Quantitative Subharmonic Breast Imaging: Subharmonic Imaging and Pressure Estimation for Monitoring Neoadjuvant Chemotherapy
Phase: NA
Conditions: Breast Cancer
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Thomas Jefferson University

0 
 
PROTOCOL 
 
 
TITLE:  Quantitative Subharmonic Breast Imaging: Subharmonic Imaging and Pressure 
Estimation for Monitoring Neoadjuvant Chemotherapy 
 
VERSION & DATE:  Version 1.5- February 20th, 2013 
 
 
PRINCIPAL INVESTIGATOR:  
Flemming Forsberg, PhD 
Professor, Department of Radiology 
Thomas Jefferson University 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: flemming.Forsberg@jefferson.edu 
Tel: 215-955-4870 
Fax: 215-955-8549 
 
KEY PERSONNEL: 
Barbara M. Cavanaugh, MD  
            Aninna Wilkes, MD 
Clinical Associate Professor of Radiology 
Clinical Assistant Professor of Radiology 
Department of Radiology 
 
 
Department of Radiology 
Thomas Jefferson University   
           Thomas Jefferson University 
 
Tiffany Avery, MD 
                                     
Assistant Professor of Medical Oncology              
Department of Medical Oncology                         
Thomas Jefferson University                            
 
 
 
 
LOCATION OF STUDY: 
Thomas Jefferson University 
925 Chestnut Street, 2th Floor and 132 South 10th Street, 7th Floor 
 
Philadelphia, PA 19107 
 
STUDY DURATION:  2 year project 
 
CONFIDENTIALITY STATEMENT 
This document is the confidential property of Thomas Jefferson University.  No part of it may be 
transmitted, reproduced, published, or used by other persons without the permission of Thomas 
Jefferson University. 
 

1 
 
SYNOPSIS 
 
Protocol Title: Quantitative Subharmonic Breast Imaging; Subharmonic Imaging and Pressure 
Estimation for Monitoring Neoadjuvant Chemotherapy 
 
Trial Objectives:  
The primary objective of this first-in-humans trial is: 
 
To evaluate the ability of 3D Subharmonic Imaging (SHI) and Subharmonic Imaging and 
Pressure Estimation  (SHAPE) to track changes in locally advanced breast cancer (LABC) angiogenesis 
and interstitial fluid pressure (IFP), respectively, by studying women undergoing neoadjuvant 
chemotherapy before as well as with around 10 % and 60% of the neoadjuvant chemotherapy 
treatment delivered and after completion of the neoadjuvant chemotherapy treatment.  Results 
will be compared to MRI and pathology. 
 
The secondary aim of this trial is: 
 
· To compare the 3D SHI depiction of breast cancer angiogenesis in humans to CD31, an 
immunohistochemical predictor of angiogenesis. 
 
Trial Design:   
This is an open-label, non-randomized, first-in-humans trial that will be conducted at one clinical 
site Thomas Jefferson University (TJU).  All subjects will undergo an unenhanced (baseline) 
imaging study followed by one infusion of Definity (Lantheus Medical Imaging, Billerica, 

2 
 
MA), and a Definity contrast-enhanced US imaging study for evaluation of a breast mass or 
breast abnormality without mass. 
 
Trial Population: This trial will consist of between 20 and 50 adult (21 years of age or older) 
female subjects who are undergoing neoadjuvant chemotherapy. 
 
Trial Procedures:  Subjects eligible for trial enrollment will be identified by the investigators 
from among TJU’s patient population who are undergoing neoadjuvant chemotherapy.  A full 
demographic profile, known drug allergies or intolerances, and review of the subject’s 
medical/surgical history will be recorded and reviewed to ensure the subject meets inclusion 
criteria.   
 
A modified Logiq 9 scanner (GE Medical Systems, Milwaukee, WI) with a broad bandwidth, 
3D, linear array (the 4D10L) will be used to acquire conventional images and subharmonic 
imaging (SHI).  The US software has been modified by GE Medical Systems on the clinical 
system, the Logiq 9 scanner, to permit acquisition of subharmonic data.  All acoustic parameters 
have been verified and are all within the current standards of the machine, making the modified 
US system a non-significant risk device.  US imaging will be performed at a transmit frequency 
of 5.8 MHz and the subharmonic obtained at 2.9 MHz.  Using this setup, acoustic pressure 
amplitudes will all be below 1.5 MPa peak negative pressure, 2.5 MPa peak positive pressure 
(MI < 0.33). 
 
A baseline US grayscale scan will be used to identify the mass or abnormal area seen by 

3 
 
mammography (or another concomitant imaging mode, such as US or MRI) and to evaluate the 
following criteria: diagnosis; size, shape, and orientation of the lesion; echogenicity compared to 
surrounding tissue.  Standard Power Doppler (PDI) of the lesion or target area will also be 
performed.  The distribution of color signals and the overall color content of the lesion will be 
evaluated by comparing the pattern and amount of color to the normal surrounding breast.  
Irregularity of the course of the vessels and anastomoses will be evaluated.  Digital clips of the 
two baseline imaging modes will be acquired   
 
The contrast administration will be an intravenous infusion of 2 vials of Definity/50ml saline 
through the subjects’ vascular port (established as part of their standard of care), with infusion 
rates of 4 to 10 ml/min (titrated to effect).  Next, we will run a power optimization algorithm to 
establish the individualized acoustic parameter settings for the case (on the first imaging study 
only).  We will use those parameters for all subsequent imaging studies to sweep the area of the 
LABC to acquire 3D, pulse inversion SHI grayscale volume data.  The digital clips and radio-
frequency (RF) data obtained from each 3D SHI injection will be transferred to a PC for off-line 
analysis.  The SHI images will be qualitatively analyzed as described above.  This protocol will 
be followed for all the contrast studies for each subject.  All subjects will be closely monitored 
by a physician during and following the study. 
 
Moreover, the 3D SHAPE results (i.e., IFP) will be compared to lesion size, tumor response, 
DFS and overall survival.  Results will also be compared to MRI enhancement kinetics and 
imaging measurements of lesion size obtained before and after completion of the chemotherapy.  
Once surgery has been performed the following variables estrogen, progesterone and HER2 

4 
 
receptor presence or absences; presence or absence of hemorrhagic, necrotic, or other component 
of the lesion (and their location if present); histological margins; lesion size; TNM staging; 
histological lesion type; presence or absence of vascular invasion; histological and cytological 
grade; presence or absence of metastases; node staging; and percentage of the lesion that was 
invasive or in situ will be collected as part of the patient’s clinical care and correlated with the 
imaging results. 
 
Statistical Methodology: The ability of the imaging tests to distinguish responders from non-
responders will be compared using ROC analysis, while the incremental validity of combining 
imaging assessments will be analyzed using logistic regression and ROC analyses. Given 40 
subjects split evenly into 20 responders and 20 non-responders, the analysis will have 80 % 
statistical power to detect a difference of 0.174 between a diagnostic test with an area under the 
ROC curve (Az) of 0.800 (the null hypothesis) and an actual Az of 0.974 using a one sample, 
two-sided z-test with a significance level of 0.05.  All analyses will be performed with Stata 12.0 
(Stata Corp., College Station, TX). 
 
1. Introduction  
 
Systemic preoperative therapy (neoadjuvant chemotherapy alone or in combination with 
endocrine therapy) is widely used in the treatment of locally advanced, primary breast cancer 
(LABC) and is considered the standard of care for these patients.1,2,3,4 Some 20 % of all primary 
breast cancers are diagnosed as LABC or are carcinomas with an unfavorable tumor-to-breast 
size ratio. In the largest study to date (>1500 patients), there was no statistical differences 

5 
 
between survival among patients that underwent neoadjuvant therapy (69%) and patients 
receiving adjuvant therapy (70%; p=0.80) after 9 years.5 Likewise, for disease free survival 
(DFS), which was 53 and 55 %, respectively, in the adjuvant and the neoadjuvant groups 
(p=0.5). These results were supported by a recent meta-analysis involving almost 4000 women, 
which found no statistical or clinical differences between neoadjuvant and adjuvant therapy arms 
associated with survival or disease progression5 and this result was recently confirmed even after 
10 years of follow-up.6 Moreover, neoadjuvant therapy is increasingly being used to treat 
operable, palpable breast cancers as well, in an attempt to avoid mastectomies and allow for 
breast conserving surgery instead.4,7,8 Neoadjuvant therapies have the potential to improve 
survival and reduce morbidity by downstaging tumors, since a reduction in the primary tumor 
volume can be used to predict a reduction in micrometastatic tumor volume and clinical 
benefit.4,9 However, monitoring the in vivo response to therapy is essential to distinguish between 
patient responders and non-responders as early as possible to allow for better, individualization 
of treatment options. A noninvasive technique that reliably determines breast cancer response to 
therapy may improve treatment outcomes.   
 
Scintimammography utilizing Tc-99m sestamibi10,11 , positron emission tomography (PET), 
computed tomography (CT) and optical imaging .12,13,14  as well as Magnetic resonance imaging 
(MRI) has  been used to monitor the response of LABC to neoadjuvant therapy.15,16,17,18,19 While 
current guidelines recommend that patients diagnosed with breast cancer are followed post-
surgery with x-ray mammography every 6 to 12 months,20 a study comparing MRI, 
mammography and ultrasound (US) imaging for evaluation of residual disease following 
neoadjuvant therapy concluded that MRI and US were equivalent and better than mammography 

6 
 
for this application.21 Also, mammography does not provide functional information and contrast-
enhanced MRI is expensive, not readily available in many communities and associated with 
nephro-toxicity.20 
 
Measuring a functional parameter such as breast tumor neovascularity with color and power 
Doppler imaging (CDI and PDI, respectively) have meet with initial success (κ=0.87) in small 
studies.22,23 However, the sensitivity of CDI and PDI can be markedly improved with 
intravenous (IV) injection of gas microbubbles as US contrast agents, as we and others have 
demonstrated in patients with LABC.24,25,26 Following our initial and somewhat disappointing 
experiences with two-dimensional (2D) contrast enhanced PDI (no statistically improvements in 
sensitivity and specificity were found with contrast,26 we embarked on a study of 55 patients 
(with 16 cancers and 38 benign lesions) utilizing contrast enhanced three-dimensional (3D) PDI 
(supported by DAMD17-97-1-7116).27 The sensitivity of contrast enhanced 3D PDI was 
significantly greater than that of standard 2D and 3D US imaging (grayscale as well as PDI; 
p<0.04). 27 Recent studies of breast tumors by our group have indicated significant correlation of 
direct pathologic vascularity assessments, such as the intra-tumoral micovascular density 
(iMVD), with ultrasonic vascularity measurements post contrast particularly for vessels 20 to 39 
µm in diameter (p<0.03).28 This indicates the potential of contrast-enhanced US to provide a 
noninvasive measure of breast tumor neovascularity. It is important, since angiogenesis may be 
an independent prognostic marker for breast cancer and, thus, an important parameter to monitor 
when assessing LABC therapy response.29,30 
 

7 
 
Moreover, solid tumors are known to have higher interstitial fluid pressures (IFPs) than 
surrounding normal tissues (10–25mmHg depending on tumor type and size),31 due to the leaky 
nature of angiogenic tumor vessels that makes the microvascular pressure (MVP) equivalent to 
the IFP.31,32,33,34 The development of tumor angiogenesis is closely related to IFP,35 as confirmed 
by results in human breast cancer patients where the IFP was significantly higher in cancers than 
in surrounding normal tissues (29 vs. -0.3mmHg; p=0.001).36 The high IFP and elevated vascular 
permeability in tumors may prevent the delivery of therapeutic drugs to large regions of the 
tumors.37 Thus, it could be hypothesized that breast cancers with high IFP would respond poorly 
to chemotherapy because of poor drug delivery. This concept is supported by a recent study in 54 
patients with LABC, which measured IFP before and after treatment with one of two 
chemotherapy agents, and found that one therapy (paclitaxel) significantly decreased the mean 
IFP (by 36%; p=0.02) whereas the other agent did not.38 While clinical outcomes are not 
available for this study, another study demonstrated that tumor IFP can predict survival in 
patients with cervix cancer, independently of other prognostic factors.39 These results suggest 
that tumor IFP may be a useful quantitative endpoint for assessing response to neoadjuvant 
therapy of LABC.  
 
Consequently, a proposal to establish a new technique for monitoring the response to 
neoadjuvant therapy through the novel and innovative use of contrast-enhanced US based 
pressure estimation to measure IFP in breast tumors was funded by the DOD (BCRP award 
W81XWH-08-1-0503). In that project we measured IFP using subharmonic pressure estimation, 
which requires the use of subharmonic signals from US contrast agents. These agents not only 
enhance the backscattered signals, but at higher acoustic pressures they also act as nonlinear 

8 
 
oscillators producing significant energy components in the received echo signals, which span the 
range of possible frequency emissions from subharmonics through ultraharmonics.25,40 Nonlinear 
bubble echoes can be separated from tissue echoes and used to create contrast specific imaging 
modalities, which allows blood flow in small vessels and capillaries (e.g., tumor angiogenesis) to 
be detected. One such modality is harmonic imaging (HI), which has been extensively 
studied.25,41 HI preferentially enhances and displays contrast signals rather than surrounding 
tissue echoes by transmitting at the fundamental transducer frequency (f0) and receiving at the 
second harmonic (2f0). HI is commercially available on most state-of-the-art US scanners. 
However, in practice HI suffers from reduced blood-to-tissue contrast due to second harmonic 
generation in tissue.39  
 
Alternatively, our group has been developing a novel contrast-specific imaging modality called 
subharmonic imaging (SHI), transmitting at double the resonance frequency (2f0) and receiving 
at the subharmonic (f0), because of the excellent suppression of tissue echoes (i.e., a much better 
signal-to-noise ratio) relative to other US contrast modes.42 Most importantly, as part of a DOD 
funded study (DAMD17-00-1-0464) conducted at Thomas Jefferson University (TJU), we have 
demonstrated, in a first-in-humans study of 14 women with 16 lesions (out of which 4 were 
cancers) that SHI can detect the slow, small volume blood flow associated with breast tumor 
angiogenesis demonstrating that subharmonic signals can be obtained in humans with clinical 
dosages of US contrast agents.43 This concept is now being explored further in a NCI funded 
clinical  trial of 3D SHI in 450 women with breast lesions (under R01 CA140338). 
 

9 
 
Microbubbles can also act as pressure sensors due to the difference in compressibility between 
the bubble and the surrounding medium.44 Our group has proposed, patented and investigated a 
novel and innovative technique called subharmonic-aided pressure estimation 
(SHAPE).45,46,47,48,49,50,51,52,53 We have demonstrated that the subharmonic signal component 
from contrast microbubbles is an excellent indicator of the hydrostatic pressure variations. 
Changes in the first, second, and subharmonic amplitudes of five different ultrasound contrast 
agents were measured in vitro at static hydrostatic pressures from 0 to 186 mmHg (i.e., the range 
of human blood pressures). Over the pressure range studied the first and second harmonic 
amplitudes reduced approximately 2 dB for all contrast agents. Over the same pressure range, the 
subharmonic amplitudes decreased by 9-14 dB and excellent linear regressions were achieved 
between the subharmonic amplitude and the hydrostatic pressure variations (r2≥0.98, 
p<0.001).45,46,48  
 
We implemented an initial, off-line version of the SHAPE algorithm on a commercial US 
scanner.49,50 SHAPE was realized on a phased array transducer (PA4-2) with a frequency range 
from 1.5-4.5 MHz and in vivo experiments were carried out in 4 canines with simultaneous 
measurements of pressure in the heart chambers performed with SHAPE and with a solid state 
pressure catheter (SPC-350; the reference standard). 
 
Using SHAPE for IFP estimation in LABC requires a high frequency linear. As an in vivo 
proof-of-concept, 5 Sinclair swine with naturally occurring melanomas,54 were studied. 
Subharmonic signals were acquired (in triplicate) in tumors and surrounding tissues with the US 
scanner using an L14-5 probe. Definity was infused at a rate of 6.25 ml/min. RF data was 

10 
 
acquired in pulse inversion grayscale mode at different transmit frequencies and acoustic power 
levels (6.7 and 10.0 MHz as well as -4, -8 and -12 dB).51 SHAPE results were compared to IFP 
measurements obtained with an invasive pressure monitor (Stryker, Berkshire, UK) as the 
reference standard. The difference between the subharmonics and the IFP in tumors and their 
surrounding tissues was most linear for 10 MHz transmission (r2≥0.67; p<0.05; Fig 6).51 The 
slope from 3 out of 4 animals was similar to each other (-0.19±0.07) and to the in vitro slope (-
0.22), while the last swine (i.e., swine 1) showed a large spread within the normal tissue IFP. 
These results provide proof-of-concept for the feasibility of using SHAPE as a noninvasive 
monitor of IFP.  
 
2. Hypothesis 
 
The main hypothesis is that subharmonic signals from US contrast microbubbles can be 
employed to image breast cancer neovascularity as well as noninvasively measure the IFP of 
breast cancer (i.e., using SHAPE) and that this information will improve the monitoring of tumor 
response to neoadjuvant therapies ultimately allowing for better, individualized treatment options 
to be selected. This in turn should result in improved clinical outcomes and greater survivability 
for women afflicted with breast cancer. 
 
 
3.  Specific Aims for the Human Clinical Trial. 
1)  To evaluate the ability of 3D SHI and SHAPE to track changes in LABC angiogenesis and 
IFP, respectively, by studying women undergoing neoadjuvant chemotherapy before as well 
as with around 10% and 60% of the neoadjuvant chemotherapy treatment delivered and after 

11 
 
completion of the neoadjuvant chemotherapy treatment and comparing results to MRI and 
pathology. 
2)  Finally, as a secondary aim, to compare the 3D SHI depiction of breast cancer angiogenesis 
in humans to CD31, an immunohistochemical predictor of angiogenesis. 
 
4.  Research Strategy   
Ultrasound Contrast Agent: We have selected the contrast agent Definity (which will be 
provided free of charge by Lantheus) for use in this project.  The route of administration and the 
dosages employed in the proposed clinical trial follow the recommendations issued by the 
manufacturer and employed by our group under an FDA issued IND (73,313).55 However, the 
FDA has yet to approve Definity for use in breast imaging and, as such this constitutes an off-
label use of the agent.  Consequently, we intend to apply to the FDA for an investigator initiated 
IND (investigational new drug) exemption to cover the use of Definity in patients with breast 
cancer undergoing neoadjuvant chemoptherapy.  
 
Definity is a sterile, non-pyrogenic suspension of liposome-encapsulated 
perfluoropropane microbubbles.25 The agent will be stored in a secure cabinet, with only the 
study investigators and research personnel having access. In vivo: The administration of the contrast 
will be done as an intravenous infusion of 2 vials of Definity/50ml saline through the subjects’ vascular 
port (established as part of their standard of care), with infusion rates of 4 to 10 ml/min (titrated to effect); 
as in our prior human trial.55 Next, we will run a power optimization algorithm to establish the 
individualized acoustic parameter settings for the case (on the first imaging study only).56  We will use 
those parameters for all subsequent imaging studies to sweep the area of the LABC to acquire 3D, pulse 
inversion SHI grayscale volume data. The digital clips and RF data obtained from each 3D SHI injection 

12 
 
will be transferred to a PC for off-line analysis. The SHI images will be qualitatively analyzed as 
described above. This protocol will be followed for all the contrast studies for each subject (except for the 
power optimization algorithm, which will only be run once for each patient). All subjects will be closely 
monitored by a physician during and following the study. 
 
Definity is well tolerated by patients. The largest safety studies published to date on the 
use of US 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 US applications.57,58,59 
However, as rare but serious cardiopulmonary reactions have been reported following the 
intravenous injection of Definity, all subjects will be closely monitored by a physician during the 
entire imaging period (as recommended by the FDA).60,61 
 
Definity Clinical Safety 
Definity is well tolerated and has been used extensively in echocardiography applications [Goldberg et al 
2001].  In pre-market clinical trials, Definity was administered to 1716 patients.  In these patients 269 
(8.4%) reported at least one adverse event.  Of these events, 26 were classified as serious including 19 
(1.1%) patients experiencing serious cardiopulmonary symptoms including eight deaths.  The deaths 
occurred several days after activated Definity administration and appear to be related to the course of 
underlying disease.  Of the 11 other serious adverse events, which appeared within days of the drug 
administration (2-15 days), all appeared to be a progression of underlying cardiac and non-cardiac 
disease.  However, a role for Definity in the initiation or course of these adverse events cannot be ruled 
out. 
 
Of the reported adverse reactions following the use of Definity the most frequently reported were 
headache (2.3%), back and renal pain (2.1%), flushing (1.1%), and nausea (1.0%).  Additional risks 

13 
 
associated with the contrast material are described in the attached Definity Product insert (Appendix B). 
All of the non-serious reported side-effects have been transient, usually lasting only a few minutes.   
 
 
Table 1. 
Selected Adverse Events Reported in ≥ 0.5% of the Subjects who Received Definity in Controlled 
Clinical Studies 
 
No. of Patients Exposed to Definity 
 
1716 
 
 
No. of Patients Reporting an Adverse Event 
269  
(8.8%) 
Central and peripheral nervous system 54  
(3.2%) 
 
Headache 
 
 
40  
(2.3%) 
 
Dizziness 
 
 
11  
(0.6%)  
Body as a Whole 
 
 
41 
(2.4%) 
 
Back/Renal Pain 
 
20  
(1.2%) 
 
Chest Pain 
 
 
13  
(0.8%) 
Digestive System 
 
 
31 
(1.8%) 
 
Nausea  
 
 
17 
(1.0%) 
Vascular (extracardiac) disorders 
19 
(1.1%) 
 
Flushing 
 
 
19 
(1.1%) 
Application Site Disorders 
 
11 
(0.6%) 
 
Injection Site Reactions  
11 
(0.6%) 
 
Additional information concerning pre-clinical and clinical experience with Definity, including 
the dosing levels and reported subject complaints, can be found in the Definity Package Insert that is 
included as Appendix B. 

14 
 
 
Patient Enrollment 
At TJU, Dr Avery (the medical oncologist on this project) and her colleagues treat more 
than 500 women with breast cancer each year out of which1-2 women/week will be treated with 
neoadjuvant chemotherapy. We expect to enroll between 20 and 50 subjects undergoing 
neoadjuvant chemotherapy (i.e., 10 – 25 women/year) in this first-in-humans project who will be 
studied with contrast US (SHI and SHAPE) before as well as with around 10 % and 60% of the 
neoadjuvant chemotherapy treatment delivered and after completion of the last chemotherapy 
treatment. Thus, a total of some 144 contrast studies will be performed in this project (range: 80 
– 200). The patients studied in this project will be women over the age of 21, who will receive an 
MRI as part of their standard of care (before and after completion of the chemotherapy). The 
MRI results together with pathology findings will be compared to the 3D SHI and SHAPE 
studies as a means for treatment monitoring. 
 
Given that these women have been diagnosed with breast cancer, they are usually very 
motivated to participate in research studies that include additional monitoring and, therefore, we 
do not expect recruitment to be an issue.  The patient population of this project will reflect the 
population demographics found at major American urban academic health centers.  The overall 
hospital demographics for TJU include 60 % Caucasian, 16 % African American, 13 % 
Hispanic, 5 % Asian, 1 % Other, and 5 % unknown patients.  Breast cancer in males accounts for 
only about 1% of cases at TJU and, thus, will not be included in the patient population.  
 
The inclusion criteria for this trial are as follows: 
• Females  
• Be diagnosed with T1 or greater LABC, any N and M0. 

15 
 
• Be scheduled for neoadjuvant chemotherapy 
• Be at least 21 years of age. 
• Be medically stable. 
• If a female of child-bearing potential, must have a negative pregnancy test. 
• Have signed Informed Consent to participate in the study. 
The exclusion criteria are:   
• Males 
• Females who are pregnant or nursing. 
• Patients with other primary cancers requiring systemic treatment. 
• Patients with any metastatic disease.   
• Patients undergoing neoadjuvant endocrine therapy. 
• Patients with known hypersensitivity or allergy to any component of Definity. 
• Patients with cardiac shunts or congenital heart defects. 
• Patients with unstable cardiopulmonary conditions or respiratory distress syndrome. 
• Patients with uncontrollable emphysema, pulmonary vasculitis, pulmonary 
hypertension or a history of pulmonary emboli. 
• Patients who have received any contrast medium (X-ray, MRI, CT or US) in the 24 
hours prior to the research US exam. 
 
US Examination Protocol.  
A baseline ultrasound grayscale (US) scan of the treated lesion will be obtained and the 
following criteria assessed: the size, shape, and orientation of the site; echogenicity compared to 
surrounding tissue. Standard PDI of the LABC will also be performed. The distribution of color 

16 
 
signals and the overall color content of mass will be evaluated by comparing the pattern and 
amount of color to the normal surrounding breast. If uniform, the color will be categorized as 
less intense, iso-intense or more intense than normal breast.  If non-uniform, the color will be 
described by location, intensity and distribution. Irregularity of the course of the vessels will be 
scored on a scale from 1.0 to 5.0 (smooth to severe irregularity) and anastomoses between 
adjacent vessels will be noted (as 1-2, 3-5 or >5 vessels connecting). The vascular pattern will 
also be graded as peripheral, radial from one or multiple sites, spotty or a combination. Digital 
clips of the two baseline imaging modes will be acquired.  
 
The contrast administration will be an intravenous infusion of 2 vials of Definity/50ml saline 
through the subjects’ vascular port (established as part of their standard of care), with infusion rates of 4 
to 10 ml/min (titrated to effect).55 Next, we will run a power optimization algorithm to establish the 
individualized acoustic parameter settings for the case (on the first imaging study only).56 We will use 
those parameters for all subsequent imaging studies to sweep the area of the LABC to acquire 3D, pulse 
inversion SHI grayscale volume data. The digital clips and RF data obtained from each 3D SHI injection 
will be transferred to a PC for off-line analysis. The SHI images will be qualitatively analyzed as 
described above. This protocol will be followed for all the contrast studies for each subject (except for the 
power optimization algorithm, which will only be run once for each patient). All subjects will be closely 
monitored by a physician during and following the study. 
 
Data Assessments.  
The pre- and then post-contrast US imaging will be evaluated for each patient at all 4 time points 
in one reading as follows: grayscale, grayscale and PDI (baseline), and grayscale and 3D SHI. 
While this may introduced some bias from pre- to post-contrast results, we considered this the 

17 
 
more realistic approach to how US contrast may be used in clinical practice.  Each case will be 
read independently by the 2 experienced co-investigators (more than 15 years of expertise in 
breast imaging) blinded to the contrast-enhanced MRI evaluations, to allow repeatability to be 
assessed. All US studies will be rated on a quasi-continuous scale from 0 to 100 ranging from 
“no change relative to pre-therapy” over “indeterminate” to “definite changes seen.” Using a 
quasi-continuous 100-point rating scale has an intuitive probabilistic interpretation and is known 
to improve the assessment of the capabilities of the imaging modes studied.62   
 
Moreover, the 3D SHAPE results (i.e., IFP estimates) will be compared to lesion size, 
tumor response, DFS and overall survival. Results will also be compared to MRI enhancement 
kinetics and imaging measurements of lesion size obtained before and after completion of the 
chemotherapy. Once surgery has been performed the following variables estrogen, progesterone 
and HER2 receptor presence or absences; presence or absence of hemorrhagic, necrotic, or other 
component of the lesion (and their location if present); histological margins; lesion size; TNM 
staging; histological lesion type; presence or absence of vascular invasion; histological and 
cytological grade; presence or absence of metastases; node staging; and percentage of the lesion 
that was invasive or in situ will be collected as part of the patient’s clinical care and correlated 
with the imaging results. Finally, the ability of 3D SHI to image angiogenesis (specifically 
fractional tumor vascularity) will be compared to specimens stained for an immunohistochemical 
predictor of angiogenesis, specifically a monoclonal antibody against the PECAM endothelial 
cell marker (anti-CD31; Dako Corporation, Carpinteria, CA). The PI has developed the 
necessary software for quantitative analysis of contrast enhanced US images and specimens 

18 
 
stained with CD31 using a histomorphometry system based on an SMZ-10A microscope (Nikon, 
Melville, NY) and ImagePro Plus software.28 
 
Statistical Analysis. Our statistical analysis will address the primary objective of this study 
by answering two major questions:   
1. Can in vivo 3D SHAPE monitor the effect of neoadjuvant chemotherapy on breast 
cancer IFP? 
2. Can in vivo 3D SHI monitor the effect of neoadjuvant chemotherapy (i.e., 
responders/non-responders) on breast cancer? 
 
The findings of SHI/SHAPE will be correlated to vascular morphology (i.e., tumor angiogenesis 
ascertained with CD31) and other pathological findings; including size, vascularity, presence or 
absence of receptors. For the pre and post-contrast enhanced comparisons, dichotomous 
parameters (e.g., responder/non-responder) and ranked data (less, iso, or more intense) will be 
analyzed with the McNemar test which measures significance of changes in related samples 
(pairwise comparisons) for each time point.  For variables in which accurate numerical 
measurement is possible (e.g., IFP and vascularity), the techniques will be compared using 
repeated measures logistical regression and ANOVA. 
 
To answer statistical question 2, the ability of the imaging tests to distinguish responders from 
non-responders will be compared using ROC analysis, while the incremental validity of 
combining imaging assessments will be analyzed using logistic regression and ROC 
analyses.62,63,64  The tests are fundamental grayscale US imaging, PDI, 3D SHI and contrast 

19 
 
enhanced MRI, while patient outcomes and pathology  will provide the reference standards.  A 
power analysis was performed using PASS 12 (NCSS, East Kaysville, UT) to estimate the 
number of cases required to produce clinically acceptable results.  A sample size of 20 
responders and 20 non-responders (i.e., approximately halfway between 20 and 50 subjects) 
achieve 80% power to detect a difference of 0.174 between a diagnostic test with an area under 
the ROC curve (Az) of 0.800 (the null hypothesis) and an actual Az of 0.974 using a one sample, 
two-sided z-test with a significance level of 0.05.  The ratio of the standard deviation of the 
responses in the two groups was assumed to be 1.00.  All analyses will be performed with Stata 
12.0 (Stata Corp., College Station, TX). 
 
 
5.  Medications 
Definity will be provided by Lantheus Medical Imaging, Billerica, MA.  An FDA Sponsor-Investigator 
IND will be obtained prior to beginning the trial. 
 
Definity is a sterile, non-pyrogenic suspension of liposome-encapsulated perfluoropropane 
microbubbles [Goldberg et al 2001; Miller & Nanda 2004].  The contrast agent is composed of a blend of 
three phospholipids contained in a matrix of sodium chloride, propylene glycol, and glycerin in water.  
The contrast agent is supplied in a vial that contains the phospholipids and perfluouropropane gas.  The 
microbubble agent is supplied in a standard-size 2 ml vial and is prepared by shaking the vial with the aid 
of a shaking device (Vialmix: ESPE, Seefeld, Germany).  Detailed resuspension instructions are provided 
in the Definity Product Insert, found in Appendix B. 
 
5.1  Administration 

20 
 
The contrast administration will be an intravenous infusion of 2 vials of Definity/50ml saline through the 
subjects’ vascular port (established as part of their standard of care), with infusion rates of 4 to 10 ml/min 
(titrated to effect).55 Next, we will run a power optimization algorithm to establish the individualized 
acoustic parameter settings for the case (on the first imaging study only).56 We will use those parameters 
for all subsequent imaging studies to sweep the area of the LABC to acquire 3D, pulse inversion SHI 
grayscale volume data. The digital clips and RF data obtained from each 3D SHI injection will be 
transferred to a PC for off-line analysis. The SHI images will be qualitatively analyzed as described 
above. This protocol will be followed for all the contrast studies for each subject (except for the power 
optimization algorithm, which will only be run once for each patient). All subjects will be closely 
monitored by a physician during and following the study. 
 
5.2  Contraindications 
Definity should not be administered to patients with known or suspected hypersensitivity to perflutren. 
The safety of Definity in patients with 1) right-to-left, bi-directional or transient right-to-left cardiac 
shunts; 2) severe emphysema, pulmonary vasculitis or a history of pulmonary emboli; 3) confirmed or 
suspected severe liver lesions; and 4) respiratory distress syndrome has not been studied.  Therefore, 
patients with any of these conditions will be excluded from the participation. 
 
5.3  Randomization 
This is a non-randomized trial; therefore, no randomization procedure is required. 
 
5.4  Blinding and Unblinding Methods 
This is an open-label trial; therefore, no blinding or unblinding procedures for the trial drug are required.  
 
5.5  Storage 

21 
 
Definity vials will be stored in a secure refrigerator, with only the study investigators and research 
personnel having access.  The study research coordinator will be responsible for drug suspension and 
inventory control. 
 
6.  Trial Procedures 
6.1 Patient Enrollment and Consent 
Subjects eligible for trial enrollment will be identified by the investigators from among TJU patient 
population who are who are undergoing neoadjuvant chemotherapy.  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.  
 
6.2  Screening Assessments 
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).  
 
6.3 Ultrasound Imaging 

22 
 
The US examinations will be performed by a qualified sonographer.  Procedures and equipment for this 
trial will be used in accordance with typical clinical procedures.  All trial procedures will be conducted in 
accordance with Good Clinical Practice.  For the ultrasound examination, the patient will be asked to lie 
in the supine position.  Acoustic coupling gel will be applied to the breast area of interest.  A baseline US 
grayscale scan will be used to identify the breast cancer seen by mammography (or another concomitant 
imaging mode such as US or MRI).  Standard PDI of the tumor will also be performed.  If the mass is too 
large to be completely imaged by the 4D10 L probe, the most vascular region seen on 2D PDI will be 
selected for the 3D SHI studies. 
 
To perform the 3D SHI imaging, US software has been modified by GE Medical Systems on the clinical 
system, the Logiq 9 scanner, to permit acquisition of subharmonic data.  All acoustic parameters have 
been verified and are all within the current standards of the machine, making the modified ultrasound 
system a non-significant risk device.  US imaging will be performed at a transmit frequency of 5.8 MHz 
and the subharmonic obtained at 2.9 MHz.  Using this setup, acoustic pressure amplitudes will all be 
below 1.5 MPa peak negative pressure, 2.5 MPa peak positive pressure (MI < 0.33). 
 
The contrast administration will be an intravenous infusion of 2 vials of Definity/50ml saline 
through the subjects’ vascular port (established as part of their standard of care), with infusion rates of 4 
to 10 ml/min (titrated to effect).55 Next, we will run a power optimization algorithm to establish the 
individualized acoustic parameter settings for the case (on the first imaging study only).56 We will use 
those parameters for all subsequent imaging studies to sweep the area of the LABC to acquire 3D, pulse 
inversion SHI grayscale volume data. The digital clips and radio frequency data obtained from each 3D 
SHI injection will be transferred to a PC for off-line analysis. The SHI images will be qualitatively 
analyzed as described above. This protocol will be followed for all the contrast studies for each subject 
(except for the power optimization algorithm, which will only be run once for each patient). All subjects 
will be closely monitored by a physician during and following the study. 

23 
 
 
6.4  Safety Monitoring 
Patients will be monitored for AEs during and immediately after contrast administration. All other 
procedures will be performed according to TJU standard of care. 
 
6.5 Safety Assessments 
Adverse events will be monitored during the entire procedure.  Specifically, the patient will be monitored 
with non-leading questions to monitor the patient for the transient side effects that are described below. 
 
6.6.1  Risks/Benefits Assessment.   
Serious cardiopulmonary and allergic reactions including fatalities have occurred during or following 
administration of Definity, causing the FDA to place a black box warning on the agent. However these 
occurrences have been rare (less than 1 in 5,000 patients). As a result, patients with cardiac shunts or 
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 rare. 
 
Clinically significant adverse effects from the administration of Definity are unlikely based on 
our previous experiences.55 The use of contrast with the new US imaging techniques is expected to 
provide significantly more information than from conventional US techniques.  This may lead to 
additional information about the characteristics of breast tumors which may be clinically relevant.   
 
To minimize and/or eliminate risks a nurse will be present during the entire procedure.  Adverse 
events will be monitored during the entire procedure.   
 

24 
 
6.6.2  Adverse Events 
An AE includes any condition that was not present prior to trial treatment, but appeared following 
initiation of trial medication; any condition that was present prior to trial treatment, but worsened during 
trial medication; or any condition, of which the subject has a history, that was not present prior to trial 
medication initiation but reappeared following administration of Definity.  This would include conditions 
that are likely to be associated with an underlying or intermittent disease (e.g., angina, flu, etc.). 
 
The subjects will be monitored for AEs during the entire procedure.  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 non-
serious AE data form, whether or not associated with the use of the trial medication.  All adverse events 
are reported to the Clinical Trial Support Office (CTSO) via the password protected Kimmel Cancer 
Center Adverse Event Reporting System. In addition all unexpected and serious adverse events (SAEs) 
are reported to the TJU IRB and to the Food and Drug Administration (FDA) if applicable.  The 
investigator is required to submit all unexpected and serious adverse events to the TJU IRB and the 
DSMB within 48 hours.  Fatal adverse events related to treatment which are unexpected must be reported 
within 24 hours to the TJU IRB and the DSMB through CTSO.  Fatalities not related to the study 
drug/device must be reported within 5 days.  
 
The AE forms will include: subject identification number and initials; subject’s date of birth, 
gender, and ethnicity; date of Definity administration; signs/symptoms and severity; date of onset; date of 
resolution or death; relationship to the study drug; action taken; concomitant medication(s) including 
dose, and route and duration of treatment.   
 
Whenever possible, the AE will be evaluated and reported as a diagnosis rather than individual 
signs and symptoms.  If a definitive diagnosis is not possible, the individual signs and symptoms will be 

25 
 
recorded.  The investigator will evaluate and note the duration, intensity, and relationship to (association 
with) the Definity administration, the action taken, and the determination of seriousness for each AE. 
 
INTENSITY OF AES 
The intensity of the AE will be characterized as mild, moderate, or severe. 
Mild AEs are usually transient, require no special treatment, and do not interfere with the subject's daily 
activities. 
Moderate AEs traditionally introduce a low level of inconvenience or concern to the subject and may 
interfere with daily activities but are usually ameliorated by simple therapeutic measures. 
Severe AEs interrupt a subject's usual daily activity and typically require systemic drag therapy or other 
treatment. 
 
When the intensity of the AE changes over time, the maximum intensity will be recorded. 
 
RELATIONSHIP TO DEFINITY ADMINISTRATION 
The relationship or association of the AE to the Definity administration will be characterized as 
"unlikely," "possible," or "probable."  A relationship assessment will be performed by the investigator to 
determine if an AE is attributable to Definity and will be recorded on a data form.  The investigator will 
refer to the Definity Package Insert for assistance in determining AE relationship. 
 
An "unlikely" relationship indicates that there is little or no chance that Definity caused the 
reported AE; other conditions, including concurrent illnesses, progression or expression of the disease 
state, or a reaction to a concurrent medication, appear to explain the reported AE. 
A "possible" relationship indicates that the association of the AE with Definity is unknown.  However, the 
AE is not reasonably attributed to any other condition. 

26 
 
A "probable" relationship indicates that a reasonable temporal association exists between the AE and 
Definity administration and, based upon the investigator's clinical experience, the association of the event 
with the trial medication seems likely. 
 
SERIOUS ADVERSE EVENTS 
A "serious" AE (SAE) is defined as a significant clinical hazard, contraindication, or precaution that: 
Results in death 
• 
Is life-threatening (In the opinion of the investigator, there is an immediate risk of death from the 
AE as it occurred. This does not include an AE that had it occurred in a more serious form may 
have caused death.) 
• 
Results in a persistent or significant temporary disability/incapacity defined as a substantial 
disruption of a person's ability to conduct normal life functions 
• 
Results in or prolongs an existing in-patient hospitalization (an overnight stay in the hospital, 
regardless of length) [Note: A hospitalization for an elective procedure or treatment which is not 
associated with an AE, hospitalization for a pre-existing condition which did not worsen, and 
hospitalization for reasons of convenience or observation, do not constitute an SAE.] 
• 
Is a congenital anomaly/birth defect (in offspring of a subject taking the trial medication, 
regardless of time to diagnosis) 
• 
Is an important medical event that may not result in death, be life-threatening, or require 
hospitalization but based upon the appropriate medical judgment, the event may jeopardize the 
subject and may require medical or surgical intervention to prevent one of the outcomes listed for 
the definition of a serious adverse experience. 
 
All unexpected and serious adverse events are reported to the TJU IRB and to FDA if applicable.  
The investigator is required to submit all unexpected and serious adverse events to the TJU IRB and the 

27 
 
data safety monitoring board within 48 hours.  Fatal adverse events related to treatment which are 
unexpected must be reported within 24 hours to the TJU IRB and the DSMB through CTSO.  
 
The written report for any SAEs that occur during the study, whether or not related to the Definity 
administration will be submitted immediately (within 24 hours) to the TJU Institutional Review Board. 
 
The designated medical monitor will review all serious and unexpected adverse events associated 
with the protocol and provide an unbiased written report of the event within 10 calendar days of the initial 
report.  At a minimum, the medical monitor will comment on the outcomes of the adverse event and 
relationship of the event to the Definity administration.   
 
A copy of the SAE will be retained on file with the respective subject's data forms.  
 
6.7  End-of-Treatment and End-of-Trial Evaluations 
6.7.1  Discontinuation of Subjects 
Subjects will be free to discontinue trial participation at any time.  The investigator will also discontinue 
any subject from the trial if, in the investigator's opinion, it is not safe for the subject to continue.  The 
date the subject is withdrawn from a treatment and/or from the trial and the reason for discontinuation 
will be recorded on the CRF. 
 
Trial participation will be considered completed if the subject has met all of the trial requirements 
that is to participate in 4 US contrast studies during the durantion of their clinical cancer treatment.   
 
If a subject's participation in the trial is interrupted for any reason (e.g., because of an AE or if the 
subject is lost to follow-up) 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 

28 
 
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 a discontinued subject. 
 
7  Data Management And Statistical Analyses 
7.1  Data Management 
Data forms will be completed for all subjects enrolled in the trial.  The patient study files will be stored in 
a secure file cabinet and maintained by the research study coordinator.  Patient study files will be kept for 
7 years after the completion of the study.   
 
The final data will be entered into a database.  The investigator will be responsible for 
management of the database.  The database will be maintained within an organized and secure directory 
system. 
 
7.2 Statistical Analyses 
7.2.1  Hypotheses: 
H1:  Our hypothesis is that subharmonic signals from US contrast microbubbles can be employed 
to image breast cancer neovascularity as well as noninvasively measure the IFP of breast cancer 
(i.e., using SHAPE) and that this information will improve the monitoring of tumor response to 
neoadjuvant therapies ultimately allowing for better, individualized treatment options to be 
selected. This in turn will result in improved clinical outcomes and greater survivability for 
women afflicted with breast cancer. 
 
 
7.2.2 Analysis of Results 
For the pre and post-contrast enhanced comparisons, dichotomous parameters (e.g., responder/non-
responder) and ranked data (less, iso, or more intense) will be analyzed with the McNemar test which 

29 
 
measures significance of changes in related samples (pairwise comparisons).  For variables in which 
accurate numerical measurement is possible (e.g., IFP and vascularity), the techniques will be compared 
using logistical regression and an ANOVA. 
 
The ability of the imaging tests to distinguish responders from non-responders will be compared 
using ROC analysis, while the incremental validity of imaging assessments will be analyzed using logistic 
regression and ROC analyses.62,63,64  The tests are fundamental grayscale US imaging, PDI, 3D SHI and 
contrast enhanced MRI , while patient outcomes and pathology  will provide the reference standards.  All 
studies will be rated on a quasi-continuous scale from 0 to 100, since this has an intuitive probabilistic 
interpretation and improves the assessment of the characterization capabilities of the imaging modalities 
compared to using a coarser 6-point scale.62  Logistical regression techniques will be used to combine the 
3 US imaging modes and MRI as well as to incorporate the quantitative parameters (i.e., SHAPE IFP 
estimates) into the US evaluation before repeating the ROC analysis. This will allow all possible 
combinations to be compared to one another (e.g., MRI versus all the US modes combined or MRI and 
grayscale US versus MRI and grayscale US and SHI, etc.).  Differences between ROC curves will be 
tested by computing Mann-Whitney statistics.  
 
All histopathological variables will be compared to imaging judgments of vascularity for the 
different modalities.  When both sets of variables are nominal, chi-square tests will be conducted.  When 
both types of variables are ordinal or continuous, correlations will be calculated.  When one type of 
variable is nominal and one continuous non-parametric rank order tests such as Mann-Whitney U-tests or 
Kruskal-Wallis tests will be performed.65  Inter- observer variability will also be determined by 
calculating the intraclass correlation coefficient.66,67  
 
Comparisons of subject outcomes (i.e., responders vs. non-responders) established by monitoring 
the treatment with the different imaging modalities will be conducted with Cox Survival analysis (against 

30 
 
the pathological reference standard).  For the US techniques where data from multiple time points are 
available, this analysis will be repeated for each time point.  All of the statistical analyses proposed for the 
human clinical trial will be repeated split by racial and ethnic groups to determine if clinically important 
race/ethnicity differences exist in the ability of 3D SHI/SHAPE to monitor breast cancer treatment. 
 
Finally, the vascularity measures obtained from the CD31 stained breast tumor specimens will be 
compared to the fractional tumor neovascularity data to determine if any correlation exists (in baseline 
PDI or contrast enhanced mode).  The existence of a linear relationship between US and pathologic data 
will be assessed using single variable linear regression techniques and reverse stepwise multiple linear 
regression analysis.   
 
All analyses and computations will be performed using Stata 12.0 (Stata Corporation, College 
Station, TX), while the study database will be designed and implemented in Filemaker Pro 10.0 
(Filemaker Inc, Santa Clara, CA).  This database will contain all patient information (except names and 
other identifiers) and the results of the various US imaging modes as well as the pathology results. 
 
 
 

31 
 
 
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