Detection of High Grade Prostate Cancer With Subharmonic Ultrasound Imaging, A Pilot Study
Phase: PHASE2
Conditions: Prostatic Neoplasm
Sponsor: Thomas Jefferson University

THOMAS JEFFERSON UNIVERSITY 
Kimmel Cancer Center 
Detection of high grade prostate cancer with subharmonic ultrasound imaging, A pilot 
study. 
Principal 
Investigators: 
Ethan J Halpern, MD       
 Flemming Forsberg, PhD 
Department of Radiology       
 Department of Radiology 
Thomas Jefferson University   
  Thomas Jefferson University 
132 South 10th Street       
  132 South 10th Street 
Philadelphia, PA  19107-5244 
  Philadelphia, PA  19107-5244 
215-955-5345       
 215-955-4870 
Co-Investigator(s): 
Edouard Trabulsi 
Department of Urology 
Leonard Gomella 
Department of Urology 
Costas Lallas 
Department of Urology 
Funding Sponsor: 
NIH R21CA202214 
Regulatory Sponsor: 
Flemming Forsberg PhD 
IND Number: 
112,241 
Study Product: 
Definity 
Protocol Number: 
15F.607 
Version: 
Date: 
1.0 
11/2015 
1.1 
11/2016 
1.2 
12/01/17 
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. 

 
 
 
 
 
2 
Study Summary 
Title 
Detection of high grade prostate cancer with subharmonic 
ultrasound imaging, A pilot study 
Short Title 
Using Contrast-enhanced Ultrasound (CE-TRUS) to Identify 
Aggressive forms of Prostate Cancer 
Protocol Number 
 
Phase 
Phase II clinical study – classified as Phase II because the 
goal is to demonstrate that imaging of the prostate with 
CEUS is improved with the use of subharmonic imaging 
Methodology/Study 
Design 
Pilot cohort study of patients scheduled for prostate biopsy 
Study Duration 
two years 
Study Center(s) 
Single-center  
Objectives 
1. To implement SHI technology on a transrectal probe 
suitable for prostate imaging and biopsy 
2. To demonstrate visualization of prostatic vascularity using 
subharmonic contrast-enhanced imaging. 
3. To provide a preliminary estimate of the diagnostic 
accuracy of contrast-enhanced subharmonic imaging for 
detection of clinically significant PCa.  
Number of 
Subjects 
55 subjects (5 controls and 50 patients)  
Diagnosis and 
Main Inclusion 
Criteria 
1. Patients who are scheduled for prostate biopsy 
2. and who are able to undergo CEUS 
Study Therapy, 
Dose, Route, 
Regimen 
The study will involve infusion of the ultrasound 
microbubble contrast agent Definity immediately prior to a 
prostate biopsy procedure. 
Duration of 
administration and 
follow-up 
The contrast administration will require 10-15 minutes, 
during which time a transrectal ultrasound study of the 
prostate will be performed, and a targeted biopsy procedure 
of the prostate will be performed. 
Reference therapy 
Conventional 12-core systematic prostate biopsy 
Statistical 
Methodology 
Computation of sensitivity and specificity for detection of 
clinically significant prostate cancer with conventional 12 
core prostate biopsy as the reference standard.  No further 
statistical testing will be performed as this is a preliminary 
study, designed to demonstrate the feasibility of 
subharmonic imaging of the prostate and to obtain an 
estimate of the diagnostic accuracy of CEUS with 
subharmonic imaging of the prostate. 

 
 
 
 
 
3 
Schema 
Subharmonic imaging software will be expanded to work 
with an endorectal probe on an FDA approved ultrasound 
imaging system 
↓ 
Patient with elevated PSA or an abnormal rectal exam, or a 
patient requiring a prostate biopsy on active surveillance 
↓ 
Scheduled for prostate biopsy 
↓ 
Has CEUS with subharmonic imaging 
↓ 
Prostate biopsy with up to 6 targeted cores based upon 
subharmonic imaging as well as prostate biopsy with 12 
standard systematic cores. 
 
 
 

 
 
 
 
 
4 
1.0 INTRODUCTION 
 
This document is a protocol for a human research study. This study is to 
be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies 
and procedures.  
 
1.1 Specific Aims and Hypothesis 
 
1. To implement SHI technology on a transrectal probe suitable for prostate 
imaging and biopsy 
2. To demonstrate visualization of prostatic vascularity during subharmonic 
contrast enhanced imaging of the prostate. 
3. To provide a preliminary estimate of the diagnostic accuracy of contrast-
enhanced subharmonic imaging for detection of prostate cancer.  
 
1.2 Background and Rationale 
 
Prostate cancer (PCa) is the most prevalent cancer among American 
males, with an estimated 3,922,600 living PCa patients.1 Biochemical screening 
with serum prostate specific antigen (PSA) results in the detection of many small 
cancers such that “the risk of being diagnosed with PCa is increasingly greater 
than the risk of dying of it”.2 Thus, although PCa will account for 27% 
(233,000/855,220) of all new cancer diagnoses among American males in 2014, 
PCa is estimated to be directly responsible for only 9.5% (29,480/310,010) of 
cancer related deaths among American males.3 PCa has been likened to the two 
faces of Janus: “one benevolent face of small, indolent tumors, abundant among 
middle-aged and elderly men” and a second “grim face of a great killer, being the 
second most common cancer-related cause of death within the EU and in the 
US”.4 This dichotomy in the progression and prognosis of PCa creates a dilemma 
for both physician and patient in deciding between definitive therapy of a 
potentially lethal disease and expectant management of a potentially indolent 
disease. Simply stated, the PCa treatment dilemma is related to our inability to 
distinguish indolent from aggressive PCa at the time of diagnosis. 
 
Two recent clinical trials have sparked an intense controversy related to 
the benefit of screening and treatment of PCa. The Prostate, Lung, Colorectal, 
and Ovarian (PLCO) Cancer Screening Trial failed to show a mortality benefit 
from screening and treatment of PCa.5 The European Randomized Study of 
Screening for PCa (ERSPC) suggests that with the current standard of care, 
1410 men must be screened and 48 additional cases of PCa treated to prevent 

 
 
 
 
 
5 
one death.6 Both studies suggest that overdiagnosis and overtreatment of 
insignificant cancer is a major drawback of PCa screening, and point to the need 
for better diagnostic tools to identify clinically significant PCa.7  Based upon the 
data from these trials, the U.S. Preventive Services Task Force (USPSTF) has 
issued a grade “D” recommendation against PSA-based screening.8 
 
Various treatment options are available to the patient with a new diagnosis 
of PCa. Aggressive therapy with surgery or radiation constitutes the traditional 
therapy for localized PCa, with the goal of treating the entire prostate to eradicate 
the cancer. However, both radical prostatectomy and radiation therapy can be 
associated with substantial morbidity, including sexual impotence, urinary 
incontinence and injury to adjacent organs.9,10 Expectant management without 
curative therapy has demonstrated favorable 15-20 year follow-up results for 
localized, low-grade PCa.11,12 Prospective cohort studies suggest that aggressive 
treatment for PCa may be deferred until there is evidence of cancer 
progression.13 For men with “favorable risk” PCa, defined as low volume disease 
(< 0.5 ml) with a Gleason score below 7, active surveillance (AS) results in 
similar survival as definitive therapy,14 and conservative management is 
associated with a 50% lower incidence of incontinence and impotence as 
compared to radical prostatectomy.15 A major barrier to the widespread adoption 
of AS is the lack of an accurate non-invasive diagnostic test to prospectively 
identify those patients with aggressive “clinically significant” cancer whose 
disease will progress without definitive therapy. Patients on AS therefore require 
a periodic invasive biopsy for tissue diagnosis. A technique that could improve 
the accuracy of diagnostic ultrasound for the diagnosis of clinically significant 
PCa would facilitate wider use of AS. 
 
 
Contrast-Enhanced Imaging of Prostate Cancer 
 
As prostate imaging is necessary for biopsy guidance in the diagnosis of 
PCa, much research has focused on optimization of imaging techniques for PCa 
detection. Our group has focused primarily on prostate imaging with microbubble 
ultrasound contrast agents, as ultrasound is the least expensive and most 
commonly used modality for guidance of prostate biopsy. When exposed to an 
ultrasound pulse at some fundamental frequency (f0), microbubbles act as 
nonlinear oscillators, generating significant energy components which span the 
range of frequency emissions from subharmonics through ultraharmonics.16 
While conventional ultrasound processes received echoes at the fundamental 
frequency (f0), contrast-enhanced ultrasound imaging is based upon received 
echo signals in the harmonic (2f0, 3f0,,etc) or subharmonic (1/2 f0, 1/3 f0,,etc) 
range. Since microbubble contrast agents remain intravascular, contrast-
enhanced transrectal ultrasound (CE-TRUS) with harmonic imaging (HI) or 
subharmonic imaging (SHI) selectively images vascularity within the prostate. 

 
 
 
 
 
6 
Pathologic studies demonstrate a clear association of microvessel density 
with the presence of PCa,17 with metastatic disease,18 with disease stage19 and 
with disease-specific survival.20,21 Although microvessels are below the 
resolution of conventional ultrasound Doppler imaging, CE-TRUS provides a 
practical solution to imaging the microvasculature. Clinical studies using HI have 
demonstrated that enhancement of the prostate with CE-TRUS is related to 
increased microvessel density,22,23 that CE-TRUS can improve detection of 
PCa,24,25 and that CE-TRUS can increase cancer detection with fewer biopsy 
cores.26,27  We review preliminary results with CE-TRUS HI and SHI below, but 
note that there are no published studies using SHI of the prostate.  
 
1.3 Study Therapy 
 
           Since the subjects in this study are scheduled to have prostate biopsy as 
part of their standard of care, the only study related test that will be performed is a 
contrast-enhanced ultrasound with subharmonic imaging. The proposed 
ultrasound contrast agent for the current study, Definity™, (Perflutren Lipid 
Microsphere, Lantheus Medical Imaging, Inc; N. Billerica, MA), is a microbubble 
contrast agent that demonstrates enhancement of the prostate on both grayscale 
and Doppler imaging. Definity is a sterile, non-pyrogenic suspension of liposome-
encapsulated perfluoropropane microbubbles. This contrast agent is composed of 
a blend of three phospholipids contained in a matrix of sodium chloride, propylene 
glycol and glycerin in water. The contrast agent is supplied in a vial that contains 
the phospholipids and perfluouropropane gas. The microbubble agent is prepared 
by shaking the standard size vial with the aid of a shaking device (Vialmix; ESPE, 
Seefeld, Germany). Based upon our previous experience with Definity, two vials 
of Definity will be mixed and diluted in 50 ml of normal saline, yielding a 
concentration of 49.4 μl/ml. For the purpose of contrast-enhanced imaging, 
Definity will be infused over approximately 10-12 minutes, during which time 
pulse inversion harmonic and flash replenishment imaging will be performed. 
Definity remains stable within the circulation for 3 to 6 minutes after infusion; all 
contrast-enhanced procedures will be completed during the infusion time.   
 
1.3 Clinical Data – Breast and Liver 
 
Feasibility studies of this new SHI mode have been conducted in vitro and 
in vivo by our group28,29 as well as others30;31,32,33,34,35;36;37;38. Researchers at 
Jefferson produced the first ever in vivo grayscale SHI images (of canine 
kidneys) using a modified US scanner. We also produced the first ever in vivo 
human grayscale SHI images, demonstrating (in 14 women with 16 breast 
lesions) that SHI can detect the slow, small volume blood flow associated with 
breast tumor angiogenesis in humans.39  

 
 
 
 
 
7 
Fig. 1 presents an 
example of a fibroadenoma 
imaged after administration 
of 
Optison 
with 
power 
Doppler imaging (PDI) and 
in SHI mode (transmitting at 
4.4 MHz and receiving at 2.2 
MHz; optimized for SHI) 
using a modified Logiq 9 
scanner 
(GE 
Healthcare, 
Milwaukee, WI).  Notice, the almost complete lack of signal from non-vascular 
tissue, and that the internal morphology of the tumor vascularity was visualized 
better with SHI than with contrast enhanced PDI.   
 
Fig. 2 shows a 
breast lesion with benign 
ductal microcalcifications 
and fibrocystic changes. 
Branching vessels within 
and around the lesion are 
depicted in finer detail 
with SHI (Fig 3b) and 
without 
the 
color 
blooming 
of 
contrast 
enhanced PDI (Fig 3a). 
Finally, SHI depicts neovessels (~20-40µm diameter40) not observed with 
conventional imaging. 
 
More recently we have focused on translating grayscale SHI into human 
clinical trials in the breast and liver41,42 and on improved processing of SHI 
images.43 We have introduced the concept 
of CMI-SHI, where the inflow of contrast 
bubbles 
in 
tumor 
neovessels 
is 
reconstructed 
by 
temporal 
maximum 
intensity projection of pixels traced from 
successive image frames.44;45 A dynamic 
cumulative maximum intensity display for 
subharmonic 
imaging 
(CMI-SHI) 
was 
developed using a new automated kernel 
tracking algorithm by identifying motion 
induced displacements. As demonstrated 
by the receiver operating characteristic 
analysis in Fig 3, the area under the curve 
for diagnosis of breast cancer was greatest 
for dynamic CMI-SHI (0.90) and was 
(a) 
 
 
 
         (b) 
Figure 2.  Benign ductal microcalcifications and 
fibrocystic changes (arrows) depicted with contrast 
PDI (a) and with SHI (b).
Figure 3.  ROC curves for four of the 
imaging modalities; (GS: grayscale). 
Figure 1.  Fibroadenoma (arrows) imaged in PDI mode 
(a) and in SHI mode (b) after injection of contrast.  
(a) 
 
 
         (b) 

 
 
 
 
 
8 
significantly better than for mammography (p = 0.031). With regard to tissue 
perfusion, we have developed a novel method for deriving quantitative estimates 
of tissue perfusion, based on in vivo grayscale SHI46 as an alternative to the well-
known destruction/reperfusion technique.47 A modified Logiq 9 scanner operating 
in SHI mode was used to measure renal perfusion. Fractional blood volumes 
were estimated from subharmonic signal intensities from blood in tissue.48,49 
Perfusion was estimated from the initial slope of the SHI time intensity curve and 
correlated with the reference standard neutron activation assay technique (r = 
0.62; p < 0.001).50 Finally, we created parametric images of SHI contrast kinetics, 
demonstrating increased perfusion in breast cancer relative to benign lesions 
(0.103 ± 0.030 vs. 0.054 ± 0.017; p = 0.0014).51 
 
1.5 Clinical Data - Prostate 
 
Two basic ultrasound technologies have been used to image microbubble 
contrast agents in the prostate: Doppler and grayscale HI. These technologies 
are summarized in a textbook by one of the PIs.52 Color and power Doppler 
imaging techniques, available on most modern ultrasound systems, utilize 
relatively high energy levels that destroy a large proportion of the microbubbles 
as they are imaged. Pulse inversion and grayscale HI are technological 
advances that together permit high resolution, low energy grayscale imaging of 
contrast agents.53,54,55,56 Various pulsing sequences can further improve 
grayscale HI of the prostatic microcirculation.57,58 Flash replenishment imaging 
was first described as a grayscale harmonic technique that can demonstrate 
tissue perfusion and may be used for blood flow estimation.59,60,61 Contrast-
Enhanced Flash Replenishment may be combined with Maximum Intensity 
Projection (CE-FR-MIP) of each pixel in the image over 2-4 seconds of low 
power image frames to demonstrate microvascular patterns associated with 
neovascularity62 and PCa.63  Whereas assessment for neovascularity with 
contrast-enhanced Doppler imaging is based upon the magnitude of contrast 
enhancement, CE-FR-MIP provides additional information based upon changes 
to the normal intraprostatic microvascular architecture (Fig. 4). 
 
Numerous trials of CE-TRUS using HI to detect PCa have been conducted 
at the Jefferson Prostate Diagnostic Center.64,65 An early study (n=100 subjects) 
demonstrated improved sensitivity for PCa detection from 38% at baseline to 
Figure 4. Gleason 7 PCa in 
the right mid-gland is invisible 
on grayscale (left image), but 
tumor vascularity is clearly 
defined 
with 
CE-FR-MIP 
imaging (right image; arrows). 

 
 
 
 
 
9 
65% after infusion of the microbubble agent Definity (Lantheus Medical Imaging, 
N Billerica, MA); p ≤ 0.004.66 Furthermore, lesions detected by contrast-
enhancement were generally larger and with a higher Gleason score – that is, 
they were the more clinically significant cancers. The last 40 subjects in this 
study were evaluated with targeted biopsy. A suspicious site identified by CE-
TRUS was 3.5 times more likely to have a positive biopsy than an adjacent site 
without contrast enhancement (p < 0.025).67 
 
A Department of Defense funded clinical trial of 301 men demonstrated 
that targeted cores based upon CE-TRUS were twice as likely to return a positive 
biopsy as systematic cores.68  Using CE-FR-MIP, our group demonstrated 
positive biopsies in 29/225 targeted cores as compared with 50/600 systematic 
cores, yielding an odds ratio of 2.0 for cancer detection with CE-TRUS (p = 
0.034).69 A recently completed National Cancer Institute (NCI) funded clinical trial 
(n = 272) confirmed increased PCa detection with CE-TRUS, and specifically 
with CE-FR-MIP (OR=2.1, p < 0.001). More significantly, CE-TRUS preferentially 
detected lesions with greater tumor volume and Gleason score ≥ 7 (p < 0.001). 
ROC analysis for detection of high volume / high grade PCa with CE-TRUS 
yielded an area under the curve of 0.90 (p = 0.001).70 Comparison of CE-TRUS 
HI with whole mount prostatectomy specimens in our DOD and NCI study 
subjects demonstrated microbubble enhancement in 93% of malignant foci. In 
summary, PCa’s visualized with CE-TRUS tend to be larger and of higher grade. 
 
In summary, clinical studies demonstrate selective detection of higher grade 
PCa with HI, based upon the increased neovascularity of these tumors. 
Nonetheless, although CE-TRUS with HI selectively detects clinically significant 
PCa, overall by-patient detection of high grade PCa with targeted biopsy based 
upon HI alone remains inferior to a 12 core systematic biopsy. This limitation is 
related, in part, to difficulty discerning enhancing foci of PCa adjacent to the 
hypervascular transition zone (see abstract in appendix). A major limitation of HI 
is related to reduced blood-to-tissue contrast resulting from second harmonic 
generation and accumulation in tissue.71 Hence, subharmonic imaging (SHI), 
transmitting at the fundamental frequency (f0) and receiving at the subharmonic 
(f0/2), becomes an attractive alternative contrast-specific imaging mode, because 
of the weaker subharmonic generation in tissue and the significant subharmonic 
scattering produced by some new contrast agents.72 As demonstrated in the 
previous section, preliminary data with SHI suggest that this technique provides a 
superior signal to background ratio for evaluation of blood flow, that SHI can 
improve depiction of neovessels, and that SHI may be useful for quantitative 
assessment of tissue perfusion in cancer. In order to optimize the detection of 
clinically significant PCa, we propose the use of SHI with CE-TRUS for targeted 
biopsy of the prostate. 
 
 
 

 
 
 
 
 
10 
1.6 Dose Rationale and Risk/Benefits 
 
Based upon our previous experience with Definity and according to the 
package insert, two vials of Definity will be mixed and diluted in 50 ml of normal 
saline, yielding a concentration of 49.4 μl/ml. For the purpose of contrast-
enhanced imaging, Definity will be infused over approximately 10-12 minutes, 
during which time pulse inversion HI and SHI will be performed. Definity remains 
stable within the circulation for 3 to 6 minutes after infusion; all contrast-
enhanced procedures will be completed during the infusion time. The risk of this 
procedure is basically the risk of an adverse reaction to Definity. The largest 
safety studies published to date on the use of ultrasound contrast agents in 
humans (involving up to 4,300,966 subjects) concluded that these agents have a 
good safety profile in both cardiac and non-cardiac applications.73,74,75,76  In our 
experience, adverse reactions to Definity include only minor sensations of 
discomfort which resolve as soon as the infusion is stopped. 
2.0 STUDY OBJECTIVES 
 
1. To implement SHI technology on a transrectal probe suitable for prostate 
imaging and biopsy 
2. To demonstrate visualization of prostatic vascularity during subharmonic 
contrast enhanced imaging of the prostate. 
3. To provide a preliminary estimate of the diagnostic accuracy of contrast-
enhanced subharmonic imaging for detection of prostate cancer.  
 
3.0 STUDY DESIGN 
3.1 General Design 
 
This study is a pilot phase II clinical trial of 55 patients (5 controls and 50 
patients) over the course of 2 years. Experimental software used for 
subharmonic imaging of other body parts will be adopted to work with the 
transrectal prostate probe of an FDA approved ultrasound system, specifically 
the IC5-9D transducer on the Logiq E9 system (GE Healthcare; Milwaukee, WI). 
In vitro testing will be performed on 5 patients to ensure that the power output of 
the transducer with the new subharmonic software is within the FDA approved 
limits for the system power output. After in vitro optimization of the subharmonic 
mode for imaging, we will conduct a pilot clinical trial of 50 patients over 2 years.  
All participants will be evaluated with contrast-enhanced transrectal ultrasound 
(CE-TRUS) prior to prostate biopsy. Subharmonic imaging of the CE-TRUS study 
will be used to direct targeted biopsy of the prostate with up to 6 biopsy cores. 
This will be followed by a conventional systematic biopsy of the prostate with12 
cores. 

 
 
 
 
 
11 
3.2 Primary Study Endpoints 
a. Demonstration of prostate vascularity with subharmonic contrast-enhanced  
imaging. Specifically, demonstration of increased prostate vascularity in patients 
with high risk, aggressive prostate cancer. 
 
b. Preliminary measures of efficacy (sensitivity and specificity) will be obtained 
for subharmonic CE-TRUS in the detection of high risk, aggressive prostate 
cancer after biopsy procedure on 50 patients. 
3.3 Secondary Study Endpoints 
n/a 
3.4 Primary Safety Endpoints 
 
No safety issues are expected from the use of subharmonic imaging, as the 
power output of the ultrasound system will be similar to its operation in 
conventional mode. No safety issues are expected from the administration of 
Definity for CEUS, as this agent has been used safely in numerous clinical trials 
and is FDA approved for intravenous administration during cardiac imaging. 
4.0 SUBJECT SELECTION AND WITHDRAWAL 
 
4.1 Inclusion Criteria 
 
1. Subject must be scheduled for a clinically indicated needle biopsy of the 
prostate based upon an elevated PSA, abnormal digital rectal examination, or 
based upon active surveillance of prostate cancer. 
2. Subject must be able and willing to give written informed consent for a 
contrast enhanced ultrasound study of the prostate. 
3. Subject must be a male at least 18 years of age when informed consent is 
obtained. 
4. Subject must have a life expectancy that exceeds the duration of the clinical 
trial. 
4.2 Exclusion Criteria 
 
1. Participant in a clinical trial involving an investigational drug within the past 30 
days. 
2. Patients with known or suspected hypersensitivity to perflutren 
3. Previous treatment for PCa. 
4. Clinically unstable, severely ill, or moribund. 
 

 
 
 
 
 
12 
4.3 Gender/Minority/Pediatric Inclusion for Research 
 
This study is only applicable to adult males, as PCa is only present in the 
adult male population. The study will include men of all races; there will be no 
exclusion based upon race or ethnicity. 
 
4.4 Subject Recruitment and Screening 
 
Fifty adult male patients will be recruited to this study by our urology co-
investigators from the population scheduled to undergo prostate biopsy at 
Thomas Jefferson University Hospital. Subjects will be screened for eligibility by 
the attending urologist. The urologist will explain the procedure and may obtain 
written informed consent from each study participant at that time or in radiology 
on the day of study and prior to any study procedures being performed. 
 
 
4.5 Early Withdrawal of Subjects 
4.5.1 When and How to Withdraw Subjects 
Any subject may withdraw from the study up until the time the CEUS study is 
performed.  However, once that study is performed, the data will be included in 
our analysis. 
5.0 STUDY DRUG/THERAPY  
5.1 Description 
This is not a therapeutic trial.  The only drug that will be administered is Definity, 
which is a microbubble ultrasound contrast agent. The risk of the procedure is 
the risk of an adverse reaction to Definity, which is considered minimal. Although 
the study participants may not benefit from the findings, future patients may 
benefit from development of a non-invasive imaging technique that can detect 
and characterize aggressive PCa.  If successful, this technique may reduce the 
number of biopsy procedures performed for prostate cancer, and it will likely be 
used to guide a limited number of targeted biopsy cores into those areas that 
demonstrate imaging characteristics of aggressive prostate cancer. 
5.2 Treatment Regimen 
Based upon our previous experience with Definity and according to the 
manufacturer’s instruction in the package insert, two vials of Definity will be 
mixed and diluted in 50 ml of normal saline, yielding a concentration of 49.4 
μl/ml. For the purpose of contrast-enhanced imaging, Definity will be infused over 

 
 
 
 
 
13 
approximately 10-12 minutes, during which time pulse inversion HI and SHI will 
be performed. 
5.3 Risks 
 
The risk of this procedure is basically the risk of an adverse reaction to Definity, 
and the risks of conventional prostate biopsy. The largest safety studies 
published to date on the use of ultrasound contrast agents in humans (involving 
up to 4,300,966 subjects) concluded that these agents have a good safety profile 
in both cardiac and non-cardiac applications.77,78,79,80  In our experience, adverse 
reactions to Definity include only minor sensations of discomfort which resolve as 
soon as the infusion is stopped. 
 
5.4 Method for Assigning Subjects to Treatment Groups 
 
n/a, as all subjects will be studied with HI and SHI. 
6.0 STUDY PROCEDURES 
 
6.1 Study Visit Schedule 
 
The CEUS study procedure will be performed during a single episode of 
care at which time the patient will undergo a clinically indicated biopsy of the 
prostate. No additional visits will be required to participate in the study. 
 
 
 
All ultrasound imaging will be performed with a commercially available 
Logiq E9 ultrasound system that provides conventional color and power Doppler 
imaging, and which will be modified to provide contrast-enhanced HI and SHI. 
Because of the very low SHI signal obtained from tissue, SHI is performed with a 
dual imaging technique that displays a conventional grayscale image alongside 
the SHI image. The conventional grayscale image is necessary to define the 
tissue anatomy and determine location within the prostate while the SHI is used 
to demonstrate vascularity and neovessels. 
 
The proposed microbubble contrast agent for the current study, Definity, 
(Perflutren Lipid Microsphere, Lantheus), is a microbubble contrast agent that 
demonstrates enhancement of the prostate on both grayscale and Doppler 
imaging. Definity remains stable within the circulation for 3 to 6 minutes after 
infusion and all contrast-enhanced procedures will need to be completed during 
the infusion time. This time constraint has not been a problem in prior studies. 
Based on our previous experience with Definity (and in accordance with the 
manufacturer’s instructions), two vials will be mixed and diluted in 50 ml of 

 
 
 
 
 
14 
normal saline, yielding a concentration of 49.4 μl/ml. A 20 gauge intravenous 
catheter will be inserted into an arm vein for administration of contrast material. 
Definity will be delivered as an infusion at an initial rate of 4 ml/min. The infusion 
rate will be titrated to achieve adequate enhancement of the prostatic 
microvasculature. Post contrast imaging will begin as soon as contrast is visible. 
The infusion of Definity will continue for approximately 10-12 minutes during 
which time the contrast-enhanced diagnostic ultrasound, the assignment of rating 
scores, and the SHI guided biopsy procedure will be performed.  
 
Study participants will be prepared for biopsy according to standard 
clinical operating procedure for subjects undergoing TRUS-guided biopsy at the 
Jefferson Prostate Diagnostic Center. Study subjects will be imaged in the 
lithotomy position with grayscale ultrasound performed to measure gland size.81 
Next, the prostate will be evaluated with both baseline and contrast-enhanced 
imaging modes. The dynamic range and imaging frequencies for grayscale, and 
color Doppler imaging will be standardized for all subjects (scanning frequency of 
7.0 MHz with dynamic range of 65 dB). The Doppler window for color imaging will 
include the entire gland. The baseline examination (~5 minutes) will consist of 
conventional grayscale imaging and color Doppler imaging. Contrast-enhanced 
imaging (~5-7 minutes) will consist of color Doppler imaging, HI and SHI. In order 
to allow comparison of the various imaging modes, a standard imaging sweep 
through the prostate will be performed in the axial plane from the base of the 
gland to the apex for each of the 2 baseline imaging modes and 3 contrast-
enhanced imaging modes.  
 
Study participants will be monitored for AEs during and 30 minutes after contrast 
administration.  
 
6.2 Study Interpretation 
 
The peripheral zone of each prostate will be rated for suspicion of PCa at 
12 sites, including medial and lateral component of each sextant. A 5-point rating 
score will be assessed in real-time by the examining physician for each 
ultrasound technique. These scores have been previously described and 
validated,82 with proven utility for predicting the risk of malignancy in prior 
studies.Error! Bookmark not defined.,Error! Bookmark not defined. A maximum of 6 sites will be 
identified as the most suspicious areas on SHI; targeted biopsy specimens will 
be obtained from these sites under ultrasound guidance during contrast-
enhanced imaging. If fewer sites are identified, fewer targeted biopsy cores may 
be obtained. 
 
The interpretation of CEUS studies will follow a 12 part extended sextant 
grid as demonstrated in the illustration below: 
 

 
 
 
 
 
15 
 
 
 
 
7.0 STATISTICAL PLAN 
7.1 Sample Size Determination 
 
The study is a pilot study, for the purpose of generating preliminary data for 
a future grant submission. The study is not powered to demonstrate any specific 
accuracy of CEUS, but rather to accumulate several demonstration cases of SHI 
in high grade prostate cancer.  
 
Based upon our past experience we expect approximately 30% of our 
patients to be positive for prostate cancer and half of these (15%) to have a 
diagnosis of high grade disease. Thus, in a population of 50 patients, the mean 
expected number of patients with high grade disease would be 7.5. Using an 
exact binomial probability distribution, the probability of finding at least 5 patients 
in this sample with high grade disease is 88%. The probability of finding 3 or 
fewer patients with high grade disease is 4.6%. The probability of not finding a 
single patient with high grade disease is 0.03%.  Thus, the sample size of n=50 
provides a greater than 95% probability of demonstrating the performance of SHI 
in at least 3 patients with high grade prostate cancer. 

 
 
 
 
 
16 
7.2 Statistical Methods 
Rough estimates of the sensitivity and specificity of SHI for high grade disease 
may be obtained if there are sufficient patients with high grade disease. No 
additional statistical analysis will be performed 
7.3 Subject Population(s) for Analysis 
All study participants will be included in the analysis. 
8.0 SAFETY AND ADVERSE EVENTS 
8.1 Definitions 
Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that 
develops or worsens in severity during the course of the study.  Intercurrent 
illnesses or injuries should be regarded as adverse events.  Abnormal 
results of diagnostic procedures are considered to be adverse events if the 
abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.   
 
A serious adverse event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
 
Important medical events are those that may not be immediately life 
threatening, but are clearly of major clinical significance. They may 
jeopardize the subject, and may require intervention to prevent one of the 
other serious outcomes noted above.  For example, drug overdose or 
abuse, a seizure that did not result in in-patient hospitalization, or intensive 
treatment of bronchospasm in an emergency department would typically be 
considered serious.  
 

 
 
 
 
 
17 
All adverse events that do not meet any of the criteria for serious should be 
regarded as non-serious adverse events.  
Adverse Event Reporting Period 
The study period during which adverse events will be reported will extend 
from the time of administration of the Definity contrast agent, up to 24 hours 
after the administration.  
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A 
preexisting condition should be recorded as an adverse event if the 
frequency, intensity, or the character of the condition worsens during the 
study period. 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a 
preexisting condition.  At the end of the study, any new clinically significant 
findings/abnormalities that meet the definition of an adverse event must also 
be recorded and documented as an adverse event.  
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until 
the events are resolved, the subject is lost to follow-up, or the adverse event 
is otherwise explained.  The investigator will instruct each subject to report 
any subsequent event(s) that the subject, or the subject’s personal 
physician, believes might reasonably be related to participation in this study.   
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization 
should be documented and reported as a serious adverse event unless 
specifically instructed otherwise in this protocol.  Any condition responsible 
for surgery should be documented as an adverse event if the condition 
meets the criteria for and adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery 
are reported as an adverse event in the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective 
surgical procedures for a preexisting condition.  Surgery should not 
be reported as an outcome of an adverse event if the purpose of the 
surgery was elective or diagnostic and the outcome was uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy 
measurement for the study. 
• Hospitalization or prolonged hospitalization for therapy of the target 
disease of the study, unless it is a worsening or increase in 
frequency of hospital admissions as judged by the clinical 
investigator. 

 
 
 
 
 
18 
8.2 Recording of Adverse Events 
 
The following subsections detail what information must be documented for each 
adverse event occurring during the time period specified in Section 8.3. 
8.2.1 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. 
 
8.2.2 Expectedness 
The PI is responsible for determining whether an AE is expected or unexpected. 
An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the 
intervention. Risk information to assess expectedness can be obtained from 
preclinical studies, the investigator’s brochure, published medical literature, the 
protocol, or the informed consent document. 
 
8.2.3 Severity of Event 
Adverse events will be graded for severity according to the Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0.  
 
8.2.4 Intervention 
Any intervention implemented to treat the adverse event must be documented for 
all adverse events. 
8.3 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 

 
 
 
 
 
19 
designee) will inquire about the occurrence of AE/SAEs since the last visit. 
Events will be followed for outcome information until resolution or stabilization. 
8.4 Reporting to IRB 
8.4.1 Unanticipated Problems 
All incidents or events that meet criteria for unanticipated problems (UAPs) as 
defined in Section 8.1.1 Unanticipated Problems require the creation and 
completion of an unanticipated problem report form (OHR-20).  
UAPs that pose risk to participants or others, and that are not AEs, will be 
submitted to the IRB on an OHR-20 form via the eazUP system within 10 working 
days of the investigator becoming aware of the event.  
UAPs that do not pose risk to participants or others will be submitted to the IRB 
at the next continuing review.  
8.4.1.1 Adverse Events  
Grade 1 AEs will be reported to the IRB at continuing review.  
Grade 2 AEs will be reported to the IRB at the time of continuing review.  
8.4.1.2 Serious Adverse Events  
SAEs will be reported to the IRB on OHR-10 forms via the electronic reporting 
system (eSAEy) according to the required time frames described below. 
Grade 3-4 AEs that are unexpected and deemed to be at least possibly related to 
the study will be reported to the IRB within 2 working days of knowledge of the 
event. 
Grade 3-4 AEs that are deemed unrelated to the study will be reported to the IRB 
within 5 working days.  
Grade 5 AEs will be reported to the IRB within one working day of knowledge of 
the event. 
All SAEs will be submitted to the IRB at continuing review, including those that 
were reported previously. 
 
8.5 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 

 
 
 
 
 
20 
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 

 
 
 
 
 
21 
 
 
 
 
8.6 Report to FDA 
In accordance with 21 CFR 212.32, sponsor-investigators of studies conducted 
under an IND must comply with following safety reporting requirements:  
 
Expedited IND Safety Reports: 
 
7 Calendar-Day Telephone or Fax Report:   
 
The Sponsor-Investigator is required to notify the FDA of any fatal or life-
threatening adverse event that is unexpected and assessed by the investigator to 
be possibly related to the use of Definity.  An unexpected adverse event is one 
that is not already described in the Investigator Brochure.   
 
Such reports are to be telephoned or faxed to the FDA within 7 calendar days of 
first learning of the event.  Each telephone call or fax transmission (see fax 
number below) should be directed to the FDA new drug review division in the 
Center for Drug Evaluation and Research or in the product review division for the 
Center for Biologics Evaluation and Research, whichever is responsible for the 
review of the IND. 
 
15 Calendar-Day Written Report:   
 
The Sponsor-Investigator is also required to notify the FDA and all participating 
investigators, in a written IND Safety Report, of any serious, unexpected AE that 
is considered possibly related to the use of Definity. An unexpected adverse 
event is one that is not already described in the Investigator Brochure. 
 
Written IND Safety Reports should include an Analysis of Similar Events in 
accordance with regulation 21 CFR § 312.32.  All safety reports previously filed 
with the IND concerning similar events should be analyzed.  The new report 
should contain comments on the significance of the new event in light of the 
previous, similar reports.   
 
Written IND safety reports with Analysis of Similar Events are to be submitted to 
the FDA and all participating investigators within 15 calendar days of first 
learning of the event.  The FDA prefers these reports on a MedWatch 3500A 
Form but alternative formats are acceptable (e.g. summary letter). 
 
FDA fax number for IND Safety Reports: 
 
1 (800) FDA - 0178 
 
All written IND Safety Reports submitted to the FDA by the Sponsor-Investigator 
must also be faxed to:  
 
Thomas Jefferson University IRB 
 

 
 
 
 
 
22 
 
IND Annual Reports 
 
In accordance with the regulation 21 CFR § 312.32, the Sponsor-Investigator 
shall within 60 days of the anniversary date that the IND went into effect submit a 
brief report of the progress of the investigation.  Please refer to Code of Federal 
Regulations, 21 CFR § 312.32 for a list of the elements required for the annual 
report.  All IND annual reports submitted to the FDA by the Sponsor-Investigator 
should be copied to the National Institutes of Heatlh as the sponsor of this study. 
 
8.5.2 Data and Safety Monitoring Committee 
Data and Safety Monitoring Committee (DSMC) is the Data and Safety Monitoring 
Board (DSMB) for the KCC. The DSMC is a multidisciplinary committee charged 
with overseeing the monitoring of safety of participants in clinical trials, and the 
conduct, progress, validity, and integrity of the data for all clinical trials at the 
Thomas Jefferson University KCC. The committee meets quarterly to review the 
progress and safety of all active research protocols that are not monitored by 
another safety and data monitoring committee or board. 
• The DSMC meets quarterly. Additional DSMC meetings are scheduled 
based on the nature and number of trials being monitored over a 
specified time period. The DSMC meets (by conference call) within 24 
hours following the notification of an unexpected adverse event felt to 
be related to the study drug.  
• Prior to each DSMC meeting, each board member, is provided a 
printout of all reported AEs and SAEs occurring during the reporting 
period for this clinical trial. The principal investigator provides a detailed 
and comprehensive narrative assessment of current adverse events to 
date, indicating their possible significance and whether these toxicities 
have affected the conduct of the trial. DSMC members are provided 
with the principal investigator’s assessment, a written report 
summarizing adverse events, safety data, and activity data observed 
during the specified time period described in each protocol, as well as 
recommendations from the Medical Monitor. A review of outcome 
results (response, toxicity and adverse events) and factors external to 
the study (such as scientific or therapeutic developments) is discussed, 
and the Committee votes on the status of each study.  
• A summary of the board’s action is sent to each investigator, the 
CCRRC and TJU IRBs. The DSMC actions may include 
recommendations/requirements that will lead to improved patient safety 
and/or efficacy, significant benefits or risks that have developed, or 
other changes determined to be necessary. The DSMC may also take 
note of slow accrual or lack of scientific progress, and refer such issues 
to the CCRRC. The DSMC provides the investigator with the rationale 
for any decision made.  
 
 

 
 
 
 
 
23 
9.0 DATA HANDLING AND RECORD KEEPING 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according 
to the requirements of the Health Insurance Portability and Accountability Act of 
1996 (HIPAA).  Those regulations require a signed subject authorization 
informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in 
this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of 
their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the 
investigator, by regulation, retains the ability to use all information collected prior 
to the revocation of subject authorization.  For subjects that have revoked 
authorization to collect or use PHI, attempts should be made to obtain permission 
to collect at least vital status (i.e. that the subject is alive) at the end of their 
scheduled study period. 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, 
or other activities in a clinical trial necessary for the reconstruction and evaluation 
of the trial.  Source data are contained in source documents   Examples of these 
original documents, and data records include: hospital records, clinical and office 
charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, 
pharmacy dispensing records, recorded data from automated instruments, copies 
or transcriptions certified after verification as being accurate and complete, 
microfiches, photographic negatives, microfilm or magnetic media, x-rays, 
subject files, and records kept at the pharmacy, at the laboratories, and at 
medico-technical departments involved in the clinical trial. 
9.3 Case Report Forms 
 
The study case report form (CRF) is the primary data collection instrument for the 
study. CRFs for the CEUS studies will be completed for each study subject, and 
are included in section 14 of this protocol. All data requested on the CRF must be 
recorded.  All missing data must be explained.  If a space on the CRF is left 
blank because the procedure was not done or the question was not asked, write 
“N/D”.  If the item is not applicable to the individual case, write “N/A”.  All entries 
should be printed legibly in black ink.  If any entry error has been made, to 
correct such an error, draw a single straight line through the incorrect entry and 
enter the correct data above it.  All such changes must be initialed and dated.  

 
 
 
 
 
24 
DO NOT ERASE OR WHITE OUT ERRORS.  For clarification of illegible or 
uncertain entries, print the clarification above the item, then initial and date it. 
 
9.4 Records Retention 
 
It is the investigator’s responsibility to retain study essential documents for at 
least 2 years after the last approval of a marketing application in their country 
and until there are no pending or contemplated marketing applications in their 
country or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product.  These documents should be 
retained for a longer period if required by an agreement with the sponsor.  In 
such an instance, it is the responsibility of the sponsor to inform the 
investigator/institution as to when these documents no longer need to be 
retained.  
 
10.0 STUDY MONITORING, AUDITING, AND INSPECTING 
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. 
 
 
 
 
11.0 ETHICAL CONSIDERATIONS 
This study is to be conducted according to US and international standards of 
Good Clinical Practice (FDA Title 21 part 312 and International Conference on 
Harmonization guidelines), applicable government regulations and Institutional 
research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted 
independent Institutional Review Board (IRB), in agreement with local legal 

 
 
 
 
 
25 
prescriptions, for formal approval of the study conduct.  The decision of the IRB 
concerning the conduct of the study will be made in writing to the investigator 
before commencement of this study.   
 
All subjects for this study will be provided a consent form that is compliant with 
local and federal regulations, describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in 
this study.  See Attachment for a copy of the Subject Informed Consent Form.  
This consent form will be submitted with the protocol for review and approval by 
the IRB for the study.  The formal consent of a subject, using the IRB-approved 
consent form, must be obtained before that subject is submitted to any study 
procedure.  This consent form must be signed by the subject or legally 
acceptable surrogate, and the investigator-designated research professional 
obtaining the consent.  
 
12.0 STUDY FINANCES 
12.1 Funding Source 
Funding for this study will be provided by an R21 grant from the NIH (R21 
CA202214). 
12.2 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). 
12.3 Subject Stipends or Payments 
n/a 
13.0 PUBLICATION PLAN 
The investigators involved in this study will be soley responsible for analysis of 
the data, and publication of any results.  It is expected that the data from this 
study will be published in a peer reviewed urology journal. 
 

 
 
 
 
 
26 
 
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