Contrast-Enhanced Ultrasound for Diagnosis and Therapy of Cholangiocarcinoma
Phase: PHASE2
Conditions: Intrahepatic Cholangiocarcinoma
Sponsor: Thomas Jefferson University

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THOMAS JEFFERSON UNIVERSITY 
Sidney Kimmel Cancer Center 
 
Contrast-Enhanced Ultrasound for Diagnosis and Therapy of Cholangiocarcinoma 
 
Principal Investigators: 
John Eisenbrey, PhD 
Associate Professor 
Department of Radiology 
Thomas Jefferson University  
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: John.Eisenbrey@jefferson.edu 
Tel: 215-503-5188 
Fax: 215-955-8549 
 
 
 
Co-Investigator(s): 
Kevin Anton, MD 
Assistant Professor 
Department of Radiology 
Thomas Jefferson University 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: Kevin.Anton@jefferson.edu 
Tel: 215-955-4998 
Fax: 215-955-6754 
 
Flemming Forsberg, PhD 
Professor 
Department of Radiology,  
Thomas Jefferson University, 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
E-mail address: flemming.forsberg@jefferson.edu 
Tel : 215-955 4870 
Fax: 215-955-8549 
 
Andrej Lyshchik, MD 
Associate Professor 
Department of Radiology,  
Thomas Jefferson University, 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: Andrej.Lyshchik@jefferson.edu 
Tel: 215-503-0587 
Fax: 215-955-8549 
 
Patrick O’Kane, MD 
Professor 
Department of Radiology,  

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Thomas Jefferson University, 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
E-mail address: Patrick.OKane@jefferson.edu 
Tel: 215-955-8749 
Fax: 215-955-8549 
 
Jesse Civan, MD 
Assistant Professor 
Department of Internal Medicine,  
Thomas Jefferson University, 
132 South 10th Street, 4th Floor 
Philadelphia, PA 19107 
E-mail address: Jesse.Civan@jefferson.edu 
Tel: (215) 955-8900 
Fax: (215) 503-2146 
 
James Posey, MD 
Professor 
Department of Medical Oncology 
Thomas Jefferson University, 
132 South 10th Street 
Philadelphia, PA 19107 
Email address: James.PoseyIII@jefferson.edu  
Tel: 215-955-8874 
 
Kibo Nam, PhD 
Research Assistant Professor  
Department of Radiology,  
Thomas Jefferson University, 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: Kibo.Nam@jefferson.edu 
Tel: 215-955-4998 
 
Alexander E. Trebelev, MD 
Associate Professor 
Department of Radiology,  
Thomas Jefferson University, 
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: Alexander.Trebelev@jefferson.edu 
 
 
Statistician: 
Scott W. Keith, PhD 
Associate Professor 
Department of Pharmacology & Experimental Therapeutics,  
Thomas Jefferson University, 
1015 Chestnut Street, 5th Floor 
Philadelphia, PA 19107 
Email: Scott.Keith@jefferson.edu 

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Tel: (215) 503-9876 
Fax: (215) 503-3804 
 
Funding Sponsor: 
NIH/NCI via R21 CA259750 
 
IND/IDE Holder: 
John Eisenbrey, PhD 
IND/IDE Number: 
126,768 
Study Product: 
Optison, an ultrasound contrast agent 
Protocol IDs: 
JeffTrial # pending 
PRC # pending 
IRB Control # pending 
 
Version Number: 
Version Date: 
1.0 
21 July, 2021 
1.1 
10 August, 2021 
1.2 
25 August 2022 
2.0 
27 June 2023 
2.1 
30 January 2025 
 
CONFIDENTIAL 
This document is confidential and the property of THOMAS JEFFERSON UNIVERSITY. 
No part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 
 

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Table of Contents  
 
Signature Page ............................................................................................................................. 9 
Statement of Compliance .............................................................................................................. 9 
List of Abbreviations .................................................................................................................... 10 
Study Summary .......................................................................................................................... 12 
1 
Introduction ........................................................................................................................... 15 
1.1 
Background Information ............................................................................................... 15 
1.2 
Rationale for the Proposed Study ................................................................................. 19 
1.3 
Correlative Studies ....................................................................................................... 21 
1.4 
Potential Risks and Benefits ......................................................................................... 21 
1.4.1 Potential Risks .......................................................................................................... 21 
1.4.2 Benefits ..................................................................................................................... 22 
2 
Study Objectives .................................................................................................................. 23 
2.1 
Objectives ..................................................................................................................... 23 
2.1.1 Primary ...................................................................................................................... 23 
2.1.2 Secondary ................................................................................................................. 23 
2.1.3 Exploratory ................................................................................................................ 23 
2.2 
Endpoints/Outcome Measures ..................................................................................... 23 
2.2.1 Primary ...................................................................................................................... 24 
2.2.2 Secondary ................................................................................................................. 24 
3 
Study Design ........................................................................................................................ 24 
3.1 
Characteristics .............................................................................................................. 24 
3.2 
Number of Participants ................................................................................................. 24 
3.3 
Duration of Therapy ...................................................................................................... 24 
3.4 
Duration of Follow Up ................................................................................................... 25 
3.5 
Treatment Assignment Procedures .............................................................................. 25 

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3.5.1 Randomization Procedures (if applicable) ................................................................ 25 
3.5.2 Masking Procedures (if applicable) ............................. Error! Bookmark not defined. 
3.6 
Study Timeline .............................................................................................................. 25 
3.6.1 Primary Completion .................................................................................................. 25 
3.6.2 Study Completion ...................................................................................................... 25 
4 
Study Enrollment and Withdrawal ........................................................................................ 25 
4.1 
Eligibility Criteria ........................................................................................................... 26 
4.1.1 Inclusion Criteria ....................................................................................................... 26 
4.1.2 Exclusion Criteria ...................................................................................................... 26 
4.2 
Gender/Minority/Pediatric Inclusion for Research ........................................................ 27 
4.3 
Strategies for Recruitment and Retention .................................................................... 27 
4.4 
Participant Withdrawal .................................................................................................. 27 
4.4.1 Reasons for Withdrawal ............................................................................................ 27 
4.4.2 Handling of Participant Withdrawals and Participant Discontinuation of Study 
Intervention ........................................................................................................................... 28 
4.5 
Premature Termination or Suspension of Study ........................................................... 28 
5 
Study Intervention ................................................................................................................ 28 
5.1 
Study Product ............................................................................................................... 28 
5.1.1 Acquisition ................................................................................................................. 28 
5.1.2 Formulation, Packaging, and Labeling ...................................................................... 28 
5.1.3 Product Storage and Stability ................................................................................... 29 
5.2 
Dosage, Preparation, and Administration ..................................................................... 29 
5.3 
Dose Modifications and Dosing Delays ........................................................................ 29 
5.4 
Study Product Accountability ........................................................................................ 29 
5.5 
Assessing Participant Compliance with Study Product Administration......................... 29 
5.6 
Concomitant Medications/Treatments .......................................................................... 29 
5.7 
Dietary Restrictions ...................................................................................................... 29 

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5.8 
Study Procedural Intervention(s) Description ............................................................... 29 
5.9 
Administration of Procedural Intervention ..................................................................... 31 
5.10 
Procedures for Training of Clinicians on Procedural Intervention................................. 31 
5.11 
Assessment of Clinician and/or Participant Compliance with Study Procedural 
Intervention .............................................................................................................................. 31 
6 
Study Schedule .................................................................................................................... 31 
6.1 
Pretreatment Period/Screening .................................................................................... 31 
6.2 
Enrollment/Baseline ...................................................................................................... 32 
6.3 
Treatment Period .......................................................................................................... 32 
6.4 
Long Term/Survival Follow-up ...................................................................................... 32 
6.5 
Withdrawal Visit/Discontinuation of Therapy ................................................................ 32 
7 
Study Procedures and Evaluations ...................................................................................... 33 
7.1 
Study Procedures/Evaluations ..................................................................................... 33 
8 
Evaluation of Safety ............................................................................................................. 33 
8.1 
Specification of Safety Parameters .............................................................................. 34 
8.1.1 Unanticipated Problems ............................................................................................ 34 
8.1.2 Adverse Events ......................................................................................................... 34 
8.1.3 Serious Adverse Events ............................................................................................ 34 
8.2 
Safety Assessment and Follow-Up ............................................................................... 35 
8.3 
Recording Adverse Events ........................................................................................... 35 
8.3.1 Relationship to Study Intervention ............................................................................ 36 
8.3.2 Expectedness ........................................................................................................... 36 
8.3.3 Severity of Event ....................................................................................................... 37 
8.3.4 Intervention ............................................................................................................... 37 
8.4 
Safety Reporting ........................................................................................................... 37 
8.4.1 Reporting to IRB ....................................................................................................... 37 
8.4.2 Reporting to SKCC DSMC ........................................................................................ 38 

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8.4.3 Reporting to Funding Sponsor .................................................................................. 40 
8.4.4 Reporting to FDA ...................................................................................................... 40 
8.4.5 Reporting of Pregnancy ............................................................................................ 40 
8.5 
Halting Rules ................................................................................................................ 41 
9 
Study Oversight .................................................................................................................... 41 
10 
Clinical Site Monitoring and Auditing ................................................................................ 41 
11 
Statistical Considerations ................................................................................................. 41 
11.1 
Study Hypotheses ........................................................................................................ 41 
11.2 
Analysis Plans .............................................................................................................. 42 
11.3 
Interim Analyses and Stopping Rules ........................................................................... 43 
11.3.1 
Safety Review ....................................................................................................... 43 
11.3.2 
Efficacy Review ..................................................................................................... 43 
11.4 
Sample Size Considerations ........................................................................................ 43 
11.4.1 
Replacement Policy ............................................................................................... 43 
11.4.2 
Accrual Estimates .................................................................................................. 43 
11.5 
Exploratory Analysis ..................................................................................................... 44 
11.6 
Evaluation of Safety ...................................................................................................... 44 
12 
Source Documents and Access to Source Data/Documents ........................................... 44 
13 
Quality Control and Quality Assurance ............................................................................ 46 
14 
Ethics/Protection of Human Participants .......................................................................... 46 
14.1 
Ethical Standard ........................................................................................................... 46 
14.2 
Institutional Review Board ............................................................................................ 46 
14.3 
Informed Consent Process ........................................................................................... 46 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) .......................... 47 
14.5 
Participant Confidentiality ............................................................................................. 47 
15 
Data Handling and Record Keeping ................................................................................. 47 

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15.1 
Data Management Responsibilities .............................................................................. 48 
15.2 
Data Capture Methods ................................................................................................. 48 
15.3 
Types of Data ............................................................................................................... 48 
15.4 
Study Records Retention .............................................................................................. 49 
15.5 
Protocol Deviations ....................................................................................................... 49 
16 
Study Finances ................................................................................................................ 49 
16.1 
Funding Source ............................................................................................................ 49 
16.2 
Conflict of Interest ......................................................................................................... 49 
16.3 
Participant Stipends or Payments ................................................................................ 49 
17 
Publication and Data Sharing Policy ................................................................................ 49 
18 
Literature References ....................................................................................................... 50 
SUPPLEMENTAL MATERIALS .................................................................................................. 54 
Appendices ................................................................................................................................. 54 
APPENDIX A: SCHEDULE OF EVENTS ................................................................................... 55 
 

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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 Investigators: 
Signed: 
 
Date: 
 
Name: 
John Eisenbrey, PhD 
Title: 
 
Associate Professor of Radiology 
Signed: 
 
Date: 
 
Name: 
Kevin Anton, MD, PhD 
Title: 
 
Assistant Professor 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/Adverse Experience 
CEUS 
Contrast-enhanced Ultrasound 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CR 
Complete Response 
CRF 
Case Report Form 
CRO 
Clinical Research Organization 
CT 
Computed Tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
DSMC 
Data and Safety Monitoring Committee 
DSMP 
Data and Safety Monitoring Plan 
ECOG 
Eastern Cooperation Oncology Group 
EMR 
Electronic Medical Records 
FDA 
Food and Drug Administration 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
GWAS 
Genome-Wide Association Studies 
HCC 
Hepatocellular Carcinoma 
HI 
Harmonic Imaging 
HIPAA 
Health Insurance Portability and Accountability Act 
HVPG 
Hepatic Venous Pressure Gradient 
IB 
Investigator’s Brochure 
ICC 
Intrahepatic Cholangiocarcinoma 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISPTA 
Spatial-peak Temporal-average Intensity 
LFT 
Liver Function Test 
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
Mechanical Index 
MOP 
Manual of Procedures 
MRI 
Magnetic Resonance Imaging 

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mRECIST 
Modified Response Evaluation Criteria in Solid Tumors 
N 
Number (typically refers to participants) 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
OLS 
Ordinary Least Squares 
PHI 
Protected Health Information 
PR 
Partial Response 
PI 
Principal Investigator 
PRC 
Protocol Review Committee 
QA 
Quality Assurance 
QC 
Quality Control 
ROI 
Region of Interest 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SDS 
Safety Data Sheet (formerly MSDS; Material Safety Data Sheet) 
SHAPE 
Subharmonic Aided Pressure Estimation 
SKCC 
Sidney Kimmel Cancer Center 
SoC 
Standard of Care 
SOP 
Standard Operating Procedure 
SPECT 
Single Photon Emission Computed Tomography 
TARE 
Transarterial Radioembolization 
TACE 
Transarterial Chemoembolization 
TJU 
Thomas Jefferson University 
UAP 
Unanticipated Problem 
UCA 
Ultrasound Contrast Agent 
UTMD 
Ultrasound-triggered microbubble destruction 
Y-90 
Yttrium-90 
 
 

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Study Summary 
Title: 
Contrast-Enhanced Ultrasound for Diagnosis and Therapy of 
Cholangiocarcinoma 
Précis: 
20 patients with biopsy-proven intrahepatic cholangiocarcinoma (ICC) 
or other non-HCC tumors in the liver will receive their standard of care 
(SoC) sub-lobar transarterial radioembolization (TARE), in addition to 
four sessions of ultrasound contrast infusion and ultrasound scanning. 
One session will be within 1 month prior to treatment and the other 
three will be at different time points after treatment. The treatment 
outcomes will be compared to a historical group of similar patients 
receiving TARE alone. The main hypotheses are that contrast-
enhanced ultrasound (CEUS) can be used to predict non-HCC 
response to TARE prior to therapy, improve the sensitivity of these 
tumors to radiotherapy, and assess treatment response earlier than 
the current clinical standard.  
Objectives: 
 
To evaluate the utility of CEUS-derived quantitative measures in 
predicting tumor response before and in the early phases after TARE 
treatment, and to evaluate the safety and efficacy of microbubble 
cavitation in improving TARE treatment.  
 
Primary:  
• 
To determine the ability of quantitative volumetric CEUS to 
predict tumoral response to TARE prior to therapy. 
 
Secondary:  
• 
To characterize the safety and preliminary efficacy of using 
localized UCA inertial cavitation to improve non-HCC tumor 
response to radioembolization. 
• 
To determine if CEUS estimated tumor perfusion and residual 
vascularity can predict non-HCC tumor response to 
radioembolization 7-14 days post treatment. 
• 
To evaluate tumoral response using the patient’s 1 month MRI 
(obtained clinically) and determine the accuracy of MR or CT 
tumor evaluation at this earlier time point. 
Exploratory: 
• 
To examine the utility of SHAPE to noninvasively monitor 
tumoral interstitial fluid pressure (IFP) and provide an early 
biomarker of radiotherapy response. 
Population: 
20 patients with intrahepatic cholangiocarcinoma (ICC) or other non-
HCC tumors receiving sub-lobar transarterial radioembolization 

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(TARE). Patients at least 18 years of age, medically stable, and have 
a previously untreated tumor greater than 1 cm but small enough to 
be visualized in the ultrasound 3D volume will be included. These 
patients will be accrued from the Hepatology department, 
Interventional Radiology, and the Multidisciplinary Liver Tumor Board 
of the Sidney Kimmel Cancer Center at Thomas Jefferson University. 
All subjects must have read, signed, and dated the consent form(s) 
approved by the institutional review board of Thomas Jefferson 
University. 
Phase: 
Pilot 
Number of Sites: 
Thomas Jefferson University, Philadelphia, PA, USA 
Description of 
Intervention: 
Five ml of activated Optison will be suspended in 50 ml of saline and 
infused through an 18- to 22-gauge angiocatheter placed in an 
antecubital vein at a rate of 120 ml/hour (dosages based on prior 
approved Optison ultrasound-triggered microbubble destruction 
(UTMD) protocol and product insert) at the time points described 
above. All imaging will be performed using a modified Logiq E10 
system with RAB6-D probe (GE Healthcare). For the second aim, a 
destructive (high mechanical index (MI)) pulse will be generated 
across the selected volume following peak enhancement (center 
frequency 2.1 MHz, pulse length 20 μs, line density 4). Acoustic 
pressure amplitudes will be adjusted as necessary to ensure inertial 
microbubble cavitation. 
Study Duration: 
24 months 
Participant 
Participation 
Duration: 
3-6 weeks 
Estimated Time to 
Complete 
Enrollment: 
36 months 

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Schematic of Study Design: 
 
  
 
Prior to  
Enrollment 
 
 
 
 
 
< 1 month  
Pre-TARE  
 
 
1-4 hours 
Post-TARE 
 
 
1 week 
Post-TARE 
 
 
2 weeks 
Post-TARE 
 
 
1-2 months 
Post-TARE 
 
 
 
3-6 months 
Post-TARE 
 
 
 
 
 
 
 
 
 
Total N: 20 
Pre-treatment MRI/CT and flow study 
 Screen potential subjects by inclusion and exclusion criteria; obtain history, document. 
Obtain informed consent. 
Quantitative volumetric contrast-enhanced ultrasound (CEUS) scan 
First follow-up MRI/CT 
Second follow-up MRI/CT 
CEUS with inertial microbubble cavitation. 
CEUS with inertial microbubble cavitation. 
CEUS with inertial microbubble cavitation. 

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1 
Introduction 
1.1 
Background Information 
 
Intrahepatic non-HCC tumors 
A variety of both primary and metastatic cancers present in the liver. Cholangiocarcinoma is an 
aggressive epithelial tumor arising from the lining of the biliary tract with high morbidity and 
extremely poor prognosis. There are three tumor subtypes defined by the anatomical region from 
which they arise – intrahepatic, peri-hilar and distal (extrahepatic) bile ducts. Approximately 10% 
of cholangiocarcinomas are intrahepatic. Intrahepatic cholangiocarcinoma (ICC) is the second 
most common primary liver malignancy after hepatocellular carcinoma (HCC), accounting for 10-
20% of primary liver tumors1. Sadly, ICC incidence and mortality have been increasing in Western 
countries over the past two decades, highlighting the need to improve therapeutic options2. 
Additionally, many non-liver cancers will first metastasis to the liver. These include (but are not 
limited to) uveal melanoma, neuroendocrine tumors, pancreatic cancers, breast cancer, and 
colorectal cancers. Management options for non-HCC tumors in the liver are limited and the 
disease generally portends a poor prognosis. Liver transplant is currently not an option and while 
curative surgical resection is the first line of therapy in patients with ICC without extension beyond 
the regional lymph nodes, it is only possible in less than 30% of cases with median disease free 
survival of 26 months1,3. The majority of patients present with locally advanced or metastatic 
disease and are treated with systemic chemotherapy or loco-regional therapies. Liver-directed 
therapies including thermal ablation, transarterial chemoembolization (TACE), and transarterial 
radioembolization (TARE) are indicated for localized tumors4,5.  
 
Transarterial radioembolization 
A meta-analysis of 12 studies in which 298 patients suffering ICC were treated with Yttrium-90 
(Y-90) radioembolization reported overall median survival of 15.5 months6. TARE can be offered 
to patients with adequate performance status according to Eastern Cooperation Oncology Group 
(ECOG) performance score, blood cell count, liver function, and kidney function4. Additionally, 
TARE is being increasingly used for the treatment of other non-HCC solid tumors in the liver 
including colorectal cancer metastasis, uveal melanoma metastasis, and neuroendocrine 
metastasis. Using this approach, radioactive microspheres are delivered via a catheter placed in 
the hepatic artery branch supplying the tumor, thereby providing a localized and sustained release 
of radiation. The microspheres used are 20-30 µm diameter glass beads containing Y-90. Y-90 is 
a pure beta emitter. It decays to stable Zirconium-90 with a half-life of 64 hours and has an 
average energy emission of 0.94 MeV. The maximum tissue penetration in the liver is 10 mm 7,8,9. 
Lobar doses range from 110-150 Gy, but radiation delivered to malignant tissue is dependent on 
distance from the yttrium source. Consequently, treatment response after radioembolization is 
between 25-60% when based on modified response criteria in solid tumors (mRECIST)9,6,10. 
 

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Ultrasound contrast agents 
Ultrasound contrast agents (UCA) are gas-filled microbubbles, encapsulated by a lipid or protein 
shell for stability. These agents are small enough (1-8 μm in diameter), to pass through the 
pulmonary capillaries, but are still restricted to the vascular system11. These microbubbles have 
been approved for use in echocardiography and also for the characterization of liver lesions in 
the United States, Europe and Asia12. UCA perfuse into the vasculature of liver tumors, and their 
wash-in/wash-out kinetics can be used to characterize liver masses (Figure 1). Both 2D and 4D 
CEUS can be acquired in hepatic tumors and used to quantify a variety of vascular parameters 
including blood flow dynamics and morphological features of the vasculature13,14. Our group has 
demonstrated the exceptional safety and accuracy of contrast-enhanced ultrasound (CEUS) for 
monitoring response to chemoembolization of HCC15. Importantly, we have shown that the 
vascular networks of HCC depicted by CEUS can be used to predict the likelihood of a response 
to TACE14. We have also shown that UCA perfuse into liver tumors post TARE, due to the fact 
that the larger Y-90 beads (20-30µm diameter) do not completely restrict blood flow. UCA can 
also be noninvasively destroyed in tissue using flash-replenishment sequences to generate 
inertial microbubble cavitation. Stable and inertial microbubble cavitation has demonstrated a 
variety of bioeffects useful for therapeutic applications16. Both stable and inertial cavitation have 
been shown to alter vascular and cellular permeability17. 
 
Ultrasound-triggered microbubble destruction (UTMD) has also been shown to sensitize tumors 
to radiotherapy by inducing vascular endothelial cell apoptosis18,19,20. This endothelial cell death 
has been attributed to increased ceramide production after interactions with inertially cavitating 
microbubbles21. Microbubble cavitation has also been shown to result in decreased tumor 
vascularity22. UTMD leads to increased DNA damage within the targeted tumor after radiotherapy, 
despite decreases in oxygenation21. Detailed parameter studies have been conducted in tumor 
xenografts, and synergistic combinations of UTMD and radiotherapy at mechanical indexes (MIs) 
> 0.8, radiation dosages of 1.8-8 Gy, and microbubble concentrations of 1-3% (v/v) have been 
explored18,19. Importantly, and yet somewhat paradoxically, UTMD in normal tissue increases 
vascular perfusion, due to cavitation-related increases in shear forces and activation of 
endothelial nitric oxide synthase23. Hence, UTMD combined with radiotherapy potentially offers a 
tumor-specific method of tissue sensitization. 
 
Optison 
The proposed agent for the current study, Optison (GE Healthcare, Princeton NJ), is a sterile non-
pyrogenic suspension of encapsulated perflutren microbubbles11,24. The contrast agent is consists 
of a human serum albumin shell with perflutren core and contains 5.0 - 8.0 x 108 microspheres/ 
ml. The microbubble agent is supplied in a standard-size 3 ml vial and is prepared by manually 
shaking the vial. Optison is currently only approved for use in echocardiography.  The agent will 
be used as an off-label indication for this study, but within the approved dosage 
recommendations.    

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Optison Clinical Safety 
Optison is well tolerated and has been used extensively in echocardiography applications11.  In 
pre-market clinical trials, Optison was administered to 279 patients.  In these patients 47 (16.8%) 
reported at least one adverse event.  Of these events, 1 was classified as serious and required 
antihistamines for hypersensitivity manifestations of dizziness, nausea, flushing, and temperature 
elevation.  No deaths were reported.  Of the reported adverse reactions following the use of 
Optison the most frequently reported were headache (5.4%), nausea and/or vomiting (4.3%), 
warm sensation or flushing (3.6%), and dizziness (2.5%).  Additional risks associated with the 
contrast material are described in the attached Optison Product insert (Appendix B).  All of the 
non-serious reported side effects have been transient, usually lasting only a few minutes.   
 
Table 1. Selected Adverse Events Reported in ≥ 0.5% of the Subjects who Received Optison in 
Controlled Clinical Studies (From Optison Product Insert). 
SELECTED ADVERSE EVENTS REPORTED IN ≥ 0.5% OF THE SUBJECTS WHO 
RECEIVED OPTISON™ IN CONTROLLED CLINICAL STUDIES   
No. of Patients Exposed to OPTISON™  
279  
No. of Patients Reporting on Adverse Event  
47 (16.8%)  
Body as a Whole  
38 (13.6%)  
Headache  
15 (5.4%)  
Warm Sensation/Flushing  
10 (3.6%)  
Chills/fever  
4 (1.4%)  
Flu-like Symptoms  
3 (1.1%)  
Malaise/Weakness/Fatigue  
3 (1.1%)  
Cardiovascular System  
12 (4.3%)  
Dizziness  
7 (2.5%)  
Chest Pain  
3 (1.1%)  
Digestive System  
12 (4.3%)  
Nausea and/or Vomiting  
12 (4.3%)  

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Nervous System  
3 (1.1%)  
Respiratory System  
5 (1.8%)  
Dyspnea  
3 (1.1%)  
Skin & Appendages  
11 (3.9%)  
Injection Site Discomfort  
3 (1.1%)  
Erythema  
2 (0.7%)  
Special Senses  
9 (3.2%)  
Altered Taste  
5 (1.8%)  
(1) Patients are counted separately within each body system. (2) The body system is 
reported if the aggregate is ≥ 0.5%. Details are not shown if the subsystem is not ≥ 
0.5%.  
 
Additional information concerning pre-clinical and clinical experience with Optison, including the 
dosing levels and reported subject complaints, can be found in the Optison Package Insert that 
is included as Appendix B. As a tool, contrast-enhanced ultrasound is well documented for the 
characterization of indeterminate liver lesions including HCC and ICC, as well as the 
identification of a variety of solid tumor metastasis within the liver including breast, 
neuroendocrine, pancreatic, and colorectal cancers12. Importantly, our group has also 
demonstrated the preliminary safety of using Optison with UTMD in patients undergoing Y-90 
TARE of hepatocellular carcinoma26.  
 
Study goal 
Our group has demonstrated the benefits of UTMD for sensitizing human HCC to radiotherapy in 
an orthotopic rat model25. More recently, we have translated this work to a first-in-humans 
randomized controlled clinical trial using localized UCA cavitation with 2D ultrasound within HCC 
vasculature to sensitize liver tumors to radiation following radioembolization (NCT# 03199274). 
An example imaging series from this trial is shown in Figure 1. Our recent interim analysis of this 
trial26 has demonstrated this approach is safe and results in promising radiosensitization. We 
intend to leverage our prior data in interventional oncology-based CEUS to improve treatment of 
ICC and other solid tumors within the liver. Consequently, the scientific premise of this work is 
that volumetric CEUS can be used to predict non-HCC tumoral response to TARE prior to therapy 
(thereby guiding selection of treatment option), improve the sensitivity of non-HCC tumors to 
radiotherapy (thereby improving tumor response), and assess treatment response earlier than the 
current clinical standard (thereby enabling earlier tumor retreatment when necessary). 

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1.2 
Rationale for the Proposed Study 
 
Contrast-enhanced Ultrasound 
 
The sensitivity and specificity of ultrasound imaging can be improved by intravenous (IV) injection 
of gas microbubbles as vascular contrast agents, which can enhance both Doppler and grayscale 
signals by up to 27 dB27,11. These contrast agents not only enhance the backscattered ultrasound 
signals, but at sufficient acoustic pressures they also act as nonlinear oscillators. These 
oscillations generate significant energy components in the received echo signals, which span the 
range of possible frequency emissions from subharmonics through ultraharmonics11,28. These 
nonlinear bubble echoes can be separated from tissue echoes and used to create contrast 
sensitive imaging modalities such as harmonic imaging (HI), which is commercially available on 
most state-of-the-art ultrasound scanners27,11.   
CEUS is one of the most important advances in ultrasonography over the last two decades. One 
example is the rapid success in detection and characterization of focal liver lesions, where CEUS 
in many studies have demonstrated similar value as Computer Tomography (CT) and Magnetic 
Resonance Imaging (MRI)29,30,31. 
 
Safety of ultrasound contrast agents 
 
Figure 1. Example series of a patient undergoing HCC radioembolization 
with UTMD. The series shows baseline MRI (A), Tc-99m MAA 
(macroaggregated albumin) SPECT-CT during treatment planning (B), 
angiography during Y90 delivery (C), B-mode ultrasound immediately post 
radioembolization (D), and peak CEUS enhancement during UTMD 2 
hours (E), 1 week (F), and 2 weeks (G) post radioembolization [Eisenbrey 
et al. 2021].  

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Ultrasound contrast agents have minimal side effects compared to CT or MRI contrast agents. 
They are not toxic, and do not influence the metabolism of the thyroid gland. The most serious 
adverse event is anaphylactic reaction. The frequency of anaphylaxis using registered ultrasound 
contrast agents is very low (0.001 % for ultrasound contrast agents versus 0.07 % for X-ray 
contrast agents)32,33. Contrast-enhanced ultrasound is well documented for the characterization 
of indeterminate liver lesions including HCC and ICC, as well as the identification of a variety of 
solid tumor metastasis within the liver including breast, neuroendocrine, pancreatic, and colorectal 
cancers12. 
 
Ultrasound-triggered microbubble destruction 
Ultrasound-triggered microbubble destruction (UTMD) has also been shown to sensitize tumors 
to radiotherapy by inducing vascular endothelial cell apoptosis18,19,20. This endothelial cell death 
has been attributed to increased ceramide production after interactions with inertially cavitating 
microbubbles21. Microbubble cavitation has also been shown to result in decreased tumor 
vascularity22. UTMD leads to increased DNA damage within the targeted tumor after radiotherapy, 
despite decreases in oxygenation21. Detailed parameter studies have been conducted in tumor 
xenografts, and synergistic combinations of UTMD and radiotherapy at MIs > 0.8, radiation 
dosages of 1.8-8 Gy, and microbubble concentrations of 1-3% (v/v) have been explored 18,19. 
Importantly, and yet somewhat paradoxically, UTMD in normal tissue increases vascular 
perfusion, due to cavitation-related increases in shear forces and activation of endothelial nitric 
oxide synthase23. Hence, UTMD combined with radiotherapy potentially offers a tumor-specific 
method of tissue sensitization.  
 
Subharmonic-aided pressure estimation 
Ultrasound contrast agents were proposed as pressure sensors for noninvasive pressure 
estimation in the cardiovascular system using a novel and innovative technique called 
subharmonic-aided pressure estimation (SHAPE)34. This method estimates internal pressure 
variations by transmitting at one frequency, receiving at its subharmonic frequency and then 
monitoring the subharmonic contrast bubble amplitude variations.  We have demonstrated that 
the subharmonic signal component from contrast microbubbles is an excellent indicator of the 
hydrostatic pressure variation.  High correlation (r2 > 0.95) between the amplitude of the 
subharmonic component and hydrostatic pressure (from 0 to 186 mmHg) was obtained for several 
contrast agents in vitro 35.  
We conducted a first-in-humans pilot study of this technique’s ability to estimate portal pressures 
in 45 adult patients undergoing transjugular liver biopsy36, which showed significantly higher 
SHAPE gradients between the portal and hepatic veins in subjects with clinically significant portal 
hypertension (HVPG > 10 mmHg) than in those with lower HVPGs (1.37 ± 0.59 dB vs. -1.68 ± 
0.27 dB, p<0.001). Recently, we have expanded on the concept of using SHAPE for portal 
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using modified GE Logic 9 systems (GE Healthcare) were very encouraging with an accuracy for 
diagnosing clinically significant portal hypertension of 95% (with a 95% confidence interval (CI): 
89%-99%, p<0.001) and achieving a sensitivity of 91% (95% CI: 88%-93%) and a specificity of 
82% (95% CI: 75%-85%). SHAPE offers the possibility of allowing pressure gradients in interstitial 
fluid pressure (IFP) in tumors to be obtained noninvasively36,38. The role of tumoral IFP in radiation 
response remains relatively understudied due to difficulties obtaining these measurements with 
traditional wick-in-needle approaches39. However, it has been proposed that tumor IFP drops 
following sufficient radiation exposure39,40,41. We hypothesize that tumoral IFP estimated via 
SHAPE can be used as a biomarker to predict tumor response to TARE therapy.  
 
1.3 
Correlative Studies 
 
Abdominal MRI/CT 3 months to two weeks before study inclusion and approximately 1-2 months 
and 3-6 months after treatment will be collected (as part of SoC). Post-treatment single-photon 
emission computed tomography (SPECT) imaging will be evaluated for all patients to evaluate if 
the initial UTMD session skews microsphere distribution in the liver. After the study period, the 
patients’ records will be monitored until progression or death is documented. Each patient’s time 
to required next treatment and overall survival will be recorded. The clinical outcomes and MRI/CT 
results will used for evaluation of responses and local primary tumor progression.  
 
1.4 
Potential Risks and Benefits 
 
Optison is well tolerated and has been used extensively in echocardiography applications11. 
Ultrasound contrast agents in general have a very favorable safety profile42,43,44 with a serious 
adverse even risk below 0.03%. In pre-market clinical trials, Optison was administered to 279 
patients.  In these patients 47 (16.8%) reported at least one adverse event. Of these events, 1 
was classified as serious and required antihistamines for hypersensitivity manifestations of 
dizziness, nausea, flushing, and temperature elevation. Additionally, contrast-enhanced 
ultrasound is well documented for the characterization of indeterminate liver lesions including 
HCC and ICC, as well as the identification of a variety of solid tumor metastasis within the liver 
including breast, neuroendocrine, pancreatic, and colorectal cancers12.  
 
1.4.1 
Potential Risks 
 
Serious cardiopulmonary and allergic reactions including fatalities have occurred during or 
following administration of Optison, causing the food and drug administration (FDA) to place a 
black box warning on the agent.  However these occurrences have been rare (less than 1 in 5,000 
patients).  As a result, patients with unstable cardiopulmonary conditions will be excluded. All 

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contrast injections will be supervised by a board certified physician.  Resuscitation equipment and 
trained personnel will be immediate proximity to the patient during each contrast-enhanced 
ultrasound exam.  Patients will also be monitored for 30 minutes after contrast administration for 
any adverse reactions.  
Clinically significant adverse effects from the administration of Optison are unlikely. This approach 
is similar to our ongoing randomized control trial using 2D UTMD to sensitize HCC to TARE. An 
interim analysis from this study was recently published and demonstrated that using 2D UTMD in 
HCC patients is both safe and effective26. Safety results from 28 patients have demonstrated no 
significant changes in vital signs pre- and post-UTMD (or significant changes in LFTs between 
patients receiving TARE alone or UTMD combined with TARE (p>0.25). In addition, no 
radiopharmaceutical activity was detected outside the liver on SPECT, ruling out non-target 
embolization in patients who received UTMD. 
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. 
Based on the available non-clinical and clinical safety data and the dosage levels of Optison that 
will be used in this study, safety concerns are minimal. The potential side effects related to Optison 
administration are described in Table 1 above. 
Optison destruction within the nontumor liver may sensitize this tissue to Y-90 beta emissions.  
However, destruction will be primarily located within the target area due to selective placement of 
the flash/replenishment ROI.  Additionally, liver parenchyma outside the treatment area will have 
limited radiation exposure due to the selective delivery of Y-90 as well as the limited penetration 
of its beta emissions. 
To minimize and/or eliminate risks a nurse will be present during the entire procedure.  Adverse 
events will be monitored during the entire procedure and for 30 minutes post injection.   
 
1.4.2 
Benefits 
 
Patients may potentially benefit from being in this study by experiencing improved tumoral 
outcomes to radiotherapy as a result of CEUS. Based on our recent animal data25 and preliminary 
human data26 shown in figure 1, we expect a 40-60% improvement in tumor response when 
radioembolization is combined with microbubble cavitation. CEUS-based data will also be 
collected as a means for predicting tumor response, but no treatment decisions will be based on 
this information.  
This study aims to define the efficacy and safety of using volumetric microbubble cavitation for 
sensitizing malignant tissue to radiotherapy and predicting treatment outcomes using CEUS-
based perfusion estimation. Once properly validated, this technique is ultimately expected to 
improve patient outcomes by locally and selectively sensitizing malignant tissue to Y90 
radiotherapy, identifying which patients would most benefit from TARE, and by enabling 
alternative forms of treatment through earlier identification of patients not responding to initial/first-

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line therapy. In addition, findings are expected to be applicable to other solid tumor applications 
in radiation oncology and microbubble cavitation research. 
2 
Study Objectives 
2.1 
Objectives 
 
To evaluate the utility of CEUS in prediction of  tumoral response to TARE prior to therapy, 
improving the sensitivity of non-HCC tumors to radiotherapy, and assessment of treatment 
response earlier than the current clinical standard. 
2.1.1 
Primary 
 
To determine the ability of quantitative volumetric CEUS to predict non-HCC tumor response to 
TARE prior to therapy. Existing volumetric parametric imaging algorithms will also be employed 
to quantify vascular heterogeneity, volumetric perfusion, and area under the curve will be 
quantified. These variables will then be compared to modified response evaluation criteria in solid 
tumors (mRECIST) findings 3-6 months post treatment to identify CEUS-derived hallmarks of 
tumor vascularity that predict treatment response. 
 
2.1.2 
Secondary 
 
• 
To characterize the safety and preliminary efficacy of using localized UCA inertial 
cavitation to improve tumoral response to radioembolization.  
• 
To determine if CEUS estimated tumor perfusion and residual vascularity 7-14 days post 
treatment can predict tumoral response to radioembolization. 
• 
To evaluate tumoral response using the patient’s 1 month MRI (obtained clinically) and 
determine the accuracy of MR or CT tumor evaluation at this earlier time point. 
 
2.1.3 
Exploratory 
 
To examine the utility of SHAPE to noninvasively monitor tumoral interstitial fluid pressure (IFP) 
and provide an early biomarker of radiotherapy response. 
 
2.2 
Endpoints/Outcome Measures 
 

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2.2.1 
Primary 
Tumor complete response (CR) and partial response (PR) on mRECIST reads of contrast-
enhanced CT or MRI obtained 3-6 months after treatment determined by two independent 
readers 
2.2.1 Secondary 
Tumor complete response (CR) and partial response (PR) on mRECIST reads of contrast-
enhanced CT or MRI obtained 3-6 months after treatment determined by two independent 
readers 
Changes in Liver Function Tests (LFTs)  
Observed and self-reported adverse events 
Time required to next treatment 
Overall survival  
2.2.2 
Exploratory 
Tumor complete response (CR) and partial response (PR) on mRECIST reads of contrast-
enhanced CT or MRI obtained 3-6 months after treatment determined by two independent 
readers  
 
3 
Study Design 
 
3.1 
Characteristics 
Patients at least 18 years of age, medically stable with a previously untreated non-HCC tumor in 
the liver greater than 1 cm but small enough to be visualized in the ultrasound 3D volume, will 
be included. These patients will be recruited from the Hepatology department, Interventional 
Radiology, and the Multidisciplinary Liver Tumor Board of the Sidney Kimmel Cancer Center at 
Thomas Jefferson University.  
 
3.2 
Number of Participants 
20 patients 
 
3.3 
Duration of Therapy 
Patients will receive a quantitative volumetric CEUS scan < 1 month before TARE therapy. 
Consequently, they will receive UTMD sessions 1-4 hours, approximately 1 week, and 
approximately 2 weeks after TARE therapy. The entire ultrasound imaging protocol will require 
approximately 1.5 hours including a 30 min observation period. 

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3.4 
Duration of Follow Up 
After the subjects have completed treatment, they will be followed through the electronic 
medical record (EMR) system for up to 1 year to document long term outcomes.  
 
3.5 
Treatment Assignment Procedures 
 
3.5.1 
Randomization Procedures (if applicable) 
This is a non-randomized trial; therefore, no randomization procedure required. 
 
3.6 
Study Timeline 
 
3.6.1 
Primary Completion 
Primary completion is anticipated in  June of 2025. 
 
3.6.2 
Study Completion 
Study completion and publication of results is anticipated in July of 2025 
 
4 
Study Enrollment and Withdrawal 
 
Patients at least 18 years of age, medically stable who have a previously untreated intrahepatic 
non-HCC tumor  greater than 1 cm but small enough to be visualized in the ultrasound 3D 
volume will be included. These patients will be recruited from the Hepatology department, 
Interventional Radiology, and the Multidisciplinary Liver Tumor Board of the Kimmel Cancer 
Center at Thomas Jefferson University.  
A sufficient number of patients will be screened in order to enroll 20 patients with non-HCC 
tumors receiving TARE over the duration of the study. 
Patients who are discontinued from the study prior to the completion of all planned procedures 
and assessments will not be replaced. 
Each subject is identified in the study by a unique subject number that is assigned when subject 
enrolls after signing the Informed Consent Form. Once assigned the subject number cannot be 
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4.1 
Eligibility Criteria 
4.1.1 
Inclusion Criteria 
 
The patients enrolled in this project will be adults over the age of 18 capable of providing written 
informed consent. Based on the study protocol and Optison contraindications, inclusion criteria 
for this trial are as follows: 
• 
Be scheduled for sub-lobar radioembolization therapy of a previously untreated non-
HCC tumor greater than 1 cm but small enough to be fully visualized in the 
ultrasound 3D volume (approximately 6 cm maximum diameter, but depth 
dependent) 
 
• 
Be at least 18 years of age. 
• 
Be medically stable. 
• 
If a female of child-bearing age, have a negative pregnancy test prior to each 
ultrasound exam. 
• 
Have signed Informed Consent to participate 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: 
Exclusion criteria for this study will consist of: 
• 
Females who are pregnant or nursing.  
• 
Patients with recent cerebral hemorrhage. 
• 
Patients with known sensitivities to albumin, blood, or blood products 
• 
Patients with known hypersensitivity to perflutren 
• 
Patients with known congenital heart defects. 
• 
Patients with severe emphysema, pulmonary vasculitis, or a history of pulmonary 
emboli. 
• 
Patients with bilirubin levels > 2 mg/dL 
 

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4.2 
Gender/Minority/Pediatric Inclusion for Research  
 
The subject population for this study will be made up of approximately 30% women based upon 
a retrospective review of the demographics of the population presenting with non-HCC tumors at 
the Oncology section of Thomas Jefferson University. 
 
No patients will be excluded on the basis of race. The patient population of this project will 
reflect the population demographics found at major American urban academic health centers 
who present with cancer of the intrahepatic bile duct. The overall hospital demographics for TJU 
include 58-66 % Caucasian, 28-30 % African American, and 2-4 % Asian, with 10-12 % 
representing Hispanic patients. 
No children will be included. The patients enrolled in the human studies portion of this project 
will be adults over the age of 18 diagnosed with non-HCC tumors, who are scheduled to receive 
sub-lobar TARE. 
 
4.3 
Strategies for Recruitment and Retention 
 
Suitable subjects referred to the Division of Interventional Radiology within the Department of 
Radiology at Thomas Jefferson University for radioembolization of non-HCC tumors. There will 
be no further recruitment of subjects.  The research coordinators for the study will invite the 
subjects to participate in the study. The study will be explained to potential subjects and, if they 
would like to participate, they will be given an informed consent to read. The consent will be 
reviewed with the patient, providing the patients time to consider participation in the study and 
ask questions about the research study. The subject will sign the consent in the presence of a co-
investigator and will receive a copy of the signed consent form. 
 
4.4 
Participant Withdrawal 
 
4.4.1 
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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4.4.2 
Handling of Participant Withdrawals and Participant Discontinuation of Study 
Intervention 
Patients who withdraw or are withdrawn from the study, will stop further UTMD procedures, as 
described by the study protocol. The reason for discontinuation shall be recorded. The 
investigator will follow up any significant adverse events related to the novel agent with 
appropriate clinical care. 
4.5 
Premature Termination or Suspension of Study 
This study may be suspended or prematurely terminated if there is sufficient reasonable cause. 
Written notification, documenting the reason for study suspension or termination, will be provided 
by the suspending or terminating party to all regulatory authorities. If the study is prematurely 
terminated or suspended, the principal investigator will promptly inform the IRB and will provide 
the reason(s) for the termination or suspension.  
Circumstances that may warrant termination include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants. 
• 
Insufficient adherence to protocol requirements. 
• 
Data that is not sufficiently complete and/or evaluable.  
• 
Determination of futility. 
5 
Study Intervention 
 
5.1 
Study Product  
One ultrasound contrast agent will be used (Optison). The agent will be used as an off-label 
indication for this study, but within the approved dosage recommendations. Optison is a sterile 
non-pyrogenic suspension of encapsulated perflutren microbubbles.  The contrast agent consists 
of a human serum albumin shell with perflutren core and contains 5.0 - 8.0 x 108 microspheres/ 
ml.   
 
5.1.1 
Acquisition 
The ultrasound contrast agent will be purchased from GE Healthcare, Princeton, NJ.  
 
5.1.2 
Formulation, Packaging, and Labeling 
The microbubble agent is supplied in a standard-size 3 ml vial and is prepared by manually 
shaking the vial.  Detailed instructions are provided in the Optison Product Insert, found in 
Appendix B. 
 

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5.1.3 
Product Storage and Stability 
Optison will be stored in a secure refrigerator, with only the study investigators and research 
personnel having access.  Unused drug and empty vials will be properly disposed of after 
reconciling the log of study drug kept at Thomas Jefferson University. 
 
 
5.2 
Dosage, Preparation, and Administration  
All contrast injections will be supervised by a board certified physician.  Resuscitation equipment 
and trained personnel will be immediate proximity to the patient during each contrast-enhanced 
ultrasound exam.  Optison will be administered by IV infusion through an 18- to 22-gauge 
angiocatheter placed in a peripheral arm vein, preferably an antecubital vein.  Five milliliters of 
Optison will be activated and suspended in a 50 ml bag of saline.  The saline/UCA mixture will 
then be infused through the IV at a rate of 120 ml/hour.  Subjects will be instructed not to move 
their arm during the administration of the UCA. 
 
5.3 
Dose Modifications and Dosing Delays 
The standard dose of Optison is 5 mL that will be activated and suspended in a 50 ml bag of 
saline.  The Optison mixture will then be infused through the IV at a rate of 120 ml/hour. The 
reported adverse event (AE) rates at the dosages employed in this study are very low, but have 
not been fully evaluated in conjunction with radioembolization. No reasons for dose modifications 
for Optison are considered. 
 
 
5.4 
Study Product Accountability 
The study coordinators at each site will keep an electronic log accounting for the use of each kit 
of Optison and individual vials based on the sign-out sheets. 
 
 
5.5 
Assessing Participant Compliance with Study Product Administration 
Compliance will be recorded through attendance for the scheduled visits. 
 
5.6 
Concomitant Medications/Treatments 
No restrictions related to concomitant medications or treatments are considered.  
 
 
5.7 
Dietary Restrictions 
No special dietary or “life-style” requirements are needed during the infusion time.   
 
5.8 
Study Procedural Intervention(s) Description 
All imaging will be performed using a modified Logiq E10 system with RAB6-D probe (GE 
Healthcare). A brief abdominal ultrasound (without contrast) will be performed prior to 

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radioembolization to ensure the target lesion is visible on ultrasound. Volumes of the mass on B-
mode and power Doppler will be acquired for later processing. 
For the pretreatment exam, 5 ml of activated Optison will be suspended in 50 ml of saline and 
infused through an 18- to 22-gauge angiocatheter placed in an antecubital vein at a rate of 120 
ml/hour (dosages based on prior approved Optison UTMD protocol and product insert). Following 
confirmation of UCA arrival, 4D CEUS data will be acquired. Imaging parameters (gain, MI, 
frequency preset) will be optimized on a patient-by-patient basis, while still acquiring adequate 
signal at the required depth. The volumetric region of interest will be placed to encompass the 
entire lesion and surrounding normal liver tissue while still being minimized enough to ensure an 
adequate volume acquisition rate45 (0.5-2 volumes/second in our experience). Every 60 seconds, 
microbubbles within the volume will be destroyed using a 4 second flash pulse (MI>1.1), followed 
by lower MI (<0.15) nondestructive imaging to monitor tumor reperfusion. Data from > 5 flash-
replenishment cycles will be obtained, allowing us to define intra-patient variability for each 
quantitative parameter. For the last 2 minutes of the infusion, a SHAPE acoustic output 
optimization algorithm will be initiated on the E10 system46. Following identification of the optimal 
acoustic output, 10 seconds of 4D SHAPE data will then be collected from both the tumor and 
surrounding liver tissue in triplicate. After complete washout of the UCA (around 10 minutes) 
baseline 4D SHAPE data at the optimal acoustic output without bubbles present will be acquired. 
This acquisition involves standard ultrasound only and can be obtained during the 30 minutes 
post-contrast observation period. 
Post treatment ultrasound exams will take place 2-4 hours post radioembolization in Interventional 
Radiology’s recovery unit prior to single photon emission computed tomography (SPECT) 
imaging, and again 7 and 14 days post treatment in Thomas Jefferson University’s Interventional 
Radiology Division. Both of these locations have full time nursing support and with patient 
physiological monitoring. Specific imaging time points are based on times when patients are 
present or returning to the hospital for regular follow up, experiences with our study enrollment in 
ongoing HCC trials, and at a time when the Y90 spheres are still active. All contrast injections will 
be supervised by a board certified physician (Drs. Anton, Civan, O’Kane, or Lyshchik) with 
resuscitation equipment in immediate proximity during CEUS exam. Patient vital signs will be 
monitored and recorded throughout the visit. Tumor characteristics (size and location) will also be 
recorded for subset analysis to study how these factors influence treatment. 
Similar to the pre-treatment exam, B-mode and Doppler volumes will be acquired for each exam 
post-TARE. Following these acquisitions, 5 milliliters of activated Optison will be suspended in 50 
ml of saline and infused through an 18- to 22-gauge angiocatheter placed in a peripheral arm vein 
at a rate of 120 ml/hour. This total Optison administration falls within the product insert dosage 
guidelines (up to 5 ml within a 10 minute period and up to 8.7 ml in any one study). Following 
peak enhancement, a destructive (high MI) pulse will be generated across the selected volume. 
This will be performed using a modified software platform (created as part of R01 CA199646) that 
can be customized for transmission of UTMD flash replenishment sequences across a volume. 
The system will be set a center frequency of 2.1 MHz, with pulse lengths of 20 μs and a line 
density of 4. Acoustic pressure amplitudes will be adjusted as necessary to ensure inertial 
microbubble cavitation. Importantly, this research software system provides range gated 

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detection of stable and inertial cavitation through analysis of received radiofrequency data and 
real time display of spatial-peak temporal-average intensity (ISPTA). For each new patient, an 
ISPTA of 300 mW/cm2 will be used as a starting point and increased in 50 mW/cm2 increments 
until inertial cavitation is detected. However, the system is also limited to not exceed any acoustic 
output limits set by the FDA and the MI will be kept well below 1.7. This acoustic output setting 
will then be used for all further UTMD sequences. UTMD sequences followed by 10 second low 
MI microbubble replenishment sequences will then be repeated across the tumor volume for the 
remainder of the infusion (approximately 20-30 sequences). Finally, for the last 30 seconds of the 
infusion, 4D SHAPE data will be acquired using the optimal acoustic output determined at 
baseline. The patient will be monitored for 30 minutes per our institutional CEUS protocol before 
being discharged (during which time the baseline 4D SHAPE data will be obtained).  
 
 
5.9 
Administration of Procedural Intervention 
Imaging will be performed an ultrasonographer with over 10 years of experience in liver imaging. 
All contrast injections will be performed by a research nurse with CEUS experience under with 
resuscitation equipment in immediate proximity. All imaging will be performed using a modified 
Logiq E10 system with RAB6-D probe (GE Healthcare).  
 
5.10 
Procedures for Training of Clinicians on Procedural Intervention 
The personnel performing the UTMD procedures are all from the Ultrasound Research lab and 
have extensive experience with CEUS and ultrasound imaging procedures. These individuals will 
all receive training by the PI in this specific protocol. 
 
5.11 
Assessment of Clinician and/or Participant Compliance with Study Procedural 
Intervention 
Compliance of the subjects will be recorded through attendance for the scheduled visits. There 
are no clinicians outside of the Ultrasound Research lab involved in administrating the UTMD 
procedure. 
 
6 
Study Schedule  
6.1 
Pretreatment Period/Screening 
Subjects eligible for trial enrollment will be identified by Drs. Anton, Gonsalves, and Civan from 
their patient population (approximately 50-60 eligible patients per year recruited across 4 
Jefferson Health campuses and treated in our main Center City Hospital in Philadelphia).  Patients 
will be accrued from the Hepatology department, Interventional Radiology, and the 

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Multidisciplinary Liver Tumor Board of the Sidney Kimmel Cancer Center at Thomas Jefferson 
University.  
 
6.2 
Enrollment/Baseline 
Study Entry visit (Day 0) 
Screening assessments will be performed when the patients are on campus for their pre-
treatment evaluation.  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 age, she will have a urine pregnancy test (the 
results of which will be made available to the subject prior to study initiation) prior to each 
ultrasound exam. A 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 coordinator will review the consent form with the patient and then the patient will be given the 
form to review. The patient, coordinator, and a study investigator will all sign the consent form. 
The patient will be given a copy of the signed consent form for their records. Pre-treatment 
volumetric CEUS exam will be performed within 1 month prior to radioembolization treatment.  
 
6.3 
Treatment Period 
Transarterial Radioembolization (TARE) treatment visit (Day 1-31) 
 
• 
A brief abdominal ultrasound (without contrast) will also be performed prior to 
radioembolization to ensure the target lesion is visible on ultrasound. 
• 
Post treatment CEUS exams with microbubble cavitation 1-4 hours after TARE  
 
Post-TARE visit 1 (Day 8-38) 
 
• 
Post treatment CEUS exams with microbubble cavitation approximately 1 week after 
TARE  
 
Post-TARE visit 2 (Day 15-45) 
 
• 
Post treatment CEUS exams with microbubble cavitation approximately 2 weeks after 
TARE  
6.4 
Long Term/Survival Follow-up  
After the subjects have completed treatment, they will be followed through the EMR system to 
for up to 1 year document long term outcomes.   
 
6.5 
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Patients who withdraw or are withdrawn from the study, will stop further CEUS procedures, as 
described by the study protocol. The reason for discontinuation shall be recorded. The 
investigators will follow any patient who develops significant adverse events. 
 
Patients may be discontinued from study treatment and assessments at any time. Specific 
reasons for discontinuing a patient for this study are: 
• 
Voluntary discontinuation by the patient who is at any time free to discontinue his/her 
participation in the study, without prejudice to further SoC treatment. 
• 
Patient lost to follow-up. 
• 
Safety reason as judged by the Principal Investigators. 
• 
Severe non-compliance to protocol as judged by the Principal Investigators. 
• 
Incorrect enrolment i.e., the patient does not meet the required inclusion/exclusion criteria 
for the study. 
• 
Disease progression and/or rapidly deteriorating ECOG performance status. 
• 
Deterioration in the patient’s condition which in the opinion of the Principal Investigator 
warrants UTMD discontinuation (to be recorded as an AE or under Investigator Discretion) 
o Patient requires dialysis at any visit 
o Deteriorating liver function  
• 
Related Serious Adverse Event 
• 
A female patient becoming pregnant  
• 
Any reason why, in the opinion of the investigator, the patient should not participate 
 
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 
 
The sponsor and principal investigator will promptly inform all investigators and the relevant 
regulatory authorities of the termination of the trial along with the reasons for such action 
 
7 
Study Procedures and Evaluations 
 
7.1 
Study Procedures/Evaluations 
• 
Medical history will be obtained from the electronic medical records (EMR) system 
and during the first study visit < 1 month before the TARE.  
• 
Volumetric CEUS exam will be obtained approximately < 1 month before the 
treatment as described in section 5.8.  
• 
Three CEUS exam with microbubble cavitation will be performed 2-4 hours, 1 week, 
and 2 weeks after treatment as described in section 5.8. 
8 
Evaluation of Safety 
 

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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 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. . All AEs, including observed 
or volunteered problems, complaints, signs or symptoms, and diagnoses, occurring from the study 
treatment will be recorded on a serious or non-serious AE data form.  Whenever possible, the AE 
will be evaluated and reported as a diagnosis rather than individual signs and symptoms. If a 
definitive diagnosis is not possible, the individual signs and symptoms will be recorded.  The 
investigator will evaluate and note the duration, intensity, and relationship to (association with) 
the treatment or imaging procedures, the action taken, and the determination of seriousness for 
each AE. The intensity of the AE will be characterized as mild, moderate, or severe. 
Optison is well tolerated and has been used extensively in echocardiography applications.  In pre-
market clinical trials, Optison was administered to 279 patients.  In these patients 47 (16.8%) 
reported at least one adverse event.  Of these events, 1 was classified as serious and required 
antihistamines for hypersensitivity manifestations of dizziness, nausea, flushing, and temperature 
elevation.  No deaths were reported.  All of the non-serious reported side effects have been 
transient, usually lasting only a few minutes. Please see table 1 and the attached Optison Product 
insert (Appendix B). 
 
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) 
• 
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• 
Results in inpatient hospitalization or prolongation of existing hospitalization 
• 
Results in a persistent or significant disability or incapacity 
• 
Results in a congenital anomaly or birth defect  
• 
An important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical 
judgment, the event may jeopardize the participant or may require intervention to 
prevent one of the outcomes listed in this definition. 
 
8.2 
Safety Assessment and Follow-Up 
The PI will follow adverse events 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. At each study visit, the investigator (or designee) will inquire about the occurrence 
of AE/SAEs since the last visit. 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 
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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 
product and the AE.  
• 
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 adverse events. Evaluation of relatedness must consider etiologies such as natural history 
of the underlying disease, concurrent illness, concomitant therapy, study-related procedures, 
accidents, and other external factors.  
The following guidelines are used to assess relationship of an event to study intervention: 
1. Related (Possible, Probable, Definite) 
a. The event is known to occur with the study intervention. 
b. There is a temporal relationship between the intervention and event onset. 
c. The event abates when the intervention is discontinued. 
d. The event reappears upon a re-challenge with the intervention. 
2. Not Related (Unlikely, Not Related) 
a. There is no temporal relationship between the intervention and event onset. 
b. An alternate etiology has been established. 
 
8.3.2 
Expectedness 
The PI 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. 

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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 adverse events. 
 
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 events will be graded by the Investigator according to the CTCAE version 5.0. A grading 
(severity) scale is provided for each AE term. 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 as described 
below. 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.  

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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 working 
days.  
Grade 5 AEs will be reported to the IRB within one working day of knowledge of the event. 
All SAEs will be submitted to the IRB at continuing review, including those that were reported 
previously. 
 
8.4.2 
Reporting to SKCC DSMC 
All AEs and SAEs, safety and toxicity data, and any corrective actions will be submitted to the 
DSMC per the frequency described in the SKCC DSMP. The report to the SKCC DSMC will also 
include any unanticipated problems that in the opinion of the PI should be reported to the DSMC. 
For expedited reporting requirements, see table below:                                        
 DSMC AE/SAE Reporting Requirements 

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8.4.3 
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.  
 
8.4.4 
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. For event reporting, any event that is both serious and unexpected 
must be reported to the Food and Drug Administration (FDA) as soon as possible and no later 
than 7 days (for a death or life-threatening event) or 15 days (for all other SAEs) after the 
investigator’s or institution’s initial receipt of the information. Thomas Jefferson University will 
submit SAE reports to the FDA. The TJU Study Site Contact is responsible for submitting SAEs 
to the funder that occur at the affiliate site.  
 
SAEs should be reported on MedWatch Form 3500A, which can be accessed at: 
http://www.accessdata.fda.gov/scripts/medwatch/  
 
MedWatch SAE forms should be sent to the FDA at: 
 
MEDWATCH 
5600 Fishers Lane 
Rockville, MD 20852-9787 
Fax: 1-800-FDA-0178 (1-800-332-0178) 
http://www.accessdata.fda.gov/scripts/medwatch/ 
 
• 
An SAE report should be completed for any event where doubt exists regarding its 
seriousness.  
• 
For studies with long-term follow-up periods in which safety data are being reported, 
include the timing of SAE collection in the protocol. 
• 
If the investigator believes that an SAE is not related to study drug, but is potentially related 
to the conditions of the study (such as withdrawal of previous therapy or a complication of 
a study procedure), the relationship should be specified in the narrative section of the SAE 
Report Form. 
• 
If only limited information is initially available, follow-up reports are required. (Note: Follow-
up SAE reports should include the same investigator term(s) initially reported.) 
 
8.4.5 
Reporting of Pregnancy 
 
Pregnancy is an exclusion criteria and women of a child-bearing potential must use adequate 
protection measures. If a subject becomes pregnant during the study they will be discontinued 
from the study. 

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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 
 
9 
Study Oversight 
In addition to the PI’s responsibility for oversight, study oversight will be under the direction of 
the SKCC’s Data and Safety Monitoring Committee (DSMC). The SKCC DSMC operates in 
compliance with a Data and Safety Monitoring Plan (DSMP) that is approved by the NCI.  
 
10 Clinical Site Monitoring and Auditing 
Clinical site monitoring and auditing is conducted to ensure that the rights of human participants 
are protected, that the study is implemented in accordance with the protocol and/or other 
operating procedures, and that the quality and integrity of study data and data collection methods 
are maintained. Monitoring and auditing for this study will be performed in accordance with the 
SKCC’s Data and Safety Monitoring Plan (DSMP) developed by the SKCC Data and Safety 
Monitoring Committee (DSMC). The DSMP specifies the frequency of monitoring, monitoring 
procedures, the level of clinical site monitoring activities (e.g., the percentage of participant data 
to be reviewed), and the distribution of monitoring reports. Some monitoring activities may be 
performed remotely, while others will take place at the study site(s). Appropriate staff will conduct 
monitoring activities and provide reports of the findings and associated action items in accordance 
with the details described in the SKCC DSMP. 
11 Statistical Considerations  
 
11.1 
Study Hypotheses 
H1: Contrast reperfusion time, degree of tumor enhancement relative to the surrounding 
parenchyma, tumoral IFP and 3D morphological features of the vasculature quantified via CEUS 
before therapy correlate with SoC mRECIST reads of non-HCC tumor patients treated with TARE.  
H2: mRECIST reads for non-HCC tumor patients receiving TARE combined with microbubble 
inertial cavitation have more complete response (CR) and partial response (PR) reads compared 
to the historical group treated with TARE alone. 
H3: Patient’s time to required next treatment and overall survival are longer for non-HCC tumor 
patients receiving TARE combined with microbubble inertial cavitation compared to the historical 
group treated with TARE alone. 

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H4: Observed and self-reported adverse events, and changes in liver function tests (LFTs) one 
month post-TARE in non-HCC tumor patients receiving TARE combined with microbubble inertial 
cavitation are similar compared to the historical group treated with TARE alone. 
H5: Values of tumor perfusion and residual vascularity quantified by CEUS 2-4 hours, 1 week, 
and 2 weeks after TARE therapy are lower in CR and PR mRECIST reads of non-HCC tumor 
patients treated with TARE.  
H6: Reductions of tumor perfusion and residual vascularity values quantified by CEUS 2-4 hours, 
1 week, and 2 weeks after TARE therapy are more frequent in CR and PR mRECIST reads of 
non-HCC tumor patients treated with TARE.  
H7: Tumoral response in mRECIST reads of MRI or CT (obtained clinically from patients treated 
with TARE) at 1 month post treatment will agree with response in their mRECIST reads at 3-6 
months.  
H8: Values of IFP quantified by SHAPE 2-4 hours, 1 week, and 2 weeks after TARE therapy are 
lower in CR and PR mRECIST reads of patients treated with TARE. 
H9: Reductions of IFP values quantified by SHAPE 2-4 hours, 1 week, and 2 weeks after TARE 
therapy are more frequent in CR and PR mRECIST reads of patients treated with TARE. 
11.2 
Analysis Plans 
In general, categorical data will be summarized as counts and percentages (or proportions) and 
continuous data will be summarized with descriptive statistics such as mean, standard deviation 
(SD), median, and range. All statistical analysis will be performed by Dr. Keith using SAS version 
9.4 (Cary, NC). All statistical tests will be conducted at a nominal α=0.05 level. For H1 (see 
Section 11.1 above), the respective relationships between the CEUS-derived contrast reperfusion 
time, degree of tumor enhancement relative to the surrounding parenchyma, tumoral IFP and 3D 
morphological features of the vasculature with the patients’ subsequent mRECIST diagnosis (3 
levels: stable disease, partial response, or complete response) will be evaluated by testing for 
mean difference in the CEUS measures between response levels by analysis of variance 
(ANOVA). Historical controls (n=20) will be identified from a pool of recently treated non-HCC 
patients at Jefferson that meet study eligibility criteria for the study and match our enrollees by 
propensity score based on a logistic regression model including patient age, tumor size, bilirubin 
and ECOG performance score and utilizing a greedy matching algorithm. Testing for the effect of 
radioembolization on tumor response in H2 will be conducted using non-parametric Mann-
Whitney U-tests of the differences in mRECIST distributions between historical controls and 
enrolled patients. The difference in time-to-event endpoints (i.e., to next treatment or death, 
respectively) between the treated and historical control patients for H3 will be estimated and 
tested using Kaplan-Meier survival methods and log-rank tests. Differences in safety data will be 
evaluated for H4 with descriptive statistics, but not statistical hypothesis tests. A similar ANOVA 
approach taken for evaluating H1 will be taken for evaluating the means of CEUS measures at 
different times (2-4 hours, 1 week, and 2 week, respectively) of tumor perfusion and residual 
vascularity values between response levels to address H5 and SHAPE IFP values for addressing 
H8, as well as the means of changes from baseline in these measure for addressing H6 and H9. 
The agreement on response between mRECIST response diagnoses at 1 month and 3-6 months 
post-TARE will be tested using McNemar’s test. 
 

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11.3 
Interim Analyses and Stopping Rules 
This is a pilot study, no interim analysis will be considered. 
 
11.3.1 
Safety Review 
Safety outcomes will be compared between the treatment group and the historical group using 
observed and self-reported adverse events, and changes in LFTs one month post-embolization 
(performed as part of clinical standard of care). LFTs will include blood tests for alanine 
aminotransferase, alkaline phosphatase, asparate aminotransferase, bilirubin, albumin, total 
protein, and prothrombin time. SPECT imaging will be evaluated for all patients by two blinded 
radiologists to evaluate if the initial UTMD session skews microsphere distribution in the liver. 
 
11.3.2 
Efficacy Review 
The primary measure of tumor response will be evaluated using mRECIST criteria as determined 
by Dr. Lyshchik and O’Kane in consensus using 3-6 month contrast enhanced MRI or CT (blinded 
to patient group). Following a wait period and randomization of the patient order, both readers will 
also evaluate tumoral response using the patient’s 1 month MRI (obtained clinically) to determine 
the accuracy of MR or CT tumor evaluation at this earlier time point and its use as an alternative 
to CEUS. Finally, each patient’s time to required next treatment and overall survival will be 
monitored, although this is a secondary outcome for this study and expected to surpass the 
timeline of this study. 
 
11.4 
Sample Size Considerations 
Sample size for this pilot study is primarily driven by patient availability and larger, multi-center 
randomized trials will likely be needed to fully demonstrate efficacy. However, for the primary 
objective, a sample size of 20 patients (of which we expect 25% having stable disease, 50% 
partial responders, and 25% complete responders) will provide 80% power to detect a 0.76 
standard deviation difference in CEUS measurement means (e.g., mean contrast perfusion time) 
between mRECIST response levels, by analysis of variance (ANOVA) with a nominal 5% type I 
error rate. Similarly, in analyses comparing the prospective treated patients to matched historical 
control patients, 20 patients per arm will provide 80% power to detect a relative effect size of 0.66 
standard deviations by t-test with a nominal 5% type I error rate (i.e., a moderately large, but 
reasonable effect given our current data in HCC patients).  
 
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 

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We estimate it will take 2 years to recruit 20 subjects for this trial. 
 
11.5 
Exploratory Analysis 
Exploratory analysis is anticipated for the associations between IFP or reductions in IFP and 
mRECIST response. The plans for that analysis are included in the statistical analysis plans 
above.  
 
11.6 
Evaluation of Safety 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or SAE. Each patient will be instructed to contact the investigator 
immediately should they manifest any signs or symptoms they perceive as serious. 
To determine the safety profile of the treatment the following safety assessments will be done: 
• 
Clinical laboratory tests 
• 
Physical examinations 
• 
Sitting vital signs, body temperature and body weight 
• 
Adverse events registration with attribution in accordance with section 8.3.1 
 
The methods for collection safety data are described below. 
Expected AE’s for the SoC TARE and the ultrasound contrast agent (i.e., Optison) will be 
recorded. 
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 
 
The medical records for each patient should contain information which is important for the 
patient’s safety and continued care and to fulfil the requirement that critical study data should be 
verifiable. To achieve this, the medical records of each patient should clearly describe at least: 

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• 
That the patient is participating in the study e.g., by including the enrollment number and the 
study code or other study identification; 
• 
Date when Informed Consent was obtained from the patient for the following and statement 
that patient received a copy of the signed and dated Informed Consent; 
• 
Results of all assessments confirming a patient’s eligibility for the study; 
• 
Diseases (past and current; both the disease studied and others, as relevant); 
• 
Surgical history, as relevant; 
• 
Treatments withdrawn/withheld due to participation in the study; 
• 
Results of all assessments performed during the study; 
• 
WHO performance status assessments conducted as part of the study; 
• 
Treatments given, changes in treatments during the study and the time points for the changes;  
• 
Visits to the clinic during the study, including those for study purposes only; 
• 
AEs and SAEs (if any) including causality assessments;  
• 
Date of, and reason for, discontinuation from study treatment;  
• 
Date of, and reason for, withdrawal from study; 
• 
Date of death and cause of death, if available; 
• 
Additional information according to local regulations and practice. 
 
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. 
The PI will perform spot checks to verify CRFs and database entries match source documents. 
Verified data is entered into a 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.  

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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 initialed. Correction fluid is not allowed. All original data collected with 
paper and pen will immediately be recorded electronically by the study coordinator within the 
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. 
Unanticipated problems and adverse events 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.  
The study will be conducted in accordance with ethical principles that have their origin in the 
Declaration of Helsinki and are consistent with ICH/Good Clinical Practice and applicable 
regulatory requirements. 
 
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.  
 
14.3 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual agreeing to participate in the 
study and continues throughout study participation. Extensive discussion of risks and possible 
benefits of study participation will be provided to participants and their families, if applicable. A 
consent form describing in detail the study procedures and risks will be given to the participant. 
Consent forms will be IRB-approved, and the participant is required to read and review the 

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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 clinical or research record.  
The investigators are responsible for giving the patients full and adequate verbal and written 
information about the nature, purpose, possible risk and benefit of the study. They will be informed 
as to the strict confidentiality of their patient data, but that their medical records may be reviewed 
for trial purposes by authorized individuals other than their treating physician.  
 
It will be emphasized that the participation is voluntary and that the patient is allowed to refuse 
further participation in the protocol whenever she/he wants. This will not prejudice the patient’s 
subsequent care. Documented informed consent must be obtained for all patients included in the 
study before they are registered in the study. This will be done in accordance with the national 
and local regulatory requirements of each site. The investigators are responsible for obtaining 
signed informed consent. 
 
A copy of the patient information and consent will be given to the patients. The signed and dated 
patient consent forms will be filed in the Investigator File binder and also scanned to be part of 
the patient’s electronic medical record at the hospital. 
 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) 
No children will be included. The patients enrolled in this study will be adults over the age of 18 
diagnosed with a non-HCC solid tumor, who are scheduled for TARE. Malignant intrahepatic bile 
duct tumors are rare in children and adolescents (0.7% of new cases are in patients younger than 
20 
years), 
and 
the 
median 
age 
of 
diagnosis 
is 
65 
years 
old 
[https://seer.cancer.gov/statfacts/html/livibd.html]. 
 
14.5 
Participant Confidentiality 
Subject identification, other than subject number, date of study and date of birth, will not appear 
in any eCRF pages or other documents given to the Sponsor. Only the PIs as well as the 
persons authorized to verify the quality and integrity of the study will have access to subject 
records where the subject can be identified. The PIs shall arrange for the secure retention of the 
patient identification and the code list. 
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 CRFs and source documentation. The monitor and/or 

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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.  
 
15.1 
Data Management Responsibilities 
Data management will be performed by the ultrasound research laboratory using paper charts 
hosted at Thomas Jefferson University in accordance with the department’s SOPs and ICH 
guidelines. The data management process will be described in the study specific data handling 
report after database closure. 
 
15.2 
Data Capture Methods 
Data management will be performed by the ultrasound research laboratory using paper charts 
hosted at Thomas Jefferson University in accordance with the department’s SOPs and ICH 
guidelines. The data management process will be described in the study specific data handling 
report after database closure. 
Data entered into the database will be validated as defined in the data validation plan. Validation 
includes, but is not limited to, validity checks (e.g. range checks), consistency checks and 
customized checks (logical checks between variables to ensure that study data are accurately 
reported) for data captured in the database and external data (e.g. laboratory data). 
Data management personnel will perform both manual CRF review and review of additional 
electronic edit checks to ensure that the data are complete, consistent and reasonable. The 
electronic edit checks will run continually throughout the course of the study and the issues will 
be reviewed manually online to determine what action needs to be taken. 
Manual queries may be written by clinical data management or study monitor. Queries will be sent 
to the investigator for resolving. All updates to queried data will be made by clinical data 
management only and all modifications to the database will be recorded in an audit trail.  
Adverse events and medical history will be coded from the verbatim description (Investigator term) 
using the Medical Dictionary for Regulatory Activities (MedDRA). Prior and concomitant 
medications and therapies will be coded according to MedDRA. 
Once the database has been completed and locked, the PI will authorize database lock and all 
electronic data will be analyzed. Subsequent changes to the database will then be made only by 
written agreement. 
The data will be stored in a dedicated and secured area at Thomas Jefferson University. 
Electronic data will be stored at the Thomas Jefferson University servers. Data will be stored in a 
de-identified manner, where each study participant is recognizable by his/her unique trial subject 
number. The data will be stored at least 15 years after study closure. 
 
15.3 
Types of Data 

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Imaging data (ultrasound, CT, and MRI), clinical and laboratory data, AEs 
 
15.4 
Study Records Retention 
The data will be stored in a dedicated and secured area at Thomas Jefferson University. 
Electronic data will be stored at the Thomas Jefferson University servers. Data will be stored in a 
de-identified manner, where each study participant is recognizable by his/her unique trial subject 
number. The data will be stored at least 15 years after study closure. 
 
15.5 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical study protocol, Good Clinical Practice, 
or Manual of Procedures requirements. The noncompliance may be on the part of the participant, 
the investigator, or study staff. As a result of deviations, corrective actions are to be developed 
by the study staff and implemented promptly. 
All deviations from the protocol must be addressed in study participant source documents and 
promptly reported to the IRB and other regulatory bodies according to their requirements.  
16 Study Finances 
 
16.1 
Funding Source 
This study is financed through a grant from the United States National Institute of Health. 
 
16.2 
Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or 
financial gain greater than the minimum allowable by their institution, etc.) must have the conflict 
reviewed by a properly constituted Conflict of Interest Committee with a Committee-sanctioned 
conflict management plan that has been reviewed and 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 
Parking or travel reimbursement will be provided to patients as needed to cover transportation 
costs of participation. The total payment will not exceed $100 per patient.  
 
17 Publication and Data Sharing Policy 

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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. 
 
18 Literature References 
1.  
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2.  
Valle JW, Borbath I, Khan SA, et al. Biliary cancer: ESMO clinical practice guidelines for 
diagnosis, treatment and follow-up. Ann Oncol. Published online 2016. 
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3.  
Bridgewater J, Galle PR, Khan SA, et al. Guidelines for the diagnosis and management 
of intrahepatic cholangiocarcinoma. J Hepatol. Published online 2014. 
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4.  
Mosconi C, Calandri M, Javle M, Odisio BC. Interventional radiology approaches for intra-
hepatic cholangiocarcinoma. Chinese Clin Oncol Vol 9, No 1 (February 2020) Chinese 
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5.  
Ray CE, Edwards A, Smith MT, et al. Metaanalysis of survival, complications, and 
imaging response following chemotherapy-based transarterial therapy in patients with 
unresectable intrahepatic cholangiocarcinoma. J Vasc Interv Radiol. Published online 
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6.  
Al-Adra DP, Gill RS, Axford SJ, Shi X, Kneteman N, Liau SS. Treatment of unresectable 
intrahepatic cholangiocarcinoma with yttrium-90 radioembolization: A systematic review 
and pooled analysis. Eur J Surg Oncol. Published online 2015. 
doi:10.1016/j.ejso.2014.09.007 
7.  
Paul SB, Sharma H. Role of Transcatheter Intra-arterial Therapies for Hepatocellular 
Carcinoma. J Clin Exp Hepatol. 2014;4(Suppl 3):S112-21. doi:10.1016/j.jceh.2014.03.048 
8.  
Houle S, Yip TK, Shepherd FA, et al. Hepatocellular carcinoma: pilot trial of treatment 
with Y-90 microspheres. Radiology. 1989;172(3):857-860. 
doi:10.1148/radiology.172.3.2549567 
9.  
Andreana L, Isgrò G, Marelli L, et al. Treatment of hepatocellular carcinoma (HCC) by 
intra-arterial infusion of radio-emitter compounds: Trans-arterial radio-embolisation of 
HCC. Cancer Treat Rev. 2012;38(6):641-649. 
doi:https://doi.org/10.1016/j.ctrv.2011.11.004 
10.  
Hyder O, Marsh JW, Salem R, et al. Intra-arterial therapy for advanced intrahepatic 
cholangiocarcinoma: a  multi-institutional analysis. Ann Surg Oncol. 2013;20(12):3779-
3786. doi:10.1245/s10434-013-3127-y 

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11.  
Goldberg BB, Raichlen JS, Forsberg F. Ultrasound Contrast Agents: Basic Principles and 
Clinical Applications.; 2001. 
12.  
Lyshchik A. Specialty Imaging: Fundamentals of CEUS.; 2019. 
13.  
Nam K, Stanczak M, Lyshchik A, et al. Evaluation of Hepatocellular Carcinoma 
Transarterial Chemoembolization using  Quantitative Analysis of 2D and 3D Real-time 
Contrast Enhanced Ultrasound. Biomed Phys Eng express. 2018;4(3):35039. 
doi:10.1088/2057-1976/aabb14 
14.  
Oezdemir I, Wessner CE, Shaw C, Eisenbrey JR, Hoyt K. Tumor Vascular Networks 
Depicted in Contrast-Enhanced Ultrasound Images as a  Predictor for Transarterial 
Chemoembolization Treatment Response. Ultrasound Med Biol. 2020;46(9):2276-2286. 
doi:10.1016/j.ultrasmedbio.2020.05.010 
15.  
Shaw CM, Eisenbrey JR, Lyshchik A, et al. Contrast-enhanced ultrasound evaluation of 
residual blood flow to hepatocellular carcinoma after treatment with transarterial 
chemoembolization using drug-eluting beads: A prospective study. J Ultrasound Med. 
Published online 2015. doi:10.7863/ultra.34.5.859 
16.  
Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. Adv 
Drug Deliv Rev. 2008;60(10):1153-1166. doi:10.1016/j.addr.2008.03.005 
17.  
Ferrara K, Pollard R, Borden M. Ultrasound microbubble contrast agents: fundamentals 
and application to gene and  drug delivery. Annu Rev Biomed Eng. 2007;9:415-447. 
doi:10.1146/annurev.bioeng.8.061505.095852 
18.  
Tran WT, Iradji S, Sofroni E, Giles A, Eddy D, Czarnota GJ. Microbubble and ultrasound 
radioenhancement of bladder cancer. Br J Cancer. 2012;107(3):469-476. 
doi:10.1038/bjc.2012.279 
19.  
Czarnota GJ, Karshafian R, Burns PN, et al. Tumor radiation response enhancement by 
acoustical stimulation of the vasculature. Proc Natl Acad Sci. 2012;109(30):E2033 LP-
E2041. doi:10.1073/pnas.1200053109 
20.  
Kim HC, Al-Mahrouki A, Gorjizadeh A, Sadeghi-Naini A, Karshafian R, Czarnota GJ. 
Quantitative ultrasound characterization of tumor cell death: ultrasound-stimulated  
microbubbles for radiation enhancement. PLoS One. 2014;9(7):e102343. 
doi:10.1371/journal.pone.0102343 
21.  
Al-Mahrouki AA, Iradji S, Tran WT, Czarnota GJ. Cellular characterization of ultrasound-
stimulated microbubble radiation enhancement in a prostate cancer xenograft model. 
DMM Dis Model Mech. Published online 2014. doi:10.1242/dmm.012922 
22.  
Wood AKW, Sehgal CM. A review of low-intensity ultrasound for cancer therapy. 
Ultrasound Med Biol. 2015;41(4):905-928. doi:10.1016/j.ultrasmedbio.2014.11.019 
23.  
Belcik JT, Mott BH, Xie A, et al. Augmentation of limb perfusion and reversal of tissue 
ischemia produced by  ultrasound-mediated microbubble cavitation. Circ Cardiovasc 
Imaging. 2015;8(4). doi:10.1161/CIRCIMAGING.114.002979 

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24.  
Miller AP, Nanda NC. Contrast echocardiography: new agents. Ultrasound Med Biol. 
2004;30(4):425-434. doi:10.1016/j.ultrasmedbio.2003.12.002 
25.  
Daecher A, Stanczak M, Liu J-B, et al. Localized microbubble cavitation-based 
antivascular therapy for improving HCC treatment response to radiotherapy. Cancer Lett. 
2017;411:100-105. doi:10.1016/j.canlet.2017.09.037 
26.  
Eisenbrey JR, Forsberg F, Wessner CE, et al. US-triggered Microbubble Destruction for 
Augmenting Hepatocellular Carcinoma  Response to Transarterial Radioembolization: A 
Randomized Pilot Clinical Trial. Radiology. 2021;298(2):450-457. 
doi:10.1148/radiol.2020202321 
27.  
Davidson BP, Lindner JR. Future applications of contrast echocardiography. Heart. 
2012;98(3):246-253. doi:10.1136/heartjnl-2011-300737 
28.  
Leighton T. The Acoustic Bubble.; 1994. 
29.  
Ran L, Zhao W, Zhao Y, Bu H. Value of contrast-enhanced ultrasound in differential 
diagnosis of solid lesions of  pancreas (SLP): A systematic review and a meta-analysis. 
Medicine (Baltimore). 2017;96(28):e7463. doi:10.1097/MD.0000000000007463 
30.  
Dietrich CF, Burmester E. Contrast-enhanced ultrasound of small focal solid pancreatic 
lesions: A must! Endosc ultrasound. 2017;6(Suppl 3):S106-S110. 
doi:10.4103/eus.eus_73_17 
31.  
Li X-Z, Song J, Sun Z-X, Yang Y-Y, Wang H. Diagnostic performance of contrast-
enhanced ultrasound for pancreatic neoplasms: A  systematic review and meta-analysis. 
Dig liver Dis  Off J Ital Soc  Gastroenterol Ital Assoc Study Liver. 2018;50(2):132-138. 
doi:10.1016/j.dld.2017.10.012 
32.  
Claudon M, Dietrich CF, Choi BI, et al. Guidelines and good clinical practice 
recommendations for Contrast Enhanced  Ultrasound (CEUS) in the liver - update 2012: 
A WFUMB-EFSUMB initiative in cooperation with representatives of AFSUMB, AIUM, 
ASUM, FLAUS and ICUS. Ultrasound Med Biol. 2013;39(2):187-210. 
doi:10.1016/j.ultrasmedbio.2012.09.002 
33.  
Goldberg M. Systemic reactions to intravascular contrast media. A guide for the  
anesthesiologist. Anesthesiology. 1984;60(1):46-56. doi:10.1097/00000542-198401000-
00010 
34.  
Forsberg F, Liu J-B, Shi WT, Furuse J, Shimizu M, Goldberg BB. In vivo pressure 
estimation using subharmonic contrast microbubble signals: proof of  concept. IEEE 
Trans Ultrason Ferroelectr Freq Control. 2005;52(4):581-583. 
doi:10.1109/tuffc.2005.1428040 
35.  
Halldorsdottir VG, Dave JK, Leodore LM, et al. Subharmonic contrast microbubble 
signals for noninvasive pressure estimation under  static and dynamic flow conditions. 
Ultrason Imaging. 2011;33(3):153-164. doi:10.1177/016173461103300301 
36.  
Eisenbrey JR, Dave JK, Halldorsdottir VG, et al. Chronic liver disease: noninvasive 
subharmonic aided pressure estimation of hepatic  venous pressure gradient. Radiology. 

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2013;268(2):581-588. doi:10.1148/radiol.13121769 
37.  
Gupta I, Eisenbrey JR, Machado P, et al. Diagnosing Portal Hypertension with 
Noninvasive Subharmonic Pressure Estimates from  a US Contrast Agent. Radiology. 
2021;298(1):104-111. doi:10.1148/radiol.2020202677 
38.  
Nam K, Eisenbrey JR, Stanczak M, et al. Monitoring Neoadjuvant Chemotherapy for 
Breast Cancer by Using Three-dimensional Subharmonic Aided Pressure Estimation and 
Imaging with US Contrast Agents: Preliminary Experience. Radiology. 2017;285(1):53-62. 
doi:10.1148/radiol.2017161683 
39.  
Znati CA, Rosenstein M, Boucher Y, Epperly MW, Bloomer WD, Jain RK. Effect of 
radiation on interstitial fluid pressure and oxygenation in a human tumor  xenograft. 
Cancer Res. 1996;56(5):964-968. 
40.  
Znati CA, Rosenstein M, McKee TD, et al. Irradiation reduces interstitial fluid transport 
and increases the collagen content  in tumors. Clin cancer Res  an Off J Am Assoc  
Cancer Res. 2003;9(15):5508-5513. 
41.  
Multhoff G, Vaupel P. Radiation-induced changes in microcirculation and interstitial fluid 
pressure  affecting the delivery of macromolecules and nanotherapeutics to tumors. Front 
Oncol. 2012;2:165. doi:10.3389/fonc.2012.00165 
42.  
Khawaja OA, Shaikh KA, Al-Mallah MH. Meta-analysis of adverse cardiovascular events 
associated with echocardiographic  contrast agents. Am J Cardiol. 2010;106(5):742-747. 
doi:10.1016/j.amjcard.2010.04.034 
43.  
Muskula PR, Main ML. Safety With Echocardiographic Contrast Agents. Circ Cardiovasc 
Imaging. 2017;10(4). doi:10.1161/CIRCIMAGING.116.005459 
44.  
Main ML, Goldman JH, Grayburn PA. Ultrasound contrast agents: balancing safety 
versus efficacy. Expert Opin Drug Saf. 2009;8(1):49-56. 
doi:10.1517/14740330802658581 
45.  
Calio BP, Lyshchik A, Li J, et al. Long Term Surveillance of Renal Cell Carcinoma 
Recurrence Following Ablation using 2D and 3D Contrast-Enhanced Ultrasound. Urology. 
2018;121:189-196. doi:10.1016/j.urology.2018.05.053 
46.  
Forsberg F, Gupta I, Machado P, et al. Contrast-Enhanced Subharmonic Aided Pressure 
Estimation (SHAPE) using Ultrasound  Imaging with a Focus on Identifying Portal 
Hypertension. J Vis Exp. 2020;(166). doi:10.3791/62050 
 
 
 
 
 

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SUPPLEMENTAL MATERIALS 
These documents are relevant to the protocol, but they are not considered part of the protocol. 
They are stored and modified separately. As such, modifications to these documents do not 
require protocol amendments. 
 
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 
Appendix B: Optison product insert 
 
 
 

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APPENDIX A: SCHEDULE OF EVENTS 
 
Procedures 
Screening & 
baseline 
(D
0)
Study Visit 1 
(Day 7 ± 14) 
Study Visit 2 
(Day 14 ± 21) 
Study Visit 3 
(Day 21 ± 28) 
Signed Consent Form 
X 
 
 
 
Assessment of Eligibility 
Criteria 
X 
 
 
 
Review of Medical/Dental 
History 
X 
 
 
 
Review of Concomitant 
Medications  
X 
X 
X 
X 
Contrast-enhanced ultrasound 
(CEUS) exam 
X 
X 
X 
X 
Ultrasound-triggered 
microbubble destruction 
(UTMD) 
 
X 
X 
X 
Physical 
Examination 
Complete 
 
 
 
 
Symptom-
Directed 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
Assessment of Adverse 
Events 
X 
X 
X 
X 
Clinical 
Laboratory 
Pregnancy test 
X 
X 
X 
X 
Chemistry