Aspirin in Adults With Nonalcoholic Fatty Liver Disease
Phase: PHASE1
Conditions: Nonalcoholic Fatty Liver, Nonalcoholic Steatohepatitis
Sponsor: Massachusetts General Hospital

 
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Aspirin for the Treatment of Nonalcoholic Fatty Liver Disease 
 
Title: Aspirin for the Treatment of Nonalcoholic Fatty Liver Disease 
NCT Number: NCT04031729 
Document: Study Protocol and Statistical Analysis Plan 
Original Document Date: 08/11/2019 
 
 
 

 
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ORIGINAL STUDY PROTOCOL 
Aspirin for Nonalcoholic Fatty Liver Disease (NAFLD):  
A Randomized Clinical Trial 
 
Version: 3 
Version Date: 08/11/2019 
 
IRB Approval Date: 08/19/2019 
Status: Final 
 
I. BACKGROUND AND SIGNIFICANCE 
A. Background. 
Nonalcoholic steatohepatitis (NASH) is an aggressive form of nonalcoholic fatty liver disease 
(NAFLD) that affects an estimated 18 million Americans, and can lead to cirrhosis, 
hepatocellular carcinoma (HCC) and death1, 2. Despite the growing prevalence and mortality of 
NASH cirrhosis2, 3, there are currently no effective FDA-approved therapies for the treatment of 
NAFLD and NASH. Considerable recent preclinical and observational data4, 5 6 demonstrate that 
long-term aspirin use is associated with significantly reduced risk for adverse hepatic events, 
including HCC and liver-related death, in unselected populations without established liver 
disease. However, whether aspirin improves liver fat and markers of inflammation in patients 
with established NAFLD is unknown. Further, the mechanisms underpinning these putative 
associations are undefined. Addressing these knowledge gaps has been highlighted as a key 
priority “to realize the full potential of aspirin in precision chemoprevention” 7.  
 
It has been recommended by subspecialty societies, including the American Association for the 
Study of Liver Disease (AASLD), that changes in liver fat content as quantified by magnetic 
resonance spectroscopy (MRS) or MRI proton density fat fraction (MRI-PDFF), can serve as an 
appropriate and clinically relevant primary outcome in early, phase 1 and 2 clinical trials of novel 
NAFLD therapeutics. Specifically, recent advances in MR-based imaging techniques now permit 
very accurate and validated means of quantifying intrahepatic lipids, via MRS and MRI-PDFF. 
Results of intrahepatic lipids (%) by MRS correlate closely with biochemical and histological 
assessment of liver triglyceride content, with an area under the receiver operating characteristic 
curve that exceeds 90%. Moreover, MRS measures global hepatic fat content across the entire 
liver (rather than from a small sample, from a liver biopsy), which is relevant as lipid content 
may be heterogeneous and thus liver biopsy could result in sampling error. Collectively, this 
robust prior evidence demonstrates that MRS (and MRI-PDFF) are robust and accurate means 
of quantifying steatosis for the primary endpoint assessment, in early-phase NAFLD 
interventional trials. In addition to these established MRI endpoints, the present study also 
includes an optional enrollment and month 6 liver biopsy, as part of the optional biopsy sub-
study, but liver biopsy is not required for the main study.  
 
II. SPECIFIC OBJECTIVES & ENDPOINTS 
We will conduct a randomized, placebo-controlled trial to assess the feasibility and efficacy of 
aspirin for reducing liver fat, inflammation and fibrosis, in 80 adults aged 18-65 with established 
nonalcoholic fatty liver disease (NAFLD), confirmed by validated imaging modalities or by a 
clinical liver biopsy within 12 months of the screening visit. 
 
Primary Efficacy Endpoint: 6-month absolute difference in % hepatic fat fraction (HFF), 
measured by 1H-MRS. 
 
Key Secondary Endpoints:  

 
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1. Relative 6-month percentage change in HFF %, by 1H-MRS.  
2. Proportion of patients with 30 percentage point or greater relative reduction in HFF, by 
1H-MRS.  
3. Absolute change in liver fat %, as measured by MRI-PDFF.  
4. Relative percentage change in liver fat %, as measured by MRI-PDFF. 
 
Non-Key Secondary Endpoints: 
5. 6-month change in alanine aminotransferase (ALT) and aspartate aminotransferase 
(AST). 
6. 6-month change in the composite of inflammation and fibrosis, measured by the cT1 
score, using LiverMultiScan 
7. 6-month change in liver stiffness measurement (LSM) of fibrosis, assessed by vibration 
controlled transient elastography 
8. 6-month changes in body weight (kg) and body mass index (BMI). 
 
Exploratory Endpoints: 
9. Changes in circulating biomarkers of aspirin target engagement  
10. Changes in levels of pro- and anti-inflammatory lipid species and plasma metabolites, 
and changes in gut microbial composition and function, that may be impacted by aspirin 
therapy 
 
Safety Endpoints: 
Safety endpoints will be assessed according to the following schedule, and will be closely 
monitored by the IRB and by the Data Safety Monitoring Board (DSMB). Criteria for individual 
and/or study discontinuation based on safety assessments are outlined in detail below (see 
Safety Monitoring). 
1. CBC: assessed at screening, baseline, and 6 months (and hematocrit additionally 
assessed at 3 months)   
2. Pregnancy testing at all visits  
 
 
III. SUBJECT SELECTION 
A. Eligibility  
Inclusion criteria:  
1. Ages 18 to 65 years  
2. NAFLD defined as confirmed hepatic steatosis (>5% intrahepatic triglycerides) by liver 
biopsy (within 12 months of screening) or by validated imaging modality (non-contrast 
CT scan, 1H-MRS, MRI-PDFF, ultrasound), in the absence of significant alcohol 
consumption (>2 drinks daily for women, >3 drinks daily for men) and other causes of 
hepatic steatosis. If relevant liver imaging (defined above) or liver biopsy has not been 
performed clinically within the prior 12 months before the screening date, liver ultrasound 
will be performed as part of the screening visit.  
 
Exclusion criteria:  
1. Other chronic liver disease, including viral hepatitis B or C infection, alpha-1 antitrypsin 
deficiency, Wilson’s disease, hemochromatosis, autoimmune hepatitis, primary biliary 
cholangitis, primary sclerosing cholangitis, or HIV infection, as defined by medical record 
review or by patient self-report, and confirmed with the treating medical provider.  
2. Alcohol intake greater than or equal to two drinks per day in women or three drinks per 
day in men.  

 
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3. Cirrhosis or any prior liver decompensation event (i.e., ascites, spontaneous bacterial 
peritonitis, hepatic encephalopathy, esophageal varices, and/or hepatorenal syndrome), 
defined by review of medical record (including clinical notes, prior liver imaging, 
procedures and prior liver biopsies where available), and by confirmation from treating 
medical provider. 
4. Severe cardiovascular disease (i.e. myocardial infarction within the last year, left 
ventricular ejection fraction of less than 40% or active angina), defined by medical record 
review or by patient self-report 
5. Severe renal disease (defined as eGFR less than 30) 
6. Pregnancy or desire to become pregnant or breastfeeding. 
7. Contraindications to aspirin use, including aspirin intolerance, prior peptic ulcer disease, 
prior GI or non-GI bleeding 
8. Thrombocytopenia  
9. Aspirin use within the preceding 3 months  
10. Current use of other antithrombotics, anticoagulants, or antiplatelet agents (not including 
non-aspirin nonsteroidal anti-inflammatory drugs, NSAIDs) 
11. Routine MRI exclusion criteria (i.e. metal implants, etc.) 
12. Bariatric surgery within the past 2 years 
13. Active cancer (i.e. currently undergoing treatment), except for non-melanoma skin 
cancer. 
 
B. Recruitment:  
We will use the following potential techniques for recruitment:  
1. Advertisements in newspapers 
2. MGH and BWH’s “Research Studies Seeking Volunteers” email 
3. RSVP for health emailing 
4. Internet posting (Craig’s List, Facebook) 
5. Mass mailings, based on demographic and geographic area 
6. Advertise in local newspapers 
7. Postering throughout the Boston metropolitan area, with close attention to recruiting 
minorities 
8. Letters to patients from Massachusetts General Hospital, Brigham and Women’s 
Hospital, North Shore Medical Center, Salem Hospital and Faulkner Hospital 
(“Partners Healthcare System”) who have been diagnosed with obesity, fatty liver 
disease and/or related conditions, based on Partners Healthcare System-approved 
search of billing records “RPDR” search. Potential subjects will be contacted after 
signed permission from a provider OR contacted directly if the potential subject has 
previously signed up for this feature. 
9. Recruitment through the MGH Fatty Liver Clinic, The Gastrointestinal Surgery Clinic 
at MGH and other clinics in which current study staff provide care. 
10. Recruitment through the MGH NAFLD Tissue Repository Studies (IRB #: 
2009P002081 and IRB #: 2009P002098) 
11. Recruitment through a list of research subjects who have previously been recruited by 
our study team and have agreed to recontact for future studies. 
12. Recruitment through materials distributed to providers who care for NAFLD patients 
outside of the Partners System. 
13. Recruitment through the Research Match (Vanderbilt Recruitment Tool) 
 
Subjects who express interest will be pre-screened via a REDCap questionnaire or phone. 
 
 

 
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IV. SUBJECT RECRUITMENT 
A. Trial Population 
Subjects responding to the initial recruitment advertisements will undergo initial screening and if 
deemed potentially eligible for participation, they will be contacted by telephone and invited to 
participate in a screening visit. Based on a conservative assumption of 60% rate of either 
screening failure or subjects declining to participate in a research study, we expect to screen 
200 adult subjects and enroll a total of 80 subjects with confirmed NAFLD without cirrhosis.  
 
B. Consent  
All subjects will receive the consent form for the study as well as the Human Subject’s Bill of 
Rights. These documents will be read by the study subjects and also reviewed carefully by the 
study subject with a clinician on the research staff, prior to participating in the study. Any 
questions, concerns, or ambiguities will be appropriately clarified by the study clinician prior to 
the patient signing consent. Potential study subjects will sign informed consent and only then 
will they begin participation in the study. If new information is ascertained during the study, we 
will modify our consent and re-consent our patients accordingly.  
 
C. Randomization  
After baseline testing, 80 subjects who meet all eligibility criteria will be randomized to one of 
two treatments for 6 months: 1) low-dose aspirin (81mg per day), or 2) identical, blinded placebo 
pills.  
 
 
V. STUDY PROCEDURES: 
 
A. Overview of Study Design. 
An overview of the trial design is shown in the figure. This study will examine the effect of low-
dose aspirin administration on intrahepatic lipid accumulation (by 1H-MRS) in 80 subjects with 
confirmed NAFLD without cirrhosis.  
 

 
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The MGH Research Pharmacy will prepare and dispense blinded, identical aspirin or placebo 
capsules to the randomized subjects, thereby maintaining double-blinding.  
 
Screening Visit: (n= an estimated 200 subjects) 
Pre-screening for eligibility will be performed, including obtaining confirmation from treating 
medical provider regarding potential eligibility, if indicated. At the screening visit, a history and 
physical (including measurement of vital signs, height and weight, and calculation of BMI) will be 
performed. A creatinine, CBC and a PT/INR will be performed. A pregnancy test will be 
performed in all premenopausal women. Individuals will be queried regarding alcohol use and 
frequency of use, as well as for history of other chronic liver disease or cirrhosis or hepatic 
decompensation. Hepatitis B surface antigen and/or Hepatitis C Ab testing will be performed in 
study subjects who have not had this, as part of their clinical care. Individuals will be carefully 
queried for all inclusion and exclusion criteria and for their eligibility to undergo MRI scanning.  
 
Finally, histology slides from prior clinically-indicated liver biopsies will be requested if/when 
available. Record requests will be initiated for slides to be obtained outside of MGH. Slides will 
be reviewed at MGH for a central pathology read. Slides will be reviewed for grading of NASH 
activity score and other NAFLD/NASH-related pathology assessments. All histology slides will 
be returned after review. 
 
 
Study Visits:  
1. Baseline and 6-month visit:  
The baseline visit will occur within 16 weeks after the screening visit. The following 
assessments will be made during the baseline visit and repeated at the month 6 visit: 
• 
Interview to determine changes in medical history since the prior visit. 
• 
Measurement of blood pressure, weight, BMI, waist-to-hip ratio  
• 
Serum or urine pregnancy testing in women of childbearing age and potential. 
• 
Fasting blood samples:  
o Comprehensive metabolic panel, complete blood count, PT/INR, insulin/glucose, 
hemoglobin A1c, inflammatory markers, lipid panel and liver function testing. 
These laboratory assessments are consistent with current recommendations for 
early-phase clinical intervention trials of NAFLD/NASH8 
o NAFLD Fibrosis Score will be calculated from standard labs and subject 
characteristics (age, BMI, blood glucose, platelet count and albumin). 
o Serum and Plasma will be collected for future measurement of lipids, bile acids, 
other metabolites including adipokines and inflammatory markers. 
• 
Additional peripheral blood (serum, plasma, and whole blood) from which we will extract 
DNA and RNA. This will permit testing (among other things) for the PNPLA3 rs738409 
genotype, as this is an established NAFLD risk locus, and testing of PNPLA3 is currently 
recommended in NAFLD/NASH clinical trials8. 
• 
Stool samples that have been collected at home will be brought with the patient on the 
Baseline Visit. A collection kit provided during the Screening Visit will allow minimal 
contact with the stool and allow easy storage of the sample until the day of the Baseline 
Visit. If the patient forgets or is unable to complete the sample collection prior to their 
appointment, they may be given the materials to mail their sample to the study staff.  
• 
MRI: 1H-MRS of the right and left hepatic lobe will be performed to quantify hepatic lipid 
content and other fat depots. LiverMultiscan sequences will also be performed for 
noninvasive analysis of hepatic inflammation and fibrosis. During the same MRI session, 

 
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MRI-PDFF will also be performed. MRI will be repeated at the 6-month visit (for a total of 
2 MRI sessions per subject). 
• 
Fibroscan to measure liver stiffness via transient elastography (ultrasound). Fibroscan 
will be repeated at the 6-month visit (for a total of 2 Fibroscans per subject).  
• 
Standard bionutrition assessment by trained research nutritionist. 
• 
Assessment of adherence (month 6 visit) by returned pill count and by self-report. 
• 
Three validated questionnaires will be administered to subjects:  
o The Paffenbarger Physical Activity questionnaire 
o The SF-36 health survey questionnaire  
o The semi-quantitative Food Frequency Questionnaire (FFQ) 
 
Total Blood Drawn: Total blood volume from the participant is below 200ml for this Baseline 
Visit and for the Month 6 visit. As this is below 300ml over at least 6 hours, this does not result 
in added risk. Normal blood donation volume is 450-500ml.   
 
2. Follow-up 3-month visit: (n=80) 
This visit will be performed during a +/- 2-week interval around the planned date, to 
accommodate potential scheduling conflicts. A physical examination and measurements of body 
weight and waist-to-hip ratio will be completed. Study subjects will be assessed for interim 
medical history, and any side effects to study drug. We will measure a hematocrit.  
 
3. Twice monthly telephone calls or voicemail messages (+/- 1 week):  
The trained clinical research coordinator, working directly with the PI, will conduct twice-monthly 
check-in telephone calls to enrolled subjects on the months when the subject does not have a 
study visit (i.e. at month 2, month 4 and month 5), using a Partners IRB-approved telephone 
script. The purpose of these calls will be to ask about medication compliance and to inquire 
about any concerns or side effects related to the study drug. All subjects will be reminded during 
this call or voicemail message that the PI (Dr. Simon) will be available by pager at all times 
during the study to answer any questions or concerns that arise. 
 
4. Early Termination Visit:  
Subjects who discontinue the study early due to an adverse event, toxicity, or personal reasons 
will be asked to complete an Early Termination visit. This visit will include all assessments that 
are performed at the final 6-month visit described above. Participants must have been on the 
study medication for at least 11 weeks in order to be eligible for completing an Early 
Termination visit.  
 
5. Remote Visits: Subjects who do not live locally will have the option to perform the 3-month 
follow-up visit remotely. This will be performed with remote labs (via Quest Diagnostics 
Locations) and medical history via phone call with a study staff member. For these remote visits, 
the study staff member will speak to the subject over the phone to review the medical history. If 
during a phone-medical history, the study staff member believes the subject may be 
experiencing side-effects related to the study medication, the PI will be notified and the subject 
will be informed that they need to come to MGH for a physical exam. For these remote visits, 
the study team will FedEx study medication to the subject in a manner approved by the 
research pharmacy as needed for resupply. The subject would bring back unused study 
medication and empty vials to the study team at the next in person visit to maintain standard 
study drug accountability logs.  
 
 

 
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B. DRUGS 
Aspirin: Aspirin 81mg tablets (generic; purchased directly by the MGH Research Pharmacy and 
made into blinded aspirin capsules that are identical to placebo capsules). 
 
Placebo: Blinded placebo capsules that are identical to the aspirin capsules will be prepared by 
the MGH Research Pharmacy (purchased directly by the MGH Research Pharmacy).  
 
Rationale for dose: Dosing is based on U.S. Preventive Services Task Force prior 
recommendations for consideration for primary CV and colorectal cancer (CRC) prevention9. 
Use of 81mg/day is also consistent with another active RCT of aspirin for CRC prevention 
(NCT02394769)10. Aspirin is not contraindicated in NAFLD. Nevertheless, bleeding with aspirin 
is dose-dependent11, so the choice to use low-dose aspirin (81mg per day daily dose) will 
optimally balance safety and efficacy.  
 
Rationale for duration: 6 months is recommended for early-phase NAFLD clinical trials by the 
AASLD8, is widely used12-14, and is sufficient to reveal changes in fat15-21, thus this trial duration 
appropriately balances efficacy and compliance. 
 
The risks of low-dose (81mg) aspirin treatment include aspirin allergy or intolerance, with or 
without associated bronchospasm, gastrointestinal (GI) upset, risk for peptic ulcer disease, and 
bleeding, including GI bleeding, intracranial bleeding and bleeding from other sites11, 22-24. We 
have taken several steps to minimize these potential risks. First, we have selected a low daily 
aspirin dose (81mg per day), which minimizes potential bleeding or ulcer-related risks, 
compared to higher doses. Second, we will exclude any subject with a personal history of 
aspirin intolerance, and anyone with a personal history indicating an increased risk for bleeding, 
including: personal history of bleeding or stroke, and/or history of coagulopathy and/or 
thrombocytopenia, and/or any subject with current use of any other anti-coagulant or 
antithrombotic medications. Third, we will conduct a 3-month visit with assessment of safety 
labs (hematocrit), as well as an interval history and physical examination to assess for any 
interval bleeding. Finally, we will conduct twice-monthly telephone check-ins that will further 
enhance safety by giving subjects additional opportunities to further discuss any issues or 
concerns. Importantly, we will ensure that all subjects are aware of and understand these risks, 
through our informed-consent process.  
 
It should be emphasized that bleeding-related risks are not felt to be elevated in individuals with 
non-cirrhotic NAFLD (defined by fibrosis stage <= 3) compared to the general population. In 
fact, recent preliminary data suggest that patients with NAFLD may have an increased risk for 
thrombotic events for which an anti-thrombotic medication may offer important benefits25. Thus, 
we do not expect daily aspirin 81mg to increase the risk of bleeding more in a population with 
NAFLD without cirrhosis, compared to the general population. Overall, we believe that the 
potential overall benefits low-dose aspirin for adults with non-cirrhotic NAFLD/NASH outweigh 
the potential risks.  
 
 
C. DEVICES AND PROCEDURES: 
Intrahepatic fat:  We will perform proton MR spectroscopy (1H-MRS) and MRI-PDFF of the right 
and left hepatic lobe to determine hepatic fat fraction, using a 3Tesla MRI device (Siemens Trio, 
Siemens Medical Systems, Erlangen, Germany) as previously described26.  Advantages of 
higher magnetic field strength are an increase in signal-to-noise ratio resulting in improved 
spatial resolution and increased chemical shift dispersion, potentially improving assessment of 

 
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other fat resonances. MRI sequences are also performed for assessment of other fat depots 
and LiverMultiScan analysis within the same session.   
 
LiverMultiScan: LiverMultiScan uses the same scanner as 1H-MRS, producing a validated, non-
invasive assessment of inflammation and fibrosis called the Liver Inflammation Fibrosis (LIF) 
score, which has increasing severity from 0-4, and which correlates with NAFLD histological 
severity27, and further predicts outcomes.28 Image processing and analysis of 1H-MRS and 
LiverMultiScan data will be completed without charge through an established collaboration that 
is already in place between the PI (Dr. Simon) and Perspectum Diagnostics, who also work 
closely with the MGH Department of Radiology. 
 
Liver function tests:  A standard liver function test panel consisting of ALT, AST, GGT, alkaline 
phosphatase, total bilirubin, direct bilirubin, indirect bilirubin, ferritin and albumin will be 
measured using a standard clinical platform. 
 
Lipids and Lipoproteins:   A fasting lipid profile, including total cholesterol, LDL, HDL and 
triglycerides, will be determined.   
 
The NAFLD fibrosis score: This score predicts the probability of fibrosis in patients with NAFLD 
using a score that uses the patient's age, body mass index, blood glucose levels, 
aminotransferase levels, platelet count, and albumin. A high NAFLD fibrosis score of >0.676 is 
associated with a probability of advanced fibrosis (F3-F4) of 82 percent (sensitivity 43 percent, 
specificity 96 percent), and a score of <-1.455 is associated with a negative predictive value of 
88 percent (sensitivity 77 percent, specificity 71 percent) based on prior studies.29, 30 
 
Fibroscan: Elastography (Fibroscan) is a rapid, non-invasive ultrasound-based modality that 
measures liver stiffness by transmission of mild amplitude and low frequency (50Hz) through the 
intercostal space using a vibrator at the skin surface. The vibration induces a shear wave 
velocity through the hepatic tissue, which is directly related to tissue stiffness and is expressed 
in kilopascals (kPa). Transient Elastography is an inexpensive, reproducible, painless, rapid <10 
minutes and easy to perform tool. It has been proven in many studies to correlate well with 
degree or severity of fibrosis and or cirrhosis.  
 
Additional Blood Samples: After the subject has signed the informed consent form, peripheral 
blood will be collected for serum, plasma and whole blood, from which we will extract DNA and 
RNA. The blood draw will occur on the day of the Baseline Visit (Visit 1). Raw samples will be 
transferred at room temperature within 24 hours of collection to the laboratory of Dr. Tracey 
Simon for sample processing and storage at -800 C.  
 
Some examples of the type of analyses that we may perform with the specimens collected are 
provided in the following paragraphs: 
 
Lipid Mediators, Inflammatory Markers and Metabolites Serum and plasma samples for 
measurement of circulating metabolites, inflammatory markers and lipids relevant to aspirin 
target engagement, including eicosanoid lipid species, SPMs, untargeted lipid metabolites and 
bile acids, will be quantified using validated and well-established liquid chromatography tandem 
mass spectrometry (LC-MS-MS) platforms for metabololipidomics profiling. Metabolomics 
profiling will be performed with Metabolon, Inc. Lipidomics profiling will be performed with our 
collaboratirs, Drs. Maddipati and Charles Serhan, with data acquired in negative ionization 
mode, and further eicosanoid and SPM quantification by multiple reaction monitoring (MRM)31, 

 
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32, according to published criteria33, 34. Dr. Maddipati together with Co-Investigator Dr. Charles 
Serhan have shown excellent reproducibility of this method for quantifying eicosanoid 
derivatives, including SPMs and other circulating lipid mediator species32, 34, with a lower limit of 
detection of ~0.1pg34. Notably, this method will also permit measure of SPM precursors (e.g. 
erythrocyte docosahexaenoic acid [DHA], eicosapentaenoic acid [EPA]), which also reflect 
dietary intake, aspirin use and hepatic DHA/EPA35, 36, and which may impact hepatic steatosis37.  
 
Stool Metagenomics: Stool samples will be collected at home on two instances (before the 
Baseline Visit and Before Visit #4). A collection kit will allow minimal contact with the stool and 
allow easy storage of the sample until the day of the study visit. If the patient forgets or is unable 
to complete the sample collection prior to their appointment, they may be given the materials to 
mail their sample to the study staff at a later date. Stool samples will be collected on a spoon 
and placed into two tubes. The tubes will be included in stool collection kit and will contain 95% 
ethanol for both future DNA/RNA extractions and metabolomic analysis. Upon receiving the 
samples, the stool in the first tube will be centrifuged and separated from the fixative. Then, both 
stool samples will be stored at -80°C in the lab of Dr. Tracey Simon and Dr. Raymond Chung. 
 
 
Genotyping  
Genomic DNA samples will be used for the identification and/or genotyping of polymorphisms 
that may predict the pathogenesis and progression of NAFLD. Additionally, polymorphisms in 
any one of many genes, including those encoding drug receptors, drug transporters, and cell 
signaling pathways, can be important determinants of clinical response. An advantage of 
developing a DNA-based diagnostic test as a clinical biomarker is that only a small sample of 
tissue (i.e. blood from a finger prick or buccal cells) is required for genotyping of a large number 
of polymorphisms. DNA-based diagnostic assays also provide a clinician the ability to perform 
high-throughput and accurate diagnostic tests for a reasonable cost.  
 
Transcriptional Profiling  
Whole-genome analyses are beneficial to research studies where the end goal is to focus on a 
small number of genes. Transcriptional profiling is an efficient tool to sort through the expression 
of thousands of genes, and to identify significant associations with disease severity or drug 
response. Another advantage of performing whole-genome analyses is that novel biological 
pathways related to the disease could be more clearly elucidated, and mechanisms of drug 
action can be revealed in a genomic context.  
 
Proteomics  
Although genotyping and transcriptional profiling can yield important pharmacogenomic results, 
a major disadvantage of these techniques is the lack of correlation between gene expression at 
the mRNA level with the amount of actual expressed protein within the cell. Proteomics 
technologies allow for the characterization of protein expression, protein-protein interactions, 
and post-translational modifications and processing that are important in disease states. 
Moreover, the ideal platform for a clinical diagnostic test is an ELISA assay to detect serum 
levels of a particular secreted protein biomarker.  
 
Serum and plasma samples will be used for proteomic and/or ELISA analysis to identify protein 
or peptide biomarkers that are associated with NAFLD outcomes.  Typically, 2-D gel 
electrophoresis will be used for initial detection and location of protein differences, in addition to 
liquid chromatography-mass spectrometry and tandem mass spectrometry for detailed peptide 

 
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mapping, characterization and identification of proteins. Multiple ELISA assays for proteins from 
biological fluids will subsequently be developed for marker validation.  
 
 
VI. APPROACH AND BIOSTATISTICAL ANALYSIS 
 
A. Experimental approach: 
A total of approximately 80 adult subjects with NAFLD (F0-3) will be enrolled after a screening 
visit at which study eligibility will be determined and confirmed. We expect to screen up to 200 
subjects in order to enroll a total of 80 subjects. Eligible subjects will be randomized to receive 
aspirin or placebo. Subjects will be studied for 6 months.  
 
B. Data Integrity: 
To ensure data entry accuracy, peer review of source documentation and case report forms in 
REDCap will be completed following MGB Study Data Management and Quality Assurance 
Plan Guidelines. Research coordinators will exchange subject files and review informed 
consents, case report forms, and adverse event logs for accuracy and completeness. 
Additionally, eCRFs in REDCap will be reviewed for accuracy and completeness using source 
data verification. 
 
C. Statistical Analysis and Power 
Data Analysis Plan: 
This statistical analysis plan will be finalized before database lock. Baseline data between 
groups will be compared using chi-squared test or Fisher’s exact test for categorical variables, 
and using ANOVA for continuous variables, with any non-normally distributed data log-
transformed prior to analysis. If significant baseline group imbalance is detected on any 
particular variable, that variable will be included as a covariate in the analyses. 
 
Absolute and relative changes in continuous outcomes between treatment groups will be 
calculated using analysis of covariance (ANCOVA), with treatment group at randomization 
included as a fixed effect, and the baseline value of the relevant outcome included as a 
covariate. Logistic regression will be used for binary outcomes (i.e., achievement of ≥30% 
relative HFF reduction). For the primary analysis, all study endpoints will be analyzed using an 
intention-to-treat approach, using data from all randomly assigned participants in the full 
analysis set. The primary analysis will assume that missing data were missing at random38, and 
data augmentation for missing observations will be conducted via multiple imputation, using all 
available non-missing data of the outcome measurement from the same treatment arm.  
 
We will also utilize data from all participants who adhere to therapy as assigned and complete 
the final, 6-month visit for study outcomes (i.e., the per protocol analysis set).   
 
We will conduct the following subgroup and sensitivity analyses for the primary and key 
secondary endpoints:  
To address potential confounding due to meaningful weight loss between baseline and month 6, 
we will exclude any participant who loses ≥3% body weight during the 6-month study period. We 
will construct separate, multivariable-adjusted regression models accounting for a priori defined 
clinical confounders, including age, sex, race/ethnicity, type 2 diabetes status at baseline, 
fasting insulin level at baseline, body weight and visceral adipose tissue (VAT) volume, as 
previously recommended for NAFLD clinical trials8. We will also repeat the analyses with 
missing data for primary and key secondary endpoints considered as nonresponses, by carrying 
forward the baseline values. We will also conduct subgroup analyses for the endpoints of 

 
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absolute change in HFF by MRS (primary endpoint) and relative HFF change by MRS (key 
secondary endpoint) in participants with significant, stage 2-3 fibrosis at baseline. 
 
Sample Size, Statistical Power & Multiplicity Testing:   
We anticipate a conservative drop-out rate of approximately 15%, thus we estimate an effective 
sample size of 68 patients. Based on prior studies of a dietary intervention for hepatic steatosis 
(Ryan et al. 39), and data from a meta-analysis of oral omega-3 supplementation and hepatic 
steatosis (Parker et al.40) we expect an effect size of 3% with the standard deviation of the 
change in liver fat of 2.45, by 1H-MRS. Applying these estimates, the probability is >90% that 
the study will detect a treatment difference at a two-sided 0.05 significance level41. The Holm 
procedure to control for multiple testing will be applied to the 4 key secondary outcomes, as 
follows: P=0.0125 for relative change in HFF by MRS, P=0.0167 for proportion achieving ≥30 
percentage point reduction in HFF by MRS, P=0.025 for absolute change in HFF by MRI-PDFF, 
and P=0.05 for relative change in hepatic fat by MRI-PDFF. For the other non-key secondary 
outcomes no multiplicity adjustments will be employed. For exploratory outcomes, no multiplicity 
adjustments will be used. 
 
Definitions of Analysis Sets: 
The full analysis set includes all 80 randomized subjects. Subjects in the full analysis set will 
contribute to the “intention-to-treat” analysis. The as-treated set will include all participants who 
adhered to therapy as assigned and completed the final, 6-month study visit for study outcomes. 
Finally, an additional analysis set for a sensitivity analysis will be constructed with missing data 
for study endpoints treated as nonresponses, by carrying forward baseline values. 
 
 
VI. RISKS AND DISCOMFORTS 
A. Potential Risks to the Subjects 
Human Subjects Involvement and Characteristics: This study will enroll 80 adults with NAFLD 
without cirrhosis (F0-3) between the ages of 18 and 65 years. Rationale for exclusions is 
discussed in the study protocol; in brief, these exclusions are designed to 1) exclude persons at 
greatest risk for significant adverse events, and 2) avoid confounding effects of other clinical 
comorbidities and/or medications.  
 
We may send de-identified samples to a third party for analysis, such as to Metabolon, or to 
Qiagen, GenomeScan, or others.  These specimens will not be linked to any individual 
identifiers associated with the patients. If we send de-identified samples to a third party, we will 
first generate a contract for this purpose through the hospital supply chain.   
 
Potential risks from study drug: 
• 
Aspirin: Subjects will be randomized to receive aspirin in the form of an 81mg capsule or 
blinded, identical placebo capsule, to be taken once daily. The risks of aspirin treatment 
include aspirin allergy or intolerance, with or without associated bronchospasm, 
gastrointestinal (GI) upset, risk for peptic ulcer disease, and bleeding, including GI bleeding, 
intracranial bleeding and bleeding from other sites11, 22-24. We have taken several steps to 
minimize these potential risks. First, we have selected a low daily aspirin dose (81mg per 
day), which minimizes potential bleeding or ulcer-related risks, compared to higher doses. 
Second, we will exclude any subject with a personal history of aspirin intolerance or aspirin 
allergy, and anyone with a personal history indicating an increased risk for bleeding, 
including: personal history of bleeding or stroke, and/or history of coagulopathy and/or 
thrombocytopenia, and/or any subject with current use of any other anti-coagulant or 
antithrombotic medications. Finally, we will conduct a 3-month visit with assessment of 

 
13 
safety labs (hematocrit), as well as an interval history to assess for any interval bleeding. 
Moreover, we will conduct twice-monthly telephone check-ins that will further enhance safety 
by giving subjects additional opportunities to further discuss any issues or concerns. 
Importantly, we will ensure that all subjects are aware of and understand these risks, 
through our informed-consent process. However, it should be noted that bleeding-related 
risks are not felt to be elevated in individuals with non-cirrhotic NAFLD/NASH, compared to 
the general population. In fact, recent preliminary data suggest that patients with 
NAFLD/NASH may have an increased risk for thrombotic events for which an anti-
thrombotic medication may offer important benefits25. Thus, we do not expect daily aspirin 
81mg to increase the risk of bleeding more in a population with non-cirrhotic NAFLD, 
compared to the general population. Overall, we believe that the potential overall benefits 
low-dose aspirin for adults with non-cirrhotic NAFLD to outweigh the potential risks.  
• 
Placebo: The risks associated with a placebo capsule include very minor intolerance or GI 
upset, however this is very infrequent and placebo pills are generally well-tolerated.  
 
Risks from blood draws: 
Blood sampling is performed in this study and there is always a very minor risk of infection, 
bruising, or syncope during a blood draw. There is also the discomfort of having one’s blood 
drawn. The blood draws will be performed by trained staff who have certificates documenting 
their ability to draw blood.  
 
Risks from Fibroscans: 
Fibroscans will be performed using FDA approved devices. There are no known foreseeable 
risks associated with exposure to Fibroscan. 
 
Risks from MRI/MR Spectroscopy:  
MRI and MR spectroscopy will be performed twice during this prospective study (baseline and 
final 6-month visit) using FDA-approved devices and pulse sequences. A standard MRI 
questionnaire will be administered to all potential study subjects, and no study subject with a 
contraindication to MR testing will be admitted to the study. There are no known foreseeable 
risks associated with exposure to MRI, provided patients are appropriately screened and there 
are no metallic implants (i.e. vascular clamps or pacemakers). All potential subjects will be 
carefully screened for the presence of such implants or any other contraindication to MRI, prior 
to each exam. No study subject with a contraindication to MR testing will be admitted to the 
study. Some subjects report some claustrophobia during MRI scans. If a patient expresses any 
discomfort whatsoever during the scan, the procedure will be aborted and not repeated without 
his/her full consent. 
 
Risks of Stool Collection: 
Stool will be collected using a stool collection kit that contains some special preservatives to 
make sure they are stable over time.  If skin comes into contact with these preservatives, they 
could irritate the skin. The preservative can simply be washed off with soap and water.  
 
Risks of Genetic Testing 
Genetic information that results from this study does not have medical or treatment importance 
at this time. However, there is a risk that information about taking part in a genetic study may 
influence insurance companies and/or employers regarding a subject’s health. To safeguard the 
privacy of our subjects, genetic information obtained in this study will not be shared or made 
available to be placed in the medical record. Taking part in a genetic study may also have a 
negative impact on family or other personal relationships.  
 

 
14 
We will in no way discourage use of any medications that are clinically indicated for the subject. 
Subjects will be discontinued from the study if they start a medication that is known to affect 
liver fat, including methotrexate, amiodarone, oral steroids or SERMs (such as tamoxifen). The 
subject will be able to continue in the study if they initiate other medications that are not known 
to affect liver fat content.  
 
B. Protection Against Risk  
In addition to the formal safety monitoring plan described below, the procedures to protect 
against or minimize potential risks include the following: (1) the assignment of unique study 
subject numbers to patients, (2) the use of these primary identifiers throughout the study, (3) 
storage of information in locked file cabinets, and (4) access limited to study personnel for these 
file cabinets and data. The blood draws will be performed by trained staff who are certified to 
draw blood. We are performing a physical examination and routine laboratory tests prior to 
allowing anyone to enter the protocol to ensure that subjects are medically stable. Close 
monitoring of patients throughout the study will ensure that adverse effects from our 
interventions. All study subjects will be instructed on how to contact study clinicians in the case 
of an emergency. The PI (Dr. Simon) will be available by pager at all times during the study to 
answer any questions or address any concerns that a subject may have. 
 
Participants will also be required to sign a consent form prior to entering into the study. All 
recruitment materials will be approved by the MGH Institutional Review Board prior to initiating 
the study.  
 
C. Anticipated Benefits to Subjects 
There is no expected benefit to participation in this study. If one is assigned to receive aspirin 
therapy, it is possible that their amount of liver fat may decrease and their cardiovascular risk 
and/or waist fat may decrease during the study. However, those effects are not promised or 
guaranteed. If benefits occur, there is no way to know how long they will last.  
Participants will have thorough physical exams while in this study. If a medical condition is 
discovered, one will be referred to your physician. It is possible that information learned from 
this study may help improve therapy for people who are obese. Further, all participants will 
receive information about their metabolic and nutritional health at the screening visit, consistent 
with current recommendations for clinical trials in patients with NAFLD/NASH8. The risk involved 
in study participation is thus thought to be balanced by the possible benefits to participants.   
 
 
IX. INDEPENDENT MONITORING OF SOURCE DATA: 
Monitoring of Source Data:  
The principal investigator will be reviewing each subject’s data periodically throughout the 
duration of the study for quality, validity and integrity assurance and for adherence to the IRB 
approved protocol. In addition, the study coordinators will monitor subject documents on an 
ongoing basis.  All source data will be organized and filed into subject binders.  Any pertinent 
notes to file regarding the subject will be documented by the study coordinator and kept in the 
applicable subject binder.  All subjects will be assigned a study identification number.   
 
Data Quality Control Monitoring:  
Data will be recorded on specified Case Report Forms retained at Massachusetts General 
Hospital through REDCap. REDCap is a secure, web-based application designed to support 
data capture for research studies. The system was developed by a multi-institutional consortium 
initiated at Vanderbilt University and designed to comply with HIPAA regulations. Data forms 
from REDCap can be automatically exported into a spreadsheet to which additional data 

 
15 
(laboratory studies, etc) can be added. Forms that have missing or inconsistent data will also be 
entered into the database, except that a missing value indicator will take the place of the 
missing or inconsistent information. A missing information form will be filled out to indicate the 
items that are missing. Any paper data forms will be filed in a dedicated locked filing cabinet 
organized by subject code number. To ensure data entry accuracy, peer review of source 
documentation and case report forms in REDCap will be completed following MGB Study Data 
Management and Quality Assurance Plan Guidelines. The data will reside on the secure 
Massachusetts General Hospital secure file server which is within the secure Partners firewall, 
and which is backed up every day, with periodic secure back-ups off-site. 
 
A physician will be available at all times during the study by pager to answer any questions a 
patient might have. The physician will arrange to immediately see every patient with a concern. 
All efforts will be made to protect the confidentiality rights of the study subjects who will be 
referred to by code numbers only. Confidentiality of the patients will always be of paramount 
importance to study investigators. No data on patients will be shared with persons other than 
those directly involved in the study, except at the documented request of the patient. Samples 
that are sent to laboratories outside of MGH will be labeled with a non-identifying numeric code.  
 
Safety Monitoring:  
A Data Safety Monitoring Board (DSMB) will be established at Massachusetts General Hospital 
consisting of independent members, who will review all study procedures, adverse events, 
protocol violations, protocol exceptions and deviations as well as study inclusion and exclusion 
criteria. The DSMB will also review safety monitoring blood tests and study data as available. 
The DSMB members will consist of independent experts in the field with experience in the 
conduct of clinical trials, statistical knowledge and independence from the direct management of 
the clinical trial. These members include Drs. Karen Miller MD (Professor of Medicine, 
Massachusetts General Hospital), Jules Dienstag MD (Professor of Medicine, Massachusetts 
General Hospital), and Dr. Charles Serhan, PhD (Professor of Anesthesiology, Brigham and 
Women’s Hospital). In addition, the PI (Dr. Simon) and the co-investigator team (Drs. Chan, 
Chung and Corey) will participate in a portion of the meeting as non-voting members. The 
DSMB and the study investigators will meet together at least once every 6-9 months, or more 
often as needed, and the chair (Dr. Miller) will be the contact person for the DSMB and will be 
responsible for overseeing the meetings, developing the agenda and summarizing the findings 
of the meeting.  
 
All treatment-related serious adverse events will be reported to the DSMB within 24h of 
occurrence. In addition, the chair may call ad hoc meetings. The Board will be provided with 
reports of serious adverse events as they occur and the chair of the committee has the 
responsibility of calling an ad hoc meeting if the type or frequency of the serious adverse events 
is of concern. In addition, an ad-hoc meeting may be called to evaluate whether a subject 
should be unblinded due to safety concerns or a serious adverse event (SAE). If a study subject 
is unblinded, the PI will not be made aware of the treatment assignment. 
 
The Board may request additional information from the Investigator in the course of its review. 
The Investigator shall not be present for part of the Board’s meeting. 
 
Safety assessments: 
3. CBC: at screening, baseline, and 6 months (and hematocrit at 3 months)   
4. Pregnancy testing at all visits  
 
The physician investigator will review all laboratory results in a timely manner. 

 
16 
 
Subjects will be discontinued if they develop: 
1. 
Bleeding event resulting in hospitalization or decrease in hematocrit by >20% from 
baseline   
2. 
Decrease in hematocrit by >20% from baseline 
3. 
Positive pregnancy test 
4. 
Severe / intolerable side effects of aspirin 
5. 
Development of malignancy  
6. 
Initiation of the following medications: methotrexate, amiodarone, systemic steroids or 
tamoxifen 
 
Safety Monitoring:  
1. Review of all adverse events (expected, unexpected and serious)  
Severity, frequency and reporting protocol adherence  
2. Review safety assessments described above  
3. Review CBC (or hematocrit) results with unblinded monitor 
4. Recruitment, enrollment (adherence to eligibility criteria)  
5. Dropout rates  
6. Protocol changes  
 
Discontinuation Guidelines:  
1. Individual:  
a. Positive pregnancy test  
b. Severe / intolerable side effects of aspirin 
c. Bleeding in the gastrointestinal tract 
d. Decrease in hematocrit by >20% from baseline 
e. Development of malignancy  
f. Initiation of the following medications: systemic steroids for >5 days, 
methotrexate, amiodarone, tamoxifen 
2. Study: 
a. Unexpected severe / frequent adverse events  
 
The major outcome choice following the data review by the Board is: 1) continuing the trial 
unchanged, or 2) modify the protocol and/or consent form, or 3) terminate the trial.  
 
The chairperson (Dr. Miller) will be responsible for overseeing the meetings, developing the 
agenda and summarizing the meeting. The chairperson is the contact person for the DSMB.  
 
The DSMB responsibilities will include: 
1. Review of the research protocol, informed consent documents and plans for safety and data 
monitoring of the study. This review is to determine the risks and benefits to research subjects, 
protection and safety of the subjects and to offer suggestions for improving the study design. 
2. Review interim data to detect adverse effects to determine if the trial should continue as 
originally designed, should be changed or should be stopped based on the data. 
3. Evaluate the progress of the trial including recruitment goals, accrual and retention of 
participants and other factors that may affect the study outcome. 
4. Protect confidentiality of the study participants, trial data and results of the monitoring.  
 
 
C. Adverse Events Reporting  

 
17 
All treatment emergent serious adverse events will be documented and reported immediately to 
the IRB at Partners HealthCare System, as well as to the DSMB. An event that is serious must 
be recorded on the case record and requires expeditious handling to comply with regulatory 
requirements. In the event that a patient becomes ill or injured as a direct result of participation 
in the research study, necessary medical care will be made available. All adverse effects will be 
reported as per Partners Healthcare System requirements. Potentially serious adverse events 
(SAEs) will be followed to resolution or stabilization and reported as SAEs if they become 
serious.  
 
A serious adverse event is defined as one that meets any one of the following criteria:  
1. Fatal or life threatening  
2. Requires inpatient hospitalization  
3. Results in persistent or significant disability or incapacity  
4. Congenital anomaly  
5. Important medical event that may jeopardize the patient or require intervention to 
prevent serious outcome  
6. Cancer  
7. Overdose  
8. Results in development of drug dependency or drug use  
 
 
 
 
 
 
 
 
 
 
 

 
18 
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Nonalcoholic Fatty Liver Disease: Practice Guidance from the American Association for 
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Simon T, Ma Y, Ludvigsson JF, Chong DQ, Giovannucci EL, Fuchs CS, Meyerhardt JA, 
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7. 
Seewaldt V. Aspirin and Chemoprevention: Have we Arrived? JAMA Oncology 2018. 
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Huang ES, Strate LL, Ho WW, et al. Long-term use of aspirin and the risk of 
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Kato H, Nagai Y, Ohta A, et al. Effect of sitagliptin on intrahepatic lipid content and 
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Cuthbertson DJ, Irwin A, Gardner CJ, et al. Improved glycaemia correlates with liver fat 
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Kirk E, Reeds DN, Finck BN, et al. Dietary fat and carbohydrates differentially alter 
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Cussons AJ, Watts GF, Mori TA, et al. Omega-3 fatty acid supplementation decreases 
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Miglio F, Rovati LC, Santoro A, et al. Efficacy and safety of oral betaine glucuronate in 
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19 
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Loomba R, Kayali Z, Noureddin M, et al. GS-0976 Reduces Hepatic Steatosis and 
Fibrosis Markers in Patients with Nonalcoholic Fatty Liver Disease. Gastroenterology 
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Lawitz EJ, Coste A, Poordad F, et al. Acetyl-CoA Carboxylase Inhibitor GS-0976 for 12 
Weeks Reduces Hepatic De Novo Lipogenesis and Steatosis in Patients With 
Nonalcoholic Steatohepatitis. Clin Gastroenterol Hepatol 2018. 
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hospitalized patients with cirrhosis due to non-alcoholic steatohepatitis. Clin Transl 
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Angulo P, Bugianesi E, Bjornsson ES, et al. Simple noninvasive systems predict long-
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31. 
Chiang N, Fredman G, Backhed F, et al. Infection regulates pro-resolving mediators that 
lower antibiotic requirements. Nature 2012;484:524-8. 
32. 
Dalli J, Serhan CN. Specific lipid mediator signatures of human phagocytes: 
microparticles stimulate macrophage efferocytosis and pro-resolving mediators. Blood 
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33. 
Dalli J, Winkler JW, Colas RA, et al. Resolvin D3 and aspirin-triggered resolvin D3 are 
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20 
35. 
Elizondo A, Araya J, Rodrigo R, et al. Effects of weight loss on liver and erythrocyte 
polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-
alcoholic fatty liver disease. Biol Res 2008;41:59-68. 
36. 
Elizondo A, Araya J, Rodrigo R, et al. Polyunsaturated fatty acid pattern in liver and 
erythrocyte phospholipids from obese patients. Obesity (Silver Spring) 2007;15:24-31. 
37. 
Scorletti E, Bhatia L, McCormick KG, et al. Effects of purified eicosapentaenoic and 
docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome* 
study. Hepatology 2014;60:1211-21. 
38. 
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39. 
Ryan MC, Itsiopoulos C, Thodis T, et al. The Mediterranean diet improves hepatic 
steatosis and insulin sensitivity in individuals with non-alcoholic fatty liver disease. 
Journal of hepatology 2013;59:138-43. 
40. 
Parker HM, Johnson NA, Burdon CA, et al. Omega-3 supplementation and non-alcoholic 
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21 
Changes to Study Protocol After Trial Initiation Date: 
 
• 
Version 4 (Amendment 2; approval date: 09/10/2019) updated the informed consent 
process descriptions. 
 
• 
Version 5 (Amendment 5; approval date 10/02/2019) increased the upper age limit from 
65 to 70 years; changed the frequency of follow-up phone calls from twice monthly to 
once monthly as recommended by the MGB QI team; and updated the funding 
information. 
 
• 
Version 6 (Amendment 8; approval date 01/06/2020) updated the format of the SF-36 
questionnaire; changed the compensation scheme for subjects, while keeping the total 
compensation for the overall trial unchanged; included the option of a serum pregnancy 
test for women of childbearing potential (in addition to urine pregnancy test); and 
included updated written instructions for stool collection kits, for subjects. 
 
• 
Version 7 (Amendment 10; approval date 09/30/2020): added an oral microbiome 
sample to the baseline and month 6 time points; updated the payment distribution 
scheme for each study visit. 
 
• 
Version 8 (Amendment 11; approval date 11/12/2020): clarified the exclusion criteria for 
thrombocytopenia by including the definition of lower platelet count threshold of 100K/uL. 
 
• 
Version 9 (Amendment 12; approval date 02/19/2021): added an optional pre-treatment 
and/or post-treatment liver biopsy. 
 
• 
Version 10 (Amendment 17; approval date 08/05/2022): added institution-wide research 
announcements as an additional method of potential recruitment. 
 
 
Changes to Statistical Analysis Plan After Trial Initiation Date: none