Clinical trial NCT01661764
Conditions: Colorectal Adenomatous Polyps
Keywords: Colorectal Neoplasm
Sponsor: Vanderbilt University
Start date: 2013-02-04; Completion date: 2018-01-23
Enrollment: 141 participants; Allocation: RANDOMIZED; Intervention model: FACTORIAL

Brief summary:
Colorectal cancer is the second leading cause of cancer-related death within the United States. Animal models and observational studies have suggested that marine-derived n-3 polyunsaturated fatty acids \[PUFA\] such as eicosapentanoic acid \[EPA\] and docosahexanoic acid \[DHA\] may reduce the risk of colorectal cancer. In addition, it may be the relative proportion of n-3 to n-6 PUFAs that best determines the chemopreventive effects of fish oils. This ratio is important because the n-6 PUFA, arachidonic acid (ARA), is converted via the cyclo-oxygenase (COX) pathway to prostaglandin E2 (PGE2), an inflammatory eicosanoid overproduced in colorectal neoplasms while EPA is converted to the anti-inflammatory prostaglandin E3 (PGE3). While the ratio of n-6 to n-3 PUFAs can be altered through dietary changes, genetic factors may also influence this ratio. Recent genetic studies have demonstrated that much of the tissue levels of ARA is determined by differences in a gene called fatty acid desaturase 1 (FADS1). FADS1 is the rate-limiting enzyme in the conversion of linoleic acid, the most commonly consumed PUFA in the Western diet, to ARA, and one particular genetic variant caller rs174537 is associated with lower fatty acid desaturase activity and subsequently lower tissue levels of ARA.

The study hypothesis is that individuals with genetically determined lower activity of FADS1 will derive greater benefit from fish oil supplementation than individuals with higher FADS1 activity because of lower tissue levels of ARA and subsequently a more favorable n-6 to n-3 PUFA ratio. To test this hypothesis the investigators will recruit 150 participants with recently identified adenomatous polyps and conduct a 6-month double blind 3 X 2 factorial randomized controlled trial. The first factor will be FADS1 genotype (GG, GT, and TT) and the second factor will be fish oil supplementation (fish oil versus placebo). The primary outcome will be the change in rectal epithelial cell growth and cell death. Secondary outcomes will include rectal epithelial cell expression of genes important in PGE2 production, rectal cell production of PGE2 and PGE3, rectal mucosal tissue levels of fatty acids, and changes in biomarkers of inflammation (C-reactive protein), adipokines (leptin, adiponectin), and markers of insulin sensitivity.

The specific aims include: 1) to determine the efficacy of fish oil supplements on rectal epithelial cell proliferation indexes and markers of rectal crypt apoptosis, and 2) to determine the effect of genetically-determined fatty acid desaturase 1 activity on fish oil supplementation for colorectal cancer chemoprevention. The investigators long-term objectives are to determine genetic factors that might influence the efficacy of fish oil supplementation in order to conduct a more definitive adenoma recurrence trial using marine-derived n-3 PUFAs. The investigators anticipate that fish oil will have anti-neoplastic effect and individuals with low FADS1 activity will have a greater response compared to individuals with high FADS1 activity

Detailed description:
1.Rationale and Specific Aims

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer related mortality in the United States. Animal and human studies have suggested that the marine-derived n-3 polyunsaturated fatty acids (PUFAs), eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), have cancer inhibitory properties while conversely, n-6 PUFAs such as arachidonic acid (ARA) may promote tumorigenesis. The mechanism behind these opposing effects is likely due to differences in the biological activity of their eicosanoids end products and their effects on chronic inflammation. Prostaglandin E2 (PGE2) is a pro-inflammatory eicosanoid that is aberrantly produced in both colorectal adenomas and cancer and is derived from ARA via the cyclo-oxygenase pathway. EPA is converted through the same pathway into prostaglandin E3, which has 4 to 7- fold less prostaglandin E receptor affinity, is less inflammatory, and may even be pro-apoptotic compared to PGE2. As such, it may be the ratio of ARA to EPA and DHA rather than the absolute levels of marine-derived n-3 PUFAs that contribute most towards their antiproliferative and pro-apoptotic effects.

The ratio of ARA to EPA + DHA can be manipulated through fish oil supplementation, however; genetic factors may play a key role on determining this ratio. Recent genome-wide association and haplotype studies have demonstrated that up to 28% of the additive variance in tissue levels of ARA is explained by variants in a single gene, fatty acid desaturase 1 (FADS1). FADS1 is the rate-limiting enzyme in the conversion of linoleic acid (LA), the most commonly consumed PUFA, to ARA, and homozygotes for the T allele (population frequency of 13%, HapMap -CEU) in rs174537 have lower fatty acid desaturase activity and subsequently lower tissue levels of ARA. While EPA can be produced in vivo from α-linolenic acid, in humans, this process is extremely inefficient and most tissue level EPA derives directly from dietary consumption of fatty fish. Thus, high activity of FAD1 and subsequently increased tissue levels of ARA may offset some of the potential benefits of dietary supplementation with fish oil. To date, no previously published studies have investigated how genetic variants that influence fatty acid desaturase activity might modify the beneficial effects of fish oil supplementation.

The investigators hypothesis is that the individuals with genetically determined lower activity of FADS1 will derive greater benefit from fish oil supplementation than individuals with higher FADS1 activity because of lower tissue levels of ARA and subsequently a more favorable ARA to EPA + DHA ratio. To test this hypothesis the investigators will recruit 150 participants with recently identified adenomatous polyps and conduct a 6-month double blind 3 X 2 factorial randomized controlled trial. The first factor will be the rs174537 genotype (GG, GT, and TT) in the FADS1 gene and the second factor will be fish oil supplementation (fish oil versus placebo). The primary study outcome will be the change from baseline in rectal epithelial cell proliferation as measured by Ki-67 labeling and rectal crypt apoptosis as measured by TUNEL. Secondary endpoints will include rectal epithelial cell expression of COX-2 and 15-PGDH, rectal cell production of PGE2 and PGE3, rectal cell fatty acid concentrations, as well as, changes from baseline in biomarkers of inflammation (C-reactive protein), adipokines (leptin, adiponectin), and markers of insulin sensitivity (HOMA-IR).

The Specific Aims for this research proposal are:

1. to determine the efficacy of fish oil supplements on rectal epithelial cell proliferation indexes and markers of rectal crypt apoptosis; and,
2. to determine the effect of genetically determined fatty acid desaturase activity on fish oil supplementation for markers of colorectal cancer risk.

The investigators long-term objectives are to determine genetic factors that might influence the efficacy of fish oil supplementation in order to conduct a more definitive adenoma recurrence trial using marine-derived n-3 PUFAs. The investigators anticipate that fish oil will have anti-neoplastic effect and individuals with low FADS1 activity will have a greater response compared to individuals with high FADS1 activity. This study will be the first to investigate the nutrigenomics of fish oil supplementation in colorectal cancer chemoprevention and may have implications beyond cancer prevention as fish oil is being actively investigated for its anti-inflammatory effects in cardiovascular and psychiatric diseases as well as diabetes mellitus and the metabolic syndrome.

2\. Recruitment and Retention

The investigators will identify eligible participants based on the inclusion criteria by reviewing study data and medical record data collected in the Tennessee Colorectal Polyp Study(TCPS). Participants still eligible after record review will be mailed an introductory letter inviting them to participate. One week after the letter is mailed; a trained interviewer from the Vanderbilt Survey Research Shared Resource (SRSR) will call the potential participants to provide more detailed information about the study, answer questions about the study, and to see if they may be interested in participating. At that time, an appointment will be made with the participant for the baseline in-person visit. After the initial visit and informed consent is obtained an interviewer from the SRSR will conduct the baseline interview survey and 24-hour dietary recalls. At the baseline visit, the investigators will re-genotype rs174535 to confirm the accuracy of the imputation process. This strategy will allow the investigators to efficiently and accurately identify appropriate candidates for our study.

Eligible subjects will present to the Vanderbilt General Clinical Research Center (GCRC) for the initial visit and baseline study procedures. Participants who are eligible for the study and provide written consent for enrollment will have blood obtained; an adipose tissue biopsy performed, and undergoes the baseline rectal mucosal biopsy procedure. Treatment assignment will be obtained from the Vanderbilt Investigational Pharmacy by a coordinator. The first dose of the study medication will be given to patients at the initial visit and the date and time recorded. This date and time will be considered the time of randomization.

3\. Randomization

Randomization will be performed according to a permuted block randomization scheme stratified on the three genotypes. Randomization will proceed within these three strata with a block size of balancing interval, varying randomly according to the outcome of a computer generated random number. This ensures that the cumulative proportion of assignments to each treatment will be balanced after each block of assignments has been made.

4\. Study Procedures

1. Data Collection

   Because outside dietary exposure to both n-6 and n-3 PUFAs could possibly confound the effect of fish oil supplementation the investigators will perform a total of four 24-hour dietary recall studies for each participant over the course of the study. At enrollment, the investigators will conduct two 24-hour dietary assessments, one on the weekday and one on the weekend, as participants' diet may differ based on the day of the week. In addition the investigators will conduct one 24-hour dietary recall at week 8 and at week 16. The investigators will use data collected from these 24-hour dietary assessments along with standard food composition tables to calculate dietary exposure to PUFAs.

   The investigators will determine adherence to study drug at each in-person visit during the study. Medication and medication changes will be recorded at these visits. Patients who initiate a new NSAID during the study will be withdrawn and an exit visit performed. Adherence to fish oil will also be determined through RBC phospholipid membrane fatty acid analysis performed at month 3 and month 6. In addition, to determine whether fish oil supplementation also influences fatty acid membrane concentration at the target tissue, the investigators will determine the change in rectal epithelial cell phospholipid membrane fatty acid concentration.
2. Fish oil Capsules

   Participants allocated to fish oil supplementation will be instructed to take three Lovaza® capsules each containing 465 mg of EPA and 375 mg DHA daily; this will provide a total daily dose of 1395 mg EPA plus 1125 mg DHA for a total daily dose of fish oil of 2.5 grams. Patients will take one tablet three times a day with meals. Lovaza® capsules are the only FDA approved preparation of fish oil, and as such, the quality of the drug is monitored and assured. Pharmacological grade fish oil capsules have the advantage of providing high concentrations of PUFAs, low levels of contaminants, such as mercury, and almost no fish odor.
3. Placebo Capsules

   The investigators will use oleic acid as a placebo. The reason for the use of oleic acid is several-fold. First, oleic acid (olive oil) capsules have a similar texture, size, color, and consistency to fish oil capsules. More importantly, oleic acid does not undergo conversion to an eicosanoid or any other metabolically active product. This is opposed to corn oil, which has also been used as a placebo in fish oil studies but is primarily linoleic acid and could subsequently increase tissue levels of ARA and confound the results of our study. Oleic acid has been used as a placebo in several prior studies of fish oil supplementation and is well tolerated.
4. Assessment Visits

Patients will attend the GCRC clinic at the baseline (initial visit), after 3 months of study treatment (mid-point visit), and after 6 months of study treatment (end visit). The study coordinator will contact participants every 4 weeks over the course of the study to encourage adherence to the study protocol. At the 3-month and 6-month visits, compliance with treatment will be monitored by capsule count and measurement of RBC phospholipid fatty acid concentrations. Adverse events will be recorded at these visits.

5\. Data management and quality control

The Vanderbilt GCRC Informatics Core will be used as a central location for data processing and analysis. Vanderbilt University has developed software tools and workflow methodology for electronic collection and management of research study data. (132) REDCap (Research Electronic Data Capture) is a secure, web-based application that provides an intuitive interface for users to enter validated data remotely (with automated data type and range checks), data manipulation audit trails and reporting, and an export mechanism for end-of-study export of data to common statistical packages.

Arms: arm 1: Eicosapentanoic acid and docosahexanoic acid arm 2: Oleic Acid arm 3: Eicosapentanoic acid and docosahexanoic acid arm 4: Oleic Acid arm 5: Eicosapentanoic acid and docosahexanoic acid arm 6: Oleic Acid
Interventions: intervention 1: Eicosapentanoic acid and docosahexanoic acid intervention 2: Oleic Acid
Intervention descriptions: intervention 1: 1395 mg EPA plus 1125 mg DHA daily for 24 weeks intervention 2: Placebo

Protocol documents:
Prot_SAP_000.pdf: 
R01CA160938 (Murff, PI) 
 
 
 
Protocol Version #: V1.2 
 
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Protocol Date: 09/04/2012 
 
 
 
 
 
 
 
 
 
Fatty Acid Desaturase Activity, Fish Oil, and Colorectal 
Cancer Prevention 
 
NCT# 01661764 
 
Harvey J. Murff, M.D., M.P.H. 
Associate Professor of Medicine 
Institute for Medicine and Public Health 
Vanderbilt Epidemiology Center 
2525 West End Avenue, Suite 600 
Nashville, TN 37203-1738 
(o) 615 936-8319 
(f) 615 936-8291 
Harvey.j.murff@vanderbilt.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Protocol Version #: V1.2 
 
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Protocol Date: 09/04/2012 
 
 
Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or 
Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 Literature Cited 
 
Appendices 
Appendix A Study Procedure Calendar 


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Arachidonic Acid 
PGE2 
COX-2 
15-PGDH 
Increases β-catenin transcriptional activity/ 
Anti-apoptotic/ 
Promotes growth through cyclin D1/ 
Promotes angiogenesis through VEGF 
1.0 
Background 
 
Colorectal cancer (CRC) is the fourth most common cancer and the second leading cause of cancer-related 
death in the United States. (1)  Despite advances in our understanding of the molecular biology of CRC, little 
progress has been made in improving survival rates for individuals diagnosed in advanced stages.(2)  Thus, 
early identification and prevention are important strategies to reduce CRC mortality.(3) 
 
Chronic Inflammation and Colorectal Cancer 
While inflammation is a necessary physiological response to 
infection and tissue injury, mounting evidence has implicated 
chronic inflammation as an initiator and promotor of 
carcinogenesis.(4)  Multiple inflammatory conditions are 
associated with increased cancer risk, and chronic infections 
can predispose individuals to cancer.  Indeed, a recent cohort 
study including over 10,000 individuals found C-reactive protein 
(CRP), an acute-phase reactant and biomarker of inflammation, 
to be associated with an increase hazard for cancer of any type 
(1.3; 95% CI 1.0-1.6) with a particularly strong association for 
CRC (HR = 1.9; 0.8-4.6).(5) 
 
Chronic inflammation can contribute to cancer via several mechanisms including inappropriate gene 
expression; epigenetic alterations; enhanced cell proliferation; and resistance to apoptosis.(6)  One link 
between cancer and inflammation involves cyclooxygenase-2, (COX-2) the inducible form of cyclooxygenase, 
which is overexpressed in multiple malignancies.(7)  In the COX pathway, arachidonic acid (ARA) is released 
from cellular membranes and then enzymatically converted into various bioactive lipid molecules including 
prostaglandins.  These molecules then exert their cellular functions through binding to cell surface receptors of 
the seven transmembrane G-coupled rhodopsin-types.  Production of prostaglandin E2 (PGE2) in particular, 
may promote carcinogenesis through stimulating cellular proliferation, inhibiting apoptosis, promoting 
angiogenesis, and/or enhancing invasiveness.(8)  (Figure 1)  Animal models of CRC have demonstrated the 
connection between tumor formation and ARA with a positive correlation between increasing tissue levels of 
ARA and increased PGE2 production with increased PGE2 production being associated with tumor number.(9-
12)  Up-regulation of COX-2 occurs in 50% of colon adenomas and 85% of colon cancers and is considered a 
key and early oncogenic event in colorectal carcinogenesis.(13) However, while COX-2 is upregulated, 15-
hydroxyprostaglandin dehydrogenase (15-PGDH), which is the normal physiological antagonist of COX-2 
and inactivates PGE2,(14) is often down regulated and is considered a tumor-suppressor gene.(15-16)   
 
Chronic inflammation and 
Obesity/Adipokines/Insulin Resistance 
 
Obesity and overweight are strong risk 
factors for CRC.(17)  Potential 
mechanisms behind this association are 
beginning to be elucidated, beginning 
with the identification of adipose tissue as 
an active endocrine organ that releases a 
wide variety of biologically active 
molecules.(18-19)  Many of these 
adipokines, such as leptin and 
adiponectin, are related to glucose homeostasis, 
insulin sensitivity, and energy balance.(20-21)  Obesity is associated with increased levels of leptin and 
decreased levels of adiponectin and reduced insulin sensitivity.(22-24)  Insulin activates the PI3K/Akt signal 
transduction pathway which increases cell proliferation and cell survival. (27) Adiponectin has insulin-
Obesity/Dysfunctional 
Adipose Tissue 
↑ Leptin 
↓ Adiponectin 
↑ Insulin/Insulin 
Resistance 
PI3-K/Akt System/mTOR 
Ras/Raf MAPK System 
AMPK/P21/P53 System 
Cell Proliferation 
Cell Survival 
Figure 1: Eicosanoid production and CRC risk 
Figure 2: Dysfunctional adipose tissue and CRC risk 


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sensitizing effects and may have anti-carcinogenic effects through the activation of the AMP-activated protein 
kinase (AMPK) system which upregulates the tumor suppressor genes p53 and p21.(25-27)  (Figure 2) 
Conversely, leptin is mitogenic and inhibits apoptosis, likely through the Ras/Raf/ mitogen-activated-protein-
kinase (MAPK) system.(27-29)  The association between insulin levels and CRC has been well described(30-
36) and epidemiological evidence is beginning to emerge suggesting that increased leptin levels(37) and 
decreased adiponectin levels may be risk factors for colorectal neoplasms although this association has not 
been consistent across studies.(37-40) 
 
Chemoprevention of Colorectal Cancer with Fish Oil 
Eicosapentanoic acid (EPA) is converted to eicosanoids through the same enzymatic pathways as ARA but 
produces series-3 prostanoids that have less inflammatory actions due to lower receptor affinities when 
compared to ARA derived series-2 prostanoids.(41-42) In Sprague-Dawley rats, PGE3 is increased with fish oil 
supplementation and has pro-apoptotic effect.(43)  Similar pro-apoptotic effects of EPA derived PGE3 have 
also been reported in human lung cancer A549 cells.(44)  Several studies have demonstrated that increased 
consumption of marine fatty fish results in an increased phospholipid membrane proportion of EPA and a 
concomitant decreased in ARA and ARA-derived eicosanoids.(45-47)  Other potential mechanisms beyond fish 
oils effect on inflammatory eicosanoids that might contribute to its chemopreventive actions includes 
downregulation of protein kinase CbII and decreased intracellular Ras levels.(48-51)  
 
Animal models of CRC can involve chemically induced tumors, cancer cell lines transplants, and transgenenic 
animals.  With regards to chemically induced tumors a meta-analysis of 14 studies of F344 or Sprague-Dawley 
rats found a consistently protective effect of fish oil modeled as either percent of total fat or estimated fat 
calories.(52)  In transplant models, fish oil inhibits the growth of transplanted CT-26 colon tumor lines.(53) 
Several small, randomized controlled trials have been performed to investigate the effect of n-3 PUFAs on 
colorectal neoplasms with most studies finding fish oil reduces cell proliferation and promote apoptosis.  (54-
60)  While most of these studies have used surrogate endpoints, such as rectal epithelial cell proliferation or 
apoptosis, a recent 6-month RCT study found that EPA supplementation (2 g per day) reduced rectal polyp 
number and size in individuals with familial adenomatous polyposis.(61)  Taken together these studies strongly 
support the CRC chemopreventive potential of fish oil. 
 
Eicosanoid Synthesis and the Importance of the n-6 to n-3 PUFA ratio 
We hypothesize that diets with a high ratio of ARA/ [EPA + DHA] result in a pro-inflammatory milieu and that 
these pro-inflammatory effects might contribute to colon carcinogenesis.  This hypothesis is supported by 
animal models of CRC, which have investigated dietary ratios of polyunsaturated fatty acids (PUFAs) and 
tumor burden.  In the ApcMin/+ mouse model of colon carcinogenesis, mice fed EPA and ARA simultaneously 
had similar tissue levels of ARA and intestinal tumor growth compared to mice feed ARA without EPA.(11)  
Boudreau et al. investigated the effect of fish oil consumption on the production of ARA derived eicosanoids on 
Sprague-Dawley rates fed at a constant ratio of n-3 to n-6 PUFAs. (62) As the investigators increased the 
absolute dose of n-3 PUFAs, they also increased the dose of n-6 PUFAs to maintain a fixed ratio.  There was 
no apparent dose effect of n-3 PUFAs on ARA-derived eicosanoids under the conditions of a fixed ratio of n-3 
to n-6. Thus, the investigators concluded that the ratio of n-3 to n-6 PUFA must be taken into account when 
investigating dietary interventions of n-3 PUFAs. 
 
One of the most compelling animal models supporting the benefits of modulating the ARA to EPA + DHA ratio 
is the FAT-1 transgenic mouse.  These mice have been engineered to carry the fat-1 gene from C. elegans 
and are able to convert n-6 PUFAs to n-3 PUFAs in vivo, a function all mammals are unable to perform.(63)  
Subsequently, FAT-1 mice preferentially convert dietary n-6 PUFAs to n-3 PUFAs.  Gravaghi et al. compare 
FAT-1 transgenic mice to wild type mice in the DSS-induced experimental colitis model.(64)  Both groups of 
mice were fed the same n-6 PUFA rich diet.  FAT-1 mice had a significant attenuation of colonic inflammation 
along with lower colonic cell ratios of n-6 to n-3 PUFA and a significant decrease in PGE2 production and COX-
2 expression. 
 


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FADS1 
α-linolenic acid  
EPA 
PGE3 
Stearidonic acid  
Eicosatrienoic acid  
COX2  
γ-linolenic acid  
DGLA  
FADS2 
Linoleic acid  
Arachidonic acid  
PGE2 
Very limited controlled trials have investigated the impact of varying amounts of PUFAs and CRC risk.  Bartram 
et al. randomized 12 volunteers to either fish oil supplementation (4.4 g n-3 fatty acids/day) versus placebo and 
found no differences in proliferation indexes after 30 days.(65)  This study was designed to determine the 
effects of fish oil supplementation in subjects eating a controlled high fat diet (50% of energy).  The null results 
of this study contrasted with an earlier study by Bartram where volunteers maintained their usual dietary fat 
intake (30% of total energy).(59)  Comparing the results of these two trials, Bartram et al., concluded that the 
ratio of n-3 to n-6 PUFAs likely plays an important mediating role in fish oils anti-proliferative effects.(65)   
 
Taken together these animal and human studies strongly emphasize the important of the ratio of n-6 PUFAs to 
n-3 PUFAs on modulating inflammation and eicosanoid production in the colon.  However while dietary 
manipulation can result in changes in tissue levels of n-6 and n-3 PUFAs, genetic factors may also play a key 
role in determining this ratio. 
 
Genetic Determinants of Polyunsaturated Fatty Acid Levels 
PUFA are chains of 18 to 22 carbon atoms that contain two or more sequential double bonds.  In the Western 
diet, linoleic acid (18:2n-6) accounts for almost 89% of total PUFA energy intake.(66)  Linoleic acid (LA) is 
metabolized to ARA (20:4n-6) through a series of desaturation and elongation reactions.  The first step in this 
metabolic pathway is fatty acid desaturase 2 (FADS2) that converts LA to γ-linolenic acid [GLA] (18:3n-
6).(67)  GLA is elongated to dihomo-γ-linolenic acid [DHLA] (20:3n-6) and then desaturated by fatty acid 
desaturase 1 (FADS1) to ARA.  Both the genes for FADS2 and FADS1 are localized in chromosome 11 
(11q12-q13.1) as a cluster.(68) While EPA can be produced in vivo from α-linolenic acid through this same 
pathway, in humans, this process is extremely inefficient and most tissue level EPA derives directly from 
dietary consumption of fatty fish.(69-71)  
 
Evidence that genetic factors could play an important role in tissue levels of PUFA comes from several 
sources.  In a study of 80 families conducted in kibbutz settlements in Israel the heritability estimated for red 
blood cell (RBC) phospholipid membrane concentrations were 0.53 ± 0.11 for ARA, 0.56 ± 0.11 for EPA, and f 
0.71 ± 0.08 for DHA, suggesting a strong genetic component influencing tissue levels of long chain PUFAs(72).  
Similarly, genomic studies have established that variants in FADS1 strongly influence RBC membrane and 
plasma levels of ARA.(73)   Three recent genome-wide association studies (GWAS) found a strong association 
between single nucleotide polymorphisms (SNPs) in FADS and plasma and RBC phospholipid PUFA levels 
(74-76)  with 18 to 28% of the additive variance in tissue ARA levels explained by FADS genotypes.(77-78)  
Tanaka et al. in a GWAS study investigating genetic factors associated with plasma and RBC PUFA levels 
found a strong association between the rs174537 SNP in FADS1 and ARA levels.(74)  In this study, 
participants in the InCHIANTI study with the GG genotype had higher plasma concentrations of ARA and this 
level decreased with increasing copy of the T allele (8.76% for GG, 7.39% for GT, 6.35% for TT, P-value 
<0.0001).  This finding was replicated in the GOLDN cohort with a similar statistically significant decrease in 
ARA concentration.(74)  This SNP is included within haplotype blocks for two prior studies that found strong 
associations between FADS1 gene variation and tissue levels of ARA.(77-78) Thus the results of familial 
aggregation studies, haplotype studies, and GWAS studies have all supported that genetic variants in FADS1 
are important determinates of tissue levels of ARA and have led some investigators to call for the “inclusion of 
FADS genotypes as well as diet and lifestyle factors in future 
randomized control trials addressing biological effects of PUFAs..”. 
(79)  
 
2.0 
Rationale and Specific Aims 
 
Colorectal cancer (CRC) is the third most common cancer and the 
second most frequent cause of cancer related mortality in the United 
States.  Animal and human studies have suggested that the marine-
derived n-3 polyunsaturated fatty acids (PUFAs), eicosapentanoic 
acid (EPA) and docosahexanoic acid (DHA), have cancer 


R01CA160938 (Murff, PI) 
 
 
 
Protocol Version #: V1.2 
 
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Protocol Date: 09/04/2012 
inhibitory properties while conversely, n-6 PUFAs such as arachidonic acid (ARA) may promote 
tumorigenesis.  The mechanism behind these opposing effects is likely due to differences in the biological 
activity of their eicosanoids end products and their effects on chronic inflammation.  Prostaglandin E2 (PGE2) 
is a pro-inflammatory eicosanoid that is aberrantly produced in both colorectal adenomas and cancer and is 
derived from ARA via the cyclo-oxygenase pathway.  EPA is converted through the same pathway into 
prostaglandin E3, which has 4 to 7- fold less prostaglandin E receptor affinity, is less inflammatory, and may 
even be pro-apoptotic compared to PGE2.  As such, it may be the ratio of ARA to EPA and DHA rather than 
the absolute levels of marine-derived n-3 PUFAs that contribute most towards their antiproliferative and pro-
apoptotic effects.   
 
The ratio of ARA to EPA + DHA can be manipulated through fish oil supplementation, however; genetic factors 
may play a key role on determining this ratio.  Recent genome-wide association and haplotype studies have 
demonstrated that up to 28% of the additive variance in tissue levels of ARA is explained by variants in a single 
gene, fatty acid desaturase 1 (FADS1).  FADS1 is the rate-limiting enzyme in the conversion of linoleic acid 
(LA), the most commonly consumed PUFA, to ARA, and homozygotes for the T allele (population frequency of 
13%, HapMap –CEU) in rs174537 have lower fatty acid desaturase activity and subsequently lower tissue 
levels of ARA.  While EPA can be produced in vivo from α-linolenic acid, in humans, this process is extremely 
inefficient and most tissue level EPA derives directly from dietary consumption of fatty fish.  Thus, high activity 
of FAD1 and subsequently increased tissue levels of ARA may offset some of the potential benefits of dietary 
supplementation with fish oil.  To date, no previously published studies have investigated how genetic variants 
that influence fatty acid desaturase activity might modify the beneficial effects of fish oil supplementation. 
 
Our hypothesis is that the individuals with genetically determined lower activity of FADS1 will derive greater 
benefit from fish oil supplementation than individuals with higher FADS1 activity because of lower tissue levels 
of ARA and subsequently a more favorable ARA to EPA + DHA ratio.  To test this hypothesis we will recruit 
150 participants with recently identified adenomatous polyps and conduct a 6-month double blind 3 X 2 
factorial randomized controlled trial.  Our first factor will be the rs174537 genotype (GG, GT, and TT) in the 
FADS1 gene and our second factor will be fish oil supplementation (fish oil versus placebo).  Our primary study 
outcome will be the change from baseline in rectal epithelial cell proliferation as measured by Ki-67 labeling 
and rectal crypt apoptosis as measured by TUNEL.  Secondary endpoints will include rectal epithelial cell 
expression of COX-2 and 15-PGDH, rectal cell production of PGE2 and PGE3, rectal cell fatty acid 
concentrations, as well as, changes from baseline in biomarkers of inflammation (C-reactive protein), 
adipokines (leptin, adiponectin), and markers of insulin sensitivity (HOMA-IR).   
 
Our Specific Aims for this research proposal are:  
 
1) to determine the efficacy of fish oil supplements on rectal epithelial cell proliferation indexes and markers of 
rectal crypt apoptosis; and,  
 
2) to determine the effect of genetically determined fatty acid desaturase activity on fish oil supplementation for 
markers of colorectal cancer risk.   
   
Our long-term objectives are to determine genetic factors that might influence the efficacy of fish oil 
supplementation in order to conduct a more definitive adenoma recurrence trial using marine-derived n-3 
PUFAs.  We anticipate that fish oil will have anti-neoplastic effect and individuals with low FADS1 activity will 
have a greater response compared to individuals with high FADS1 activity.  Our study will be the first to 
investigate the nutrigenomics of fish oil supplementation in colorectal cancer chemoprevention and may have 
implications beyond cancer prevention as fish oil is being actively investigated for its anti-inflammatory effects 
in cardiovascular and psychiatric diseases as well as diabetes mellitus and the metabolic syndrome. 
 
3.0 
Animal Studies and Previous Human Studies 
 


R01CA160938 (Murff, PI) 
 
 
 
Protocol Version #: V1.2 
 
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Protocol Date: 09/04/2012 
Please see above for relevant description of animal studies and previous human studies. 
 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion Criteria: 
1) ≥ 40 and < 80 years of age 
2) History of 1 or more adenomatous polyps 
3) Consent to be contacted for future studies 
4) Participants with known genotype for rs174535 in FADS1 
 
Exclusion Criteria: 
1) Previously resected colorectal cancer 
2) Congestive heart failure 
3) Current metabolic or life-threatening disease 
4) Currently taking fish oil supplements 
5) Allergic to fish products 
6) Diagnosis of inflammatory bowel disease 
7) Diagnosis of any cancer (except non-melanoma skin cancer) 
8) Diagnosis of cirrhosis 
9) Diagnosis of kidney disease requiring dialysis 
10) Pregnant or breast feeding 
11) Previous partial of total colectomy 
12) Inability to come to Vanderbilt GRCR for research procedures 
 
5.0 
Enrollment/Randomization 
 
Recruitment and Retention 
We will identify eligible participants based on the inclusion criteria by reviewing study data and medical record 
data collected in the TCPS.  Participants still eligible after record review will be mailed an introductory letter 
inviting them to participate.  One week after the letter is mailed; a trained interviewer from the SRSR will call 
the potential participants to provide more detailed information about the study, answer questions about the 
study, and to see if they may be interested in participating.  At that time if the participant appears interested 
verbal consent will be obtained and the SRSR interviewer will administer the baseline survey and the first 48 
hour dietary recall interview.  At the end of the first 48-hour dietary recall interview, an appointment will be 
made with the participant for the baseline in-person visit.  After this initial visit,informed consent is obtained.  At 
the baseline visit, we will re-genotype rs174535 to confirm the accuracy of our imputation process.  This 
strategy will allow us to efficiently and accurately identify appropriate candidates for our study. 
 
Eligible subjects will present to the Vanderbilt General Clinical Research Center (GCRC) for the initial visit and 
baseline study procedures.  Participants who are eligible for the study and provide written consent for 
enrollment will have blood obtained; an adipose tissue biopsy performed, and undergoes the baseline rectal 
mucosal biopsy procedure.  Treatment assignment will be obtained from the Vanderbilt Investigational 
Pharmacy by a coordinator.  The first dose of the study medication will be given to patients at the initial visit 
and the date and time recorded.  This date and time will be considered the time of randomization.   
 
Randomization 
Randomization will be performed according to a permuted block randomization scheme stratified on the three 
genotypes.  Randomization will proceed within these three strata with a block size of balancing interval, varying 
randomly according to the outcome of a computer generated random number.  This ensures that the 
cumulative proportion of assignments to each treatment will be balanced after each block of assignments has 
been made.  
 


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Side Effect 
Drop Out 
TCPS adenoma cases 
Introduction Letter 
1/3 GG, 1/3 GT, 1/3 TT 
Enrollment 
50 Cases with  
GG Genotype 
50 Cases with  
GT Genotype 
50 Cases with  
TT Genotype 
Two 24 Hour Recall Dietary Assessments 
(1 Weekday, 1 Weekend)  
Randomization 
Baseline Collection of Rectal Biopsy 
Blood and Adipose tissue  
 24 Hour Recall Dietary Assessments 
(Week 8)  
Fish Oil Supplementation 
25 with GG 
25 with GT 
25 with TT 
Placebo 
25 with GG 
25 with GT 
25 with TT 
 24 Hour Recall Dietary Assessments 
(Week 16)  
 Midpoint Collection of Blood 
(Week 12)  
 End: 24 Hour Recall Dietary Assessment 
Collection of Rectal Biopsy and Blood  
6.0 
Study Procedures 
 
Data Collection 
Because outside dietary exposure to both n-6 and n-3 PUFAs could possibly confound the effect of fish oil 
supplementation we will perform a total of three 48-hour dietary recall studies for each  participant over the 
course of the study.  At enrollment, we will conduct the initial 48-hour dietary assessments.(93-94)  In addition 
we will conduct one 48-hour dietary recall at week 12 and at week 24.  We will use data collected from these 
48-hour dietary assessments along with standard food composition tables to calculate dietary exposure to 
PUFAs.  We have chosen to use 48-hour dietary recalls as opposed to food frequency questionnaires as 48-
hour dietary recalls are considered the most accurate survey based method of determining food exposure.(95)  
We will determine adherence to study drug at each in-person visit during the study.  Medication and medication 
changes will be recorded at these visits.    Adherence to fish oil will also be determined through RBC 
phospholipid membrane fatty acid analysis performed at month 3 and month 6.  Prior studies have confirmed 
that this measure is a valid method to determine compliance with fish oil supplementation and have 
documented increased levels of erythrocyte phospholipid membrane concentrations of EPA in subjects 
randomized to fish oil supplements. (96-100)  In addition, to determine whether fish oil supplementation also 
influences fatty acid membrane concentration at our target tissue, we will determine the change in rectal 
epithelial cell phospholipid membrane fatty acid concentration. 
 
Fish oil Capsules 
Participants allocated to fish oil supplementation will be instructed to 
take three Lovaza® capsules each containing 465 mg of EPA and 
375 mg DHA daily; this will provide a total daily dose of 1395 mg 
EPA plus 1125 mg DHA for a total daily dose of fish oil of 2.5 grams.  
Patients will take one tablet three times a day with meals.  The 
rationale for this dose is that it has successfully lowered indexes of 
rectal mucosal proliferation in previously trials and is well tolerated 
without any reported adverse effects.(54, 56) Prior studies have 
demonstrated that 2.5 mg of fish oil daily is able to decrease the 
rectal epithelial cell ratio of ARA to EPA + DHA by 49 to 57%.(54, 
56)  Lovaza® capsules are the only FDA approved preparation of 
fish oil, and as such, the quality of the drug is monitored and 
assured.  Pharmacological grade fish oil capsules have the 
advantage of providing high concentrations of PUFAs, low levels of 
contaminants, such as mercury, and almost no fish odor.   
 
Placebo Capsules 
We will use oleic acid as our placebo.  The reason for the use of 
oleic acid is several-fold.  First, oleic acid (olive oil) capsules have a 
similar texture, size, color, and consistency to fish oil capsules.  
More importantly, oleic acid does not undergo conversion to an 
eicosanoid or any other metabolically active product.  This is 
opposed to corn oil, which has also been used as a placebo in fish 
oil studies but is primarily linoleic acid and could subsequently 
increase tissue levels of ARA and confound the results of our study.  
Oleic acid has been used as a placebo in several prior studies of 
fish oil supplementation and is well tolerated.(54-56)   
 
Assessment Visits 
Patients will attend the GCRC clinic at the baseline (initial visit), after 3 months of study treatment (mid-point 
visit), and after 6 months of study treatment (end visit).  The study coordinator will contact participants every 
4 weeks over the course of the study to encourage adherence to the study protocol.  At the 3-month and 6-
Figure 3: Study Flow Diagram 


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month visits, compliance with treatment will be monitored by capsule count and measurement of RBC 
phospholipid fatty acid concentrations.  Adverse events will be recorded at these visits.   
 
Primary Endpoint and its Determination 
The primary outcome of interest is rectal epithelial cell proliferation, as measured by Ki67 (mib-1) labeling and 
apoptosis as measured by TUNEL (TdT-mediated dUTP Nick-End Labeling).  At the initial visit and the end 
visit, eight mucosal biopsies will be taken 30 cm from the anal margin with 2.4 mm disposable biopsy forceps 
(Cook Medical, Charlotte, NC).  Biopsies will be taken from normal appearing mucosa.  Four biopsies will be 
formalin-fixed and paraffin embedded and processing will begin within 1 hour of collection.  Four additional 
biopsies will be snap frozen in liquid nitrogen and stored at -70˚C until analysis of mucosal fatty acids.  Rectal 
biopsies offer numerous advantages to sigmoid colonic biopsies as no bowel preparation is required and no 
analgesia or sedation is required.   
 
Serial sections of 5 µm thick will be prepared from the rectal tissue.  The sections will be placed sequentially 
on positive-charged slides and numbered from 1 up to 26.  The sections numbered 1, 11, and 21 will be used 
for H&E staining.  For each biomarker, we will stain three sections spaced 50 µm apart to avoid double 
counting of crypts in biopsy specimens.  Thus, for example, sections 2, 12, and 22 may be used for one 
biomarker but adjoining sections (e.g. 2-4) may not be used for the same biomarker.  We will collect multiple 
biopsies and prepare multiple sections for quality control or to repeat assays in case of failure.  Since we 
produce tissue sections that are 34 microns thick, literally hundreds of sections could be prepared from any 
tissue block that is 2.4 mm thick.  If there is remaining tissue, the block face will be re-sealed with paraffin to 
prevent degradation of tissue components through oxidation. Sections will also be coated with a thin layer of 
paraffin.  All blocks and slides will be stored in a vacuum chamber at 4°C in a cold room for long-term storage. 
 
Expression of Ki-67 in colon epithelial cells will be detected following the standard IHC protocol of EnVision™+ 
System, HRP (DAKO).  After deparaffinization of the tissue slides, antigen retrieval is performed by heating the 
slides with a pressure cooker in the optimized buffer solutions: Retrievagen A pH6.5 (BD Pharmingen) for Ki-
67 and using the primary antibodies of mouse monoclonal anti-Ki-67(BD Biosciences Pharmingen). The 
TUNEL  assay is conducted to measure apoptosis of colon epithelium using DeadEnd Colorimetric TUNEL 
System (Promega).  After all fields of each sample are measured, the final immunoreaction indices are 
generated automatically by setting algorithms as ‘‘total positive area / total nuclear area.  Apoptotic activity is 
also scored using standard morphologic criteria applied to H&E stained sections. Briefly, a total of 24 to 30 
longitudinal crypts at three section levels are scored per biopsy. The cells are considered apoptotic if the 
shrinkage of the cell from its neighbors, chromatin condensation and nuclear fragmentation (karyorrhexis) are 
presented.  Cells manifesting these criteria are included in apoptosis scores only when observed in isolated, 
single cells not associated with an inflammatory response. The number of apoptotic cells for each crypt is 
combined to calculate a mean apoptosis score per crypt for each specimen 
 
All biomarkers will be scored using imaging analysis.  We use a system composed of an Olympus BX40 
microscope, a Retiga FAST 1394 color digital camera and BioQuant NOVA Prime imaging software (BioQuant, 
Nashville, TN).  The video images are captured using a 10X objective lens under a constant state of exposure 
control.  Approximately 24-30 well-oriented longitudinal crypts at three section levels per biopsy specimen are 
quantified.  The distribution of positive cells in the upper, middle, and basal zones is evaluated separately by a 
zonal quantitative analysis procedure established in our lab.  When each field is measured, region of interest 
tools are used to exclude blank, folding, hemorrhage, necrosis, poorly stained, and stromal areas, whereas 
threshold tools are used to precisely define and measure total epithelial area, total nuclear area, positively 
stained area, and average gray density.  After all fields of each sample are measured, the final immunoreaction 
indices are generated automatically by setting algorithms as ‘‘total positive area / total nuclear area’’ for nuclear 
staining markers.   
 
Rationale for Using Ki-67 and TUNEL as a Primary Endpoint 


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The rationale for using cell proliferation and apoptosis labeling indexes is five-fold.  First, there is an 
association between proliferation and apoptosis indexes and colorectal adenomas and cancer.(101-106)  
Second, intra-observer correlations of blinded recounts are reported at 0.80 or greater suggesting good 
reproducibility for Ki-67 labeling.(107) Third, for TUNEL, the balance between crypt proliferation and apoptosis 
is important in colorectal cancer tumorigenesis and TUNEL methodology to date has been the most employed 
biomarker of colon crypt apoptosis.(108-109)  Fourth, proliferation, and apoptosis indexes have been 
commonly used in prior studies investigating fish oils versus placebo.  Finally, the use of proliferation and 
apoptosis indexes allows for a smaller sample size and shorter duration of study than using an outcome such 
as adenoma recurrence. (107)  This will allow a more efficient experiment to test our hypotheses that fish oil 
will reduce markers associated with an increase risk of colorectal cancer before performing the more resource 
intense trial using adenoma recurrence as an outcome. 
 
Samples and Laboratory Measurements 
At the initial visit, mid-point visit, and end visit participants will be asked to provide 10 ml venous blood for 
measurements of erythrocyte phospholipids fatty acid content, to determine our secondary biomarker 
endpoints, and to provide plasma and serum that will be stored.  Over the entire course of the study this will 
result in the collection of 30 ml of blood.  At the initial visit, subjects will undergo an adipose tissue biopsy of 
the upper buttock to obtain approximately 20 mg of adipose tissue for fatty acid analysis.  Adipose tissue 
reflects dietary exposure to fatty acids over a period of 2-3 years and is considered the best biomarker of long 
term dietary fatty acid exposure.(110)  Survey studies have found that research volunteers consider this 
procedure no more or less uncomfortable when compared to venipuncture.(134) 
 
Secondary Endpoints- Immunohistochemistry: 
The rationale for including the secondary outcome measurements of COX-2 and 15-PGDH is several fold.  Our 
secondary markers includes expression levels of two growth factors that are often aberrantly expressed in the 
early stages of CRC and are important factors in the production of eicosanoids.(111)  COX-2 converts ARA to 
PGE2 and 15-PGDH is the key enzyme responsible for the biological inactivation of these eicosanoids.(112)  
Thus, aberrantly production of PGE2 could be related to over-expression of COX-2 or inactivation of 15-PGDH 
and indeed both of these findings have been described as important contributors toward colorectal 
carcinogenesis.(111)  Expression of COX-2 and 15-PGDH in rectal epithelial cells will be detected following the 
standard IHC protocol of EnVision™+ System, HRP (DAKO).  After deparaffinization of the tissue slides, 
antigen retrieval is performed by heating the slides with a pressure cooker in the optimized buffer solutions: R-
buffer A for COX-2 and Tris-glycine for 15-PGDH, The primary antibodies of mouse monoclonal anti-COX-2 
and rabbit polyclonal anti-HPGD are applied with proper work dilutions.  After all fields of each sample are 
measured, the final immunoreaction indices are generated automatically by setting algorithms as ‘‘total positive 
area / total epithelial area X average density’’ for cytoplasmic/membranous staining markers (COX-2, 15-
PGDH). 
 
Determination of Erythrocyte and Rectal Cell Phospholipid Membrane Fatty Acid Analysis 
Lipids will be extracted using the method of Folch-Lees.(113) Fatty acid methyl esters are identified by 
comparing the retention times to those of known standards.  Inclusion of the internal standard, dipentadecanoyl 
phosphatidylcholine (C15:0), permits quantitation of phospholipid amount in the sample. The lowest level of 
detection for individuals’ fatty acids is less than 0.5% of the total profile.  In our laboratory, the inter-assay 
coefficient of variation (CV) is 1.1% and the intra-assay CV is 1.2% for measuring the mass of erythrocyte 
phospholipids.  The variation for the percent fatty acid is less than 1% 
 
Determination of C-reactive protein: 
The rationale for including measures of CRP into the study design is two-fold.  First, CRP is a well-known and 
validated biomarker of systemic inflammation, which has been associated with an increased risk for many 
cancers. (5, 114)  Second, prior literature has suggested that fish oil supplementation may affect the 
production of CRP however; this finding has not been entirely consistent and may be modified by genetic 
factors.  (114-116) Plasma samples will be transported from the GCRC Core Laboratory.  CRP measurements 


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were performed using the High Sensitivity C-Reactive Protein Reagent (Pointe Scientific, Inc, Canton, MI) by 
means of latex particle enhanced immunoturbidimetric assay (ITA).  The minimal detectable concentration of 
CRP is 0.1 mg/L.  The coefficients of variation are 1.99 to 5.74% for within day samples and 1.23 to 6.97% for 
day-to-day sampling. 
 
Determination of Adipokines 
The rationale for determining the effect of fish oil supplementations on adipokines is two-fold.  First, emerging 
evidence has suggested that these adipose-derived hormones may be an important factor linking obesity with 
CRC risk. (19, 27, 37-38, 117-118)  As such, we plan to determine baseline levels of these factors in 
individuals at increased risk for CRC and determine if levels of these factors can be manipulated through fish 
oil supplementation.  Second, although not entirely consistent, several studies have suggested that fish oil 
supplementation may reduce leptin levels while increasing adiponectin levels and thus reduce obesity-related 
chronic inflammation.(119-126)  Measurement of adipokines levels will be performed in the Vanderbilt 
Hormone Assay and Analytical Services Core, which has over 30 years experience in measuring circulating 
levels of hormones.  Leptin and adiponectin will be assayed via radioimmunoassay (RIA) using a double 
antibody procedure.  Final analysis is accomplished by quantifying the bound radioactive counts with a 
Packard Gamma counter connected to a computerized data reduction station.  
 
Determination of Insulin Sensitivity: 
The rationale for including the homeostasis model assessment-insulin resistance (HOMA-IR) within our study 
design is that the HOMA-IR is strongly correlated to euglycemic clamp estimates of insulin sensitivity in 
individuals with normal glucose tolerance, individuals with impaired glucose tolerance and individuals with 
Type 2 diabetes mellitus.(91, 127-128)  The HOMA-IR independently predicts the development of diabetes.  
(129-130) Diabetes mellitus and elevated insulin levels are well-described risk factors for colorectal cancer 
(131) and knowledge as to whether FADS1 activity might modify the effect of fish oil on glucose homeostasis 
could be relevant towards both prevention of colorectal cancer and metabolic diseases.  We will calculate 
HOMA-IR using a method described by Matthews et al.(91)  The HOMA-IR derives an estimate of insulin 
sensitivity from the mathematical modeling of fasting plasma glucose and insulin concentrations. The formula 
is [fasting glucose*fasting insulin]/22.5.  Plasma glucose will be determined based on the Hexokinase method 
measuring the absorbance of NADPH spectrophotometrically and measures on the Hitachi 911 analyzer using 
Roche Diagnostics regents (Indianapolis, IN).  Plasma insulin level will be determined by radioimmunoassay 
(RAI) using a double antibody procedure.  Both plasma glucose and plasma insulin determination will be 
performed in the GCRC Core Laboratory that has extensive experience in determining plasma glucose and 
insulin levels for research studies. 
 
 
Data management and quality control 
The Vanderbilt GCRC Informatics Core will be used as a central location for data processing and analysis.  
Vanderbilt University has developed software tools and workflow methodology for electronic collection and 
management of research study data. (132)  REDCap (Research Electronic Data Capture) is a secure, web-
based application that provides an intuitive interface for users to enter validated data remotely (with automated 
data type and range checks), data manipulation audit trails and reporting, and an export mechanism for end-of-
study export of data to common statistical packages.   
 
 
7.0 
Risks 
 
Potential Risks 
The potential risk to the subjects related to the drawing of venous blood, adipose tissue biopsy, the biopsy 
procedure for rectal tissue, and the use of fish oil supplements.   
 
Sampling of venous blood includes risk such as bruising, bleeding, and infection.  These complications are 
uncommon and phlebotomy is a routine part of general clinical care. 


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Adipose tissue biopsies are associated with several risks.  We will obtain a subcutaneous adipose biopsy from 
the upper buttock with a 16-gauge needle and disposable syringe.  These procedures have been successfully 
used in multiple nutritional epidemiological studies as well as clinical trials, are well tolerated and do not require 
local anesthetics.  These procedures do not require any specialized equipment and can be performed with 
standard 10-ml vacutainer tube and butterfly needle.  Survey studies have found that research volunteers 
consider this procedure to be no more or less uncomfortable compared to venipuncture.(134)  The risks 
associated with this procedure are similar to those of venipuncture and include bruising, bleeding and infection.   
 
Rectal mucosal biopsies are associated with several potential risks.  There is a risk of patient discomfort 
associated with the insertion of the anoscope.  However, this discomfort is generally mild, such that when 
anoscopy is utilized in clinical care, for example visualizing internal hemorrhoids, sedation and analgesia is not 
required. In addition, no bowel preparation is needed reducing the risk of abdominal discomfort associated with 
cathartic agents.  Infection and bleeding are also potential risk associated with rectal biopsy procedures.  
Infection however should be uncommon as all equipment, including the anoscope, obturator and surgical 
forceps are sterile and disposable so there is no risk of contamination between subjects.  Bleeding is 
minimized by the shallow depth of the biopsy and small circumference of the biopsy.  When bleeding does 
occur with the procedure it is generally well controlled through the application of gentle pressure or silver 
nitrate. 
 
Omega-3 fatty acids have been part of the human diet for millennia and have uncommon and generally trivial 
side effects.  In 1997 the Food and Drug Administration rules that an intake of up to 3 g/day of marine omega-3 
fatty acids are Generally Recognized as Safe (GRAS)(135)  (http://www.epa.gov/fedrgstr/EPA-
IMPACT/2002/February/Day-26/i4327.htm). This ruling specifically considered the possible effects of fish oil on 
bleeding time, glycemic control, and LDL cholesterol.  Fish oil supplements have been used in several large 
randomized controlled studies of cardiac patients.(136) In some trials doses as high as 12 g/d for durations of 
2 years have been well tolerated.(137)  The most common side effect of fish oil is a fishy after-taste which is 
less of a problem with pharmaceutical grade supplements.  Minor gastrointestinal symptoms occur in 5 percent 
of patients. (135) Fish oil has been approved by the FDA for treatment of hypertriglyceridemia. 
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or 
Others 
 
Data and Safety Monitoring Plan 
 
Adverse event (AE) grading and attribution scale 
For the purposes of this study and adverse event is defined as any untoward medical occurrence in a subject, 
not necessarily having a causal relationship with the study.  A serious adverse event (SAE) is any untoward 
medical occurrence that a) results in death, b) is life-threatening, c) requires inpatient hospitalization or 
prolongation of existing hospitalization, d) results in persistent or significant disability/incapacity, or e) is a 
congenital anomaly/birth defect.  AE’s are graded as Mild (no limitation of usual activities), Moderate (some 
limitation) or Severe (inability to carry out usual activities) and attributes according to the relationship to the 
study drug and/or procedure as Not related, Unlikely, Possible, Probable, or Definite.   
 
Plan for unanticipated AE reporting 
SAEs and unanticipated AEs will be reported immediately by telephone to the Vanderbilt University IRB, the 
Nashville VA IRB, the GCRC and the DSMB.  An Adverse Event Report form will be completes and returned to 
the VU and VA IRB, GCRC, and DSMB within 5 working days. 
 
Plan for annual reporting of AEs 
Annual reports are submitted to the VU and VA IRBs and will contain a) the number of adverse events and an 
explanation of how each event was handled, b) the number of complaints and how each complaint was 


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handled, c) the number of subject withdrawals and an explanation of why the subject withdrew or was 
withdrawn, and d) the number of protocol violations and how each was handled. 
 
Plan for safety review  
Patients will be monitored by both a study physician and a nurse during rectal biopsy procedures.  Subjects will 
be monitored for 1 hour after the procedure is complete within the GCRC to ensure to immediate adverse 
events and will be contacted by the Research Assistant over the next 24 hours to assess for pain or any other 
symptoms which might be possible post-procedure complications. 
 
9.0 
Study Withdrawal/Discontinuation 
 
Participants may be withdrawn from the study if they are not able to provide blood, urine or rectal samples, or 
they are not able to take their supplements as instructed. 
 
If the intervention proves clearly harmful, the study will be stopped ahead of schedule.  Furthermore, a single 
life-threatening condition related to the intervention, or a single life-threatening event from biospecimen 
collection, would cause consideration for study termination.   Moderate complications occurring in more than 
one subject from any protocol would likewise cause consideration for study termination. 
 
10.0 Statistical Considerations 
 
General Approach 
Patients will be followed for a maximum of 6 months and will complete the study on the last day of treatment 
after rectal mucosal biopsy.  The following categories of patients may fail to complete follow-up: (1) patients 
who discontinue the study before 6 months due to adverse events or other reasons not related to the endpoint 
will be considered withdrawals, (2) patients who are observed at least once during follow-up but die or do not 
return for subsequent visit or do not return for scheduled visits, (3) patients who die before a scheduled visit or 
do not return for any scheduled visits.   
 
Approach to Specific Aim 2 
Hypothesis 
Specific Aim 2 will test the hypothesis that genetically-determined fatty acid desaturase activity will modify the 
effect of fish oil supplementation on biomarkers of colorectal cancer risk  
 
General Approach for Specific Aims 2  
The 150 patients enrolled in Specific Aim 1 will provide data for Specific Aims 2.  Thus, the inclusion/exclusion 
criteria, recruitment and retention, randomization, study design, data collection, intervention, determination of 
the end points, and assessment visits have been described in Specific Aim 1.   
 
Statistical Analysis: Specific Aim 1 and Aim 2 
General approach 
The study is a double-blinded 3 X 2 factorial randomized controlled trial where patients will be randomized to 
the fish oil intervention and placebo group within each genotype group.  The primary outcomes are rectal 
epithelial cell proliferation measured by Ki-67 labeling and rectal crypt apoptosis measured by TUNEL.  The 
main secondary outcome variables will include: rectal mucosal expression of COX-2 and 15-PGDH, production 
of PGE2 and PGE3, rectal mucosal tissue levels of fatty acids, and changes in biomarkers of inflammation (C-
reactive protein), adipokines (leptin, adiponectin), and markers of insulin sensitivity.   
 
In Aim 1, the primary analysis will compare baseline and 6-month Ki-67 proliferative indexes and TUNEL 
labeling indexes in participants allocated to fish oil supplements compared to those allocated to placebo.  In 
Aim 2, the primary analysis will compare baseline to 6-month Ki-67 proliferation indexes and TUNEL labeling 
indexes in participants in FADS1 genotypes (GG, GT, and TT) allocated to fish oil supplements.  The 


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secondary analysis will follow a similar plan as the primary outcome with the secondary outcomes of rectal 
mucosal tissue expression of COX-2 and 15-PGDH and the rectal epithelial cell production of PGE2 and PGE3, 
serum levels of C-reactive protein, leptin, adiponectin, and HOMA-IR scores. 
 
Statistical analysis plan  
Linear regression models will be used to test whether there are differences in the primary outcomes between 
the fish oil intervention group and placebo group (Aim 1) as well as the interaction between the two groups and 
FADS1 genotypes (Aim 2) after controlling for the baseline measurement for each outcome and potential 
confounding factors.  For Aim 2, we will also test differences in the primary outcomes among the genotypes 
within the fish oil intervention group.  For our secondary analyses, a similar regression modeling approach will 
be employed to test differences in all the outcomes among comparison groups described above.  The potential 
confounders included in the models will be polyp type, size, and location for primary adenoma as well as body 
mass index, age, total energy intake, intakes of other nutrients (average of four 24-hour recalls), and baseline 
PUFA levels derived from adipose tissue fatty acid biomarkers. 
 
Descriptive statistics, including means, standard deviation, and ranges for continuous variables, as well as 
percents and frequencies for categorical variables, will be presented.  Standard graphing and screening 
techniques will be used to detect outliers and to ensure data accuracy.  Distributions of continuous outcomes 
will be assessed for normality.  If normality is violated, an appropriate data transformation will be employed. 
Hypotheses will be tested at the level of α=0.05. This data analysis plan will be carried out using statistical 
software SAS® (Cary, North Carolina) or statistical package R (R Development Core Team, 2008).  
 
Sample size estimation and power analysis  
Our sample size estimates are derived using previously described clinical trial information using similar primary 
outcomes and doses of fish oil supplements.  Courtney et al found a change in the mean level of Ki67 labeling 
of -5.86 (SD ± 8.8) for individuals allocated to fish oil supplements (n=14) and 3.62 (SD  8.0) for the placebo 
group (n=14).(57, 60)  With a sample size of 22 per group for each genotype would provide at least 90% of 
power to detect 1 SD (8.4) difference in ΔKi67 between the two groups within each genotype with type I error 
rate = 5%.  Assuming approximately 10% of drop-out rate, the total of 150 patients (25 patients per group per 
genotype  2 groups  3 genotypes = 150) would provide excellent power to detect a clinically significant 
difference between the intervention and placebo groups. 
 
11.0 Privacy/Confidentiality Issues 
 
Confidentiality and ethical considerations will be addressed as follows.  All identifying documents, data and 
specimens collected as a result of this study will be retained by the investigator.  Access to this material will be 
available only to the research investigator and his staff.  Paper (hard) copies of study documents will be kept in 
a locked file cabinet.  Electronic copies of documents will be stored in a password protected database on a 
secured server.  If results of this study are to be published, only code numbers will be used for identification.  
Participants will not be identified by name. 
 
12.0 Follow-up and Record Retention 
 
Hard copies of all records will be stored in locked filing cabinets located in locked office space. Electronic 
databases are located in restricted access folders within the Vanderbilt University Medical Center computer 
network. Access to database is restricted by password or permission-based access. All identifying information 
will be removed and/or deleted from the participant records at 30 years following the conclusion of the 
investigation. Biological samples are coded and retained in a locked location with access limited to specific 
study personnel. DNA extracted will be stored for 30 years. After this period DNA samples will be destroyed. 
 
13.0 Literature Cited: 


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1. 
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009 Jul-
Aug;59(4):225-49. 
2. 
Krause WF, DuBois RN. The molecular basis for prevention of colorectal cancer. Clin Colorectal 
Cancer. 2001 May;1(1):47-54. 
3. 
Wilson SJ, L. Coussens, C. Hanna, K., editor. Cancer and the Enviroment: Gene-Enviromental 
Interactions. Washington, D.C.: National Academy Press; 2002. 
4. 
Kundu JK, Surh YJ. Inflammation: gearing the journey to cancer. Mutat Res. 2008 Jul-Aug;659(1-2):15-
30. 
5. 
Allin KH, Bojesen SE, Nordestgaard BG. Baseline C-Reactive Protein Is Associated With Incident 
Cancer and Survival in Patients With Cancer. J Clin Oncol. 2009 Mar 16. 
6. 
Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002 Dec 19-26;420(6917):860-7. 
7. 
Wang MT, Honn KV, Nie D. Cyclooxygenases, prostanoids, and tumor progression. Cancer Metastasis 
Rev. 2007 Dec;26(3-4):525-34. 
8. 
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Appendix A Study Procedure Calendar 
 
Table D summarizes proposed research tasks during a 5-year study plan.   
 
 
Timeline 
 
Year 1 
Year 2 
Year 3 
Year 4 
Year 5 
Study 
Phase 
Task 
3 
6 
9 
1
2 
1
5 
1
8 
2
1 
2
4 
2
7 
3
0 
3
3 
3
6 
3
9 
4
2 
4
5 
4
8 
5
1 
5
4 
5
7 
6
0 
Run-In 
Refine proposal-PI 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Maintain IRB status-PI 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Order supplies-Core 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Order Medication 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient 
Recruitment 
Pre-recruitment  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Begin clinical recruitment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment visits 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biomarker analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DSMB meetings 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Statistical 
Analysis 
Prepare analytical 
database 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Manuscripts 
Anticipate 3 or more-PI 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Archival 
Archive datasets