Clinical trial NCT03297112
Conditions: Adnexal Mass
Sponsor: Sidney Kimmel Cancer Center at Thomas Jefferson University
Start date: 2017-08-01; Completion date: 2018-09-04
Enrollment: 12 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This pilot clinical trial studies how well contrast-enhanced subharmonic ultrasound imaging works in improving the characterization of adnexal masses in patients undergoing surgery. Contrast-enhanced subharmonic ultrasound imaging uses high-frequency sound waves to produce images of internal organs and when combined with an ultrasound agent such as perflutren lipid microspheres, may help improve imaging and management of adnexal masses.

Detailed description:
PRIMARY OBJECTIVES:

I. To develop qualitative subharmonic imaging (SHI) or SHI-derived quantitative biomarkers.

II. To generate pilot data for a study to evaluate if they improve the characterization of benign and malignant adnexal masses compared to standard ultrasound or contrast enhanced magnetic resonance imaging (MRI) or the risk of malignancy index (RMI).

SECONDARY OBJECTIVES:

I. To compare the SHI depiction of adnexal masses' neovascularity in humans to intra-tumoral microvascular density (iMVD) obtained from CD31 an immunohistochemical marker of angiogenesis.

Arms: arm 1: Patients receive perflutren lipid microspheres IV. After 15 minutes, patients receive perflutren lipid microspheres IV again over 5 minutes and undergo contrast-enhanced subharmonic ultrasound imaging over 60 minutes.
Interventions: intervention 1: Perflutren Lipid Microspheres intervention 2: Contrast-Enhanced Subharmonic Ultrasound Imaging
Intervention descriptions: intervention 1: Given IV intervention 2: Undergo contrast-enhanced subharmonic ultrasound imaging

Protocol documents:
Prot_SAP_000.pdf: 
 
1
PROTOCOL 
 
TITLE: SUBHARMONIC CONTRAST ULTRASOUND FOR IMPROVED CHARACTERIZATION 
OF ADNEXAL MASSES – A Pilot Study 
 
VERSION & DATE: Version 1.5 – May 12, 2016 
 
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: 
Ji-Bin Liu, MD  
Andrej Lyshchik, MD, PhD  
John Eisenbrey, PhD 
Research Professor  
Assistant Professor  
 
Research Assistant Professor  
Department of Radiology 
Department of Radiology 
 
Dept of Radiology 
Thomas Jefferson Univ 
Thomas Jefferson Univ 
 
Thomas Jefferson Univ  
 
 
Norman Rosenblum, MD, PhD 
Professor 
Department of Obstetrics and Gynecology 
Thomas Jefferson Univ 
 
 
Medical Monitor: 
TBN 
 
Research Coordinator: TBN 
 
 
Sonographer:  Maria Stanczak, MS, RDMS 
 
 
LOCATION OF STUDY: 
Thomas Jefferson University 
 
132 South 10th Street, 7th Floor  
Philadelphia, PA 19107  
 
 
STUDY DURATION: 2 years 
 
 
 
 
 
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. 
 
 


 
2
Contents 
SYNOPSIS .................................................................................................................................................................... 3 
1. INTRODUCTION ..................................................................................................................................................... 6 
1.1 Background .......................................................................................................................................................... 6 
1.1.1  Definity Clinical Safety ............................................................................................................................... 9 
1.2  Rationale ........................................................................................................................................................... 10 
2.  TRIAL OBJECTIVES ............................................................................................................................................ 10 
3.  TRIAL DESIGN ..................................................................................................................................................... 10 
3.1  Trial Duration ................................................................................................................................................... 11 
4.  TRIAL POPULATION .......................................................................................................................................... 11 
4.1  Inclusion Criteria .............................................................................................................................................. 12 
4.2  Exclusion Criteria ............................................................................................................................................. 12 
5.  MEDICATIONS ..................................................................................................................................................... 13 
5.1  Contraindications .............................................................................................................................................. 14 
5.2  Randomization .................................................................................................................................................. 14 
5.3  Blinding and Unblinding Methods ................................................................................................................... 14 
5.4  Storage .............................................................................................................................................................. 15 
6.  TRIAL PROCEDURES ......................................................................................................................................... 15 
6.1  Screening Assessments ..................................................................................................................................... 15 
6.2  Treatment Administration ................................................................................................................................. 15 
6.3  Trial Assessments ............................................................................................................................................. 16 
6.3.1  Medical History ......................................................................................................................................... 16 
6.3.2 Ultrasound Imaging .................................................................................................................................... 16 
6.4  Efficacy Assessments ....................................................................................................................................... 18 
6.5  Safety Assessments ........................................................................................................................................... 19 
6.5.1  Risks/Benefits Assessment. ....................................................................................................................... 19 
6.6.2  Adverse Events .......................................................................................................................................... 21 
6.7  End-of-Treatment and End-of-Trial Evaluations .............................................................................................. 24 
6.7.1  Discontinuation of Subjects ....................................................................................................................... 24 
7.  DATA MANAGEMENT AND STATISTICAL ANALYSES .............................................................................. 25 
7.1  Data Management ............................................................................................................................................. 25 
7.2  Statistical Analyses ........................................................................................................................................... 25 
7.2.1  Hypotheses ................................................................................................................................................. 25 
7.2.2  Analysis of Results .................................................................................................................................... 26 
7.2.3  Sample Size Considerations....................................................................................................................... 26 
7.2.4 Disposition of Software .............................................................................................................................. 27 
8.  REFERENCES ....................................................................................................................................................... 28 
APPENDIX A - INVESTIGATOR OBLIGATIONS ................................................................................................. 32 
APPENDIX B – Definity PACKAGE Insert ............................................................................................................... 34 


 
3
 
SYNOPSIS 
 
Protocol Title: subharmonic contrast ultrasound for improved characterization of adnexal masses 
 
Trial Objectives: The primary objective of this trial is: 
 
 
To develop qualitative subharmonic imaging (SHI) or SHI-derived quantitative biomarkers to 
generate pilot data for a study to evaluate if they improve the characterization of benign and 
malignant adnexal masses compared to standard ultrasound or contrast enhanced magnetic 
resonance imaging (MRI) or the risk of malignancy index (RMI). 
 
and a secondary objective is: 
 
To compare the SHI depiction of adnexal masses’ neovascularity in humans to intra-tumoral 
microvascular density (iMVD) obtained from CD31 an immunohistochemical marker of 
angiogenesis. 
 
Trial Design: This is an open-label, non-randomized pilot study that will be conducted at one clinical site, 
Thomas Jefferson University (TJU).  Enrolled patients scheduled for subsequent surgery of an adnexal mass 
will receive a bolus IV injection followed 15 minutes later by a continuous infusion of the ultrasound 
contrast agent Definity® (Lantheus Medical Imaging, N Billerica, MA).  Ultrasound imaging will be 
performed using an Logiq E9 scanner with a C10-3v endovaginal (EV) transducer (GE Healthcare, 
Waukesha, WI) operating in SHI mode.  Results will be compared to MRI assessments and pathology 
obtained as part of the subjects’ standard of care. 
 
Trial Population:  This trial will consist of up to 45 adult women (21 years of age or older) at TJU, who 
have an adnexal mass and are scheduled for surgery based on their morphological characteristics (on 
standard imaging), CA125 levels and menopausal status. 
 
Trial Procedures:  Subjects eligible for trial enrollment will be identified by the co-investigator, Dr. 
Rosenblum, from his patient population of subjects scheduled for surgery of an adnexal mass.  The research 
coordinator for this study will explain the study to the patients.  After consenting to participate, a full 
demographic profile, known drug allergies or intolerances, and review of the subject’s medical/surgical 


 
4
history will be recorded.   
 
Prior to a subject undergoing surgery of an adnexal mass, a baseline grayscale scan of the lesion will be 
obtained, while power Doppler imaging (PDI) will be performed to select the area with the greatest 
vascularity.  Two contrast injections will be administered.  The first injection will involve pulse inversion 
grayscale EV SHI of the area of greatest vascularity within the adnexal mass (established by baseline PDI).  
There will be a 15 minute pause between injections to allow for complete contrast clearance.  For the second 
injection, Definity will be infused over 5 minutes and subharmonic destruction/reperfusion SHI imaging 
performed in multiple imaging planes covering the adnexal mass.   
 
The pre- and then post-contrast US imaging will be read independently by the 2 experienced co-
investigators blinded to the surgical results and contrast-enhanced MRI evaluations.  Then, sensitivity, 
specificity and accuracy will be calculated for all imaging modalities (baseline and SHI as well as MRI) 
and compared to the RMI used to select patients for surgery [Ferrazzi et al. 1997; 2005; Moolthiya & 
Yuenyao 2009].  Off-line SHI time intensity curves (TICs) will be created on a pixel by pixel basis and TIC 
parameters will be compared to the lesion characterization by the other imaging modalities with pathology 
as the reference standard.  Finally, the ability of EV SHI to image angiogenesis (specifically fractional 
tumor vascularity) will be compared to so called “hot spots” within surgical specimens stained for an 
immunohistochemical predictor of angiogenesis (the endothelial cell marker CD31).   
 
Statistical Methodology:  As this project involves a pilot study of a new US imaging mode (SHI) in a 
completely new application, no power analysis can be performed a priori.  Nonetheless, our statistical 
analysis will address the following two major questions: 
 
1. Can EV SHI biomarkers improve the characterization of benign and malignant adnexal masses 
compared to standard US or contrast enhanced MRI or other parameters? 
2. Do SHI ovarian neovascularity measurements correlate with iMVD as a marker of angiogenesis? 


 
5
As a pilot study this project is not powered to provide definitive answers to the questions presented above, 
but rather is a first step towards establishing feasibility and to provide data for a future more exhaustive 
study. 
 
All analyses and computations will be performed using NCSS/PASS 2008 and Stata 12.0 (Stata 
Corporation, College Station, TX), while the study database will be designed and implemented in REDCap 
(Vanderbilt University, Nashville, TN).  This database will contain all patient information (except names 
and other identifiers), including results of surgeries, MRI and SHI measurements as well as other clinical 
variables. 


 
6
1. INTRODUCTION 
 
In the United States there will be an estimated 22,240 new cases of ovarian cancer in 2013 leading to an 
estimated 14,030 deaths for a mortality rate of 63 % or 5 % of all female cancer deaths [Siegel et al. 2013].  
This makes ovarian cancer one of the ten most common types of cancer in women and an estimated 1 in 71 
women in the United States will develop ovarian cancer in their lifetime [Siegel et al. 2013].  World-wide 
ovarian cancer is the sixth most commonly diagnosed cancer accounting for 4 % of all female cancers.  
Women diagnosed with disease confined to the ovary (stage I), require less morbid surgical intervention, 
have a significantly improved quality of life, and most importantly have an overall 5-year survival 
approximating 90 % [Jinawath & Shih 2011].  Regrettably, almost 75 % of women are diagnosed with 
advanced disease, which greatly increases mortality (up to 85 %), which clearly indicates the inadequacy 
of a pelvic examination and conventional endovaginal (EV) ultrasound (US) imaging; as the first-line 
imaging test for evaluating adnexal masses [Fishman et al. 2005; Mohaghegh & Rockall 2012; Moyle et al. 
2010].  Hence, earlier and accurate characterization of adnexal masses is a clinical imperative in order to 
improve patient’s survival and imaging techniques play a critical role in fulfilling this goal [Mohaghegh & 
Rockall 2012; Moyle et al. 2010]. 
 
1.1 Background 
 
Among the early changes that differentiate normal from cancerous tissues is the process by which the tumor 
develops its blood supply, tumor angiogenesis [Folkman 1990; Li 2000].  In addition, angiogenic vascular 
morphology, often quantified as the intra-tumoral microvessel density (iMVD), is an independent predictor 
of malignant disease; including in the ovaries [Brawer et al. 1992; Labiche et al. 2009; Palmer et al. 2007; 
Weidner et al. 1991; Weind et al. 1998; Wang et al. 2011].  Angiogenesis is a prerequisite for tumor growth 
greater than 2 mm3 and is also a key factor of metastasis by allowing the ingrowth of vessels into a tumor 
providing a pathway for systemic dissemination via the blood or lymph system [Holmgren et al. 1995; 
O'Reilly et al. 1994].  Therefore, tumor angiogenesis is an important marker for imaging adnexal masses.  
The opportunity for noninvasive imaging of tumor angiogenesis is strengthened by the fact that vessels 


 
7
form a substantial portion of the mass of malignant tumors, contributing up to 10 % of the total volume of 
the tumor [Jain 1994].  Currently adnexal masses are imaged with standard EV US, with contrast enhanced 
MRI or computed tomography (CT) as well as, more rarely, with positron emission tomography (PET) 
[Bazot et al. 2008; Fleischer et al. 2012; Mohaghegh & Rockall 2012; Moyle et al. 2010].  While overall 
accuracies of 88 to 93 % have been reported for the latter modalities [Bazot et al. 2008; Mohaghegh & 
Rockall 2012], they are quite expensive and employ contrast agents which can produce marked adverse 
reactions, while CT also exposes patients to significant radiation.   
 
Ultrasound imaging has great potential in providing clinically relevant information related to angiogenesis 
by measuring ovarian tumor flow and vascular volume in real time and when combined with gas 
microbubbles to perform contrast-enhanced ultrasound (CEUS) results improve even further [Fleischer et 
al. 2010; 2012; Goldberg et al. 2001].  Moreover, microbubble-based ultrasound contrast agents not only 
enhance the backscattered signals, but at higher acoustic pressures they also act as nonlinear oscillators 
producing significant energy components in the received echo signals [Goldberg et al. 2001; Lauterborne 
1976].  These nonlinear bubble echoes can be separated from tissue echoes and used to create contrast 
specific ultrasound imaging modalities.  Several previous studies have addressed the use of CEUS for 
benign and malignant ovarian tumors [Fleischer et al. 2008; 2010; 2012; Orden et al. 2003; Marret et al. 
2004; Testa et al. 2005; Wang et al. 2011].  Results show that CEUS can characterize blood flow kinetics 
in ovarian tumors, but suffers from reduced blood-to-tissue contrast due to second harmonic generation in 
tissue, which will inevitably degrade the final outcomes [Hamilton & Blackstock 1998].  
 
Alternatively, our group has been developing a new contrast-specific imaging modality: 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., much better SNR) relative to other US contrast 
modes [Forsberg et al. 2000].  Feasibility studies of SHI have been conducted in vitro and in vivo by our 
group [Bhagavatheeshwaran et al. 2004; Dave & Forsberg 2009; Dave et al. 2010; 2011; 2012a; 2012b; 


 
8
2012c; 2012d; Eisenbrey et al. 2011a; 2011b; 2011c; 2012; Forsberg et al. 2000; 2006; 2007; Shi et al. 
1999; Sridharan et al. 2013; 2015] and by others [Chomas et al. 2002; Faez et al. 2011a; 2011b; Goertz et 
al. 2005; 2006; 2007; Helfield et al. 2012; Needles et al. 2008; Shankar et al. 1998].  We have demonstrated, 
in a first-in-humans study of 14 women with 16 breast lesions that SHI can detect the slow, small volume 
blood flow associated with tumor angiogenesis [Dave et al. 2010; Eisenbrey et al. 2011b; 2011c; Forsberg 
et al. 2007].  Moreover, unlike the gadolineum chelates used as MRI contrast agents, gas-filled contrast 
bubbles are pure intravascular tracers and flow through the body differently, which enables CEUS to depict 
tumor vascularity differently from MRI.  Moreover, SHI can be used for quantitative perfusion estimation 
(in ml/min/g), which has been a long sought after clinical goal and is potentially useful for the 
characterization of malignant vs. benign lesions [Forsberg et al. 2006; Sridharan et al. 2013].  We have 
created SHI parametric images of ultrasound contrast kinetic data acquired in our breast pilot study 
[Eisenbrey et al 2011b].  This innovative technique will be expanded to adnexal masses in this project.   
 
Based on our previous in vitro and in vivo studies and since it is one of only three ultrasound contrast agents, 
which are commercially available and approved by the FDA for use in humans (in echocardiography) in 
the United States, we have selected the contrast agent Definity (Lantheus Medical Imaging) 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 the FDA.  However, the FDA has yet to approve Definity 
for use in gynecological 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 adnexal masses. Definity is a sterile, non-pyrogenic suspension 
of liposome-encapsulated perfluoropropane microbubbles [Goldberg et al. 2001; Miller & Nanda 2004].  
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 [Dolan 2009; Main et al. 2008; 2009;.Wei et al. 2008]. 
 


 
9
1.1.1  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 (AE).  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 AEs, 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 AEs 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 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%) 
 


 
10
Additional information concerning pre-clinical and clinical experience with Definity, including the dosing 
levels and reported subject complaints, can be found in section 6.5 and the Definity Package Insert that is 
included as Appendix B. 
 
1.2  Rationale 
 
Our fundamental hypothesis is that subharmonic signals from ultrasound contrast microbubbles can be 
employed to improve the characterization of adnexal masses as benign or malignant (either through direct 
imaging or through quantitative SHI biomarkers).  Hence, we propose the development of a novel and 
innovative ultrasound based technology for the noninvasive evaluation of adnexal masses (i.e., contrast 
enhanced SHI) in order to improve the management of ovarian cancer. 
 
2.  TRIAL OBJECTIVES 
 
 
Trial Objectives: The primary objective of this trial is: 
 
 
To develop qualitative subharmonic imaging (SHI) or SHI-derived quantitative biomarkers and to 
generate pilot data for a study to evaluate if they improve the characterization of benign and 
malignant adnexal masses compared to standard ultrasound or contrast enhanced magnetic 
resonance imaging (MRI) or the risk of malignancy index (RMI). 
 
and a secondary objective is: 
 
To compare the SHI depiction of adnexal masses’ neovascularity in humans to intra-tumoral 
microvascular density (iMVD) obtained from CD31 an immunohistochemical marker of 
angiogenesis. 
 
 
3.  TRIAL DESIGN 
 
This is an open-label, non-randomized pilot study that will be conducted at one clinical site, Thomas 
Jefferson University (TJU).  Enrolled patients scheduled for subsequent surgery of an adnexal mass will 
receive a bolus IV injection followed 30 minutes later by a continuous infusion of the ultrasound contrast 
agent Definity.  Contrast ultrasound imaging will be performed using an EPIQ scanner with a broad 


 
11
bandwidth C10-3v EV transducer operating in SHI mode.  Results will be compared to MRI assessments 
and pathology obtained as part of the subjects’ standard of care. 
 
A full demographic profile, known drug allergies or intolerances, and review of the subject’s 
medical/surgical history will be recorded.  If the subject is of childbearing potential, she will have a urine 
pregnancy test (the results of which will be made available to the woman prior to study initiation). 
 
 
3.1  Trial Duration 
 
Individual participation in this trial will be limited to one ultrasound imaging study prior to their surgery 
for removal of an adnexal mass (as part of the standard of care at TJU).  The entire ultrasound imaging 
protocol will require approximately one hour.   
 
Subject recruitment is expected to last 18 months (February, 2016 – July, 2017).  Analysis and publication 
of results are expected to take an additional month (August, 2017).   
 
4.  TRIAL POPULATION 
 
Trial Population:  This trial will consist of up to 45 adult women (21 years of age or older) at TJU, who 
have an adnexal mass and are scheduled for surgery based on their morphological characteristics (on 
standard imaging), CA125 levels and menopausal status. 
 
Subjects eligible for trial enrollment will be identified by the co-investigator, Dr. Rosenblum, from his 
patient population of subjects scheduled for surgery of an adnexal mass.  The research coordinator for this 
study will explain the study to the patients.  The patient will be given time to consider the risks and benefits 
of the study and ask questions about participation.  The coordinator will review the consent form with the 
woman and then the patient will be given the form to review.  The patient, coordinator, and a study 
investigator will all sign the consent form.  The patient will be given a copy of the signed consent form for 


 
12
their records. 
 
4.1  Inclusion Criteria 
All subjects accepted for this trial must fulfill all the following criteria: 
 
Be diagnosed with an adnexal mass. 
 
Be scheduled for surgery to remove the adnexal mass. 
 
Be at least 21 years of age. 
 
Be clinically stable. 
 
If a female of child-bearing potential, must have a negative pregnancy test. 
 
Be conscious and able to comply with study procedures. 
 
Have read and signed the IRB-approved Informed Consent form for participating in the study. 
 
4.2  Exclusion Criteria 
 
Subjects who fulfill any of the following conditions or who have had the following procedures will 
be excluded from this trial:  
 
Females who are pregnant or nursing. 
 
Patients who have received an investigational drug in the 30 days before study drug 
administration, or will receive one within 72 h afterwards. 
 
Patients with known or suspected right-to-left, bi-directional, or transient right-to-left cardiac 
shunts. 
 
Patients with pulmonary hypertension or unstable cardiopulmonary conditions. 
 
Patients currently on chemotherapy or with other primary cancers requiring systemic or 
hepatic loco-regional treatment. 
 
Patients who are clinically unstable, patients who are seriously or terminally ill with a life 
expectancy of less than 1 month, and patients whose clinical course are unpredictable.  For 
example: 
 
Patients on life support or in a critical care unit. 
 
Patients with unstable occlusive disease (e.g., crescendo angina). 
 
Patients with clinically unstable cardiac arrhythmias, such as recurrent ventricular 
tachycardia. 
 
Patients with uncontrolled congestive heart failure (NYHA Class IV). 
 
Patients with recent cerebral hemorrhage. 
 
Patients who have undergone surgery within 24 hours prior to the study sonographic 
examination. 
 
Patients with a history of anaphylactic allergy to Definity, manifested by one or more of the 
following symptoms: generalized urticaria, difficulty in breathing, swelling of the mouth and 
throat, hypotension, or shock.   
 
Patients with congenital heart defects. 
 
Patients with severe emphysema, pulmonary vasculitis, or a history of pulmonary emboli. 
 
Patients with respiratory distress syndrome. 
 
Patients with thrombosis within the splenic vein. 
 
Subject identification will be maintained with a study specific alphanumeric code including the institution 
(TJU), patient number (001 and onwards) and the patient’s initials.   


 
13
 
5.  MEDICATIONS 
 
Definity will be provided by Lantheus Medical Imaging (N Billerica, MA).  An investigator initiated IND 
(investigational new drug) application will be submitted to the FDA to cover the use of Definity in patients 
with adnexal masses (since Definity is currently only approved for use in echocardiography).  Studies will 
not commence until regulatory approval has been obtained from the FDA as well as the IRB committee for 
TJU. 
 
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).  The vial contains a clear, colorless, sterile, non-
pyrogenic, hypertonic liquid, which upon activation with the aid of a Vialmix, provides a homogeneous, 
opaque, milky white injectable suspension of perflutren lipid microspheres.  After activating the contents 
of the vial in a Vialmix, each ml of the milky white suspension contains a maximum of 1.2 x 1010 perflutren 
lipid microspheres, and about 150 µl/ml (1.1 mg/ml) octafluoropropane.  The mean diameter range is 1.1 
µm to 3.3 µm with 98% microspheres having size less than 10 µm. The maximum microsphere diameter is 
20 µm.  Detailed resuspension instructions are provided in the Definity Product Insert, found in Appendix 
B. 
 
Definity is 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 [Dolan 2009; Main et al. 2008; 
2009;.Wei et al. 2008].  However, as rare but serious cardiopulmonary reactions have been reported 


 
14
following the intravenous injection of Definity, all subjects will be closely monitored by a physician during 
the entire imaging period [FDA Alert 2007; 2008]. 
 
Briefly, Definity will be administered by bolus IV injection through an 18 to 20 gauge angiocatheter placed 
in a peripheral arm vein, preferably an antecubital vein.  Subjects will be instructed not to move their arm 
during the administration of the contrast agent.  All subjects will receive an initial bolus injection at a steady 
rate not to exceed 1 ml/s with doses of 1.3 to 1.5 ml.  Each bolus injection of Definity will be followed with 
a flush of 10 ml of normal saline.  There will be a 15 minute pause between injections to allow for complete 
contrast clearance.  For the second injection, we will infuse up to 1.5 ml of Definity mixed in 25 ml of 
saline over 5 minutes (which is the approved dose and concentration for infusion of Definity). 
 
Definity will be stored in a secure cabinet, with only the study investigators and research personnel having 
access.  New vials must be prepared for each subject's contrast administration.  Unused drug and empty 
vials will be properly disposed of after reconciling in the log of study drug. 
 
5.1  Contraindications 
 
Definity should not be administered to patients with known or suspected right-to-left, bi-directional or 
transient right-to-left cardiac shunts, by intra-arterial injection, or to patients with known hypersensitivity 
to perflutren.  Patients with any of these characteristics will be excluded from the study.  Additional 
exclusion criteria are listed in section 4.2. 
 
5.2  Randomization 
 
This is a non-randomized trial; therefore, no randomization procedure is required. 
 
5.3  Blinding and Unblinding Methods 
 


 
15
This is an open-label trial; therefore, no blinding or unblinding procedures for the trial drug are required. 
 
5.4  Storage 
 
Definity vials will be stored in a locked refrigerator, with only the study investigators and research 
personnel having access.  The study researchers will be responsible for drug reconstitution and inventory 
control.  Activated Definity should be used within 5 minutes, but once activated the vials can be carried 
around to be used again for a period of 12 hours by reactivating with 10 seconds of hand agitation.  After a 
period of 12 hours from the initial activated time, unused activated vials will be discarded. 
 
6.  TRIAL PROCEDURES 
 
6.1  Screening Assessments 
All subjects will receive a written consent form and a verbal explanation of the study by an investigator 
and/or the research study coordinator and will be asked to sign the written informed consent.  Study 
participants will have the presence of inclusion criteria (section 4.1) and absence of exclusion criteria 
(section 4.2) verified by providing a comprehensive medical history (and a urine pregnancy test in the case 
of women of child bearing potential).  
 
6.2  Treatment Administration 
 
Administration of Definity will be performed under direct supervision of a medical doctor.  Emergency 
services (e.g., a crash cart) will be available within the hospital in case of any acute adverse reactions.  
Additionally, resuscitation equipment and trained CPR personnel will be in immediate proximity to the 
patient during Definity administration.   
 
Briefly, Definity microbubbles will be activated with a Vialmix device using a full 45-second activation 
cycle to ensure consistent microbubble size, as per manufacturer’s recommendation.  A needle will be 


 
16
inserted into an activated Definity vial to be used as an air vent.  An 18 to 20 gauge needle will be attached 
to 3 or 5 cc syringe and inserted into the vial to withdraw 1.3 to 1.5 ml of activated Definity.  All subjects 
will receive an initial bolus injection at a steady rate not to exceed 1 ml/s with doses of 1.3 to 1.5 ml.  Each 
bolus injection of Definity will be followed with a flush of 10 ml of normal saline.  Following a 15 minutes 
interval a dose of one vial of activated Definity, mixed in 25 ml of saline, will be prepared for each subject 
and infused through a peripheral vein at a rate sufficient to provide diagnostic intra-tumoral contrast 
visibility.  Infusions will be performed as described in the “Instructions for Use” provided by the 
manufacturer.  All materials and supplies used for the infusion procedure will be identical to those described 
by the manufacturer.  Up to 1.5 ml of activated Definity per vial will be extracted (as described above) and 
added to 25 ml of preservative-free saline.  The IV bag will then be gently squeezed to evenly distribute the 
microbubbles and an infusion will be initiated.  As per the manufacturer’s recommendation the infusion 
rate may vary between 4 ml/min to 10 ml/min and in this study the infusion rate will be maintained within 
this range but will be adjusted to provide diagnostic intra-tumoral contrast visibility. 
 
6.3  Trial Assessments 
 
6.3.1  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 woman is 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).  
Emergency services (e.g., a crash cart) will be available within the hospital in case of any acute adverse 
reactions.  Adverse events will be monitored during the entire procedure. 
 
6.3.2 Ultrasound Imaging 
 
The ultrasound 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.  A modified Logiq E9 scanner with a C10-3v EV transducer 


 
17
(GE Healthcare) operating in SHI mode will be used to obtain subharmonic radiofrequency (RF) data from 
the adnexal mass within the ovary and/or fallopian tube.  Both the experimental machine and software will 
be labeled as an investigational device with a GE provided label prior to beginning any clinical experiments 
(21 CRF 812.5).   
 
A baseline grayscale ultrasound scan of the 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 
mass will also be performed to select the area with the greatest vascularity.  The distribution of color signals 
and the overall color content of the mass will be evaluated by comparing the pattern and amount of color 
to normal tissue on the contra-lateral side.  If uniform, the color will be categorized as less intense, iso-
intense or more intense than normal tissues.  If non-uniform, the color will be described by location, 
intensity and distribution.  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, spotty or a combination.  Digital 
clips of the two baseline imaging modes will be acquired.  
 
Two contrast injections will be performed.  The first injection will involve pulse inversion grayscale EV 
SHI (in a dual display format utilizing at a transmit frequency of 5.8 MHz and the subharmonic obtained at 
2.9 MHz [Eisenbrey et al. 2011a; Sridharan et al. 2015]) of the area of greatest vascularity within the 
adnexal mass (as established by baseline PDI) followed by IV administration of 1.3 to 1.5 ml of Definity 
and acquisition of the contrast enhancement covering baseline, wash-in and peak enhancement to complete 
wash-out (typically 1-2 minutes).  There will be a 15 minute pause between injections to allow for complete 
contrast clearance.  For the second injection, we will infuse up to 1.5 ml of Definity mixed in 25 ml of 
saline over 5 minutes and then perform subharmonic destruction/reperfusion SHI imaging in multiple 
imaging planes covering the adnexal mass through the entire period of enhancement (in practice 3-4 minutes 
of equilibrium contrast concentration); similar to our previous work [Linden et al. 2007; Shi et al. 2001].  
The digital clip(s) obtained from the SHI injection will be transferred to a PC for off-line analysis.  The 
SHI images will be qualitatively analyzed as described above.  It should be noted, that all subjects will be 


 
18
closely monitored by a physician during the study [FDA Alert 2007; 2008].   
 
6.4  Efficacy Assessments 
 
The pre- and then post-contrast ultrasound imaging will be evaluated for each patient as follows: grayscale, 
grayscale and PDI (baseline), and grayscale and SHI.  While this may introduced some bias from pre- to 
post-contrast results, we considered this the more realistic approach to how CEUS may be used in clinical 
practice.  Each case will be read independently by the 2 experienced co-investigators (15+ years of expertise 
in CEUS imaging) blinded to the surgical results and contrast-enhanced MRI evaluations, to allow 
repeatability to be assessed.  All ultrasound studies will be rated on a quasi-continuous scale from 0 to 100 
ranging from “no lesion seen (no findings)” over “definitely benign,” “indeterminate” to “definitely 
malignant.”  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 [Wagner et al. 
2001].  The SHI clips will be processed to produce CMI-SHI images [Dave & Forsberg 2009; Dave et al 
2010], which will also be read by the blinded readers.  Finally, sensitivity, specificity and accuracy will be 
calculated for all imaging modalities (baseline and SHI as well as MRI) and compared to the RMI values 
used to select patients for surgery [Ferrazzi et al. 1997; 2005; Moolthiya & Yuenyao 2009].   
 
Off-line SHI TICs will be created on a pixel by pixel basis.  Parametric images will be generated showing 
time–to-peak, perfusion rate, peak intensity (calculated in dB as the ratio of the peak to the baseline values) 
and area under the curve (AUC) in the area of greatest vascularity as well as parameters across the mass 
(based on the data from second infusion) [Eisenbrey et al. 2011b].  The TIC parameters will be compared 
to the lesion characterization of the other imaging modalities with pathology as the reference standard.  It 
is our hypothesis that these new SHI based biomarkers will improve our capabilities to characterize ovarian 
cancers.  Results will also be compared to MRI enhancement kinetics and imaging assessments obtained as 
part of the subjects’ standard of care.   
 


 
19
Dr. Rosenblum will mark the transaxial plane of the lesion by sutures upon excision of the adnexal mass, 
so the specimen can be serially sectioned in the transaxial plane and immunohistochemical staining 
performed to determine the following variables: tumor type (benign or malignant); if malignant is the cancer 
invasive or a tumor of low metastatic potential; histological lesion type (epithelial, stromal or germ cell), 
RMI as well as staging and other markers will be collected as part of the patient’s clinical care and correlated 
with the imaging results.  Finally, the ability of EV SHI to image angiogenesis (specifically fractional tumor 
vascularity) will be compared to so called “hot spots” within surgical specimens stained for an 
immunohistochemical predictor of angiogenesis, specifically a monoclonal antibody against the CD31 
endothelial cell marker (Dako Corporation, Carpinteria, CA).  The principal investigator of this project has, 
as part of previously NIH and DOD funded projects, developed the necessary software for quantitative 
analysis of CEUS images and specimens stained with CD31 using a histomorphometry system based on an 
Labophot-2 microscope (Nikon, Melville, NY) and ImagePro Plus software [Forsberg et al. 2008]. 
 
6.5  Safety Assessments 
Emergency services (e.g., a crash cart) will be available within the hospital in case of any acute adverse 
reactions.  AEs will be monitored during the entire procedure.  Specifically, the patient will be monitored 
with non-leading questions to monitor the patient for the occurrence of transient side effects that are 
described below. 
 
6.5.1  Risks/Benefits Assessment. 
 
The known risks from the administration of Definity are minimal.  Definity is well tolerated and has been 
used extensively in echocardiography applications.  A total of 1716 subjects were evaluated in pre-market 
clinical trials of Definity.  Of these patients 144 (8.4%) reported at least one treatment-related adverse 
reaction.  The most common AEs were reported in the Central and peripheral nervous system (3.1%), Body 
as a Whole (2.4%) and Gastrointestinal system (1.8%).  The most common events were headache (2.3%), 
back and renal pain (1.2%), flushing (1.1%) and nausea (1.0%).  All of the non-serious reported side-effects 
have been transient, usually lasting only a few minutes.  


 
20
 
There were 26 serious AEs and 15 (0.9%) subjects discontinued because of an AE.  Nineteen (1.1%) 
suffered serious cardiopulmonary adverse events including eight deaths. The deaths occurred several days 
after Definity administration and appear to be related to the course of underlying disease.  Of the 11 other 
serious AEs, 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 AEs cannot be ruled out. As a result of post market analysis, the FDA added a black box 
warning to Definity in 2008 for patients with unstable cardiac or respiratory conditions.  In one of the largest 
retrospective multicenter study evaluating the effect of Definity administration in 66,164 cases, no deaths 
were reported attributable to the drug use [Wei et al. 2008].  The rate of severe allergic reactions for either 
cardiac or abdominal ultrasound imaging following Definity was 0.0006% [Wei et al. 2008]. It was 
concluded that the severe AE rate following the use of ultrasound contrast agents is no greater and may be 
lower, than the reported event rate for other contrast agents used in other cardiac imaging studies [Wei et 
al. 2008].  
 
A prospective, open-label, nonrandomized, multicenter phase 4 surveillance registry was conducted at 15 
clinical sites in the USA to assess the risk for adverse cardiopulmonary events during or within a 30 minute 
period after administering Definity to patients between February 2008 and April 2009 [Weiss et al. 2012].  
In this study, 1053 patients were administered Definity and no deaths, serious AEs and other significant 
AEs were reported [Weiss et al. 2012].  The overall drug related adverse event rate was only 3.5% and 
within these cases, 96.5% of the AEs were of mild or moderate intensity [Weiss et al. 2012].  Based on 
study by Weiss et al [2012], the FDA concluded that deaths or serious AEs are “unlikely to occur at a rate 
of more than 0.3% when Definity is used according to recommendations” [FDA 2011]. 
 


 
21
The use of an intravenous needle and the fluids given through the needle may cause minor discomfort, 
bleeding under the skin (bruise), and possible infection at the site of needle insertion. 
 
Clinically significant adverse effects from the administration of Definity are unlikely.  The use of contrast 
with the new ultrasound imaging techniques is expected to provide significantly more information than 
conventional ultrasound techniques.  This may lead to a non-invasive method for characterization of 
adnexal masses.  
 
To minimize and/or eliminate risks a nurse will be present during the entire procedure.  Emergency services 
(e.g., a crash cart) will be available within the hospital in case of any acute adverse reactions. Adverse 
events will be monitored during the entire procedure.   
 
The risk benefit ratio is low.  Based on the available nonclinical and clinical safety data and the anticipated 
dose levels of Definity that will be used in this study, safety concerns are minimal.  The potential side 
effects related to Definity administration are described above and listed in greater detail in the investigator’s 
brochure. 
 
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 


 
22
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. GE Healthcare will be 
informed of all AEs. In addition all unexpected and serious AEs are reported to the TJU IRB and to the 
FDA.  The investigator is required to submit all unexpected and serious AEs to the TJU IRB within 48 
hours, and to GE Healthcare. Fatal AEs related to treatment which are unexpected must be reported within 
24 hours to the TJU IRB and to GE Healthcare. Fatalities not related to the study drug/device must be 
reported within 5 days.  The PI of the study has previous experience running ultrasound clinical trials and 
will serve as the study sponsor. He will be responsible for ensuring all FDA requirements are met and all 
AE are properly reported. 
 
The AE forms will include: subject identification number and initials; subject’s date of birth, gender, and 
ethnicity; name of the institution, 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 recorded.  
The investigator(s) 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 drug therapy or other 


 
23
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 investigator brochure 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. 
 
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 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 


 
24
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 serious AE.] 
 
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. 
 
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.  The medical monitor will also indicate whether he concurs with 
the details of the report provided by the principal investigator. 
 
A copy of the serious AE 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 following trial requirements: 
 
 
Has received one bolus injection and one infusion of Definity 
 
Has undergone the complete ultrasound imaging study as described in this protocol 
 
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 for any reason 


 
25
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 coordinators at TJU.  Patient study files will be 
kept for 7 years after the completion of the study.  The PI of the study has previous experience running 
ultrasound clinical trials and will serve as the study sponsor.  He will be responsible for ensuring all FDA 
requirements are met and all AE are properly reported. 
 
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:  EV SHI will significantly differentiate malignant from benign adnexal masses using surgical pathology 
as the reference standard.  As an initial end-point we speculate that an area under the ROC curve of 0.75 or 
better will be achieved by EV SHI. 
H2:  EV SHI biomarkers will improve the characterization of benign and malignant adnexal masses 
compared to standard ultrasound or contrast enhanced MRI or other clinical parameters.  As an initial end-
point we speculate that an area under the ROC curve of 0.75 or better will be achieved by EV SHI, which 
will be better than low-end estimates of the accuracy of standard ultrasound, contrast enhanced MRI and 
other clinical parameters (which is on the order of 0.70 to 0.72). 


 
26
H3:  A correlation coefficient of 0.70 will be achieved between in vivo SHI ovarian neovascularity 
measurements and iMVD. 
 
7.2.2  Analysis of Results 
The findings of SHI will be correlated to vascular morphology (i.e., tumor angiogenesis ascertained with 
CD31) and other imaging and pathological findings; including size, vascularity, presence or absence of 
receptors and RMI values.  The techniques will be compared using an analysis of variance (ANOVA) with 
method (i.e., baseline ultrasound imaging, SHI, or MRI) as the dependent variable and outcome as the 
independent variables.  For the pre and post-contrast enhanced comparisons, dichotomous parameters (e.g., 
benign/malignant) and ranked data (less, iso, or more intense) will be analyzed with the McNemar test.  The 
ability of the imaging tests to distinguish benign from malignant masses will be compared using ROC 
analysis, while the incremental validity of imaging diagnosis and morphological characteristics as well as 
serum biomarkers (i.e., RMI) will be analyzed using logistic regression and ROC analyses [DeLong et al. 
1988; Metz 1986; Wagner et al. 2001].  Differences between ROC curves will be tested by computing 
Mann-Whitney statistics.   All of the statistical analyses proposed for this 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 SHI to characterize adnexal masses (although the limited sample size of this pilot study 
makes it unlikely that such differences – if any exist - will be detectable).   
 
All analyses and computations will be performed using NCSS/PASS 2008 and Stata 12.0 (Stata 
Corporation, College Station, TX), while the study database will be designed and implemented in REDCap 
(Vanderbilt University, Nashville, TN).  This database will contain all patient information (except names 
and other identifiers), including pathology, MRI and SHI measurements as well as other clinical variables. 
 
7.2.3  Sample Size Considerations 
 
As this project involves a pilot study of a new ultrasound imaging mode (i.e., SHI) in a completely new 
application, no power analysis can be performed a priori.  As a pilot study this project is not powered to 


 
27
provide definitive answers to the hypotheses presented above (cf., section 7.2.1), but rather is a first step 
towards establishing feasibility and to provide data for a future more exhaustive study.  The planned sample 
size of 45 subjects will provide important information on the precision of the anticipated correlation 
coefficient of 0.70 between in vivo SHI ovarian neovascularity measurements and iMVD such that the 95% 
confidence interval would be (0.51, 0.82). 
 
The Gynecological Oncology Division at TJU evaluate more than 500 women with adnexal masses each 
year out of which around 1 woman/week will require surgical intervention based on their morphological 
characteristics (on standard imaging), CA125 levels and menopausal status [Ferrazzi et al. 1997; 2005; 
Moolthiya & Yuenyao 2009].  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.  We anticipate enrolling approximately 45 subjects scheduled for surgery in this project 
(with some 15-20 % expected to be cancers), which means we are aiming for a recruitment rate of 
approximately 58% (45/78). 
 
7.2.4 Disposition of Software 
 
Investigational software containing the modified SHI mode will be removed from the EPIQ scanner upon 
completion of the study to avoid the investigational device being used for any unapproved investigations. 
At the conclusion of the study, the developed software will be saved on CD for future experimentation. 
These investigations may ultimately further the development of SHI technology as a cancer imaging system. 
However, under no circumstances with the software be used in a clinical environment (i.e., for human 
studies) without GE consent. This will be ensured by the use of a GE controlled password for installation 
of experimental software which is currently in use and will expire at the conclusion of the study. 
 
 


 
28
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APPENDIX A - INVESTIGATOR OBLIGATIONS 
 
 
A. 
Institutional Review Board (IRB) and Human Subjects Research Review Board (HSRRB) 
Review/Approval 
 
The protocol and informed consent for this study must be reviewed and approved by an appropriate IRB 
and HSRRB prior to enrollment of participants in the study.  It is the responsibility of the investigator to 
assure that all aspects of the ethical review are conducted in accordance with FDA Regulations 21 CFR 
Part 56.  A letter documenting the IRB and HSRRB approval which specifically identifies the 
study/protocol must be obtained by the investigator prior to initiation of the study.  Amendments to the 
protocol will be subject to the same requirements as the original protocol.  The HSRRB must review and 
approve each modification to the study prior to implementation. 
 
A progress report with a request for re-evaluation and re-approval will be submitted by the investigator to 
the IRB and HSRRB at intervals required by the IRB, and not less than annually.  
 
After completion or termination of the study, the investigator will submit a final report to the IRB. This 
report should include: deviations from the protocol, the number and types of participants evaluated, the 
number of participants who discontinued (with reasons), results of the study, if known, and all AEs, 
including deaths. 
 
 
B. 
Informed Consent 
 
Signed, written informed consent which conforms to FDA Regulation 21 CFR Part 50, must be obtained 
from each participant prior to entering the study.  Each participant will be provided a written consent form 
and verbal information in an understandable manner which describes the nature and duration of the study.  
The research study coordinator or the investigator will conduct the informed consent interview in a private 
examination room.  The potential subject will be allowed to discuss the study with the investigator, research 
study coordinator, or any persons who may have accompanied the potential subject.  Additionally, the 
participant must be allowed adequate time to consider the potential risks and benefits associated with his 
participation in the study.  The research study coordinator will sign the informed consent as the person 
conducting the consent interview.   
 
 
C. 
Data Reporting and Data Forms  
 
Data reflecting participant's experiences with the study will be recorded on CRFs by the investigator.  
 
 
D. 
Records Retention 
 
All records pertaining to the conduct of the clinical study, including CRFs, informed consent forms, source 
documents, and other study documentation must be retained for seven (7) years after the end of the study. 
 
Other study documentation includes all protocols and amendments, drug supply receipt, dispensing and 
final disposition records, IRB correspondence and approvals, signed consent forms, a blank copy of study 
consent forms, Form 1572, curriculum vitae or biosketches of members of the research team including the 
medical monitor, HSRRB correspondence and approval, and Statement of Investigator forms. 
 


 
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Source documents include all original records of observations, results, and activities necessary to 
reconstruct and evaluate the study.  Source documents include but are not limited to laboratory reports, 
electrocardiogram tracings, X-ray films, ultrasound images, subject diaries, subject progress notes, hospital 
charts, appointment books, radiologic reports or pharmacy records, and any other records or reports of 
procedures performed during the study.  Source documents also may include copies of the CRF or sponsor 
supplied worksheets when original information is recorded directly onto these forms. 
 
Whenever possible, an original recording of an observation should be retained as the source document.  
However, a photocopy of a record is acceptable provided it is legible and is a verified copy of the original 
document. 
 
 
E. 
Deviation from the Protocol 
 
The investigator will not deviate from the protocol without prior written approval from the IRB and the 
HSRRB.  In medical emergencies, the investigator will use medical judgment and remove the participant 
from immediate hazard.  The HSRRB and the IRB will be notified regarding the type of emergency and 
course of action taken.  Any other changes to or deviations from the protocol will be made as an amendment 
to the protocol.  The amendment must be submitted for review and approval to the local IRB and the 
HSRRB for review and approval. 
 
 
F. 
Roles and Responsibilities of Study Personnel 
 
Flemming Forsberg, Ph.D., Professor of Radiology and Director of Ultrasound Physics, will serve as 
Principal Investigator on this project.  He will be responsible for the scientific goals of the project.  Dr. 
Forsberg will oversee patient recruitment, informed consent, ultrasound studies, and the data entry and 
statistical analyses.  He will also supervise the SHI data acquisition from patients.  Dr. Forsberg will also 
prepare any manuscript(s) resulting from this grant.   
 
Ji-Bin Liu, MD, Research Professor of Radiology will assist with the animal studies and interpret 
ultrasound images from animals as well as humans. 
 
Andrej Lyshchik, MD, PhD, Assistant Professor of Radiology will interpret ultrasound images from 
animals as well as humans and advise on clinical issues. 
 
Norman Rosenblum, MD, .PhD, Professor of Obstetrics and Gynecology will assist with the patient 
recruitment, interpret surgical findings and advise on clinical issues. 
 
John Eisenbrey, PhD, Research Assistant Professor of Radiology will assist with the animal studies, aid in 
patient recruitment, informed consent, ultrasound studies, and the data entry and statistical analyses. 
 
 
 
Signature of PI:  
 
 
 
 
 
 
 
 
Flemming Forsberg, PhD 
 
 
 
 


 
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APPENDIX B – DEFINITY PACKAGE INSERT