 
Study Product: 
Perflutren lipid microspheres; DEFINITY 
 
Protocol IDs: 
JeffTrial # 14825 
PRC # pending 
IRB Control # 20G605 
 
Version Number: 
Version Date: 
2.0 
2019-08-21 
3.0 
2019-09-03 
3.1 
2019-09-23 
3.2 
2020-03-25 
3.3 
2020-06-16 
4.0 
2021-04-20 
 
CONFIDENTIAL 
This document is confidential and the property of THOMAS JEFFERSON UNIVERSITY. 
No part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 
 

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Table of Contents  
 
Signature Page ............................................................................................................................. 8 
Statement of Compliance .............................................................................................................. 8 
List of Abbreviations ...................................................................................................................... 9 
Study Summary .......................................................................................................................... 10 
1 
Introduction ........................................................................................................................... 13 
1.1 
Background Information ............................................................................................... 13 
1.2 
Rationale for the Proposed Study ................................................................................. 13 
1.3 
Correlative Studies ....................................................................................................... 14 
1.4 
Potential Risks and Benefits ......................................................................................... 14 
1.4.1 Potential Risks .......................................................................................................... 14 
1.4.2 Benefits ..................................................................................................................... 15 
2 
Study Objectives .................................................................................................................. 15 
2.1 
Objectives ..................................................................................................................... 15 
2.1.1 Primary ...................................................................................................................... 15 
2.1.2 Secondary ................................................................................................................. 15 
2.2 
Endpoints/Outcome Measures ..................................................................................... 15 
2.2.1 Primary ...................................................................................................................... 15 
2.2.2 Secondary ................................................................................................................. 16 
3 
Study Design ........................................................................................................................ 16 
3.1 
Characteristics .............................................................................................................. 16 
3.2 
Number of Participants ................................................................................................. 16 
3.3 
Duration of Therapy ...................................................................................................... 16 
3.4 
Duration of Follow Up ................................................................................................... 16 
3.5 
Study Timeline .............................................................................................................. 16 
3.5.1 Primary Completion .................................................................................................. 17 

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3.5.2 Study Completion ...................................................................................................... 17 
4 
Study Enrollment and Withdrawal ........................................................................................ 17 
4.1 
Eligibility Criteria ........................................................................................................... 17 
4.1.1 Inclusion Criteria ....................................................................................................... 17 
4.1.2 Exclusion Criteria ...................................................................................................... 17 
4.2 
Gender/Minority/Pediatric Inclusion for Research ........................................................ 18 
4.3 
Strategies for Recruitment and Retention .................................................................... 18 
4.4 
Participant Withdrawal .................................................................................................. 18 
4.4.1 Reasons for Withdrawal ............................................................................................ 18 
4.4.2 Handling of Participant Withdrawals and Participant Discontinuation of Study 
Intervention ........................................................................................................................... 19 
4.5 
Premature Termination or Suspension of Study ........................................................... 19 
5 
Study Intervention ................................................................................................................ 19 
5.1 
Study Product ............................................................................................................... 19 
5.2 
Study Product Description ............................................................................................ 20 
5.2.1 Acquisition ................................................................................................................. 20 
5.2.2 Formulation, Packaging, and Labeling ...................................................................... 20 
5.2.3 Product Storage and Stability ................................................................................... 20 
5.3 
Dosage, Preparation, and Administration ..................................................................... 21 
5.4 
Dose Modifications and Dosing Delays ........................................................................ 21 
5.5 
Study Product Accountability ........................................................................................ 21 
5.6 
Dietary Restrictions ...................................................................................................... 21 
5.7 
Administration of Procedural Intervention ..................................................................... 21 
5.8 
Procedures for Training of Clinicians on Procedural Intervention................................. 21 
5.9 
Assessment of Clinician and/or Participant Compliance with Study Procedural 
Intervention .............................................................................................................................. 22 
6 
Study Schedule .................................................................................................................... 22 

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6.1 
Pretreatment Period/Screening .................................................................................... 22 
6.2 
Enrollment/Baseline ...................................................................................................... 22 
6.3 
Treatment Period .......................................................................................................... 22 
6.4 
End of Treatment Study Procedures ............................................................................ 23 
6.5 
Withdrawal Visit/Discontinuation of Therapy ................................................................ 23 
7 
Study Procedures and Evaluations ...................................................................................... 23 
7.1 
Study Procedures/Evaluations ..................................................................................... 23 
7.2 
Laboratory Procedures/Evaluations ............................................................................. 23 
7.2.1 Clinical Laboratory Evaluations ................................................................................. 23 
8 
Evaluation of Safety ............................................................................................................. 23 
8.1.1 Unanticipated Problems ............................................................................................ 23 
8.1.2 Adverse Events ......................................................................................................... 24 
8.1.3 Serious Adverse Events ............................................................................................ 24 
8.2 
Safety Assessment and Follow-Up ............................................................................... 24 
8.3 
Recording Adverse Events ........................................................................................... 25 
8.3.1 Relationship to Study Intervention ............................................................................ 25 
8.3.2 Expectedness ........................................................................................................... 25 
8.3.3 Severity of Event ....................................................................................................... 25 
8.3.4 Intervention ............................................................................................................... 26 
8.4 
Safety Reporting ........................................................................................................... 26 
8.4.1 Reporting to IRB ....................................................................................................... 26 
8.4.2 Reporting to SKCC DSMC ........................................................................................ 26 
8.4.3 Reporting to Funding Sponsor .................................................................................. 28 
8.4.4 Reporting to FDA ...................................................................................................... 28 
8.4.5 Reporting of Pregnancy ............................................................................................ 28 
8.5 
Halting Rules ................................................................................................................ 29 

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9 
Study Oversight .................................................................................................................... 29 
10 
Clinical Site Monitoring and Auditing ................................................................................ 29 
11 
Statistical Considerations ................................................................................................. 29 
11.1 
Study Hypotheses ........................................................................................................ 29 
11.2 
Analysis Plans .............................................................................................................. 29 
11.3 
Sample Size Considerations ........................................................................................ 30 
11.3.1 
Replacement Policy ............................................................................................... 30 
11.3.2 
Accrual Estimates .................................................................................................. 30 
12 
Source Documents and Access to Source Data/Documents ........................................... 30 
13 
Quality Control and Quality Assurance ............................................................................ 32 
14 
Ethics/Protection of Human Participants .......................................................................... 32 
14.1 
Ethical Standard ........................................................................................................... 32 
14.2 
Institutional Review Board ............................................................................................ 32 
14.3 
Informed Consent Process ........................................................................................... 32 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) .......................... 32 
14.5 
Participant Confidentiality ............................................................................................. 33 
14.6 
Future Use of Stored Specimens and Other Identifiable Data...................................... 33 
15 
Data Handling and Record Keeping ................................................................................. 33 
15.1 
Data Management Responsibilities .............................................................................. 33 
15.2 
Data Capture Methods ................................................................................................. 33 
15.3 
Types of Data ............................................................................................................... 34 
15.4 
Study Records Retention .............................................................................................. 34 
15.5 
Protocol Deviations ....................................................................................................... 34 
16 
Study Finances ................................................................................................................ 34 
16.1 
Funding Source ............................................................................................................ 34 
16.2 
Conflict of Interest ......................................................................................................... 34 

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16.3 
Participant Stipends or Payments ................................................................................ 35 
17 
Publication and Data Sharing Policy ................................................................................ 35 
18 
Literature References ....................................................................................................... 35 
SUPPLEMENTAL MATERIALS .................................................................................................. 37 
Appendices ................................................................................................................................. 37 
APPENDIX A: SCHEDULE OF EVENTS ................................................................................... 38 
 

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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 US federal 
regulations and ICH guidelines. 
Principal Investigator: 
Signed: 
 
Date:  
Name:  
Kibo Nam, PhD 
Title:  
Research Assistant Professor 
Statement of Compliance 
This study will be conducted in accordance with the International Conference on Harmonisation 
guidelines for Good Clinical Practice (ICH E6), the Code of Federal Regulations on the 
Protection of Human Subjects (45 CFR Part 46), and Thomas Jefferson University research 
policies 
 
 
 

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List of Abbreviations 
AE 
Adverse Event/Adverse Experience 
CARPA 
Complement Activation Related Pseudo Allergy  
CEUS 
Contrast-enhanced Ultrasound 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DSMC 
Data and Safety Monitoring Committee 
DSMP 
Data and Safety Monitoring Plan 
FDA 
Food and Drug Administration 
ICH 
International Conference on Harmonisation 
IFP 
Interstitial Fluid Pressure 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
LABC 
Locally Advanced Breast Cancer 
N 
Number (typically refers to participants) 
NAC 
Neoadjuvant Chemotherapy 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
PI 
Principal Investigator 
PRC 
Protocol Review Committee 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SDS 
Safety Data Sheet (formerly MSDS; Material Safety Data Sheet) 
SHAPE 
Subharmonic Aided Pressure Estimation 
SKCC 
Sidney Kimmel Cancer Center 
TJU 
Thomas Jefferson University 
UAP 
Unanticipated Problem 
 
 
 
 
 
 
 
 
 
 

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Study Summary 
Title: 
Interstitial Fluid Pressure Estimation in Breast Cancer using 3D 
Subharmonic Signals from Contrast-enhanced Ultrasound 
Précis: 
Breast cancer patients scheduled for a breast biopsy will undergo 3D 
Contrast-Enhanced Ultrasound (CEUS) exam with a Logiq E9 or 
E10 scanner (GE Healthcare, Milwaukee, WI) using the ultrasound  
contrast agent Definity (Lantheus Medical Imaging, Billerica, MA). 
After CEUS, the measurements of interstitial fluid pressure (IFP) in 
the tumor and its surrounding tissue (i.e., two sites) using an intra-
compartmental pressure monitoring system (Stryker, Newbury, 
Berkshire, UK) will be performed guided by conventional ultrasound. 
Objectives: 
 
Primary: To determine if the pressure estimates from 3D 
Subharmonic Aided Pressure Estimation (SHAPE) technique is 
correlated with the direct measurements of IFP in breast tumors and 
the surrounding tissue  
 
Secondary: To determine if the pressure estimates from 3D SHAPE 
technique have diagnostic information to detect the malignancy of 
breast tumors 
Population: 
The patient population of this study will reflect the demographics of 
female breast cancer incidence in the city of Philadelphia. The 
overall demographics include 30% Caucasian, 29% African 
American, 20% Asian, and 21% Hispanic patients.  
The sample size of this study was determined to be 25 subjects as a 
pilot study requiring invasive pressure measurements. The patients 
enrolled in this project will be female over the age of 21 scheduled 
for a breast biopsy. The exclusion criteria include patients with 
known hypersensitivity or allergy to any component of Definity as 
well as patients with unstable cardiopulmonary conditions or 
respiratory distress syndrome. 
Phase: 
Pilot 
Number of Sites: 
One; Thomas Jefferson University 
Description of 
Intervention: 
Patients will first undergo baseline ultrasound imaging. B-mode 
measurements and sweeps of breast tumor in the transverse and 
sagittal planes will be acquired. Following baseline imaging, patients 
will have an administration of ultrasound contrast agent.  
The ultrasound contrast administration will be an intravenous 
infusion of 2 vials of Definity/50ml saline will be administered as an 
IV infusion via a peripheral vein (preferably the antecubital vein), with 

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infusion rates of 4 to 10 ml/min (titrated to effect) through an 18-22 
gauge angiocatheter. During the contrast infusion, 3D CEUS data 
will be collected.  
Study Duration: 
15 months 
Participant 
Participation 
Duration: 
The participation duration is about 80-90 minutes (including 30 
minute-long monitoring time) in a single visit. 
Estimated Time to 
Complete 
Enrollment: 
15 months 

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Schematic of Study Design: 
Prior to  
Enrollment 
 
 
 
 
Visit 1 
On the day  
Of a Biopsy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Total 25: Obtain informed consent. Screen potential subjects by inclusion 
and exclusion criteria; obtain history, document. 
Conventional ultrasound imaging will be performed to examine the tumor 
followed by CEUS exam. Before collecting CEUS data, Definity will be 
administered IV and the acoustic settings will be optimized using a built-in 
software in a modified Logiq E9 or E10 scanner. The 3D SHAPE data will 
be collected using the determined acoustic settings during the infusion. 
Then, we wait for the clearance of Definity and another set of SHAPE data 
will be collected without Definity keeping the same acoustic settings. 
The subject will have local anesthesia procedure and IFP will be measured 
from the breast tumor and its surrounding tissue using an 18G single-use 
needle connected to Stryker pressure monitoring system. The 
measurement procedure will be guided by ultrasound imaging. The 
measurements will be documented as well as their locations. 
Compare the results from 3D 
SHAPE data to IFP measurements 
and find the correlation 

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1 Introduction 
1.1 
Background Information 
It has been shown that solid tumors have higher interstitial fluid pressure (IFP) 
compared to normal tissue. Proliferating tumor cells produce solid stress that 
compresses blood and lymphatic vessels causing vessel leakiness and lack of 
functional lymphatics. As a result, IFP becomes significantly elevated in solid tumors 
[Fukumura et al. 2007, Milosevic et al. 1999]. A study showed that solid tumors had 
higher IFP compared to normal tissue by 10-25 mmHg using mice with four human 
tumor cell lines [Stohrer et al. 2000].  Nathanson et al. [1994] measured IFP in the 
operating room in 25 patients undergoing excision breast biopsy under local anesthetic 
for diagnostic purposes. They found that invasive ductal carcinomas’ mean IFP was 29 
mmHg, while those of normal breast tissue, benign tumor, noninvasive carcinomas, and 
tumor with other benign breast conditions were between -0.3-3.6 mmHg. Additionally, 
increased IFP prevents an effective uptake of therapeutic agents and reduces the 
efficacy of cancer therapy [Heldin et al. 2004]. Patients with high IFP had a greater 
chance to recur after radiotherapy and die of progressive disease than those with low 
IFP [Milosevic et al. 2001]. Also early decreases in IFP during chemotherapy were 
highly correlated with late decreases in breast tumor volume in an animal study [Ferretti 
et al. 2009]. Thus, it can be clinically helpful to measure IFP in breast cancer patients.  
1.2 
Rationale for the Proposed Study 
The IFP of breast tumors can be directly measured invasively using a wick-in-needle 
(WIN) technique, which employs a catheter connected to a transducer to record the 
pressures [Taghian et al. 2005]. However, this invasive method is difficult to apply in 
patients and not standard of care. Contrast-enhanced ultrasound (CEUS) has shown 
potential to estimate tumor IFP in animals [Halldorsdottir et al. 2014]. CEUS utilizes 
microbubbles, which not only enhance the backscattered signals, but at higher acoustic 
pressures also act as nonlinear oscillators producing multiple harmonics of the 
fundamental frequency. Microbubbles can also act as pressure sensors, due to the 
difference in compressibility between the bubble and the surrounding medium. Our 
group has developed the fundamental concept of subharmonic (half of fundamental 
frequency) aided pressure estimation (SHAPE) using CEUS and demonstrated its 
potential to non-invasively measure IFP [Halldorsdottir et al. 2014]. The SHAPE method 
estimates IFP using the inverse relationship between the CEUS signal magnitude at the 
subharmonic frequency and IFP. This inverse relationship varies with the applied 
acoustic pressure and an individual optimization of the acoustic setting for SHAPE is 
therefore needed [Shi et al. 1999]. Recently, we conducted a pilot study of 3D SHAPE’s 
ability to monitor treatment response in women undergoing neoadjuvant chemotherapy 
(NAC) for locally advanced breast cancer (LABC) [Nam et al. 2017], showing that the 
subharmonic signal difference in the tumor relative to the normal tissue could predict 
treatment response after administration of 10% of therapy regimen (i.e., 1 cycle of 
neoadjuvant regimen). This study did not measure IFP directly, therefore the correlation 
between IFP and subharmonic signal magnitude has not been studied yet. Thus, in this 

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study, we will determine if the 3D SHAPE can estimate IFP in breast tumors and the 
surrounding normal breast tissue noninvasively. 
1.3 
Correlative Studies 
Non-invasively, diffusion-weighted MRI and dynamic contrast-enhanced (DCE) MRI 
have been suggested as alternative methods for estimating tumor IFP [Hompland et al. 
2014]. However, this technique has not been widely adopted in the clinic. Moreover, 
MRI is expensive and may not be tolerated by all patients, including those with 
claustrophobia, metal implants, and compromised renal function. 
1.4 
Potential Risks and Benefits 
This study is composed of two parts: Contrast Enhanced Ultrasound (CEUS) with 
ultrasound contrast agent Definity and invasive Interstitial Fluid Pressure (IFP) 
measurements using Stryker compartment pressure monitoring system (Kalamazoo, 
MI).  
1.4.1 
Potential Risks 
Serious cardiopulmonary and allergic reactions including fatalities have occurred during 
or following administration of Definity. However, these occurrences have been rare (less 
than 1 in 50,000 patients). As a result, patients with unstable cardiopulmonary 
conditions will be excluded. The majority of adverse events from Defnity were mild to 
moderate in severity. Transient side effects that have been described as possibly 
related to Definity administration include headache (2.3%), back and renal pain (2.1%), 
flushing (1.1%) and nausea (1.0%). Hypersensitivity reactions to perflutren may occur, 
although extremely rare. 
In a meta-analysis (which included 110,500 patients) [Khawaja et al. 2010] performed 
early after the FDA safety alerts in 2007, the incidence of serious allergic and 
anaphylactoid reactions immediately after the ultrasound contrast agent administration 
was estimated at 0.009% and 0.004%, respectively. These very rare but severe allergic 
reactions are secondary to a recently described variant of the type1 hypersensitivity 
reaction known as Complement Activation Related Pseudo Allergy, or CARPA 
reactions. [Szebeni 2005]. 
In a retrospective multicenter study, Wei et al [2008] reported on the safety of Definity 
(n=66164 doses) and Optison (n=12219 doses)- these included >10000 doses 
administered to critically ill patients in the intensive care unit or to patients with 
presumed ischemic chest pain. Severe reactions likely attributable to the contrast 
agents occurred in 8 patients (0.01%), and 4 of these were anaphylactoid reactions. 
Interestingly, all of the severe reactions occurred in outpatients, indicating that the FDA 
warning on contrast administration in patients with severe cardiopulmonary conditions 
likely would not have mitigated risk in these patients. 
CARPA reactions may be mild to moderate (sneezing, tingling sensation, urticaria, or 
pruritis) or severe (wheezing, angioedema, cyanosis, and anaphylactic shock). CARPA 

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reactions seem more common when a large bolus of lipid shelled contrast agent is 
injected, and slow continuous infusions of diluted contrast likely afford significant 
protection against these reactions. 
The use of an intravenous needle and the fluids given through the needle may cause 
minor discomfort, bleeding under the skin (bruise), fainting, and possible infection at the 
site of needle insertion. These risks are minimized by having nursing staff trained in 
placing IV lines perform these procedures in a safe, aseptic manner. The subjects’ 
alternative is not to participate in this study and have only their scheduled clinical 
procedure(s) performed. 
The invasive pressure measurement procedure (with 18G needle) has low risk similar to 
a stereotactic breast biopsy (mostly with 14G or larger needles). The risks includes: 
bruising and swelling of the breast, infection of the measurement site, and soreness at 
the needle injection. Also, there is low risk of infection and pain and they could be 
minimized by strict sterile technique and local lidocaine infiltration, respectively. The 
subjects’ alternative is not to participate in the study.    
1.4.2 
Benefits 
We do not expect any direct benefit for subjects enrolled in this study. The long-term 
benefits of this study will be the development of a noninvasive, safe, accurate IFP 
estimator. Additionally, it may lead to a novel functional predictor of malignancy in 
breast tumors.  
2 Study Objectives 
2.1 
Objectives 
This study is designed to compare the direct measurements of IFP in the breast tumor 
and its surrounding tissue with the values obtained from 3D SHAPE data (i.e., CEUS 
data). 
2.1.1 
Primary 
The primary objective of the study is to determine if the 3D SHAPE results can be used 
to estimate IFPs in breast tumor and its surrounding tissue.  
2.1.2 
Secondary 
The secondary objective of the study is to determine if the malignancy of breast tumor 
can be predicted by the 3D SHAPE results. 
2.2 
Endpoints/Outcome Measures 
2.2.1 
Primary 
A Pearson’s correlation coefficient above 0.5 will be considered a successful outcome.  

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2.2.2 
Secondary 
We will use the results from the biopsy as the reference standard. The significance level 
of Generalized linear mixed logistic or GEE logistic modeling as well as the Mann-
Whitney or Student’s t-test will be 0.05. 
3 Study Design 
3.1 
Characteristics 
This is an open-label, non-randomized, human trial that will be conducted at one clinical 
site, Thomas Jefferson University. Before IFP measurement, all participants will have an 
unenhanced (baseline) ultrasound imaging study followed by CEUS imaging study with 
a single infusion of Definity. Then, they will have the IFP measurements taken by 
Stryker compartment pressure monitoring system from a breast tumor and its 
surrounding tissue with ultrasound imaging guide.    
3.2 
Number of Participants 
We plan to enroll 25 participants. 
3.3 
Duration of Therapy 
The total infusion of Definity will be about 15 minutes. 
3.4 
Duration of Follow Up 
All participants will have a 30-minute monitoring period after the administration of 
Definity.  
3.5 
Study Timeline 
The period of this study is 15 months and the primary and secondary studies will be 
completed at the same time. 
Year 1 
(a) Begin enrolling patients (Quarter 1) 
(b) Active patient enrollment (Quarter 2-4)  
(c) 3D SHAPE data analysis (Quarter 2-4) 
 
Year 2 
(a) Active patient enrollment (Quarter 5)  
(b) 3D SHAPE data analysis (Quarter 5) 
(c) Statistical analysis and preparation for publications (Quarter 5) 
 

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3.5.1 
Primary Completion 
The primary study will completed after 15 months. 
3.5.2 
Study Completion 
The study will completed after 15 months. 
4 Study Enrollment and Withdrawal 
4.1 
Eligibility Criteria 
4.1.1 
Inclusion Criteria 
Individuals must meet all of the following inclusion criteria in order to be eligible to 
participate in the study: 
1. Provide signed and dated informed consent form 
2. Be conscious, willing and able to comply with all study procedures and be 
available for the duration of the study  
3. Female, at least 21 years old 
4. Be scheduled for a breast biopsy (BIRAD 4, 4A, 4B, 4C or 5) 
5. Patient with an at least 1 cm mass located at <3cm depth, approachable by 2.5 
inch needle 
6. Be medically stable as determined by the investigator 
7. If a female of child-bearing potential, must have a negative urine pregnancy test 
within 24 hours prior to administration of Definity. 
  
4.1.2 
Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation 
in this study: 
1. Males 
2. Females who are pregnant or nursing 
3. Patients who are medically unstable, patients who are seriously or terminally ill, 
and patients whose clinical course is unpredictable. For example:   
a. Patients on life support or in a critical care unit. 
b. Patients with unstable occlusive disease (e.g., crescendo angina). 
c. Patients with clinically unstable cardiac arrhythmias, such as recurrent 
ventricular tachycardia. 

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d. Patients with uncontrolled congestive heart failure (NYHA Class IV). 
e. Patients with recent cerebral hemorrhage. 
f. Patients who have undergone surgery within 24 hours prior to the study 
sonographic examination. 
4. Patients with known hypersensitivity or allergy to any component of Definity. 
5. Patients with unstable cardiopulmonary conditions or respiratory distress 
syndrome. 
6. Patients with uncontrollable emphysema, pulmonary vasculitis, pulmonary 
hypertension or a history of pulmonary emboli. 
4.2 
Gender/Minority/Pediatric Inclusion for Research  
We include only female 21 years of age or older regardless of race and speaking language. 
Since this study does not provide direct benefit to subjects, we determined the age limit of 21 so 
no state law requires parent’s consent for our subject to participate in the study.  In 2019, an 
estimated 268,600 new cases of invasive breast cancer are expected to be diagnosed in 
women while about 2,670 new cases of invasive breast cancer are expected to be diagnosed in 
men. The lifetime risk (up to age 95 and older) of breast cancer for women in US is about 13%. 
However, this risk varies by race and ethnic group: white- 13%, Black- 12%, Asian/Pacific 
Islander- 11%, Hispanic- 10%, American Indian/Alaska Native- 8%.  
4.3 
Strategies for Recruitment and Retention 
Subjects eligible for trial enrollment will be identified by the investigators from the TJU 
patient population who are scheduled for a breast biopsy. An investigator or research 
coordinator will explain the study to the patient. The patient will be given time to 
consider the risks and benefits of the study and ask questions about participation. The 
consent form will be reviewed with the patient and then the patient will be given the form 
to review. If consent interview is conducted by a coordinator, a study investigator will 
then discuss the study with the subject and answer any additional questions. The 
patient, person conducting study interview (if applicable), and a study investigator will all 
sign the consent form. The patient will be given a copy of the signed consent form for 
her records. 
Screening assessments will be performed within 24 hours prior to the administration of 
Definity. Trial participants will have the presence of inclusion criteria and absence of 
exclusion criteria verified by providing a medical history. A full demographic profile, 
known drug allergies or intolerances, and a review of the subject’s medical/surgical 
history will be recorded. If the subject is a woman of childbearing potential, she will have 
a urine pregnancy test (the results of which will be made available to the subject prior to 
study initiation). 
4.4 
Participant Withdrawal 
4.4.1 
Reasons for Withdrawal 

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Participants are free to withdraw from participation in the study at any time upon 
request. 
An investigator may terminate a study participant’s participation in the study if: 
 Any clinical adverse event (AE), laboratory abnormality, or other medical 
condition or situation occurs such that continued participation in the study would 
not be in the best interest of the participant. 
 The participant meets an exclusion criterion (either newly developed or not 
previously recognized) that precludes further study participation. 
4.4.2 
Handling of Participant Withdrawals and Participant Discontinuation of 
Study Intervention 
The date the subject is withdrawn from the trial and the reason for discontinuation will 
be recorded on the CRF. If a subject's participation in the trial is interrupted for any 
reason (e.g., because of an AE) and the subject has met the criteria described above 
for completing the trial, the subject's trial participation will be considered completed. If a 
subject's trial participation is interrupted for any reason by the subject's or investigator's 
choice and the subject has not met all of the criteria listed above, then the subject will 
be considered as an incomplete participant.  
4.5 
Premature Termination or Suspension of Study 
This study may be suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or termination, 
will be provided by the suspending or terminating party to investigators, funding agency, 
the Investigational New Drug (IND) sponsor, and regulatory authorities. If the study is 
prematurely terminated or suspended, the principal investigator will promptly inform the 
IRB and will provide the reason(s) for the termination or suspension.  
Circumstances that may warrant termination include, but are not limited to: 
 Determination of unexpected, significant, or unacceptable risk to participants. 
 Insufficient adherence to protocol requirements. 
 Data that is not sufficiently complete and/or evaluable.  
 Determination of futility. 
5 Study Intervention 
5.1 
Study Product  
This study uses the ultrasound contrast agent, Definity (Lantheus Medical Imaging, 
Billerica, MA).  

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5.2 
Study Product Description 
Definity is a sterile, injectable cardiovascular ultrasound contrast agent comprised of 
lipid-coated echogenic microbubbles (majority diameter of 1-4 micrometers) filled with 
octafluoropropane gas and approved by the FDA for use in cardiology. 
5.2.1 
Acquisition 
Definity will be provided by Lantheus, the manufacturer, directly to Thomas Jefferson 
University. 
5.2.2 
Formulation, Packaging, and Labeling 
Component 
Concentration 
Chemical Abstracts 
Service 
Glycerin 
>10% 
56-81-5 
Lipid Blend SG896 
<1% 
N/A 
Water 
>50% 
7732-18-5 
Perfluoropropane 
<1% 
76-19-7 
Propylene Glycol 
>1% 
57-55-6 
Sodium Chloride 
<1% 
7647-14-5 
Disodium 
orthophosphate 
<1% 
7782-85-6 
Sodium phosphate 
monobasic 
<1% 
10049-21-5 
Definity is supplied as a single use 2 ml clear glass vial containing clear liquid in 
packages of 4 and 16 single-use vials. It is labeled for the use in patients with 
suboptimal echocardiograms to opacity the left ventricular chamber and to improve the 
delineation of the left ventricular endocardial border. In this study, Definity will be used 
off-label with FDA approval under an IND (no. 112,241). 
5.2.3 
Product Storage and Stability 
Definity needs to be stored between 2-8°C (36°-46°F). The microbubbles are stable and 
it is necessary to activate them before use. 

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5.3 
Dosage, Preparation, and Administration  
Definity will be at room temperature to warm before starting the activation procedure. 
Definity will be activated by shaking the vial for 45 seconds using the VIALMIX® 
apparatus (Lantheus Medical Imaging, North Billerica, MA) as suggested by the 
manufacturer. Two vials of Definity (3 ml in total) suspended in 50 ml saline will be 
administered as an IV infusion via a peripheral vein (preferably the antecubital vein), 
with infusion rates of 4 to 10 ml/min (titrated to effect). 
5.4 
Dose Modifications and Dosing Delays 
The recommended infusion dose for the labeled echocardiograms is 1.3 ml and it was 
modified to 3 ml for this study in accordance with our prior experience in CEUS studies 
[Sridharan et al. 2013, Nam et al. 2017]. This dose typically provides 5 to 8 minutes of 
enhancement. Definity is reported to be well tolerated by patients. The largest safety 
studies published to date on the use of ultrasound contrast agents in humans (involving 
up to 4,300,966 subjects) concluded that these agents have a good safety profile in 
both cardiac and abdominal ultrasound applications [Main et al. 2009, Dolan et al. 2009, 
Wei et al. 2008]. However, as rare but serious cardiopulmonary reactions have been 
reported following the IV administration of Definity, all subjects will be monitored by a 
physician during the entire imaging period (as recommended by the FDA). 
5.5 
Study Product Accountability 
The agent will be stored in a secure refrigerator, with only the study investigators and 
research personnel having access. The study researchers will be responsible for 
inventory control. The manufacturer will distribute Definity directly to TJU. Any unused 
product will be disposed at the study site. 
5.6 
Dietary Restrictions 
There is no dietary restrictions for the study. 
5.7 
Administration of Procedural Intervention 
Definity will be administered as an IV infusion via a peripheral vein (preferably the 
antecubital vein), with infusion rates of 4 to 10 ml/min (titrated to effect) through an 18 to 
22 gauge angio-catheter.  
5.8 
Procedures for Training of Clinicians on Procedural Intervention 
The staff in this study are trained for the administration of Definity from our previous 
studies. If there are clinicians who are not familiar with Definity, we will train them before 
their participation in the study. If the last training case is found appropriate, the clinical 
site will be allowed to proceed with the study and training cases will be included in the 
study analysis. If not, the site personnel will be re-trained. 

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5.9 
Assessment of Clinician and/or Participant Compliance with Study 
Procedural Intervention 
The assessment of clinician and participant compliance with the procedural intervention 
will be subject to a strict QA review by the study coordinator and investigators at TJU.  
6 Study Schedule  
6.1 
Pretreatment Period/Screening 
Patients will be identified and approached from TJU Breast Imaging Center (by 
investigators). An investigator or research coordinator will explain the study to the 
patient. The patient will be given time to consider the risks and benefits of the study and 
to ask questions about participation. A consent form will be reviewed with the patient. 
Study participants will have the presence of inclusion criteria and absence of exclusion 
criteria verified by providing a medical history. A full history and physical examination 
will be obtained from the patient’s referring physician. If the history and physical 
examination note does not provide adequate information about the patient, the subjects 
will be assessed for their eligibility by their referring physician before enrollment into the 
study. If the subject is a woman of childbearing potential, she will have a urine 
pregnancy test 24 hours prior to the CEUS study (the results of which will be made 
available to the subject prior to study initiation). 
6.2 
Enrollment/Baseline 
Enrollment/ Visit day (before a biopsy) 
 Obtain and document consent from participant on study consent form. 
 Verify inclusion/exclusion criteria.  
 Obtain demographic information, medical history, and medication history.  
 Obtain the previous results of physical and imaging tests if available. 
 If woman of childbearing potential, urine pregnancy test within 24 hours prior to 
receiving Definity (CEUS exam) 
 
6.3 
Treatment Period 
On the visit day  
 Perform Baseline Ultrasound imaging. 
 Administer Definity (IV infusion) 
o Determine the optimized acoustic settings for 3D SHAPE imaging  
o Perform CEUS exam and collect 3D SHAPE data 

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 Wait for clearance of Definity 
o Collect 3D SHAPE data 
 
Local anesthesia procedures will be performed using simple Lidocaine 1%, or 
combined with 1:100,000 Epinephrine (EPI) before the IFP measurements.  
 Take a single IFP measurement each from the breast tumor and its surrounding 
tissue using Stryker compartment pressure monitoring system with ultrasound 
imaging guide 
Participants will be monitored for 30 minute following the administration of Definity.  
 
6.4 
End of Treatment Study Procedures 
We will collect the results from a breast biopsy obtained as standard of care. 
6.5 
Withdrawal Visit/Discontinuation of Therapy 
If participant withdraws early or investigator terminates participant’s participation, no 
additional visits or evaluations will be required or offered to the participant. 
7 Study Procedures and Evaluations 
7.1 
Study Procedures/Evaluations 
 Ultrasound, without contrast 
 Definity administration 
 CEUS exam 
 IFP measurements 
 
7.2 
Laboratory Procedures/Evaluations 
7.2.1 
Clinical Laboratory Evaluations 
There is no clinical laboratory procedure for this study, except for the urine pregnancy 
test for women of childbearing potential. We will only collect the results from histology, 
pathology, and imaging provided as standard of care to evaluate the malignancy of the 
breast tumor. 
8 Evaluation of Safety 
8.1.1 
Unanticipated Problems 

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Unanticipated problems (UAPs) include, in general, any incident, experience, or 
outcome that meets the following criteria: 
 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 participant population being studied; 
UAPs are considered to pose risk to participants or others when they suggest that the 
research places participants or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
8.1.2 
Adverse Events 
An adverse event is any untoward or unfavorable medical occurrence in a human 
participant, including any abnormal sign (for example, abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the participant’s 
participation in the research, whether or not considered related to the participant’s 
participation in the research. 
8.1.3 
Serious Adverse Events 
A serious adverse event (SAE) is one that meets one or more of the following criteria: 
 Results in death 
 Is life-threatening (places the participant at immediate risk of death from the 
event as it occurred) 
 Is disabling or incapacitating 
 Results in inpatient hospitalization or prolongation of existing hospitalization 
 Results in a persistent or significant disability or incapacity 
 Results in a congenital anomaly or birth defect  
 An important medical event that may not result in death, be life threatening, or 
require hospitalization may be considered an SAE when, based upon appropriate 
medical judgment, the event may jeopardize the participant or may require 
intervention to prevent one of the outcomes listed in this definition. 
8.2 
Safety Assessment and Follow-Up 
The subjects will be monitored for AEs and SAEs during the entire procedure and 30 
minute monitoring window. All AEs, including observed or volunteered problems, 
complaints, signs or symptoms, and diagnoses, occurring from the initiation of Definity 
dosing until the completion of the Definity administration will be recorded on a serious or 

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non-serious AE data form, whether or not associated with the use of the trial 
medication.   
8.3 
Recording Adverse Events 
The following subsections detail what information must be documented for each 
adverse event occurring during the time period specified in Section 8.2 Safety 
Assessment and Follow-Up.  
8.3.1 
Relationship to Study Intervention 
The relationship to study intervention or study participation must be assessed and 
documented for all adverse events. Evaluation of relatedness must consider etiologies 
such as natural history of the underlying disease, concurrent illness, concomitant 
therapy, study-related procedures, accidents, and other external factors.  
The following guidelines are used to assess relationship of an event to study 
intervention: 
1. Related (Possible, Probable, Definite) 
a. The event is known to occur with the study intervention. 
b. There is a temporal relationship between the intervention and event onset. 
c. The event abates when the intervention is discontinued. 
d. The event reappears upon a re-challenge with the intervention. 
2. Not Related (Unlikely, Not Related) 
a. There is no temporal relationship between the intervention and event 
onset. 
b. An alternate etiology has been established. 
8.3.2 
Expectedness 
The PI or Co-Investigator is responsible for determining whether an AE is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency 
of the event is not consistent with the risk information previously described for the 
intervention. Risk information to assess expectedness can be obtained from preclinical 
studies, the investigator’s brochure, published medical literature, the protocol, or the 
informed consent document. 
8.3.3 
Severity of Event 
Adverse events will be graded for severity according to the Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0.  

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8.3.4 
Intervention 
Any intervention implemented to treat the adverse event must be documented for all 
adverse events. 
8.4 
Safety Reporting  
8.4.1 
Reporting to IRB 
All incidents or events that meet criteria for unanticipated problems (UAPs) as defined in 
Section 8.1.1 Unanticipated Problems require the creation and completion of an 
unanticipated problem report form (OHR-20).  
UAPs that pose risk to participants or others, and that are not AEs, will be submitted to 
the IRB on an OHR-20 form via the eazUP system within 5 working days of the 
investigator becoming aware of the event.  
UAPs that do not pose risk to participants or others will be submitted to the IRB at the 
next continuing review.  
8.4.1.1 Adverse Events  
Grade 1 AEs will be reported to the IRB at continuing review.  
Grade 2 AEs will be reported to the IRB at the time of continuing review.  
8.4.1.2 Serious Adverse Events  
SAEs will be reported to the IRB on OHR-10 forms via the electronic reporting system 
(eSAEy) according to the required time frames described below. 
Grade 3-4 AEs that are unexpected and deemed to be at least possibly related to the 
study will be reported to the IRB within 2 working days of knowledge of the event. 
Grade 3-4 AEs that are deemed unrelated to the study will be reported to the IRB within 
5 working days.  
Grade 5 AEs will be reported to the IRB within one working day of knowledge of the 
event. 
All SAEs will be submitted to the IRB at continuing review, including those that were 
reported previously. 
8.4.2 
Reporting to SKCC DSMC 
All AEs and SAEs, safety and toxicity data, and any corrective actions will be submitted 
to the DSMC per the frequency described in the SKCC DSMP. The report to the SKCC 
DSMC will also include any unanticipated problems that in the opinion of the PI should 
be reported to the DSMC. 

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For expedited reporting requirements, see table below:                                            
DSMC AE/SAE Reporting Requirements 
 

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8.4.3 
Reporting to Funding Sponsor 
Serious adverse events that occur with commercially available agents/devices are 
reported through Food and Drug Administration. The manufacturer (Lantheus) will also 
receive reports. 
8.4.4 
Reporting to FDA 
The investigators will report in an IND safety report any suspected adverse reaction to 
study treatment (i.e., including active comparators) that is both serious and unexpected 
(21 CFR 312.32(c)(1)(i)) as soon as possible and no later than 7 days (for a death or 
life-threatening event) or 15 days (for all other SAEs) after the investigator’s or 
institution’s initial receipt of the information. Before submitting an IND safety report, the 
investigators needs to ensure that the event meets all three of the definitions: 
 Suspected adverse reaction 
 Serious 
 Unexpected 
SAEs will be reported on MedWatch Form 3500A, which can be accessed at: 
http://www.accessdata.fda.gov/scripts/medwatch/. 
MedWatch SAE forms will be sent to the FDA at: 
 
MEDWATCH 
5600 Fishers Lane 
Rockville, MD 20852-9787 
Fax: 1-800-FDA-0178 (1-800-332-0178) 
http://www.accessdata.fda.gov/scripts/medwatch/ 
 
If the adverse event does not meet all three of the definitions, it will not be submitted as 
an IND safety report. 
In addition, the investigators will identify in each IND safety report all IND safety reports 
previously submitted to FDA concerning a similar suspected adverse reaction and will 
analyze the significance of the suspected adverse reaction in light of previous, similar 
reports or any other relevant information (21 CFR 312.32(c)(1)). The analysis will 
include similar reports from all INDs held by the sponsor and any other relevant 
information known to the sponsor (21 CFR 312.32(c)(1)). Investigators will evaluate a 
suspected adverse reaction in the context of other related reports or adverse events, 
including those that occurred in the placebo or active comparator group and those that 
occurred in pre- and postmarketing studies. 
8.4.5 
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If the subject is a woman of childbearing potential, she will have a urine pregnancy test 
prior to each CEUS study. 
8.5 
Halting Rules 
Should the observed rate of AE/SAE at any of the scheduled interim analyses exceed 
25%, study enrollment may be suspended so that the study committee may consider 
whether a change in protocol is necessary. 
9 Study Oversight 
In addition to the PI’s responsibility for oversight, study oversight will be under the 
direction of the SKCC’s Data and Safety Monitoring Committee (DSMC). The SKCC 
DSMC operates in compliance with a Data and Safety Monitoring Plan (DSMP) that is 
approved by the NCI.  
10 Clinical Site Monitoring and Auditing 
Clinical site monitoring and auditing is conducted to ensure that the rights of human 
participants are protected, that the study is implemented in accordance with the protocol 
and/or other operating procedures, and that the quality and integrity of study data and 
data collection methods are maintained. Monitoring and auditing for this study will be 
performed in accordance with the SKCC’s Data and Safety Monitoring Plan (DSMP) 
developed by the SKCC Data and Safety Monitoring Committee (DSMC). The DSMP 
specifies the frequency of monitoring, monitoring procedures, the level of clinical site 
monitoring activities (e.g., the percentage of participant data to be reviewed), and the 
distribution of monitoring reports. Some monitoring activities may be performed 
remotely, while others will take place at the study site(s). Appropriate staff will conduct 
monitoring activities and provide reports of the findings and associated action items in 
accordance with the details described in the SKCC DSMP. 
11 Statistical Considerations  
11.1 Study Hypotheses 
The main hypothesis is that the 3D subharmonic signals in the tumor and its 
surrounding tissue are correlated with the IFP values in them. The secondary 
hypothesis is that a marker from 3D subharmonic signals can predict the malignancy of 
breast tumor. 
11.2 Analysis Plans 
Radio frequency (RF) data from 3D SHAPE will be transferred to a PC for off-line 
analysis. 3D RF data from SHAPE will be composed of multiple 3D volumes (5 to 21 
volumes) over time. Each 3D volume will consist of multiple 2D data sets (about 79 
slices), which will be collected in the elevational direction. All data processing will be 

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performed using Matlab (R2015b; Mathworks, Natick, MA). Two regions of interest 
(ROI) will be selected in each elevational plane for SHAPE: one within the tumor and 
the other in the surrounding tissues (available top and/or bottom region of tumor). The 
ROIs of the tumor and surrounding area will be selected as a free form on the images 
generated from RF data (i.e., raw data before post-processing for on-screen display). 
The ROIs will be selected by the sonographer or radiologist. Then, a Fast Fourier 
Transform will be computed for each A-line within the ROI to obtain the frequency 
spectrum. The maximum magnitude from the resultant frequency spectrum will be 
extracted from each pre- and during infusion data sets and the ratio will be calculated 
for each ROI. If both top and bottom regions are selected as a surrounding area ROI, 
then the ratios will be averaged to one value. Finally, the obtained subharmonic signal 
ratios from the tumor and the surrounding area will be compared to the direct IFP 
measurements. Pearson’s correlation coefficients will be computed between SHAPE 
results and direct IFP measurements by Stryker compartment pressure monitoring 
system. These data will also be rigorously analyzed for agreement using Bland-Altman 
analysis (Bland & Altman, 1999) among tumor and healthy tissue samples. To explore if 
the relationship between SHAPE results and IFP is confounded by or might depend on 
study variables collected (e.g.’s, patient characteristics, tissue/tumor characteristics, 
etc.) linear mixed effects or generalized estimating equations modeling will be used. 
Generalized linear mixed logistic or GEE logistic modeling of tumor vs. non-tumor 
samples will be used to explore the potential for using SHAPE results as a novel 
functional predictor of breast tumor malignancy. Additionally, the subharmonic signal 
ratio from the tumor will be used to predict the malignancy of breast tumor using a 
biopsy result as a gold standard. The Mann-Whitney or Student’s t-test will be 
performed depending on the distribution of the results. 
11.3 Sample Size Considerations 
The aim of the study is to determine the correlation between SHAPE results and direct 
measurements of IFP. The aim is to get significant result (p < 0.05) with sufficient power 
(80%) to detect at least correlation coefficient of 0.5-0.6. Therefore, the required sample 
size for this study is 19 -29 based on the formula for calculation is based on two-tailed 
test [Guenther 1977]. Thus, we chose 25 patients as our sample size.  
11.3.1 Replacement Policy 
The patients who consent for the study participation but do not undergo the CEUS exam 
or IFP measurements will be replaced.  Also, unsuccessful training cases will be 
replaced. 
11.3.2 Accrual Estimates 
 At the Jefferson-Honickman Breast Imaging Center, a breast biopsy is performed on 
more than 500 women each year. We expect that 25 subjects can be enrolled within 15 
months. 
12 Source Documents and Access to Source Data/Documents 

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Study staff will maintain appropriate medical and research records for this study, in 
compliance with ICH E6, and regulatory and institutional requirements for the protection 
of confidentiality of participant information. Study staff will permit authorized 
representatives of SKCC and regulatory agencies to examine (and when required by 
applicable law, to copy) research records for the purposes of quality assurance reviews, 
audits, and evaluation of the study safety, progress and data validity.  

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13 Quality Control and Quality Assurance 
3D SHAPE data including ultrasound imaging and CRFs will be subject to a strict QA 
review by the study coordinator and PI at TJU. Results of the QA review will be shared 
on the quarterly basis with additional training provided, if necessary.   
14 Ethics/Protection of Human Participants  
14.1 Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report: Ethical Principles and Guidelines for the 
Protection of Human Subjects of Research, as drafted by the US National Commission 
for the Protection of Human Subjects of Biomedical and Behavioral Research (April 18, 
1979) and codified in 45 CFR Part 46 and/or the ICH E6.  
14.2 Institutional Review Board 
The protocol and informed consent form will be submitted to the IRB for review and 
approval. Approval of both the protocol and the consent form must be obtained before 
any participant is enrolled. Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented in the study.  
14.3 Informed Consent Process 
Informed consent is a process that is initiated prior to the individual agreeing to 
participate in the study and continues throughout study participation. Extensive 
discussion of risks and possible benefits of study participation will be provided to 
participants and their families, if applicable. A consent form describing in detail the study 
procedures and risks will be given to the participant. Consent forms will be IRB-
approved, and the participant is required to read and review the document or have the 
document read to him or her. The investigator or designee will explain the research 
study to the participant and answer any questions that may arise. The participant will 
sign the informed consent document prior to any study-related assessments or 
procedures. Participants will be given the opportunity to discuss the study with their 
surrogates or think about it prior to agreeing to participate. They may withdraw consent 
at any time throughout the course of the study. A copy of the signed informed consent 
document will be given to participants for their records. The rights and welfare of the 
participants will be protected by emphasizing to them that the quality of their clinical 
care will not be affected if they decline to participate in this study. The consent process 
will be documented in the clinical or research record. For non-English speaking 
participants, a translator fluent in both languages, will present and explain the study and 
full consent form at the time of the consent discussion. In addition, an IRB approved 
short consent form, translated in the subject’s language will be provided to the subject. 
All parties will sign the translated consent form. 
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The subject population of this study will be 100% women over 21 years old. Children, 
and pregnant women will be excluded based on the benefit and risk assessment. No 
patient will be excluded based on race.  
14.5 Participant Confidentiality 
Participant confidentiality is strictly held in trust by the investigators, study staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to any study information relating to participants. 
The study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be 
released to any unauthorized third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all 
study documents and records required to be maintained by the investigator, including 
but not limited to, medical records (office, clinic, or hospital) for the study participants. 
The clinical study site will permit access to such records. 
14.6 Future Use of Stored Specimens and Other Identifiable Data  
No specimens or other identifiable data will be stored for future use.  
15 Data Handling and Record Keeping 
The investigators are responsible for ensuring the accuracy, completeness, legibility, 
and timeliness of the data reported. All source documents must be completed in a neat, 
legible manner to ensure accurate interpretation of data. The investigators will maintain 
adequate case histories of study participants, including accurate case report forms 
(CRFs), and source documentation. 
15.1 Data Management Responsibilities 
Data collection and accurate documentation are the responsibility of the study staff 
under the supervision of the investigator. All source documents and laboratory reports 
must be reviewed by the study team and data entry staff, who will ensure that they are 
accurate and complete. Unanticipated problems and adverse events must be reviewed 
by the investigator or designee.  
15.2 Data Capture Methods 
All patient data will be captured electronically via excel file in a password protected 
computer and image and 3D SHAPE data will be stored in the secured computer hard 
drive.  Image and 3D SHAPE data will be de-identified. Copies of study documents will 
have personal identifying information retracted and replaced with study patient ID 
number. Originals of the study documents with personal identifying information will be 
retained at TJU. All this information will be available for audit. In addition, de-identified 
study data may be shared with study co-sponsors GE Healthcare and Lantheus Medical 
Imaging. 

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15.3 Types of Data 
The following data will be collected: 
 Results of clinical examination before the enrollment  
 Breast biopsy schedule and results 
 De-identified 3D SHAPE (RF) data, Stryker IFP measurements, and B-mode 
images from the study 
15.4 Study Records Retention 
Study records will be maintained for at least three years from the date that the grant 
federal financial report (FFR) is submitted to the NIH. 
Study documents must be retained for a minimum of 2 years after the last approval of a 
marketing application in an ICH region and until there are no pending or contemplated 
marketing applications in an ICH region or until at least 2 years have elapsed since the 
formal discontinuation of clinical development of the investigational product. These 
documents will be retained for a longer period, however, if required by local regulations. 
No records will be destroyed without the written consent of the sponsor, if applicable. It 
is the responsibility of the sponsor to inform the investigator when these documents no 
longer need to be retained. 
15.5 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical study protocol, Good Clinical 
Practice, or Manual of Procedures requirements. The noncompliance may be on the 
part of the participant, the investigator, or study staff. As a result of deviations, 
corrective actions are to be developed by the study staff and implemented promptly. 
All deviations from the protocol must be addressed in study participant source 
documents and promptly reported to the IRB and other regulatory bodies according to 
their requirements.  
16 Study Finances 
16.1 Funding Source 
This study will be financed through a grant from the US National Institute of Health. 
16.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) 
must have the conflict reviewed by a properly constituted Conflict of Interest Committee 
with a Committee-sanctioned conflict management plan that has been reviewed and 

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approved by the study sponsor prior to participation in this study. All Jefferson 
University Investigators will follow the TJU Conflicts of Interest Policy for Employees 
(107.03). 
16.3 Participant Stipends or Payments 
At the completion of the study (a single visit for CEUS and IFP measurements), the 
participants will receive a preloaded “clincard” for the amount of  $100.   
17 Publication and Data Sharing Policy 
This study will comply with the NIH Public Access Policy, which ensures that the public 
has access to the published results of NIH funded research. It requires scientists to 
submit final peer-reviewed journal manuscripts that arise from NIH funds to the digital 
archive PubMed Central upon acceptance for publication. 
The International Committee of Medical Journal Editors (ICMJE) member journals have 
adopted a clinical trials registration policy as a condition for publication. The ICMJE 
defines a clinical trial as any research project that prospectively assigns human 
participants to intervention or concurrent comparison or control groups to study the 
cause-and-effect relationship between a medical intervention and a health outcome. 
Medical interventions include drugs, surgical procedures, devices, behavioral 
treatments, process-of-care changes, and the like. Health outcomes include any 
biomedical or health-related measures obtained in patients or participants, including 
pharmacokinetic measures and adverse events. The ICMJE policy requires that all 
clinical trials be registered in a public trials registry such as ClinicalTrials.gov, which is 
sponsored by the National Library of Medicine. Other biomedical journals are 
considering adopting similar policies. The ICMJE does not review specific studies to 
determine whether registration is necessary; instead, the committee recommends that 
researchers who have questions about the need to register err on the side of 
registration or consult the editorial office of the journal in which they wish to publish. 
U.S. Public Law 110-85 (Food and Drug Administration Amendments Act of 2007 or 
FDAAA), Title VIII, Section 801 mandates that a "responsible party" (i.e., the sponsor or 
designated principal investigator) register and report results of certain "applicable 
clinical trials:"  
Trials of Drugs and Biologics: Controlled, clinical investigations, other than Phase I 
investigations, of a product subject to FDA regulation;  
NIH grantees must take specific steps to ensure compliance with NIH implementation of 
FDAAA. 
18 Literature References 
Bland JM, Altman DG (1999). "Measuring agreement in method comparison studies". 
Statistical Methods in Medical Research. 8 (2): 135–60. 

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Ferretti S, Allegrini PR, Becquet MM, McSheehy PM. Tumor interstitial fluid pressure as 
an early-response marker for anticancer therapeutics. Neoplasia 2009; 11:874-881.  
 
Fukumura D, Jain RK. Tumor microenvironment abnormalities: causes, consequences, 
and strategies to normalize. J Cell Biochem 2007; 101:937-949. 
 
Guenther WC. Desk calculation of probabilities for the distribution of the sample 
correlation coefficient. The American Statistician 1977; 31:45-58. 
 
Halldorsdottir VG, Dave JK, Eisenbrey JR, et al. Subharmonic aided pressure 
estimation for monitoring interstitial fluid pressure in tumours –in vitro and in vivo proof 
of concept. Ultrasonics 2014; 54:1938-1944.  
 
Heldin CH, Rubin K, Pietras K, Ostman A. High interstitial fluid pressure-an obstacle in 
cancer therapy. Nat Rev Cancer 2004; 4:806-813. 
 
Khawaja OA, Shaikh KA, Al-Mallah MH. Meta-analysis of adverse cardiovascular events 
associated with echocardiographic contrast agents. AmJ Cardiol 2010;106:742–747. 
 
Milosevic MF, Fyles AW, Hill RP. The relationship between elevated interstitial fluid 
pressure and blood flow in tumors: a bioengineering analysis. Int J Radiat Oncol Biol 
Phys 1999; 43:1111-11239. 
 
Milosevic M, Fyles A, Hedley D, et al. Interstitial fluid pressure predicts survival in 
patients with cervix cancer independent of clinical prognostic factors and tumor oxygen 
measurements. Cancer Res 2001; 61:6400-6405.  
 
Nam K, Eisenbrey JR, Stanczak, et al. Monitoring neoadjuvant chemotherapy for breast 
cancer by using three-dimensional subharmonic aided pressure estimation and imaging 
with US contrast agents: preliminary experience. Radiology 2017; 285:53-62. 
 
Nathanson SD, Nelson L. Interstitial fluid pressure in breast cancer, benign breast 
conditions, and breast parenchyma. Ann Surg Oncol. 1994 Jul; 1:333-338. 
 
Shi WT, Forsberg F, Raichlen JS, Needleman L, Goldberg BB. Pressure dependence of 
subharmonic signals from contrast microbubbles. Ultrasound Med Biol 1999; 25:275-
283.  
 
Sridharan A, Eisenbrey JR, Liu JB, et al. Perfusion estimation using contrast enhanced 
three-dimensional subharmonic ultrasound imaging: an in vivo study. Invest Radiol 
2013; 48:654-660. 
 
Stohrer M, Boucher Y, Stangassinger M, Jain RK. Oncotic pressure in solid tumors is 
elevated. Cancer Res 2000; 60:4251-4255.  
 

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Szebeni J. Complement activation-related pseudoallergy: a new class of drug-induced 
acute immune toxicity. Toxicology. 2005;216:106–121. 
 
Taghian AG, Abi-Raad R, Assaad SI, et al. Paclitaxel decreases the interstitial fluid 
pressure and improves oxygenation in breast cancers in patients treated with 
neoadjuvant chemotherapy: clinical implications. J Clin Oncol 2005; 23:1951-1961.  
 
Wei K, Mulvagh SL, Carson L, et al. The safety of deFinity and Optison for ultrasound 
image enhancement: a retrospective analysis of 78,383 administered contrast doses. J 
Am Soc Echocardiogr. 2008;21:1202–1206. 
 
 
 
 
 
SUPPLEMENTAL MATERIALS 
These documents are relevant to the protocol, but they are not considered part of the 
protocol. They are stored and modified separately. As such, modifications to these 
documents do not require protocol amendments. 
 
Case Report Forms (CRFs) 
 
 
Appendices 
The following documents are officially affiliated with the protocol and will be submitted to the IRB 
as a part of the protocol. As such, changes to these items require a protocol amendment. 
Appendix A: Schedule of Events 
 
 
 

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APPENDIX A: SCHEDULE OF EVENTS 
 
Procedures 
Screening  
Study Visit  
Signed Consent Form 
X 
 
Assessment of Eligibility 
Criteria 
X 
X 
Review of Medical History 
X 
 
Definity Administration 
 
X 
CEUS exam 
 
X 
Ultrasound without contrast  
X 
Assessment of Adverse 
Events 
 
 X 
IFP Measurements 
 
X 
Pregnancy test (for women 
of childbearing potential)1 
 X 
 X 
1. Urine pregnancy test within 24 hours prior to administration of Definity