Intravital Microscopy (IVM) in Patients With Peritoneal Carcinomatosis (PC)
Phase: NA
Conditions: Peritoneal Carcinomatosis
Sponsor: Mayo Clinic

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TITLE: 
 
Intravital Microscopy (IVM) in patients with peritoneal carcinomatosis 
Study Number:  
 
Initial Date:  
Principal Investigator: Emmanuel Gabriel, MD, PhD 
Mayo Clinic Florida 
4500 San Pablo Road 
Jacksonville, FL 32224 
904-953-3722 
Gabriel.Emmanuel@mayo.edu 
Confidentiality Statement 
Any and all information presented in this document shall be treated as confidential and shall 
remain the exclusive property of the party(ies) mentioned above. The use of such confidential 
information must be restricted to the recipient for the agreed purpose and must not be disclosed, 
published, or otherwise communicated to any unauthorized persons, for any reason, in any form 
whatsoever without the prior written consent of the party(ies) above. 

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SYNOPSIS: Intravital Microscopy (IVM) in patients with peritoneal carcinomatosis 
PI: Emmanuel Gabriel MD, PhD        
Co-PI: Sanjay P. Bagaria, MD 
 
Michael B. Wallace, MD 
 
Keith L. Knutson, PhD 
Statistician: Zhuo Li, MS 
 
Study Center: Mayo Clinic Florida 
 
Concept and Rationale: Intravital microscopy (IVM) allows real-time, direct visualization of 
microscopic blood vessels and calculation of blood flow. Our group has used IVM in mouse 
models to characterize aberrant blood vessel structure surrounding tumor implants and the 
associated impaired lymphocyte trafficking along these tortuous vessels.[1] These preclinical 
studies have been translated to human intravital microscopy (HIVM) in a pilot study that 
analyzed the tumor-associated microvasculature at primary melanomas.[2] The feasibility of 
HIVM was 90% at the primary tumor. As an extension of this completed trial, Dr. Gabriel had 
implemented an on-going clinical trial (NCT02857374, Intravital Microscopy in Identifying 
Tumor Vessels in Patients with Stage IB-IIIC Melanoma Undergoing Sentinel Lymph Node 
Biopsy) that investigates the role of IVM in analyzing the vessels associated with sentinel lymph 
nodes. This proposed trial at Mayo Clinic Florida will further determine the usefulness of IVM in 
human subjects, now applying it to patients with deeper sites of tumor, i.e. peritoneal 
carcinomatosis. 
 
In these clinical trials, fluorescein was used to augment visualization of tumor-associated vessels. 
It is a commonly used and safe intravital dye. Similarly, indocyanine green (ICG) is a commonly 
used dye and will be also used in this study. Due to its heavier molecular weight compared to 
fluorescein, ICG allows additional analysis of vessel permeability. Although it is also considered 
a safe intravital dye, small studies report a higher risk of adverse events (AEs) associated with its 
use in patients with hepatic or renal dysfunction. 
 
In this clinical trial proposal, the following aims will be addressed: 
 
1. Determine the feasibility of HIVM in characterizing the tumor-associated 
microvasculature in patients with peritoneal carcinomatosis. 
2. To correlate these findings to the HIVM observations at the primary site, extent of 
carcinomatosis, tumor response, and survival outcomes.  
 
 
Objectives: 
 
Primary objective(s): To determine the feasibility of performing HIVM in patients with 
peritoneal carcinomatosis during standard course of treatment (cytoreductive surgery with 

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hyperthermic intraperitoneal chemotherapy, or CRS-HIPEC). A successful intravital microscopic 
observation will include the ability to complete each of the following: 
 
1. Identify and measure vessels associated with peritoneal tumor implants 
2. Determine vessel density per 10x field 
3. Visualize vital dyes within the vessels [fluorescein and indocyanine green (ICG)] 
4. Calculate the blood flow velocity of the vessels and tissue penetration of fluorescein 
and ICG as markers of vessel permeability.  
 
 
Secondary objective(s):  
1. Compare the microscopic observation of the tumor-associated vessels with normal 
tissue (peritoneal surface) in each individual subject. 
2. Correlate the microscopic observations of the tumor-associated vessels with pathologic 
grade of tumor implants.  
3. To correlate the microscopic observation of the microvasculature with tumor-specific 
and overall survival. 
 
 
Study Design: This is a non-randomized, single center, study of HIVM observation in subjects 
with peritoneal carcinomatosis undergoing CRS-HIPEC. 
 
All subjects will meet the inclusion and exclusion criteria prior to participation in this study. 
Standard evaluation by the Preoperative Anesthesia clinic at Mayo Clinic Florida may include 
additional testing such as EKG, chest x-ray, and/or pulmonary function tests. 
 
The study will be performed in two parts. Part 1 will enroll 10 patients with an expected 
feasibility rate of 70%. Whereas the feasibility rate in primary melanoma was 90%, the expected 
feasibility rate for the peritoneal surface may be lower as the site is in an anatomically deeper 
location. If Part 1 is not met (i.e. in three or more patients), the primary objective would not be 
considered technically feasible, and then the study will be stopped. If Part 1 is fulfilled, then the 
study will continue with an additional 20 patients enrolled (total of 30 patients). Accrual is 
expected to take 2.5 years. 
 
 
Inclusion criteria: 
1. Age ≥ 18 years of age. 
2. ECOG Performance Status of ≤ 2. 
3. Measurable tumor on the peritoneal surface by direct visualization requiring surgical 
resection in the OR. 
4. Peritoneal carcinomatosis that meets indications for CRS-HIPEC. Sites of origin for tumor 
include appendix, colon, stomach, and primary peritoneal surface malignancies such as 
peritoneal mesothelioma. 

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5. Subject must understand the investigational nature of this study and sign an Independent 
Ethics Committee/Institutional Review Board approved written informed consent. 
6. Subject must have a skin prick test pre-operatively (at the time of the preoperative visit and 
after signed informed consent for entry into this clinical trial is given) to determine any 
sensitivity to fluorescein. 
 
Exclusion criteria: 
1. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations. 
2. Renal dysfunction as defined as a GFR < 45. 
3. Liver dysfunction as defined by Child-Pugh score > 5, or LFT’s 1.5x above normal range. 
4. Any known allergy or prior reaction to fluorescein or ICG or a positive skin prick test to 
fluorescein. 
5. Pregnant or nursing female subjects, determined preoperatively with a urine pregnancy test. 
6. Unwilling or unable to follow protocol requirements. 
7. Any condition which in the Investigators’ opinion deems the patient unsuitable (e.g., 
abnormal EKG). 
8. Any condition that excludes CRS-HIPEC as the standard of care (e.g. high disease burden 
where alternative treatments like systemic chemotherapy would be preferred). 
 
 
Primary and Secondary Endpoints/Criteria for Evaluation: 
 
Primary endpoint: A patient will be deemed a success if each of the following parameters were 
measured: 
1. Identify tumor implant associated vessels and measure vessel diameters 
2. Determine vessel density per 10x field 
3. Visualize vital dyes within the tumor-associated vessels (fluorescein and green ICG) 
4. Calculate the blood flow velocity of the tumor-associated vessels and tissue 
penetration of fluorescein and ICG as markers of vessel permeability.  
 
Secondary endpoints: 
1. Post-operative comparison of the microvasculature of peritoneal carcinomatosis with 
normal tissue (peritoneum) in each individual subject using vessel diameters, 
vessel density, detection of intravital dye and flow rates.  
2.  Post-operative correlation of the microvasculature with pathologic features of the 
tumor implants (i.e. tumor grade) at the time of the final pathology report (5-7 
days after surgery). 
3. Post-operative correlation of the microscopic observation of the tumor 
microvasculature tumor-specific and overall survival. 
 
 

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Subject Name: ________________________________________________________________ 
Medical Record No.: ___________________________________________________________ 
Title: Intravital Microscopy (IVM) in patients with peritoneal carcinomatosis 
INCLUSION CRITERIA 
Y 
N 
N/A 
All answers must be “YES or “N/A” for subject enrollment. 
Date 
 
 
 
1. Age ≥ 18 years of age. 
 
 
 
 
2. Have an ECOG Performance Status of ≤ 2. Refer to Appendix C. 
 
 
 
 
3. Have measurable disease in the peritoneum by direct visualization (visible 
lesion typically > 0.5 cm in maximal diameter). 
 
 
 
 
4.     Carcinomatosis that meets indications for CRS-HIPEC. 
 
 
 
 
5.    Subject must understand the investigational nature of this study and sign an 
Independent Ethics Committee/Institutional Review Board approved 
written informed consent form prior to receiving any study related 
procedure. 
 
 
 
 
6.     A negative skin-prick test to fluorescein. 
 
 
EXCLUSION CRITERIA 
Y 
N 
N/A 
All answers must be “NO” or “N/A” for subject enrollment 
Date 
 
 
 
1. Uncontrolled intercurrent illness including, but not limited to, ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 
pectoris, cardiac arrhythmia, or psychiatric illness/social situations. 
 
 
 
 
2. Renal dysfunction as defined as a GFR < 45. 
 
 
 
 
3. Liver dysfunction as defined by Child-Pugh score > 5, or LFT’s 1.5x above 
normal range. 
 
 
 
 
4. Any known allergy or prior reaction to fluorescein or ICG or a positive skin 
prick test to fluorescein. 
 
 
 
 
5. Pregnant or nursing female subjects, determined preoperatively with a urine 
pregnancy test. 
 
 
 
 
6. Unwilling or unable to follow protocol requirements. 
 
 
 
 
7. Any condition which in the Investigators’ opinion deems the patient 
unsuitable (e.g., abnormal EKG). 
 
 
 
 
8. Any condition that excludes CRS-HIPEC as the standard of care for the 
patient. 
 
 
Study participant meets all entry criteria: 
 
 
Yes 
 
No 
Investigator Signature: ___________________________________ Date: ___________

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1 
BACKGROUND 
Intravital microscopy (IVM) is the microscopic observation of living tissue in real time. IVM has 
been successfully applied to the investigation of the tumor microvasculature of human primary 
melanoma.[3] It has been shown that tumor vessels lack the sequential hierarchy of normal 
vessels such that arterioles, capillaries, and venules typically cannot be discriminated within 
tumor tissues.[4, 5] This disorganization of aberrant tumor vessels was demonstrated in a 
previous feasibility clinical trial.[2] These tumor-associated vessels were characterized by 
irregular diameters, aberrant branching patterns, abnormal blood flow rates, and anastomotic 
structures. More specifically, the human tumor-associated vessel diameters observed in real time 
in vivo were larger than those predicted either by in vivo mouse models or by human 
immunohistochemistry (IHC) on tumor specimens analyzed ex vivo.[3] These characteristics 
could have profound influence on the delivery of agents (i.e., chemotherapy or cellular 
immunotherapy) to the tumor microenvironment.[6]  
 
In order to obtain this data, human intravital microscopy (HIVM) was used first in melanoma 
primary tumors. Intravital microscopy (IVM) has also been used to directly examine the 
hemodynamic properties of tumor microvessels in preclinical mouse models.[1, 7-9] These 
studies were primarily based on the analysis of blood vessels and leukocytes labeled in situ with 
fluorescent tracking dyes (e.g., fluorescein or ICG). Recent mouse studies in B16 murine 
melanoma have used IVM to demonstrate that the abnormal, tortuous vascular structure in 
tumors represents a bottleneck to adoptive cell transfer immunotherapy because of poor 
trafficking of cytotoxic effector T cells at this site.[1] Others have used different forms of IVM 
to observe lymphatics or metastatic disease in real time in mouse models.[10, 11] There has also 
been reported real time imaging of human tumor and lymphatics using different techniques such 
as multiphoton imaging, high resolution ultrasound, or optical coherence tomography.[12, 13] To 
date, there have been no studies of IVM in the setting of human peritoneal carcinomatosis. 
 
The detection of blood flow parameters (including vessel diameter, flow rates, vessel density, 
and fluorescent markers of tissue diffusion) in tumor implants of patients with peritoneal 
carcinomatosis may have utility in characterizing the locoregional spread of disease and in 
predicting drug uptake and clinical response in these tumor implants. The overall goal will be to 
characterize, for the first time, vasculature associated with peritoneal carcinomatosis in real-time. 
It is anticipated that this novel approach has the potential to generate a more complete picture of 
the tumor-associated locoregional vasculature. The investigation of tumor-associated vessels in 
real time may lead to a better understanding of factors influencing systemic drug efficacy. 
 
1.1 
STUDY DISEASE – PERITONEAL CARCINOMATOSIS 
Peritoneal carcinomatosis can occur in a variety of malignancies, including those of 
gastrointestinal (GI) origin, the genitourinary (GU) tract, and primary peritoneal mesothelioma. 
It is estimated to affect up to 13-17 % of patients with GI malignancies.[14, 15] Current 
treatments include systemic chemotherapy and regional therapies, namely as cytoreductive 

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surgery with hyperthermic peritoneal chemotherapy (CRS-HIPEC). This treatment modality can 
be associated with significant response and typically requires the expertise found in high-volume 
performing institutions (including Mayo Clinic Florida) in order to provide the infrastructure 
essential to performing this procedure. Significant oncologic benefits of CRS-HIPEC have been 
reported for certain malignancies.[16-19] The categorization of CRS-HIPEC has recently 
changed by the National Comprehensive Cancer Network (NCCN) from being an investigational 
approach to being a considered approach for the appropriate patient.[20] Despite these advances, 
current treatments for peritoneal carcinomatosis still generate heterogeneous responses, and 
many patients will not benefit from CRS-HIPEC or systemic chemotherapy, or may experience 
limited responses. As such, additional therapies for carcinomatosis are strongly needed. The 
relevance of this clinical trial in addressing this current need is the investigation of tumor vessels 
associated with carcinomatosis as a prognostic and ultimately therapeutic target for treatment. 
 
1.2 
DIAGNOSTIC INTERVENTION - FLUORESCEIN 
Drug Information - Fluorescein is delivered intravenously during the course of this observation. 
Fluorescein is an FDA approved drug for visual angiography. The package insert is shown in 
Appendix A. A single dose consists of a 5 mL vial of 25% fluorescein and costs $3.90.  
 
Fluorescein has a very rare incidence of anaphylactic reaction (1 in 222,000), but a skin-prick 
test may allow these patients to be recognized.[21] Therefore, any reported sensitivity to 
fluorescein will be considered an exclusion criteria as well as a positive skin-prick test performed 
during the routine preoperative visit (at least 1 week prior to surgery). The skin-prick test will be 
performed at the time of the preoperative visit if the patient decides to enter the clinical trial and 
signs the informed consent. Patients that are not eligible for the study or do not give consent to 
participate in the study will not undergo a skin-prick test. 
  
The skin-prick test will be performed by the Principal Investigator using a sterile Duotip® lancet 
(Lincoln Medical, USA) in 3 separate areas of skin on the forearm. The skin-pricks will include 
4 microliters of 10% fluorescein, histamine chloride (10 mg/ml) [positive control], and 50% 
saline solution [negative control] administered in areas of skin prepped with an alcohol pad. 
After 30 minutes, the appearance of a wheal greater than 3 mm in diameter is considered a 
positive test result in the context of the appropriate positive and negative control. If a positive 
test is noted, the patient will no longer be eligible for this study. For comfort and relief of any 
skin irritation/pruritis, the patient will be offered an anti-histamine (fexofenadine [Allegra®] 
30 mg po x 1) following the skin-prick test if necessary. 
 
The package insert for fluorescein (which is a commonly used agent in the OR and readily 
available to the anesthesiologist in their PYXIS system and does not require delivery from 
pharmacy or an order to pharmacy) is attached in the Appendix section. 
 
The fluorescein will be provided by the study.  It will be ordered and sent to surgery for the 
patient at the time of the planned microscopic observation. 
 

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1.2.1 PRECLINICAL STUDIES WITH FLUORESCEIN 
Murine tumors have been investigated with the use of intravital microscopy in an attempt to 
evaluate blood vessel characteristics, blood flow patterns, and interactions with immune cell 
subsets.[1, 7, 9] Fluorescein conjugated to Dextran has been employed for several intravital 
microscopy studies[1, 22] and allows for a more precise assessment of the tumor vasculature[1, 
7]. 
 
Preclinical laboratory studies have established that fluorescein can be used to image the 
microvasculature in B16 mouse melanoma tumors in the similar doses used clinically. 
Fluorescein is not expected to have a direct intervention on tumor biology. The ability to study 
tumor microvasculature by using IVM does not require a window chamber, and human IVM 
techniques have provided highly reproducible and consistent measurements. There have been 
several published animal studies which have demonstrated IVM of the lymphatic system in 
addition to blood vessels.[10, 23] 
 
1.2.2 CLINICAL STUDIES WITH FLUORESCEIN 
This protocol intends to apply the commonly used intraoperative fluorescent agent, fluorescein, 
to further characterize blood vessels associated with peritoneal carcinomatosis during the course 
of CRS-HIPEC. It is anticipated that fluorescein will be readily detectable in the microscopic 
field of observation following intravenous fluorescein injection. This is expected to be similar to 
a previous trial NCT01886235, which utilized fluorescein to study the microvasculature of 
melanoma primary tumors, and an on-going trial that uses fluorescein to visualize vessels 
associated with sentinel lymph nodes (NCT02857374). 
 
Fluorescein is not expected to have a direct intervention on tumor biology. Fluorescein is 
routinely used during the course of surgery for a variety of indications (e.g. intraoperative 
assessment of bowel or organ perfusion, vessel angiography of the retina) and is deemed non-
toxic with no long term side effects. 
 
Clinically, fluorescein is most commonly used for ophthalmologic procedures including 
fluorescein angiography with intravenous injection. Fluorescein angiography is performed by 
injecting fluorescein sodium dye as a bolus into a peripheral vein. The normal adult dosage is 
500 mg, and is typically packaged in doses of 5 mL of 10% or 2 mL of 25%. For pediatric 
patients, the dose is adjusted to 35 mg per 10 pounds of body weight.[24] 
 
Upon entering the circulation, approximately 80% of the dye molecules bind to plasma proteins, 
which significantly reduces fluorescence because the free electrons that form this chemical bond 
are subsequently unavailable for excitation.[25] The remaining unbound or free fluorescein 
molecules fluoresce when excited with light of the appropriate wavelength. With a molecular 
weight of 376, fluorescein diffuses freely out of all capillaries except those of the central nervous 
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The dye is metabolized by the kidneys and is eliminated through the urine within 24 to 36 hours 
of administration. During this period of metabolism and elimination, fluorescein has the potential 
to interfere with clinical laboratory tests that use fluorescence as a diagnostic marker.[26, 27] To 
avoid any false readings, it may be prudent to schedule clinical lab tests either before the 
angiogram, or postpone testing for 1 or 2 days to allow sufficient elimination of the dye. Side 
effects of intravenous fluorescein include discoloration of the urine for 24 to 36 hours and a 
slight yellow skin discoloration that fades within a few hours. Nursing mothers should be 
cautioned that fluorescein is also excreted in human milk,[28] for this reason, nursing mothers 
will be excluded from this study. 
 
Use of fluorescein sodium may be contraindicated in patients with history of allergic 
hypersensitivity to fluorescein. Although generally considered safe for patients receiving 
dialysis, one manufacturer of fluorescein suggests using half the normal dose in dialyzed 
patients.[29] There are no known risks or adverse reactions associated with pregnancy, but most 
practitioners avoid performing fluorescein angiography in pregnant women, especially in their 
first trimester.[30-32] 
 
Historically, adverse reactions occur in 5% - 10% of patients and range from mild to severe.[33-
39] Anecdotal evidence suggests a lower incidence of reaction in recent years and the first large 
study conducted in over a decade seems to confirm that, reporting a frequency of adverse 
reaction of just over 1%.[40] Continued improvements in manufacturing processes and 
implementation of tighter pharmacopeial standards are credited with this reduction and may lead 
to lower rates of reaction in the future.[41] 
 
Transient nausea and occasional vomiting are the most common reactions and require no 
treatment. These mild reactions typically occur 30 – 60 seconds after injection and last for about 
1 to 2 minutes. Fortunately, they seldom compromise the diagnostic quality of the angiogram. 
The incidence of nausea and vomiting seems to be related to the volume of dye and rate of 
injection. A relatively slow rate of injection often reduces or eliminates this type of reaction but 
can adversely affect image quality and alter arm-to-retina circulation times. Premedication with 
promethazine hydrochloride or proclorperazine may prevent or lessen the severity of nausea and 
vomiting in patients with a history of previous reactions to fluorescein, but is rarely needed and 
one study noted a higher frequency of these reactions in patients that had been premedicated.[37] 
Some patients report a strong taste sensation or hypersalivation following injection of 
fluorescein. 
 
Moderate reactions occur less frequently, affecting less than 2% of patients that undergo 
angiography. Allergic reactions such as pruritus or urticaria can be treated with antihistamines, 
but any patient who experiences these symptoms should be observed carefully for the possible 
development of anaphylaxis. The advisability of performing angiograms in patients with a 
history of allergic reaction to fluorescein should be considered carefully, as allergic sensitization 
to the dye can increase with each subsequent use. Patients with previous history of mild allergic 
reaction to fluorescein can be pre-treated with an antihistamine, such as diphenhydramine, 

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30 - 40 minutes prior to any subsequent angiograms to limit allergic response, although this may 
not prevent serious reactions.[42] 
More severe reactions are rare, but include laryngeal edema, bronchospasm, anaphylaxis, tonic-
clonic seizure, myocardial infarction, and cardiac arrest.[43-47] The overall risk of death from 
fluorescein angiography has been reported as 1 in 222,000.[37] Although life-threatening 
reactions during angiography are rare, angiographic facilities and personnel should be properly 
equipped and prepared to manage serious reactions to the procedure. A resuscitative crash cart 
and appropriate agents to treat severe reactions should be readily available including epinephrine 
for intravenous or intramuscular use, soluble corticosteroids, aminophylline for intravenous use, 
oxygen, and airway instrumentation. It is generally recommended that a physician be present or 
available during angiographic procedures. 
 
Extravasation of fluorescein dye during the injection can be a serious complication of 
angiography. With a pH of 8 to 9.8, fluorescein infiltration can be quite painful. If fluorescein 
dye extravasates, cold compresses should be placed on the affected area for 5 to 10 minutes, and 
the patient should be reassessed until edema, pain, and redness resolve. Serious complications 
are more likely to occur when large amounts of dye extravasate. Sloughing of the skin, localized 
necrosis, subcutaneous granuloma, and toxic neuritis have been reported following extravasation 
of fluorescein.[48-50] To avoid these problems, continual observation of the injection site during 
the course of the injection and monitoring the patient for pain is recommended. Accidental 
arterial injections are rare, but can be quite painful. The dye remains concentrated and stains the 
effected extremity with little or no dye reaching the retinal vasculature. With proper technique, 
these complications of injection can usually be avoided. 
 
1.3 
RISKS AND/OR BENEFITS 
Risks – low risk of reaction to fluorescein as described above. Otherwise, no increased risk of 
surgical procedure anticipated. There are inherent risks to CRS. Studies have characterized the 
complications for which patients undergoing CRS-HIPEC are at risk, including anastomotic leak, 
ileus and obstruction, and neutropenia.[51-53] However, these risks are not expected to be 
increased due to the use of fluorescein. 
 
Benefits – depending upon collected findings and correlation with clinical outcome, IVM may 
offer prognostic information and contribute to staging classifications for peritoneal 
carcinomatosis with the intent of potentially guiding treatment decisions in the future. 
 
1.4  
DIAGNOSTIC INTERVENTION – INDOCYANINE GREEN (ICG) 
 
Drug Information – Indocyanine green (ICG or IC-Green) is the second intravenous (IV) dye to 
be administered in this clinical trial. The ICG will be administered after the fluorescein has been 
observed to washout from the tumor associated vasculature, which can be directly monitored 
during HIVM. Following this, the ICG will be injected. ICG injection after fluorescein injection 
is favored due to the characteristics of these dyes, with ICG being heavier and more albumin 

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bound than fluorescein. ICG would be expected to stay within the intravascular compartment 
longer than fluorescein with estimates of up to 20-30 minutes. It is therefore a more optimal 
utilization of time to administer the dyes in this order, because the reverse order (ICG prior to 
fluorescein) may result in an increased waiting time for the ICG to dissipate from the circulation. 
 
ICG was approved by the FDA in November 2007 for use in the study of cardiac output 
determination, hepatic function and liver blood flow measurement, and ophthalmic 
angiography.[54, 55] However, it has been used in many different clinical applications, including 
those listed above, for over 50 years. 
 
It is supplied by Akorn, Inc. under the brand name IC-GREEN®. In this study, a single unit for a 
given patient consists of a 25 mg vial to which 5 ml of aqueous solvent (sterile water) is added, 
thereby reconstituting the drug to 5 mg/ml. Further dilutions are achieved using sterile saline, 
making the final solution slightly hypo-osmolar. 
 
Administered dosages vary for different investigative purposes. Based on hepatic function 
studies, the dose to be administered is calculated for 0.5 mg/kg of body weight.[55] The total 
dose of ICG should not exceed 2 mg/kg. The dose used for the purposes of vessel evaluation 
have been reported to be much lower (e.g. 0.5 mg - 5 mg per patient).[56] Administration of 5 
mg is the recommended dose for cardiac dilution studies for adults.[55] Therefore, it is 
anticipated that the dose used for this evaluation is well below the maximum recommended dose. 
The dose of 5 mg ICG would be an appropriate amount for this study. 
 
The drug is supplied as a kit containing six 25 mg vials and six 10 ml Aqueous Solvent ampules.  
The cost of a single kit is $508.76. 
 
ICG is an iodide-containing, water soluble tricarbocyanine dye that fluoresces when exposed to 
infrared light (800 nm).[57] Compared to Fluorescein, the molecular weight of ICG is over 
double at 774.98 g/mol (Fluorescein molecular weight is 332.31 g/mol). The package insert and 
the Material Safety Data Sheet are shown in the Appendix B.  
 
Following IV injection, ICG is rapidly bound to plasma proteins of which albumin is the main 
carrier (95%).[55, 58] The drug undergoes no significant extrahepatic or enterohepatic 
circulation; it is taken up mainly by hepatic parenchymal cells and excreted into bile.[55, 59] It 
has a plasma half-life of approximately 3-4 minutes.[55, 58] Due to its high binding to albumin 
and significantly greater molecular weight than Fluorescein, ICG is expected to persist within the 
tissues for a longer period of time resulting from a slower washout. This characteristic would 
have several advantages to the study of HIVM in tumor associated vessels, including increased 
imaging times, more extensive characterization of blood vessel architecture, and more accurate 
calculation of blood flow velocities. 
 
The ICG will be provided by the study. It will be ordered and sent to the operating room during 
surgery at the time of the planned HIVM observation. The dose to be administered will be 
calculated prior to the surgery based on the patient’s most accurate and current weight. It will be 

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injected through the IV by a physician member of the surgical or anesthesia team. Surgeons and 
anesthesiologists have experience with the IV administration of ICG during other procedures, 
such as injection during plastic surgery reconstructive procedures to assess tissue viability. 
 
Similar to Fluorescein, ICG has a rare incidence of anaphylactic reaction (0.05 – 0.15%). It does 
contain an iodide component, which accounts for the allergy potential of ICG.  Therefore, 
patients with a known allergy to iodine or contrast dye will be excluded from the study. Patients 
who have received iodine based contrast computerized tomography (CT) scans with IV iodine 
contrast dyes would be unlikely to have an iodine allergy, particularly if multiple scans have 
been performed. During the staging of patients with peritoneal carcinomatosis, routine use of 
imaging is routinely recommended to investigate the degree of metastasis. Thus, it is extremely 
unlikely that a patient who has received IV iodine contrast dye during a CT scan will have an 
allergic reaction to ICG. 
 
 
1.4.1 PRECLINICAL STUDIES WITH ICG 
 
Similar to animal studies with Fluorescein, ICG has been used to study blood flow in several 
different models.[60-62] ICG has been successfully used in these studies to characterize the 
vasculature associated with the tumor microenvironment. 
 
 
1.4.2 CLINICAL STUDIES WITH ICG 
 
Similar to Fluorescein, ICG is not expected to have a direct intervention on tumor biology. ICG 
has been used during the course of surgery and other procedures for widespread indications and 
is FDA approved. In general, it is well tolerated, non-toxic, and has no long term side effects. 
 
Clinically, ICG is most commonly used in the assessment of liver function,[63-65] ophthalmic 
angiography,[66-68] and the analysis of cardiac function with increasingly novel applications in 
cardiac studies.[69, 70] It is typically administered by a member of the anesthesia team during 
cases or procedures in the operating suite under general anesthesia. In addition to these 
applications, ICG has been used in a variety of other studies in a safe manner. ICG has been used 
to map sentinel lymph nodes (SLN) in several malignancies,[71] including melanoma,[56, 72, 
73] breast,[74-76] and colon cancer.[77] These studies have not reported any major adverse 
reactions or toxicities resulting from exposure to ICG. While the doses of ICG used in these 
studies varies, the recommended dose per the manufacturer is calculated on the basis of 0.5 
mg/kg of body weight.[55]  
 
Other applications of ICG have been studied in the field of plastics surgery. ICG has been given 
in patients to determine the perfusion and viability of perforator flaps.[78, 79] Similar to the 
studies in malignancy and SLN, the fluorescent properties of ICG have been utilized and shown 
to be effective in other studies. ICG has also been used to identify and target esophageal 
varices.[80] Further, during neurovascular operations, ICG has been readily administered by 

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anesthesiologists during intraoperative videography of aneurysm clippings and carotid bypass 
procedures.[81, 82] 
 
As discussed in the previous section, the use of ICG is contraindicated in patients with a history 
of allergic hypersensitivity to iodine. There have been rare but reported cases of anaphylaxis and 
death due to allergic reactions during procedures using ICG.[25, 83, 84] Therefore, patients with 
a history iodine allergy will be excluded from our clinical trial. Other adverse reactions have 
been reported with the use of ICG in patients with allergic reactions.[85] 
 
A higher risk of adverse reactions has been reported in small studies in patients with liver 
disease, chronic kidney disease, and uremia.[84] Although the manufacturer does list these 
conditions as contraindications, patients with uremia or liver disease will be identified through a 
thorough history and physical, and screened with a basic metabolic panel (which includes a 
serum creatinine and blood urea nitrogen or BUN) and liver function tests (LFT’s). These 
patients will be informed of the potential increased risk for side effects and may be excluded 
from the study. 
 
The safety of ICG has been assessed in pregnancy, although there exist no strong level of 
evidence (i.e. randomized controlled trials) to determine conclusively the safety of ICG in 
pregnant patients. As such, it is labeled by the FDA as pregnancy category C. There do exist 
retrospective series and case reports that have safely utilized ICG in the pregnant patient. ICG 
has been used in the study of ophthalmic angiography during preeclampsia.[86] A survey of 
ophthalmologists who use ICG in the study of retinal vessels showed that 89% of the surveyed 
ophthalmologists have encountered at least one pregnant patient who required either ICG or 
Fluorescein angiography, and of these 24% withheld ICG angiography.[87] Regarding nursing 
mothers, it is unknown where ICG is excreted in human milk and can have any adverse effects in 
the newborn or infant period. Due to the limited data, pregnant and nursing mothers will be 
excluded from the trial in a similar manner with regard to the use of Fluorescein. A routine day 
of surgery pregnancy test is performed for pre-menopausal patients. 
 
Historically, the rate of adverse reactions to ICG are low. A study of over 1900 cases of ICG 
video angiography cases in the ophthalmologic setting reported the incidence of mild, moderate 
and severe reactions to be 0.15%, 0.2% and 0.05%, respectively.[84] ICG was administered IV 
in these cases. The death rate after ICG injection has been cited as 1 in 333,333.[84] 
 
Transient nausea and vomiting, and local discomfort, are the most common reactions and require 
no treatment. These mild reactions typically occur shortly after injection and are transient.[84]  
 
Moderate reactions such as pruritus or urticaria can be treated with antihistamines, but any 
patient who experiences these symptoms will be observed carefully for the possible development 
of anaphylaxis. These symptoms have been successfully treated with anithistamines, and were of 
short duration.[84] In the event of severe reactions, assessment and treatment will be 
implemented similar to protocol used in Fluorescein adverse reactions. 
 

14 
 
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Severe reactions include anaphylactic reactions. The dose of ICG has not been well correlated to 
the severity of symptoms. These symptoms include severe generalized pruritis, diaphoresis, 
weakness, agitation, and syncope.[84, 88] Signs include hypotension and hemodynamic 
instability. In extreme cases, death may result. Although life-threatening reactions during 
angiography are rare, angiographic facilities and personnel should be properly equipped and 
prepared to manage serious reactions to the procedure. A resuscitative crash cart and appropriate 
agents to treat severe reactions will be readily available (including epinephrine for intravenous or 
intramuscular use, soluble corticosteroids, aminophylline for intravenous use, oxygen, and 
airway instrumentation). It is generally recommended that a physician be present or available 
during angiographic procedures. 
 
Local toxicity has been described for ICG, but largely in the setting of topical application during 
ophthalmologic procedures.[89, 90] ICG has widespread use as a vital stain during macular 
surgery. It is hypothesized that the local irritant effects combined with the hypo-osmolar tonicity 
over prolonged exposure times (over 10 minutes) may contribute to local toxicities to the 
macula, potentially resulting in decreased visual acuity, optic disc changes, and visual field 
deficits.[91] However, in our application of ICG for HIVM, local effects should be minimized 
through IV injection and dilution as it enters the blood stream. These toxicities have not been 
reported in studies where ICG has been used intravenously for liver and cardiac function. Mild 
discomfort has been associated with extravasation.[84] Care will be taken to minimize injection 
within the soft tissue and limit extravasation of ICG. 
 
1.5 
RISKS AND/OR BENEFITS 
 
Risks – similar to the section on fluorescein, low risk of reaction to ICG as described above. 
Otherwise, no increased risk to the surgical procedure anticipated. 
 
Benefits – depending upon collected findings and correlation with clinical outcome, IVM may 
offer prognostic information and improve the staging classification of peritoneal tumors with the 
intent of potentially guiding treatment decisions in the future. 
 
2 
RATIONALE 
These studies will elucidate the hemodynamic properties of microvessels associated with 
peritoneal carcinomatosis. In order to model the effects of tumor-associated vessel function on 
patient outcomes, direct examination of the microvasculature in these patients is necessary to 
ascertain if the preclinical model systems have accurately recapitulated the clinical setting. Thus, 
the current proposed trial represents an opportunity to support the development of interventions 
to improve patient treatment by extending the application of IVM to the locoregional tumor 
microenvironment. 
 

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3 
OBJECTIVES 
3.1 
PRIMARY OBJECTIVE - PART I (10 PATIENTS): 
To determine the feasibility of performing HIVM in patients with peritoneal carcinomatosis 
during standard course of treatment (cytoreductive surgery with hyperthermic intraperitoneal 
chemotherapy, or CRS-HIPEC). A successful intravital microscopic observation will include the 
ability to complete each of the following: 
 
1. Identify and measure vessels associated with peritoneal tumor implants 
2. Determine vessel density per 10x field 
3. Visualize vital dyes within the vessels [fluorescein and indocyanine green (ICG)] 
4. Calculate the blood flow velocity of the vessels and tissue penetration of fluorescein 
and ICG as markers of vessel permeability.  
 
3.2 
SECONDARY OBJECTIVES - PART II (20 PATIENTS): 
1. Compare the microscopic observation of the tumor-associated vessels with normal 
tissue (peritoneal surface) in each individual subject. 
2. Correlate the microscopic observations of the tumor-associated vessels with pathologic 
grade of tumor implants.  
3. To correlate the microscopic observation of the microvasculature with tumor-specific 
and overall survival. 
 
3.3    PRIMARY AND SECONDARY ENDPOINTS: 
 
Primary endpoint: A patient will be deemed a success if each of the following parameters were 
measured: 
1. Identify tumor implant associated vessels and measure vessel diameters 
2. Determine vessel density per 10x field 
3. Visualize vital dyes within the tumor-associated vessels (fluorescein and green ICG) 
4. Calculate the blood flow velocity of the tumor-associated vessels and tissue 
penetration of fluorescein and ICG as markers of vessel permeability.  
 
Secondary endpoints: 
1. Post-operative comparison of the microvasculature of peritoneal carcinomatosis with 
normal tissue (peritoneum) in each individual subject using vessel diameters, 
vessel density, detection of intravital dye and flow rates.  
2.  Post-operative correlation of the microvasculature with pathologic features of the 
tumor implants (i.e. tumor grade) at the time of the final pathology report (5-7 
days after surgery). 

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3. Post-operative correlation of the microscopic observation of the tumor 
microvasculature tumor-specific and overall survival. 
 
4 
METHODOLOGY 
4.1 
Study Design 
This is an open-label, non-randomized, single center, study of IVM observation in conjunction 
with fluorescein and ICG in subjects with peritoneal carcinomatosis undergoing CRS-HIPEC. 
The first part is a Pilot study of feasibility. 
 
Subjects will be treated on an inpatient basis. All subjects will meet the inclusion and exclusion 
criteria summarized in Section 5.1 and Section 5.2 prior to participation in this study. Standard 
evaluation by the Preoperative Anesthesia clinic may include additional testing such as EKG, 
chest x-ray, and/or pulmonary function tests, but this is performed based upon their established 
guidelines.  If any of these tests preclude a wide excision in the operating room, then the patient 
is excluded from this study. 
 
4.2   STUDY METHODS 
During the course of treatment for peritoneal carcinomatosis while the patient is under 
anesthesia, CRS-HIPEC will be performed. Surgical exposure is performed through a midline 
incision and the assistance of retractors as shown in the technical schema (Appendix D). 
 
The IVM technology utilized at Mayo Clinic currently consists of a high resolution confocal 
endomicroscope (Appendix E). This apparatus has the ability to provide single cell resolution 
and high quality images of the microvasculature. While the typical application of this device is to 
investigate GI mucosal surfaces (i.e. esophagus or colon), it can be readily and easily applied to 
any surface. It can be sterilized or used with a sterile sleeve in order to interface with the 
peritoneal surface. 
 
The microscope will be moved into position next to the OR table by the operating surgeon. The 
microscope will be covered with a standard sterile drape similar to that used for cords associated 
with other intraoperative devices (NeoProbe or intraoperative ultrasound). The only exposed area 
will be the microscopic objective located at the tip of the endoscope, which will be made sterile. 
This microscopic objective will come into close contact with the peritoneal surface for 
microscopic observation. Once the microscopic objective is in proper position, the epifluorescent 
light source will be turned on and digital video recording will commence. All video images will 
be captured to the hardware directly attached to the microscope (Appendix E). All data will be 
backed up on a password-protected hard drive in the Principal Investigator’s laboratory.  
 
Following several observations over various portions of the peritoneal surface, 1 ampule (5 mL) 
25% fluorescein will be injected intravenously and observation will continue until loss of 

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fluorescence. IVM is continuously performed at the time the fluorescent agent is injected and 
video is recorded in real-time. The fluorescein is almost immediately visible within the 
microscopic field, and the vessels are quickly outlined in great detail. The fluorescence lasts for a 
few minutes (2-3 minutes) and then either fades or begins to permeate through the tissue. The 
extravasation of dye will be determined by visualizing fluorescence outside of the defined 
vessels for a total time of 5 minutes.  The distance from the vessels will be measured, recorded, 
and expressed as a function of time (extravasation rate) in an analysis performed off-line from 
the digitally recorded video. 
 
Following the dissipation of the fluorescein from the visual field, 5 mg of ICG will be injected 
intraveneously. The observation will repeat in a similar manner, now with the use of ICG. 
Tumor-associated vasculature will be observed in order to record the similar vessel 
characteristics. ICG is expected to persist within the vasculature for an extended period of time, 
thereby allowing further characterization of the field. It is not necessary to wait for the ICG to 
dissipate from the field as this may take up to 20-30 minutes due to its high level of albumin 
binding. 
 
At the completion of the observation period with ICG, the microscope will be removed and 
sterilized between uses. This would complete the observation period, which is anticipated to take 
15 - 20 minutes total time. The observation will take place during the course of standard surgery 
and is not anticipated to delay or significantly lengthen the duration of the procedure. For 
example, other portions of the procedure will be continued during the observation period 
(e.g., setting up the circuit required for HIPEC). The tumor implants will be removed and 
undergo standard pathology evaluation. The patient will recover in the PACU per standard 
protocol. 
 
Technical difficulties with the microscope apparatus (e.g. malfunction of the software or 
structural damage to any of the microscope components) or unforeseen events during the course 
of surgery that are unrelated to the study intervention but that result in the termination of the 
surgery (e.g. adverse reaction to anesthetic prior to administration of intravital dye or 
hemodynamic instability from a complication of the surgery) will not be considered failures of 
the primary objective. In these circumstances, the subject will be considered to be off-study and 
be replaced with a subsequent patient. 
 
Off-line analysis of digitally recorded live video will be performed using parameters and 
statistical methods that have been developed in our preclinical imaging studies.[1] Lumenal 
cross-sectional diameter (D) of vessels and velocity (V) of dye labeled cells will be measured in 
off-line observations. Wall shear rate () will be calculated as 8 (V ÷ D).[92]  Vessel density will 
be determined by measuring the calculated blood vessel area as a percent of the total visual field 
area. If visible, the uptake of fluorescein or ICG will be measured as a diffusion rate (distance 
from tumor vessel over time) and as a percent of the total tumor field observed (percent of visual 
field expressed as surface area with dye detected). Blood flow velocity will be determined by the 
equation Q = (RBC velocity/1.6) x (d/2) x pi.  
 

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These data will be calculated and recorded in a spread sheet that will also be saved onto a 
password-protected hard drive in the Principal Investigator’s laboratory. Additionally, all data 
will be de-identified and provided to the Biostatistician. Generated data and the raw video 
images will be stored for review at any time to satisfy any audit of the collected data. This saved 
data will readily allow the determination of a successful observation including the identification 
of tumor vessels, the associated vessel measurements, vessel density, and dye enhanced imaging 
acquisition. 
 
TARGET ACCRUAL AND STUDY DURATION 
A maximum of 30 subjects [Part I (10 patients) and Part II (20 patients)] will be enrolled. The 
number of subjects required is a function of the expected feasibility. Accrual is expected to take 
up to 2.5 years. 
 
5 
SUBJECT SELECTION 
Patients who are scheduled to undergo CRS-HIPEC in the operating room who can provide 
informed consent, have a good performance status (ECOG ≤ 2), and have no known allergy or 
prior reaction to fluorescein, ICG or iodine, are eligible. 
5.1 
INCLUSION CRITERIA 
1. Age ≥ 18 years of age. 
2. Have an ECOG Performance Status of ≤ 2. Refer to Appendix C. 
3. Have measurable disease in the peritoneum by direct visualization (visible lesion typically 
> 0.5 cm in maximal diameter). 
4.   Carcinomatosis that meets indications for CRS-HIPEC. 
5.    Subject must understand the investigational nature of this study and sign an Independent 
Ethics Committee/Institutional Review Board approved written informed consent form prior 
to receiving any study related procedure. 
6.    A negative skin-prick test to fluorescein. 
 
5.2 
EXCLUSION CRITERIA 
1. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 

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psychiatric illness/social situations. 
2. Renal dysfunction as defined as a GFR < 45. 
3. Liver dysfunction as defined by Child-Pugh score > 5, or LFT’s 1.5x above normal range. 
4. Any known allergy or prior reaction to fluorescein or ICG or a positive skin prick test to 
fluorescein. 
5. Pregnant or nursing female subjects, determined preoperatively with a urine pregnancy test. 
6. Unwilling or unable to follow protocol requirements. 
7. Any condition which in the Investigators’ opinion deems the patient unsuitable (e.g., 
abnormal EKG). 
8. Any condition that excludes CRS-HIPEC as the standard of care for the patient. 
 
5.3 
INCLUSION OF WOMEN AND MINORITIES 
Both men and women and members of all races and ethnic groups are eligible for this study. 
 
5.4 
SUBJECT WITHDRAWAL 
Any time prior to the planned surgical procedure, the patient may withdraw from this 
observational study for any reason without prejudice. 
 
6 
STUDY PROCEDURES 
6.1 
BASELINE EVALUATIONS 
Standard presurgical assessment with labs and studies as determined by the preoperative 
Anesthesia clinic based upon their established guidelines. 
The following will be performed within 1 week prior to the planned microscopic observation: 
 
Informed consent: Must be completed prior to receiving any study-related procedures. 
 
Medical history 
 
Physical examination, including vital signs (i.e., temperature, heart rate, respiratory rate, 
blood pressure, body weight, and height). 
 
If determined to be eligible for the study and after giving informed consent, a skin-prick test 
to determine sensitivity and risk of anaphylaxis to fluorescein and a patch test for ICG will be 

20 
 
Version 2.0, 15 March 2018 
performed by the Principal Investigator. For testing allergic response to fluorescein, a sterile 
Duotip® lancet (Lincoln Medical, USA) is used in 3 separate areas of skin on the forearm. 
The skin-pricks will include 4 microliters of 10% fluorescein, histamine chloride (10 mg/ml) 
[positive control], and 50% saline solution [negative control] administered in areas of skin 
prepped with an alcohol pad. After 30 minutes, the appearance of a wheal greater than 3 mm 
in diameter is considered a positive test result in the context of the appropriate positive and 
negative control. If a positive test is noted, the patient will no longer be eligible for this 
study. For comfort and relief of any skin irritation/pruritis, the patient will be offered an anti-
histamine (fexofenadine [Allegra®] 30 mg po x 1) following the skin-prick test if necessary. 
The patient will also be screened for a history of allergic reaction to iodine or iodinated 
contrast dyes. 
 
If indicated by anesthesia pre-op assessment (standard operating procedure for pre-op clinic) 
- Hematology [i.e., CBC (with or without differential, auto or manual), ANC, and platelets]. 
 
A complete metabolic panel (CMP): chloride, Co2, potassium, sodium, BUN, glucose, 
calcium, creatinine, total protein, albumin, total bilirubin, alkaline phosphatase, AST, ALT, 
A/G ratio, BUN/creatinine ratio, osmol (Calc), anion gap). 
 
If indicated by anesthesia pre-op assessment (standard operating procedure for the pre-op 
clinic) – 12-lead electrocardiogram, chest x-ray, and/or pulmonary function tests if indicated.  
These tests are not strictly required to support eligibility for this study, but if indicated and 
performed, may exclude the patient from receiving a general anesthetic and therefore, a wide 
excision performed in the operating room (thereby excluding the patient from this study). 
 
Pregnancy test (urine) in females of childbearing potential. Females who have undergone 
surgical sterilization or who have been postmenopausal for at least 2 years are not considered 
to be of childbearing potential. The standard operating procedure for all females of 
childbearing potential is to obtain a urine HCG (or a serum HCG if urine unable to be 
obtained). If the pregnancy test is positive, the patient is excluded from the study. 
 
6.2 
EVALUATIONS PERFORMED AT END OF TREATMENT 
 
Standard Post Anesthesia Care Unit (PACU) protocols for patient recovery from surgery. 
Patients will be continuously monitored for any reaction to either dye both intraoperatively 
and during the recovery period. Adverse events will be recorded throughout the time of entry 
into the operating room until the post-operative follow up. If the patient is transported 
directly to the Intensive Care Unit (ICU), these protocols will also be performed. 
 

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6.3 
POST-TREATMENT FOLLOW-UP EVALUATIONS 
 
Standard follow-up and safety evaluations from surgery includes a post-op visit at 2-3 weeks 
and scheduled follow-up based upon final staging of the patient’s cancer. Follow-up will be 
based upon current NCCN guidelines. 
 
 
 
Study Calendar 
 
 
Pre-operative Visit 
Surgery 
Post-operative Visit 
History and Physical 
X 
 
 
Informed consent 
X 
 
 
Fluorescein  
skin prick test 
X  
 
 
Tests as medically 
indicated 
X 
 
 
 
 
 
 
CRS-HIPEC with 
intra-op IVM 
 
X  
 
Post-surgical 
monitoring in 
recovery and 
inpatient stay 
 
X 
 
 
 
 
 
Review of pathology  
 
 
X  
Routine cancer 
surveillance 
 
 
X  
 
 
At the completion of the surgery, if gross tumor is entirely debulked or ablated, then patients 
are considered disease free. Treatment response will be based upon the presence of a 
recurrence of tumor at either the primary site, locoregional recurrence (peritoneum) or 
metastatic sites.  
 
The time to recurrence will determine the standard length of clinical follow up. However, for 
this study, a 10-year limit will be placed on clinical data collection during the standard 
clinical follow up.  After a patient is enrolled, the duration of data collection will end at 10 
years from the time of microscopic observation (surgery).  These data (time of recurrence 

22 
 
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and/or survival) will be correlated with findings from the one-time, initial IVM observation 
for the defined 10-year period of data collection.   
 
The surveillance data (no evidence of disease, disease recurrence, death) will be stored in a 
secured database and matched to the microscopic observation data.  These data will serve as 
the basis for statistical comparisons of microscopic findings and clinical outcomes  to be 
performed by our study statistician as described in section 9. 
 
6.4 
PATHOLOGY 
Standard pathologic evaluation (no deviation from current standard practice of pathology 
department) will be performed. 
 
7 
SAFETY EVALUATION 
7.1 
ADVERSE EVENTS 
7.1.1 DEFINITION 
An adverse event or adverse experience (AE) is any untoward medical occurrence associated 
with the use of a drug in humans, whether or not considered drug related. Therefore, an AE can 
be ANY unfavorable and unintended sign (including an abnormal laboratory finding), symptom, 
or disease temporally associated with the use of a medicinal (investigational) product, whether or 
not considered related to the medicinal (investigational) product (attribution of ‘unrelated’, 
‘unlikely’, ‘possible’, ‘probable’, or ‘definite’). 
An AE is considered “unexpected” if it is not listed in the investigator brochure or is not listed at 
the specificity or severity that has been observed; or if an investigator brochure is not required or 
available, is not consistent with the risk information described in the general investigational plan 
in other study-related documents. “Unexpected” also refers to adverse events that are mentioned 
in the investigator brochure (or other study-related documents) as occurring with a class of drugs 
or as anticipated from the pharmacological properties of the drug, but are not specifically 
mentioned as occurring with the particular study drug. 
Adverse events will be recorded in this study for the purpose of safety monitoring and adverse 
events are anticipated to be absent or unlikely. 
 
7.1.2 GRADING AND RELATIONSHIP TO DRUG  
The descriptions and grading scales found in the CTEP Version 4 of the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) will be utilized for AE reporting. CTEP 
Version 4 
of 
the 
CTCAE 
is 
identified 
and 
located 
at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. AEs not covered by 

23 
 
Version 2.0, 15 March 2018 
specific criteria should be reported with common medical terminology and graded according to 
definitions provided in the CTCAE Version 4. 
AEs NOT listed in the NCI-CTCAE, the severity of AEs will be graded as follows: 
 
Grade 1 – Mild: Event barely noticeable to subject; no limitation in activity, no minimal 
medical intervention/therapy required. 
 
Grade 2 – Moderate: Event of a sufficient severity to make participant uncomfortable. ADL 
limited; no or minimal medical intervention/therapy required. 
 
Grade 3 – Severe: Event causes significant discomfort/limitation in activity. May be such 
severity that the subject cannot continue on study. Severity may cause cessation of treatment. 
Treatment for symptoms may be given and/or subject hospitalized. 
 
Grade 4 - Life Threatening: Event places the participant at immediate risk of death from 
the reaction as it occurred. Significant medical intervention/therapy required; hospitalization 
or hospice care probably. 
 
Grade 5 - Death. 
 
The relationship of event to study drug will be documented by the Investigator as follows: 
 
Unrelated: The event is clearly related to other factors such as the subject’s clinical state, 
other therapeutic interventions or concomitant drugs administered to the subject. 
 
Unlikely: The event is doubtfully related to investigational agent(s). The event was most 
likely related to other factors such as the subject’s clinical state, other therapeutic 
interventions, or concomitant drugs. 
 
Possible: The event follows a reasonable temporal sequence from the time of drug 
administration, but could have been produced by other factors such as the subject’s clinical 
state, other therapeutic interventions or concomitant drugs. 
 
Probable: The event follows a reasonable temporal sequence from the time of drug 
administration, and follows a known response pattern to the study drug. The event cannot be 
reasonably explained by other factors such as the subject’s clinical state, therapeutic 
interventions or concomitant drugs. 
 
Definite: The event follows a reasonable temporal sequence from the time of drug 
administration, follows a known response pattern to the study drug, cannot be reasonably 
explained by other factors such as the subject’s condition, therapeutic interventions or 
concomitant drugs; AND occurs immediately following study drug administration, improves 
upon stopping the drug, or reappears on re-exposure. 
 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the adverse event. Reasonable possibility means there is evidence to suggest a causal 
relationship between the drug and the adverse event, such as: 

24 
 
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 
A single occurrence of an event that is uncommon and known to be strongly associated with 
drug exposure (e.g., angioedema, hepatic injury, Stevens-Johnson Syndrome). 
 
One or more occurrences of an event that is not commonly associated with drug exposure, 
but is otherwise uncommon in the population exposed to the drug. 
 
An aggregate analysis of specific events observed in a clinical research study that indicates 
those events occur more frequently in the drug treatment group than in a concurrent or 
historical control group. 
An AE for which there is a reasonable possibility that the drug caused the adverse event includes 
those assessed as having a Possible, Probable, or Definite causality assessment. 
 
7.1.3 REPORTING ADVERSE EVENTS 
Table 1. 
Guidelines for Routine Adverse Event Reporting for Pilot Studies 
(Regardless of Expectedness) 
Attribution 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Unrelated 
X 
X 
X 
X 
X 
Unlikely 
X 
X 
X 
X 
X 
Possible 
X 
X 
X 
X 
X 
Probable 
X 
X 
X 
X 
X 
Definite 
X 
X 
X 
X 
X 
All subjects are evaluated at baseline and any abnormalities are documented. 
 
Worsening abnormalities and/or worsening of a pre-existing condition (e.g., diabetes, 
migraine that has increased in severity, frequency or duration of the condition, or an 
association with significantly worse outcome) can become an AE or serious adverse event 
and must be documented and followed. 
 
A persistent AE is one that extends continuously, without resolution between treatment 
cycles/courses. Reporting of persistent AEs is as follows: 
a) Routine reporting: The AE must be reported only once unless the grade becomes 
more severe in a subsequent cycle/course. If the grade becomes more severe the 
AE must be reported again with the new grade. 
b) Expedited Reporting: the AE must be reported only once unless the grade 
becomes more severe in the same or a subsequent cycle/course. 
 
A recurrent AE is one that occurs and resolves during a cycle/course of therapy and then 
reoccurs in a later cycle/course. Reporting of recurrent AEs is as follows: 
a) Routine Reporting: An AE that resolves and then recurs during a subsequent 
cycle/course must be reported by the routine procedures. 

25 
 
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b) Expedited Reporting: An AE that resolves and then recurs during a subsequent 
cycle/course does not require reporting unless the grade increases and 
hospitalization is associated with the recurring AE. 
 
At each evaluation, the Investigator will determine whether any AEs have occurred by 
evaluating the subject’s signs, symptoms, and current laboratory and/or other test results. 
 
All AEs (expected or unexpected), including worsening of a pre-existing conditions 
(increased severity, frequency or duration of the condition and/or associated with 
significantly worse outcomes) which occur during the specified collection period, whether 
observed by the Investigator or by the subject, and whether or not thought to be related to 
study drug, will be documented in detail and followed until resolution, stabilization, death, or 
the start of new treatment. 
 
Whenever possible record a diagnosis for signs and symptoms of a common syndrome 
(i.e., cough, sneezing, runny nose would be reported as an upper respiratory infection). 
All AEs for Pilot studies must be entered on the Adverse Event eCRF page, using the CRS 
EXPeRT database. 
 
7.2 
SERIOUS ADVERSE EVENTS 
7.2.1 DEFINITION 
A serious adverse event (SAE) is any adverse event (experience) that in the opinion of either the 
investigator or sponsor results in ANY of the following: 
 
Death. 
 
A life-threatening adverse event (experience). Any AE that places a subject or subject, in the 
view of the Investigator or sponsor, at immediate risk of death from the reaction as it 
occurred. It does NOT include an AE that, had it occurred in a more severe form, might have 
caused death. 
 
Inpatient hospitalization or prolongation of existing hospitalization (for > 24 hours). 
 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
 
A congenital anomaly or birth defect. 
 
Important Medical Event (IME) that, based upon medical judgment, may jeopardize the 
subject or subject and may require medical or surgical intervention to prevent one of the 
outcomes listed above. 
Elective hospital admission (including planned treatment for progressive disease or a planned 
protocol procedure), admission for socioeconomic reasons, and death due to tumor progression 
will not be considered an SAE. 

26 
 
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All SAEs will be collected from the date of subject or subject signing consent until 30 days after 
the last dose of study drug and/or procedure. 
 
7.2.2 REPORTING SERIOUS ADVERSE EVENTS 
AE may be identified as an Unanticipated Problem by the Investigator. Please refer to 
Section 7.3.2 for details on reporting Unanticipated Problems. 
The SAE Form should be completed with all available information, including a brief narrative 
describing the adverse event and any other relevant information. If applicable, the narrative must 
also include identification of similar reports and an analysis of the significance of the AE. 
 
7.2.3 FOLLOW-UP FOR SERIOUS ADVERSE EVENTS 
All SAEs will be followed until resolution, stabilization, death, or the start of new treatment. 
 
7.3 
UNANTICIPATED PROBLEMS 
7.3.1 DEFINITION 
An Unanticipated Problem (UP) is any incident, experience, or outcome that meets all of the 
following criteria: 
 
Unexpected (in terms of nature, severity, or frequency) given: 
o The research procedures that are described in the study-related documents, 
including study deviations, as well as issues related to compromise of subject 
privacy or confidentiality of data. 
o The characteristics of the subject population being studied. 
 
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). 
 
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 
and if in relation to an AE is also deemed Serious per Section 7.2. 
 
7.3.2 REPORTING UNANTICIPATED PROBLEMS 
SAEs that are serious, unexpected and for which there is a reasonable possibility that the study 
drug caused the adverse event are reported on the Unanticipated Problem Form and submitted to 
CRS Compliance Office within 24 hours of becoming aware of the Unanticipated Problem. 

27 
 
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Within 5 business days, an Unanticipated Problem Form should be completed with all available 
information, including a brief narrative describing the unanticipated problem and any other 
relevant information. 
Within 5 business days of becoming aware of new information about the Unanticipated Problem, 
submit this updated information to CRS Compliance with an updated UP Form. 
 
8 
DATA AND SAFETY MONITORING 
The Mayo Clinic Data and Safety Monitoring Board will assess the progress of the study, the 
safety data, and critical efficacy endpoints. The DSMB will review the study annually and will 
make recommendations that include but not limited to; (a) continuation of the study, (b) 
modifications to the design (c) or termination of the study. 
 
9 
STATISTICAL METHODOLOGY 
Initially ten patients will be treated to determine the feasibility of HIVM for peritoneal 
carcinomatosis. This first part of the study is a pilot study of feasibility. If seven or more patients 
are deemed a success by meeting all of the criteria below, then HIVM will be deemed feasible in 
the study of peritoneal carcinomatosis. Criteria include the ability to complete each of the 
following: 
1. Identify and measure vessels associated with peritoneal tumor implants 
2. Determine vessel density per 10x field 
3. Visualize vital dyes within the vessels [fluorescein and indocyanine green (ICG)] 
4. Calculate the blood flow velocity of the vessels and tissue penetration of fluorescein 
and ICG as markers of vessel permeability. 
 
If the true success rate is 60%, the probability of deeming HIVM feasible is 63%; if the true 
success rate is 70%, then the probability of deeming HIVM feasible is 88%; and if the true 
success rate is 90%, then the probability of deeming HIVM feasible increases to 99%. 
 
If HIVM is deemed feasible, then an additional 20 patients will be accrued to this study in order 
to address the following:  
1. Post-operative comparison of the microvasculature of peritoneal carcinomatosis with 
normal tissue (peritoneum) in each individual subject using vessel diameters, 
vessel density, detection of intravital dye and flow rates.  
2.  Post-operative correlation of the microvasculature with pathologic features of the 
tumor implants (i.e. tumor grade) at the time of the final pathology report (5-7 
days after surgery). 
3. Post-operative correlation of the microscopic observation of the tumor 
microvasculature tumor-specific and overall survival. 
 
An abnormal HIVM observation is defined by any of the following criteria:  

28 
 
Version 2.0, 15 March 2018 
1. Aberrant vessel structure compared to normal tissue, which includes tortuosity of the 
vessel wall or a closed loop formation. 
2. Aberrant vessel branching patterns compared to normal tissue, which includes erratic 
and disorganized branching points. 
3. Blood vessel density that is less than or greater than one standard deviation of the 
blood vessel density observed in normal tissue. 
4. Inability to detect flow or intravital dye within a vessel. 
5. Average blood flow rate through observed vessel that is less than one standard 
deviation of the average blood flow rate through a normal vessel. 
 
A matched analysis will be performed for observations of microvasculature between the 
peritoneal tumor implants and normal tissue. For dichotomous variable comparisons between 
vessel structure (normal vs aberrant), vessel branching patterns (normal vs aberrant), or detection 
of intravital dye (present vs absent), a McNemar’s test will be performed. For continuous 
variable comparisons between blood vessel density and average blood flow rate, a paired t test 
will be performed. 
 
Associations between characteristics for the baseline (normal peritoneal microvasculature 
observed surrounding the tumor implant site) and tumor implant itself will be depicted with 
graphics, correlations, and model results. 
 
Adverse event and complication rates will be described using upper one-sided 95% Clopper 
Pearson confidence limits.  
 
Overall survival will be defined as the duration of time from diagnosis to time of death.  Disease 
specific survival will be defined as the time from CRS-HIPEC to death due to recurrence. 
Overall survival and disease specific survival will be generated using standard Kaplan-Meier 
methodology. 
 
A maximum of 30 subjects will be enrolled in this study (initially 10 to determine feasibility and 
then 20 additional subjects if deemed feasible). The accrual rate is estimated at one subject per 
month, and the estimated study duration is 30 months. 
 
9.1 
RANDOMIZATION 
This is a nonrandomized study. No randomization scheme will be generated. Subjects will be 
assigned to a treatment group based on their date of qualification for participation. 
 
9.2 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Patients of all racial and socioeconomic demographics having met the criteria for study entry will 
be eligible for this protocol. Descriptive statistics (as appropriate: n, percent, mean, median, min 
and max) will be used to summarize demographic and baseline characteristics. 

29 
 
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10 ETHICAL AND REGULATORY STANDARDS 
10.1 ETHICAL PRINCIPLES 
This study will not be initiated until the protocol and informed consent document(s) have been 
reviewed and approved by a properly constituted Institutional Review Board (IRB) or 
Independent Ethics Committee (IEC). Each subject (or legal guardian) shall read, understand, 
and sign an instrument of informed consent prior to performance of any study-specific 
procedure. It is the responsibility of the investigator to ensure that the subject is made aware of 
the investigational nature of the treatment and that informed consent is given. 
The Investigator is responsible for the retention of the subject log and subject records; although 
personal information may be reviewed by authorized persons, that information will be treated as 
strictly confidential and will not be made publicly available. The investigator is also responsible 
for obtaining subject authorization to access medical records and other applicable study specific 
information according to Health Insurance Portability and Accountability Act regulations (where 
applicable). 
This study will be conducted in compliance with all applicable laws and regulations of the state 
and/or country and institution where the subject is treated, in accordance with the Declaration of 
Helsinki, Good Clinical Practice, and according to the guidelines in this protocol, including 
attached appendices. 
 
10.2 INFORMED CONSENT 
The Investigator is responsible for obtaining written consent from each subject or the subject's 
legally authorized representative in accordance with ICH-GCP guidelines using the approved 
informed consent form, before any study specific procedures (including screening procedures) 
are performed. The informed consent form acknowledges all information that must be given to 
the subject according to ICH-GCP, including the purpose and nature of the study, the expected 
efficacy and possible side effects of the treatment(s), and specifying that refusal to participate 
will not influence further options for therapy. Any additional information that is applicable to the 
study must also be included. Additional national or institutionally mandated requirements for 
informed consent must also be adhered to. The subject should also be made aware that by 
signing the consent form, processing of sensitive clinical trial data and transfer to other countries 
for further processing is allowed. 
The Investigator shall provide a copy of the signed consent form to the subject and the signed 
original shall be maintained in the Investigator File. A copy of the signed consent form must be 
filed in the subject file. At any stage, the subject may withdraw from the study and such a 
decision will not affect any further treatment options. 
 

30 
 
Version 2.0, 15 March 2018 
11 STUDY RESPONSIBILITIES 
11.1 DATA COLLECTION 
Data entry into the database is to be completed in a timely fashion (approximately within 
28 days) after the subject’s clinic visit. If an AE is considered serious it is captured on both the 
Adverse Event page and the Serious Adverse Event Form, which is handled in an expedited 
fashion. 
Data management activities will be performed using EXPeRT. EXPeRT is a suite of software 
tools that enables the collection, cleaning and viewing of clinical trial data. CRS data 
management will design the study-specific database and facilitate its development by the 
EXPeRT Information Technology team. Once the database design is approved by the 
Investigator, Statistician, and Clinical Research Coordinator, the database will be put into 
production and data entry can begin. Data can be entered and changed only by those with the 
rights to do so into the eCRFs. EXPeRT is compliant with all relevant technical aspects of 
relevant GCP guidelines. 
 
The system can generate accurate copies of stored data and audit trail information in human 
readable form. 
 
System access is limited to authorized individuals through the controlled assignment of 
unique ID and password combinations. 
 
The system is designed to periodically force users to change their passwords and verifies that 
user ID and password combinations remain unique. 
 
The system automatically generates a permanent time-stamped audit trail of all user 
interactions. 
When data entry is complete, data management will review the data and will query any missing, 
incomplete, or invalid data points for resolution by the Clinical Research Coordinator and 
Investigator. Once all queries have been resolved, the data can be released to the statistician for 
analysis. 
 
11.2 MAINTENANCE OF STUDY DOCUMENTS 
Essential documents should be retained for 6 years from the study termination date. These 
documents could be retained for a longer period, however, if required by the applicable local 
regulatory requirements or by an agreement with Mayo Clinic. If, for any reason, the Investigator 
desires to no longer maintain the study records, they may be transferred to another institution, 
another investigator, or to Mayo Clinic upon written agreement between the Investigator and 
Mayo Clinic. 
 

31 
 
Version 2.0, 15 March 2018 
12 ADMINISTRATIVE RULES 
12.1 REVISIONS TO THE PROTOCOL 
Mayo Clinic may make such changes to the protocol as it deems necessary for safety reasons or 
as may be required by the U.S. FDA or other regulatory agencies. Revisions will be submitted to 
the IRB/ERC for written approval before implementation. 
 
12.2 TERMINATION OF THE STUDY 
It is agreed that, for reasonable cause, either the Investigators or the Sponsor, Mayo Clinic may 
terminate this study, provided a written notice is submitted within the time period provided for in 
the Clinical Trial Agreement. In addition, Mayo Clinic may terminate the study at any time upon 
immediate notice if it believes termination is necessary for the safety of subjects enrolled in the 
study. 
 
12.3 CONFIDENTIALITY 
Any data, specimens, forms, reports, video recordings, and other records that leave the site will 
be identified only by a participant identification number (Participant ID, PID) to maintain 
confidentiality. All records will be kept in a limited access environment. All computer entry and 
networking programs will be done using PIDs only. Information will not be released without 
written authorization of the participant. 
 
 

32 
 
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14 APPENDICES 
Appendix A: Fluorescein Drug Package Insert. 
Fluorescein Package Insert 
 
 
 
 
 

 
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39 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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40 
Appendix B: Indocyanine Green Drug Package Insert. 
 
 
 

 
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41 
Appendix C. ECOG Performance Status Scores. 
Description 
Status 
Fully active, able to carry on all pre-disease performance without restriction. 
0 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary 
nature, e.g., light housework, office work. 
1 
Ambulatory and capable of all self-care but unable to carry out any work activities. 
2 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 
3 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 
4 
Dead 
5 
 
 
 
 

 
Version 2.0, 15 March 2018 
42 
Appendix D. Technical Schema of exposure for IVM of peritoneal carcinomatosis. 
 
 
 
A. Depiction of retraction and exposure of the peritoneal cavity during cytoreductive surgery with 
hyperthermic intraperitoneal chemotherapy (CRS‐HIPEC). 
 
B. Tumor implants can be present along the surface of the peritoneum itself or on organs within the 
abdominal compartment. In this case, the small bowel also has tumor implants. This can also be observed 
with IVM. 
 

 
Version 2.0, 15 March 2018 
43 
Appendix E. Intravital endomicroscope used for performing the human IVM observation. 
 
A. Images depicting the intravital 
endomicroscope to be used in this 
study. 
The 
fiberoptic 
objective 
is 
deployed at the tip of the endoscope. 
Bottom insets show a complete view 
(left) and zoomed in view (right) of the 
microscope. The hardware is directly 
attached 
to 
the 
endomicroscope 
apparatus and records images/videos 
for post‐hoc analysis. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A 
 
 
 
B. Example of images of colonic 
mucosa generated using the 
IVM 
endomicroscope. 
High 
quality 
resolution 
can 
be 
achieved at the single cell level. 
This photo does not show the 
addition 
of 
intravital 
dyes 
(fluorescein or ICG). These dyes 
will be used for imaging of 
peritoneal tumor implants. 
 
 
 
 
 
 
 
B