A Feasibility Study to Evaluate the Combination of Electromagnetic Tracking and Optical Imaging With Indocyanine Green (ICG) for Hepatic Biopsies
Phase: NA
Conditions: Hepatocellular Carcinoma
Sponsor: National Institutes of Health Clinical Center (CC)

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Abbreviated Title: ICG for Hepatic Biopsies 
Version Date: 09/09/2025 
 
Abbreviated Title: ICG for Hepatic Biopsies 
Protocol #: 20CC0039 
Version Date: 09/09/2025 
NCT Number: Pending 
Title: A Feasibility Study to Evaluate the Combination of Electromagnetic Tracking and              
Optical Imaging with Indocyanine Green (ICG) for Hepatic Biopsies 
 
NIH Principal Investigator: Peter Pinto M.D. 
Urologic Oncology Branch 
National Cancer Institute 
Building 10, Room 2-5952 
10 Center Drive 
Bethesda, MD 20892 
Phone: 240-858-3700 
E-mail: pp173u@nih.gov 
Investigational Agents: N/A 
Commercial Products: 
 
Autoclavable 
Camera Head 
(device) 
UreteroRenoscope/ 
Ureteroscope 
(device) 
Stryker® 
IRF Light 
Source and 
SafeLight 
Cable 
(device) 
PercuNav 
and 
Uronav 
(device) 
Indocyanine 
Green 
(ICG) 
(contrast) 
Sponsor: 
N/A 
Manufacturer: 
Karl Storz 
Endoscopy 
Imaging, Inc 
Karl Storz 
Endoscopy 
Imaging, Inc. 
Stryker 
Philips 
Generic 
 
 
 

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TABLE OF CONTENTS  
TABLE OF CONTENTS ................................................................................................................ 2 
STATEMENT OF COMPLIANCE ................................................................................................ 6 
1 
PROTOCOL SUMMARY ...................................................................................................... 7 
1.1 
Synopsis ........................................................................................................................... 7 
1.2 
Schema ............................................................................................................................. 9 
1.3 
Schedule of Activities (SOA) ......................................................................................... 10 
2 
INTRODUCTION ................................................................................................................ 11 
2.1 
Study Rationale .............................................................................................................. 11 
2.2 
Background .................................................................................................................... 11 
2.3 
Preliminary Studies ........................................................................................................ 13 
2.3.1 
Phantom study to measure ICG signal intensity relative to distance and ICG 
concentration in tissue........................................................................................................... 13 
2.3.2 
ICG uptake in a murine model of focal hepatic malignancy .................................. 15 
2.3.3 
Xenograft Fluorescence Microscopy of ICG .......................................................... 16 
2.3.4 
Handheld miniaturized optical imaging device and the point-of care optical 
imaging system ..................................................................................................................... 17 
2.3.5 
Results of pilot clinical trial for hepatic lesion sampling with optical guidance .... 17 
2.3.6 
Electromagnetic navigation for liver biopsies ........................................................ 19 
2.3.7 
Combination EM and OMI System ........................................................................ 22 
2.3.8 
Handheld OMI ........................................................................................................ 24 
2.3.9 
Point-of-care (POC) Optical Molecular Imager (OMI) .......................................... 24 
2.3.10 EM tracking ............................................................................................................ 25 
2.3.11 Software .................................................................................................................. 26 
2.4 
Risk/Benefit Assessment ................................................................................................ 26 
2.4.1 
Known Potential Risks ............................................................................................ 26 
2.4.2 
Known Potential Benefits ....................................................................................... 27 
2.4.3 
Assessment of Potential Risks and Benefits ........................................................... 27 
3 
OBJECTIVES AND ENDPOINTS ...................................................................................... 27 
4 
STUDY DESIGN.................................................................................................................. 29 
4.1 
Overall Design................................................................................................................ 29 
4.2 
Scientific Rationale for Study Design ............................................................................ 29 
5 
STUDY POPULATION ....................................................................................................... 30 

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5.1 
Inclusion Criteria ............................................................................................................ 30 
5.2 
Exclusion Criteria ........................................................................................................... 31 
5.3 
Inclusion of Vulnerable Participants .............................................................................. 31 
5.3.1 
Inclusion of children ............................................................................................... 31 
5.3.2 
Pregnant Women ..................................................................................................... 31 
5.3.3 
Decisionally Impaired ............................................................................................. 31 
5.4 
Lifestyle Considerations ................................................................................................. 31 
5.4.1 
Disease Community Engagement ........................................................................... 31 
5.5 
Screen Failures ............................................................................................................... 31 
5.6 
Strategies for Recruitment and Retention ...................................................................... 32 
5.6.1 
Costs ........................................................................................................................ 32 
5.6.2 
Compensation ......................................................................................................... 32 
6 
STUDY INTERVENTION ................................................................................................... 32 
6.1 
Study Interventions Administration ............................................................................... 32 
6.1.1 
Study Intervention Description ............................................................................... 33 
6.1.2 
In-procedure biopsy ................................................................................................ 33 
6.1.3 
Ex vivo tissue sample analysis outside of the procedure (point of care) ................ 33 
6.1.4 
Dosing and Administration ..................................................................................... 33 
6.2 
Preparation/Handling/Storage/Accountability ............................................................... 43 
6.2.1 
Acquisition and Accountability .............................................................................. 43 
6.2.2 
Formulation, Appearance, Packaging, and Labeling .............................................. 43 
6.2.3 
Product Storage and Stability.................................................................................. 43 
6.2.4 
Preparation .............................................................................................................. 43 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL..................................................................................... 43 
7.1 
Discontinuation of Study Intervention ........................................................................... 43 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................... 44 
8 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 44 
8.1 
Screening Procedures ..................................................................................................... 44 
8.2 
Registration Procedures.................................................................................................. 44 
8.3 
Treatment Assignment and Randomization/Stratification Procedures Cohorts ............. 44 
8.4 
Efficacy Assessments ..................................................................................................... 45 

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8.4.1 
Biospecimen Evaluations ........................................................................................ 45 
8.5 
Adverse Events and Serious Adverse Events ................................................................. 45 
8.5.1 
Definition of Adverse Event ................................................................................... 45 
8.5.2 
Definition of Serious Adverse Events (SAE) ......................................................... 46 
8.5.3 
Classification of an Adverse Event ......................................................................... 46 
8.5.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 47 
8.5.5 
Adverse Event Reporting ........................................................................................ 48 
8.5.6 
Serious Adverse Event Reporting ........................................................................... 48 
8.6 
Unanticipated Problems ................................................................................................. 48 
8.6.1 
Definition of Unanticipated Problems (UP) ........................................................... 48 
8.6.2 
Unanticipated Problem Reporting........................................................................... 49 
8.6.3 
NIH Intramural IRB Reporting of IND Safety Reports .......................................... 49 
9 
STATISTICAL CONSIDERATIONS.................................................................................. 49 
9.1 
Statistical Hypothesis ..................................................................................................... 49 
9.2 
Sample Size Determination ............................................................................................ 49 
9.3 
Populations for Analyses ................................................................................................ 50 
9.3.1 
Evaluable for toxicity .............................................................................................. 50 
9.3.2 
Evaluable for objective response ............................................................................ 50 
9.3.3 
Evaluable Non-Target Disease Response ............................................................... 51 
9.4 
Statistical Analyses ........................................................................................................ 51 
9.4.1 
General Approach ................................................................................................... 51 
9.4.2 
Analysis of the Primary Endpoints ......................................................................... 51 
9.4.3 
Analysis of the Secondary Endpoints ..................................................................... 52 
9.4.4 
Safety Analyses ....................................................................................................... 52 
9.4.5 
Baseline Descriptive Statistics ................................................................................ 52 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS ......................................... 53 
10.1 
Informed Consent Process .......................................................................................... 53 
10.1.1 Consent Procedures and Documentation ................................................................ 53 
10.1.2 Participation of Subjects Who Are/Become Decisionally Impaired ....................... 53 
10.1.3 Telephone Consent.................................................................................................. 53 
10.2 
Study Discontinuation and Closure ............................................................................ 54 
10.3 
Confidentiality and Privacy ........................................................................................ 54 

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10.4 
Future use of Stored Specimens and Data .................................................................. 54 
10.5 
Safety Oversight ......................................................................................................... 54 
10.6 
Clinical Monitoring .................................................................................................... 54 
10.7 
Quality Assurance and Quality Control ...................................................................... 55 
10.8 
Data Handling and Record Keeping ........................................................................... 55 
10.8.1 Data Collection and Management Responsibilities ................................................ 55 
10.8.2 Study Records Retention......................................................................................... 55 
10.9 
Protocol Deviations .................................................................................................... 56 
10.9.1 NIH Definition of Protocol Deviation .................................................................... 56 
10.9.2 Unanticipated Problem............................................................................................ 56 
10.10 
Publication and Data Sharing Policy .......................................................................... 56 
10.10.1 Human Data Sharing Plan ....................................................................................... 56 
10.11 
COLLABORATIVE AGREEMENTS ....................................................................... 56 
10.11.1 Cooperative Research and Development Agreement (number pending) ............... 56 
10.11.2 Material Transfer Agreement (number pending) .................................................... 57 
10.12 
Conflict of Interest Policy........................................................................................... 57 
11 ABBREVIATIONS .............................................................................................................. 58 
12 REFERENCES ..................................................................................................................... 60 
 
 
 

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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (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 to the study. In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
 

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1 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS 
Title: 
A Feasibility Study to Evaluate the Combination of Electromagnetic 
Tracking and Optical Imaging with Indocyanine Green (ICG) for Hepatic 
Biopsies 
Study Description: 
This is a pilot study to evaluate the feasibility of the combination of optical 
molecular imaging (OMI) and electromagnetic (EM) tracking for use 
during biopsy of intrahepatic lesions to assess the concordance between 
fluorescence ICG and histopathology for the diagnosis of liver cancer. 
Objectives: 
 
Primary Objective: 
• 
To evaluate the feasibility of using both optical molecular imaging 
technique and electromagnetic tracking during standard liver 
biopsy. 
 
Secondary Objectives: 
1. To assess concordance of ICG fluorescence signal at the in vivo 
site of biopsy with the presence or absence of malignancy, as 
defined by pathology 
2. To assess, in biopsy tissue, the concordance of ICG florescence 
signal ex vivo (outside of the body with camera) with the presence 
or absence of malignancy as defined by pathology 
Endpoints: 
Primary Endpoint: 
• 
The determination of ICG fluorescent signal at the in-vivo site of 
biopsy using combination OMI and EM tracking will be based on 
Target-to-Background (TBR) such that TBR of ≥2 will produce a 
malignant diagnosis and TBR will produce a benign diagnosis. 
 
Secondary Endpoints: 
1. To evaluate the agreement between histopathology and ICG 
fluorescent signal in-vivo biopsy site using combined OMI and EM 
tracking using histopathology results as the gold standard. 
2. To evaluate the agreement between histopathology and ICG 
fluorescent signal of the specimen ex-vivo using a combination of 
OMI and EM tracking. 
Study Population: 
Patients ≥ 18 years of age whose imaging findings suggest liver cancer 
(hepatocellular carcinoma) or other metastatic liver malignancy for whom 
image-guided percutaneous biopsy is separately indicated or planned to be 
performed. 
Phase: 
Non-randomized, Open Label, prospective pilot study. 
Description of 
Sites/Facilities 
Enrolling Participants: 
Recruitment and investigational procedures will take place at the NIH 
Clinical Research Center in the Department of Radiology, Special 
Procedures.  
Description of Study 
Intervention: 
This is a non-randomized, prospective pilot study enrolling 77 patients with 
liver foci suspicious for cancer by invitation only. Patients will be referred 
by their NIH primary care team and will need to be registered to an existing 
NIH clinical study prior to enrolling in this study. 
Liver biopsy will be performed 24 hours after administration of clinically 
approved ICG 0.5 mg/kg IV. Biopsy will be performed using EM 

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technology and evaluating OMI as a bystander in liver cancer biopsies. The 
fluorescence signal of the tissue cores will be evaluated ex vivo and the 
signal will be correlated to histopathological characteristics of the tissues 
Study Duration: 
We anticipate that 100% of the projected enrollment will be completed by 4 
years. Data collection (including correlative pathology) will be completed 
by 4 years. We anticipate global primary and secondary analysis along with 
the final report will be completed in 5 years. 
Participant Duration: 
Participants will be considered “Off Treatment” upon completion of the 
hepatic biopsy and four (4) to six (6) hour post procedure monitoring 
period. Participants will be considered “Off Study” within one (1) ± two (2) 
months post correlative pathological assessment to result. 
 
 
 

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1.2 
SCHEMA 
 
Review baseline labs, 
allergies, and active illness 
as part of clinical 
assessment per 
departmental guidelines. 
Review H&P and imaging. 
Sign consent.  
Administer 0.5 mg/kg ICG 
up to 40mg roughly 24hrs 
prior to scheduled biopsy. 
. 
Measure ICG 
epifluorescence from liver 
parenchyma, target lesion, 
and core sample. Submit core 
sample for standard 
pathologic analysis. 
 
Once patients are referred 
to the study, initiate 
screening procedures and 
assess patients’ eligibility.  
Correlate and evaluate 
Pathology report  
Perform 
biopsy in IR 

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1.3 
 SCHEDULE OF ACTIVITIES (SOA) 
Table 1. Study Elements 
Procedures 
Screening 
Pre-Procedure 
Study Day 1 
Intervention 
Off Treatment 
Biopsy and post 
procedure 
monitoring 
completed 
Off Study  
1±2 months for 
all correlative 
pathological 
assessments 
Informed consent a 
X 
 
 
 
 
Review of imaging 
X 
X 
 
 
 
Review of medical 
history 
X 
 
 
 
 
Physical examination 
X 
 
 
 
 
Review of laboratory 
values 
X 
 
 
 
 
ICG Administration  
 
X 
 
 
 
Percutaneous Liver 
Biopsy 
 
 
X 
 
 
Adverse Event 
monitoring 
 
X 
X 
 
 
Remove from protocol 
therapy 
 
 
 
X 
 
Remove from study 
follow up 
 
 
 
 
X 
Completion of 
histopathology 
assessment 
 
 
 
 
X 
a. Informed consent obtained prior to screening procedures. 

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2 
 INTRODUCTION 
2.1 
STUDY RATIONALE 
We propose a pilot study to determine the feasibility of using optical molecular imaging (a low 
cost, real-time, high resolution imaging discipline) for minimally invasive liver biopsy guidance 
in patients with diagnosed or suspected HCC or metastatic CRC. Patients who require a 
clinically indicated or separately IRB approved research liver biopsy would after consent, have 
the OMI performed during biopsy but not as the primary directorate to the biopsy site. The 
standard of care imaging and cognitive registration would be employed for navigation or 
guidance of needle and OMI would act as a bystander. Nonetheless, data from OMI imaging 
would be obtained and evaluated with ex vivo POC OMI and histopathology. Correlations from 
these studies might support a future larger clinical trial, with different goals that might not 
exclude efficacy and safety, or even allow for guidance of the needle if separately IRB-approved. 
2.2 
BACKGROUND  
The advent of advanced imaging techniques including multi-phase contrast enhanced CT and 
MRI has improved the sensitivity for detection of focal hepatic lesions. However, focal lesions 
smaller than 3cm often lack specific features to allow for a reliable non-invasive 
characterization. Even molecular imaging modalities such as FDG-PET may be of limited utility 
given the intrinsic spatial resolution of PET as well as the heterogeneous background uptake of 
FDG by liver parenchyma. For this reason, the number of biopsies for small hepatic lesions has 
increased over the past decade (1). Compared to non-invasive imaging and serologic tests, 
biopsy is more sensitive and specific in diagnosing malignancy (2). 
Optical molecular imaging is a low cost, real-time, high resolution imaging discipline that holds 
promise for minimally invasive procedure guidance. Optical molecular imaging as a field 
encompasses a vast array of imaging modalities; one such modality is the imaging of 
exogenously administered organic fluorochromes. For example, ICG is a common clinically used 
and FDA-cleared optical molecular imaging agent that fluoresces in the near infrared (NIR) and 
localizes with high target-to-background ratios to HCC and metastases from colorectal 
carcinoma CRC (5-8), whereas non-tumorous hepatic tissue clears the ICG. Recently the intra-
operative imaging of ICG for the detection of intrahepatic malignancy has been demonstrated 
(6). Indeed, OMI maybe superior to standard visual inspection and intraoperative ultrasound for 
the detection of focal hepatic lesions during surgical resection. 
Though still in its nascent stage, optical molecular imaging has the potential to make a 
tremendous impact in IR. With numerous fluorescently labeled molecular markers in preclinical 
and clinical development (not subject of this study), the opportunities to perform procedures 
guided by highly sensitive and specific molecular beacons of disease will continue to grow. In 
the context of percutaneous biopsy, optical imaging probes could improve operator confidence 
and decrease the risk of sampling error, which in turn would reduce the clinical concern of false 
negative following a benign biopsy, although this is speculative. Moreover, the improved 
operator confidence could enable sampling of smaller lesions, thereby potentially providing a 
diagnosis and appropriate management plan to patients earlier in the course of disease. Although 
speculative, given the molecular specificity of future fluorescent imaging agents, a quantitative 
assessment of the degree of molecular probe uptake could provide an “in vivo histology” 

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diagnosis, potentially without requiring tissue sampling, a feature that may be an important 
consideration for lesions in locations where obtaining a core specimen may be dangerous. 
However, due to the short distance passage of light, the optical technique alone is not efficient at 
localizing a lesion if more than 0.5cm away from the target. Electromagnetic tracking (EM) and 
fusion co-display, on the other hand, can ensure the close proximity of the needle tip to lesion, by 
referencing and displaying the needle in relation to the pre-procedural imaging -CT or MRI. EM 
tracking for registration of preoperative images has been utilized during neurosurgery, 
orthopedic surgery, as well as in image-guided percutaneous biopsy at NIH for over 15 years. 
The PhD and MD researchers at NIH-CC-CIO have extensive experience in clinical use of a 
minimally-invasive image guidance navigation system that integrated electromagnetic tracking 
of needles and US probes to allow registration, fusion and co-display with 3D imaging data, to 
improve target visualization during needle-based procedures in interventional radiology. 
Multiple vendors and FDA cleared systems are currently marketed under the same basic 
principles (UroNav, PercuNav, Koelis, MIMS, Hitachi, Siemens, General Electric, Esaote 
BioSound, Imactis, Veran, Superdimension, Stealth Medtronic, Covidien Johnson and Johnson, 
Cascination to name a few). Multiple studies show that this technology can improve basic needle 
biopsy accuracy and minimize risks, complications and error during image guided percutaneous 
or transrectal biopsy. 
In this “bystander” study for data acquisition and feasibility, we propose combining these two 
intrinsically synergistic, recent advances in the field of interventional radiology, in a feasibility 
study for data acquisition alone. “Bystander” is defined as a term to describe the study performed 
in the background during a standard procedure, performed in a very similar fashion to the 
standard methodology and workflow, while data will be gathered as described in this protocol. 
The final determination of malignancy will come from histopathology testing, accepted as the 
gold standard; that pathologic evaluation will proceed in the standard fashion Neither safety nor 
efficacy is a goal of this feasibility pilot study. However, the long range futuristic goals of the 
general approach may facilitate a better understanding of the perspective and scientific rationale 
underlying this feasibility pilot study. The following is not part of the study, and thus should not 
be factored in to the risk determination of this study, as it does not describe current method of 
use of this study outlined above and in the study protocol. Although not the subject of this study, 
the long range speculative goal might be to provide added information to potentially guide (in 
the future) intra-tumoral sampling towards the most viable and aggressive lesion components, or 
towards cancerous tissue instead of normal tissue, in order to attempt to improve the biopsy yield 
(but not in this study). In such a speculative future use, the EM navigation might navigate the 
biopsy needle generally close to suspicious (previously imaged) lesions; while the optical 
imaging devices provide real-time small scale, near field, feedback to assess whether needle 
cores are appropriately aimed and located precisely within small hepatic tumors, as opposed to 
normal liver or regenerative nodules. Again, this is not within the scope or methods for use of 
this study, and is provided for informational 

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2.3 
PRELIMINARY STUDIES 
A series of preclinical feasibility studies were 
performed of the combination optical and 
electromagnetic system, including phantom, 
tissue and in vivo animal were conducted, 
which included phantom ex vivo work, and in 
vivo animal trials. Separate distinct human in 
vivo clinical trials have been conducted of 
each technology in distinct trials: 1. EM 
tracking for biopsy (NIH CC) and 2. Optical 
imaging with ICG for liver biopsy 
(Massachusetts General Hospital). Prior work 
has included validation studies, semi-
quantitative studies, software verification 
studies, accuracy studies, and ICG calibration 
testing.  
2.3.1 
Phantom study to measure ICG 
signal intensity relative to distance and ICG 
concentration in tissue.  
A phantom was designed to measure ICG 
fluorescence signal intensity through varying 
thicknesses of liver tissue and at different ICG 
concentrations. The phantom was composed of 
two separate compartments; the upper 
compartment consisted of a cuvette filled with 
25 mm of a homogenate of normal mouse liver 
tissue. The transparent bottom of the cuvette 
was in direct contact with a 1-mm layer of ICG 
mixed with liver tissue homogenate. Different 
concentrations of ICG mixture in liver tissue 
homogenate were created by using serial 
dilution (0.0008, 0.004, 0.02, and 0.1 mg/L of 
ICG) and were placed in the compartment 
below the cuvette. A 1.6-mm–diameter fiber-
optic pediatric cystoscope-urethroscope was 
inserted into the cuvette. The pediatric 
cystoscope-urethroscope (Thorlabs, Newton, 
NJ) and withdrawn by increments of 1.25 mm 
from the cuvette dye interface. The light guide 
of the pediatric cystoscope-urethroscope was 
connected to a 785-nm fiber-coupled laser (BWTEK, Lubeck, Germany), and the eyepiece was 
connected through an 800-nm bandpass filter (Semrock, Rochester, NY) to an NIR camera 
(Allied Vision Technologies, Stadtroda, Germany). With each withdrawal of the pediatric 
Figure 1. Graphs from a phantom 
experiment of ICG fluorescence as a 
function of concentration and distance 
through liver. (A) There is marked ICG 
fluorescence above background levels 
through 5-10 mm of homogenized liver 
tissue. (B) ICG fluorescence is measurable 
over two orders of magnitude of ICG 
concentration through liver tissue. arb = 
arbitrary units. 

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Figure 2. Stryker® IRF L10 Light Source 
and Safelight Cable generated a stronger 
fluorescence signal when tested with ICG 
contrast compared to the original device 
B&W Tek BWF1 laser system. 
cystoscope-urethroscope, still images were recorded in triplicate with a 50-msec exposure for 
each ICG concentration.  
To reduce the variability of measurement, a circular region of interest with an area of 20,000 
square pixels was placed in the center of each image by using a standard image analysis software 
package (Image J; U.S. National Institutes of Health, Bethesda, MD). Signal intensities were 
measured independently and the mean of the triplicate measurements was used as the mean 
signal intensity. ICG fluorescence was detectable through 10 mm of homogenized tissue, 
suggesting that lesions within approximately 1 cm of the imaging system are potentially 
detectable. There was a monotonically increasing relationship between ICG concentration and 
fluorescence intensity over a range of ICG concentrations the spanned two orders of magnitude. 
The BW & TEK 785 nm class IIIb laser system that was used for pre-clinical testing is not 
approved for use in human subjects, therefore an alternative system that complies with the FDA 
requirements was tested for substitute use in the in vivo component of the clinical trial. The 
camera of The ICG imaging system was tested in water and ICG solutions using (1) the original 
B&W Tek BWF1 laser generator (laser class IIIb) and (2) Stryker® IRF Light Source and 
Safelight Cable (ENV mode). The camera of the imaging system did not show presence of ICG 
in water regardless of which light source (or laser generator) to use. In ICG solutions, the camera 
showed stronger signals with the Stryker® IRF Light Source and Safelight Cable than the 
original BW&TEK laser generator. The experiment showed that the ENV mode of Stryker L10 
light source is at least as good as the original BW&TEK laser generator which was already 
validated in ex vivo and in vivo animal experiments. 
      
 
 
  
      
 
 
Stryker® IRF L10 Light Source and Safelight Cable is an FDA 510 (k) cleared device that 
complies with IEC 60825-1:2007 and 21 CFR, Subchapter J, Parts 1040.10 and 1040.11. The 
808 nm wavelength is the peak excitation wavelength for ICG and is the optimal excitation 
wavelength for the use proposed in the study. The wavelength, power output and the laser mode 
(continuous or pulsating) can be adjusted by the user in the Endoscopic Near-Infrared 
Visualization (ENV) mode. Additionally, this device is designed to ensure electromagnetic 
Figure 3. ICG contrast testing with B&W 
Tek BWF1 laser system. This device is not 
approved for use in human subjects. 

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compatibility with other electrical medical devices and complies with IEC 60601-1-2 
requirements for EMC with other devices. In summary, L10 Light Source by Stryker is the ideal 
device for the in vivo component of the proposed study. 
2.3.2 ICG uptake in a murine model of focal hepatic malignancy 
A murine model of focal hepatic malignancy was generated by injecting human colorectal cancer 
cells (HT-29;ATCC, Manassas, VA) into the livers of athymic nude mice (nu/nu; Taconic, 
Germantown, NY). Three weeks later, 0.5mg/kg ICG was injected intravenously via tail vein 
injection. The mice were then sacrificed at 3, 6, and 24 hours post-injection, and surface 
reflectance NIR imaging of the excised livers was performed (Carestream, New Haven, CT). 
ICG localizes to the intrahepatic lesions with high TBRs (Figure 2 and Figure 3), with a TBR of 
approximately 4.0 at 24 hours (Figure 4). Of note, the injected ICG dose of 0.5mg/kg is 
equivalent to the manufacturer recommended dose. 
 
Figure 4. A, B) Intrahepatic xenografts demonstrate avid ICG update in a murine model. Gross 
appearance of the focal tumor within the liver (A) with elevated ICG update relative to liver on 
surface reflectance fluorescence imaging (B). C) ICG update in focal hepatic tumors over time. 
TBR for ICG update within intrahepatic tumors relative to adjacent liver parenchyma is maximal 
at 24 hours. D) Core biopsy sample from a preclinical hepatic tumor. Fluorescence signal from 
the tumor reflects accumulation of ICG.  

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2.3.3 
Xenograft Fluorescence 
Microscopy of ICG 
A model of focal hepatic malignancy was 
generated by injecting human hepatocellular 
carcinoma cells (HuH-7, Japanese Collection of 
Research Bioresources) into the livers of athymic 
nude mice. Four weeks later, 0.5mg/kg ICG was 
injected 
intravenously via 
tail vein injection. 
The mice were 
then sacrificed at 
24 hours post- 
njection. The 
excised livers 
were stored at 
−80°C. Two serial 
cryostat sections 
were cut from the tissue samples for fluorescence microscopy (10-μm section) and hematoxylin-
eosin staining (50-μm section). The fluorescence microscopy was performed using laser 
scanning confocal microscopy (LSM 5 PASCAL; Zeiss, Oberkochen, Germany) with an 
excitation wavelength of 633 nm and 100× magnification. 
The histologic specimens were evaluated qualitatively to determine tumor-liver boundaries.  
Histologic findings in the tumor are remarkable for densely packed tumor cells with high 
nuclear-cytoplasmic ratios, an appearance in contrast to the adjacent normal hepatic sinusoidal 
architecture. Fluorescence microscopy similarly shows a sharp delineation between malignant 
and normal tissue, with increased ICG uptake in the xenograft. Notably, focal areas of ICG 
pooling are present in the biliary canaliculi in the adjacent liver—an expected finding given the 
hepatobiliary excretion of ICG (Figure 5). 
Figure 5. Hematoxylin-eosin-stained and fluorescence microscopy images 
of orthotopic hepatocellular tumor xenograft developed from HuH-7 cells. 
(A) H&E staining at 100x shows hepatocellular tumor with densely packed 
cells, and high nuclear-cytoplasmic ratios. The tumor-liver margin 
demonstrates the transition from normal to malignant cells. (B) The sharp 
tumor-liver margin is evident at ICG fluorescence imaging (100x), with 
increased ICG update within the tumoral cells. Of note, pools of ICG within 
the hapatic parenchyma reflect normal excretion of ICG into biliary 
canaliculi. 

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2.3.4 Handheld miniaturized optical imaging device and the point-of care optical imaging 
system 
A model of focal hepatic malignancy was 
generated by injecting human hepatocellular 
carcinoma cells (HuH-7, Japanese 
Collection of Research Bioresources) into 
the livers of athymic nude mice. Four weeks 
later, 0.5mg/kg ICG was injected 
intravenously via tail vein injection. The 
mice were then sacrificed at 24 hours post-
injection. The excised livers were stored at 
−80°C. Two serial cryostat sections were 
cut from the tissue samples for fluorescence 
microscopy (10-μm section) and 
hematoxylin-eosin staining (50-μm section). 
The fluorescence microscopy was 
performed using laser scanning confocal 
microscopy (LSM 5 PASCAL; Zeiss, 
Oberkochen, Germany) with an excitation 
wavelength of 633 nm and 100× 
magnification. 
The histologic specimens were evaluated 
qualitatively to determine tumor-liver 
boundaries. 
Histologic findings in the tumor are remarkable for densely packed tumor cells with high 
nuclear-cytoplasmic ratios, an appearance in contrast to the adjacent normal hepatic sinusoidal 
architecture. Fluorescence microscopy similarly shows a sharp delineation between malignant 
and normal tissue, with increased ICG uptake in the xenograft. Notably, focal areas of ICG 
pooling are present in the biliary canaliculi in the adjacent liver—an expected finding given the 
hepatobiliary excretion of ICG (Figure 6). 
2.3.5 Results of pilot clinical trial for hepatic lesion sampling with optical guidance 
Partners at MGH recently conducted the first-in-man trial of clinical liver biopsies using 
percutaneous OMI following injection of ICG (Figure 7). They recruited 6 patients for this pilot 
study [1]. The median size of the targeted lesions was 16 mm (range, 10–21 mm). Four of five 
biopsies (80%) yielded a positive pathologic diagnosis, and one biopsy specimen showed benign 
liver parenchyma. 
Figure 6. A) Schematic demonstrates design 
of handheld OMI device. B) Photograph of 
device, demonstrating handheld OMI system 
consists of a miniature endoscope coupled to a 
high resolution 12-bit camera. C) Handheld 
device advanced through a standard 17-gauge 
introducer needle US-guided percutaneous 
focal liver biopsy to perform real-time 
measurements of ICG localization. D) Point-
of-care OMI system for acquisition of high 
resolution ICG fluorescence images of biopsy 
cores for real time assessment. 

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Figure 7. Focal hepatic lesions on, A, MR image and, B, US image. C, 18F FDG PET scan 
shows avid lesions suspected of being malignancy. Intraprocedural optical molecular images 
confirmed the accuracy of the biopsy needle placement, by measuring increased fluorescence, E, 
in the target lesion relative to, D, normal liver. Optical images of the core biopsy specimen show 
visualized area of, F, focally increaed ICG localization, G, in the core specimen. Final pathologic 
analysis of the core specimes revealed a granulomatous process. 
 
Intraprocedural imaging of ICG fluorescence 
intensity at the tip of a standard introducer 
needle and within core biopsy specimens was 
feasible and added less than 5 minutes of 
procedure time in all patients. ICG was found 
to localize with TBRs greater than 2.0 (range, 
2.4–13.4; median, 7.9) in all target lesions 
including primary HCC, metastatic CRC, and a 
non-neoplastic granulomatous process (Figure 
8). 
The difference between ICG fluorescence 
intensity between normal liver and focal 
hepatic lesions across the study cohort 
approached significance (P = .06). No trial-
specific adverse events were observed up to 7 
days following the procedures. We have shown that ICG imaging using these systems is a robust 
Figure 8. Plot of normalized ICG 

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adjunct to conventional image guidance for patients undergoing biopsy of primary HCC, hepatic 
CRC metastasis, and non-neoplastic inflammatory processes, as well as thermal ablations. 
 
Figure 9. NIH Clinical Center setup for an ultrasound/computed tomography –guided biopsy 
procedure with electromagnetic tracking guidance. FG: electromagnetic filed generator, NS: 
navigation. 
2.3.6 Electromagnetic navigation for liver biopsies 
Electromagnetic (EM) tracking for registration of preoperative images during procedures maps 
conventional US and CT images for “fusion guidance” [7-19]. Over almost the past 2 decades, 
the NIH navigation system has integrated electromagnetic tracking of needles and US probes 
with custom software to allow registration and fusion with three-dimensional images to improve 
target visualization during needle-based procedures (Figure 9 and Figure 10). 
We have used anthropomorphic multimodality injectable interventional phantoms such as Model 
57 (CIRS, Norfolk, VA) for a qualitative feasibility studies of EM tracking to demonstrate this 
application in different clinical scenarios using different imaging modalities to locate the lesion.  
For instance, in a feasibility study over a decade ago, using EM tracking alone to navigate to a 
simulated hot PET lesion using 18F containing fiducials, PET/MR data set was acquired and the 
PET scan DICOM images were used to navigate a tracked needle to the internal target. DICOM 
images were transferred to navigation software and a tracked 22-gauge needle (with a sensor coil 
embedded in the tip) was placed toward the internal target with electromagnetic tracking 
guidance. MR scan verification was performed. 

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PET and MR datasets were also successfully loaded and registered to the phantom for navigation 
within metabolic and functional datasets. An internal radiopharmaceutical target was located 
with electromagnetic tracking with a tracked needle (Figure 11). This technique has been used 
successfully as a part of an NIH clinical trial for 15 years, and has also been commercialized or 
duplicated by at least 20 companies. 
 
 
 
 
 
 
 
 

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Figure 10. Electromagnetic tracking system components and 
setup: (a) electromagnetic tracking system set up and rationale. 
 (b) Components of image-guided system incorporating 
electromagnetic tracking for abdominal interventions. 
A swine model was used to evaluate the feasibility of EM tracking 
alone. Our studies showed that prospective use of the EM navigation 
system allows needle and electrode placement with higher confidence 
in all cases and provides valuable target information that is unavailable 
by conventional image guidance in most cases. For instance in one of 
our decade-old studies, the tracking technology enabled 22 needle and 
electrode placements in 19 procedures; these 22 placements would 
have had a high risk of technical failure with conventional image 
guidance only, owing to lack of target localization [11]. These were 
primarily cases in which the target lesions were visible only on an 
arterial phase CT scan or on prior PET or PET/CT scan and were then 
brought into the procedure by image registration with the navigation 
CT (Figure 11).  
The technology was especially enabling for liver targets that were 
visible only during brief arterial phase enhancement on contrast-
enhanced CT scan. The additional information allowed completion of 
biopsies in less time than they would have taken with conventional 
guidance, based on the number of failed needle positioning attempts 
with CT guidance only.  
Figure 11. Use of pre-procedural 
PET and MR for needle 
navigation in a phantom. (A) T2-
weighted MR image of phantom 
with gadolinium fiducials 
(arrows). (B) CT/PET image of 
interventional phantom with 6 
mCi of 18F fiducials (arrows) 
could also be used for needle 
navigation. (C) Tracked needle 
position (small arrow) is 
displayed near internal target 
(large arrow) from pre-
procedural PET during tracked 
needle insertion. 

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Figure 12. Navigation display at the NIH Clinical Center (A-C). Display showing the virtual 
needle aligned with the target identified on arterial phase-enhanced computed tomography scan. 
(D) Fiducial markers were placed the patient’s skin to facilitate registration process. E) Non-
contrast-enhanced confirmation scan of needle position does not show the target lesion. F) After 
rigid registration of the confirmation scan (pseudocolored) with the arterial contrast-enhanced 
scan (gray-scale background) in the vicinity of the target lesion, the correct alignment of the 
needle with the target is confirmed. 
Additionally, fusion guidance significantly improves angle recent study, the addition of needle 
and US tracking improved needle path off-target error from 17.8 mm ± 17.1 to 3.3 mm ± 3.1 and 
changed the insertion angle by 13.3 degrees ± 6.5. We showed that registration and use of pre-
procedural diagnostic or metabolic images enabled identification and localization of targets and 
had a direct impact on outcomes in interventions [11]. 
2.3.7 Combination EM and OMI System 
The combined integrated platform provided simultaneous optical and EM feedback during 
simulated ex vivo and real in vivo animal biopsy. EM was used to place the needle roughly near 
the ICG target, and the optical signal was used to fine-tune and verify precise tip location at the 
optical source in phantoms and in live Woodchucks with liver tumors (data on file). The ICG 
signal was predictive of tumor presence on histology biopsy samples using a combined system. 
 

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Figure 13. Color means ICG present = tumor 
 
Figure 14. White light  Optical POC box ICG imaging 
 
Top row = no ICG = normal liver 
Bottom row = ICG present = tumor 

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2.3.8 Handheld OMI 
The OMI is a handheld endoscopic instrument consisting of an FDA-cleared and clinically used 
pediatric cystoscope-urethroscope (Model 27033 AA; Karl Storz, Tuttlingen, Germany) 
illuminated by a safelight cable attached to an 808 nm laser excitation source (Stryker IRF L10 
Light Source and Safelight Cable) with an adjustable user feature in the Endoscopic Near-
Infrared visualization (ENV) mode that can pass coaxially through the introducer of a standard 
17-gauge core biopsy system (see Figure 6).  
The eyepiece of the pediatric cystoscope-urethroscope is attached to high-definition endoscopic 
camera system sensitive to the visible and infrared spectrums. The optical image is transferred 
from the surgical site to the camera unit which is attached to the camera head (Mako; Allied 
Vision Technologies) and the light output is by a filtered band-pass filter that is compatible with 
the laser excitation source.  
The imaging pediatric cystoscope-urethroscope tip is the only component that is in direct contact 
with the subject and will be sterilized according to manufacturer’s guidelines, which the NIH 
Clinical Center sterilization have confirmed they can do as prescribed by the manufacturer in a 
standard fashion. The remainder of the handheld device is remote from the patient, but can be 
placed in sterile plastic surgical cover. The Stryker IRF L10 Light Source and Safelight Cable 
system are part of the Stryker® Infrared Fluorescence (IRF) Imaging System, which is an 
endoscopic illumination and imaging system for real-time high definition (HD) visible light and 
near-infrared dye fluorescence imaging of indocyanine green (ICG) during minimally invasive 
surgery/ procedures. The system complies with IEC 60825-1:2007 and 21 CFR, Subchapter J 
Parts 1040.10 and 1040.11. This device is designed to ensure electromagnetic compatibility with 
our electrical medical devices and complies with IEC 6060-1-1-2 requirements for EMC with 
other devices. 
2.3.9 Point-of-care (POC) Optical Molecular Imager (OMI) 
This is a camera in a black box taking images of the ex vivo tissue using a BWF1 laser to excite 
light to see the ICG fluorescence. Specifically, the core biopsy tissue specimen is placed in a 
sterile container and removed to a windowless room adjacent to the Interventional Radiology 
(IR) suite. The tissue is placed inside a 2 foot tall black box on a benchtop with the laser. The 
laser does not come in contact with the patient, nor in the same room as the patient, at any time. 
(Figure 15). 
The BWF1 laser generator from the medical laser series will be used with the point of care OMI, 
which does not occur in vivo, and occurs remote from the patient and not in the procedure room. 
This system takes pictures of the ex vivo biopsy core itself with special lenses and filters, to 
display a signal of whether ICG is present in the tissue sample. Although speculative and not part 
of this study, this assessment might in the future, facilitate examination of future biopsy tissue 
quality or might point towards the absence or presence of tumor (although no such claims or 
hypotheses are made in this study). 
In the POC epifluorescence OMI system, the fluorescence excitation light is provided by a 450-
mW, 785-nm laser (Edmund Optics). Upon the absorption of laser light, the ICG in the vessels is 
excited and emits infrared light at a different wave length. The camera system captures the 
infrared emission, processes the image and displays it on a surgical display. Emitted fluorescent 
light is collected by a close focus video zoom lens (Edmund Optics) and filtered by a band-pass 

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filter. The filtered fluorescent light is imaged 
with a near-infrared-optimized high temporal- 
and high-spatial resolution camera (Mako; 
Allied Vision Technologies) (Figure 2).  A 
new filter slider, which allows for direct 
visualization of the sample by the POC 
system, is used for adjusting the sample in the 
field of view of the camera. A custom-built 
dark cabinet for t the POC eliminates or 
reduces signal from stray photons in the 
environment. (Figure 15 and Figure 16). 
 
 
 
 
 
 
Figure 16. Outside imaging – the black box contents a. stage b. camera c. laser input to excite 
ICG for imaging. 
2.3.10 EM tracking 
Aurora (Northern Digital Inc, Waterloo, Ontario, Canada) EM tracking system is used for device 
tracking. A small electromagnetic field generator is mounted on a modified passive support and 
positioning arm, and produces an ultra-low electromagnetic field (<70T) enabling simultaneous 
tracking of the several sensor coils in the inner needle stylet at the beginning of the biopsy, 
within a work volume of ~ 500mm3. Commercial and FDA-cleared “PercuNav” needles 
(Philips) have stylets that contain sensor coils in the needle tips. These needles allow for tracking 
and mapping location coordinates in relation to the pre-procedural CT or MRI when in the 
Camera 
Laser 
Stage 
Lens 
Laser generator
 
Figure 15. Black Box and contents for sample 
evaluation. 

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presence of an EM field generator and a tracking system. During the biopsy (as described 
above), after both inner and outer biopsy needles are placed in the liver, the coaxial inner stylet is 
removed and replaced with the OMI scope. The outer needle remains in the liver after the inner 
stylet is removed, and provides a cannula lumen, through which is placed the scope (for data 
sampling of fluorescent signal). This occurs once along the way to the target liver lesion - likely 
in normal liver- and the process is repeated after advancement of the original needle and stylet to 
the target lesion, during the standard clinically-indicated biopsy (as described above). After 
optical ICG signal data acquisition, the scope is removed and a standard spring-loaded 
disposable FDA-cleared biopsy gun takes a tissue specimen from the same location as would 
have been biopsied in the absence of this study. Thus the FDA-cleared outer cannula introducer 
allows insertion of the biopsy needles and the pediatric cystoscope-urethroscope via the same 
outer needle / cannula, using a coaxial approach. 
2.3.11 Software 
The EM and optical display software components analyze the EM and optical data and signal 
outside of the body, and the information is not used to guide the needle placement or the biopsy 
sample. The commercial EM needle, described above, and the commercial EM field generator 
and its commercial advanced programming interface are analyzed by custom software in order to 
co-display the local optical signal as well as the EM tracking fusion biopsy location in relation to 
a pre-procedural CT or MRI image. This information will be analyzed post-procedure, but is not 
used for navigation of the device. This system and multiple commercial fusion biopsy platforms 
(UroNav & PercuNav, Philips, for example) have been in clinical use on IRB-approved protocols 
at NIH for 15 years. These NIH research studies are currently approved under an IDE exemption. 
The OMI/EM system in its entirety has been thoroughly tested in phantoms as well as live 
animals (woodchuck liver cancer tumors for biopsy). This in vivo testing, included the EM 
tracking / fusion display components, the OMI pediatric cystoscope-urethroscope, and the 
combined signal display. This custom software platform incorporates EM fusion co-display (that 
tracks, fuses, and displays the needle with respect to CT images). The system also combines this 
EM fusion co-display navigation with the software that adjusts the camera (optical signal) 
functions from the pediatric cystoscope-urethroscope, and co-displays the OMI camera feed 
(ICG dye) in real time. DICOM images are loaded onto workstation computer by network 
transfer directly from the DICOM server/client. The graphical user interface includes tri-planar 
reconstructed views (sagittal, coronal, and axial) next to the images. The coil sensor in the inner 
stylet guider needle tip gives position and orientation to reformat and display the corresponding 
CT display planes. The device location within the registered image is updated in real-time at 10 
frames per second. Thus both EM and optical information provide position and ICG levels, 
respectively, in real time simultaneously. This information however, will NOT be used to guide 
the biopsy needle to a sample location for biopsy or data sampling. The optical and EM data will 
be analyzed after the procedure and do not impact the biopsy sampling location. 
2.4 
RISK/BENEFIT ASSESSMENT 
2.4.1 Known Potential Risks 
Liver biopsy is an invasive technique, with a well-described risk profile, and will be performed 
as clinically indicated and per the standards of the medical procedure. The biopsy procedure will 
be performed on the patients who have already been enrolled in other studies and required liver 

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image-guided biopsies, and we anticipate less than 5 minutes of added procedural time per 
participant using our designed biopsy device. We will exclude the patients who are not eligible 
for a routine image-guided biopsy, to minimize the adverse events. In case of any reactions, or 
detection of any potential risks to the participants or any predicted or un-predicted adverse 
events, the principal investigator will stop the procedure and all the adverse events will be 
reported in 24 hours. All the collected samples and data will be coded to maximize the 
participant’s privacy. The records will be kept in locked drawers or password-protected 
electronic devices to ensure the safety of participants. 
2.4.2 Known Potential Benefits 
Patients with focal hepatic lesions need biopsies for accurate diagnosis, and the ones with small 
focal hepatic lesions approximately l< 3 cm required more accurate methods to guide the biopsy 
needle into the lesion. Accurate diagnosis of HCC or cancer is important in these patients since 
these patients are usually candidates for focal ablation and subsequent liver transplantation. 
Optical molecular imaging has been reported as a promising method for better detection of the 
tumors. Our null hypothesis is that there is discordance greater than 20% between the 
fluorescence signal during biopsy and histopathology. We anticipate that with the combination of 
both optical molecular imaging guidance and electromagnetic guidance, we can address issues of 
tumor heterogeneity, by guiding intra-tumoral sampling towards the most viable and aggressive 
lesion components to improve the biopsy yield. 
2.4.3 Assessment of Potential Risks and Benefits 
Since the selected participants are referred from other NIH research protocols that require an 
image-guided biopsy, they will be assessed for any risk of a percutaneous biopsy under 
anesthesia or conscious sedation per standard clinical care. Individuals eligible for biopsy based 
on standard clinical criteria and who have no contraindications to receiving ICG will be referred 
to this study and enrolled. 
Participants will not be offered biopsy for the sole purpose of participation in this trial. 
Participation in this trial will not significantly alter clinical care. The procedure in this study 
requires intravenous administration of the FDA-approved agent (ICG) 24 hours before the 
intervention, and the biopsy procedure will be performed with FDA-cleared devices used as 
biopsy guides, which help the routine clinically used needle to enter the small lesion in the liver. 
Therefore, we anticipate that the participants in this study would be at minimal risk for using 
these devices compared to the ones who undergo a routine image-guided biopsy. 
3 
OBJECTIVES AND ENDPOINTS  
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
To evaluate the feasibility of 
using both the optical molecular 
imaging technique and EM 
Malignancy determination will be 
based on TBR: TBR≥2 will 
produce a malignant diagnosis, 
The primary endpoint 
is the concordance 
between 
histopathology 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
tracking during standard liver 
biopsies. 
and TBR<2 will produce a benign 
diagnosis. 
outcomes and Target 
to Background Ratio 
(TBR) in vivo and ex 
vivo with a handheld 
OMI and PIC. 
Concordance is 
defined as a TBR ≥2. 
TBR will be 
calculated by 
dividing the mean 
fluorescence intensity 
within the lesion by 
the mean 
fluorescence intensity 
within the adjacent 
liver parencyhyma. 
This endpoint was 
chosen because 
fluorescence 
microscopy similarly 
shows a sharp 
delineation between 
malignant and normal 
tissue, with increased 
ICG uptake in 
malignant cells in 
preclinical models. 
This additive marker 
aids in better 
diagnostic procedures 
to diagnose liver 
cancer. 
Secondary 
 
 
To assess the concordance of 
ICG fluorescence signal at the in 
vivo site of biopsy with the 
presence or absence of 
malignancy, as defined by 
pathology. 
ICG fluorescent signal at the in-
vivo site of biopsy using a 
combination of OMI and EM 
tracking 
We are assessing 
concordance with the 
histopathology and 
measured TBR from 
two different 
environments with 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
To assess the concordance of 
ICG fluorescence signal ex vivo 
(outside of body with camera) of 
biopsy with the presence or 
absence of malignancy, as 
defined by pathology. 
ICG fluorescent signal ex vivo of 
the specimen obtained using a 
combination of OMI and EM 
tracking. 
approved devices. 
We expect that they 
will match but we 
will need to confirm. 
4 
STUDY DESIGN 
4.1 
OVERALL DESIGN 
This is a non-randomized, open-label prospective pilot study to evaluate the feasibility of the 
combination of optical molecular imaging (OMI) and EM tracking to guide liver biopsies and to 
detect and measure fluorescent ICG during intrahepatic biopsies. The primary endpoint is the 
concordance between the histopathology and measured ICG signal (typically TBR by handheld 
OMI for HCC). Concordance will be evaluated by confirming that all 4 of the following exist: 
1.Diagnostic tissue specimen is obtained for conventional pathology analysis, defined by 
pathologist report of diagnostic liver lesion tissue 2. Imaging confirms that biopsy was taken 
from region desired 3. An ICG optical signal is obtained by ICG apparatus 4. EM tracking fusion 
image displays a location of biopsy. 
Based on our preliminary data a TBR cut off value of 2.0 is typically considered positive for 
presence of malignant pathology. We expect that fluorescence intensity measurements will be 
able to accurately differentiate between benign and malignant lesions. This initial study will 
enroll 77 participants who have focal hepatic lesions suspicious for HCC or other metastatic liver 
disease, and for whom based on their primary research protocol, are directed to have a 
percutaneous diagnostic and or research biopsy. 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
Liver cancer is the sixth most common malignancy worldwide. This includes hepatocellular 
carcinoma (HCC) as the most common type of primary liver neoplasm and CRC as the major 
source of metastasis to the liver. The incidence of HCC and CRC is increasing rapidly, 
particularly in Western countries. Imaging plays a pivotal part in the diagnosis of HCC, 
intrahepatic colorectal cancer (CRC), and other liver malignancies; contrast-enhanced cross 
sectional imaging can often accurately diagnose the tumor non-invasively. Those lesions that are 
unable to be characterized non-invasively, however, require biopsy for tissue confirmation of 
malignancy. By providing a definitive diagnosis, focal image guided biopsy is an indispensable 
component of the care of patients with cirrhosis. Biopsy can also determine the suitability of 
minimally invasive therapies for HCC, intrahepatic CRC, and other liver malignancies such as 
thermal ablation or chemoembolization. 
Biopsy is not considered a “perfect” test, and a negative biopsy does not exclude the diagnosis of 
liver malignancies such as HCC or metastatic CRC (3), due to the potential for sampling error. 
Small hepatic lesions less than 3cm can pose unique biopsy challenge. MRI visible liver lesions 
are not always visible by ultrasound or CT, the two available modalities in IR and thus may be 

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“invisible” to the Interventional Radiologist during the procedure. Patient habitus or underlying 
liver disease can hinder ultrasound detection of small lesions vis a vie overlying adipose tissue in 
obese patients and the echogenicity of hepatic parenchyma in cirrhotic patients. In CT-guided 
procedures, an important limitation is the beam-hardening artifact caused by the biopsy needle. 
At the critical moment when the biopsy needle is in the vicinity of the lesion, this artifact can 
obscure the lesion and decrease the Interventionalist’s confidence in the accurate location of the 
biopsy target. Indeed, in a retrospective study, up to 45% of small hepatic lesions were 
insufficiently visualized with the biopsy needle in place (4). The negative predictive value (NPV) 
in one study was only 63%; that is, 37% of patients with benign biopsies were subsequently 
found to have malignant lesions at the attempted site of biopsy. In a large series from a partner 
hospital / institution, the NPV for biopsy of small hepatic lesions was 72% (1): over a quarter of 
the benign biopsies were in fact false negatives. Confidence in the accuracy of a focal hepatic 
biopsy is essential to diagnosis, staging, and triage of care, as a false negative biopsy can have a 
devastating impact on a patient’s outcome. Multiple attempts have been made to improve biopsy 
accuracy. For example, some institutions perform cytological “wet reads” by examining the 
biopsy tissue under a microscope, while the patient is still on the scanner table. However, this 
additional step adds a considerable amount of procedural and sedation time and may not always 
predict the final biopsy result. 
We propose a trial to determine the feasibility of using optical molecular imaging (a low cost, 
real-time, high resolution imaging discipline) for minimally invasive liver biopsy guidance in 
patients with diagnosed or suspected HCC or metastatic CRC. Patients who require a clinically 
indicated or separately IRB approved research liver biopsy would after consent, have the OMI 
performed during biopsy but not as the primary directorate to the biopsy site. The standard of 
care imaging and cognitive registration would be employed for navigation or guidance of needle 
and OMI would act as a bystander. Nonetheless, data from OMI imaging would be obtained and 
evaluated with ex vivo POC OMI and histopathology. Correlations from these studies might 
support a future larger clinical trial, with different goals that might not exclude efficacy and 
safety, or even allow for guidance of the needle if separately IRB-approved. 
5 
STUDY POPULATION 
The target study population is patients with focal hepatic lesions with imaging features 
suspicious for hepatocellular carcinoma or other metastatic liver disease. A radiologist will 
review patients’ imaging for disease appropriate for biopsy. The decision to refer the patient to 
enroll in this study may occur between during tumor boards or other clinical care meeting where 
the primary care team, consults with a CC Interventional Radiologist/ AI or PI, Chief of 
CC/Interventional Radiology. 
5.1 
INCLUSION CRITERIA 
In order be eligible to participate in this study, an individual must meet all of the following 
criteria: 
• Patients must have imaging findings consistent with hepatocellular carcinoma or other 
liver neoplasms or metastasis, for whom image-guided percutaneous biopsy is planned 
as clinically indicated or IRB-approved under a separate research protocol.  
• Patients must have at least one lesion that can readily be biopsied per Principal 
Investigator. 

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• Age >18 years.  
• Patients must have the ability to understand and the willingness to sign a written 
informed consent document. 
• Evidence of post-menopausal status or negative urinary or serum pregnancy test for 
female pre-menopausal patients. Women will be considered post-menopausal if they 
verbally report amenorrhea for 12 months without an alternative medical cause, or have 
had surgery or received chemicals to induce menopause. 
5.2 
EXCLUSION CRITERIA 
• History of hypersensitivity reactions to Indocyanine Green (ICG), iodinated contrast, or 
sulfur-containing compounds.  
• Pregnant women and nursing mothers are excluded from this study because of exposure 
to radiation from CT scanning associated with the biopsy 
• Uncontrolled intercurrent illness including, but not limited to, ongoing or active  
infection, symptomatic congestive heart failure, unstable angina pectoris, uncontrolled 
cardiac arrhythmia, or psychiatric illness/social situations that, in the opinion of the 
Principal Investigator, would limit compliance with study requirements 
5.3 
INCLUSION OF VULNERABLE PARTICIPANTS 
5.3.1 Inclusion of children 
HCC is rarely seen in children therefor we will enroll participants > 18 years of age. Children are 
excluded from this study. 
5.3.2 Pregnant Women 
Pregnant women and nursing mothers are excluded from this study because of exposure to 
radiation from CT scanning associated with the biopsy. 
5.3.3 Decisionally Impaired 
Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent 
may be necessary and there is a possibility, that subjects could become decisionally impaired. In 
these cases, the subject will be taken off study. 
5.4 
LIFESTYLE CONSIDERATIONS 
5.4.1 Disease Community Engagement 
The research framework of disease community engagement is not applicable to the proposed 
study. This feasibility study is at its earliest stages. The anticipated results may not be globally 
useful yet for the target population of the clinical trial. 
5.5 
SCREEN FAILURES 
Recruitment for this protocol is by invitation only; as such referral to this protocol is dependent 
on the evaluation of the investigator of the primary protocol. Individuals who do not meet the 
criteria for participation in this study (screen failure) may be considered for rescreen at the 
discretion of the PI/ referral team. However, patients who have screened, found eligible, signed a 
consent, and assigned a unique study ID but become ineligible consequently for various reasons 

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prior to the biopsy, will be considered inevaluable. These patients will be considered a “screen 
failure” and his/ her unique study ID will be counted towards overall total accrual. 
5.6 
STRATEGIES FOR RECRUITMENT AND RETENTION 
This study will be “Enrolling by Invitation Only”. Patients will be referred by their NIH primary 
care team and will need to be registered to an existing NIH clinical study prior to enrolling in this 
study. Any patient who is referred and found eligible for a standard liver biopsy will be able to 
screen for this study. 
5.6.1 Costs 
Potential participants will not incur any costs associated with this protocol. 
5.6.2 Compensation 
Eligible subjects who sign a consent form will be compensated when ICG is administered in 
accordance with NIH guidelines (Table 2).  
All remuneration will be directed to be paid to the participant. These funds are intended to 
compensate for time away from work that maybe required to participate in this study and time 
spent receiving ICG. Payment will be sent after the biopsy procedure. Travel will not be 
provided or compensated for. We will also not provide remuneration for meals and lodging. If a 
participant withdraws from study, no future compensation will be made, but prior compensation 
will not be revoked. 
Table 2. Subject Reimbursement Schedule 
* Inconvenience unit = $10 
6 
STUDY INTERVENTION 
6.1 
STUDY INTERVENTIONS ADMINISTRATION 
ICG is a clinical fluorescent imaging contrast agent and will be ordered from the CC Pharmacy. 
Incidence of 1% to 10%: diaphoresis, headache, pruritus, skin discoloration at the intravenous 
site. In subjects allergic to iodine anaphylactic reactions have occurred. The total dose to be 
administered will not exceed 40 mg per day. Doses of up to 5 mg/kg have been administered 
routinely in Japan without adverse reactions. All adverse events experienced by participants will 
be collected from the time ICG is administered through the end of the procedure. Participants 
continuing to experience toxicity will be assessed until toxicity has resolved or deemed 
irreversible. 
 
Inconvenience units* 
Pay for 
inconvenience  
Time (h) Pay for time  Total pay 
 
Contrast IC/Oral 
2 
$20 
3 
$40 
$60 
Peripheral IV 
1 
$10 
N/A 
$0 
$10 
Screening 
3 
$30 
N/A 
$0 
$30 
TOTAL 
 
 
 
 
$100 
NOTE: 
Patient will be paid $100 when he or she received ICG. 

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6.1.1 Study Intervention Description 
The study has 2 steps: 
a) In-procedure biopsy 
b) Ex vivo tissue sample analysis outside of procedure (point of care) 
6.1.2 In-procedure biopsy 
Tumor biopsy is divided into several stages or parts, described below. The first step is the 
standard of care preparation of the site and imaging of the liver, infiltration of local anesthesia 
followed by placement of the PercuNav coaxial needle by ultrasound guidance and EM tracking 
and co-display into the liver parenchyma. The next step requires a very short pause to thread that 
pediatric scope inside the PercuNav sheath. This is done by removing the PercuNav inner stylet 
from the hollow sheath. Once the scope is in the sheath, it is advanced in order to allow for 
signal sampling from normal liver. After acquiring images from normal liver, the scope is 
removed and once again the PercuNav needle inner stylet is reinserted, and the needle is 
advanced and manipulated into the target lesion in the usual standard fashion with standard 
clinical image-guidance. When the target is reached, the same process ensues, with exchange of 
the inner stylet for the scope, and subsequent repeat ICG OMI imaging occurs again. Finally, the 
PercuNav stylet is removed and replaced with a standard spring-deployed biopsy needle, and 
tissue core biopsy sampling occurs. Except for the two pauses, the biopsy procedure occurs as 
per usual medical practice, with standard clinical image guidance determining the location of 
biopsy tissue sampling, as is standard practice for many decades at NIH and around the globe. 
Thus, in summary, in order to record the data (optical study has 2 steps). 
6.1.3 Ex vivo tissue sample analysis outside of the procedure (point of care) 
The second step of the study (Point of Care = POC) involves assessment of the tissue sample 
after it is removed from the body in the usual fashion, thus does not introduce risk to the patient, 
as it occurs outside of the procedure room itself. The sample is not perturbed, thus is totally 
analyzable after the study in the standard pathology fashion. 
6.1.4 Dosing and Administration 
6.1.4.1 Pre-Procedure and ICG Administration 
ICG was first FDA cleared in 1959. ICG (IC-Green™ Akorn, Inc. Buffalo Grove, IL) (ICG 
C43H47N2NaO6S2 Molecular Weight 775) is a water-soluble, tricarbocyanine dye routinely 
used in dilution studies, hepatic and cardiac function studies and ophthalmic retinal fluoroscopy 
(20). ICG contains not more than 5% sodium iodide. ICG is indicated for determining cardiac 
output, hepatic function and liver blood flow, and for ophthalmic angiography. In this study, ICG 
may be used to roughly determine local tissue hepatic function, after the procedure, for data 
correlation. 
ICG has been shown to localize to HCC and intrahepatic tumors 24-72 hours post-injection with 
high target to background ratios. Doses of ICG up to 5 mg/kg have been administered without 
adverse reactions. All patients will receive a dose of ICG 0.5mg/kg, with a maximum dose of 
40mg, 18-30 hours (roughly 24 hours) prior to the scheduled procedure. Nurses from 3SE Day 
Hospital will administer ICG as per drug indications. 

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After receiving ICG, all patients will be monitored for 30 minutes following the injection to 
identify any acute adverse reaction, after which point they will be free to leave without any 
dietary or activity restrictions. Patients will receive contact information prior to injection and 
biopsy. See Section 6.1.4 for ICG administration procedure. 
The usual fluoroscopy dose is 5 - 40 mg (adult dosage). ICG has been used to identify and 
monitor ocular melanoma (21), retinal metastases, and in the photodynamic therapy of cutaneous 
Kaposi’s sarcoma (22). The recommended dose of up to 40 mg of dye in 1-2 mL of aqueous 
solvent is safe and in routine clinical use. The maximum dose for this trial will be 40mg ICG. 
The LD50 after IV administration in rodents is 50 – 80 mg/kg. A bolus of ICG can be 
administered through any venous access. Immediately following the bolus dose of dye, a 5 mL 
bolus of sodium chloride 0.9% is given; this regimen delivers a spatially limited dye bolus of 
optimal concentration to the arterial and arteriolar vasculature. Excitation of ICG occurs at 805 
nm with emission maximum at 835 nm. In Japan, ICG is routinely administered to evaluate 
hepatic function prior to hepatic resection, as ICG is almost entirely and rapidly excreted via the 
hepatobiliary system. ICG is also used to evaluate vascular lesions of the gastrointestinal tract 
(e.g. esophageal varices) using NIR endoscopy systems. Recently, ICG, administered 
intravenously, has also been used in NIR endoscopy systems to successfully differentiate 
superficial gastric tumors from invasive cancers (23). ICG has also been shown to localize to 
hepatocellular carcinoma and intrahepatic colorectal cancer metastases 24 – 72 hours post-
injection with high target to background ratios. Doses of ICG up to 5 mg/kg have been 
administered without adverse reactions. The abnormal liver tissue retains ICG, which does not 
washout and is thus detectable with OMI. See Section 6.2 for ICG preparation and 
administration. 
Administration of ICG will be limited to the day prior to the period in which the biopsy is 
scheduled. 
6.1.4.2 Intra-procedure and ICG fluorescence measurement 
On the day of the biopsy, patients will be brought to the biopsy suite in the Interventional 
Radiology department, Clinical Center, NIH. After standard preparation for the biopsy, skin 
adhesive/removable fiducials (Figure 18) will be placed on the patient’s abdomen for 
registration with electromagnetic imaging (EM). Using EM guidance static images (MRI, CT, 
PET) are fused to real time imaging (ultrasound, cone beam CT) and a PercuNav needle tracker 
that registers with EM is placed and directed towards the target lesion. The PercuNav needle is a 
coaxial needle with tracking capability. The PercuNav needle tracker (Figure 17) has a stylet 
and a hollow core sheath that allows passage of a biopsy needle and an optical guided pediatric 
cystoscope-urethroscope. There will be at least 3 or more measurements of the ICG fluorescence 
signal attempted. 
 

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The procedure will follow a standard percutaneous liver biopsy using a PercuNav coaxial 
introducer and tracker along with a commercial needle biopsy device (e.g., Temno). The 
PercuNav commercial coaxial needle tracker devices have 2 parts – a blunt hollow cannula and a 
sharp inner needle stylet that punctures the skin and has a wire at its very end that connects to the 
EM tracking device. In this case, the PercuNav needle tracker provides the access to the skin and 
the biopsy needle. 
The procedure for obtaining measurements of the ICG in vivo has 2 parts: a) In-procedure biopsy 
b) Ex vivo tissue sample analysis outside of procedure (point of care).  
The biopsy is divided into several stages or parts. The first step (biopsy) allows for signal 
sampling by a commercial cystoscope-urethroscope just from the tip of a commercial coaxial 
needle cannula, during a percutaneous biopsy. This step is done by pausing during the standard 
coaxial percutaneous image-guided biopsy procedure, in order to allow for signal sampling from 
normal liver on the way to the target lesion. The standard needle inner stylet is then replaced, and 
the needle is manipulated into the target lesion in the usual standard fashion, followed by a 
repeat sample via the standard outer cannula introducer, after the endoscope is replaced a second 
time into the outer needle cannula (this time, with the tip residing in the target lesion). Except for 
the 2 pauses, the biopsy procedure occurs as per usual medical practice, with image guidance 
determining the location of biopsy tissue sampling, as is standard practice for many decades at 
NIH and around the globe. Thus, in summary, in order to record the data (optical signal) from 2 
locations, there are 2 brief pauses, [no more than several minutes during a 20-60 minute 
procedure], where the pediatric cystoscope-urethroscope samples optical signal at 1) normal liver 
on the way to target lesion, and 2) from the target lesion itself (Figure 19 and Figure 20). 
Prior to sending the biopsy tissue to anatomic pathology for clinical diagnosis, the tissue will 
undergo the second part of the study via the Point of Care (POC) system. This optical imaging 
device a BWF1 laser generator from the medical laser series will be used with the point of care 
OMI, which occurs away from the patient, outside the procedure room. This system takes 
pictures of the ex vivo biopsy core itself, and may, at some point in the future, contribute as a 
“wet-read” by providing placement information as a cytology technician might.  
A study physician will assist the operator and perform all manipulations of the imaging system. 
This ex vivo process occurs after the tissue is removed from the body in the usual fashion. 
Figure 18. Skin adhesives/ removable 
fiducials 
 
Figure 17. Philips PercuNav needle tracker 

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Neither the patient nor IR staff are exposed to laser energy, as it occurs outside of the procedure 
room itself.  After completion of the biopsy, participants will recover as per standard clinical 
practice. Tissue will not be stored or specifically obtained for this study. 
 
Figure 19. Percutaneous biopsy procedure flow with ICG signal measurement using a coaxial 
biopsy needle device, a tracked Percunav introducer/ stylet needle, and a pediatric cystoscope-
urethroscope (“commercial scope”). 
 

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Figure 20. Demonstration on a phantom model of the insertion of the pediatric scope into the 
PercuNav Coaxial needle introducer. 
B.Advancing the 
scope to the first 
area of imaging 
(phantom liver). 
A.Inserting 
the pediatric 
scope 
through the 
co-axial 
introducer 
(after the 
stylet has 
been 
removed). 
C. Take the 
first image. 
E. 2nd image 
after further 
advancement 
of the needle  
 
F. Full image of 
all parts in 
process with the 
electromagnetic 
generator in 
view 
D. Removing the 
scope.  

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6.1.4.3 Post-procedure care 
After completion of the procedure, the patients will recover as per standard clinical practice. 
Participants will be closely monitored for typically 4-6 hours after the procedure as part of their 
standard post liver biopsy clinical care. All pathological specimens will be evaluated by the NCI 
Laboratory of Pathology, who will classify the histology of the lesion according to standard 
criteria. After review, the results of the pathological analysis will be associated with the 
participant’s code. 
6.1.4.4 Duration of follow up 
Participants will typically be closely observed for 4-6 hours after the biopsy procedure as part of 
their standard clinical care. Adverse event monitoring will be performed up to the completion of 
the biopsy monitoring period. 
6.1.4.5 Study Device Description and Device Use 
We will be using an FDA-cleared optical imaging device and EM tracking hardware devices for 
the proposed study. The software is not specifically FDA cleared, but the same platform has been 
extensively used in NIH Clinical Trials dating back to ~2003 in many thousands of patients. The 
hardware and software components are based upon the design of multiple pre-clinical studies as 
well as clinical imaging systems that we have previously constructed and tested in patients (EM 
at NIH and OMI at MGH). The modular design of the system allows for the seamless 
interchange of a variety of imaging catheters, expanding the depth and breadth of possible 
applications. The eye-piece of the Safelight cable imaging catheter connects to a laparoscopic or 
endoscopic camera and captures the infrared emission, processes the image in high resolution 
and displays it on a surgical display in real-time (Mako camera, Allied Vision Technologies, 
Stadtroda, Germany). 
 
 

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Figure 21. The OMI apparatus labeled, the coaxial needle (PercuNav) and the pediatric scope. 
The laser system used for imaging ICG in vivo is a Stryker® IRF L10 Light Source and 
Safelight Cable which has an FDA 510K clearance and is approved for clinical use. This system 
has class 1M lasers at 808 and 830 nm. The 808 nm wavelength is the peak excitation 
wavelength for ICG and is the optimal excitation wavelength for the proposed study. The 
wavelength, power output and the laser mode (continuous or pulsating) can be by adjusted by the 
user in the Endoscopic Near-Infrared Visualization (ENV) mode. This system complies with IEC 
60825-1:2007 and 21 CFR, Subchapter J, Parts 1040.10 and 1040.11. Additionally, this device is 
designed to ensure electromagnetic compatibility with other electrical medical devices and 
complies with IEC 60601-1-2 requirements for EMC with other devices.  
Image display and camera functions are controlled on a laptop by a custom software program. 
We will use a clinically approved pediatric cystoscope-urethroscope (Model 27033 AA, Karl 
Storz, Tuttlingen, Germany) for the optical assessment. This component was selected due to its 
small outerdiameter, allowing it to pass through the introducer of a standard 17-gauge core 
biopsy system. The FDA cleared imaging catheter / scope and the FDA cleared biopsy needle 
will be the only components that are in contact with the patient. This scope piece will be 
autoclaved after each patient examination according to manufacturer’s guidelines, and the 
remainder of the handheld device will be wrapped in a sterile drape. Sterilization of components 
will be performed based on manufacturer’s recommendation 
(http://www.urologicservices.com/documentation/karl-storz-general-reprocessing-
instructions.pdf) in consultation with the CC Central Sterile Service. 
We will also be using a BWF1 laser manufactured by BW&Tek with the point of care system for 
ex vivo testing, which will occur outside of the procedure room. The excitation light is provided 
by a 450mW, 785nm laser. Fluorescent light collected by the imaging catheter is filtered to 
exclude reflected excitation light by a bandpass filter. The eyepiece of the fiber optic imaging 
catheter connects to a video endoscopic camera which can acquire high resolution, 12 bit images 
in real time. This laser system as part of the point of care device that will analyze tissue samples 
only, will not be near the patient, and will be located in a room separate from the clinical suite 
where the patient is having the biopsy. This laser system is not approved for in vivo use, 
therefore will only be used ex vivo, and will only be used to test for fluorescence signal after the 
tissue is obtained and transported to the ex vivo system for analysis. 
6.1.4.6 Laser Safety 
The NIH approved laser safety protocol and processes will be used only in reference to the B&W 
Tek BWF5 Coupled Fiber Laser System. A laser safety training SOP and process that is specific 
to this laser has been developed and approved by (CDR Dennis House), Laser Safety Officer 
who also conducts the NIH Laser Safety equipment inspections and audits. Persons using the 
laser will be trained on safety, as well as use. Laser safety training sessions will be conducted in 

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adherence with NIH policies and guidelines for safe use of laser systems. The laser waves are 
emitted only in the black box. The room in which the box is kept, will be windowless and the 
single external door will be placarded with signs “Laser in use”, and laser safety goggles will be 
used by operating personnel and all personnel in the procedure room.  
Point of Contact for Laser Safety: 
Julie Peretti, RN 
Research Nurse Specialist/CIO 
10 Center Dr. 
Building 10 Rm. 1C305 
MSC 1182 
Bethesda, Maryland, 20892 
301-451-3287 
jperetti@cc.nih.gov  
The use of class 3b and 4 lasers is regulated by the Occupational Health and Safety 
Administration (OSHA). The laser will be registered with the NIH Laser Safety Program and 
reported to the Division of Occupational Health and Safety, NIH, as are all class 3b and 4 lasers. 
(http://www.ors.od.nih.gov/sr/dohs/Documents/NIH%20Laser%20Safety%20Program.pdf).  
Users will be trained in laser safety and laboratory-specific safe operating procedures will be 
followed. Additional information about the laser safety program is available at: 
http://www.ors.od.nih.gov/sr/dohs/HealthAndSafety/IH/Pages/ih_laser.aspx.  
6.1.4.7 Handheld Ocular Molecular Imaging (OMI) device 
This is the FDA cleared endoscopic device that is placed through the standard outer cannula 
introducer (outer part of the FDA cleared coaxial needle system). At no time does an 
experimental device touch the patient, in any way. The standard flexible and blunt-tip pediatric 
cystoscope-urethroscope “stylet/camera” is placed inside the introducer cannula to the 
predetermined length, in order to sample the tissue just immediately at the tip of the outer needle 
(see Figure 6). This tip of the pediatric cystoscope-urethroscope is like a non-sharp blunt felt-tip 
pen tip that cannot pierce the skin or tissue with normal forces of gentle manual manipulation, as 
is intended. It is a hand-held device used by an experienced interventional radiology physician 
who is certified and credentialed to practice (image guided therapy) interventional radiology. 
The camera and safelight cable are never inserted into the patient’s tissue beyond the ~ 1mm tip 
of the 17 Gauge outer cannula coaxial needle. The tissue (just beyond the pediatric cystoscope-
urethroscope tip in the lesion) is sampled by the standard spring-loaded biopsy needle.  
Liver biopsy is an invasive technique, with a well-described risk profile, and will be performed 
as clinically indicated and per the standards of the medical procedure. The 2 pauses to take signal 
sample with standard devices during the biopsy, is of non-significant risk (or minimal risk) in the 
opinion of the Principal Investigator the research team and others during the institutional 
scientific review process. The FDA-cleared pediatric cystoscope-urethroscope is not normally 
used via this needle cannula, tube, or lumen, but it is compatible.  
The OMI is a handheld endoscopic instrument consisting of an FDA-cleared and clinically used 
pediatric cysto-urethroscope (Model 27033 AA; Karl Storz, Tuttlingen, Germany) illuminated by 
a safelight cable attached to an 808 nm laser excitation source (Stryker IRF L10 Light Source 

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and Safelight Cable) with an adjustable user feature in the Endoscopic Near-Infrared 
visualization (ENV) mode that can pass coaxially through the introducer of a standard 17-gauge 
core biopsy system (Figure 6). 
The eyepiece of the pediatric cystoscope-urethroscope is attached to high-definition endoscopic 
camera system sensitive in the visible and infrared spectrum. (Stryker 1588 Camera Control Unit 
and Camera Head). The optical image is transferred from the surgical site to the camera unit by 
the aforementioned pediatric cysto-urethroscope which are attached to the camera head (Mako; 
Allied Vision Technologies) and the light output is by a filtered band-pass filter that is 
compatible with the laser excitation source. Expanded details of the device is described in 
Section 2.3.8. 
6.1.4.8 Point-of-care (POC) Optical Molecular Imager (OMI) 
This is a “camera in a black box” taking images of the ex vivo tissue using a BWF1 laser to 
excite light to see the ICG fluorescence. Specifically, the core biopsy tissue specimen is placed 
in a sterile container and removed to a windowless room adjacent to the IR suite. The tissue is 
placed inside a 2 foot tall black box on a benchtop with the laser. The laser does not come in 
contact with the patient, nor is it in the same room as the patient at any time. Details about the 
POC device is found in Section 2.3.9. 
6.1.4.9 Non-significant Risk Determination Criteria 
The proposed study meets the definition of a non-significant risk device and is subject to 
abbreviated IDE regulations as stated in 21 CFR Part 812.2(b): 
Requirements for labeling the device (§812.5)  
Maintaining IRB approval of the study as a Non-significant risk study (§56)  
Ensuring all subjects provide informed consent (§50) 
Monitoring the conduct and compliance of all sub-investigators and evaluating all adverse device 
effects to determine whether the study is safe to continue (§812.46 [a & b]) 
Maintaining records of the following (§812.140[b][4] and [5]): 
a) basic device information 
b) brief explanation of why the device is non-significant risk (See Below) 
c) all adverse events, whether anticipated or unanticipated 
Making reports of the following (§812.150[b][1-3] & [5-10]):  
a) continuing review as required by the IRB 
b) deviations from the investigational plan to the IRB  
c) final report upon study termination/completion to IRBs  
d) device (or parts) recall/returns/repairs  
Restrictions on promoting (812.7[a]), charging subjects for the device (812.7[b]), prolonging an 
investigation (812.7[c]), and stating a device's safety/efficacy (812.7[d]) 

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6.1.4.10 Brief explanation of why the device is non-significant risk and applicability of 
abbreviated IDE regulations 
Regulatory issues for this study can be separated into invasive biopsy procedure risk, specific 
device risk and overall study risk. The biopsy risk will be undertaken regardless of this study, as 
it is clinically indicated or separately approved under a separate IRB approved protocol. 
However, because the research is related to the invasive biopsy procedure, the risk involving this 
invasive procedure pre-exists.  
Devices used are FDA cleared, but the pediatric cystoscope-urethroscope is being used off-label, 
since its FDA indication-for-use clearance does not directly specify use through a cannula needle 
prior to liver biopsy sampling, during the overall liver biopsy procedure. The outer needle 
cannula is a sort of “lumen”, but the use described is away from the original intended use.   
The FDA’s abbreviated IDE regulations are applicable to the study because although the devices 
to be used have received 510(k) FDA clearances, the research study itself in which they are to be 
used is related to an invasive procedure (regardless of it being clinically indicated). Thus, the 
research study involves an “invasive sampling” and has inherent risks associated with it. Despite 
the invasive sampling, we are confident that this research will not add significant risk because 
the added time required for the signal sampling (ICG signal) is short, and this added time will be 
a small fraction of the overall time required for a standard biopsy using the standard tools used 
for NIH biopsy. This is minimal added risk, if definable at all. The optical and molecular signal 
acquired from the tissue will be obtained via the coaxial biopsy cannula (through the outer 
biopsy needle conduit / cavity). Thus the study meets the IDE abbreviated requirements of the 
FDA, and the risk determination is non-significant risk (sub-category minimal risk).  
The study determines the overall research determination and we will adhere to the FDA 
abbreviated IDE regulations per 21 CFR Part 812.2(b). 
• This study will be using FDA 510(k) cleared devices. See APPENDICES for FDA 510(k) 
clearance letters to Karl Storz Imaging, Inc, Akorn, Inc., and Philips Invivo Corporation 
for permission to market and outlines for Indications for Use 
• FDA 510 (k) Clearances for Karl Storz Imaging, Inc. The following Karl Storz Imaging, 
Inc. devices are FDA 510(k) cleared: 
1) Karl Storz Uretero-Renoscope/ Ureteroscope (Appendix A) 
a. June 24, 1994: FDA Clearance K940464/A 
2) Karl Storz-Endoscope Sterilization Trays (Appendix B) 
b. August 18, 2009: FDA Clearance K090818 
• This study will be using FDA 510(k) cleared devices. See attached FDA 510(k) clearance 
letters to Karl Storz Imaging, Inc, Akorn, Inc., and Philips Invivo Corporation for 
permission to market and outlines for Indications for Use. The following Karl Storz 
Imaging, Inc. devices are FDA 510(k) cleared: 
1) Karl Storz Uretero-Renoscope/ Ureteroscope (Appendix A) 
a. June 24, 1994: FDA Clearance K940464/A 
2) Karl Storz-Endoscope Sterilization Trays (Appendix B) 
b. August 18, 2009: FDA Clearance K090818 

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6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY  
6.2.1 Acquisition and Accountability 
ICG is a clinical fluorescent imaging contrast agent and will be ordered from the CC Pharmacy). 
6.2.2 Formulation, Appearance, Packaging, and Labeling 
(https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/011525s017lbl.pdf)  
Injection: 25 mg freeze-dried powder in vials supplied with 10 ml aqueous solvent NDC 7478-
701-02 (pH 5.5 – 6.5). Manufactured as IC-Green™ Akorn, Inc. Buffalo Grove, IL. 
PREPARATION: PHARMACY PREPARATION INSTRUCTIONS (AS PER ICG PACKAGE 
INSERT): One or two vials of ICG 25 mg will be dissolved with 2 ml diluent, provided by the 
manufacturer (yielding ICG in solution at 12.5 mg/mL concentration). The CC Pharmacy will 
prepare the ICG for infusion. 
Incidence of 1% to 10%: diaphoresis, headache, pruritus, skin discoloration at the intravenous 
site. In subjects allergic to iodine anaphylactic reactions have occurred. The total dose to be 
administered will not exceed 40 mg per day. Doses of up to 5 mg/kg have been administered 
routinely in Japan without adverse reactions. All adverse events experienced by participants will 
be collected from the time ICG is administered through the end of the procedure. Participants 
continuing to experience toxicity will be assessed until toxicity has resolved or deemed 
irreversible. 
6.2.3 Product Storage and Stability 
Storage: Store at 15° to 25° C (59° to 77°).  
Stability: Indocyanine Green for Injection USP is unstable in aqueous solution and must be used 
within 6 hours. However, the dye is stable in plasma and whole blood so that samples obtained in 
discontinuous sampling techniques may be read hours later. Sterile techniques should be used in 
handling the dye solution as well as in the performance of the dilution curves. If a precipitate is 
present, discard the solution. 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/011525s017lbl.pdf.  
6.2.4 Preparation 
The CC Pharmacy will prepare ICG for infusion”. The drug is supplied in a kit (NDC 17478-
701-02) containing six 25 mg IC-GREEN vials and six 10mL Aqueous Solvent ampules. NDC 
17478-701-25 IC-Green vial. 25 mg fill in 50 mL vial. NDC 17478-701. 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL  
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
Off-Treatment Criteria: 
• Completion of biopsy and biopsy observation period (typically no more than a 6-hour 
stay in the recovery area after the biopsy procedure) 
• Participant requests to be withdrawn from the biopsy procedure. 

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• Allergic reaction (as described in section 8.5) and serious adverse event that is definitely 
related to the research. 
• Unacceptable toxicity per PI  
• Investigator discretion 
• Loss of capacity to provide informed consent 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Off-Study Criteria: 
• 1 ± 2 months of the completed biopsy (for all correlative pathological assessment of 
biopsy specimen availability) 
• Participant requests to be withdrawn from study 
• Death 
8 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 
SCREENING PROCEDURES 
The target study population is patients with focal hepatic lesions with imaging features 
suspicious for hepatocellular carcinoma or other metastatic liver disease. A radiologist will 
review patients’ imaging for disease appropriate for biopsy. The decision to refer the patient to 
enroll in this study may occur between during tumor boards or other clinical care meeting where 
the primary care team, consults with a CC Interventional Radiologist/ AI or PI, Chief of 
CC/Interventional Radiology. 
The research team will review and verify the following available information prior to patient 
registration on the protocol: 
1. Clinical evaluation by referring team/ primary care team 
2. Laboratory tests (blood work) 
3. Radiology tests with report of focal lesions within the liver and confirmed or suspicious 
HCC other liver neoplasms or metastasis 
8.2 
REGISTRATION PROCEDURES 
The Principal Investigator, along with key research personnel, will screen and enroll all eligible 
patients on the study. The screening and enrollment process will be documented in CRIS. All 
patients enrolled in the study will receive a unique patient identifier and will be entered in the 
study’s screening and enrollment logs which are kept in our research database. The study 
research database will be maintained in an electronic folder in a larger central shared drive with a 
secure password and Clinical Center network back-up, in accordance with NIH CC policy. 
8.3 
TREATMENT ASSIGNMENT AND RANDOMIZATION/STRATIFICATION PROCEDURES 
COHORTS 
Cohorts 
Number 
Name 
Description 
1 
Patients 
Patients scheduled for liver biopsy 

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Arms 
Number 
Name 
Description 
1 
Interventional therapy 
Liver biopsy with optical imaging and EM tracking guidance 
8.4 
EFFICACY ASSESSMENTS 
8.4.1 Biospecimen Evaluations 
We will be using concordance with pathology as gold standard by measuring TBR (See 
Statistical section). ICG measurements to obtain the TBR will be done in vivo and ex vivo inside 
the IR suite. Specimens for pathology will be evaluated for quality and for diagnosis by NCI 
Laboratory of Pathology. No biospecimens will be collected and stored specifically for our 
research. 
8.5 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
To comply with the abbreviated IDE regulations 21 CFR 812.2(b), records concerning adverse 
device effects should include anticipated and unanticipated events and complaints.  
Exceptions to adverse event data collection: 
• Expected events related to the clinical biopsy procedure (for example, but incomplete 
list): 
o Pain or bruising at the biopsy site. 
o Increased redness, swelling or bloody or serosanguinous drainage at the biopsy 
site. 
o Events directly related to local anesthetic administration such as nausea, 
dizziness, numbness or tingling. 
o Self-resolved transient hypotension or bradycardia.  
o Diaphoresis 
o Headache 
o Pruritus 
o Skin discoloration at the intravenous site. 
8.5.1 Definition of Adverse Event 
8.5.1.1 Adverse Event 
Any untoward medical occurrence in a human subject, including any abnormal sign (for 
example, abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in research, whether or not considered related to the 
subject’s participation in the research. 
8.5.1.2 Adverse Device Effect 
An adverse device effect (ADE) is an AE related to the use of our devices under study: the Karl 
Storz devices, Stryker® IRF Light Source and Safelight Cable, UroNav, or PercuNav. 

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8.5.1.3 Unanticipated Adverse Device Effect 
Defined as any serious adverse effect on health or safety or any life-threatening problem or death 
caused by, or associated with, a device, if that effect, problem, or death was not previously 
identified in nature, severity, or degree of incidence in the investigational plan or  other 
unanticipated serious problem associated with a device that relates to the rights, safety and 
welfare of subjects. 
8.5.2 Definition of Serious Adverse Events (SAE) 
8.5.2.1 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in the following:  
• death 
• serious deterioration of the subject’s health, leading to: 
o life-threatening illness or injury, or 
o permanent impairment of a body structure or function, or 
o in-patient or prolonged hospitalization, or 
o medical or surgical intervention to prevent life-threatening illness or injury or 
permanent impairment of a body structure or function 
8.5.2.2 Serious Adverse Device Effect (SADE) 
A serious adverse device effect (SADE) is an ADE that results in any of the consequences 
characteristic of an SAE. An unanticipated SADE (USADE) is a SADE which is not anticipated 
by the risk analysis, while an anticipated SADE (ASADE) is a SADE which is anticipated by the 
risk analysis. 
8.5.3 Classification of an Adverse Event 
For adverse events (AEs) not included in the protocol defined grading system, the following 
guidelines will be used to describe severity. 
8.5.3.1 Severity of the Event 
• Mild – Events require minimal or no treatment and do not interfere with the participant’s 
daily activities.  
• Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating. Of note, the term “severe” does not necessarily equate to “serious” 
8.5.3.2 Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to study intervention assessed by the 
investigator who examines and evaluates the participant based on temporal relationship and 
his/her clinical judgment. The degree of certainty about causality will be graded using the 
categories below. In a clinical trial, the study product must always be suspect.  

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• Definitely Related – There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to study intervention 
administration and cannot be explained by concurrent disease or other drugs or 
chemicals. The response to withdrawal of the study intervention (dechallenge) should be 
clinically plausible. The event must be pharmacologically or phenomenologically 
definitive, with use of a satisfactory rechallenge procedure if necessary. 
• Probably Related – There is evidence to suggest a causal relationship, and the influence 
of other factors is unlikely. The clinical event, including an abnormal laboratory test 
result, occurs within a reasonable time after administration of the study intervention, is 
unlikely to be attributed to concurrent disease or other drugs or chemicals, and follows a 
clinically reasonable response on withdrawal (dechallenge). Rechallenge information is 
not required to fulfill this definition. 
• Potentially Related – There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after administration of the trial medication). 
However, other factors may have contributed to the event (e.g., the participant’s clinical 
condition, other concomitant events). Although an AE may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be 
upgraded to “probably related” or “definitely related”, as appropriate. 
• Unlikely to be related – A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to study intervention administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time after 
administration of the study intervention) and in which other drugs or chemicals or 
underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments). 
• Not Related – The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There must 
be an alternative, definitive etiology documented by the clinician.] 
8.5.3.3 Expectedness  
The Principal Investigator will be responsible for determining whether an adverse event (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 
study intervention. 
8.5.4 Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study visits and interviews of a study participant presenting 
for medical care, or upon review by a study monitor. 
As this is a feasibility study, only grade 3 and above adverse events that are possibly, probably 
and definitely related to the research procedure (i.e., use of biopsy assisting devices and ICG 
administration) will be recorded, and reported if applicable. Given the decades’ long use of the 
ICG as an approved agent, and the dose range used, which is often less than what is used 
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All applicable AEs as described above, including local and systemic reactions not meeting the 
criteria for SAEs will be captured on the appropriate case report form (CRF). Information to be 
collected includes event description, time of onset, clinician’s assessment of severity, 
relationship to study product (assessed only by those with the training and authority to make a 
diagnosis), and time of resolution/stabilization of the event. All recordable AEs occurring while 
on study must be documented appropriately.  All reportable AEs will be followed to adequate 
resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
8.5.5 Adverse Event Reporting  
Adverse event monitoring will be performed from the time the consent is obtained until the 
patient is off treatment (4-6 hours post biopsy procedure). Adverse event reporting will be 
performed during this timeframe. Any reported Grade 3 and above adverse events that are 
possibly, probably, and definitely related to the research procedure, will be followed until 
resolution or stabilization. 
8.5.6 Serious Adverse Event Reporting  
The study investigator shall complete an Unanticipated Adverse Device Effect Form and submit 
to the study sponsor and to the reviewing Institutional Review Board (IRB) as soon as possible, 
but in no event later than 7 calendar days after the investigator first learns of the effect. The 
study sponsor is responsible for conducting an evaluation of an unanticipated adverse device 
effect and shall report the results of such evaluation to the Food and Drug Administration (FDA) 
and to all reviewing IRBs and participating investigators within 10 working days after the 
sponsor first receives notice of the effect. Thereafter, the sponsor shall submit such additional 
reports concerning the effect as FDA requests. 
8.6 
UNANTICIPATED PROBLEMS 
8.6.1 Definition of Unanticipated Problems (UP) 
Any incident, experience, or outcome that meets all of 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 Institutional Review 
Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; and 
• Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and 
• Suggests that the research places participants or others (which many include research 
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greater risk of harm (including physical, psychological, economic, or social harm) than 
was previously known or expected. 
8.6.2  Unanticipated Problem Reporting  
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board 
(IRB) as per Policy 801. 
8.6.3 NIH Intramural IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NIH Intramural IRB. 
9 
STATISTICAL CONSIDERATIONS 
Three medical procedures will be conducted to diagnose liver lesions as benign/malignant 
(negative/positive): 
(1) Histopathology, using well-established standard procedures. 
(2) ICG fluorescent signal at the in-vivo site of biopsy using a combination of OMI and EM 
tracking.  
(3) ICG fluorescent signal ex vivo of the specimen obtained using a combination of OMI and 
EM tracking. 
For (2) and (3), malignancy determination will be based on TBR: TBR≥2 will produce a 
malignant diagnosis, and TBR<2 will produce a benign diagnosis. 
One main objective is to evaluate the agreement between (1) and (2), and between (1) and (3), 
with (1) considered as the gold standard. Since determinations are dichotomous, Cohen’s kappa 
will be used to assess agreement. 
9.1 
STATISTICAL HYPOTHESIS 
In terms of concordance, i.e. the proportion of EM+optical imaging and histopathology test 
results that match, the null hypothesis is that concordance ≤ 80%, and that the expected 
concordance (or concordance worth detecting) is 90%. Based on a positivity prevalence of 80%, 
i.e. 80% true malignant and 20% true benign, an 80% concordance can produce a kappa value 
ranging from 0 to 0.55; and a 90% concordance can produce a kappa value ranging from 0.62 to 
0.74. Based on these numbers, the null and alternative hypotheses for this study are set in terms 
of kappa as follows: 
• H0: kappa ≤ 0.4  
• H1: kappa > 0.4 
9.2 
SAMPLE SIZE DETERMINATION 
Based on the null kappa value k0=0.4, a kappa value k1=0.7 worth detecting, alpha (Type I error) 
equal to 0.05, 80% power, and a prevalence (true positive rate) of 80%, the resulting sample size 
from a one-sided test is 77. 

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Generally, kappa=0.4 is considered as fair/moderate agreement, and kappa=0.7 as substantial 
agreement.1 With an 80% true positive rate, kappa=0.4 corresponds to an 86% sensitivity and 
55% specificity, and kappa=0.7 corresponds to a 93% sensitivity and 80% specificity 
With data on 77 cases, it is worthwhile determining the optimal cut-off point for TBR to confirm 
whether TBR=2 is optimal. This can be done based on Youden’s index in receiver operating 
characteristic (ROC) analysis. 
9.3 
POPULATIONS FOR ANALYSES 
The target study population is patients with focal hepatic lesions with imaging. Features are 
suspicious for hepatocellular carcinoma or other liver neoplasm. Patients whose imaging findings 
suggest liver cancer (hepatocellular carcinoma) or other metastatic liver cancer for whom image-
guided percutaneous biopsy is planned. 
Accurate diagnosis of HCC and other liver malignancies is important in these patients since these 
patients may be candidates for liver transplantation or bridging transarterial chemoembolization 
or palliative focal ablation with systemic chemotherapy. Eligible patients demonstrate a focal 
hepatic lesion for which percutaneous image-guided biopsy is planned. The subject selection 
based on the race, ethnicity, sex and age has been considered based on the reported statistics of 
the HCC population. 
Recruitment of the participants will be through the referring clinicians at NIH. The accrual will 
extend to different race and ethnicities and both sex. Since the safety of EM methods and ICG is 
not reported in children, we will enroll participants > 18 years to avoid any possible adverse 
events in children 
9.3.1 Evaluable for toxicity  
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study. The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 
4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40).  
Incidence of 1% to 10%: diaphoresis, headache, pruritus, skin discoloration at the intravenous 
site. In subjects allergic to iodine, anaphylaxis reactions have occurred. The total dose 
administered will not exceed 40 mg per day. Of note, doses of up to 5 mg/kg have been 
administered as routine in Japan without adverse reactions. All adverse events experienced by 
participants will be collected from the time ICG is administered, through the study, and until the 
patient is off treatment. 
9.3.2 Evaluable for objective response  
The metric to evaluate where ICG localizes is TBRs and will be calculated by dividing the mean 
fluorescence intensity within the lesion by the mean fluorescence intensity within the adjacent 
 
1 Landis, J.R. & Koch, G.G. (1977). "The measurement of observer agreement for categorical data". 
Biometrics, 33 (1): 159–174. 

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liver parenchyma with in vivo and ex vivo samples. All specimens will be sent to NCI 
Laboratory of Pathology for diagnosis and research as defined by the patient’s primary protocol. 
9.3.3 Evaluable Non-Target Disease Response 
The intervention (guided biopsy) is performed at a single point, and will not alter disease burden. 
Thus, no patients will be classified as evaluable for non-target disease response.  
9.4 
STATISTICAL ANALYSES 
9.4.1 General Approach 
All patients who enroll in the study and complete the biopsy procedure will be evaluable for an 
objective response. Response is defined as localization of the target lesion and lends information 
regarding the feasibility of using the optical imaging device and EM tracking tool to guide liver 
biopsies. Fluorescence microscopy showing a clear delineation between malignant and normal 
tissue is also an objective response. 
The main intervention of the study is the ability to improve biopsy guidance. Please note that 
standard pathology results of the hepatic biopsies will be part of routine clinical care and will be 
the responsibility of the NIH primary team to review and to provide appropriate follow up and 
clinical management. The tracking and fluorescence results will not be shared with participants, 
as these results are unlikely to have direct effect clinically on patient care. In addition to routine 
histological analysis, additional analysis for specific molecular markers will be performed in the 
NCI Laboratory of Pathology, as clinically indicated. However, all study-specific results are 
bench studies and will not be used in patient care decision making, thus we do not anticipate 
obtaining any incidental findings since the intervention is technical in nature regarding the 
biopsy procedure itself.  
The main intervention of the study is the ability to improve biopsy guidance. Please note that 
standard pathology results of the hepatic biopsies will be part of routine clinical care and will be 
the responsibility of the NIH primary team to review and to provide appropriate follow up and 
clinical management. The tracking and fluorescence results will not be shared with participants, 
as these results are unlikely to have direct effect clinically on patient care. In addition to routine 
histological analysis, additional analysis for specific molecular markers will be performed in the 
NCI Laboratory Pathology, as clinically indicated. However, all study-specific results are bench 
studies and will not be used in patient care decision making, thus we do not anticipate obtaining 
any incidental findings since the intervention is technical in nature regarding the biopsy 
procedure itself. 
9.4.2 Analysis of the Primary Endpoints 
Completion of primary end and secondary endpoint analysis – The majority of data analysis can 
be performed on a per patient basis, thus analysis will be ongoing along with enrollment and 
does not need to wait until all subjects are enrolled to begin analysis.  
Final Report - All primary and secondary data analysis along with completion of the final study 
report will be completed within one month of data collection completion. We anticipate that 
100% of the projected enrollment will be completed by 4 years. Data collection (including 
correlative pathology) will be completed by 4 years. We anticipate global primary and secondary 
analysis along with the final report will be completed in 5 years. 

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9.4.3 Analysis of the Secondary Endpoints 
Concordance will be evaluated by confirming that all 4 of the following exist:  
1. Diagnostic tissue specimen is obtained for conventional pathology analysis, defined by 
pathologist report of diagnostic liver lesion tissue  
2. Imaging confirms that biopsy was taken from region desired  
3. An ICG optical signal is obtained by ICG apparatus  
4. EM tracking fusion image displays a location of biopsy. 
9.4.4 Safety Analyses 
As this is a feasibility study, only grade 3 and above adverse events that are possibly, probably 
and definitely related to the research procedure (i.e., use of biopsy assisting devices and ICG 
administration) will be recorded, and reported if applicable. Given the decades’ long use of the 
ICG as an approved agent, and the dose range used, which is often less than what is used 
clinically, we do not anticipate any toxicity.  
To comply with the abbreviated IDE regulations 21 CFR 812.2(b), records concerning adverse 
device effects should include anticipated and unanticipated events and complaints.  
Exceptions to adverse event data collection: 
• Expected events related to the clinical biopsy procedure (for example, but incomplete 
list)  
o Pain or bruising at the biopsy site. 
o Increased redness, swelling or bloody or serosanguinous drainage at the biopsy 
site. 
o Events directly related to local anesthetic administration such as nausea, 
dizziness, numbness or tingling. 
o Self-resolved transient hypotension or bradycardia.  
o Diaphoresis 
o Headache 
o Pruritus 
o Skin discoloration at the intravenous site. 
9.4.5 Baseline Descriptive Statistics 
The following will be collected while the patient remains on the study: 
• Demographics – age, ethnicity, sex, diagnosis 
• Imaging pertaining to liver disease diagnosis 
• Lesion information– included but not limited to size, number, location 
• Intra-procedural details (included but not limited to) 
o start and end time of the procedure 
o intraprocedural scans used (ex: Ultrasound, Cone beam CT or Fluoroscopy) 
o ICG fluorescence signal (Target to Background/ TBR measurement)  
• Post-procedural information  
o Specimen information – cores or fine needle aspirations, location specimen was 
collected, histopathology and diagnosis, H&E and immunohistochemistry (IHC) 
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The following device information will be collected and kept in the study’s regulatory binder 
while the study is ongoing:  
• Basic device information – labeling of the device should include the name and place of 
business of the manufacturer (in accordance with 21 CFR 801.1), the quantity of the 
contents if applicable and the following statement, “CAUTION – Investigational device. 
Limited by Federal law to investigational use.” The label should also describe all relevant 
contraindications, hazards, adverse effects, interfering substances or devices, warnings, 
and precautions.  
• Any information regarding a return, repair or disposition of the device or any units of the 
device. 
10 REGULATORY AND OPERATIONAL CONSIDERATIONS 
10.1 INFORMED CONSENT PROCESS 
10.1.1 Consent Procedures and Documentation 
All patients who are being considered for this trial will undergo informed consent prior to being 
enrolled in the trial, as well as evaluation by multi-disciplinary review. A forthright and detailed 
discussion of the biopsy options available to the patient and family members, shall occur at the 
time of consent. The experimental nature of the treatment, its theoretical advantages and 
disadvantages, and an overview of the procedure and anticipated convalescence are presented. 
The Informed Consent document is given to the patient and they are asked to review it, make 
notes and follow-up with a phone call to the physician or nurse investigator to have any 
additional questions answered prior to considering treatment on protocol. 
When the patient is admitted to the Clinical Center for treatment, the PI, or AI will perform the 
consenting process. 
The Investigator will obtain written informed consent from each patient. The informed consent 
form will contain all the Essential Elements of Informed Consent set forth in 21CFR, Part 50, the 
ICH Guideline for Good Clinical Practice, and the terms of the Declaration of Helsinki. Copies 
of the signed document should be given to the patient and filed in the Investigator’s study file, as 
well as the patient’s medical record if in conformance with Standard Operating Procedures. 
10.1.2 Participation of Subjects Who Are/Become Decisionally Impaired 
Adults unable to give consent are excluded from enrolling in the protocol as outlined in section 
5.3.3. 
10.1.3 Telephone Consent 
The informed consent document will be sent to the subject. An explanation of the study will be 
provided over the telephone after the subject has had the opportunity to read the consent form. 
The subject will sign and date the informed consent.  
The original informed consent document will be sent back to the consenting investigator who 
will sign and date the consent form with the date the consent was returned. 
A fully executed copy will be returned via mail for the subject’s records. 

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The informed consent process will be documented on a progress note by the consenting 
investigator. 
The investigator will confirm that, when required, written legally effective consent has been 
obtained prior to initiating any study interventions. 
10.2 STUDY DISCONTINUATION AND CLOSURE 
Below are the criteria to be met before the study could be discontinued: 
• Completion of data collection time – The intervention, a hepatic biopsy guided with 
novel combination of devices, is concluded the same day as the scheduled biopsy. All 
correlative pathologic assessment of biopsy specimens will occur within 1 ± 2 months of 
the completed biopsy. We expect to complete all data collection for a single subject 
within 1 ± 2 months of the study procedure.  
• Completion of primary end and secondary endpoint analysis – The majority of data 
analysis can be performed on a per patient basis, thus analysis will be ongoing along with 
enrollment and does not need to wait until all subjects are enrolled to begin analysis.  
• Final Report - All primary and secondary data analysis along with completion of the final 
study report will be completed within one month of data collection completion. We 
anticipate that 100% of the projected enrollment will be completed by 4 years. Data 
collection (including correlative pathology) will be completed by 4 years. We anticipate 
global primary and secondary analysis along with the final report will be completed in 5 
years. 
• Plan of action if study fails to meet accrual milestones - If the study fails to meet accrual 
milestones, we will review our recruitment strategy and consider changing the status to  
“Open – Recruiting” to improve accrual. 
10.3 CONFIDENTIALITY AND PRIVACY 
All the collected samples and data will be coded to maximize the participant’s privacy. The 
records will be kept in locked drawers or password-protected electronic devices to ensure the 
safety of subjects. 
10.4 FUTURE USE OF STORED SPECIMENS AND DATA 
Stored specimens are not anticipated with this protocol. 
10.5 SAFETY OVERSIGHT 
Safety oversight will be performed through the NIH Clinical Center’s Quality Assurance 
Program. 
10.6 CLINICAL MONITORING 
The clinical research team will meet on a regular basis when patients are actively receiving an 
intervention on the trial to discuss each patient.  
All data will be collected in a timely manner and reviewed by the principal investigator or a lead 
associate investigator. Adverse events will be reported as required above. Any safety concerns, 
new information that might affect either the ethical and or scientific conduct of the trial, or 
protocol deviations will be immediately reported to the IRB using iRIS.  

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Accrual and safety data will be monitored by the principal investigator, who will provide 
oversight to the conduct of this study. The PI will continuously evaluate implementation of the 
protocol for any unusual or unpredicted complications that occur and will review the data for 
accuracy and completeness.  
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff. 
10.7 QUALITY ASSURANCE AND QUALITY CONTROL 
The NIH Clinical Center’s Quality Assurance Program will conduct study monitoring at least 
annually or more frequently as required per 21 CFR 812.46. Patient consent documents, primary 
outcome and safety laboratory results and diagnostic test results will be monitored for accuracy, 
correct dating, and agreement between case report forms and source documents. All regulatory 
reports, reviews and amendments, adverse events and problem reports related to study, along 
with investigator credentials, training records, and the delegation of responsibility log will also 
be reviewed during monitoring visits. Monitoring visits will also include evaluation of conduct 
and compliance of all sub-investigators. Any major findings will be summarized in writing and 
reported to the study PI who will be responsible for submitting the monitoring report to the IRB. 
10.8 DATA HANDLING AND RECORD KEEPING 
10.8.1 Data Collection and Management Responsibilities 
Completion of data collection time – The intervention, a hepatic biopsy guided with novel 
combination of devices, is concluded the same day as the scheduled biopsy. All correlative 
pathologic assessment of biopsy specimens will occur within 1 ± 2 months of the completed 
biopsy. We expect to complete all data collection for a single subject within 1 ± 2 months of the 
study procedure.  
Completion of primary end and secondary endpoint analysis – The majority of data analysis can 
be performed on a per patient basis, thus analysis will be ongoing along with enrollment and 
does not need to wait until all subjects are enrolled to begin analysis.  
Accrual data will be monitored by the Principal Investigator, who will provide oversight to the 
conduct of this study. As this is a feasibility study, safety data collection is minimal and will be 
concentrate on ICD dye administration side effects, if any. The PI will continuously evaluate 
implementation of the protocol for any unusual or unpredicted complications that occur and will 
review the data for accuracy and completeness. 
10.8.2 Study Records Retention 
Study record retention will conform to regulatory requirements as per 21CFR 312.57. The 
investigator must maintain the required records for a period of two years after the date the 
investigation is completed or terminated or the records are no longer required to support a PMA 
or PDP, whichever date is later. 

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10.9 PROTOCOL DEVIATIONS 
It is the responsibility of the investigator to use continuous vigilance to identify and report 
deviations to the NIH Institutional Review Board as per Policy 801. All deviations must be 
addressed in study source documents, reported to Clinical Center Program Official. The 
investigator is responsible for knowing and adhering to the reviewing IRB requirements. 
10.9.1 NIH Definition of Protocol Deviation 
A protocol deviation is any changed, divergence, or departure from the IRB-approved research 
protocol.  
• Major deviations: Deviations from the IRB approved protocol that have, or may have the 
potential to, negatively impact the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study. 
• Minor deviations: Deviations that do not have the potential to negatively impact the 
rights, safety or welfare of subjects or others, or the scientific integrity or validity of the 
study. 
10.9.2 Unanticipated Problem 
Any incident, experience, or outcome that: 
• Is unexpected in terms of nature, severity, or frequency in relation to  
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; other study documents, and 
(b) the characteristics of the subject population being studied; AND 
• Is related or possibly related to participation in the research; AND 
• Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
10.10 PUBLICATION AND DATA SHARING POLICY 
10.10.1Human Data Sharing Plan 
De-identified human data generated in this research for future research may be shared with the 
following: 
• In an NIH-funded or approved public repository clinicaltrials.gov 
• In BTRIS 
• In publication and/ or public presentations at the time of publication or shortly thereafter  
De-identified data may be shared with approved outside collaborators under appropriate 
agreements and upon IRB approval. 
10.11 COLLABORATIVE AGREEMENTS 
10.11.1Cooperative Research and Development Agreement (number pending) 
The study devices, PercuNav and Uronav, are provided by the company, Philips under a 
Collaborative Agreement [Cooperative Research and Development Agreement (CRADA)]. 

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10.11.2Material Transfer Agreement (number pending) 
Coded (linked) data will be shared with Umar Mahmood, MD at Massachusetts General Hospital 
under MTA. Decryption code to re-link data will not be shared with Dr. Mahmood.  
10.12 CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed 
and managed. Furthermore, persons who have a perceived conflict of interest will be required to 
have such conflicts managed in a way that is appropriate to their participation in the design and 
conduct of this trial. The study leadership in conjunction with the NIH Clinical Center has 
established policies and procedures for all study group members to disclose all conflicts of 
interest and will establish a mechanism for the management of all reported dualities of interest. 
 
 

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11 ABBREVIATIONS 
3D 
Three Dimentional 
AE 
Adverse Event 
ADE 
Adverse Device Effect 
ASADE 
Anticipated Serious Adverse Device Effect 
CC 
Clinical Center 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
CRC 
Colorectal Cancer 
CRF 
Case Report Form 
DHHS 
Department of Health and Human Services 
DICOM 
Digital Imaging and Communications in Medicine 
EM Tracking Electromagnetic Tracking 
FDA 
Food and Drug Administration 
FDG 
Fludeoxyglucose  
GMP 
Good Manufacturing Practices 
HCC 
Hepatocellular Cancer 
HD 
High Definition 
HIPAA 
Health Insurance Portability and Accountability Act  
ICG 
Indocyanine Green 
ICH 
International Conference on Harmonisation  
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IR 
Interventional Radiology 
IRB 
Institutional Review Board 
IRF 
Infrared Fluorescence 
IV 
Intravenous  
MR 
Magnetic Resonance 
NCI 
National Cancer Institute 
NIH  
National Institutes of Health 
IC 
Institute or Center 
NIR 
Near Infrared 
OHRP 
Office for Human Research Protections 
OMI 
Optical Molecular Imaging 
OSHA 
Occupational Health and Safety Administration 
PET 
Positron Emission Tomography 
PI 
Principal Investigator 
POC 
Point of Care 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SADE 
Serious Adverse Device Effect 

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SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
TBR 
Tumor to Background Ratio 
UP 
Unanticipated Problem 

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