The Prevalence of Nonalcoholic Fatty Liver Disease (NAFLD) in Adults
The Prevalence of Nonalcoholic Fatty Liver Disease (NAFLD) in Adults
Nonalcoholic fatty liver disease (NAFLD) is a global health concern with a suspected
 increasing prevalence due to the rise in obesity and diabetes mellitus. The vast majority of
 patients will have isolated steatosis or steatosis with mild inflammation that is very
 unlikely to progress in severity. However, about 25% of patients with NAFLD have
 non-alcoholic steatohepatitis (NASH), the more aggressive form of the disease that is
 associated with fibrosis progression and potential risk for cirrhosis and end-stage liver
 disease complications. Additionally, multiple studies have demonstrated an association
 between NAFLD and the presence of coronary artery disease by either coronary CT angiography
 (CCTA) or coronary artery calcium (CAC) score. Cardiovascular disease is the most important
 cause of mortality in patients with the entire spectrum of NAFLD. In the era of advanced
 imaging and functional vascular assessment it is possible that novel risk assessments are
 poised to refine overall prognostic estimation in this population. Multiple analyses have
 suggested that NAFLD is an independent and strong predictor of significant CAD independent of
 cardiovascular risk factors, including a significant burden of high risk CCTA findings in one
 analysis of symptomatic patients in the emergency department. Given the multiple metabolic
 derangements inherent in the NAFLD population, endothelial dysfunction is also an important
 contributor to global cardiovascular dysfunction. Furthermore, data suggests that patients
 with NAFLD may be at increased risk of adenomatous polyp formation and colorectal
 adenocarcinoma. In addition, it is suboptimal to require a liver biopsy to diagnose NASH.
 Recent imaging advances have made it possible to assess liver fibrosis but have yet to be
 fully studied in NAFLD. The purpose of this study is to assess the current prevalence and
 severity of NAFLD in adult subjects. Secondary endpoints include correlation to new vascular
 function (cine scan of the abdominal aorta) and echocardiographic imaging modalities
 available at BAMC and to circulating biomarker panels as well as to determine the prevalence
 and severity of CAD by multidetector coronary CT angiography with subject outcomes being
 monitored prospectively. Additionally, correlation of NAFLD diagnosis to colonoscopy findings
 will be performed.
In this prospective study, adult patients ranging in age from 18-80, routinely seen in the
 Brooke Army Medical Center Gastroenterology clinics will be eligible for enrollment.
 Approximately 950 adult patients will be enrolled. In phase I the prevalence of NAFLD and
 NASH will be determined, non-invasive imaging techniques for the diagnosis of NAFLD and NASH
 will be explored, and the relationship between colon polyps and NAFLD will be evaluated.
 Phase II will be offered to participants that qualified for a liver biopsy. In phase II the
 prevalence and severity of CAD, endothelial dysfunction, and diastolic dysfunction within the
 population of subjects with NAFLD will be determined.

 Phase I:

 After obtaining informed consent and confirming that the subject has met inclusion and
 exclusion criteria for the study, the study will employ a questionnaire to be completed by
 the subject. This questionnaire will include data such as race and ethnic group, past medical
 history, current medications, etc. The subject will then have routine labs (liver function
 tests, fasting insulin, glucose, and a pregnancy test for all female subjects), FibroScan®
 and MRE/MRI done to assess for the presence or absence of hepatic steatosis, inflammation,
 fibrosis, iron quantification and diameter and luminal characteristics of the abdominal
 aorta. Subjects will be called by the PI or an AI to return to the clinic for additional
 study procedures to include a core liver biopsy if they are identified to have one or more of
 the following:

 -  greater or equal to 5% steatosis on MRI or

 -  liver stiffness greater than 7 kPa on Fibroscan®, or

 -  evidence of fibrosis on MRE or

 -  LIF score of 2.0 or greater

 The PI/AI will let the subject know that he/she qualifies for further diagnostic testing as
 part of the trial and will discuss tests results with them in detail.

 FibroScan examination will be performed to assess liver stiffness according to the
 manufacturer's recommendation and in accordance with the device's FDA clearance. The device
 is equipped with two probes (M and XL) dedicated to different morphology and operating at
 different measurement depths. The M probe operates between 25 and 65 mm below the skin
 surface and the XL probe operates between 35 and 75 mm. The device's software automatically
 recommends which probe is the most appropriate during examination based on the real time
 assessment of the skin to liver capsule distance (automatic probe selection tool). The
 FibroScan used in this study will be equipped with a software allowing, in addition to liver
 stiffness, measurement of the Controlled Attenuation Parameter (CAP) which is related to
 liver steatosis with different algorithm. This additional software feature is FDA approved.
 Operators will be trained and certified by the manufacturer or its local representative.
 Recommendation from the Automatic Probe Selection tool will be followed. At least ten valid
 measurements should be obtained. De-identified acquisition files will be reviewed by the
 device's manufacturer (Echosens) to confirm the operator's choice of measurement depths and
 eventually change them as well as analyze the additional ultrasonic signals.

 In addition to the Fibroscan, a second non-invasive imaging technique, MRE/MRI Liver
 MultiScan for detecting steatosis, inflammation and liver fibrosis or stiffness will be
 conducted. This data will be used to correlate with the presence of NAFLD/NASH. MRI-Liver
 MultiScan is a post MRI processing software. It involves a specialized magnetic resonance
 imaging (MRI) technique/sequences that measures fat, iron and fibrosis in the liver using T1
 mapping. The procedure has no need for intravenous contrast. LiverMultiScan is currently
 approved by the FDA for use with Siemens 3T MRI with E11 software. However, this MRI model is
 not being used at BAMC. Therefore, the use of LMS locally will conducted under an IDE
 exemption for diagnostic devices (21 CFR 812.2(c))(3). The LMS software will not be used to
 clinically diagnose subjects. Rather, data will be collected and analyzed by the site and
 manufacturer (Perspectum) with the knowledge that MRI is not performed in accordance with LMS
 labeling.

 BAMC Radiology will work in collaboration with Perspectum to complete the post processing
 review of the MRI image data to correlate with the presence of NAFLD/NASH. The BAMC Radiology
 Department AI's will de-identify the image data (remove all PHI), load on a secure server and
 have Perspectum Diagnostics complete the post-processing interpretations.

 The MR elastography scan will be conducted in conjuncture with the MRI scan and
 post-processed by AIs within the BAMC Radiology Department. In addition, AIs in the BAMC
 Radiology Department will conduct a safety read of all scans. The results of the MRE and the
 safety read will be placed in the subject's electronic medical record.

 Participants that qualify for a core liver biopsy based upon the results from the FibroScan
 and MRE/MRI LiverMultiScan will be asked to return to specimen collection for additional
 blood work prior to the liver biopsy. This will consist of a complete blood count (CBC),
 coagulation panel, fasting lipid panel, GGT, vitamin D level, renal function panel, ferritin,
 hemoglobin A1C, and chronic hepatitis panel.

 Subjects will then undergo a percutaneous liver biopsy to establish diagnosis of NASH. This
 will occur in the Gastroenterology Clinic at Brooke Army Medical Center. The liver tissue
 will be submitted to BAMC Pathology Department for evaluation by an AI, an experienced
 hepatopathologist. The pathology results will be used to identify subjects who have the more
 severe form of NAFLD, NASH. The pathologist will provide an overall diagnostic interpretation
 based on the criteria reported by Brunt et al. and by the NAFLD Activity Score (NAS).
 According to this scoring system, the degree of steatosis and inflammatory activity is
 measured using a zero to eight scale (steatosis=0-3; lobular inflammation=0-3;
 ballooning=0-2). The NAS is the unweighted sum of steatosis, lobular inflammation, and
 hepatocellular ballooning scores. The stage of fibrosis will be assessed using a zero to four
 scale (0=no fibrosis; 1=mild/moderate zone three perisinusoidal fibrosis, or
 portal/periportal fibrosis only; 2= perisinusoidal and portal/periportal fibrosis; 3=
 bridging fibrosis; 4= cirrhosis). Additionally, at the time of liver biopsy, 4 additional
 unstained slides will be prepared for morphometry analysis by Echosens. The liver
 histological diagnosis will be established by an experienced pathologist blinded to the
 clinical characteristics of the subjects, radiographic studies and the serum biomarkers. The
 outside expert pathologist will use the same scoring system as the local pathologist. If
 there is a discrepancy between the two pathology results for a subject the final
 determination will be made by the local BAMC pathologist. An addendum will be placed in the
 electronic medical record by the BAMC pathologist. The subject will be informed of the change
 and documentation of the new diagnosis and any further treatments will be made in AHLTA.

 Echosens will use the liver biopsy slides to correlate the FibroMeter™ NAFLD/VCTE test with
 liver fibrosis and the presence of NAFLD and NASH. FibroMeter™ is a non-invasive diagnostic
 test that evaluates the level of fibrosis in the liver using patient clinical data (age and
 weight) and a proprietary selection of standard clinical lab results including platelets,
 AST, ALT, ferritin, glucose and Alpha-2-macroglobulin levels. The results of these locally
 run tests will be entered into the Echosens FibroMeter website for FibroMeter NAFLD/VCTE
 calculation. This test is approved in Europe and provides means for detection of significant
 fibrosis, detection of cirrhosis (screening for esophageal varices and hepatocellular
 carcinoma) and a quantitative estimate of fibrosis staging, thereby providing opportunity for
 serial monitoring of fibrosis in suspected cases. The FibroMeter test result will then be
 used by Echosens to correlate with the morphometry analysis. All results are identified with
 only the subject study number. Results from the FibroMeter test will not be used to support a
 FDA marketing application. Additionally, the results of the FibroMeter will not be used
 clinically nor to determine the performance of any subsequent research procedure. The results
 will not be placed in the subject's medical records.

 An additional 5 ml of whole blood and 5 ml of serum will be collected and stored by a
 research coordinator, nurse, research assistant, or investigator for analysis of specific
 biomarkers to include Hyaluronic acid, CK-18 (M30), FGF-21, Mac-2BP, FAS, AFP, YKL-40,
 Alpha-2-macroglobulin and a panel of 46 fatty acids during the liver biopsy appointment.
 Phlebotomies will be performed in Hepatology Research. The stored serum will be kept in the
 BAMC Gastroenterology Clinic in a -80 degree freezer and batch shipped to an outside lab
 periodically for analysis. Specimens will be identified by a designated research code only
 available to the local study team, thereby removing any personal identifiers on specimens
 prior to shipment to an outside lab.

 In order to address aim 3, subjects enrolled in the study who have undergone or will undergo
 colonoscopy during study participation as part of their routine clinical care will have their
 AHLTA/CHCS records reviewed and those with colon polyps or colon masses will be evaluated
 against their imaging study findings. The correlation between colon polyp number/severity and
 the presence of NAFLD will be ascertained.

 Subjects qualifying for a liver biopsy will be referred to the study team members in the
 Cardiology clinic with the option to enroll in Phase II of the protocol.

 Phase II:

 Phase II will consist of three cardiology procedures.

 1. Coronary CT angiography: All subjects who meet inclusion criteria for a percutaneous
 liver biopsy, are consented to phase II, and do not have known CAD defined as prior
 percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) or have
 not undergone CCTA within the previous 12 months, will undergo a CCTA with CAC score.
 Subjects undergoing this portion of the analysis will need a renal function panel within
 30 days of the scan date with a glomerular filtration rate (GFR) of ≥ 60 mls/min/1.73m2
 by the Cockcroft-Gault equation, no prior history of an allergic reaction to intravenous
 CT contrast, and no contraindications to administration of sublingual nitroglycerin and
 nodal blocking agents. All CCTA scans will be analyzed by a cardiologist with level III
 ACC/ACR certified imaging expertise in accordance with the Society of Cardiovascular
 Computed Tomography (SCCT) guidelines. Scans will be performed in accordance with SCCT
 guidelines. Studies will be obtained utilizing a 128-slice dual source scanner with a
 high pitch, single heart beat image acquisition capabilities (Somatom Definition Flash
 CT®, Siemens, Erlagen, Germany). CAC scanning will occur first at a standard tube
 voltage of 120 keV and 3 mm slice acquisition and CareDose® modulation of tube current
 enabled for all scans. CT technologist will be responsible for contacting the on-call CT
 imaging specialist for CAC score ≥400 prior to proceeding to CCTA acquisition.
 Continuation to CCTA will be at the discretion of the CT imaging specialist. Tube
 voltage and image acquisition protocoling will be selected by the CT technologist in
 consultation with the on-call cardiac CT imaging specialist in accordance with the
 flowsheet depicted in Appendix 3. Tube voltage of 120keV will be used for BMI > 30 and
 lowered to 100keV for BMI ≤ 30. CareDose® modulation of tube current will be enabled for
 all scans. Pretreatment with nodal blocking agents will be as follows: for HR < 70 bpm
 and systolic blood pressure (SBP) > 100 mm Hg will be given 50mg orally, for HR > 70 bpm
 and SBP > 100 mm Hg will be given 100mg orally, and nodal blocker will be held for
 patients with HR < 50 bpm or SBP < 100 mm Hg at the time of evaluation. Additional nodal
 blocker will be administered following initial dosing at the discretion of the cardiac
 imaging specialist. All subjects with SBP > 90 mm Hg and without a history of
 phosphodiesterase inhibitor usage in the preceding 72 hours received nitroglycerin spray
 0.4-0.8 mg within 2 minutes of scan acquisition. High-pitched, prospective,
 ECG-triggered helical scanning will be performed with heart rate (HR) < 60 bpm and HR
 variability < 5 beats or with HR < 60 bpm at the discretion of the imaging specialist.
 The remaining scans will be performed using prospective, ECG triggering acquisition
 centered at 70%-phase with between 10-20% padding (40-80% of the R-R interval).
 Visapaque® intravenous (IV) contrast will be used for all scans. A triphasic injection
 protocol consisting of 75mL of IV contrast followed by 40 mL of 50% contrast mixed with
 normal saline chased with normal saline at a flow rate of 5 mL/second will be used for
 all scans with total IV contrast volume of 105-125 mL. Contrast flow rates will be
 increased to 6 ml/second for patients with BMI > 30. Coronary artery stenosis by CCTA
 will be determined on a per-patient and per-vessel basis using an 18 segment model in
 accordance with SCCT guidelines for interpretation. The major epicardial coronary
 arteries (left main, left anterior descending, left circumflex, and right coronary
 arteries) will be graded for severity of CAD, defined as obstructive CAD (>50%
 stenosis), non-obstructive CAD (≤50% stenosis), or no CAD (vessels free of angiographic
 evidence of disease), respectively. The posterior descending artery will be included in
 the left circumflex or right coronary artery groups depending on its origin. The subject
 will have the option of declining the CCTA and CAC scan. Subjects with known CAD as
 defined on recent prior CCTA (within last 12 months) will not undergo CCTA, but their
 CAD history and/or prior CCTA results will be included in the analysis.

 2. Transthoracic Echocardiography (TTE): All subjects who meet inclusion criteria for a
 percutaneous liver biopsy and consented to phase II will undergo an assessment of
 cardiovascular diastolic performance measured using 2D and 3D transthoracic
 echocardiography (TTE) with Doppler technology. The TTE will be performed during a
 scheduled outpatient time slot at the convenience of the patient.

 3. EndoPAT: Endothelial function will be measured in subjects who qualify for liver biopsy
 and consent to phase II using the EndoPAT 2000®.. Every effort will be made to conduct
 the EndoPat test on the same day as the CCTA for subject convenience. The EndoPAT 2000®
 uses digital pulse amplitude tonometry. The EndoPAT device comprises a pneumatic
 plethysmograph that applies uniform pressure the surface of the distal finger.
 Throughout the test, the inflation pressure of the device will be set at 10 mmHg below
 the diastolic blood pressure or 70 mmHg, whichever is lower. Baseline pulse amplitude
 measurements will be taken for 5 minutes. Arterial flow will then be transiently
 interrupted by inflating a pneumatic cuff placed on the patient's forearm. The cuff will
 be inflated to 200 mmHg or 60 mmHg plus the systolic blood pressure, whichever is
 higher. The pulse amplitude will be recorded for 5 minutes during the occlusion phase.
 The blood pressure cuff is then deflated and post test measurements are recorded for 5
 additional minutes. Pre and post test pulse amplitudes are compared to assess the degree
 of reactive hyperemia.

 Subjects found to have evidence of coronary artery calcium, endothelial dysfunction,
 diastolic dysfunction and/or presence of non-calcific atherosclerosis on CCTA with maximal
 stenosis < 50% will be referred to the cardiology clinic for assessment of cardiovascular
 risk and aggressive medical and lifestyle risk factor modification. Patients with stenosis ≥
 50% will also be referred to the cardiology clinic to be evaluated further for the need for
 additional ischemic testing at the discretion of the treating physician.
Inclusion Criteria:

 -  Phase I:

 1. Male and female patients (≥18 years of age to 80)

 2. Eligible for care at Brooke Army Medical Center

 Phase II:

 1. Met the criteria for qualification for a percutaneous liver biopsy and completed Phase
 I

 2. Eligible for care at Brooke Army Medical Center

 Exclusion Criteria:

 -  Phase I:

 1. Patients with excessive alcohol use will be excluded as defined as >21 units of
 alcohol/week for men and 14 units of alcohol/week for women over a 2 year time
 frame. One drink "unit" or one standard drink is equivalent to a 12-ounce beer, a
 4-ounce glass of wine, or a 1-ounce shot of hard liquor.

 2. Patients with prior history of liver disease to include chronic hepatitis B or C,
 hemochromatosis, Wilson's disease, autoimmune hepatitis, primary biliary
 cirrhosis, primary sclerosing cholangitis, HIV, or prior documentation of NAFLD.

 3. Patients on medications known to cause fatty liver disease: tamoxifen,
 corticosteroids, amiodarone, methotrexate, valproic acid

 4. Patients carrying an implantable active medical device such as a pacemaker or a
 defibrillator

 5. Pregnant women

 Phase II:

 1. CCTA/CAC only: GFR <60 mls/min/1.73m2 or IV contrast dye allergy

 2. CCTA/CAC only: contraindications to atrioventricular (AV) nodal blocking agents (high
 degree AV block without permanent pacemaker, asthma, allergy to nodal blocking
 agents).

 3. Known CAD defined as previous PCI or CABG (Note: Subjects with CCTA within the past 12
 months will not be excluded from study and repeat scan will not be needed but the
 results of that previous scan will be included in the prevalence and severity
 analysis.)