Conditions: Cervical Carcinoma Malignant Female Reproductive System Neoplasm Vaginal Carcinoma Vulvar Carcinoma
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 9
Healthy volunteers: no

Brief summary:
This clinical trial compares the use of ultrasound for the detection of sentinel lymph nodes (SLNs) to the standard of care (which varies depending on cancer treatment). The ultrasound contrast agent is called Sonazoid and it consists of tiny gas-filled bubbles about the side of red blood cells. Diagnostic procedures, such as ultrasound with Sonazoid, may help identify more SLNs than standard of care in patients with cervical, vaginal, or vulvar cancer.

Detailed description:
PRIMARY OBJECTIVES:

I. To determine the concordance between lymphosonography and the standard of care in the identification sentinel lymph nodes (SLNs) in patients with cervical, vaginal or vulvar cancer.

II. To determine if lymphosonography can identify more SLNs with metastatic deposits in patients with cervical, vaginal or vulvar cancer when compared to the standard of care.

OUTLINE:

Patients receive perflubutane microbubble (Sonazoid) injections subdermally and then undergo ultrasound over 30 minutes before standard of care cancer treatment.

After completion of study intervention, patients are followed up for 30 days.

Arms:
arm 1: Patients receive perflubutane microbubble injections subdermally and then undergo ultrasound over 30 minutes before standard of care cancer treatment.

Interventions: intervention 1: Perflubutane Microbubble intervention 2: Contrast-Enhanced Ultrasound
Intervention descriptions: intervention 1: Given subdermally intervention 2: Undergo ultrasound

Protocol document:
Prot_SAP_000.pdf: 
Genital Cancer SLN 
 
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THOMAS JEFFERSON UNIVERSITY 
Sidney Kimmel Cancer Center 
 
Detection of Sentinel Lymph Nodes in Female Lower Genital Tract Cancer Patients with 
Contrast-Enhanced Ultrasound Imaging 
 
Principal Investigator: 
Ji-Bin Liu, MD 
Department of Radiology 
Thomas Jefferson University  
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
215-955-4862 
Co-Investigator(s): 
 
Andrej Lyshchik, MD, PhD 
Department of Radiology 
Thomas Jefferson University  
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107  
215-503-0587 
Norman G. Rosenblum, MD, PhD 
Department of Obstetrics and Gynecology 
Thomas Jefferson University  
833 Chestnut Street 
Philadelphia, PA 19107 
215-955-6200 
Scott D. Richard, MD 
Department of Obstetrics and Gynecology 
Thomas Jefferson University  
833 Chestnut Street 
Philadelphia, PA 19107 
215-955-5000 
 
Flemming Forsberg, PhD 
Department of Radiology 
Thomas Jefferson University 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
215-955-4870 
 
 
 
 
 
 


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Priscilla Machado, MD 
Department of Radiology 
Thomas Jefferson University 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
215-955-4279 
 
Aaron Shafer, MD 
Department of Obstetrics and Gynecology 
Thomas Jefferson University 
833 Chestnut Street 
Philadelphia, PA 19107 
215-955-5000 
 
Lindsey E. Minion, MD 
Department of Obstetrics and Gynecology 
Thomas Jefferson University  
833 Chestnut Street 
Philadelphia, PA 19107 
215 955-5000 
Statistician: 
Tingting Zhan, PhD 
Biostatistics 
1015 Chestnut Street 
Suite 520 
Philadelphia, PA 19107 
215-955-3788 
Funding Sponsor: 
National Institute of Health (NIH): R21 CA249870. 
9000 Rockville Pike  
Bethesda, Maryland 20892 
301-945-7573 
IND/IDE Holder: 
Flemming Forsberg, PhD 
Department of Radiology 
Thomas Jefferson University  
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
215-955-4870 
IND/IDE Number: 
Insert IND or IDE number, if applicable 
127,419 (amendment pending) 
Study Product: 
Sonazoid™ (perfluorobutane) Powder for Injection. 
Protocol IDs: 
Jeff Trial # 16886 (if applicable) 
PRC # pending 
IRB Control #21F.624 
 
Version Number: 
Version Date: 
Version 1.0 
12/17/20 
Version 2.0 
02/23/2021 
Version 2.1 
06/21/2021 


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Version 3.0 
3/29/2023 
Version 4.0 
11/29/2023 
 
CONFIDENTIAL 
This document is confidential and the property of THOMAS JEFFERSON UNIVERSITY. 
No part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 
 
 
 
 
 
 
Signature Page .......................................................................................................................... 8 
Statement of Compliance ........................................................................................................... 8 
List of Abbreviations ................................................................................................................... 9 
Study Summary ........................................................................................................................11 
1 
Introduction .........................................................................................................................13 
1.1 
Background Information ..............................................................................................13 
1.2 
Rationale for the Proposed Study ...............................................................................16 
1.3 
Potential Risks and Benefits .......................................................................................17 
1.3.1 Potential Risks.........................................................................................................17 
1.3.2 Benefits ...................................................................................................................17 
1.3.3 Risk-Benefit Ratio ....................................................................................................18 
2 
Study Objectives .................................................................................................................18 
2.1 
Objectives ...................................................................................................................18 
2.1.1 Hypothesis ..............................................................................................................18 
2.1.2 Primary Objectives ..................................................................................................18 
2.2 
Endpoints/Outcome Measures ....................................................................................18 
2.2.1 Primary Endpoints ...................................................................................................19 
2.2.2 Secondary Endpoints ..............................................................................................19 


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3 
Study Design ......................................................................................................................19 
4 
Study Enrollment and Withdrawal .......................................................................................19 
4.1 
Eligibility Criteria .........................................................................................................20 
4.1.1 Inclusion Criteria ......................................................................................................20 
4.1.2 Exclusion Criteria ....................................................................................................20 
4.2 
Strategies for Recruitment and Retention ...................................................................21 
4.3 
Participant Withdrawal ................................................................................................21 
4.3.1 Reasons for Withdrawal ..........................................................................................21 
4.3.2 Handling of Participant Withdrawals and Participant Discontinuation of Study 
Intervention .........................................................................................................................22 
4.4 
Premature Termination or Suspension of Study ..........................................................22 
5 
Study Intervention ...............................................................................................................22 
5.1 
Study Product .............................................................................................................22 
5.2 
Study Product Description ...........................................................................................22 
5.2.1 Acquisition ...............................................................................................................23 
5.2.2 Formulation, Packaging, and Labeling .....................................................................23 
5.2.3 Product Storage and Stability ..................................................................................23 
5.3 
Dosage, Preparation, and Administration ....................................................................23 
5.4 
Dose Modifications and Dosing Delays .......................................................................24 
5.5 
Study Product Accountability ......................................................................................24 
5.6 
Dietary Restrictions .....................................................................................................24 
5.7 
Study Procedural Intervention(s) Description ..............................................................24 
5.8 
Administration of Procedural Intervention ....................................................................25 
5.9 
Administration of Procedural Intervention ....................................................................25 
5.10 
Procedures for Training of Clinicians on Procedural Intervention ................................26 
6 
Study Schedule ..................................................................................................................26 
6.1 
Screening ...................................................................................................................26 


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6.2 
On Study Period .........................................................................................................26 
6.3 
End of Treatment Study Procedures ...........................................................................26 
6.4 
Withdrawal Visit/Discontinuation of Therapy ...............................................................26 
7 
Study Procedures and Evaluations .....................................................................................27 
7.1 
Study Procedures/Evaluations ....................................................................................27 
8 
Evaluation of Safety ............................................................................................................27 
8.1 
Specification of Safety Parameters .............................................................................27 
8.1.1 Unanticipated Problems ..........................................................................................27 
8.1.2 Adverse Events .......................................................................................................27 
8.1.3 Serious Adverse Events ..........................................................................................28 
8.2 
Safety Assessment and Follow-Up .............................................................................29 
8.3 
Recording Adverse Events ..........................................................................................29 
8.3.1 Relationship to Study Intervention ...........................................................................30 
8.3.2 Expectedness ..........................................................................................................30 
8.3.3 Severity of Event .....................................................................................................30 
8.3.4 Intervention .............................................................................................................30 
8.4 
Safety Reporting .........................................................................................................30 
8.4.1 Reporting to IRB ......................................................................................................31 
8.4.2 Reporting to SKCC DSMC ......................................................................................32 
8.4.1 Reporting to Funding Sponsor .................................................................................32 
8.4.1 Reporting to FDA .....................................................................................................32 
8.4.1 Reporting of Pregnancy ...........................................................................................32 
8.5 
Halting Rules ..............................................................................................................33 
9 
Study Oversight ..................................................................................................................33 
10 
Clinical Site Monitoring and Auditing ..............................................................................34 
11 
Statistical Considerations ...............................................................................................34 


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11.1 
Study Hypotheses .......................................................................................................34 
11.2 
Analysis Plans ............................................................................................................35 
11.3 
Interim Analyses and Stopping Rules..........................................................................35 
11.3.1 
Safety Review ......................................................................................................35 
11.3.2 
Efficacy Review ...................................................................................................35 
11.4 
Sample Size Considerations .......................................................................................35 
11.4.1 
Replacement Policy .............................................................................................36 
11.4.2 
Accrual Estimates ................................................................................................36 
11.5 
Exploratory Analysis ...................................................................................................36 
11.6 
Handling Screen Failure/Subject Discontinuation ........................................................36 
12 
Source Documents and Access to Source Data/Documents ..........................................36 
13 
Quality Control and Quality Assurance ...........................................................................37 
14 
Ethics/Protection of Human Participants .........................................................................37 
14.1 
Ethical Standard .........................................................................................................37 
14.2 
Institutional Review Board ...........................................................................................37 
14.3 
Informed Consent Process ..........................................................................................37 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) ..........................38 
14.5 
Participant Confidentiality............................................................................................38 
15 
Data Handling and Record Keeping ...............................................................................39 
15.1 
Data Management Responsibilities .............................................................................39 
15.2 
Data Capture Methods ................................................................................................39 
15.3 
Types of Data .............................................................................................................39 
15.4 
Study Records Retention ............................................................................................39 
15.5 
Protocol Deviations .....................................................................................................40 
16 
Study Finances ...............................................................................................................41 
16.1 
Funding Source ..........................................................................................................41 


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This study is financed through a grant from the U.S. National Institute of Health (NIH): R21 
CA249870. .............................................................................................................................41 
16.2 
Conflict of Interest .......................................................................................................41 
16.3 
Participant Stipends or Payments ...............................................................................41 
17 
Publication and Data Sharing Policy ...............................................................................41 
18 
Literature References .....................................................................................................42 
Appendices ...............................................................................................................................43 
 


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Signature Page 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and 
according to local legal and regulatory requirements and applicable US federal 
regulations and ICH guidelines. 
Principal Investigator: 
Signed: 
 
Date:  
 
Name: 
Ji-Bin Liu, MD 
Title:  
Professor, Department of Radiology 
Statement of Compliance 
This study will be conducted in accordance with the International Conference on Harmonisation 
guidelines for Good Clinical Practice (ICH E6), the Code of Federal Regulations on the 
Protection of Human Subjects (45 CFR Part 46), and Thomas Jefferson University research 
policies 
 
 
 


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List of Abbreviations 
 
AE 
Adverse Event 
CEUS 
Contrast-enhanced Ultrasound 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CPR 
Cardiopulmonary Resuscitation 
CRF 
Case Report Form 
CRO 
Clinical Research Organization 
CT 
Computed Tomographic 
CTCAE 
Common Terminology Criteria for Adverse Events 
FDA 
Food and Drug Administration 
DSMB 
Data Safety Monitoring Board 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HEPS 
Hydrogenated Egg Phosphatidyl Serine 
HIPAA 
Health Insurance Portability and Accountability Act 
HSRRB 
Human Subjects Research Review Board 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
IV 
Intravenous 
LC 
Lymphatic channel 
LN 
Lymph Node 
MOP 
Manual of Procedures 
MRI 
Magnetic Resonance Imaging 
N 
Number (typically refers to participants) 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
PET 
Positron Emission Tomographic 
PHI 
Protected Health Information 
PFB 
Perfluorobutane 
PI 
Principal Investigator 


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PRC 
Protocol Review Committee 
QA 
Quality Assurance 
QC 
Quality Control 
RES 
Reticuloendothelial System 
SAE 
Serious Adverse Event 
SKCC 
Sidney Kimmel Cancer Center 
SOP 
Standard Operating Procedure 
SLN 
Sentinel Lymph Node 
TJU 
Thomas Jefferson University 
TJUH 
Thomas Jefferson University Hospital 
UAP 
Unanticipated Problem 
UCA 
Ultrasound Contrast Agent 
US 
Ultrasound 
 
 


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Study Summary 
 
Title:  
Detection of sentinel lymph nodes in female lower genital tract 
cancer patients with contrast-enhanced ultrasound imaging 
Summary: 
This is an open-label, non-randomized trial that will be conducted at 
Thomas Jefferson University (TJU).  This study will compare the use 
of lymphosonography for sentinel lymph node (SLN) detection to the 
standard of care for lymphatic mapping that varies depending on the 
patient’s staging (lymph node (LN) dissection and/or sequential PET-
CT). The patients will undergo an ultrasound examination to locate the 
tumor where a baseline grayscale and color Doppler ultrasound scan 
of the lesion will be obtained. The ultrasound contrast agent Sonazoid 
(GE Healthcare, Oslo, Norway) will be administrated in 4 aliquots at 
12, 3, 6, and 9 o’clock positions around the primary tumor, with 0.25 
ml for each aliquot for a total dose of 1.0 ml. Real time contrast 
enhanced ultrasound imaging (CEUS) will then be performed to 
identify the number, location and course of the lymphatic channels 
(LCs) drainage from the tumor to the SLNs. All LNs that demonstrate 
contrast-enhancement will be considered SLNs and their locations, 
depth beneath the skin surface and size (measured in three 
orthogonal dimensions) will be recorded and compared to the 
standard of care (i.e., to blue dye, pathology or PET-CT). An Aplio 
i800 scanner (Canon Medical Systems, Tustin, CA) with a curvilinear 
(8C1), a linear (18L5) and endovaginal (11C3) probes with CEUS 
capabilities will be used during the study. 
 
Objectives: 
 
 
 
• 
Primary: 
To 
determine 
the 
concordance 
between 
lymphosonography and the standard of care in the 
identification SLNs in patients with cervical, vaginal or vulvar 
cancer.  
• 
Secondary: To determine if lymphosonography can identify 
more SLNs with metastatic deposits in patients with cervical, 
vaginal or vulvar cancer when compared to the standard of 
care. 
 
 
 


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Population: 
40 adult women, who have a diagnosis of cervical, vaginal or vulvar 
cancer, will be enrolled in this clinical trial at TJU. 
 
Phase: 
pilot 
Number of Sites: 
(1) Thomas Jefferson University (TJU) 
Description of 
Intervention: 
A single-site, open-label, non-randomized, pilot study of 
lymphosonography (i.e., CEUS of subdermal microbubbles) 
compared to standard of care assessments ((i.e., blue dye, 
pathology or PET-CT). 
Study Duration: 
2 years 
Participant 
Participation 
Duration: 
The entire CEUS imaging protocol for the clinical trial will require up 
to 30 minutes of scanning.  
Estimated Time to 
Complete 
Enrollment: 
Subject recruitment for the clinical trial is expected to last 24 months 
(January 2024-December 2025).  


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Schematic of Study Design: 
Prior to  
Enrollment 
 
 
 
 
 
 
 
 
 
 
 
 
Ultrasound Study  
 
 
 
 
 
 
SoC 
 
 
 
1 Introduction 
1.1 Background Information 
In the United States there will be an estimated 24,600 new cases of female lower genital tract 
cancer (cervical, vaginal and vulvar) in 2018 leading to an estimated 6,700 deaths for a mortality 
rate of 27 % or 21 % of all female genital cancer deaths [Siegel et al. 2016]. The treatment 
approach varies according to the stage of the disease, and in the majority of the cases, the 
treatment protocols are similar between all 3 types of cancer. Early stages of disease (stage Ia 
and Ib for cervical and vulvar cancers, stage I for vaginal cancer) are treated with surgical 
intervention, and prior to the surgery the patients undergo PET-CT to determine lymph node (LN) 
metastatic infiltration. This parameter is used to determine the necessity of surgical LN dissection. 
However, the majority of women are diagnosed with advanced disease (stages 2, 3 and 4), where 
the treatment of choice is chemotherapy and/or radiotherapy, with the use of PET-CT to determine 
LN metastatic infiltration pre- and post-treatment. 
Detection of subclinical malignancy in draining LNs is important in the management of a variety 
of malignancies including melanoma, breast, colon and other cancers, where the most important 
LN to evaluate is the sentinel lymph node (SLN), the first node to receive afferent lymphatic 
Total 40: Obtain informed consent. Screen potential subjects by inclusion and 
exclusion criteria; obtain history, document. 
Surgery/Path 
Assessment 
Lympho-
sonography 
PET-CT 
Assessment 
Lympho-
sonography 
Early stage disease 
1.0 mL of Sonazoid injected 
around tumor 
Late stage disease 
1.0 mL of Sonazoid injected 
around tumor 


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drainage through lymphatic channels (LCs) from the primary tumor [Morton & Chan 2000]. 
Various techniques and imaging agents have been developed to map lymphatic drainage from 
tumors, including injection of blue dye, indocyanine green with near-infrared fluorescence imaging 
and injection of radiopharmaceuticals (radioisotopes) followed by evaluation with a gamma 
camera (i.e., lymphoscintigraphy) or intraoperatively with a gamma probe (isotope mapping) 
[Goldfarb et al. 1998, Gimenez et al. 2001]. These surgical resection techniques and also the use 
of PET-CT to diagnosis and monitor of female lower genital tract cancers are in general accurate 
techniques, but pitfalls do exist. For example, the use of blue dye to identify SLNs requires surgical 
dissection that, in some cases, can be extensive (and multicentric) and all surgical procedures 
have inherent potential complications [Gimenez er al. 2001]. Radioactive materials make use of 
small particles and due to their size they may actually pass through the SLNs resulting in drainage 
into secondary LNs potentially resulting in a more extensive resection than might otherwise be 
needed [Goldfarb et al. 1998,] or a change in the staging of the disease, upgrading to a more 
advanced disease than the reality.   
PET-CT systems combine positron emission tomographic (PET) scanners and computed 
tomographic (CT) scanners, which provides images of both anatomic and functional information 
in a single study. However, this combination result in PET/CT examinations, especially those that 
include a diagnostic CT study, with increased patient radiation exposure compared to stand-alone 
CT or PET examinations (as the effective dose is a combination of the dose from PET and the 
dose from CT). Studies, such as Huang et al. 2009, were conducted to determine the potential 
risk from radiation exposure, and the risk-benefit ratios were assessed. The conclusion was that 
total effective dose from each PET/CT study was about five to thirteen times (12-31 mSy) the 
worldwide average effective dose from background radiation over 1 year, which is estimated to 
be about 2.4 mSv [Huang et al. 2009]. In addition, patients with cancer often undergo multiple 
PET/CT examinations for response assessment and treatment monitoring, and given that survival 
rates are markedly improved nowadays, this increases the concern that in time this cumulative 
radiation dose can increase the chance of radiation exposure induced cancer. Therefore, the 
development a non-radiation based imaging alternative is necessary. 
Diagnostic ultrasound (US) imaging has been used to evaluate LNs for benign disease as well as 
metastases, however conventional ultrasound cannot be used for lymphatic mapping, since 
mapping requires administration of a tracer. This paradigm changed with the development of 
stable (i.e., encapsulated), gas-filled microbubbles as ultrasound contrast agents (UCAs) 
[Goldberg et al. 2001]. UCA microbubbles have particle sizes of 2-8 µm that, after intravenous 
administration, serve as vascular tracers typically providing 20-25 dB of echo enhancement for 3-
10 minutes [Goldberg et al. 2001]. Also there are tissue-specific UCAs, which are taken-up by the 
reticuloendothelial system (RES) [Forsberg et al. 2002]. One such tissue-specific UCA is 
Sonazoid (GE Healthcare, Oslo, Norway) composed of a lipid stabilized suspension of 
perfluorobutane microbubbles with a median diameter of 2.4-3.5 µm [Sontum 2008]. This agent 
is taken up by macrophages of the normal RES in the liver and spleen when injected intravenously 
[Forsberg et al. 2002].  
The initial proof of concept that some UCAs are taken up by LNs (in rabbits) and can be localized 
using CEUS was reported by investigators at University of California, San Diego [Mattrey et al. 


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1999]. Our subsequent investigations, using a swine model with naturally occurring melanoma 
tumors and funded by the NCI, established that the accuracy of SLN detection was 81.8% for 
lymphosonography, which was significantly higher than the 63.2% achieved with 
lymphoscintigraphy (p < 0.0001) based on imaging 351 SLNs in 63 swine [Goldberg et al. 2011].   
The clinical translation of lymphosonography is being actively pursued in two different ongoing, 
NCI-supported clinical trials, one designed to investigate the diagnostic utility of 
lymphosonography for SLN identification in women scheduled for surgical excision of a malignant 
breast mass and the other to investigate the diagnostic utility of lymphosonography for SLN 
identification in patients undergoing endoscopic ultrasound guided biopsy of esophageal cancer. 
By injecting a UCA into the peritumoral tissues and following its uptake in the LCs using CEUS, 
our group can identify the specific location and number of SLNs draining from the primary tumor. 
The results will be compared to the established techniques currently in use. Lymphosonography 
used for SLN detection has several potential advantages. Lymphosonography does not require 
ionizing radiation (a growing concern in medicine) and has markedly better spatial resolution as 
well as anatomical information, which would allow for more precise SLN localizations; also the 
portability of US equipment lends itself to use at the patient's bedside, within the surgical suite or 
elsewhere as necessary. Lymphosonography can be used to clearly delineate adjacent LNs and 
to identify tumor involvement using conventional US criteria [Goldberg et al. 2011], which is not 
possible with the use of a gamma probe or by lymphoscintigraphy. Finally, the improvement in 
SLN detection in patients with breast cancer (from 1.67 to 2.16 SLNs/patient) achieved by using 
the tissue-specific lymphoscintigraphy agent Tilmanocept [Tokin et al. 2012], indicates that the 
much larger improvement seen with lymphosonography and the RES-specific UCA Sonazoid (3.2 
SLNs/patient) has the potential to change the clinical paradigm for SLN identification. Hence, this 
project is a natural translation of our experiences with lymphosonography in breast and 
esophageal cancer patients and it aims to shift the clinical paradigm on mapping and identification 
of SLNs in women with lower genital tract cancer.  
The safety of subdermal injections of Sonazoid was determined in a safety study conducted by 
our group [Machado et al. 2018]. The study assessed the safety and tolerability of subdermal 
administration of two dosages Sonazoid (1 and 2 ml) in the breasts of healthy, female volunteers 
with screening and baseline assessments/procedures that included informed consent, medical 
history, demography, physical examination, vital signs and a focused physical exam as well as 
recording of any adverse events (AEs). Only minor local and insignificant adverse experiences 
confined to the injection site and surrounding area were encountered by the volunteers, local 
redness was seen in 83% of the cases, local pain in 33% of the cases and local bruising in 25% 
of the cases [Machado et al. 2018]. All adverse experiences (Table 1) were completely resolved 
without any intervention by the time the study was completed.  
Table 1. Adverse experiences divided by volunteers and by dosage. 
Adverse experience 
Volunteers (n=12) 
1 ml (n=12) 
2 ml (n=12) 
Redness 
10 (83%) 
10 (83%) 
7 (58%) 
Pain 
4 (33%) 
2 (17%) 
3 (25%) 
Bruise 
3 (25%) 
2 (17%) 
1 (8%) 
Burning sensation 
2 (17%) 
1 (8%) 
2 (17%) 


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Numbness 
1 (8%) 
1 (8%) 
1 (8%) 
Tingling sensation 
1 (8%) 
0 (0%) 
1 (8%) 
 
Sonazoid Intravenous Clinical Safety Trials 
Prior to the development of lymphsonography and the subdermal use of UCA, clinical trials were 
done to evaluate the safety of intravenous use of Sonazoid.  
Sonazoid was administered intravenous (IV) in clinical trials in 1699 patients.  In these patients 
404 (23.8%) reported at least one AE, while 12 of 62 (19.4%) of patients receiving a placebo 
reported AEs. 
Forty-three (2.5%, 43/1699) subjects experienced serious adverse events (SAEs), including 13 
deaths.  None of these events were considered by the investigators to be related to Sonazoid.  All 
the SAEs were considered to be caused by the underlying disease or related treatment.  In 
addition, there were no clinically significant trends in laboratory tests, vital signs, ECGs, or 
physical examination findings.  The most commonly noted AEs were headache (3.6%, 62/1699), 
chest pain (2.3%, 39/1699), abdominal pain (1.5%, 25/1699), diarrhea (1.5%, 25/1699), and 
nausea (1.6%, 28/1699). The majority of AEs were mild to moderate in severity (92.6%, 652/704). 
The proposed agent for the current study, Sonazoid® (GE Healthcare, Oslo, Norway), is a sterile 
non-pyrogenic suspension of lipid stabilized perfluorobutane microbubbles for contrast-
enhancement, with a median diameter between 2.4 and 3.5 µm [Sontum 2008].  The FDA has yet 
to approve Sonazoid for human use in this country.  Consequently, we intend to amend our current 
investigator initiated IND (#127,419) exemption from the FDA covering the subdermal use of 
Sonazoid to also cover female lower genital cancers. This IND was originally set up for breast 
cancer and was subsequently amended to also include esophageal cancers. 
1.2 Rationale for the Proposed Study 
Women with cervical, vaginal or vulvar cancers will undergo surgical resection and/or 
chemotherapy and/or radiotherapy of their cancer (depending on their staging). The SLN mapping 
for this group of patients is done as part of the staging process using PET-CT, for patients with 
stage I that undergo surgical resection there is an additional SLN mapping procedure performed 
using blue dye. The standard of care for the surgical group of patients is LN dissection usually 
bilateral given the midline anatomical location of the female lower genital tract. Patients with 
tumors of higher stages that undergo chemotherapy and/or radiotherapy undergo systematic 
PET-CT for the continuing SLN staging during treatment. However, each of these methods has 
potential limitations that can impact the detection of SLNs and the accuracy of disease staging.  
Our group has demonstrated that CEUS after subdermal administration of a tissue-specific 
ultrasound contrast agent such as Sonazoid, can be used to noninvasively map lymphatic 
drainage and localize SLNs (so called “lymphosonography”).  Our NIH funded investigations using 
the Sinclair swine model with naturally occurring melanomas have confirmed that CEUS is 
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The fundamental hypothesis of this project is that lymphosonography can be used to identify SLNs 
in patients with cervical, vaginal or vulvar cancers with better success rates than the standard of 
care.  In summary, this study aims to determine the clinical potential of lymphosonography for 
noninvasive SLN mapping via subdermal injection of tissue-specific UCA in 40 patients with 
cervical, vaginal or vulvar cancer and compare the use of lymphosonography to the standard of 
care. 
1.3 Potential Risks and Benefits 
The known risks from the subdermal administration of Sonazoid are minimal.  Only minor local 
and non-significant adverse experiences confined to the injection site and surrounding area were 
encountered by the volunteers, local redness was seen in 83% of the cases, local pain in 33% of 
the cases and local bruising in 25% of the cases. 
UCAs in general are very safe with serious anaphylactoid-type reactions reported for intravenous 
injection at a rate of less than 0.01% [Dietrich et al., 2020]. 
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 
(although that is extremely unlikely).  Clinically significant adverse effects from the administration 
of Sonazoid are unlikely.  Hence, the use of subdermal injection of contrast with the contrast-
specific ultrasound imaging technique is expected to provide significantly more information than 
conventional ultrasound techniques.  This may lead to a non-invasive method for identification of 
SLNs.  
The cervical, vaginal or vulvar cancer treatment is being performed as a part of the patient’s 
clinical care and therefore, the risks that are associated with the treatment are not related to the 
research.  The subjects’ alternative is not to participate in this study and have only the scheduled 
standard of care treatment performed.   
1.3.1 Potential Risks 
To protect patient information, data forms will be completed for all subjects enrolled in the trial.  
The subject study files will be stored in a secure file cabinet and maintained by the research study 
coordinator.  Study files will be kept for 7 years after the completion of the study.  The PI of the 
study (Dr. Forsberg) and his co-investigators have previous experience running ultrasound clinical 
trials and the PI will serve as the study sponsor.  He will be responsible for ensuring all AEs are 
properly reported. 
The final data will be entered into a database implemented in REDCap (Vanderbilt University, 
Nashville, TN). The investigators will be responsible for management of the database. The 
database will be maintained within an organized and secure directory system. Subject 
identification will be maintained with a study specific alphanumeric code (GYN), patient number 
(01 and onwards) and the patient’s initials.   
1.3.2 Benefits 


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We do not expect any direct benefit for subjects enrolled in this study. The long-term 
benefits of this study will be the clinical use of a non-radiation based imaging alternative 
to identify SLNs in patients with cervical, vaginal or vulvar cancers.  
1.3.3 Risk-Benefit Ratio 
The risk benefit ratio is low. Based on the available nonclinical and clinical safety data 
and the anticipated dose levels of Sonazoid that will be used in this study, safety concerns 
are minimal. The potential side effects related to Sonazoid administration via subdermal 
injection are described above and listed in greater detail in the investigator’s brochure. 
2 Study Objectives 
2.1 Objectives 
This study aims to determine the clinical potential of lymphosonography for noninvasive SLN 
mapping in 40 patients with cervical, vaginal or vulvar cancer and compare the use of 
lymphosonography to the standard of care. 
2.1.1 Hypothesis 
The fundamental hypothesis of this project is that lymphosonography can be used to identify SLNs 
in patients with cervical, vaginal or vulvar cancers with better success rates than the standard of 
care.  
Primary hypothesis: Does lymphosonography identify more SLNs in patients with cervical, 
vaginal or vulvar cancer than the standard of care?  The fundamental hypothesis is that 
on average lymphosonography will detect 20 % more SLNs than the standard of care. 
Secondary hypothesis: Does lymphosonography identify more SLNs with metastatic 
deposits in patients with cervical, vaginal or vulvar cancer compared to the standard of 
care?  The fundamental hypothesis is that approximately 20% more cancerous SLNs will 
be detected with CEUS. 
2.1.2 Primary Objectives  
The primary objectives of this trial are: 
1. To determine the concordance between lymphosonography and the standard of care in 
the identification SLNs in patients with cervical, vaginal or vulvar cancer.  
 
2. To determine if lymphosonography can identify more SLNs with metastatic deposits in 
patients with cervical, vaginal or vulvar cancer when compared to the standard of care.   
2.2 Endpoints/Outcome Measures 
The findings of lymphosonography will be compared with other imaging and pathological findings 
when available; including vascularity as well as the clinical outcome as determined by their 


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clinician. A full demographic profile, known drug allergies or intolerances, and review of the 
subject’s medical/surgical history will be recorded.   
Each case will be read independently by two experienced co-investigators blinded to the clinical 
outcomes to allow repeatability to be assessed. 
2.2.1 Primary Endpoints  
The number of SLNs identified by lymphosonography as well as blue dye or PET-CT will be 
determined for each subject. 
2.2.2 Secondary Endpoints  
The locations, depth beneath the skin surface and size (measured in three orthogonal 
dimensions) of each SLN will be recorded  
The presence or absence of metastatic deposits in the SLNs will be obtained by pathology (when 
available). 
3 Study Design 
This is an open-label, non-randomized trial that will be conducted at TJU.  This study will compare 
the use of lymphosonography for SLN detection to the standard of care lymphatic mapping that 
varies depending on the patient’s staging (LN dissection and/or sequential PET-CT). The patients 
will undergo an ultrasound examination to locate the tumor where a baseline grayscale and color 
Doppler ultrasound scan of the lesion will be obtained. Sonazoid will be administrated via 
subdermal injection in 4 aliquots at 12, 3, 6, and 9 o’clock positions around the primary tumor, 
with 0.25 ml for each aliquot for a total dose of 1.0 ml. Real time CEUS will then be performed to 
identify the number, location and course of the LCs drainage from the tumor to the SLNs. All LNs 
that demonstrate contrast-enhancement will be considered SLNs and their locations, depth 
beneath the skin surface and size (measured in three orthogonal dimensions) will be recorded 
and compared to the standard of care (i.e., to blue dye, pathology or PET-CT). An Aplio i800 
scanner (Canon Medical Systems, Tustin, CA) with a curvilinear (8C1), a linear (18L5) and 
endovaginal (11C3) probes with CEUS capabilities will be used during the study.  
The findings of lymphosonography will be correlated with other imaging and pathological findings 
when available; including clinical and surgical outcomes as determined by their clinician. A full 
demographic profile, known drug allergies or intolerances, and 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). 
4 Study Enrollment and Withdrawal 


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This clinical trial will consist of 40 adult women (18 years of age or older), who have been 
diagnosed with cervical, vaginal or vulvar cancer and are seen in our Department of Gynecology 
Oncology at TJU. 
Subjects eligible for clinical trial enrollment will be identified by the surgical co-investigators, Dr. 
Rosenblum, Dr. Richard and Dr. Minion from their patient population of women with cervical, 
vaginal or vulvar cancer seen in the Department of Gynecology Oncology at TJU. The research 
coordinator for this study will explain the study to the subjects. The subject will be given time to 
consider the risks and benefits of the study and ask questions about participation. The coordinator 
will review the informed consent form with the subject and then the subject will be given the form 
to review. The subject, coordinator, and a study investigator will all sign the consent form.  The 
subject will be given a copy of the signed consent form for their records.   
Individual participation in the clinical trial will be limited to one lymphosonography study to be 
realized during their standard of care cancer treatment determined by their physician according 
with staging. The entire CEUS imaging protocol for the clinical trial will require up to 30 minutes 
of scanning.   
Subject recruitment for the clinical trial is expected to last 24months (January 2024 - December 
2025). Analysis and publication of results are expected to take an additional 6 months.   
Subject identification will be maintained with a study specific alphanumeric code (GYN), patient 
number (01 and onwards) and the patient’s initials.   
4.1 Eligibility Criteria 
4.1.1 Inclusion Criteria 
Individuals must meet all of the following inclusion criteria in order to be eligible to participate in 
the study: 
• 
Be female.  
• 
Be diagnosed with cervical, vaginal or vulvar cancer. 
• 
Be at least 18 years of age. 
• 
If of child-bearing potential, must have a negative pregnancy test 
• 
Be able to comply with study procedures. 
• 
Have read and signed the IRB-approved Informed Consent form for participating in the 
study. 
4.1.2 Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation in this 
study: 
• 
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 hours afterwards. 


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• 
Patients who are medically unstable, patients who are seriously or terminally ill, and 
patients whose clinical course is unpredictable.  For example: 
• Patients on life support or in a critical care unit. 
• Patients with clinically unstable cardiac arrhythmias, such as recurrent 
ventricular tachycardia. 
• Patients with recent cerebral hemorrhage. 
• Patients who have undergone surgery within 24 hours prior to the study 
sonographic examination. 
• 
Patients with congenital heart defects. 
• 
Patient with a known allergy to Sonazoid (including an anaphylactic allergy to eggs or 
egg products). 
4.2 Strategies for Recruitment and Retention 
Potential research subjects will be identified by a member of the patient’s care treatment team, 
the investigator or a research team member.  Investigators will then screen the patient’s medical 
records to determine the subject eligibility for study participation and discuss the study with the 
patient and their potential for enrolling in the research study. Consenting patients will be screened 
based on the inclusion/exclusion criteria above. 
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.  
Trial participants will have the presence of inclusion criteria and absence of exclusion criteria 
verified by providing a medical history. A full demographic profile, known drug allergies or 
intolerances, and a review of the subject’s medical/surgical history will be recorded. If the subject 
is a woman of childbearing potential, she will have a urine pregnancy test (the results of which 
will be made available to the subject prior to study initiation).  
4.3 Participant Withdrawal 
4.3.1 Reasons for Withdrawal 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may terminate a study participant’s participation in the study if: 
• 
Any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant. 
• 
The participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation. 


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4.3.2 Handling of Participant Withdrawals and Participant 
Discontinuation of Study Intervention 
Trial participation will be considered completed if the subject has met all of the following trial 
requirements: 
 
Has received one set of subdermal injections of Sonazoid 
 
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) and 
the subject has met the criteria described above for completing the trial, the subject's trial 
participation will be considered completed. If a subject's trial participation is interrupted for any 
reason by the subject's or investigator's choice and the subject has not met all of the criteria listed 
above, then the subject will be considered a discontinued subject. 
4.4 Premature Termination or Suspension of Study 
This study may be suspended or prematurely terminated if there is sufficient reasonable cause. 
Written notification, documenting the reason for study suspension or termination, will be provided 
by the suspending or terminating party to the IRB and the FDA. If the study is prematurely 
terminated or suspended, the principal investigator will promptly inform the IRB and will provide 
the reason(s) for the termination or suspension.  
Circumstances that may warrant termination include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants. 
• 
Insufficient adherence to protocol requirements. 
• 
Data that is not sufficiently complete and/or evaluable.  
• 
Determination of futility. 
5 Study Intervention 
5.1 Study Product  
The UCA Sonazoid consisting of gas-filled microbubbles will be used in this study. Our existing 
FDA Investigator IND (# 127,419) on subdermal injections of Sonazoid will be amended to include 
this project. This IND was originally set up for breast cancer and was subsequently amended to 
also include esophageal cancers. Studies will not commence until regulatory approval has been 
obtained from the FDA as well as the IRB committee for TJU.  The clinical trial will also be 
registered with ClinicalTrials.gov. 
5.2 Study Product Description 
Sonazoid is a sterile non-pyrogenic suspension of microspheres of lipid stabilized perfluorobutane 
(PFB) for contrast-enhancement, with a median diameter between 2.4 and 3.5 µm and less than 


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0.1 % larger than 7 µm. Sonazoid is formulated as a powder for injection consisting of lyophilized 
sucrose entrapping hydrogenated egg phosphatidyl serine (HEPS) PFB microspheres under a 
PFB headspace.  Each milliliter of Sonazoid contains roughly 1.2 billion microspheres. 
5.2.1 Acquisition 
Sonazoid will be provided by GE Healthcare (Oslo, Norway) free of charge (similar to our other 
protocols involving this UCA). 
5.2.2 Formulation, Packaging, and Labeling 
Sonazoid is supplied as a dry powder within 10 mL sealed vials.  The headspace of the vials 
contains PFB.  A single vial with 16 µL of Sonazoid microbubbles (2 mL) will be prepared for each 
subject by resuspending each vial in 2 mL of injection grade water supplied by GE. Each kit will 
be labeled with the study number as well as vial lot and batch numbers. All materials and supplies 
used for the subdermal injection procedure will be identical to those described in GE Healthcare’s 
current Sonazoid IND. 
 
Packages will contain the following medical grade, sterile and non-pyrogenic components: 
• 
SonazoidTM Powder for Injection, 16 µL microbubbles per vial;   
• 
Sterile water for reconstitution of SonazoidTM  
• 
CODAN Chemospike (contains 0.20 µm air filter and 5 µm liquid filter) 
5.2.3 Product Storage and Stability 
Sonazoid will be stored in a secure cabinet, with only the study investigators and research 
personnel having access. The study researchers will be responsible for drug reconstitution and 
inventory control. New vials must be prepared for each subject's contrast administration and will 
be used immediately upon reconstitution. Sonazoid is stable for 4 hours after reconstitution.  If 
the agent is suspended and not used within 4 hours it will be discarded. 
5.3 Dosage, Preparation, and Administration  
• 
Perforate the stopper of the Sonazoid vial with the provided CODAN Chemospike. 
• 
Remove protective cap from the syringe port of the Chemospike. 
• 
Using a 2-mL syringe, add 2 mL sterile water through the Chemospike.  
• 
With the syringe remaining attached to the Chemospike, immediately shake the product 
for 1 minute to ensure a homogeneous product. 
• 
Withdraw the product into the syringe and re-inject the product back into the vial again. 
This is to avoid dilution of the product due to the dead-space volume in the Chemospike. 
• 
Remove the syringe from the syringe port and reattach the protective cap. The 
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Each subject will receive a 1 ml subdermal dose of Sonazoid divided into four individual aliquots 
at four locations (12, 3, 6, and 9 o’clock) around the primary tumor using 20-22 gauge cannula.  
5.4 Dose Modifications and Dosing Delays 
Not applicable for this study. 
5.5 Study Product Accountability 
A log of study drug will be kept by the research coordinator on this study. All vials released for 
studies will be recorded. Unused drug and empty vials will be properly disposed of after 
reconciling in the log of study drug. 
5.6 Dietary Restrictions 
No special dietary or “life-style” requirements are applicable. 
5.7 
Study Procedural Intervention(s) Description 
Screening Assessments  
Screening assessments will be performed within 4 weeks prior to the administration of Sonazoid.  
All subjects will receive a written consent form and a verbal explanation of the trial by the 
investigator and/or the research study coordinator and will be asked to sign the written informed 
consent. Participants will have the presence of inclusion criteria and absence of exclusion criteria 
(cf., sections 4.1.1 and 4.1.2) verified by providing a comprehensive medical history, which will 
include screening assessments for their cervical, vaginal or vulvar cancer diagnosis. 
Medical History 
A full demographic profile, known drug allergies or intolerances, and a review of the subject’s 
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a urine pregnancy test (the results of which will be made available to the subject prior to study 
initiation).  
5.8 
Administration of Procedural Intervention 
Administration of Sonazoid 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 will be in immediate proximity to the 
patient.  Patients will be monitored for AEs post Sonazoid treatment for a minimum of one hour. 
Trained CPR personnel and resuscitation equipment will be in attendance during this monitoring 
period. A dose of one vial with 16 µl of Sonazoid microbubbles (2 ml total after resuspension) will 
be prepared for each subject by resuspending each vial in 2 ml of injection grade water supplied 
by GE (as described in section 5.3).  All materials and supplies used for the infusion procedure 
will be identical to those described in GE Healthcare’s current Sonazoid IND. Each subject will 
receive 1 ml subdermal dose of Sonazoid divided into four individual aliquots of 0.25ml at four 
locations (12, 3, 6, and 9 o’clock) around the primary tumor using a 20-22 gauge cannula.   
Ultrasound Imaging (lymphosonography) 
The ultrasound examinations will be performed by a qualified sonographer or physician. 
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. An Aplio i800 
scanner with a curvilinear (8C1), a linear (18L5) and an endovaginal (11C3) probe CEUS will be 
used during the study. The patients will undergo an ultrasound examination to locate the tumor 
where a baseline grayscale and color Doppler ultrasound scan of the lesion will be obtained. 
Sonazoid will be administrated via subdermal injection in 4 aliquots at 12, 3, 6, and 9 o’clock 
positions around the primary tumor, with 0.25 ml for each aliquot adding to a total dose of 1.0 ml. 
Real time CEUS will then be performed to identify the number, location and course of the LCs 
drainage from the tumor to the SLNs. All LNs that demonstrate contrast-enhancement will be 
considered SLNs and their locations, depth beneath the skin surface and size (measured in three 
orthogonal dimensions) will be recorded and compared to the standard of care (i.e., to pathology 
or PET-CT). Digital clips of the ultrasound study will be acquired. 
Contraindications 
Sonazoid should not be administered to patients with known or suspected hypersensistivity to 
egg phosphatidyl serine. Patients with a history of anaphylactic allergy to eggs or egg products 
will be excluded from the study.  
Safety Assessments 
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. 
5.9 
Administration of Procedural Intervention 
Each subject will receive a 1 ml subdermal dose of Sonazoid divided into four individual aliquots 
at four locations (12, 3, 6, and 9 o’clock) around the primary tumor using 20-22 gauge cannula by 
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sonographers, RNs, basic scientists and research coordinators). We anticipate each session will 
take approximately 45 min out of which around 25 min will be the actual scanning time.  
5.10 
Procedures for Training of Clinicians on Procedural Intervention 
The personnel performing the lymphosonography procedures are all from the Ultrasound 
Research lab or Gynecological Oncology and have extensive experience with injections as well 
as CEUS and ultrasound imaging procedures. These individuals will all receive training by the PI 
in this specific protocol. 
6 Study Schedule  
6.1 Screening 
Assess patient eligibility, complete appropriate screening procedures and evaluations prior to 
study entry.  
• 
Obtain informed consent 
• 
A qualitative urine pregnancy test, for female patients of childbearing potential.  
 
6.2 On Study Period 
The ultrasound examinations will be performed by a qualified sonographer or physician. 
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. An Aplio i800 
scanner with a curvilinear (8C1), a linear (18L5) and an endovaginal (11C3) probe CEUS will be 
used during the study. The patients will undergo an ultrasound examination to locate the tumor 
where a baseline grayscale and color Doppler ultrasound scan of the lesion will be obtained. 
Sonazoid will be administrated in 4 aliquots at 12, 3, 6, and 9 o’clock positions around the primary 
tumor, with 0.25 ml for each aliquot adding to a total dose of 1.0 ml. Real time CEUS will then be 
performed to identify the number, location and course of the LCs drainage from the tumor to the 
SLNs. All LNs that demonstrate contrast-enhancement will be considered SLNs and their 
locations, depth beneath the skin surface and size (measured in three orthogonal dimensions) 
will be recorded and compared to the standard of care (i.e., to pathology or PET-CT). Digital clips 
of the ultrasound study will be acquired. 
6.3 End of Treatment Study Procedures 
Trial participation will be considered completed if the subject has met all of the following trial 
requirements: 
• 
Has received one set of subdermal injections of Sonazoid 
• 
Has undergone the complete ultrasound imaging study as described in this protocol 
6.4 Withdrawal Visit/Discontinuation of Therapy 
If a subject's participation in the trial is interrupted for any reason (e.g., because of an AE) and 
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participation will be considered completed. If a subject's trial participation is interrupted for any 
reason by the subject's or investigator's choice and the subject has not met all of the criteria listed 
above, then the subject will be considered a discontinued subject. 
7 Study Procedures and Evaluations 
7.1 Study Procedures/Evaluations 
As outlined in section 5.7: 
• 
Medical history 
• 
Lymphosonography 
8 Evaluation of Safety 
8.1 Specification of Safety Parameters 
8.1.1 Unanticipated Problems 
Unanticipated problems (UAPs) include, in general, any incident, experience, or outcome that 
meets the following criteria: 
• 
unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the IRB-approved research 
protocol and informed consent document; and (b) the characteristics of the participant 
population being studied; 
UAPs are considered to pose risk to participants or others when they suggest that the research 
places participants or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. 
8.1.2 Adverse Events 
An adverse event (AE) is any untoward or unfavorable medical occurrence in a human participant, 
including any abnormal sign (for example, abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the participant’s participation in the research, 
whether or not considered related to the participant’s participation in the research. 
The safety of subdermal injections of Sonazoid was determined in a safety study conducted by 
our group [Machado et al. 2018]. The study assessed the safety and tolerability of subdermal 
administration of two dosages Sonazoid (1 and 2 ml) in the breasts of healthy, female volunteers 
with screening and baseline assessments/procedures that included informed consent, medical 
history, demography, physical examination, vital signs and a focused physical exam as well as 
recording of any adverse events (AEs). Only minor local and insignificant adverse experiences 
confined to the injection site and surrounding area were encountered by the volunteers, local 
redness was seen in 83% of the cases, local pain in 33% of the cases and local bruising in 25% 
of the cases [Machado et al. 2018]. All adverse experiences were completely resolved without 


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any intervention by the time the study was completed as shown in Table 1 (repeated below for 
convenience).  
Sonazoid Intravenous Clinical Safety Trials 
Prior to the development of lymphsonography and the subdermal use of UCA, clinical trials were 
done to evaluate the safety of intravenous use of Sonazoid.  
 
Table 1. Adverse experiences divided by volunteers and by dosage. 
Adverse experience 
Volunteers (n=12) 
1 ml (n=12) 
2 ml (n=12) 
Redness 
10 (83%) 
10 (83%) 
7 (58%) 
Pain 
4 (33%) 
2 (17%) 
3 (25%) 
Bruise 
3 (25%) 
2 (17%) 
1 (8%) 
Burning sensation 
2 (17%) 
1 (8%) 
2 (17%) 
Numbness 
1 (8%) 
1 (8%) 
1 (8%) 
Tingling sensation 
1 (8%) 
0 (0%) 
1 (8%) 
 
Sonazoid was administered intravenous (IV) in clinical trials in 1699 patients.  In these patients 
404 (23.8%) reported at least one AE, while 12 of 62 (19.4%) of patients receiving a placebo 
reported AEs. 
Forty-three (2.5%, 43/1699) subjects experienced serious adverse events (SAEs), including 13 
deaths.  None of these events were considered by the investigators to be related to Sonazoid.  All 
the SAEs were considered to be caused by the underlying disease or related treatment.  In 
addition, there were no clinically significant trends in laboratory tests, vital signs, ECGs, or 
physical examination findings.  The most commonly noted AEs were headache (3.6%, 62/1699), 
chest pain (2.3%, 39/1699), abdominal pain (1.5%, 25/1699), diarrhea (1.5%, 25/1699), and 
nausea (1.6%, 28/1699). The majority of AEs were mild to moderate in severity (92.6%, 652/704). 
8.1.3 Serious Adverse Events 
A serious adverse event (SAE) is one that meets one or more of the following criteria: 
• 
Results in death 
• 
Is life-threatening (places the participant at immediate risk of death from the event as it 
occurred) 
• 
Is disabling or incapacitating 
• 
Results in inpatient hospitalization or prolongation of existing hospitalization 
• 
Results in a persistent or significant disability or incapacity 
• 
Results in a congenital anomaly or birth defect  


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• 
An important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical 
judgment, the event may jeopardize the participant or may require intervention to 
prevent one of the outcomes listed in this definition. 
8.2 Safety Assessment and Follow-Up 
The PI will follow AEs with start dates occurring any time after informed consent is obtained until 
7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study participation. Events will 
be followed for outcome information until resolution or stabilization. 
8.3 Recording Adverse Events 
The following subsections detail what information must be documented for each AE occurring 
during the time period specified in Section 8.2 Safety Assessment and Follow-Up. If the patient 
has experienced AE(s), the investigator will record the following information in the AE log: 
• 
The nature of the event(s) will be described by the investigator in precise standard medical 
terminology (i.e. not necessarily the exact words used by the patient). 
• 
The duration of the event will be described in terms of event onset date and event ended 
data. 
• 
The intensity of the AE will be described according to Common Terminology Criteria for 
Adverse Events version 5.0 (CTCAE), for details see National Cancer Institute home page 
http:\evs.nci.nih.gov\ftp1\CTCAE\About.html of the AE. The CTCAE displays Grades 1 
through 5 with unique clinical descriptions of severity for each AE based on this general 
guideline: 
• 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
• 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living.  
• 
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self 
care activities of daily living.  
• 
Grade 4: Life-threatening consequences; urgent intervention indicated. 
• 
Grade 5: Death related to AE. 
• 
The Causal relationship of the event to the study medication will be assessed as one of the 
following: 
• 
Unrelated: There is not a temporal relationship to investigational product 
administration (too early, or late, or investigational product not taken), or there is 
a reasonable causal relationship between noninvestigational product, concurrent 
disease, or circumstance and the AE. 
• 
Unlikely: There is a temporal relationship to investigational product 
administration, but there is not a reasonable causal relationship between the 
investigational product and the AE. 
• 
Possible: There is reasonable causal relationship between the investigational 
product and the AE. Dechallenge information is lacking or unclear. 
• 
Probable: There is a reasonable causal relationship between the investigational 
product and the AE. The event responds to dechallenge. Rechallenge is not 
required. 
• 
Definite: There is a reasonable causal relationship between the investigational 
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• 
The Expectedness of the AE 
• 
Action taken 
• 
The outcome of the AE – whether the event is resolved or still ongoing. 
8.3.1 Relationship to Study Intervention 
The relationship to study intervention or study participation must be assessed and documented 
for all AEs. Evaluation of relatedness must consider etiologies such as natural history of the 
underlying disease, concurrent illness, concomitant therapy, study-related procedures, accidents, 
and other external factors.  
The following guidelines are used to assess relationship of an event to study intervention: 
1. Related (Possible, Probable, Definite) 
a. The event is known to occur with the study intervention. 
b. There is a temporal relationship between the intervention and event onset. 
c. The event abates when the intervention is discontinued. 
d. The event reappears upon a re-challenge with the intervention. 
2. Not Related (Unlikely, Not Related) 
a. There is no temporal relationship between the intervention and event onset. 
b. An alternate etiology has been established. 
8.3.2 Expectedness 
The PI is responsible for determining whether an AE is expected or unexpected. An AE will be 
considered unexpected if the nature, severity, or frequency of the event is not consistent with the 
risk information previously described for the intervention. Risk information to assess 
expectedness can be obtained from preclinical studies, the investigator’s brochure, published 
medical literature, the protocol, or the informed consent document. 
8.3.3 Severity of Event 
Adverse events will be graded for severity according to the Common Terminology Criteria for 
Adverse Events (CTCAE) version 5.0.  
8.3.4 Intervention 
Any intervention implemented to treat the AE must be documented for all AEs. 
8.4 Safety Reporting  
All adverse events and serious adverse events that should be reported as defined in section 8.1.1 
will be recorded in the patient's CRF. All adverse reaction will be reported according to the CT-3; 
detailed Guidance on the collection, verification and presentation of adverse event/reaction 
reports arising from clinical trials on medicinal products for human use. Link: 
 


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https://ec.europa.eu/health//sites/health/files/files/eudralex/vol-
10/2011_c172_01/2011_c172_01_en.pdf 
 
All events will be graded by the Investigator according to the Common Terminology Criteria for 
Adverse Events (CTCAE) version 5.0. A grading (severity) scale is provided for each AE term. 
SAEs must be reported within 24 hours after the site has gained knowledge of the SAE to the 
IRB, and the FDA. Every SAE must be documented by the investigator on the SAE pages (to be 
found as part of the CRF). The SAE Report Form must be completed and signed. SAEs and 
possible SAEs will be reported to the regulatory authorities and IEC according to local regulations 
and followed-up until the resolution of the event. The initial report shall promptly be followed by 
detailed, written reports if necessary. The initial and follow-up reports shall identify the trial 
subjects by unique code numbers assigned to the latter.  
 
The sponsor keeps detailed records of all SAEs reported by the investigators and performs an 
evaluation with respect to causality and expectedness. Based on, among other, SAE reports the 
sponsor will evaluate whether the risk/benefit ratio associated with study is changed. We will in 
all cases follow the ICH Harmonised Guideline Integraded Addendum to ICH E6(R1): Guideline 
for Good Clinical Practise ICH E6(R2) ICH Consensus Guideline. Link: https://ichgcp.net/411-
safety-reporting/  
 
8.4.1 Reporting to IRB 
8.4.1.1 Unanticipated Problems 
All incidents or events that meet criteria for unanticipated problems (UAPs) as defined in Section 
8.1.1 Unanticipated Problems require the creation and completion of an unanticipated problem 
report form (OHR-20).  
UAPs that pose risk to participants or others, and that are not AEs, will be submitted to the IRB 
on an OHR-20 form via the eazUP system within 10 working days of the investigator becoming 
aware of the event.  
UAPs that do not pose risk to participants or others will be submitted to the IRB at the next 
continuing review.  
8.4.1.2 Adverse Events  
Grade 1 AEs will be reported to the IRB at continuing review.  
Grade 2 AEs will be reported to the IRB at the time of continuing review.  
8.4.1.3 Serious Adverse Events  
SAEs will be reported to the IRB on OHR-10 forms via the electronic reporting system (eSAEy) 
according to the required time frames described below. 
Grade 3-4 AEs that are unexpected and deemed to be at least possibly related to the study will 
be reported to the IRB within 2 working days of knowledge of the event. 
Grade 3-4 AEs that are deemed unrelated to the study will be reported to the IRB within 5 
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Grade 5 AEs will be reported to the IRB within one working day of knowledge of the event. 
All SAEs will be submitted to the IRB at continuing review, including those that were reported 
previously. 
8.4.2 Reporting to SKCC DSMC 
All AEs and SAEs, safety and toxicity data, and any corrective actions will be submitted to the 
DSMC per the frequency described in the SKCC DSMP. The report to the SKCC DSMC will also 
include any unanticipated problems that in the opinion of the PI should be reported to the DSMC. 
For expedited reporting requirements, see Table 2 below:                                             
8.4.1 Reporting to Funding Sponsor 
Once a year throughout the clinical trial, the sponsor will provide the Competent Authority with a 
listing of all suspected serious adverse reactions which have occurred over this period and a 
report of the subjects' safety. Annual reports are submitted in accordance with ICH guideline E2F 
- Note for guidance on development safety update reports.  
8.4.1 Reporting to FDA 
The PI and the SKCC regulatory team will be responsible for submitting annual reports 
to the FDA on the progress of the study. 
8.4.1 Reporting of Pregnancy 
Pregnancy is an exclusion criteria. 
Table 2: DSMC AE/SAE Reporting Requirements 


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8.5 Halting Rules 
The whole trial may be discontinued at the discretion of the PI or the sponsor in the event of any 
of the following: 
• 
Occurrence of AEs unknown to date in respect of their nature, severity and duration 
• 
Medical or ethical reasons affecting the continued performance of the trial 
 
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In addition to the PI’s responsibility for oversight, study oversight will be under the direction of the 
SKCC’s Data and Safety Monitoring Committee (DSMC). The SKCC DSMC operates in 
compliance with a Data and Safety Monitoring Plan (DSMP) that is approved by the NCI.  
10 Clinical Site Monitoring and Auditing 
Clinical site monitoring and auditing is conducted to ensure that the rights of human participants 
are protected, that the study is implemented in accordance with the protocol and/or other 
operating procedures, and that the quality and integrity of study data and data collection methods 
are maintained. Monitoring and auditing for this study will be performed in accordance with the 
SKCC’s Data and Safety Monitoring Plan (DSMP) developed by the SKCC Data and Safety 
Monitoring Committee (DSMC). The DSMP specifies the frequency of monitoring, monitoring 
procedures, the level of clinical site monitoring activities (e.g., the percentage of participant data 
to be reviewed), and the distribution of monitoring reports. Some monitoring activities may be 
performed remotely, while others will take place at the study site(s). Appropriate staff will conduct 
monitoring activities and provide reports of the findings and associated action items in accordance 
with the details described in the SKCC DSMP. 
11 Statistical Considerations  
The fundamental hypothesis of this project is that lymphosonography can be used to identify SLNs 
in patients with cervical, vaginal or vulvar cancers with greater success than the standard of care. 
For this project the standard of care is either blue dye injections (it the subject goes to surgery) 
or PET-CT results (if the subject goes to chemotherapy) as part of their management. 
In this study we do not focus on whether an SLN is correctly identified, as there is no way of 
knowing that (unless the subject goes for surgery and the SLN has metastatic deposits but his 
will only be the case for a small subset) and the number of SLNS not identified by 
lymphosonography or by the standard of care will, for obvious reasons, be unknown. Hence, we 
presume all identified SLN are correctly identified. 
11.1 Study Hypotheses 
Primary hypothesis: Does lymphosonography identify more SLNs in patients with cervical, vaginal 
or vulvar cancer than the standard of care? The fundamental hypothesis is that on average 
lymphosonography will detect 20% more SLNs than the standard of care. 
Secondary hypothesis: Does lymphosonography identify more SLNs with metastatic deposits in 
patients with cervical, vaginal or vulvar cancer compared to the standard of care?  The 
fundamental hypothesis is that approximately 20% more cancerous SLNs will be detected with 
CEUS. 
 
H1: On average lymphosonography will detect 20% more SLN than the standard of care (blue 
dye or PET-CT). 
H10 The mean number of SLNs per subject identified by lymphosonography is less than 
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H1a The mean number of SLNs per subject identified by lymphosonography is greater 
than 120% of those identified by standard of care. 
 
H2: On average 20% more cancerous SLNs will be detected by lymphosonography than by 
standard of care (i.e., blue dye) using pathology as the reference standard.  
H20 The % of metastatic SLNs identified by lymphosonography is less than 120% of that 
identified by standard of care. 
H2a The % of metastatic SLNs identified by lymphosonography is greater than 120% of 
that identified by standard of care. 
11.2 Analysis Plans 
Thomas Jefferson University Hospital cares for approximately 200 patients with female lower 
genital tract cancer per year.  The patients studied in this project will be women over the age of 
18 who have a diagnosis of cervical, vaginal or vulvar cancer. The patient population of this project 
will reflect the population demographics found at major American urban academic health centers.  
The overall TJUH demographics include 60 % Caucasian, 16 % African American, 13 % Hispanic, 
5 % Asian, 1 % Other, and 5 % unknown patients. The goal of this pilot study is to enroll 40 
patients over 2 years. Given that these women have been diagnosed with cancer, they are usually 
very motivated to participate in research studies that include additional evaluations and, therefore, 
we do not expect recruitment to be a major issue.  
For the primary hypothesis the number of SLN detected by CEUS will be compared to the number 
of SLN detected by the standard of care (i.e., by blue dye or by PET-CT) using paired t-test or 
paired Wilcoxon test (depending on whether the data is normal distributed or not). 
For the secondary hypothesis the percentage of SLNs with metastatic deposits identified by 
lymphosonography and by blue dye will be pairwise compared after eliminating the SLNs 
identified by both methods using two-sample t-test or Wilcoxon test (again after testing for 
normalcy). The histopathological assessment of the surgically removed LNs will serve as the 
reference standard. 
11.3 Interim Analyses and Stopping Rules 
None. 
11.3.1 Safety Review 
The investigator is responsible for the detection and documentation of events meeting the criteria 
and definition of an AE or an SAE. 
11.3.2 Efficacy Review 
See section 11.2 
11.4 Sample Size Considerations 
The sample size analysis is based on the primary aim of this study (i.e., on Aim 1) and assumes 
that on average lymphosonography will detect 20% more SLN than the standard of care (blue dye 
or PET-CT]. Hence, a power analysis was performed using NCSS/PASS 2019 (NCSS, East 
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0.5 (i.e., assuming the 20% more detected SLN with CEUS versus the standard of care 
techniques, and twice of standard deviation of paired difference) at 5% significance level using a 
two-sided paired t-test. 
11.4.1 Replacement Policy 
Subjects that withdraw from the study will not be replaced.  If a sufficiently high number of subjects 
withdraw, the protocol will be amended to allow for greater enrollment. 
11.4.2 Accrual Estimates 
40 adult women, who have a diagnosis of cervical, vaginal or vulvar cancer, will be enrolled in 
this clinical trial at TJU. 
11.5 Exploratory Analysis 
The statistical analyses above will be repeated split by cancer type, but no firm conclusions are 
expected given this is a pilot study with a limited sample size. 
11.6 Handling Screen Failure/Subject Discontinuation 
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.   
If a subject's participation in the trial is interrupted for any reason (e.g., because of an AE) and 
the subject has met the criteria described above for completing the trial, the subject's trial 
participation will be considered completed. If a subject's trial participation is interrupted for any 
reason by the subject's or investigator's choice and the subject has not met all of the criteria listed 
above, then the subject will be considered a discontinued subject. 
12 Source Documents and Access to Source Data/Documents 
Study staff will maintain appropriate medical and research records for this study, in compliance 
with ICH E6, and regulatory and institutional requirements for the protection of confidentiality of 
participant information. Study staff will permit authorized representatives of SKCC and regulatory 
agencies to examine (and when required by applicable law, to copy) research records for the 
purposes of quality assurance reviews, audits, and evaluation of the study safety, progress and 
data validity.  
Source Data Verification 
The investigator will be visited on a regular basis by the Study Monitor. Monitoring will include 
source data verification (SDV) and discuss the progress of the study.  SDV is confirmed by 
comparing completed CRFs with matched source documentation in subject’s research binder. 
Monitors will also review drug usage logs according to European GCP. The PI will perform spot 
checks to verify CRFs and database entries match source documents. Verified data is entered 


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into a REDCap computer database by the study coordinator from the completed CRF’s for 
statistical evaluation at Thomas Jefferson University.  
The monitor and/or regulatory authorities will be allowed audits at the investigation site and source 
data verification in which case a review of those parts of the hospital records relevant to the study 
may be required.  
13 Quality Control and Quality Assurance 
Case report forms (CRF) will be provided for the recording of all data. Data will be recorded 
directly and legibly onto the record forms, in blue/black ink. The signature of the investigator will 
attest the accuracy of the data on each CRF. If any assessments are omitted, the reason for such 
omissions will be noted on the CRFs. Corrections, with the reason for the corrections, should be 
made legibly, dated and initialled. Correction fluid is not allowed. All original data collected with 
paper and pen will immediately be recorded electronically by the study coordinator within the 
RedCap database.  
Data collection and accurate documentation are the responsibility of the study staff under the 
supervision of the investigator. All source documents and laboratory reports must be reviewed by 
the study team and data entry staff, who will ensure that they are accurate and complete. UAPs 
and AEs must be reviewed by the investigator or designee. The monitor and/or regulatory 
authorities will be allowed audits at the investigation site and source data verification in which 
case a review of those parts of the hospital records relevant to the study may be required. 
14 Ethics/Protection of Human Participants  
14.1 Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research, as drafted by the US National Commission for the Protection of Human 
Subjects of Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 
and/or the ICH E6.  
14.2 Institutional Review Board 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval. Approval of both the protocol and the consent form 
must be obtained before any participant is enrolled. Any amendment to the protocol will require 
review and approval by the IRB before the changes are implemented in the study.  
 
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Informed consent is a process that is initiated prior to the individual agreeing to participate in the 
study and continues throughout study participation. Extensive discussion of risks and possible 
benefits of study participation will be provided to participants and their families, if applicable. A 
consent form describing in detail the study procedures and risks will be given to the participant. 
Consent forms will be IRB-approved, and the participant is required to read and review the 
document or have the document read to him or her. The investigator or designee will explain the 
research study to the participant and answer any questions that may arise. The participant will 
sign the informed consent document prior to any study-related assessments or procedures. 
Participants will be given the opportunity to discuss the study with their surrogates or think about 
it prior to agreeing to participate. They may withdraw consent at any time throughout the course 
of the study. A copy of the signed informed consent document will be given to participants for their 
records. The rights and welfare of the participants will be protected by emphasizing to them that 
the quality of their clinical care will not be adversely affected if they decline to participate in this 
study. The consent process will be documented in the esearch record.  
14.4 Exclusion of Women, Minorities, and Children (Special Populations) 
The subject population of this study will be 100% women over 18 years old. Children, and 
pregnant women will be excluded based on the benefit and risk assessment. No patient will be 
excluded based on race. We include only female 18 years of age or older regardless of race and 
speaking language. Since this study does not provide direct benefit to subjects, we determined 
the age limit of 18 so no state law requires parent’s consent for our subject to participate in the 
study.  
14.5 Participant Confidentiality 
Participant confidentiality is strictly held in trust by the investigators, study staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological samples 
and genetic tests in addition to any study information relating to participants. 
The study protocol, documentation, data, and all other information generated will be held in strict 
confidence. No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all study 
documents and records required to be maintained by the investigator, including but not limited to, 
medical records (office, clinic, or hospital) for the study participants. The clinical study site will 
permit access to such records. 
Data forms will be completed for all subjects enrolled in the trial.  The subject study files will be 
stored in a secure file cabinet and maintained by the research study coordinator.  Study files will 
be kept for 7 years after the completion of the study. The PI of the study (Dr. Forsberg) and his 
co-investigators have previous experience running ultrasound clinical trials and the PI will serve 
as the study sponsor.  He will be responsible for ensuring all AEs are properly reported. 
The final data will be entered into a database implemented in REDCap (Vanderbilt University, 
Nashville, TN). The investigators will be responsible for management of the database. The 
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15 Data Handling and Record Keeping 
The investigators are responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. All source documents must be completed in a neat, legible manner 
to ensure accurate interpretation of data. The investigators will maintain adequate case histories 
of study participants, including accurate case report forms (CRFs), and source documentation.  
All analyses and computations will be performed using NCSS/PASS 2008 and Stata 15.1 (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 the cancer treatment, pathology, CEUS 
and PET-CT as well as the clinical variables. 
15.1 Data Management Responsibilities 
Data collection and accurate documentation are the responsibility of the study staff under the 
supervision of the investigator. All source documents and laboratory reports must be reviewed by 
the study team and data entry staff, who will ensure that they are accurate and complete. The 
investigator or designee must review unanticipated problems and deviations.  
15.2 Data Capture Methods 
Data reflecting participant's experiences with the study will be recorded on CRFs by the 
investigator. 
All patient data will be captured electronically via REDCap (Vanderbilt University, Nashville, TN) 
in a password protected computer and image data will be stored in the secured computer hard 
drive. Image data will be de-identified. Copies of study documents will have personal identifying 
information retracted and replaced with study patient ID number. Originals of the study documents 
with personal identifying information will be retained at TJU. All this information will be available 
for audit.  
15.3 Types of Data 
We are committed to respecting patient’s privacy and to keeping their personal health information 
confidential. The personal health information includes the information in Health care records and 
information that can identify the patient. For example, personal information may include name, 
address, phone number, social security number, and medical information.  
The personal health information that may be collected, used, and shared for this research 
includes: 
• 
Information from medical records  
• 
Demographic information such as name, gender, birth date, ethnicity, medical history 
and health care providers 
• 
Physical examinations, procedures, imaging tests, labs, medical conditions and 
medications 
• 
Information collected about any research related injury 
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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. 
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 surgical 
reports, PET-CT studies, ultrasound images, subject progress notes, hospital charts, 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. 
15.5 Protocol Deviations 
A protocol deviation is any noncompliance with the protocol, International Conference on 
Harmonization (ICH), Good Clinical Practice (GCP) and protocol-specific guidelines. The 
deviation (any activity conducted outside the parameters established by the protocol) may be 
either on the part of the participant, the investigator, or the study site staff and may or may not 
pose a risk to participants or others or may affect the integrity of the data obtained from the study.   
The risk posed by the deviation, to the study or the study participant gives rise to an Unanticipated 
problem (UAP). It is crucial to document the deviation/unanticipated problem in the protocol 
deviation log (Appendix D) and submitted to the IRB as per the sites regulations.  As a result of 
deviations, corrective actions are to be developed and implemented promptly.   
UAPs and protocol deviations that pose risk to participants or others, and that are not AEs, or that 
affect study integrity will be submitted to the IRB via the <eazUP system> within 5 working days 
of the investigator becoming aware of the event.  
UAPs and protocol deviations that do not pose risk to participants or others and do or do not affect 
study integrity must be entered in the deviation log (Appendix D) and submitted to the IRB at the 
next continuing review.  
GE Healthcare will be informed of all AEs within 48 hours. In addition all unexpected SAEs are 
reported to the TJU IRB, the SKCC DSMB and to the FDA. 
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The relationship or association of the AE to the Sonazoid 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 Sonazoid and will be recorded on a data form.  
The investigator will refer to the Sonazoid investigator brochure for assistance in determining AE 
relationship. 
An "unlikely" relationship indicates that there is little or no chance that Sonazoid 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 Sonazoid 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 Sonazoid administration and, based upon the investigator's clinical experience, the 
association of the event with the trial medication seems likely. 
16 Study Finances 
16.1 Funding Source 
This study is financed through a grant from the U.S. National Institute of Health (NIH): R21 
CA249870. 
16.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or 
financial gain greater than the minimum allowable by their institution, etc.) must have the conflict 
reviewed by a properly constituted Conflict of Interest Committee with a Committee-sanctioned 
conflict management plan that has been reviewed and approved by the study sponsor prior to 
participation in this study. All Jefferson University Investigators will follow the TJU Conflicts of 
Interest Policy for Employees (107.03). 
16.3 Participant Stipends or Payments 
Participants will not receive payment for participating in this study. 
17 Publication and Data Sharing Policy 
Upon study completion and finalization of the study report the results of this study will either be 
submitted for publication and/or posted in a publicly assessable database of clinical study results. 
The results of this study will also be submitted to the Competent Authority and the Ethics 
Committee according to USA national regulations. 
All personnel who have contributed significantly with the planning and performance of the study 
(Vancouver convention 1988) may be included in the list of authors.  


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18 Literature References 
Forsberg F, Piccoli CW, Liu JB, Rawool NM, Merton DA, Mitchell D, Goldberg BB.  Hepatic 
tumor detection: MR imaging and conventional US versus pulse-inversion harmonic US of 
NC100100 during its reticuloendothelial system-specific phase.  Radiology 2002; 222:824–829. 
Dietrich CF, et al. Guidelines and good clinical practice recommendations for contrast-enhanced 
ultrasound (CEUS) in the liver-update 2020 WFUMB in cooperation with EFSUMB, AFSUMB, 
AIUM, and FLAUS. Ultrasound Med Biol. Jul 22:S0301-5629(20)30200-3, (2020). doi: 
10.1016/j.ultrasmedbio.2020.04.030. Online ahead of print. 
Gimenez J, Botella-Estrada R, Hernandez D, et al. Anaphylaxis after peritumoral injection of 
sulphan blue 1% for identification of the sentinel node in lymphatic mapping of the breast. Eur J 
Surg 2001;167:921-923. 
Goldberg BB, Raichlen JS, Forsberg F (eds.).  Ultrasound Contrast Agents: Basic Principles 
and Clinical Applications (2nd Ed). Martin Dunitz Ltd., England, 2001. 
Goldberg BB, Merton DA, Liu JB, Forsberg F, Zhang K, Thakur M, Schulz S, Schanche R, 
Murphy GF, Waldman SA.  Contrast-enhanced ultrasound imaging of sentinel lymph nodes after 
peritumoral administration of Sonazoid in a melanoma tumor animal model.  J Ultrasound Med 
2011; 30:441–453. 
Goldfarb LR, Alazraki NP, Eshima D, Eshima LA, Herda SC, Halkar RK: Lymphoscintigraphic 
identification of sentinel lymph nodes: clinical evaluation of 0.22-micron filtration of Tc-99m 
sulfur colloid. Radiology 1998;208:505-509. 
Huang B, Law MW-M, Khong P-L. Whole-body PET/CT scanning: estimation of radiation dose 
and cancer risk. Radiology. 2009;251(1):166-74. 
Machado P, Stanczak M, Liu JB, Moore JN, Eisenbrey JR, Needleman L, Kraft WK, Forsberg F. 
Subdermal Ultrasound Contrast Agent Injection for Sentinel Lymph Node Identification: An 
Analysis of Safety and Contrast Agent Dose in Healthy Volunteers. Journal of ultrasound in 
medicine : official journal of the American Institute of Ultrasound in Medicine. 2018;37(7):1611-
20. PMID: 29205451; PMCID: PMC5988650. 
Mattrey R, Kono Y, Baker K, Peterson T, editors. Sentinel lymphnode detection with 
microbubble ultrasound contrast. Radiology; 1999: RADIOLOGICAL SOC NORTH AMER 20TH 
AND NORTHAMPTON STS, EASTON, PA 18042 USA. 
Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, Stein KD, Alteri R, Jemal A. 
Cancer treatment and survivorship statistics, 2016. CA: a cancer journal for clinicians. 
2016;66(4):271-89. 
Morton D, Chan A.  The concept of sentinel node localization:  How it started.  Semin Nucl Med 
2000; 30:4-10. 
Sontum PC.  Physicochemical characteristics of Sonazoid, a new contrast agent for ultrasound 
imaging.  Ultrasound Med. Biol., 2008; 34:824-833. 


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Tokin CA, Cope FO, Metz WL, et al.  The efficacy of Tilmanocept in sentinel lymph mode 
mapping and identification in breast cancer patients: a comparative review and meta-analysis of 
the ⁹⁹mTc-labeled nanocolloid human serum albumin standard of care.  Clin Exp Metastasis. 
2012; 29:681-686. [PMID:  22729510] 
Appendices 
The following documents are officially affiliated with the protocol and will be submitted to the IRB 
as a part of the protocol. As such, changes to these items require a protocol amendment. 
Appendix A: Schedule of Events 
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 


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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. 
 
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 UEA procedure 
reports, MRI studies, ultrasound images, subject progress notes, hospital charts, 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 


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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, PhD, 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 
CEUS data acquisition from patients.  Dr. Forsberg will also prepare any manuscript(s) 
resulting from this grant.   
 
Norman G. Rosenblum, MD, PhD Professor of Obstetrics and Gynecology will assist with 
the patient recruitment, UCA injections and advise on clinical issues. 
 
Scott D. Richard, MD, Associate Professor of Obstetrics and Gynecology will assist with 
the patient recruitment, UCA injections and advise on clinical issues. 
 
Lindsey E. Minion, MD, Associate Professor of Obstetrics and Gynecology will assist with 
the patient recruitment, UCA injections and advise on clinical issues. 
 
Andrej Lyshchik, M.D., Ph.D, Assistant Professor of Radiology will aid in patient 
recruitment, informed consent, ultrasound studies, and advise on clinical issues. 
 
Ji-Bin Liu, M.D., Research Professor of Radiology will assist with the patient recruitment, 
interpret ultrasound images and advise on clinical issues  
 


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Priscilla Machado, MD, Project Manager will perform ultrasound studies and will also 
prepare any manuscript(s) resulting from this project.