PREPARE: PRevention Using EPA Against coloREctal Cancer
Phase: PHASE1
Conditions: Colorectal Adenoma, Colorectal Cancer, Endoscopic Surgery, Eicosapentaenoic Acid, Gastrointestinal Microbiome
Sponsor: Massachusetts General Hospital

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DF/HCC Protocol #: 19-402
TITLE:  PREPARE: PRevention using EPA against coloREctal cancer
Coordinating Center:
Massachusetts General Hospital
*Principal Investigator (PI):
Andrew T. Chan, M.D., M.P.H.
Massachusetts General Hospital
ACHAN@partners.org 
 
Other Investigators:
Mingyang Song, MD, ScD *
Harvard T.H. Chan School of Public Health
Massachusetts General Hospital
MIS911@mail.harvard.edu
Peter J. Carolan, M.D.
Massachusetts General Hospital
PCAROLAN@partners.org 
Daniel C. Chung, M.D.
Massachusetts General Hospital
CHUNG.DANIEL@mgh.harvard.edu 
Francis P. Colizzo III, M.D.
Massachusetts General Hospital
FCOLIZZO@mgh.harvard.edu 
David A. Drew, Ph.D.*
Massachusetts General Hospital
DADREW@mgh.harvard.edu  
Manish Gala, M.D.
Massachusetts General Hospital
MGALA@mgh.harvard.edu 
Hamed Khalili, M.D., M.P.H.
Massachusetts General Hospital
HKHALILI@mgh.harvard.edu 
Norman S. Nishioka, M.D.
Massachusetts General Hospital
NNISHIOKA@mgh.harvard.edu 

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James M. Richter, M.D.
Massachusetts General Hospital
JRICHTER@mgh.harvard.edu 
Kyle D. Staller, MD
Massachusetts General Hospital
KSTALLER@mgh.harvard.edu  
Joseph C. Yarze, MD
Massachusetts General Hospital
JYARZE@mgh.harvard.edu 
Wenjie Ma, ScD*
Massachusetts General Hospital
WMA6@mgh.harvard.edu 
Daniel Pratt, MD
Massachusetts General Hospital
DSPRATT@mgh.harvard.edu 
Amandeep Singh, MD, PhD
Massachusetts General Hospital
ASINGH@mgh.harvard.edu  
Long Nguyen, MD
Massachusetts General Hospital
LNGUYEN24@mgh.harvard.edu
Brian Jacobson, MD, M.P.H
Massachusetts General Hospital
BJACOBSON@PARTNERS.ORG
Jay Luther, MD
Massachusetts General Hospital
JLUTHER1@mgh.harvard.edu
Braden Kuo, MD
Massachusetts General Hospital
BKUO@mgh.harvard.edu 
Michael Dougan, MD, PhD
Massachusetts General Hospital
MICHAEL_DOUGAN@mgh.harvard.edu 
*Not responsible for patient care

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Statistician:
 
Bernard Rosner, PhD
Brigham and Women’s Hospital
STBAR@channing.harvard.edu 
Clinical Research Coordinators:
Elizabeth Prezioso
Massachusetts General Hospital
EPREZIOSO@mgh.harvard.edu
Trenton Reinicke
Massachusetts General Hospital
TREINICKE@mgh.harvard.edu
Jacqueline Woo
Massachusetts General Hospital
JEWOO@mgh.harvard.edu
Janavi Sethurathnam
Massachusetts General Hospital
JSETHURATHNAM@partners.org
Madeline Koehn
Massachusetts General Hospital
mkoehn@mgh.harvard.edu
Clinical Research Project Manager:
Marina Magicheva-Gupta
Massachusetts General Hospital
MMAGICHEVA-GUPTA@mgh.harvard.edu 
NCI-Supplied Agent(s): N/A
Other Agent(s):  AMR101 (VASCEPA), manufactured by and obtained from Amarin Pharma, 
Inc.
Study Exempt from IND Requirements per 21 CFR 312.2(b).  
Protocol Type / Version # / Version Date: Amendment 9 / Version 8 / 12/10/2021

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SCHEMA
PREPARE: PRevention using EPA against coloREctal cancer 
Adults (18-80 years) who have had a prior colonoscopy at Massachusetts General Hospital 
AND at least 1 adenoma was removed.

Participants have no diagnosis of inherited colorectal cancer syndromes (i.e. FAP), no fish oil 
use at any dose more than once per week w/in the last month, no medical contraindications, and 
are not pregnant/breastfeeding.

Determine Eligibility (see phone script)

Eligibility Confirmation
Provation review of most recent endoscopy report, and accompanying pathology report.

Registration in OnCore by the study staff
Investigating physician receives written informed consent from participant

Initial (Baseline/Pre-treatment) Visit
1. Lifestyle questionnaire; 2. Nutritional survey; 3. Flexible sigmoidoscopy (24 biopsies of 
normal colorectal mucosa, one stool sample); 4. Blood; 5. Urine

Intervention
Duration: minimum = 8 weeks, maximum = 12 weeks
AMR101, 8 capsules (4 grams) per day

Final (Post-treatment) Visit
1. Lifestyle questionnaire; 2. Nutritional survey; 3. Flexible sigmoidoscopy (24 biopsies of 
normal colorectal mucosa, one stool sample); 4. Blood; 5. Urine

Primary Endpoint:
Marine omega-3 fatty acid composition in tissue
Secondary Endpoints: Effects on colorectal cancer biomarkers: 
a.) Microbiome alterations (stool); b.) Metabolomics (stool); c.) Gene expression by RNA-seq 
(tissue); Urinary PGE-M.

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TABLE OF CONTENTS
SCHEMA.........................................................................................................................................4
1.
OBJECTIVES......................................................................................................................7
1.1
Study Design............................................................................................................7
1.2
Primary Objectives...................................................................................................7
1.3
Secondary Objectives...............................................................................................7
2.
BACKGROUND .................................................................................................................7
2.1
Study Disease(s).......................................................................................................7
2.2
IND Agent(s) ...........................................................................................................7
2.3
Other Agent(s) .........................................................................................................8
2.4
Rationale ..................................................................................................................9
2.5
Correlative Studies Background ..............................................................................9
3.
PARTICIPANT SELECTION...........................................................................................10
3.1
Eligibility Criteria..................................................................................................11
3.2
Exclusion Criteria ..................................................................................................12
3.3
Inclusion of Women and Minorities ......................................................................13
4.
REGISTRATION PROCEDURES ...................................................................................13
4.1
General Guidelines for DF/HCC Institutions ........................................................13
4.2
Registration Process for DF/HCC Institutions.......................................................14
4.3
General Guidelines for Other Investigative Sites ..................................................14
4.4
Registration Process for Other Investigative Sites.................................................14
5.
TREATMENT PLAN........................................................................................................14
5.1
Treatment Regimen................................................................................................14
5.2
Pre-Treatment Criteria ...........................................................................................15
5.3
Agent Administration.............................................................................................15
5.4
Definition of Dose-Limiting Toxicity (DLT) ........................................................15
5.5
General Concomitant Medication and Supportive Care Guidelines......................15
5.6
Criteria for Taking a Participant Off Protocol Therapy.........................................15
5.7
Duration of Follow Up...........................................................................................16
5.8
Criteria for Taking a Participant Off Study ...........................................................16
6.
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................17
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................18
7.1
Expected Toxicities................................................................................................18
7.2
Adverse Event Characteristics...............................................................................19
7.3
Expedited Adverse Event Reporting......................................................................19
7.4
Expedited Reporting to Hospital Risk Management .............................................20
7.5
Routine Adverse Event Reporting .........................................................................20

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8.
PHARMACEUTICAL INFORMATION..........................................................................20
8.1
AMR101 ................................................................................................................20
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................22
9.1
Biomarker Studies..................................................................................................22
9.2
Laboratory Correlative Studies..............................................................................30
9.3
Special Studies.......................................................................................................30
10.
STUDY CALENDAR .......................................................................................................30
11.
MEASUREMENT OF EFFECT .......................................................................................31
12.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................31
12.1
Data Reporting.......................................................................................................31
12.2
Data Safety Monitoring..........................................................................................32
12.3
Multicenter Guidelines...........................................................................................32
12.4
Collaborative Agreements Language.....................................................................32
13.
STATISTICAL CONSIDERATIONS...............................................................................32
13.1
Study Design/Endpoints ........................................................................................33
13.2
Sample Size, Accrual Rate and Study Duration ....................................................33
13.3
Stratification Factors..............................................................................................34
13.4
Interim Monitoring Plan ........................................................................................34
13.5
Analysis of Primary Endpoints..............................................................................34
13.6
Analysis of Secondary Endpoints..........................................................................34
13.7
Reporting and Exclusions ......................................................................................36
14.
PUBLICATION PLAN .....................................................................................................36
APPENDIX A
VIOSCREEN OVERVIEW...........................................................................45

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1.
OBJECTIVES
1.1
Study Design
Within the gastroenterology practice of Massachusetts General Hospital (MGH), we will conduct 
a prospective, single-arm clinical trial to measure the effects of daily 4-gram eicosapentaenoic 
acid (EPA), through treatment with AMR101 (VASCEPA, icosapent ethyl) on urine, stool and 
tissue biomarkers associated with colorectal cancer. 
1.2
Primary Objectives
To measure the effect of daily 4-gram AMR101 treatment on fatty acid composition in colorectal 
tissue among individuals with a history of colorectal adenoma.
1.3
Secondary Objectives
To measure the effect of EPA treatment on the gut microbiome and metabolome, urinary 
prostaglandin metabolites (PGE-M) and gene expression profile of colorectal tissue among 
individuals with a history of colorectal adenoma.
2.
BACKGROUND
2.1
Study Disease(s)
Colorectal cancer is the third most common cancer and third leading cause of cancer-related deaths 
in the U.S. In 2016, an estimated 95,270 new cases of colorectal cancer will be diagnosed and 
49,190 people will die from the disease.1 Age is strongly associated with colorectal cancer 
incidence, with more than a third of all colorectal cancer deaths occurring in the population aged 
80 years or older. The death rates are higher in black populations and lowest in Asian individuals. 
Men have a slightly higher lifetime probability of receiving a colorectal cancer diagnosis (5.0%) 
compared to women (4.7%).2
Nationally, incidence rates have been declining over the past two decades; and at a slightly higher 
rate over the last 10 years compared to the 1990s. This is largely believed to be due to increasing 
use of screening methods and improved risk factor profile. Still the public health concern remains 
as colorectal cancer ranks third among all cancer deaths with only an approximate 65% five-year 
survival rate, necessitating further advances in cancer prevention.2
2.2
IND Agent(s)
N/A; Exempt.
This study will investigate the effects of standard-dose EPA treatment using the FDA-approved 
drug AMR101, and meets the five exemption criteria as laid out by 21 CFR 312.2(b):

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
The drug product is lawfully marketed in the United States.
The drug AMR101 used for the study is FDA approved and lawfully marketed in the 
United States.  

The investigation is not intended to be reported to FDA as a well-controlled study in 
support of a new indication and there is no intent to use it to support any other significant 
change in the labeling of the drug.
The end points of the study are mechanistic biomarkers related to EPA and are not 
used in any clinical context.

In the case of a prescription drug, the investigation is not intended to support a significant 
change in the advertising for the drug.
The study aims to investigate the potential anti-cancer mechanisms of EPA and is not 
intended to support any change in the advertising for the drug AMR101.

The investigation does not involve a route of administration, dose, patient population, or 
other factor that significantly increases the risk (or decreases the acceptability of the risk) 
associated with the use of the drug product (21 CFR 312.2(b)(1)(iii)).
AMR101 used in the proposed study is made of ethyl esters of the omega-3 fatty acid, 
eicosapentaenoic acid (EPA), which are natural substances found in the oil of certain 
fish, and will be orally administered in a standard dose (4 g/day) in people who are 
being evaluated for colorectal cancer.   

The investigation is conducted in compliance with the requirements for review by an IRB 
(21 CFR part 56) and with the requirements for informed consent (21 CFR part 50).
The protocol will be submitted to the Dana Farber/Harvard Cancer Center IRB, who 
will also review the informed consent documents.

The investigation is conducted in compliance with the requirements of § 312.7 (i.e., the 
investigation is not intended to promote or commercialize the drug product).
The study is not intended to promote or commercialize any drug product.
Our application for an IND exempt has been approved by the Division of Oncology Products 2 
(DOP2) of the FDA (see the attached formal letter, IND 141418. Please note that the name of the 
study has been changed since receipt of the IND approval). 
2.3
Other Agent(s)
AMR101
AMR101 (icosapent ethyl) is a prescription medicine for adults to lower blood levels of 
triglycerides. It is supplied as a liquid-filled gel capsule for oral administration. The standard 
dose is 4 g per day (administered as 8 half-gram capsules). Each 0.5-gram capsule of AMR101 
contains 0.5 gram of icosapent ethyl, which is an ethyl ester of the omega-3 fatty acid 
eicosapentaenoic acid (EPA). 
EPA possesses triglyceride-lowering and anti-inflammatory activities, and has been suggested to 
be beneficial for a variety of health outcomes in adults, including coronary heart disease,3 stroke,4 
type 2 diabetes,5 depression,6 and inflammatory bowel disease,7 although the data are not 
univocal.8 Increasing evidence supports the anticancer effect of EPA.9 

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Besides EPA, AMR101 capsules also contain the following inactive ingredients: tocopherol, 
gelatin, glycerin, maltitol, sorbitol, and purified water.
2.4
Rationale
Colorectal cancer is the third leading cause of cancer-related deaths in the US.1 Substantial data 
support the benefit of EPA for colorectal cancer prevention and treatment,10 which may be mainly 
due to the immunomodulatory and anti-inflammatory activity of EPA.11 Increasing data support 
that gut microbes are pivotal in integrating dietary cues with host immunity, and that disturbances 
in the gut microbiota may promote colorectal cancer by breakdown of intestinal immune 
homeostasis. Dietary fat composition is a major driver of the gut microbial community structure 
and EPA has been shown to enrich the gut bacteria that possess immunoprotective activities. These 
data together our hypothesis that EPA modulates the gut microbiota to preserve colorectalic 
immune homeostasis and suppress colorectal cancer. In the current study, we propose to 
experimentally test this hypothesis by assessing the effect of EPA supplement on the gut 
microbiota - host immune interaction. 
2.5
Correlative Studies Background
2.5.1
EPA and colorectal cancer
Several lines of evidence support the benefit of EPA for colorectal cancer prevention and 
treatment.10 In a meta-analysis of 3 prospective cohort studies and 8 case-control studies, high 
levels of EPA are associated with lower risk of colorectal cancer in a dose-dependent manner.12 
In an RCT of patients with familial adenomatous polyposis, EPA supplement of 2 g daily for 6 
months reduces the number and size of polyps by 20-30%, an effect comparable to that by the 
FDA-approved prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2) 
inhibitors.13 
2.5.2
Immune mechanisms underlying the anticancer effect of EPA
The anticancer effect of EPA may be related to its multifaceted immunomodulatory activity 
mediated by alterations in lipid raft structure and changes in fatty acid composition of cell 
membranes.14 These changes alter gene expression and the activities of several key immune 
signaling pathways, such as PTGS2 and nuclear factor kappa-light-chain-enhancer of activated B 
cells (NF-κB), and modify downstream metabolite production, including a decrease in 
proinflammatory eicosanoids (e.g., prostaglandin E2; PGE2) and an increase in pro-resolving 
lipid mediators (e.g., resolvin and protectin).14-19 Of note, PGE2 has been suggested as one of the 
critical pathways that govern tumor-mediated immune dysfunction and contributes to a shift in 
the tumor microenvironment from anti-tumor responses to immunosuppressive responses.20 On 
the other hand, increasing data suggest that EPA may revert the immune suppression mediated 
by Treg cells.21,22 Treatment of Tregs with EPA decreases the immune suppressive activity by 
downregulating inhibitory precursors.21 In experimental colorectal cancer models, fish oil exerts 
its antitumor effect by altering Tregs and their cytokine repertoire (e.g., reduction of IL-10 
expression).22 In an RCT of 60 patients with solid tumors, supplementation of EPA increased the 

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ratio of T-helper cells to T-suppressor cells in the peripheral blood of the malnourished patients, 
and prolonged the survival of all the patients.23 
Our recent observational study indicated that high EPA intake was associated with lower risk of 
colorectal cancer that is infiltrated with a high density of FOXP3+ T cells, but not tumors with 
low densities of FOXP3+ T cells.24 Similar benefit was observed when EPA and DHA were 
examined separately. Because FOXP3 is a prerequisite transcription factor for the 
immunosuppressive function of Treg cells, we further examined the in vitro effect of EPA on the 
suppressive activity of colorectal Treg cells against proliferation of T effector (Teff) cells.24 
Consistent with our human findings, we found that EPA treatment decreased the suppressive 
activity of FOXP3+ Treg cells in a dose-dependent manner. Of note, the suppressive effect of 
EPA leveled off at around 50 uM, which is within the normal range of EPA in human plasma 
samples, highlighting the human relevance of these experimental findings.
2.5.3
Gut microbiome in relation to EPA and colorectal cancer 
Increasing data suggest that the gut microbiota play an important role in colorectal cancer 
development and progression, possibly by influencing both local and systemic immune 
response.25 Among all potential shaping factors, diet is particularly important for the gut 
microbial composition and function. Compared to other types of fat, EPA has been associated 
with enhanced intestinal microbiota diversity and EPA-rich diet ameliorates omega-6 fatty acids- 
or antibiotics-induced dysbiosis.26-29 Animal studies indicate that EPA supplements increase the 
abundance of anti-inflammatory bacteria, such as lactic acid-producing bacteria (mainly 
Lactobacillus and Bifidobacterium), and decrease the abundance of immunosuppressive and pro-
inflammatory bacteria, such as F. nucleatum, Escherichia coli, and Akkermansia. Administration 
of Bifidobacterium has been shown to improve patients’ response to cancer immunotherapy,30 
whereas increased abundance of F. nucleatum has been linked to dampened antitumor immune 
response and poor prognosis of colorectal cancer.31-33 Therefore, it is possible that the beneficial 
effect of EPA for colorectal cancer may be mediated by modulation of the gut microbial 
composition and function that in turn shape the host immune response.  
 
3.
PARTICIPANT SELECTION
Patients that meet the eligibility criteria will be identified through investigators during their routine 
clinical practice, supplemented by a periodic query of the MGH endoscopy (Provation) and 
pathology database. Patients also may be identified using Natural Language Processing clinical 
software that queries medical records. The MGH gastroenterologist that performed the procedure 
or saw the patient for outpatient care as follow-up to the patient’s colonoscopy procedure will be 
notified and asked for permission to contact their patient. We plan to recruit patients who have 
undergone a colonoscopy by an MGH gastroenterologist either in the ambulatory clinic or for an 
endoscopic procedure. In some cases, these physicians may not be members of the study team. 
However, non-study physicians will not perform study-related procedures. 
For appropriate patients, we will identify their preference for contact about research studies 
(Research Opportunities Direct to You [RODY] status) through queries of the Research Patient 
Data Registry (RPDR). Participants with a RODY YES response will be contacted by a letter 
(attached recruitment letter for RODY YES patients) from the study PI. Patients with a RODY 

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NO response or without RODY designation will be contacted by a different letter (attached 
recruitment letter for RODY NO patients). If the colonoscopy was performed at MGH, after an 
adenoma is resected from a patient at MGH, all gastroenterologists routinely contact their 
patients by mail with the results of their pathology. The recruitment letter will be included with 
the pathology results when possible, or it will be sent separately after the pathology report. If the 
colonoscopy and polypectomy were performed at another hospital, the MGH gastroenterologist 
will confirm the prior diagnosis of an adenoma through review of pathology reports. Their 
treating MGH gastroenterologist, regardless of whether the physician is study personnel, will 
sign the letter. If no response is received to this recruitment letter within two weeks (10 business 
days) a follow-up phone call or email will be placed by a research assistant/study coordinator 
(phone script/sample email attached). In some cases, the treating gastroenterologist may 
approach the patient following the colonoscopy that identified a polyp if he/she feels the patient 
would be a good candidate for the study. In these cases, the patients may not receive a 
recruitment letter as the primary method of contact. Instead, they will be directly contacted by 
phone or email (see call script/sample email) from a member of the research team prior to 
mailing the letter (if requested). Potential participants will have additional opportunities to ask 
any questions by contacting an investigator or a trained research coordinator. If the participant is 
interested in participating, eligibility will be assessed by the study coordinator (see script) and 
confirmed by the investigator through an additional check of the patients’ medical record (see 
“Investigator Representation for Review of Protected Health Information Preparatory to 
Research” form). If eligibility is confirmed, the patient will be scheduled for their initial study 
visit and registered with OnCore by the study staff. The informed consent form will be mailed to 
the patient prior to the initial visit so that he or she can read it in detail, if requested by the 
participant. Eligible participants will provide written informed consent at the baseline visit prior 
to participation in the study. The consenting process will be performed by one of the trial’s 
clinical investigators. 
3.1
Eligibility Criteria 
Participants must meet the following criteria on screening examination to be eligible to 
participate in the study:
3.1.1
Underwent screening or surveillance colonoscopy with removal of at least one adenoma; 
3.1.2
Age 18-80 years. 
This study will only include adult participants because colorectal carcinogenesis in children 
is more likely to be related to a cancer predisposition syndrome with distinct biological 
mechanisms compared with sporadic colorectal cancer in adults. Patients over age 80 will 
not be enrolled since the benefits and risks of AMR101 over the age of 80 have not yet 
been well-characterized.

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3.1.3
The effects of AMR101 on the developing human fetus are unknown.  For this reason, 
women of child-bearing potential must agree to use adequate contraception (hormonal or 
barrier method of birth control; abstinence) prior to study entry and for the duration of 
study participation.  Should a woman become pregnant or suspect she is pregnant while 
she is participating in this study, she should inform her treating physician immediately.
3.1.4
Subjects must be able and willing to follow study procedures and instructions.
3.1.5
Ability to understand and the willingness to sign a written informed consent document.
3.2
Exclusion Criteria
Participants who exhibit any of the following conditions at screening will not be eligible for 
admission into the study.
3.2.1
Currently using or have used any fish oil supplement at any dose more than once per week 
within the last month.
3.2.2
Regularly consuming more than three servings of fish per week.
3.2.3
History of allergic reactions attributed to fish or compounds of similar chemical or biologic 
composition to omega-3 fatty acid. 
3.2.4
Diagnosis of inflammatory bowel disease, liver or kidney disease, bleeding diathesis
3.2.5
Any prior diagnosis of gastrointestinal cancer (including esophageal, small intestine, 
colon, pancreatic), or any diagnosis of other cancers (with the exception of non-
melanoma skin) in which there has been any active treatment within the last three years.
3.2.6
Known diagnosis of Familial Adenomatous Polyposis (FAP) or Hereditary Non-Polyposis 
Colorectal Cancer (HNPCC, Lynch Syndrome).
3.2.7
Any adenoma that was not completely removed during previous colonoscopy.
3.2.8
Known bleeding tendency/condition (e.g. von Willebrand disease) or history of peptic ulcer 
or gastrointestinal bleed requiring hospitalization, endoscopic complications, or 
contraindication to colonoscopy.
3.2.9
Current use of anticoagulant therapies, including Heparin, Warfarin, Dalteparin sodium, 
Bivalirudin, Argatroban, Lepirudin, Heparin Sodium, Heparin/Dextrose, and an 
unwillingness or inability to discontinue anticoagulants.
3.2.10 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 that would limit compliance with study requirements.

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3.2.11 Inability or unwillingness to abstain from non-protocol use of fish oil supplements or to 
provide blood, urine or stool samples or colon biopsies during the study.
3.2.12 Participants who are receiving any other investigational agents.
3.2.13 Inability or unwillingness to swallow pills.
3.2.14 Pregnant or breastfeeding.
The effects of AMR101 on the developing human fetus are unknown. For this reason, 
women of child-bearing potential must agree to use adequate contraception (hormonal or 
barrier method of birth control; abstinence) prior to study entry and for the duration of 
study participation. Should a woman become pregnant or suspect she is pregnant while she 
is participating in this study, she should inform her treating physician immediately. 
Similarly, lactating women are excluded from this study because there is an unknown but 
potential risk of adverse events in nursing infants secondary to treatment of the mother 
with AMR101. Consequently, breastfeeding should be discontinued if the mother is 
enrolled on the study.
3.2.15 Known positive test for human immunodeficiency virus (HIV), hepatitis C virus, or 
acute or chronic hepatitis B infection.
Participants with these infections are ineligible because they are at increased risk of 
significant complications in the perioperative period, and because fresh tissue from 
patients with these infections cannot be harvested for research purposes, per 
institutional policy. Appropriate studies will be undertaken in participants receiving 
combination antiretroviral therapy when indicated.
3.3
Inclusion of Women and Minorities
Women and minorities will be eligible for this study without alteration in eligibility criteria. 
Enrollment of these underrepresented populations to this trial will be encouraged.
4.
REGISTRATION PROCEDURES
4.1
General Guidelines for DF/HCC Institutions
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registrations must occur prior to the initiation of protocol therapy. Any participant not 
registered to the protocol before protocol therapy begins will be considered ineligible and 
registration will be denied.
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist.
The study staff will register the participant in OnCore.

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Following registration, participants may begin protocol-specific therapy and/or intervention. 
Issues that would cause treatment delays should be discussed with the Overall Principal 
Investigator (PI). If the subject does not receive protocol therapy following registration, the 
subject must be taken off-study in the CTMS (OnCore) with an appropriate date and reason 
entered.  
4.2
Registration Process for DF/HCC Institutions
Applicable DF/HCC policy (REGIST-101) must be followed.  
4.3
General Guidelines for Other Investigative Sites
N/A.
4.4
Registration Process for Other Investigative Sites
N/A.
5.
TREATMENT PLAN
5.1
Treatment Regimen
After study registration, eligible participants will be assigned unique study identification numbers. 
The investigator will contact the MGH Research Pharmacy for drug dispensation. The MGH 
Research Pharmacy will dispense study capsules, obtained from Amarin Pharma (Bedminster, NJ), 
containing 0.5-gram of AMR101. The assigned dosage will not change over the course of the study 
unless dose reduction is indicated (See Section 6). The first dose of the study medication will be 
given to patients after the initial flexible sigmoidoscopy. Participants will be expected to take 4 
capsules orally, twice a day, until the return for their final visit (minimum 8 weeks, maximum 12 
weeks). The final visit, 8-12 weeks from the baseline visit, will be scheduled during or before the 
baseline visit. The MGH Research Pharmacy will assign the participant three bottles of AMR101 
each containing 240 capsules (12 weeks, 8 capsules/day) and participants will return any unused 
capsules and the bottle to the study staff at their final visit. The bottle will be labeled with their 
participant ID. The participant number must be recorded on the drug dispensation form  and in the 
participants case report form (CRF). These numbers are unique to each participant and must not 
be re-assigned. Remaining capsules will be counted as a measure of compliance, the number 
recorded and then the remaining capsules will be immediately destroyed. Weekly calls will be 
used to monitor adherence and adverse events. Patients who initiate fish oil supplements during 
the study will be withdrawn and an exit visit performed.
Compliance will be assessed based on drug diary and pills counts, as well as blood measurement 
of EPA levels. Reported adverse events and potential risks are described in Section 7. No 
investigational or commercial agents or therapies may be administered with the intent to treat the 
participants. The principal investigator will review subjects’ data periodically throughout the 
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approved protocol and will address any data-related issues that might arise.
5.2
Pre-Treatment Criteria
N/A
5.3
Agent Administration
5.3.1
AMR101
Participants will self-administer eight AMR101 capsules by mouth (4 capsules twice a day), and 
will be instructed to keep a drug diary. The dose should be taken with food and a full glass of 
water. If a daily dose is missed, the dose should be skipped. A dose is considered missed if more 
than 24 hours has elapsed since the prior dose.
5.4
Definition of Dose-Limiting Toxicity (DLT) 
N/A
5.5
General Concomitant Medication and Supportive Care Guidelines
Throughout the study, treating investigators may prescribe concomitant medications or treatments 
deemed necessary to provide adequate supportive care. However, participants may not receive: 

Other investigational agents;

Anticoagulant therapies, including Heparin, Warfarin, Dalteparin sodium, Bivalirudin, 
Argatroban, Lepirudin, Heparin Sodium, Heparin/Dextrose, and other anticoagulant 
therapies;

Any supplements that contain EPA.
All prescribed and non-prescription concomitant medications that are ingested, applied, or injected 
on an ongoing basis from signing of the informed consent, as well as changes in such concomitant 
medications, and any new concomitant medication taken while the participant is on study, should 
be recorded in the medical record. Concomitant medications for medically significant adverse 
events, which are ongoing at the end of study treatment and considered related to study treatment, 
should be followed until the adverse event is resolved or considered stable.
5.6
Criteria for Taking a Participant Off Protocol Therapy
Duration of study drug administration will be at minimum 8 weeks and no more than 12 weeks 
from the initial visit. Duration of administration will equal the number of days between initial and 
final visit. Study drug administration will continue until the final visit or until one of the following 
criteria applies:

Intercurrent illness that prevents further administration of study drug

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
Unacceptable adverse event(s)

Participant demonstrates an inability or unwillingness to comply with the oral 
medication regimen and/or compliance requirements

Participant decides to withdraw from the protocol therapy

General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator

Participant self-administers any additional non-study fish oil supplements.
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the eCRF. Alternative care options will be discussed with the participant.
When a participant is removed from protocol therapy and/or is off of the study, the relevant Off-
Treatment/Off-Study information will be updated in OnCore.
In the event of unusual or life-threatening complications, participating investigators must 
immediately notify the Overall PI, Dr. Andrew Chan at (617) 726-3212 or 
ACHAN@mgh.harvard.edu
5.7
Duration of Follow Up
Participants will be monitored closely until they complete the study. Participants who have 
completed the study will be those that have returned for the final visit and returned all necessary 
study materials including unused capsules, pill bottle and questionnaires. Participants will be 
followed for one month by phone after removal from protocol therapy or until death, whichever 
occurs first.  Participants removed from protocol therapy for unacceptable adverse event(s) will be 
followed until resolution or stabilization of the adverse event.
5.8
Criteria for Taking a Participant Off Study
Participants will be removed from study when any of the following criteria apply:

Lost to follow-up

Withdrawal of consent for data submission

Participant receives any concomitant medication or treatment included in the list in 5.5 
describing medications or treatments the participant may NOT receive

Participant demonstrates an inability or unwillingness to comply with the study regimen 
and/or compliance requirements

Participant decides to withdraw from the protocol therapy

General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator

Death

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The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the eCRF.
The study team will ensure Off Treatment/Off Study information is updated in OnCore in 
accordance with DF/HCC policy REGIST-101.
6.
DOSING DELAYS/DOSE MODIFICATIONS
Dose reduction will be made as indicated in the following table(s). The descriptions and grading 
scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) 
version 5.0 will be utilized for dose reduction. A copy of the CTCAE version 5.0can be 
downloaded from the CTEP website
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
When the study staff is informed of a GI-related adverse event, dose reduction may be 
considered, according to the following dose de-escalation schema. 
Dose Level
AMR 101 Dose
Initial Dose
4 grams/day, for the full course of the 
intervention
Level -1
2 grams/day, for the rest of the intervention 
period
 
Criteria for treatment reduction and suggested guidelines for the management of GI toxicities 
related to AMR 101 are summarized below. These general guidelines may be modified at the 
discretion of the investigator based on best clinical judgment at that time, but the reason for these 
reductions must be documented in the medical record. Any GI toxicities related to AMR 101 
should be managed according to standard medical practice. These dose reductions are guidance 
for clinicians for use of this standard therapy.
Participants who report GI-related adverse events including but not limited to the adverse events 
described in the tables below will be offered dose reduction as well as the option to withdraw 
from the study.
Nausea
Management/Next Dose for AMR 101
≤ Grade 1
Reduce to dose level -1
Grade 2
Reduce to dose level -1
Grade 3
Reduce to dose level -1
Grade 4
Reduce to dose level -1
Vomiting
Management/Next Dose for AMR 101
≤ Grade 1
Reduce to dose level -1
Grade 2
Reduce to dose level -1
Grade 3
Reduce to dose level -1

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Vomiting
Management/Next Dose for AMR 101
Grade 4
Reduce to dose level -1
Diarrhea
Management/Next Dose for AMR 101
≤ Grade 1
Reduce to dose level -1
Grade 2
Reduce to dose level -1
Grade 3
Reduce to dose level -1
Grade 4
Reduce to dose level -1
Constipation
Management/Next Dose for AMR 101
≤ Grade 1
Reduce to dose level -1
Grade 2
Reduce to dose level -1
Grade 3
Reduce to dose level -1
Grade 4
Reduce to dose level -1
Stomach pain
Management/Next Dose for AMR 101
≤ Grade 1
Reduce to dose level -1
Grade 2
Reduce to dose level -1
Grade 3
Reduce to dose level -1
Grade 4
Reduce to dose level -1
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Section 7.2) will determine whether the event requires expedited reporting in 
addition to routine reporting.
7.1
Expected Toxicities
AMR101 may produce a hypersensitivity reaction in individuals with known hypersensitivity to 
shellfish/fish. Because AMR101 contains ethyl esters of omega-3 fatty acid, patients taking other 
anticoagulants may be at risk to develop a prolonged bleeding time; the risk of active bleeding is 
unknown.
7.1.1
Adverse Events List(s)
7.1.1.1
Adverse Event List(s) for AMR101
AMR101 is usually well tolerated. According to Micromedex, arthralgia was one of the most 
commonly reported adverse events associated with the use of AMR101 occurring in 2.3% (14/622) 
of patients compared with 1% (3/109) in placebo in pooled data from 2 double-blind clinical trials, 
which included patients with triglyceride values of 200 to 2000 mg/dL. An additional adverse 
effect from clinical studies was oropharyngeal pain. Mild to moderate GI-related adverse events 

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that include stomach pain, nausea, loss of appetite, constipation, or diarrhea have also been 
reported by participants on this and other studies.13
7.2
Adverse Event Characteristics

CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
5.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 5.0.  A copy of the CTCAE version 5.0 can be 
downloaded from the CTEP web site 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Q
uick_Reference_8.5x11.pdf

For expedited reporting purposes only:  
-
AEs for the agent(s) that are listed above should be reported only if the adverse event 
varies in nature, intensity or frequency from the expected toxicity information which 
is provided.
-
Other AEs for the protocol that do not require expedited reporting are outlined in the 
next section (Expedited Adverse Event Reporting) under the sub-heading of Protocol-
Specific Expedited Adverse Event Reporting Exclusions.

Attribution of the AE:
-
Definite – The AE is clearly related to the study treatment.
-
Probable – The AE is likely related to the study treatment.
-
Possible – The AE may be related to the study treatment.
-
Unlikely – The AE is doubtfully related to the study treatment.
-
Unrelated – The AE is clearly NOT related to the study treatment.
7.3
Expedited Adverse Event Reporting 
7.3.1
Investigators must report to the Overall PI any serious adverse event (SAE) that occurs 
after the initial dose of study treatment, during treatment, or within 30 days of the last 
dose of treatment on the local institutional SAE form. 
7.3.2
DF/HCC Expedited Reporting Guidelines
Investigative sites within DF/HCC will report AEs directly to the DFCI Office for 
Human Research Studies (OHRS) per the DFCI IRB reporting policy. 
7.3.3
Protocol-Specific Expedited Adverse Event Reporting Exclusions
For this protocol only, the AEs/grades listed below do not require expedited reporting to 
the Overall PI or the DFCI IRB.  However, they still must be reported through the routine 
reporting mechanism (i.e. case report form).

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CTCAE 
SOC
Adverse 
Event
Grade
Hospitalization/ 
Prolongation of 
Hospitalization
Attribution
Comments
N/A
7.4
Expedited Reporting to Hospital Risk Management
Participating investigators will report to their local Risk Management office any participant 
safety reports or sentinel events that require reporting according to institutional policy. 
7.5
Routine Adverse Event Reporting
All Adverse Events must be reported in routine study data submissions to the Overall PI on the 
toxicity case report forms.  AEs reported through expedited processes (e.g., reported to the 
IRB, FDA, etc.) must also be reported in routine study data submissions.
8.
PHARMACEUTICAL INFORMATION
A list of the adverse events and potential risks associated with the investigational agent 
administered in this study can be found in Section 7.1.
8.1
AMR101
8.1.1
Description
The active ingredient of AMR101 is EPA ethyl ester (icosapent ethyl). The molecular formula is 
C22H34O2, and its molecular weight is 330.51. Its structural formula is:
Icosapent ethyl is de-esterified during absorption into the active metabolite EPA. EPA is 
absorbed in the small interesting through the thoracic duct lymphatic system into the circulation. 

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The Tmax of EPA was about 5 hours following oral administration of icosapent ethyl. EPA is 
metabolized in the liver by beta-oxidation into acetyl Coenzyme A, which is converted into 
energy via the Krebs cycle. Cytochrome P450-mediated metabolism is a minor pathway of 
elimination. The total plasma clearance of EPA at steady state is 684 mL/hr. The elimination 
half-life of EPA is about 89 hours.
8.1.2
Form
The trial AMR101 (icosapent ethyl) capsules are manufactured and supplied by Amarin Pharma 
(Bedminster, NJ) as 0.5-gram amber-colored soft-gelatin capsules. The capsules will be ordered 
by and shipped to the MGH Research Pharmacy where they will be packaged in three pill bottles 
each containing 240 capsules, which are sufficient for 12-week supply where 8 capsules are 
taken daily.
8.1.3
Storage and Stability
AMR101 capsules should be stored at room temperature. When stored under this condition, it 
has been indicated that the product is stable and non-reactive.
8.1.4
Compatibility
N/A
8.1.5
Handling
N/A
8.1.6
Availability
AMR101 capsules are manufactured by Amarin Pharma, Inc. and commercially available in the 
US as VASCEPA (icosapent ethyl). Ordering and packaging of AMR101 will be handled by the 
MGH Research Pharmacy free of charge to the participants. 
8.1.7
Preparation
AMR101 will be supplied by Amarin Pharma and packaged by the MGH Research Pharmacy in 
identical three bottles with a count of 240 capsules in each bottle.
8.1.8
Administration
AMR101 will be administered orally with 4 capsules twice a day at approximately the same 
times of day.
8.1.9
Ordering
AMR101 is a commercially available agent obtained from Amarin Pharma. It is the same agent 

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as that used for the EPA for Metastasis Trial 2 (EMT2), a randomized trial of omega-3 fatty 
acid among 448 patients with colorectal cancer liver metastases. AMR101 will be supplied by 
Amarin Pharma and stored in the MGH research pharmacy per institutional guidelines. The 
research pharmacist will be responsible for dispensing the study medication to participants. 
8.1.10 Accountability
The investigator, or a responsible party designated by the investigator (i.e., research 
pharmacist), must maintain a careful record of the inventory and disposition of the agent using 
the NCI Drug Accountability Record Form (DARF) or another comparable drug accountability 
form.  (See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.)
8.1.11 Destruction and Return
At the end of the study, unused supplies of AMR101 should be destroyed according to 
institutional policies. Destruction will be documented in the Drug Accountability Record Form. 
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES
9.1
Biomarker Studies
9.1.1
Background
We hypothesize that EPA modulates the gut microbiota to preserve gut immune homeostasis and 
suppress colorectal cancer. The proposed study will assess the influence of EPA treatment on the 
tissue fatty acid composition, the gut microbiome and metabolome, gene expression in colorectal 
cancer tissue, and urinary PGE-M. In doing so, we aim to provide causality for the anticancer 
mechanisms of EPA established by other studies. We and others have put forth considerable 
efforts to determine measurable biomarkers implicated in the microbial and immune mechanisms 
linking EPA and colorectal cancer. A discussion of these biomarkers and their significance is 
provided below:
9.1.1.1
EPA in colorectal tissue
Increasing data suggest that the anticancer effect of EPA relates to its incorporation into the cell 
membrane phospholipids and alteration of the structure and function of the lipid rafts.34,35 High 
intake of EPA results in increased incorporation of EPA and replacement of arachidonic acid 
(AA) in membrane phospholipids. This alteration has two consequences: 1) suppressed 
biosynthesis of AA-derived inflammatory eicosanoids in favor of EPA-derived prostanoids; 2) 
enhanced competition with omega-6 fatty acids for cyclooxygenases that leads to higher 
production of anti-inflammatory lipid mediators. Given the critical role of inflammation in 
colorectal cancer development and progression, EPA-induced shift from pro-inflammatory to 
anti-inflammatory metabolite profiles may protect against tumorigenesis.36 Moreover, EPA can 
modify membrane lipid rafts, which are lipidic microdomains consisting mainly of 
sphingomyelin, cholesterol, and glycerophospholipids that hold many signaling proteins.37 These 

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microdomains are involved in a myriad of cellular functions, such as signal transduction, 
membrane trafficking, neuronal differentiation, and entry of pathogens and toxins into the cells. 
EPA has been shown to target a number of lipid raft proteins, thereby modulating the signaling 
pathways critical for tumor growth and metastasis. In particular, given the role of lipid rafts as 
platforms for cell activation in the immune system,38 animal and in vitro studies indicate that 
EPA can influence intracellular signaling in T lymphocytes by modification of the architecture of 
lipid rafts.39-41 However, the relevance of these findings to the immunomodulatory effect of EPA 
in human cancer remains to be determined. Therefore, to establish the mechanistic foundation for 
testing downstream immune modulation, we will assess the effect of AMR101 treatment on the 
fatty acid composition in colorectal tissues as the primary endpoint. 
9.1.1.2
EPA and the gut microbiome
Increasing data indicate that the gut microbiota mediates the health effects of dietary components 
by shaping the intestinal and systemic immune response.42-45 As summarized in Table 2, both 
animal and human studies support the role of EPA in influencing the gut microbial composition 
and function. Compared to other types of fat, EPA have been associated with enhanced intestinal 
microbiota diversity and EPA-rich diet ameliorates ω-6 PUFAs- or antibiotics-induced 
dysbiosis.26-29 Animal studies indicate that EPA supplements increase the abundance of anti-
inflammatory bacteria, such as lactic acid-producing bacteria (mainly Lactobacillus and 
Bifidobacteria), and decrease the abundance of immunosuppressive and pro-inflammatory 
bacteria, such as F. nucleatum, Escherichia coli, and Akkermansia. Likewise, changes in the gut 
microbiota have been linked to CRC, with an enrichment of F. nucleatum, Escherichia coli, and 
Enterobacteriaceae; and a depletion of Bifidobacterium and Streptococcus in CRC patients.46-54 
Microbes may affect carcinogenesis by influencing DNA damage repair,55-57 cell 
proliferation,58,59 host metabolism,60-62 and immune responses.31,63-65 Moreover, recent data show 
that commensal bacteria, such as Bifidobacteria, may modulate tumor immune 
microenvironment and improve the efficiency of CTLA-4 or PD-L1 blockade.30,66,67
Table 2. Summary of EPA-related gut microbial changes in human and animal models and their link to host 
immunity and CRC
Bacteria
Mechanistic link to host immunity and CRC 
Increased by EPA
Lactobacillus27,68
Probiotics with anti-inflammatory and anticancer activity16,69,70
Bifidobacteria12, 16, 17
Probiotics with anti-inflammatory activity;71-74 improve immunotherapy 
efficacy30
Butyrivibrio75
Produce short-chain fatty acids that have immunomodulatory and anticancer 
effect60,76,77
Decreased by EPA
F. nucleatum78
Generate a tumor-permissive microenvironment and promote CRC31,47,59,79
Escherichia coli78,80
Some pathogenic strains are associated with inflammatory bowel disease and 
CRC81,82
Akkermansia75,83
Impair gut barrier integrity, induce inflammation and CRC84,85
9.1.1.3
EPA, microbiota, and stool metabolomics

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Anaerobic gut bacteria, including some species of Lactobacillus, have been implicated in 
polyunsaturated fatty acid saturation, a detoxifying mechanism that transforms bacterial growth-
inhibiting polyunsaturated fatty acids into less toxic fatty acids, such as hydroxyl fatty acids.86-92 
These microbial metabolites may help preserve intestinal barrier integrity, reduce oxidative 
stress, and lower inflammation.93,94 Interestingly, Lactobacillus is selectively enriched by EPA 
(Table 2), suggesting a reciprocal mechanism by which gut microbes adapt to host dietary 
change with functional consequences for host health. Moreover, a cross-feeding effect has been 
noted between human Bifidobacterium, which produces lactate and acetate, and the butyrate-
producing species, such as Eubacterium rectale, which convert lactate to butyrate.95-97 Butyrate, 
a short-chain fatty acid, has potent anti-inflammatory98 and potential anti-CRC properties.61,99 
In addition to microbial products, host cell metabolites can be shed into the gut lumen, including 
a series of EPA-derived eicosanoids (e.g., PGE3, lipoxin, leukotriene [LT], thromboxane [TX], 
and hydroxyeicosatetraenoic acid [HETE]) and lipid mediators (including resolvin, protectin, and 
maresin) that possess anti-inflammatory activities and may mediate the anticancer effect of 
EPA.100-105 Of note, some lipid mediators, such as resolvin, may alter the gut microbial 
composition by stimulating epithelial expression of microbial regulators, such as intestinal 
alkaline phosphatase (IAP),106 a transmissible factor with lipopolysaccharides (LPS)-detoxifying 
activities.78  Thus, stool metabolomics provides a critical tool to examine the products and 
functionality of the diet – gut microbiota – host interaction. 
9.1.1.4
Gene expression profile – immune and inflammatory pathways
Pro-inflammatory cytokines such as TNF-α and IL-6 are central players in colon cancer through 
activation of the pro-inflammatory transcription factors NF-κB and STAT3.107,108 EPA have 
multifaceted roles in immune regulation.15 High EPA intake has been associated with lower 
circulating inflammatory markers (e.g., TNFα and IL6).109,110 In a pilot study of women at high 
risk for breast cancer, EPA supplements decreased breast tissue expression of chemokine C-C 
motif chemokine ligand 2 (CCL2),111 a cancer-promoting cytokine.112 As described above, our 
prior study indicated that EPA might protect against colon cancer through downregulation of the 
immunosuppressive Treg cells.24 However, despite these data, human data linking EPA to 
colorectal cancer-related immune and inflammatory pathways are lacking. Therefore, to fill this 
knowledge gap and extend our prior findings, we will examine the effect of EPA treatment on 
the gene expression profile of colorectal tissue. We hypothesize that EPA treatment reduces the 
gene expression of inflammatory cytokines and chemokines (e.g., TNF, IL6 and CCL2).
9.1.1.5.
Urinary PGE-M
A mechanism underlying EPA’s immunomodulatory effect may be related to reduced synthesis 
of prostaglandin E2 (PGE2) by tumor epithelial cells and/or tumoral stromal cells.35 PGE2 
promotes tumor cell survival and is higher in cancer cells than in normal cells.113  PGE2 
contributes to a shift in the tumor microenvironment from anti-tumor to tumor-permissive.20 
PGE2 may promote immunosuppression by inducing FOXP3 expression in naïve T cells114-116 
and inducing differentiation of myeloid-derived suppressor cells (MDSCs). Both 
animal17,18,117,118 and human RCTs14,119 indicate that EPA treatment reduces circulating and 
colonic tissue PGE2 levels. A clinical trial showed that EPA treatment reduced urinary 
prostaglandin metabolites (PGE-M), a marker of systemic PGE2 production, by 27.6% compared 
with a 27.9% increase in the placebo group.120 

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9.1.2
Study design
Using our gastroenterology practice population, we will implement a prospective clinical trial to 
measure the effect of EPA on tissue fatty acid composition, the gut microbiome and metabolome, 
gene expression profile, and urinary PGE-M. At MGH, we will target 80 individuals over a two 
year period. Eligible patients will have had a previous colonoscopy at MGH and had at least one 
adenoma removed during the previous procedure. Eligible patients must meet all eligibility 
requirements and none of the exclusion criteria as outlined in Section 3: Eligibility criteria. 
9.1.2.1
Prior to the initial visit
No bowel preparation will be necessary for the procedure since the sigmoidoscope will only be 
advanced to the distal sigmoid colon.
9.1.2.2
Initial (baseline) Visit
At the initial visit,  the study physician will obtain written, informed consent for the study as well 
as a standard clinical consent for a flexible sigmoidoscopy. Following OnCore registration, 
participants will be asked to complete with a study coordinator a brief lifestyle questionnaire as 
well as a nutritional survey on a research iPad via VioScreenTM, a validated web-based dietary 
assessment tool developed by Viocare, Inc (See appendix for VioScreen security overview, the 
assessed food list, and sample screens).121 Patients will undergo measurements of height, weight, 
waist and hip circumference and provide a blood and urine specimen. A study gastroenterologist 
will then perform a flexible sigmoidoscopy, advancing to the level of the distal sigmoid colon. No 
more than a total of 24 mucosal biopsies will be taken from the rectum and sigmoid and 
immediately placed in collection tubes. In the MGH GI Unit, we routinely perform endoscopic 
biopsies regardless of concurrent AMR101 use, a practice consistent with recommended 
guidelines.122 A study of the safety of multiple endoscopic biopsies in research subjects from a 
National Institutes of Health series found that performing large numbers of endoscopic biopsies 
(mean number = 38.2 ± 15.6 biopsies per procedure) are “well tolerated and appears to have no 
more than minimal risk without appreciably increasing the risk of otherwise routine endoscopy.”123 
Furthermore, there is no statistically significant association between risk of complications and the 
number of biopsies, type of procedure (flexible sigmoidoscopy vs. colonoscopy), colonic location 
of biopsy, operator, or polypectomy.123 The number of biopsies is also consistent with our existing 
study protocols and has never been associated with any adverse events (See: “Endoscopy Protocol: 
Tissue Specific Immunity Against HIV-1”; PI: Kwon, Ragon Institute). We have estimated that 
24 biopsies will be necessary to complete the proposed analyses. The RNAseq experiments 
proposed require a minimum of 4 pinch biopsies for each sample. The additional biopsies will be 
required for validation experiments based on the results of genomic and metagenomic analyses 
using targeted sequencing approaches. Due to the small number of cells obtained from pinch 
biopsies and the input requirements for these assays, we may use tissue culture techniques to 
expand cell populations. This will allow us to perform these comprehensive analyses without 
additional burden to the participants (i.e. increasing tissue yields by using larger or more biopsies). 
During flexible sigmoidoscopy, stool will be aspirated through the endoscope or using a Roth net 
and snap-frozen. . Following the visit, questionnaire data will be transferred to the Partners secure 

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REDCap electronic database system. Also, REDCap will be used to store data from the study 
including endoscopy data, medical history information, sample collection, and more.
Participants will be provided with $100 (US) compensation and free parking for up to 4 hours for 
this initial visit.  The final visit will be scheduled with the patient at his or her convenience.
9.1.2.3
Final Visit
Participants will return for a second and final visit between 8 and 12 weeks from their initial visit. 
An abbreviated lifestyle questionnaire and a full nutritional survey via VioScreen will be 
administered to update information from the baseline visit. Participants will also provide blood 
and urine samples and undergo a second flexible sigmoidoscopy procedure with mucosal biopsies. 
A bowel preparation will not be necessary for the follow-up flexible sigmoidoscopy. Up to 24 
mucosal biopsies will be taken, as described for the baseline visit. A stool specimen will also be 
collected, as previously described. 
Participants will be provided with an additional $100 (US) compensation and free parking for up 
to 4 hours for this final visit. Total compensation will equal $200 (US) for successful completion 
with the study. 
9.1.3
Collection and Processing of Biospecimens
Immediately following each flexible sigmoidoscopy, urine specimens will be aliquoted and 
blood specimens will be centrifuged into plasma and buffy coat. Stool specimens will be stored 
in a cryovial and immediately frozen. Colon biopsies will be immediately frozen for future 
RNA-seq analysis. Yield from these procedures typically exceeds requirements for RNA-seq. 
Thus, any excess colon tissue will be banked for future studies. All aliquots of stool, urine, 
plasma, buffy coat, and tissue will be frozen at -80°C until analysis. We may access FFPE blocks 
of polyps/adenomas removed during the participant’s qualifying colonoscopy with 
accompanying pathology reports to correlate our findings with tissue-specific markers in the 
original adenoma.
Blood samples will be collected in two-6 mL EDTA/purple top tubes at each visit, stored on ice 
until processing and freezing (within 3 hours of collection). Immediately upon receipt, urine 
specimens will be aliquoted into 1.2 mL aliquots and stool samples will be homogenized, 
aliquoted into 100 mg aliquots, and frozen for long-term storage. Fresh tissue will be collected in 
the pathology laboratory immediately following resection, transported on wet ice and 
immediately flash frozen in liquid nitrogen. We may access archival formalin-fixed, paraffin-
embedded (FFPE) blocks of any biopsies or tissue specimens collected during the participant’s 
procedures that led to their diagnosis of colon cancer if the diagnostic procedures occurred at 
MGH. If the diagnostic procedures occurred at an external site, formal requests in writing will be 
made to the corresponding pathology department to release and ship tissue to study staff. We will 
request whole tissue blocks or 10-15 unstained slides. All samples will be transported to the 
laboratory of Dr. Andrew Chan and labeled with a study identification number, the date, and the 
type of specimen. Dr. Chan’s laboratory will supervise all storage of coded specimens. All 
endpoint analysis will occur at the end of the trial as described for each endpoint below:

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9.1.4
Tissue EPA Analysis (primary endpoint)
Fatty acid will be extracted from the biopsy tissues using the established protocol.124 We will use 
gas chromatography-mass spectrometry to measure EPA content in to assess the incorporation of 
AMR101 into local tissue.
9.1.4.1
Collection of Specimens
Mucosal biopsy samples will be collected at both the initial and final visit.
9.1.4.2
Handling of Specimens
Mucosal biopsy specimens will be stored frozen and sent in batches to the Harvard T.H. Chan 
School of Public Health (HSPH) for fatty acid analysis. 
9.1.4.3
Shipping of Specimens
Mucosal biopsy specimens will be sent on dry ice to Dr. Frank Sacks at the following address:
Dr. Frank Sacks
Department of Nutrition
Harvard T.H. Chan School of Public Health 
665 Huntington Avenue
Boston, Massachusetts, 02115
9.1.4.4
Site performing study
Dr. Sacks Laboratory at HSPH.
9.1.5
Gut microbiome analysis
We will perform metagenomic and metatranscriptomic sequencing of microbial DNA and RNA 
on pre- and post-treatment stool samples to examine the biomolecular mechanisms by which gut 
microbial activity may be altered or respond to AMR101 treatment. Stool samples will be sent, 
coded, to the Broad Institute for processing and analysis.
9.1.5.1
 Collection of Biospecimens
Stool will be collected using a Roth net or aspirated through the endoscope during flexible 
sigmoidoscopy at both the initial and final visit.
9.1.5.2
 Handling of Biospecimens
An aliquot (~200 mg) of stool will be put in tubes provided by the Broad Institute and sent back 
for processing and analysis. The remaining stool will be frozen in a 15 mL conical tube at -80°C. 

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9.1.5.3
Shipping of Specimens
Stool specimens will be shipped on dry ice overnight to:
Broad Institute
Attn: BSP platform
301 Binney Street Lab 5076
Cambridge, MA 02142
617-714-8952
9.1.5.4
Site Performing Study
The Broad Institute
9.1.6
Stool metabolomics analysis
We will perform non-targeted global metabolomics and lipidomics analysis on pre- and post-
treatment stool samples to examine the changes in the stool metabolite profile. Stool samples 
will be sent, coded, to Metabolon, Inc for processing and analysis. The global metabolomics 
analysis uses ultra-high performance liquid chromatography/tandem mass spectrometry 
(UHPLC-MS/MS) and GC/MS platforms.125-127 The lipidomics analysis uses the TrueMassTM 
Complex Lipid Panel that can provide data of both quantitative composition and complete 
speciation for over 1,110 individual lipid species.128
9.1.6.1
 Collection of Biospecimens
As described in 9.1.5.1.
9.1.6.2
 Handling of Biospecimens
An aliquot (~200 mg) of stool will be put in tubes provided by the Metabolon, Inc and sent back 
for processing and analysis. The remaining stool will be frozen in a 15 mL conical tube at -80°C. 
9.1.6.3
Shipping of Specimens
Stool specimens will be shipped on dry ice overnight to:
Metabolon, Inc 
617 Davis Drive, Suite 100
Morrisville, NC  27560
Phone: 919.572.1711
Fax: 919.572.1721
9.1.6.4
Site Performing Study

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Metabolon, Inc
9.1.7
Red Blood Cell EPA measurement
EPA will be extracted from the baseline and post-treatment blood samples using the established 
protocol129 and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to 
assess circulating EPA levels. Red blood cell (RBC) membrane EPA content is widely accepted 
as the surrogate biomarker of EPA content in other tissues, including colon.129-131
9.1.7.1
Collection of Biospecimens
Blood samples will be collected at both the initial and final visits.
9.1.7.2
Handling of Biospecimens
Two blood samples in 6 mL EDTA/purple top tubes will be collected at each visit for a total of 
24 mLs and stored on ice until processing.  
9.1.7.3
Processing of Biospecimens
Blood will be spun in a centrifuge at 2000 x g for 20 minutes at 4°C within three hours of 
venipuncture (or in accordance with manufacturer instructions). After centrifugation, isolated red 
blood cells will be stored at -80°C until fatty acid extraction, buffy coat and plasma will be 
aliquoted and stored at -80ºC for future research purposes. 
9.1.7.4
Shipping of Specimens
Blood samples will be processed and stored in the laboratory of Dr. Andrew Chan at MGH. A 
1.2 mL aliquot of red blood cells will be shipped on dry ice overnight to Dr. Frank Sacks at the 
following address:
Dr. Frank Sacks
Department of Nutrition
Harvard T.H. Chan School of Public Health 
665 Huntington Avenue
Boston, Massachusetts, 02115
9.1.7.5
Site Performing Study
Sacks Laboratory, HSPH
9.1.8
RNA sequencing of tumor tissue
We will perform RNA-seq analysis to profile gene expression in the mucosal tissue collected 
before and after AMR101 treatment using our validated platform (Illumina Transcriptome 
Capture protocol) at the Broad Institute.

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9.1.8.1
Collection of Biospecimens
As described in 9.1.4.1.
9.1.8.2
Handling of Biospecimens
As described in 9.1.4.2.
9.1.8.3
Processing of Biospecimens
Tissue will be flash-frozen in liquid nitrogen within 30 minutes of collection and stored at -80ºC 
for future research purposes.  At the conclusion of the trial, one portion will be processed to 
extract RNA using the RNAeasy micro kit. Total RNA or converted cDNA libraries will be 
stored at -80ºC prior to sending for sequencing. 
9.1.8.4
Shipping of Specimens
RNA/cDNA samples will be sent on ice to the Broad Institute for RNA-seq analysis at the 
following address:
Broad Genomics
Attn: Genomics Platform
320 Charles St.
Cambridge, MA, 02141.
9.1.8.5
Site Performing Study
The Broad Institute
9.1.9
Analysis of Urinary PGE-M
We will use mass spectroscopy to measure PGE-M in single batches of pre- and post-treatment
urine in the Eicasonoid core laboratory of Dr. Ginger Milne at Vanderbilt University.
9.1.9.1
Collection of Biospecimens
Urine samples will be collected at both the initial and final visit.
9.1.9.2
Handling of Biospecimens
Samples will be placed in a refrigerator within 2 hours of collection, and transferred to -80°C
within 4 days of collection. Urine will be split into 1.2 mL aliquots in eppendorf tubes prior to
freezing. Samples will be stored at -80°C until analysis.

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9.1.9.3
Shipping of Specimens
A 1.2 mL aliquot of each urine sample will be shipped on dry ice overnight to Dr. Ginger Milne
at the following address:
Attn: Ginger L. Milne, Ph.D.
Vanderbilt University Medical Center
561 Preston Research Building
Nashville, TN 37232-6602 USA
9.1.9.4
Site Performing Study
Eicasonoid Core Laboratory (Ginger Milne), Vanderbilt University
9.2
Laboratory Correlative Studies
N/A
9.3
Special Studies
N/A
10. STUDY CALENDAR
Initial 
Visit
W
k
1
W
k
2
W
k
3
W
k
4
W
k
5
W
k
6
W
k
7
W
k
8
W
k
9
Wk
10
Wk
11
Wk
12
Final 
Visita 
AMR101 Daily
Taken twice daily until final visit
Return 
unused 
drugs
Informed consent
X
Demographics
X
X
Lifestyle 
Questionnaire
X
X
Nutritional Survey
X
X
Height
X
Weight
X
Waist & Hip 
Circumference
X

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Flexible 
Sigmoidoscopy
X
X
Tissue Biopsy 
Specimens 
Collected
X
X
Blood Samples
X
X
Urine Samples
X
X
Stool Samples
X
X
Drug Diary
X
Used daily until final visit
X
Drug Compliance 
Callsb
X
X
X
X
X
X
X
X
X
X
X
X
Adverse event 
evaluationb
X
X
X
X
X
X
X
X
X
X
X
X
X
a: Final visit will be scheduled during the initial visit and will occur a minimum of 8 weeks 
and maximum of 12 weeks after the initial visit.
b:  Once weekly, participants will be contacted by phone to monitor adherence to drug 
administration and check for adverse events.
11. MEASUREMENT OF EFFECT
This study only uses laboratory-based endpoints to measure the effect of AMR101 treatment. 
There are no clinically observable metrics that will be used as a primary or secondary endpoint or 
primary or secondary effect measure in this study. The primary efficacy endpoint is EPA levels in 
mucosal tissue. A detailed discussion of laboratory endpoints is included in section 9.
12. DATA REPORTING / REGULATORY REQUIREMENTS
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements).
12.1
Data Reporting
12.1.1 Method
The Primary Investigator and the study team will collect, manage, and perform quality checks on 
the data for this study using RedCap.
12.1.2 Responsibility for Data Submission

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N/A
12.2
Data Safety Monitoring
The Primary Investigator and the study team will regularly review and monitor toxicity and 
accrual data from this study. The study team is composed of GI physicians, study coordinators, 
pharmacists and biostatisticians with direct experience in cancer clinical research.  Information 
that raises any questions about participant safety will be addressed with the IRB. 
The Primary Investigator will review study protocol periodically throughout the duration of the 
study for quality, validity and integrity assurance. Information to be provided to the Primary 
Investigator may include: up-to-date participant accrual; current dose level information; DLT 
information; all grade 2 or higher unexpected adverse events that have been reported; summary 
of all deaths occurring within 30 days of intervention for Phase I or II protocols; for gene therapy 
protocols, summary of all deaths while being treated and during active follow-up; any response 
information; audit results, and a summary provided by the study team. Other information (e.g. 
scans, laboratory values) will be provided upon request. 
12.3
Multicenter Guidelines
N/A
12.4
Collaborative Agreements Language
N/A
13. STATISTICAL CONSIDERATIONS
This study is a single-arm clinical trial that will measure the effects of EPA treatment on specific 
biomarkers related to EPA incorporation, gut microbiota, gut metabolome, and immune response. 
In doing so, we aim to provide causality for the microbial and immune mechanisms that potentially 
mediate the beneficial effect of EPA for colorectal cancer prevention. We and others have put forth 
considerable effort to determine measurable biomarkers implicated in colorectal carcinogenesis. 
A comprehensive discussion of these biomarkers and their significance is provided in Section 9. 
We will recruit 80 participants who recently underwent removal of their adenomatous polyps at 
MGH to conduct a single-arm trial of EPA (AMR101, 4 g/day) treatment for 2 months. The 
rationale for selection of this EPA preparation is because it 1) is FDA approved; 2) has good 
comparability across production lots; 3) has been previously used in several trials of EPA.111,132 
The rationale for selection of this dose is because it is 1) the prescription dose approved by FDA; 
2) consistent with other RCTs of EPA in colorectal neoplasia;13,120,133 and 3) can provide the best 
balance of efficacy and safety. 
13.1
Study Design/Endpoints
This study is a single-arm trial of EPA treatment in patients with a history of colorectal adenoma. 

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The single-arm design is chosen for the following reasons: 1) Based on our prior experience, 
short-term dietary supplementation is unlikely to lead to substantial changes in lifestyle. 2) Any 
major lifestyle changes will be documented by the detailed lifestyle questionnaires and 
nutritional surveys administered at both the baseline (pre-intervention) and final (post-
intervention) visits to allow for statistical adjustment in the analysis. Moreover, as described in 
the protocol, we will measure EPA content in red blood cells to further assess compliance and 
changes in omega-3 fatty acid intake. 3) Given the high dose (4 g/day) of highly purified and 
specially formulated omega-3 fatty acid used in the study (AMR101), even modest changes in 
lifestyle is unlikely to introduce significant confounding on the intervention effect of interest. For 
example, one serving (3 oz.) of Atlantic salmon only contains 1-2g of marine omega-3 fatty 
acids, and individuals have to consume at least 3 servings of salmon per day to achieve the 
intervention dose used in the study. 4) Given the fishy odor and oily appearance of the study 
drug, our MGH Research Pharmacy is unable to provide a visually identical, blinded placebo 
capsule for the study drug. Therefore, given all of these considerations as well as the biomarker-
based nature of the study, we believe that the single-arm design is the best use of the limited 
resources for the study.
The primary aim is to assess the effect of AMR101 treatment on our primary efficacy endpoint, 
mucosal tissue EPA. A detailed discussion of the background of this biomarker is provided in 
Section 9.1.1.1. Briefly, we will determine the effect of AMR101 at standard dose on mucosal 
tissue EPA content through pre- and post-treatment comparison. The rationale for selection of 
this primary endpoint is to specifically test the effect of EPA on colonic tissue, which will build 
the mechanistic foundation for testing downstream endpoints within the local environment. 
Additional endpoints will include the gut microbiome, metabolome, urinary PGE-M and gene 
expression profile of mucosal tissue. Our study design for each of these endpoints is 
comprehensively discussed in Section 9. Statistical considerations relating to these endpoints are 
provided in the following sections. 
13.2
Sample Size, Accrual Rate and Study Duration
Patients will be accrued in one stage with no early stopping rules. The total sample size is 80 
patients and we expect accrual to be 6-7 participants per month and complete the enrollment 
process within one year. This accrual rate is based upon accruals under our recently completed 
IRB-approved protocol (Protocol # 14-496; Dr. Chan, PI) for a similar trial of aspirin in patients 
with history of colorectal adenoma. Each patient will be followed for at minimum 8 weeks and at 
maximum 12 weeks. This total sample size accounts for participant drop out, as endoscopy studies 
typical experience a drop-out rate of approximately 10%.
The sample size is based upon the primary efficacy endpoint of M3PUFA supplement at 4 g/d 
increasing mucosal MO3PUFA composition from pre- to post-treatment period by a mean of 3.0%, 
according to previous studies.13,134 The null hypothesis is H0: Δpost – Δpre = 0 versus HA: Δpost – Δpre 
≠ 0, where Δpre and Δpost are the measurement of mucosal MO3PUFA composition before and after 
the intervention, respectively. Based on prior studies, we assume the standard deviation (SD) of 
9.0% for the paired difference in MO3PUFA measurement. With 80% power and the significance 
level of 0.05 in a two-sided paired t-test, we will require 73 participants to detect the mean change 
of 3.0% in MO3PUFA level from pre- to post-treatment period. To account for possible drop out 

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at 10%, we plan to enroll 80 patients.
Our accrual target rates for ethnic and racial minorities will likely be consistent with the rate at 
which we see these minorities in the gastroenterology practice at MGH. (See table on next page)
Accrual Targets
Sex/Gender
Ethnic Category
Females
Males
Total
Hispanic or Latino
4
+
4
=
8
Not Hispanic or Latino
36
+
36
=
72
Ethnic Category: Total of all subjects
40
+
40
=
80
Racial Category
American Indian or Alaskan Native
1
+
1
=
2
Asian
2
+
2
=
4
Black or African American
4
+
4
=
8
Native Hawaiian or other Pacific 
Islander
1
+
1
=
2
White
32
+
32
=
64
Racial Category: Total of all subjects
40
+
40
=
80
13.3
Stratification Factors
N/A
13.4
Interim Monitoring Plan
N/A
13.5
Analysis of Primary Endpoints
Analysis of mucosal tissue EPA (primary endpoint): Our primary biostatistician, Dr. Rosner, will 
supervise statistical analyses assessing the change in EPA levels in mucosal tissue after AMR101 
treatment, using a paired t-test. In secondary analyses, we will use mixed linear regression 
models to adjust for duration of treatment and other major factors that may influence the gut 
microbiome, including aspirin use, antibiotic use, and major dietary and lifestyle patterns (e.g., 
fiber intake, physical activity, body mass index, smoking, and alcohol intake). A robust variance 
estimate will be used to eliminate any normality assumptions for the residuals.
13.6
Analysis of Secondary Endpoints
Microbiome analysis: Dr. Huttenhower will supervise the gut microbiome analysis. Stool 
specimens 
collected 
before 
and 
after 
treatment 
will 
undergo 
metagenomics 
and 
metatranscriptomics sequencing to assess the influence of EPA intervention on the gut microbial 

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composition and function. We will use the computational tools developed by Dr. Huttenhower, a 
leading authority in microbiome data analysis (http://huttenhower.sph.harvard.edu/biobakery).  
We will associate treatment status with the microbial operational taxonomic units (OTUs) 
determined by MetaPhlAn135 using biomarker discovery tests: LEfSe (Linear Discriminant 
Analysis Effect Size),136 a univariate class comparison method; and MaAsLin (Multivariate 
Analysis by Linear models),137 a multivariate test that finds associations between clinical metadata 
and microbial abundance or function after adjusting for other covariates. Metatranscriptomic data 
will be analyzed using HUM138AnN,139 a software package designed to efficiently and accurately 
define the abundance of microbial pathways within the community, and then linked to treatment 
status by linear mixed effects model. We expect to find that, after EPA treatment, study participants 
will have increased gut bacterial diversity, experience beneficial changes in microbiota composition, 
and demonstrate decreased transcriptional activity of inflammatory pathways that have been 
previously correlated with an increased risk of colon cancer. 
Analysis of gene expression profiles: Dr. Huttenhower will supervise gene expression analysis. 
Tissues collected after treatment will undergo RNA-sequencing analysis to assess the influence 
of EPA intervention on gene expression profile of colon cancer. Samples are quantified and 
RNA quality is evaluated using Agilent's Bioanalyzer 2100. The percentage of fragments with a 
size greater than 200 nucleotides (DV200) are calculated; samples with a DV200 score < 30% are 
included, as the likelihood of success is dramatically reduced. 100ng of RNA is used as the input 
for first strand cDNA synthesis using Superscript III reverse transcriptase (Life Technologies) 
and Illumina’s TruSeq Stranded Total RNA Sample Prep Kit. Synthesis of the second strand of 
cDNA is followed by indexed adapter ligation. Subsequent PCR amplification enriches for 
adapted fragments. The amplified libraries are quantified using a Qubit assay (Life 
Technologies) and assessed for quality on an Agilent Technologies 2100 Bioanalyzer (DNA 
1000 chip). 200ng of each cDNA library is combined into two 4-plex pools. Illumina’s Coding 
Exome Oligos that target the exome are added and hybridized on a thermacycler. Following 
hybridization, streptavidin beads are used to capture probes that are hybridized. Two wash steps 
effectively remove any non-specifically bound products. These hybridization, capture, and wash 
steps are repeated to assure high specificity. A second round of amplification enriches the 
captured libraries. qPCR is performed on the pooled libraries and normalized to 2nM. The 
normalized, pooled libraries are loaded onto a HiSeq2500 for a target of 50 million 2x76bp 
paired reads per sample. RNA-Seq libraries will be analyzed using Tophat, Cufflinks140 and 
other new statistical packages to identify consistent differences in the end-of-treatment changes 
in transcript levels between tissues before and after EPA treatment at pre-determined FDRs on 
the order of 0.01. Stringent tests for statistically significant differences in RNA levels between 
samples are built into the algorithms for RNA-seq analysis.
Analysis of stool metabolomics: We will use both the non-targeted Global Metabolomics and the 
TrueMass™ Complex Lipid Panel (Metabolon, Inc.) to examine the effect of intervention on the 
stool metabolite profile.  The Global Metabolomics platform quickly and accurately identifies 
and quantitates more than 1,000 metabolites with less than 4.5% median process variability and 
is compatible with almost any sample type. The TrueMass™ Complex Lipid Panel provides 
absolute quantitation of 14 lipid classes, including principle phospholipid, sphingolipid and 
neutral lipid classes. It also provides molecular species concentrations and complete fatty acid
composition of each lipid class, thereby offering unparalleled insight into the lipidome. The 

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resultant data will be imported into Surveyor Web Tools (Metabolon, Inc.) for intuitive data 
visualization and easier interpretation. We will also use the MetaboLync® Client Portal provided 
by Metabolon, Inc. to perform comprehensive, in-depth analysis, such as the heat map tool, the 
pathway enrichment tool, and the Pathway Visualizations Tool. The treatment effect on 
metabolites (log-transformed whenever needed) will be first assessed by paired t-test and then by 
linear mixed effects model that includes treatment status (pre/post-treatment), duration of 
treatment, and other major factors that may influence the metabolomics, including major dietary 
and lifestyle patterns (e.g., fiber intake, physical activity, body mass index, smoking, and alcohol 
intake).
Analysis of Urinary PGE-M: We will measure PGE-M (11a-hydroxy-9, 15-dioxo-2,3,4,5-
tetranor-prostane 1,20-dioic acid) levels in baseline/pre-treatment and post-treatment urine 
samples using liquid chromatography-mass spectrometry (LC/MS) as previously described.141 
0.5mL of each urine specimen will be stabilized by conversion to the O-methyloxime derivative 
and purified by C18 solid phase extraction with subsequent addition of the O-methyloxime 
derivatized deuterium-labeled internal standard (custom synthesis). Liquid chromatography (LC) 
will be performed on an Acquity BEH C18 column (2.0 × 50 mm, 1.7μm particle, Waters 
Corporation, Milford, MA, USA) connected to a Waters Acquity I-Class UPLC system and 
delivered to a Waters Xevo TQ-S Micro triple quadrupole mass spectrometer (Waters 
Corporation, Milford, MA, USA). 
 
13.7
Reporting and Exclusions
Participants who never start protocol therapy or do not return for final flexible sigmoidoscopy and 
sample collection will be considered inevaluable and will be excluded from all analyses. 
13.7.1 Evaluation of Toxicity
N/A
13.7.2 Evaluation of the Primary Efficacy Endpoint
The primary endpoints will be determined for all patients who complete the final flexible 
sigmoidoscopy and sample collection, regardless of whether the patient complied with study 
drug use.144
13.7.3 Evaluation of the Secondary Endpoints
The secondary endpoints will be determined for all patients who complete the final flexible 
sigmoidoscopy and sample collection, regardless of whether the patient complied with study 
drug use.144
14. PUBLICATION PLAN
The results should be made public within 24 months of reaching the end of the study. The end of 
the study is the time point at which the last data items are to be reported, or after the outcome 

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data are sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate 
and Study Duration. If a report is planned to be published in a peer-reviewed journal, then that 
initial release may be an abstract that meets the requirements of the International Committee of 
Medical Journal Editors. A full report of the outcomes should be made public no later than three 
(3) years after the end of the study. 

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REFERENCES
1.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-
30.
2.
Siegel R, Desantis C, Jemal A. Colorectal cancer statistics, 2014. CA: a cancer journal 
for clinicians. 2014;64(2):104-117.
3.
Del Gobbo LC, Imamura F, Aslibekyan S, et al. omega-3 Polyunsaturated Fatty Acid 
Biomarkers and Coronary Heart Disease: Pooling Project of 19 Cohort Studies. JAMA 
Intern Med. 2016;176(8):1155-1166.
4.
Larsson SC, Orsini N, Wolk A. Long-chain omega-3 polyunsaturated fatty acids and risk 
of stroke: a meta-analysis. European journal of epidemiology. 2012;27(12):895-901.
5.
Forouhi NG, Imamura F, Sharp SJ, et al. Association of Plasma Phospholipid n-3 and n-6 
Polyunsaturated Fatty Acids with Type 2 Diabetes: The EPIC-InterAct Case-Cohort 
Study. PLoS Med. 2016;13(7):e1002094.
6.
Grosso G, Pajak A, Marventano S, et al. Role of omega-3 fatty acids in the treatment of 
depressive disorders: a comprehensive meta-analysis of randomized clinical trials. PLoS 
One. 2014;9(5):e96905.
7.
Calder PC. Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or 
pharmacology? Br J Clin Pharmacol. 2013;75(3):645-662.
8.
Sanders TA. Protective effects of dietary PUFA against chronic disease: evidence from 
epidemiological studies and intervention trials. Proc Nutr Soc. 2014;73(1):73-79.
9.
Nabavi SF, Bilotto S, Russo GL, et al. Omega-3 polyunsaturated fatty acids and cancer: 
lessons learned from clinical trials. Cancer Metastasis Rev. 2015;34(3):359-380.
10.
Cockbain AJ, Toogood GJ, Hull MA. Omega-3 polyunsaturated fatty acids for the 
treatment and prevention of colorectal cancer. Gut. 2012;61(1):135-149.
11.
Calder PC. n-3 fatty acids, inflammation and immunity: new mechanisms to explain old 
actions. The Proceedings of the Nutrition Society. 2013;72(3):326-336.
12.
Yang B, Wang FL, Ren XL, Li D. Biospecimen long-chain N-3 PUFA and risk of 
colorectal cancer: a meta-analysis of data from 60,627 individuals. PLoS One. 
2014;9(11):e110574.
13.
West NJ, Clark SK, Phillips RK, et al. Eicosapentaenoic acid reduces rectal polyp 
number and size in familial adenomatous polyposis. Gut. 2010;59(7):918-925.
14.
Bartram HP, Gostner A, Scheppach W, et al. Effects of fish oil on rectal cell 
proliferation, mucosal fatty acids, and prostaglandin E2 release in healthy subjects. 
Gastroenterology. 1993;105(5):1317-1322.
15.
Calder PC. Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms 
and clinical relevance. Biochim Biophys Acta. 2015;1851(4):469-484.
16.
Piazzi G, D'Argenio G, Prossomariti A, et al. Eicosapentaenoic acid free fatty acid 
prevents and suppresses colonic neoplasia in colitis-associated colorectal cancer acting on 
Notch signaling and gut microbiota. International journal of cancer Journal international 
du cancer. 2014;135(9):2004-2013.
17.
Jiang Y, Djuric Z, Sen A, et al. Biomarkers for personalizing omega-3 fatty acid dosing. 
Cancer Prev Res (Phila). 2014;7(10):1011-1022.
18.
Calviello G, Di Nicuolo F, Gragnoli S, et al. n-3 PUFAs reduce VEGF expression in 
human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-
1alpha induction pathway. Carcinogenesis. 2004;25(12):2303-2310.

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
40
19.
Nowak J, Weylandt KH, Habbel P, et al. Colitis-associated colon tumorigenesis is 
suppressed in transgenic mice rich in endogenous n-3 fatty acids. Carcinogenesis. 
2007;28(9):1991-1995.
20.
Wang D, DuBois RN. An inflammatory mediator, prostaglandin E2, in colorectal cancer. 
Cancer journal. 2013;19(6):502-510.
21.
Yessoufou A, Ple A, Moutairou K, Hichami A, Khan NA. Docosahexaenoic acid reduces 
suppressive and migratory functions of CD4+CD25+ regulatory T-cells. Journal of lipid 
research. 2009;50(12):2377-2388.
22.
Renuka, Agnihotri N, Singh AP, Bhatnagar A. Involvement of Regulatory T Cells and 
Their Cytokines Repertoire in Chemopreventive Action of Fish Oil in Experimental 
Colon Cancer. Nutrition and cancer. 2016;68(7):1181-1191.
23.
Gogos CA, Ginopoulos P, Salsa B, Apostolidou E, Zoumbos NC, Kalfarentzos F. Dietary 
omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and 
prolong survival for severely ill patients with generalized malignancy: a randomized 
control trial. Cancer. 1998;82(2):395-402.
24.
Song M, Nishihara R, Cao Y, et al. Marine omega-3 Polyunsaturated Fatty Acid Intake 
and Risk of Colorectal Cancer Characterized by Tumor-Infiltrating T Cells. JAMA 
oncology. 2016.
25.
Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell host & microbe. 
2014;15(3):317-328.
26.
Ghosh S, Molcan E, DeCoffe D, Dai C, Gibson DL. Diets rich in n-6 PUFA induce 
intestinal microbial dysbiosis in aged mice. Br J Nutr. 2013;110(3):515-523.
27.
Ghosh S, DeCoffe D, Brown K, et al. Fish oil attenuates omega-6 polyunsaturated fatty 
acid-induced dysbiosis and infectious colitis but impairs LPS dephosphorylation activity 
causing sepsis. PLoS One. 2013;8(2):e55468.
28.
Kaliannan K, Wang B, Li XY, Bhan AK, Kang JX. Omega-3 fatty acids prevent early-
life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity. Int J Obes 
(Lond). 2016.
29.
Patterson E, RM OD, Murphy EF, et al. Impact of dietary fatty acids on metabolic 
activity and host intestinal microbiota composition in C57BL/6J mice. Br J Nutr. 2014:1-
13.
30.
Sivan A, Corrales L, Hubert N, et al. Commensal Bifidobacterium promotes antitumor 
immunity and facilitates anti-PD-L1 efficacy. Science. 2015;350(6264):1084-1089.
31.
Kostic AD, Chun E, Robertson L, et al. Fusobacterium nucleatum potentiates intestinal 
tumorigenesis and modulates the tumor-immune microenvironment. Cell host & microbe. 
2013;14(2):207-215.
32.
Mima K, Nishihara R, Qian ZR, et al. Fusobacterium nucleatum in colorectal carcinoma 
tissue and patient prognosis. Gut. 2015.
33.
Mima K, Sukawa Y, Nishihara R, et al. Fusobacterium nucleatum and T Cells in 
Colorectal Carcinoma. JAMA Oncol. 2015;1(5):653-661.
34.
Yaqoob P. The nutritional significance of lipid rafts. Annu Rev Nutr. 2009;29:257-282.
35.
Larsson SC, Kumlin M, Ingelman-Sundberg M, Wolk A. Dietary long-chain n-3 fatty 
acids for the prevention of cancer: a review of potential mechanisms. The American 
journal of clinical nutrition. 2004;79(6):935-945.
36.
Wang D, Dubois RN. Eicosanoids and cancer. Nature reviews Cancer. 2010;10(3):181-
193.

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
41
37.
Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 
2000;1(1):31-39.
38.
Janes PW, Ley SC, Magee AI, Kabouridis PS. The role of lipid rafts in T cell antigen 
receptor (TCR) signalling. Semin Immunol. 2000;12(1):23-34.
39.
Stulnig TM, Berger M, Sigmund T, Raederstorff D, Stockinger H, Waldhausl W. 
Polyunsaturated fatty acids inhibit T cell signal transduction by modification of 
detergent-insoluble membrane domains. J Cell Biol. 1998;143(3):637-644.
40.
Zeyda M, Staffler G, Horejsi V, Waldhausl W, Stulnig TM. LAT displacement from lipid 
rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated 
fatty acids. J Biol Chem. 2002;277(32):28418-28423.
41.
Zeyda M, Szekeres AB, Saemann MD, et al. Suppression of T cell signaling by 
polyunsaturated fatty acids: selectivity in inhibition of mitogen-activated protein kinase 
and nuclear factor activation. J Immunol. 2003;170(12):6033-6039.
42.
Tilg H, Moschen AR. Food, immunity, and the microbiome. Gastroenterology. 
2015;148(6):1107-1119.
43.
Dorrestein PC, Mazmanian SK, Knight R. Finding the missing links among metabolites, 
microbes, and the host. Immunity. 2014;40(6):824-832.
44.
Sharon G, Garg N, Debelius J, Knight R, Dorrestein PC, Mazmanian SK. Specialized 
metabolites from the microbiome in health and disease. Cell Metab. 2014;20(5):719-730.
45.
Maslowski KM, Mackay CR. Diet, gut microbiota and immune responses. Nat Immunol. 
2011;12(1):5-9.
46.
Castellarin M, Warren RL, Freeman JD, et al. Fusobacterium nucleatum infection is 
prevalent in human colorectal carcinoma. Genome Res. 2012;22(2):299-306.
47.
Kostic AD, Gevers D, Pedamallu CS, et al. Genomic analysis identifies association of 
Fusobacterium with colorectal carcinoma. Genome Res. 2012;22(2):292-298.
48.
Ahn J, Sinha R, Pei Z, et al. Human gut microbiome and risk for colorectal cancer. J Natl 
Cancer Inst. 2013;105(24):1907-1911.
49.
Tahara T, Yamamoto E, Suzuki H, et al. Fusobacterium in colonic flora and molecular 
features of colorectal carcinoma. Cancer Res. 2014;74(5):1311-1318.
50.
Chen W, Liu F, Ling Z, Tong X, Xiang C. Human intestinal lumen and mucosa-
associated microbiota in patients with colorectal cancer. PLoS One. 2012;7(6):e39743.
51.
McCoy AN, Araujo-Perez F, Azcarate-Peril A, Yeh JJ, Sandler RS, Keku TO. 
Fusobacterium is associated with colorectal adenomas. PLoS One. 2013;8(1):e53653.
52.
Allali I, Delgado S, Marron PI, et al. Gut Microbiome Compositional and Functional 
Differences between Tumor and Non-tumor Adjacent Tissues from Cohorts from the US 
and Spain. Gut microbes. 2015:0.
53.
Feng Q, Liang S, Jia H, et al. Gut microbiome development along the colorectal 
adenoma-carcinoma sequence. Nature communications. 2015;6:6528.
54.
Nakatsu G, Li X, Zhou H, et al. Gut mucosal microbiome across stages of colorectal 
carcinogenesis. Nature communications. 2015;6:8727.
55.
Guerra L, Guidi R, Frisan T. Do bacterial genotoxins contribute to chronic inflammation, 
genomic instability and tumor progression? The FEBS journal. 2011;278(23):4577-4588.
56.
Cuevas-Ramos G, Petit CR, Marcq I, Boury M, Oswald E, Nougayrede JP. Escherichia 
coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. 
Proc Natl Acad Sci U S A. 2010;107(25):11537-11542.
57.
Nougayrede JP, Homburg S, Taieb F, et al. Escherichia coli induces DNA double-strand 

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
42
breaks in eukaryotic cells. Science. 2006;313(5788):848-851.
58.
Sears CL. Enterotoxigenic Bacteroides fragilis: a rogue among symbiotes. Clin Microbiol 
Rev. 2009;22(2):349-369, Table of Contents.
59.
Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW. Fusobacterium nucleatum 
promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via 
its FadA adhesin. Cell host & microbe. 2013;14(2):195-206.
60.
Singh N, Gurav A, Sivaprakasam S, et al. Activation of Gpr109a, receptor for niacin and 
the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. 
Immunity. 2014;40(1):128-139.
61.
Donohoe DR, Holley D, Collins LB, et al. A gnotobiotic mouse model demonstrates that 
dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-
dependent manner. Cancer discovery. 2014;4(12):1387-1397.
62.
Belcheva A, Irrazabal T, Robertson SJ, et al. Gut microbial metabolism drives 
transformation of MSH2-deficient colon epithelial cells. Cell. 2014;158(2):288-299.
63.
Hu B, Elinav E, Huber S, et al. Microbiota-induced activation of epithelial IL-6 signaling 
links inflammasome-driven inflammation with transmissible cancer. Proc Natl Acad Sci 
U S A. 2013;110(24):9862-9867.
64.
Warren RL, Freeman DJ, Pleasance S, et al. Co-occurrence of anaerobic bacteria in 
colorectal carcinomas. Microbiome. 2013;1.
65.
Irrazabal T, Belcheva A, Girardin SE, Martin A, Philpott DJ. The multifaceted role of the 
intestinal microbiota in colon cancer. Mol Cell. 2014;54(2):309-320.
66.
Iida N, Dzutsev A, Stewart CA, et al. Commensal bacteria control cancer response to 
therapy by modulating the tumor microenvironment. Science. 2013;342(6161):967-970.
67.
Vetizou M, Pitt JM, Daillere R, et al. Anticancer immunotherapy by CTLA-4 blockade 
relies on the gut microbiota. Science. 2015;350(6264):1079-1084.
68.
Caesar R, Tremaroli V, Kovatcheva-Datchary P, Cani PD, Backhed F. Crosstalk between 
Gut Microbiota and Dietary Lipids Aggravates WAT Inflammation through TLR 
Signaling. Cell metabolism. 2015;22(4):658-668.
69.
Li J, Sung CY, Lee N, et al. Probiotics modulated gut microbiota suppresses 
hepatocellular carcinoma growth in mice. Proc Natl Acad Sci U S A. 2016.
70.
van Baarlen P, Wells JM, Kleerebezem M. Regulation of intestinal homeostasis and 
immunity with probiotic lactobacilli. Trends in immunology. 2013;34(5):208-215.
71.
Furrie E, Macfarlane S, Kennedy A, et al. Synbiotic therapy (Bifidobacterium 
longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative 
colitis: a randomised controlled pilot trial. Gut. 2005;54(2):242-249.
72.
Veiga P, Gallini CA, Beal C, et al. Bifidobacterium animalis subsp. lactis fermented milk 
product reduces inflammation by altering a niche for colitogenic microbes. Proc Natl 
Acad Sci U S A. 2010;107(42):18132-18137.
73.
O'Mahony L, McCarthy J, Kelly P, et al. Lactobacillus and bifidobacterium in irritable 
bowel syndrome: symptom responses and relationship to cytokine profiles. 
Gastroenterology. 2005;128(3):541-551.
74.
Saez-Lara MJ, Gomez-Llorente C, Plaza-Diaz J, Gil A. The role of probiotic lactic acid 
bacteria and bifidobacteria in the prevention and treatment of inflammatory bowel 
disease and other related diseases: a systematic review of randomized human clinical 
trials. Biomed Res Int. 2015;2015:505878.
75.
Pusceddu MM, El Aidy S, Crispie F, et al. N-3 Polyunsaturated Fatty Acids (PUFAs) 

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
43
Reverse the Impact of Early-Life Stress on the Gut Microbiota. PLoS One. 
2015;10(10):e0139721.
76.
Chang PV, Hao L, Offermanns S, Medzhitov R. The microbial metabolite butyrate 
regulates intestinal macrophage function via histone deacetylase inhibition. Proc Natl 
Acad Sci U S A. 2014;111(6):2247-2252.
77.
Sharon G, Garg N, Debelius J, Knight R, Dorrestein PC, Mazmanian SK. Specialized 
metabolites from the microbiome in health and disease. Cell metabolism. 2014;20(5):719-
730.
78.
Kaliannan K, Wang B, Li XY, Kim KJ, Kang JX. A host-microbiome interaction 
mediates the opposing effects of omega-6 and omega-3 fatty acids on metabolic 
endotoxemia. Sci Rep. 2015;5:11276.
79.
Gur C, Ibrahim Y, Isaacson B, et al. Binding of the Fap2 protein of Fusobacterium 
nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack. 
Immunity. 2015;42(2):344-355.
80.
Li Q, Zhang Q, Wang C, et al. Fish oil enhances recovery of intestinal microbiota and 
epithelial integrity in chronic rejection of intestinal transplant. PLoS One. 
2011;6(6):e20460.
81.
Bonnet M, Buc E, Sauvanet P, et al. Colonization of the human gut by E. coli and 
colorectal cancer risk. Clin Cancer Res. 2014;20(4):859-867.
82.
Martin HM, Campbell BJ, Hart CA, et al. Enhanced Escherichia coli adherence and 
invasion in Crohn's disease and colon cancer. Gastroenterology. 2004;127(1):80-93.
83.
Noriega BS, Sanchez-Gonzalez MA, Salyakina D, Coffman J. Understanding the Impact 
of Omega-3 Rich Diet on the Gut Microbiota. Case Rep Med. 2016;2016:3089303.
84.
Zackular JP, Baxter NT, Iverson KD, et al. The gut microbiome modulates colon 
tumorigenesis. mBio. 2013;4(6):e00692-00613.
85.
Ijssennagger N, Belzer C, Hooiveld GJ, et al. Gut microbiota facilitates dietary heme-
induced epithelial hyperproliferation by opening the mucus barrier in colon. Proc Natl 
Acad Sci U S A. 2015;112(32):10038-10043.
86.
Kishino S, Takeuchi M, Park SB, et al. Polyunsaturated fatty acid saturation by gut lactic 
acid bacteria affecting host lipid composition. Proc Natl Acad Sci U S A. 
2013;110(44):17808-17813.
87.
Kishino S, Ogawa J, Yokozeki K, Shimizu S. Metabolic diversity in biohydrogenation of 
polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid 
production. Appl Microbiol Biotechnol. 2009;84(1):87-97.
88.
Hirata A, Kishino S, Park SB, Takeuchi M, Kitamura N, Ogawa J. A novel unsaturated 
fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus. J 
Lipid Res. 2015;56(7):1340-1350.
89.
Desbois AP, Smith VJ. Antibacterial free fatty acids: activities, mechanisms of action and 
biotechnological potential. Appl Microbiol Biotechnol. 2010;85(6):1629-1642.
90.
Sakurama H, Kishino S, Mihara K, et al. Biohydrogenation of C20 polyunsaturated fatty 
acids by anaerobic bacteria. J Lipid Res. 2014;55(9):1855-1863.
91.
Druart C, Bindels LB, Schmaltz R, et al. Ability of the gut microbiota to produce PUFA-
derived bacterial metabolites: Proof of concept in germ-free versus conventionalized 
mice. Mol Nutr Food Res. 2015;59(8):1603-1613.
92.
Druart C, Neyrinck AM, Vlaeminck B, Fievez V, Cani PD, Delzenne NM. Role of the 
lower and upper intestine in the production and absorption of gut microbiota-derived 

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
44
PUFA metabolites. PLoS One. 2014;9(1):e87560.
93.
Furumoto H, Nanthirudjanar T, Kume T, et al. 10-Oxo-trans-11-octadecenoic acid 
generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is 
cytoprotective against oxidative stress. Toxicol Appl Pharmacol. 2016;296:1-9.
94.
Miyamoto J, Mizukure T, Park SB, et al. A gut microbial metabolite of linoleic acid, 10-
hydroxy-cis-12-octadecenoic acid, ameliorates intestinal epithelial barrier impairment 
partially via GPR40-MEK-ERK pathway. J Biol Chem. 2015;290(5):2902-2918.
95.
Flint HJ, Duncan SH, Scott KP, Louis P. Links between diet, gut microbiota composition 
and gut metabolism. Proc Nutr Soc. 2015;74(1):13-22.
96.
Belenguer A, Duncan SH, Calder AG, et al. Two routes of metabolic cross-feeding 
between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human 
gut. Appl Environ Microbiol. 2006;72(5):3593-3599.
97.
Duncan SH, Louis P, Flint HJ. Lactate-utilizing bacteria, isolated from human feces, that 
produce butyrate as a major fermentation product. Appl Environ Microbiol. 
2004;70(10):5810-5817.
98.
Arpaia N, Rudensky AY. Microbial metabolites control gut inflammatory responses. 
Proc Natl Acad Sci U S A. 2014;111(6):2058-2059.
99.
O'Keefe SJ. Diet, microorganisms and their metabolites, and colon cancer. Nat Rev 
Gastroenterol Hepatol. 2016;13(12):691-706.
100.
Buczynski MW, Dumlao DS, Dennis EA. Thematic Review Series: Proteomics. An 
integrated omics analysis of eicosanoid biology. J Lipid Res. 2009;50(6):1015-1038.
101.
Konkel A, Schunck WH. Role of cytochrome P450 enzymes in the bioactivation of 
polyunsaturated fatty acids. Biochim Biophys Acta. 2011;1814(1):210-222.
102.
Serhan CN, Chiang N. Resolution phase lipid mediators of inflammation: agonists of 
resolution. Curr Opin Pharmacol. 2013;13(4):632-640.
103.
Serhan CN. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 
2014;510(7503):92-101.
104.
Janakiram NB, Mohammed A, Rao CV. Role of lipoxins, resolvins, and other bioactive 
lipids in colon and pancreatic cancer. Cancer Metastasis Rev. 2011;30(3-4):507-523.
105.
Wang D, Dubois RN. Eicosanoids and cancer. Nature reviews Cancer. 2010;10(3):181-
193.
106.
Campbell EL, MacManus CF, Kominsky DJ, et al. Resolvin E1-induced intestinal 
alkaline phosphatase promotes resolution of inflammation through LPS detoxification. 
Proc Natl Acad Sci U S A. 2010;107(32):14298-14303.
107.
Lasry A, Zinger A, Ben-Neriah Y. Inflammatory networks underlying colorectal cancer. 
Nat Immunol. 2016;17(3):230-240.
108.
West NR, McCuaig S, Franchini F, Powrie F. Emerging cytokine networks in colorectal 
cancer. Nat Rev Immunol. 2015;15(10):615-629.
109.
Pischon T, Hankinson SE, Hotamisligil GS, Rifai N, Willett WC, Rimm EB. Habitual 
dietary intake of n-3 and n-6 fatty acids in relation to inflammatory markers among US 
men and women. Circulation. 2003;108(2):155-160.
110.
Navarro SL, Kantor ED, Song X, et al. Factors Associated with Multiple Biomarkers of 
Systemic Inflammation. Cancer Epidemiol Biomarkers Prev. 2016;25(3):521-531.
111.
Fabian CJ, Kimler BF, Phillips TA, et al. Modulation of Breast Cancer Risk Biomarkers 
by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Postmenopausal Women. 
Cancer Prev Res (Phila). 2015;8(10):922-931.

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
45
112.
Popivanova BK, Kostadinova FI, Furuichi K, et al. Blockade of a chemokine, CCL2, 
reduces chronic colitis-associated carcinogenesis in mice. Cancer Res. 2009;69(19):7884-
7892.
113.
Chulada PC, Thompson MB, Mahler JF, et al. Genetic disruption of Ptgs-1, as well as 
Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res. 2000;60(17):4705-
4708.
114.
Baratelli F, Lin Y, Zhu L, et al. Prostaglandin E2 induces FOXP3 gene expression and T 
regulatory cell function in human CD4+ T cells. J Immunol. 2005;175(3):1483-1490.
115.
Lee SY, Choi HK, Lee KJ, et al. The immune tolerance of cancer is mediated by IDO 
that is inhibited by COX-2 inhibitors through regulatory T cells. J Immunother. 
2009;32(1):22-28.
116.
Mandapathil M, Szczepanski MJ, Szajnik M, et al. Adenosine and prostaglandin E2 
cooperate in the suppression of immune responses mediated by adaptive regulatory T 
cells. J Biol Chem. 2010;285(36):27571-27580.
117.
Gravaghi C, La Perle KM, Ogrodwski P, et al. Cox-2 expression, PGE(2) and cytokines 
production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of 
fat-1 mice. J Nutr Biochem. 2011;22(4):360-365.
118.
Piazzi G, D'Argenio G, Prossomariti A, et al. Eicosapentaenoic acid free fatty acid 
prevents and suppresses colonic neoplasia in colitis-associated colorectal cancer acting on 
Notch signaling and gut microbiota. Int J Cancer. 2014;135(9):2004-2013.
119.
Murff HJ, Shrubsole MJ, Cai Q, et al. A randomized, controlled trial of fish oil 
supplementation on eicosanoid production in patients at risk for colorectal cancer. Paper 
presented at: AACR Special Conference: Colorectal Cancer: From Initiation to 
Outcomes; September 17-20, 2016, 2016; Tampa, FL.
120.
Cockbain AJ, Volpato M, Race AD, et al. Anticolorectal cancer activity of the omega-3 
polyunsaturated fatty acid eicosapentaenoic acid. Gut. 2014;63(11):1760-1768.
121.
Kristal AR, Kolar AS, Fisher JL, et al. Evaluation of web-based, self-administered, 
graphical food frequency questionnaire. J Acad Nutr Diet. 2014;114(4):613-621.
122.
Committee ASoP, Anderson MA, Ben-Menachem T, et al. Management of 
antithrombotic agents for endoscopic procedures. Gastrointest Endosc. 2009;70(6):1060-
1070.
123.
Yao MD, von Rosenvinge EC, Groden C, Mannon PJ. Multiple endoscopic biopsies in 
research subjects: safety results from a National Institutes of Health series. 
Gastrointestinal endoscopy. 2009;69(4):906-910.
124.
Courtney ED, Matthews S, Finlayson C, et al. Eicosapentaenoic acid (EPA) reduces crypt 
cell proliferation and increases apoptosis in normal colonic mucosa in subjects with a 
history of colorectal adenomas. Int J Colorectal Dis. 2007;22(7):765-776.
125.
Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E. Integrated, nontargeted 
ultrahigh performance liquid chromatography/electrospray ionization tandem mass 
spectrometry platform for the identification and relative quantification of the small-
molecule complement of biological systems. Anal Chem. 2009;81(16):6656-6667.
126.
Goedert JJ, Sampson JN, Moore SC, et al. Fecal metabolomics: assay performance and 
association with colorectal cancer. Carcinogenesis. 2014;35(9):2089-2096.
127.
Pfalzer AC, Nesbeth PD, Parnell LD, et al. Diet- and Genetically-Induced Obesity 
Differentially Affect the Fecal Microbiome and Metabolome in Apc1638N Mice. PloS 
one. 2015;10(8):e0135758.

NCI Protocol #: N/A
DF/HCC Protocol #: 19-402
Protocol Version Date: 12/10/2021
46
128.
Irvin MR, Zhi D, Aslibekyan S, et al. Genomics of post-prandial lipidomic phenotypes in 
the Genetics of Lipid lowering Drugs and Diet Network (GOLDN) study. PloS one. 
2014;9(6):e99509.
129.
Watson H, Cockbain AJ, Spencer J, et al. Measurement of red blood cell 
eicosapentaenoic acid (EPA) levels in a randomised trial of EPA in patients with 
colorectal cancer liver metastases. Prostaglandins Leukot Essent Fatty Acids. 
2016;115:60-66.
130.
Fekete K, Marosvolgyi T, Jakobik V, Decsi T. Methods of assessment of n-3 long-chain 
polyunsaturated fatty acid status in humans: a systematic review. Am J Clin Nutr. 
2009;89(6):2070S-2084S.
131.
Harris WS, Thomas RM. Biological variability of blood omega-3 biomarkers. Clin 
Biochem. 2010;43(3):338-340.
132.
Fabian CJ, Kimler BF, Phillips TA, et al. Modulation of Breast Cancer Risk Biomarkers 
by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Premenopausal Women. 
Cancer Prev Res (Phila). 2015;8(10):912-921.
133.
Sorensen LS, Rasmussen HH, Aardestrup IV, et al. Rapid incorporation of omega-3 fatty 
acids into colonic tissue after oral supplementation in patients with colorectal cancer: a 
randomized, placebo-controlled intervention trial. JPEN J Parenter Enteral Nutr. 
2014;38(5):617-624.
134.
Hillier K, Jewell R, Dorrell L, Smith CL. Incorporation of fatty acids from fish oil and 
olive oil into colonic mucosal lipids and effects upon eicosanoid synthesis in 
inflammatory bowel disease. Gut. 1991;32(10):1151-1155.
135.
Truong DT, Franzosa EA, Tickle TL, et al. MetaPhlAn2 for enhanced metagenomic 
taxonomic profiling. Nat Methods. 2015;12(10):902-903.
136.
Segata N, Izard J, Waldron L, et al. Metagenomic biomarker discovery and explanation. 
Genome biology. 2011;12(6):R60.
137.
Morgan XC, Tickle TL, Sokol H, et al. Dysfunction of the intestinal microbiome in 
inflammatory bowel disease and treatment. Genome biology. 2012;13(9):R79.
138.
Colditz GA, Wei EK. Preventability of cancer: the relative contributions of biologic and 
social and physical environmental determinants of cancer mortality. Annu Rev Public 
Health. 2012;33:137-156.
139.
Abubucker S, Segata N, Goll J, et al. Metabolic reconstruction for metagenomic data and 
its application to the human microbiome. PLoS Comp Biol. 2012;8(6):e1002358.
140.
Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis 
of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562-578.
141.
Barnard ME, Beeghly-Fadiel A, Milne GL, et al. Urinary PGE-M Levels and Risk of 
Ovarian Cancer. Cancer Epidemiology Biomarkers &amp;amp; Prevention. 
2019;28(11):1845.
142.
Pagliaccia F, Habib A, Pitocco D, et al. Stability of urinary thromboxane A2 metabolites 
and adaptation of the extraction method to small urine volume. Clin Lab. 2014;60(1):105-
111.
143.
Pradelles P, Grassi J, Maclouf J. Enzyme immunoassays of eicosanoids using 
acetylcholine esterase as label: an alternative to radioimmunoassay. Analytical 
Chemistry. 1985;57(7):1170-1173.
144.
Bertagnolli MM, Eagle CJ, Zauber AG, et al. Five-year efficacy and safety analysis of the 
Adenoma Prevention with Celecoxib Trial. Cancer Prev Res (Phila). 2009;2(4):310-321.

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APPENDIX A
                   VIOSCREEN OVERVIEW
Product Background
VioScreen is a scientifically validated, dietary assessment tool that generates an accurate analysis 
of a subject’s dietary behavior in about 20 minutes.
The assessment allows for the evaluation of subjects against a typical North American diet. This 
graphical online assessment consists of approximately 1,200 food images. Subjects are shown a 
set of foods within a food group from which they select only foods consumed at least once a 
month over the past three months. For each food identified, the system prompts the subject to 
select the frequency and portion size typically consumed. When needed, VioScreen branches to 
collect more relevant data. For example, if the subject lists “coffee,” VioScreen prompts to see if 
sugar or milk is added and, if so, what kind of milk.
Subject responses are digitally stored in real time, verified for completeness, and analyzed to 
create an accurate and detailed nutritional profile with over 160 nutrient and food components, 
top foods with key nutrients, current and recommended eating patterns, Body Mass Index and 
Estimated Energy Requirement. Responses and analysis are immediately accessible to the 
researcher via VioScreen’s administrative interface. This straight through process eliminates the 
need for lengthy and costly interviews or error prone paper-based questionnaire processing.
VioScreen’s administrative interface is not just for data access, it makes the management of the 
entire process easy. Researchers are able to define organizations and protocols, assign user roles, 
import subject data, view results and export results to a standard file format for import to 
analytical software.
Hosting Environment & Backup
All data is stored in the latest version of Microsoft SQL Server database. The web server and 
database server reside on their own secure environments separated from other servers through a 
secure router implementation. All server access requires individual user level access via VPN, 
each user is required to update their password after 45 days, and all access is logged and 
reported. The application and database is backed up through highly secure cloud based services. 
The servers are cloud based and each server’s operating system (Windows OS) is updated as new 
patches are released. HIPAA compliance is supported through the use of tools like Idera's SQL 
Server Compliance Manager for auditing sensitive data, monitoring alerts, and potential threat 
detection. Any unauthorized access to any environment or application is reported and tracked 
within the Windows OS.
System & Data Access
VioScreen is web-based application so there is nothing to install or manage. It has interactive 
data input and retrieval over the internet via transport layer security so the only requirement is an 
internet connection and access to www.vioscreen.com. All access to the application is secured 
through encrypted password access and each session is timed out after 15 minutes of inactivity 

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for both subjects and researchers.
Upon account setup new customers will receive a study administrator account. This account 
allows the study administrator to do many things including manage their account, set privileges, 
define protocols, create local administrators and create subjects. As part of the protocol 
definition, the study administrator is able to create subjects by a unique SubjectID to ensure 
participant confidentiality. All data is available in real-time to researchers via the export 
functionality available via the administrative functionality. Data is exported to a .csv format for 
easy import to any analysis software.
VioScreen Nutritional Assessment Food List
This document lists foods that are included as part Viocare’s nutritional assessment. This is an 
electronic assessment that is administered online and includes integrated decision tree logic that 
prompts users to answer additional relevant questions (i.e. by choosing coffee, the system will ask 
if cream or sugar is used).
Food List
• Sweetened cereal (such as Apple Jacks®, Cocoa Puffs®, Fruit Loops®, Lucky Charms®, Trix®)
• Highly-fortified cereal (such as Total®, Product 19® and Smart Start®)
• Mostly or all whole grain cereal (such as Cheerios®, Grape-Nuts, Quaker Natural Granola®, 
Raisin Bran and Shredded Wheat)
• Standard cereal (such as Corn Flakes, Kellogg's Crispix® and Rice Krispies®)
• What type of milk or creamer did you put on cold cereals
• Whole grain or partial whole grain cooked cereal such as regular oatmeal (rolled oats), cream of 
brown rice, Roman Meal®
• Standard cooked cereals, such as cream of wheat, instant or quick-cooking oatmeal, cream of 
rice, grits, Malt-O-Meal®
• What type of milk or creamer did you usually use to cook the cereal?
• What type of milk or creamer did you usually put on cooked cereals?
• Pancakes, French toast and waffles
• Muffins, scones, croissants and biscuits
• White breads, including bagels, rolls and English muffins
• Whole grain breads, including whole grain bagels, rolls and English muffins
• Cornbread and corn muffins
• Corn tortillas
• Flour tortillas
• Eggs
• Egg Whites
• Tofu or soy breakfast sausage or other breakfast meat
• Bacon and breakfast sausage
• Tofu or soy cold cuts, hot dogs or deli meat substitutes
• Hot dogs and sausage, low or reduced fat
• Hot dogs and sausage, regular, not low fat
• Ham, turkey and low fat lunch meats

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• Bologna, salami, Spam® and all other regular lunch meats
• Beef, pork, ham and lamb
• Veggie soy or tofu burgers or ground meat substitute
• Hamburgers, meatloaf and other ground meat
• Liver, chicken liver and organ meats
• Soy or tofu chicken or turkey
• Fried chicken, including nuggets and tenders
• Chicken and turkey, roasted, grilled, broiled or stewed
• Fried fish, fish sandwiches and fried shrimp and oysters
• Shrimp, oysters, crab and lobster
• White fish, broiled or baked, such as cod, halibut and snapper
• Dark fish, broiled or baked, such as salmon, trout and mackerel
• Canned tuna and tuna salad
• Packaged mixed dishes with soy or tofu
• Stew, pot pies, curries and casseroles with meat and chicken
• Chili with meat and beans
• Spaghetti, lasagna or other pasta with tomato and meat sauces (whole grain, regular)
• Spaghetti, lasagna or other pasta with tomato sauce (no meat) (whole grain, regular)
• Spaghetti and other pasta with oil, cheese or cream sauce, including macaroni and cheese
• Pizza (whole grain, regular)
• Asian-style (stir-fried) noodles and rice, such as chow mein, fried rice and Pad Thai
• Japanese noodles, such as Udon and Soba
• Burritos, tacos, tostados and quesadillas
• Enchiladas and tamales
• Tofu (all types), including low fat, flavored, marinated and smoked
• Tempeh (all types)
• Vegetable, minestrone and tomato soup
• Cream soups, such as chowders, potato and cheese
• Bean soups, such as pea, lentil and black bean
• Chicken noodle and other broth soups
• Miso soup
• Ramen noodle soup
• Cottage cheese and ricotta cheese (regular, low fat, nonfat)
• Soy cheese
• Sour cream (regular, low, nonfat)
• Cheese (regular, low, nonfat)
• Soy yogurt
• Greek Yogurt (sweetened, regular, low fat, nonfat)
• Yogurt (plain, sweetened, regular, low fat, nonfat)
• Salad greens (lettuce and spinach)
• Fresh tomatoes
• Carrots (raw)
• Green peppers and green chilies (raw)
• Red peppers and red chilies (raw)
• Avocado and guacamole
• Coleslaw

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• Potato, macaroni and pasta salads
• Salad dressing
• Broccoli
• Cauliflower, cabbage and brussels sprouts
• Green beans and string beans
• Carrots (cooked)
• Green peas
• Corn and hominy
• Red peppers and red chilies (cooked)
• Green peppers and green chilies (cooked)
• Onions and leeks
• Summer squash and zucchini
• Winter squash, such as acorn and butternut
• Yams and sweet potatoes
• Kale, mustard greens and collards (cooked)
• Spinach, swiss chard and beet greens (cooked)
• French fries, fried potatoes and hash browns
• Potatoes (boiled, baked or mashed)
• Cooked soybeans or edamame
• Refried beans
• Baked beans, lima beans or all other beans, including chili made without meat
• White Rice
• Brown rice, cracked wheat and other whole grains
• Noodles as a side dish
• Cheese sauce and cream sauce
• Meat gravies
• Ketchup
• Salsa (as dip or on food)
• Mayonnaise and mayonnaise-type spreads
• Salt (in cooking or at the table)
• Soy sauce, tamari, teriyaki sauce, Szechwan sauce and natto
• Fresh garlic
• Apples, applesauce and pears
• Bananas
• Peaches, nectarines and plums
• Apricots - fresh or canned
• Apricots (dried)
• Raisins, prunes and other dried fruit (not apricots)
• Oranges, grapefruit and tangerines (not juice)
• Strawberries, blueberries and other berries
• Cantaloupe, melon and mango (in season)
• Watermelon and red melon
• Grapes, fresh
• Pineapple, fresh and canned
• Cherries, fresh
• Other fruit, including papaya, fruit cocktail or mixed fruit salads

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• Soy ice cream
• Other soy desserts such as cheesecake
• Low fat ice cream, frozen yogurt, sherbet or other low or nonfat frozen desserts
• Ice cream and milkshakes
• Pudding, custard and flan
• Doughnuts, pies and pastries
• Cookies or cakes
• Chocolate, candy bars, and toffee
• Life Savers, licorice, jelly beans or other hard candies
• Low or nonfat salty snacks such as pretzels and low or nonfat potato, tortilla and corn chips
• Regular potato, tortilla chips, corn chips and puffs
• Plain popcorn (no butter) or low fat microwave popcorn
• Microwave popcorn (Buttered and regular)
• Soy crackers
• Crackers, such as saltines and SnackWell's (low and nonfat)
• Regular crackers, such as Ritz and Wheat Thins
• Roasted soy nuts and soy nut butter
• Nuts, seeds, peanuts or peanut butter
• Whole grain crackers (low fat and regular)
• Sports bar and meal replacement bar containing soy
• Meal replacement drinks and shakes containing soy or soy protein [SOY]
• Granola bars and cereal bars such as Nutri-Grain Bars
• Sports or meal replacement bars such as Power Bars and Cliff Bars
• Meal replacement drinks and shakes such as Slim-Fast, Ensure and Carnation Instant Breakfast
• Milk as a beverage
• Latte, cappuccino, mocha or hot chocolate
• Coffee (not lattes, cappuccino or mochas)
• Sweet tea and bottled sweetened tea drinks
• Other teas, including black, green and herbal teas
• Sugar
• Diet soft drinks
• Regular soft drinks (not diet)
• Water (tap, bottled and sparkling)
• Tomato juice, V-8 and other vegetable juice
• Orange juice and grapefruit juice
• Other 100% fruit juice such as apple, grape and cranberry
• Fruit drinks fortified with Vitamin C, such as Hi-C, Fruitopia, and Kool-Aid
• Beer (all types)
• Red Wine
• White or rosé wine
• Liquor and mixed drinks

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VioScreen Diet Assessment

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