Endoscopic Ultrasound(EUS)-Guided TRUCUT Biopsy (EUS-TCB) of Suspected Nonalcoholic Fatty Liver Disease(NAFLD.)

Non-alcoholic Fatty Liver Disease (NAFLD) is an alcohol-like liver disease that occurs in
individuals who do not consume excessive alcohol. Similar to alcohol-induced liver disease,
NAFLD encompasses a spectrum of liver damage, ranging from non-alcoholic fatty liver (NAFL)
on the most clinically benign end of the spectrum to cirrhosis on the opposite extreme where
most liver-related morbidity and mortality occur. In NAFL, the liver is steatotic but
generally healthy. Nonalcoholic steatohepatitis (NASH) is a more severe form of liver damage
than NAFL because there is increased hepatocyte death in addition to steatosis. The liver
injury in NASH triggers a repair response that sometimes leads to cirrhosis.1-3 The
architecture of cirrhotic livers is distorted by regenerating nodules and fibrous tissue.
Cirrhosis results when fibrosis disrupts the normal liver architecture, carving the
parenchyma into nodules of hepatocytes that are separated from each other by bands of
connective tissue. Some individuals with NAFLD develop cirrhosis. As in other types of liver
disease, progression to this advanced stage of liver damage from NAFLD is generally thought
to require many years, perhaps decades. Because most liver-related morbidity and mortality
occur in cirrhotic individuals, researchers are trying to understand the basis for
inter-individual differences in the tendency to develop cirrhosis. Exiting blood tests and
imaging modalities have limited sensitivity for detecting liver steatosis, steatohepatitis
and cirrhosis. In addition, there is no test that can distinguish NAFLD from other causes of
fatty liver disease. Thus, at the present time, NAFLD is a diagnosis of exclusion.

Results from two large population-based studies are extremely useful in defining the
prevalence of NAFLD in the general US adult population.4,5 The Dallas Heart Study used liver
imaging to assess the prevalence of fatty liver in over 2200 adults. The overall prevalence
of fatty liver in that study was 31% with a range of 24-45% of the population demonstrating
hepatic steatosis. Of note, most individuals with elevated liver fat content did not
demonstrate aminotransferase elevations. The latter finding helps to explain why NHANES III,
another large population-based study, suggested a much lower prevalence of NAFLD in the
general US adult population. In NHANES III, serum aminotransferases were used to assess
NAFLD prevalence in over 15,000 adults and that approach suggested that 5.5% of the
population has NAFLD. Based on the results of the Dallas Heart Study it is likely that the
approach used in NHANES III underestimated the true prevalence of NAFLD in US adults. In any
case, the net results from these two studies suggest that between 5.5 to 31% of the adult US
population has NAFLD. To appreciate the significance of this finding, it is useful to
compare the prevalence of NAFLD to that of hepatitis C, which is thought to exist in 1.8% of
the US population.6 NAFLD is three to ten times more common than hepatitis C-related liver
disease in the US.

Population-based studies and case series demonstrate strong associations between NAFLD and
several conditions, including obesity, type 2 diabetes, and dyslipidemia, that comprise the
metabolic syndrome.4,5,7-13 This suggests that the prevalence of NAFLD is likely to be much
greater in these high risk populations. Studies of morbidly obese patients undergoing
gastric bypass surgery support this concept. In most of these individuals, liver disease was
unsuspected pre-operatively. However, intraoperative liver biopsies consistently demonstrate
NAFLD. In four recent series that included over 600 subjects, the prevalence of NAFL
(steatosis) ranged from 30-90% and NASH (steatohepatitis) was documented in 33-42%. Thus,
over two-thirds of morbidly obese patients undergoing gastric bypass surgery have NAFL/NASH.
Although overt manifestations of portal hypertension likely excluded patients as candidates
for elective abdominal surgery, these four surgical series consistently identified
individuals with hepatic fibrosis. Well-established cirrhosis was relatively unusual,
occurring in 1-2% of the group. However, advanced fibrosis was noted in 12% and a third had
unexplained periportal fibrosis. There is also a high prevalence of NAFLD is high in
populations of patients with type 2 diabetes mellitus. A recent survey of 100 non-alcoholic
patients with type 2 diabetes used ultrasonography to screen for NAFLD and demonstrated
fatty liver in half of the group. Subsequent liver biopsy revealed NAFL in 12% and NASH in
87% of those with ultrasonographic evidence of fatty liver. Fibrosis or cirrhosis were noted
in 20%.14 In patients with metabolic syndrome and hyperlipidemia, fatty liver was detected
in 50% of 95 adults using ultrasonography to screen for unsuspected hepatic steatosis.15

Similar to other types of chronic liver disease, liver-related morbidity and mortality are
dictated by the severity of liver damage. This concept explains observations that NAFL has a
relatively benign prognosis, whereas NAFLD-related cirrhosis may be complicated by portal
hypertension and hepatocellular carcinoma, eventuating in death from liver disease.2 Studies
disease.2 Studies in the US and France independently demonstrated 3 easily-assessed clinical
parameters that identify individuals with cryptogenic hepatitis who are likely to have
advanced fibrosis on liver biopsy. These parameters are older age (greater than 45-50
years), overweight or obese body mass index, and type 2 diabetes. The probability of having
bridging fibrosis or cirrhosis on liver biopsy is approximately 66% in individuals with
cryptogenic hepatitis who are older and either overweight/obese or diabetic.16,17
Identification of individuals who have NASH with fibrosis has important prognostic
implications. Follow-up of patients with NASH and fibrosis demonstrates that almost 30% of
these individuals become cirrhotic within 5-10 years. In contrast, only about 3% of
individuals with milder forms of non-alcoholic fatty liver disease develop cirrhosis after
more than a decade of follow-up. Therefore, NASH with fibrosis progresses to cirrhosis both
more consistently and more rapidly than simple NAFL.2,13

In order to provide optimal therapy for individuals with NAFLD, their liver disease must be
diagnosed. The diagnostic evaluation of an individual who is suspected of having NAFLD has 3
goals. The first goal is to establish fatty liver disease as the etiology of the liver
disease. Unfortunately, no specific serologic marker for NAFLD has been identified yet.
Moreover, all of the current diagnostic tests for NAFLD have some limitation. Standard
imaging tests such as ultrasonography and CT scan, may under- or over-estimate hepatic fat
content.1,18 Moreover, detecting liver fat by abdominal imaging can neither distinguish
alcoholic- from non-alcoholic-fatty liver disease, nor exclude other types of liver disease
that might co-exist with hepatic steatosis. The second goal is to confirm the specific type
of fatty liver disease, alcoholic fatty liver disease (AFLD) or non-alcoholic fatty liver
disease (NAFLD). These two entities cannot be distinguished by histologic parameters but
only by assessment of drinking habits of the patient. The third and arguably most important,
goal of the diagnostic work-up is to establish the clinical severity and prognosis of the
liver disease. Because NAFLD is common and often generates few manifestations of liver
disease, it is necessary to have a high index of suspicion to identify individuals who might
have NAFLD. This includes individuals with the metabolic syndrome (at least 2 of the
following disorders: obesity, type 2 diabetes, hypertension and dyslipidemia) or detection
of fatty liver on an imaging study of the abdomen. Imaging studies or laboratory tests may
point to advanced liver disease but early cirrhosis and NAFLD without fibrosis cannot be
reliably diagnosed without liver biopsy.

At this time, liver biopsy remains the "gold standard" for staging the severity of liver
damage in NAFLD and all other types of chronic liver disease.2 This biopsy is traditionally
performed percutaneously with or without ultrasound guidance. Needle entry for biopsy is
typically into the right lobe below the diaphragm between the ribs in the right mid-axillary
line. Alternatively, this may be done by an anterior approach into the left lobe. However,
there are potential limitations with percutaneous biopsy including sampling error19,20
(which may not be representative of the hepatic architecture) and complications (pain,
bleeding and puncture of another organ, not obtaining liver tissue). Due to anatomical
considerations, some patients are not even candidates for liver biopsy by the percutaneous
approach. In this case, biopsy may be performed by the transjugular method. This technique
involves use of conscious sedation followed by needle access of the jugular vein. A wire is
then passed through the heart and into a branch of a hepatic vein to guide a needle biopsy
of the liver. Similar to percutaneous biopsy, this procedure is safe when performed by
experienced operators. However, complications including neck hematoma, hepatic arteriovenous
fistula, liver capsular puncture, intraperitoneal hemorrhage and death have been reported by
this technique.21,22 Regardless of the approach, liver biopsy incurs monetary cost,
including fees for the facility, equipment, hepatologist and pathologist, as well as for
processing the tissue sample itself. On the other hand, the expense of a liver biopsy is
roughly equivalent to that of magnetic resonance imaging (MRI), which is the most sensitive
radiologic test for quantifying liver fat,fat; an MRI is inferior to biopsy for detecting
liver inflammation and fibrosis.23

Alternative methods for acquisition of a core biopsy of the liver may be helpful in patients
in whom percutaneous and transjugular liver biopsies would be difficult, risky or
contraindicated. Endoscopic ultrasound (EUS)-guided fine needle aspiration (EUS-FNA) has
been used for over 15 years at Indiana University Medical Center and other hospitals to
sample lymph nodes and masses in the posterior mediastinum, upper abdomen and pelvis.24-29
In 2003, we published our experience of EUS-FNA of the liver using a 22-gauge needle in 77
patients without complications and to date this remains the largest series reported in the
literature.30 Since that report, we have performed this procedure in over 100 patients also
without a known complication (not published). In 2003, the FDA approved development of a
19-gauge Trucut biopsy (TCB) device (Wilson-Cook Medical, Inc.; Winston-Salem, North
Carolina) that provides a histologic tissue sample for use with EUS. The initial reported
use of this device demonstrated its safety and utility for biopsy of major upper abdominal
organs (including the liver) in a swine model.31 Its subsequent use for sampling of
gastrointestinal stromal tumors (GISTs), pancreatic masses and mediastinal lymph nodes in
humans has also been reported.312-35 We have also studied its use in the diagnosis of
nonfocal chronic pancreatitis.36 At Indiana University, we our EUS group have used this
device in over 30 patients off protocol for various accepted indications without known
complications (not published). The use of this EUS-TCBdevice for liver biopsy has been
reported in abstract form in only two patients with suspected metastatic malignancy.37 No
complications were reported in these two patients. Its use for suspected benign disease of
the liver has not been reported to date.

Inclusion Criteria:

1. Patients between the ages of 18 and 70 years old referred to Indiana University
Hospital for outpatient upper EUS for clinical purposes (e.g., pancreatic disease).

2. Increased hepatic enzymes (AST or ALT) above the upper limit of normal within the
past 3 months. These values at University Hospital are ALT>45 U/L and AST> 41 U/L but
may vary among laboratories at different institutions.

3. Evidence of fatty liver (as detected by an increase in echogenicity or brightness of
the liver) by EUS exam.

4. Liver biopsy for suspected NAFLD is clinically indicated as determined by Dr Naga
Chalasani, one of our staff hepatologists who is an expert in NAFLD.

Exclusion Criteria:

1. Inpatient status.

2. Normal liver enzyme tests (AST, ALT).

3. Previous or current alcohol abuse (≥3 drinks/day for men; ≥2 drinks/day for women)

4. Planned liver biopsy within 30 days for known or suspected liver disease.

5. Suspected metastatic lesion in the liver by any previous imaging study or discovered
during EUS exam.

6. EUS-FNA of the liver is required for another reason (i.e. suspected metastatic
lesion)

7. Newly diagnosed, currently treated or suspected malignancy. (In this case, the
diagnosis of NAFLD would not likely impact patient care or survival). Patients with a
previously diagnosed malignancy that is in remission will be considered eligible.

8. Previously diagnosed NAFLD including steatosis or NASH (in this situation, another
biopsy is not beneficial to the patient).

9. Chronic liver disease including Hepatitis B, Hepatitis C, Wilson's disease,
alpha-1-antitrypsin disease, autoimmune hepatitis.

10. Known or suspected cirrhosis.

11. Esophageal or gastric varices by previous imaging or discovered during EUS.

12. Previous liver surgery including resection or transplant.

13. Thrombocytopenia (platelets <150,000)

14. Anemia (hemoglobin <10 gm/dL).

15. Coagulopathy (INR>1.2 or aPTT > 35 secs).

16. Use of coumadin or other anticoagulants.

17. Use of aspirin or nonsteroidal antinflammatory product within 7 days of EUS.

18. Total bilirubin >2.0 mg/dL and dilated bile duct on previous imaging study.

19. Inability to provide informed consent.

20. Pregnancy or suspected pregnancy.