REVIEW

In Search of New Biomarkers for

Nonalcoholic Fatty Liver Disease
Ting-Ting Chan, M.R.C.P., and Vincent Wai-Sun Wong, M.D.

Nonalcoholic fatty liver disease (NAFLD) affects 15% to
40% of the general adult population and has become an
important cause of cirrhosis and hepatocellular carci-
noma. Liver biopsy was traditionally the primary method
to assess the severity of NAFLD. Features of interest
include hepatic steatosis, necroinflammation, and fibro-
sis. In particular, nonalcoholic steatohepatitis (NASH), the
more progressive form of NAFLD, is defined as the pres-
ence of steatosis, lobular inflammation, and ballooning.1
In natural history studies, fibrosis, and to a lesser extent
NASH, correlates with long-term survival and liver-related
complications.2
However, liver biopsy is invasive and poorly accepted
by patients. It is also unrealistic to perform repeated
biopsies in routine practice. Biomarkers are therefore
needed for prognostication and disease monitoring.
Although not currently achieved, biomarkers that reflect
how a patient feels, functions, or survives can be used
as surrogate endpoints in clinical trials. The latter can
greatly improve the current drug development process.
In this short review, we describe currently available bio-
markers and highlight new developments in this field.
BIOMARKERS OF STEATOSIS
The diagnosis of NAFLD depends on the detection of
hepatic steatosis. In contrast, the degree of steatosis cor-
relates poorly with liver injury and clinical outcomes.2 In
fact, steatosis often decreases as a patient’s condition
progresses to cirrhosis. For the same reason, although
some early-phase studies used noninvasive tests of stea-
tosis to evaluate new treatments, it is unclear whether

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating char-
acteristic curve; CAP, controlled attenuation parameter; FIB-4, Fibrosis-4; GGT, gamma-glutamyl transpeptidase; 1H-MRS, proton
magnetic resonance spectroscopy; MRI, magnetic resonance imaging; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic
steatohepatitis.
From the Department of Medicine and Therapeutics and State Key Laboratory of Digestive Disease, The Chinese University of Hong
Kong, Hong Kong.
This study was supported by the General Research Fund of the Research Grant Council of Hong Kong (project reference 477813).
Potential conflict of interest: V. Wong has received lecture fees from Echosens.
Received 2 May 2016; accepted 15 May 2016

Contract grant sponsor: Research Grant Council of Hong Kong; contract grant number: 477813.
View this article online at wileyonlinelibrary.com
C 2016 by the American Association for the Study of Liver Diseases
V

19 | CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD
REVIEW
TABLE 1. SERUM BIOMARKERS AND PREDICTION MODELS OF STEATOSIS
Tests Description
Fatty liver index A formula comprising body mass index, triglycerides,
and GGT
NAFLD liver fat score A formula comprising metabolic syndrome, type 2 dia-
betes, fasting serum insulin, AST, and AST/ALT
SteatoTest A proprietary formula comprising a2-macroglobulin,
haptoglobin, apolipoprotein A1, total bilirubin, GGT,
fasting glucose, triglycerides, cholesterol, and ALT,
adjusted for patient’s age, sex, weight, and height
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transpeptidase.
this approach can select good candidates for further
development.
Table 1 summarizes the serum biomarkers and predic-
tion models of steatosis. These models use routine clini-
cal parameters and/or biomarkers to predict the presence
of NAFLD. Apart from clinical application, it is possible to
retrospectively calculate these scores from existing co-
horts for NAFLD research.
Because of its availability and moderate accuracy,
abdominal ultrasonography is the primary test to diagnose
NAFLD in routine practice (Table 2). In recent years, con-
trolled attenuation parameter (CAP) by FibroScan has also
been developed for steatosis measurement.3 The measure-
ment is based on the fact that the amplitude of ultrasound
waves is attenuated more rapidly in a fatty liver. CAP can
also be measured with liver stiffness simultaneously, allow-
ing evaluation of the severity of NAFLD at the same time.
Magnetic resonance imaging (MRI)–based techniques
such as proton magnetic resonance spectroscopy (1H-
MRS) and MRI-proton density fat fraction are probably the
most accurate noninvasive tests to quantify liver fat and
are sensitive to changes in steatosis over time. The cost
and availability of the techniques are the main challenges.
BIOMARKERS OF STEATOHEPATITIS
NASH is the active form of NAFLD and the target for
treatment. The development of NASH biomarkers is
therefore important for selecting patients for treatment
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New Biomarkers for NAFLD Chan and Wong
Usage and Limitations
 Simple and inexpensive
 Suitable for epidemiological studies
 Inadequate distinction of the severity of steatosis
 Suitable for epidemiological studies
 Insulin not routinely done
 Inadequate distinction of the severity of steatosis
 Suitable for epidemiological studies and individual diagnosis
 Relatively expensive
 Involves biomarkers that are not routinely done
and also for monitoring treatment response. Compared
with steatosis and fibrosis, noninvasive tests of NASH are
less well developed. Serum cytokeratin 18 fragment
reflects hepatocyte apoptosis and is one of the most
extensively validated NASH biomarkers. Its overall accu-
racy is modest.4 Other evaluated biomarkers include
other cell death markers (e.g., soluble Fas, intact cytoker-
atin 18), adipokines (e.g., adiponectin, tumor necrosis
factor-a, interleukin-6, adipocyte fatty acid–binding pro-
tein), metabolic markers (e.g., homeostasis model assess-
ment of insulin resistance, fibroblast growth factor 21),
and inflammatory markers (e.g., C-reactive protein).
Because NASH is a multisystem disease and the relative
contribution of different disease processes varies from
patient to patient, these biomarkers tend not to perform
as well when validated in independent cohorts. Com-
bined biomarker panels are probably required.
BIOMARKERS OF FIBROSIS
Like other chronic liver diseases, NAFLD can also pro-
gress to advanced fibrosis and cirrhosis starting at perisi-
nusoidal regions. Patients with NASH-related cirrhosis
have similar mortality as those with cirrhosis from other
causes.5 It is therefore important to detect and assess
severity of fibrosis in NFALD, to allow a window of com-
plication screening and treatment evaluation.
Table 3 summarizes the serum biomarkers and predicted
models of fibrosis. The NAFLD fibrosis score and Fibrosis-4
(FIB-4) index use easily accessible clinical parameters and
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REVIEW New Biomarkers for NAFLD Chan and Wong

TABLE 2. RADIOLOGICAL TESTS OF STEATOSIS

Tests Description Usage and Limitations

Abdominal  Widely available and relatively inexpensive

ultrasonography  Can screen for hepatocellular carcinoma at the same time
 Moderate distinction of the severity of steatosis
 Operator dependent
 May miss steatosis <20%
 Performs less well in patients with body mass index >40 kg/m2
CAP Can be done together with liver stiffness  Quick procedure, relatively inexpensive after the initial installment
measurement by FibroScan  Moderate distinction of the severity of steatosis
 Objective and reproducible
 Detect steatosis >10%
 Machine may be unavailable at some centers
 Performs less well in patients with body mass index >35 kg/m2
1
MRI H-MRS or MRI-estimated  Good distinction of the severity of steatosis
proton density fat fraction  Objective and reproducible
 Detects steatosis as low as <1% and sensitive to change over time
 Not affected by obesity or ascites
 Can screen for hepatocellular carcinoma at the same session
 Expensive; service may not be available at some centers

TABLE 3. SERUM BIOMARKERS AND PREDICTION MODELS OF FIBROSIS

Tests Description Usage and Limitations

NAFLD fibrosis score A formula comprising age, hyperglycemia, body mass
index, platelet count, albumin, and AST/ALT ratio
FIB-4 index A formula comprising age, platelet count,
AST, and ALT
FibroTest A proprietary formula comprising a2-macroglobulin,
apolipoprotein A1, haptoglobin, total bilirubin, and
GGT
Enhanced liver fibrosis panel A proprietary formula comprising hyaluronic acid, pro-
collagen III amino-terminal peptide, tissue inhibitor
of matrix metalloproteinase 1, and age
 Easily accessible clinical parameters
 Simple and inexpensive
 Needs independent adjustment of body mass index
across ethnic groups
 Easily accessible clinical parameters
 Simple and inexpensive
 Highest AUROC for F3-4 diseases among studies
 Validated in Asian population
 Commercially available but more expensive
 Involves biomarkers that are not routinely done
 Affected by other causes of hyperbilirubinemia and
elevated GGT, e.g., hemolysis, alcohol intake
 Commercially available but more expensive
 Good distinction of the severity of fibrosis
 Involves biomarkers that are not routinely done

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristic curve;
GGT, gamma-glutamyl transpeptidase.

are therefore inexpensive. FibroTest and the enhanced liver A number of radiological tests have been validated for
fibrosis panel instead include special biomarkers that are NAFLD in recent years (Table 4). Transient elastography
not commonly measured at routine clinical practice. by FibroScan is the most widely available tool for liver

21 | CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016 An Official Learning Resource of AASLD
REVIEW
TABLE 4. RADIOLOGICAL TESTS OF FIBROSIS
Tests Description
Transient elastography Using FibroScan to measure the velocity of
shear wave in liver parenchyma and thus
liver stiffness measurement
Acoustic radiation force impulse Measure attenuation of ultrasound wave
energy as a result of tissue absorption
and scattering
Shear wave elastography Generates shear wave directly at liver
parenchyma with ultrasound beam
Magnetic resonance elastography MRI of shear wave propagation
Multiparametric MRI Using T1 mapping for fibrosis/inflammation
imaging; T2 for liver iron quantification
and 1H-MRS for liver fat quantification
stiffness measurement. It is reproducible and highly accu-
rate for cirrhosis and less so for significant fibrosis. While
liver stiffness measurement may fail in obese subjects
with a body mass index greater than 30 kg/m2, the suc-
cess rate can still approach 80-90% when the XL probe
is used.6 Acoustic radiation force impulse accesses the
internal energy vibration, whereas shear wave elastogra-
phy differs from transient elastography in terms of direct
generation of waveforms at liver parenchyma. Magnetic
resonance elastography is superior in distinction of differ-
ent fibrosis staging but limited by availability and cost.7
Recently, the multiparametric MRI has been developed to
measure hepatic steatosis, iron content, inflammation,
and fibrosis at the same time, but its applicability in
NAFLD patients requires further studies.8
GENETIC BIOMARKERS
Studies using next-generation sequencing have identi-
fied a few genes associated with NAFLD, among which
22 | CLINICAL LIVER DISEASE, VOL 8, NO 1, JULY 2016
New Biomarkers for NAFLD Chan and Wong
Usage and Limitations
 Highly reproducible
 Validated across patients with different liver diseases
 Inaccurate in patients with acute hepatitis, congestive heart failure, and
biliary obstruction
 Performs less well in obese subjects with body mass index >35 kg/m2
when using traditional M probe; XL probe can enhance accuracy
 Can perform hepatocellular carcinoma screening at the same time
 Not affected by external factors, e.g., obesity, ascites
 Higher variability because of small region of interest
 Can perform hepatocellular carcinoma screening at the same time
 Operators can choose suitable areas of examination interest, e.g., free of
blood vessels or focal lesions
 Not affected by external factors, e.g., obesity, ascites
 Good diagnostic accuracy and distinction of different fibrosis stages than
ultrasound-based techniques
 Not affected by obesity or ascites
 Allow simultaneous structural examination and 1H-MRS for hepatic stea-
tosis assessment
 Limited by cost and availability
 Less operator dependent
 Quick
 Good diagnostic accuracy in patients with mixed liver diseases
 Can allow structural examination and hepatocellular carcinoma screening
 Limited NAFLD data
patatin-like phospholipase domain-containing protein 3
(PNPLA3) and transmembrane 6 superfamily member 2
(TM6SF2) have been most extensively validated across
different ethnicities.9,10 These gene polymorphisms are
associated with both hepatic steatosis and liver injury,
but not with increased cardiovascular risk. Currently,
these genetic biomarkers remain as research tools; their
clinical application is yet to be defined.
FUTURE PERSPECTIVES
Most biomarkers were tested against liver histology in
cross-sectional studies. Their performance as monitoring
tools is largely unknown. Because there is now an
increasing number of clinical trials on NASH, this would
be a good opportunity to develop biomarkers that can
be used serially to monitor for disease progression and
assess treatment response. In the future, changes in bio-
markers must be validated against patients’ symptoms,
functions, and survival.
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REVIEW
CONCLUSION
Abdominal ultrasonography remains the primary test
for the diagnosis of NAFLD, after excluding other liver
diseases as appropriate. Serum biomarkers of fibrosis and
physical measurement of liver stiffness can be used to
exclude advanced fibrosis. The development of bio-
markers of NASH should now be a research priority. This
would ultimately reduce the need for liver biopsy and
improve patient care.
CORRESPONDENCE
Vincent Wong, Department of Medicine and Therapeutics, 9/F, Clini-
cal Sciences Building, Prince of Wales Hospital, 30-32 Ngan Shing
Street, Shatin, Hong Kong. E-mail: wongv@cuhk.edu.hk
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