GLUTAMINE SYNTHETASE AS PROMISING

DIAGNOSTIC BIOMARKER FOR EARLY

HEPATOCELLULAR CARCINOMA

DETECTION

OF

Angkasa Ramatuan Hamdan1, Sri Hudaya Widihastha1

1
Students of Faculty of Medicine, Faculty of Medicine, University of Padjadjaran
Abstract
Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third
most frequent cause of cancer-related death in the world. The principle of
treatment of the cancer itself requires an accurate diagnostic tool, because the
death mainly caused by the late diagnosis that cause the patient to be found in a
state unoperable and ineffective treatment stage. Since the discovery in 1964,
Alpha Feto Protein has been identified as a useful diagnostic tool in the diagnosis
of HCC. Unfortunately, the sensitivity for HCC are in the range of 25% -60%, and
specifications are also quite low because it was detected also in patients with
cirrhosis and chronic hepatitis. Since then, several studies were performed to find
appropriate biomarkers as an early diagnostic tool for HCC. However, the
discovery of the substance is difficult because biomarkers in HCC should be able
to distinguish the situation in patients with cirrhosis and chronic hepatitis or
inflammation.
One substance that is considered promising is glutamine synthetase
(GS). Glutamine synthetase, one of the enzymes identified to be regulated by
nuclear -catenin, may be a candidate that contributes to enhanced malignancy of
HCCs. In the pathogenesis of HCC, serves GS catalyzes the formation of
glutamine from glutamate and ammonia into glutamine, which will then be used
as energy HCC. As a diagnostic tool of HCC, GS has some advantages of which
are believed to have an accuracy exceeding AFP in HCC diagnostic tool. In
addition, GS is also found with higher levels than in patients with cirrhosis and
chronic inflammation, making it useful to confirm the presence of HCC as a
complication of cirrhosis and chronic hepatitis. This literature review is aimed to
provide a profound comprehension on the prospect of GS as an advanced strategy
in diagnosing Hepatocellular Carcinoma, so in the future, the management can be
performed in patients with HCC as early as possible.

Keywords: hepatocellular carcinoma, diagnostic tools, GS, AFP

1.

INTRODUCTION

Liver cancer is the sixth most founded cancers in the world (1). By gender, liver
cancer is the fifth highest in men and seventh in women (2). Moreover, Southeast
Asian countries and sub-Saharan Africa has a high prevalence of liver cancer
because of the number of transmission of viral hepatitis B. Indonesia itself is a
country with the third highest prevalence of liver cancer in the world (3).
Therefore, the understanding, detection, and treatment of liver cancer is one of the
important discourse in dealing with its complications and lowering mortality
caused by liver cancer in Indonesia.
Liver cancer refers to cancers originating from the liver. There are several types of
liver cancer and of all, hepatocellular carcinoma (HCC) is the most commonly
found. About 4 out of 5 people suffering from liver cancer in adults are diagnosed
with hepatocellular carcinoma (4).
More than 40 years ago, serum glycoprotein Alpha-fetoprotein has been known
and used as a biomarker for the diagnosis of hepatocellular carcinoma. Fetal yolk
sac and fetal liver produce high levels of AFP in the end it comes down to <10
ng / dl within 300 days after birth. Therefore, the increase in serum AFP may
indicate a pathological disorder that most likely form of malignancy (5).
Unfortunately, the use of AFP as the determination of staging, course of treatment,
and prognosis in hepatocellular carcinoma is still optional. The reason is because
the increase in serum AFP only occurs in about 50% -75% of patients (5). Thus,
determining the stage at diagnosis of hepatocellular carcinoma should involve the
use of chest X-Ray (alternatively CT scans) and CT scan (Alternatively MRI) of
the abdomen.

Today, the use of Ultra Sonography which is easy and inexpensive, has been used
more widespread. The limitations of using the Ultra Sonography also appear to be
related to the ability of the medical expert and the patients condition. The state of
cirrhosis patients, for example, which is the biggest cause of hepatocellular
carcinoma can decrease sensitivity and positive predictive value to 35% -15% (5).
This indicates that there is a lack of reliability of imaging to diagnose
hepatocellular carcinoma when confronted with patients with a variety of
circumstances.
Moreover, with the use of current diagnostic patterns, there are major problems
with nearly all patients diagnosed at a late stage of disease. It has a relation with
the fact that the accuracy of the tools that has been used for the diagnosis of
hepatocellular carcinoma at an early stage is very small. As a result, patients
diagnosed with hepatocellular carcinoma are in the late stages where effective
treatment and surgery is not an option (6).
During the 40 years since the use of AFP as a biomarker that has been accepted
globally, efforts to discover biomarkers as early diagnosis of hepatocellular
carcinoma is still underway (5). The terms of a substance in order to be accepted
into a biomarker is that it should achieve a high level of accuracy. Achieving high
accuracy can increase the probability of diagnosis prior to deployment so that the
rate of healing HCC may be increased. Second, the substance becomes specimens
should be easily taken and not invasive. Third, the effectiveness of the cost should
be considered (7).

The development of hepatocellular carcinoma biomarker itself contains several

obstacles and tricks to overcome. First, hepatocellular carcinoma generally occurs
together or even be a complication of liver inflammation or cirrhosis before, so
the markers were found to be able to distinguish between these two conditions.
Second, related to the problem specification, an emerging biomarkers such as AFP
not only increased when there is cancer in the liver, but also the organ cancers
originating from the same endodermal lining the liver diverticulum (5). Thus, a
cancer that originates from the stomach, pancreas, and biliary tree can also have
high levels of AFP.
Currently, the development of early diagnostic biomarker of HCC become a
promising research. One substance that we will discuss is the glutamate synthetase
(GS). GS is an enzyme that catalyzes glutamine from glutamate and ammonia. GS
in general is found in the brain, kidney and liver (8).
Based on research conducted by Medina et al, GS is increasing in serum HCC
patients (9). It is associated with its own function which catalyze the formation of
GS gluthamine, where gluthamine is used as an energy source for cancer cells
(10-12). Further research showed that the level of GS also increased along with
the occurrence of chronic inflammation such as cirrhosis, possibly due to a role in
protein metabolism secondary to a mutation of b-catenin in the liver (13). Thus,
GS levels are higher when the emergence of HCC as a complication of cirrhosis.
Furthermore, with the use of immunohistochemistry in liver tissue of patients, it
was found that although the levels of GS in early stage HCC high, but there is an

increase of stage 1-4 HCC. This shows not only the possibility of GS as an early
diagnostic marker but also as a prognostic tool for HCC (14).
As described above, the first requirement for a substance to be a biomarker is that
a substance must achieve high accuracy in the diagnosis of disease. In this case,
the GS has a high level even in the early stage of HCC. For their examination, GS
is used as a diagnostic tool in the immunohistochemistry examination of the
patient's serum, which again qualifies as a non invasive properties biomarker.
Not only the mentioned above, the benefits of GS compared with other markers,
especially the AFP as a diagnostic tool for HCC commonly used, is shown
through correlation with cirrhosis and inflammation due to a virus as the major
cause of HCC. On the use of AFP, presence of cirrhosis will lower specificity and
positive predictive value to 35% -15% (5). This is in contrast with the presence of
GS were significantly increased when compared with patients with cirrhosis (14).
The objective of this literature review is to find out the prospect of GS as the
advanced strategy in diagnosing hepatocellular carcinoma. This review will
benefit other researchers by providing a profound comprehension for extensive
research on GS for early diagnostic tool. Furthermore, the research on this topic is
expected to offer a significant alteration regarding stagnant development of
hepatocellular carcinoma. So, in the future hopefully GS would be considered as
an early diagnostic tool for hepatocellular carcinoma.

2.

LITERATURE REVIEW

2.1

Definition and Staging of Hepatocelullar Carcinoma

Based on the definition of the American Cancer Society, liver cancer is a cancer
that originally came from the liver (4). Approximately 30 million people in
Indonesia suffered from hepatitis B and C virus and 15 million of these groups
will experience disease progression to Chronic Liver Disease (CLD). Thus, 10%
of this amount is expected to liver cancer (3).
In general, there are many types of liver cancer and the most common type of
liver cancer is hepatocellular carcinoma. Each year, hepatocellular carcinoma is
diagnosed in nearly more than half a million people worldwide and approximately
20,000 new cases are found in the United States. In fact, patients with
hepatocellular carcinoma are common in developing countries, particularly in the
area of hepatitis B virus endemic such as Southeast Asia and sub-Saharan Africa.
Indonesia itself is a country with the third highest prevalence of liver cancer in the
world. Hepatocelullar carcinoma is most common in adults. About 4 out of 5
types of liver cancer that develops in adults is hepatocellular carcinoma (4). This
cancer is also known as hepatoma and may have different patterns of development
(4)
a) Some come from changes in single cells grow bigger. But in the late phase
of the disease, it has spread to other parts of the liver
b) The second type occurs with a small change in spread of cancer nodules in
the liver. Occurs as in patients with cirrhosis (chronic liver damage) and
not only comes from one cell to undergo malignant.

In the understanding of hepatocellular carcinoma, cancer staging is critical

determination. Not only for the understanding of the disease itself, cancer staging
can be used for the determination of appropriate therapy, determination of
prognosis, and to exchange information among health professionals in order to get
rid of the ambiguity. In the last decade, the classification that mostly used is
classification of AJCC staging criteria / TNM system. Classification include the
relationship of the status of morphology, vascular invasion, metastases, and
fibrosis. The main purpose of staging hepatocellular carcinoma is to determine the
course of treatment and severity of disease. (21)

Tab. 1. Types of classification of Hepatocelullar carcinoma

2.2

Pathophysiology of Hepatocelullar Carcinoma

The pathophysiology of hepatocellular carcinoma has not been definitively

elucidated and is clearly a multifactorial event. In 1981, after Beasley linked HBV

infection to hepatocellular carcinoma development, the cause of hepatocellular

carcinoma was thought to have been identified. (15)
However, subsequent studies failed to identify HBV infection as a major
independent risk factor, and it became apparent that most cases of hepatocellular
carcinoma developed in patients with underlying cirrhotic liver disease of various
etiologies, including patients with negative markers for HBV infection and who
were found to have HBV DNA integrated in the hepatocyte genome.
Inflammation, necrosis, fibrosis, and ongoing regeneration characterize the
cirrhotic liver and contribute to hepatocellular carcinoma development. In patients
with HBV, in whom hepatocellular carcinoma can develop in livers that are not
frankly cirrhotic, underlying fibrosis is usually present, with the suggestion of
regeneration. By contrast, in patients with HCV, hepatocellular carcinoma
invariably presents, more or less, in the setting of cirrhosis. This difference may
relate to the fact that HBV is a DNA virus that integrates in the host genome and
produces HBV X protein that may play a key regulatory role in hepatocellular
carcinoma development; (16) an RNA virus replicates in the cytoplasm and does
not integrate in the host DNA.
The disease processes, which result in malignant transformation, include a variety
of pathways, many of which may be modified by external and environmental
factors and eventually lead to genetic changes that delay apoptosis and increase
cellular proliferation.
Recent analysis has sought to elucidate the genetic pathways that are altered
during hepatocarcinogenesis. (17) Among the candidate genes involved, the p53,

PIKCA, and -catenin genes appear to be the most frequently mutated in patients
with hepatocellular carcinoma. Additional investigations are needed to identify the
signal pathways that are disrupted, leading to uncontrolled division in
hepatocellular carcinoma. Two pathways involved in cellular differentiation
(ie, Wnt--catenin, Hedgehog) appear to be frequently altered in hepatocellular
carcinoma. Up-regulated WNT signaling appears to be associated with
preneoplastic adenomas with a higher rate of malignant transformation.
While various nodules are frequently found in cirrhotic livers, including
dysplastic and regenerative nodules, no clear progression from these lesions to
hepatocellular carcinoma occurs. Prospective studies suggest that the presence of
small-cell dysplastic nodules conveyed an increased risk of hepatocellular
carcinoma, while large-cell dysplastic nodules were not associated with an
increased risk of hepatocellular carcinoma. Evidence linking small-cell dysplastic
nodules to hepatocellular carcinoma includes the presence of conserved
proliferation markers and the presence of nodule-in-nodule on pathologic
evaluation. This term describes the presence of a focus of hepatocellular
carcinoma in a larger nodule of small dysplastic cells. (18)
Recent work speculated that hepatocellular carcinoma develops from hepatic stem
cells that proliferate in response to chronic regeneration caused by viral injury
(19). The cells in small dysplastic nodules appear to carry markers consistent with
progenitor or stem cells.

2.3

Diagnosis of Hepatocelullar Carcinoma

In the diagnosis of hepatocellular carcinoma, a doctor can perform history taking,

physical examination, and laboratory tests. In history taking, the most important
information that must be obtained is about whether patients belonged to the high
risk of hepatocellular carcinoma such as drug users, have a family with a history
of hepatitis B infection, and alcohol drinkers. (21)
The presentation of hepatocellular carcinoma has evolved significantly over the
past few decades. While, in the past, hepatocellular carcinoma generally presented
at an advanced stage with right upper quadrant pain, weight loss, and signs of
decompensated liver disease, currently the diagnosis of hepatocellular carcinoma
is often used in ways noninvasive imaging tests. In patients with cirrhosis and a
focal hepatic mass greater than 2 cm, it can be diagnosed by using CT and MRI.
The presence of radiological changes in inspection method is associated with
increased vascularization of the hepatocellular carcinoma. (21)
Until today, there are only a few biomarkers are available for the diagnosis and
monitoring

of

HCC.

By

its

sensitivity,

specificity,

and

operability,

alphafetoprotein (AFP) cannot fulfill the requirements of an ideal tumor marker,

although it remains the best one clinically available for HCC. With lesions 1-2 cm,
the presence of alpha-fetoprotein level of 400 ng per millimeter or higher is also
highly indicated the presence of hepatocellular carcinoma. But unfortunately,
during early HCC, asymptomatic patients with negative serum markers (AFP) are
usually not diagnosed until the advanced stages. (21)

Early diagnosis of HCC can greatly improve the outcome, with better long-term
survival and reduced recurrence risk for operative treatment. The only potentially
curative therapies depend on detection and resection of small HCCs. Because the
purely morphological concept of small HCC (diameter 3 cm) encompasses early
HCC, small HCCs possess most of the characteristics of early HCCs. Therefore,
selecting small single-nodule HCCs might be suitable for studies on the diagnosis
of, or therapy for, early HCC. GS could be potential markers for HCC, and GS
could be a novel serum marker for early HCC, especially in patients with low AFP
levels (200 ng/ml). (22)
2.4

Management and Prognosis of Hepatocelullar Carcinoma

Aggressive surgery or a liver transplant can successfully treat small or slowgrowing tumors if they are diagnosed early. However, few patients are diagnosed
early.
Chemotherapy delivered straight into the liver with a catheter can help, but it will
not cure the disease. Radiation treatments in the area of the cancer may also be
helpful. However, many patients have liver cirrhosis or other liver diseases that
make these treatments more difficult.
Sorafenib tosylate (Nexavar), an oral medicine that blocks tumor growth, is now
approved for patients with advanced hepatocellular carcinoma.
The usual outcome is poor, because only 10 - 20% of hepatocellular carcinomas
can be removed completely using surgery. If the cancer cannot be completely
removed, the disease is usually deadly within 3 to 6 months. This is partially due
to late presentation with large tumours. (23)

2.5

Alpha Fetoprotein as Biomarker Hepatocelullar Carcinoma

AFP is a 72 kDa onco-fetal glycoprotein with a size of 591 aminoacids (24). It is

normally synthesized during fetal life, first in the yolk sac and then in fetal liver;
its synthesis is normally repressed in adults (25). High levels of AFP are observed
during adulthood only under certain conditions, such as pregnancy, the presence
of some neoplasias (e.g. HCC, gastric carcinoma, testicular carcinoma, lung
cancer and pancreatic cancer) and some non-neoplastic disorders such as chronic
hepatitis (26).
Although serum AFP levels have been shown to increase in association with
several carcinomas, this parameter has only been employed as a tumoral marker
for HCC (27, 28). The present investigation showed that serum AFP levels,
measured with SUMA technology, were significantly higher in patients aged 50
years or older, in patients with viral HC, and in patients with HCC.
The results coincide with previous reports investigating the influence of viral
infections on AFP concentrations. Increased levels of AFP are detected in viral
hepatitis patients with no detectable HCC. Taking into account the results from
different publications, it can be estimated that 10 to 43% of persons with a chronic
HCV infection will have increases in this serum marker (26). Searching for an
explanation to this finding, several studies have demonstrated a correlation
between increased serum AFP levels and the degree of inflammation observed in
liver biopsies (29).
The investigations examining the usefulness of AFP as a screening and
surveillance tool have not been directly comparable: their design and the

characteristics of the studied population (type of viral infection, severity of liver

disease, demographics) have differed. This fact alone explains the wide variability
of their results, with sensitivities ranging from 41 to 69% and specificities
between 75 and 94% (30).
The association between serum AFP and HCC has been widely examined and
described by a large number of groups (31). Regardless, its sensitivity and
specificity for diagnosing HCC are variable, with figures ranging from 39 to 73%
and 65 to 96%, respectively (30), depending on factors such as the specific assay
used, the design of the study, the characteristics of the study population, and the
designated cut-off level (32). From the study that conducted by Hernandez, J. Et
al showed that the sensitivity of AFP determinations for diagnosing HCC was
68.18%; the specificity values obtained were 92.17%; 45.45% for the positive
predictive values (PPV); and 96.80% for the negative predictive values (NPV).
Youdens index was low, at 0.60. (33)
A publication by Trevisiani et al. (30) has proposed that the best diagnostic
threshold level for AFP ranges from 16 to 20 ng/mL. Using a threshold of 20
ng/mL, specificity was indeed high (89.4%), but sensitivity was only 60%; this
would produce such a significant number of false negative diagnoses for a disease
with the implications of HCC that the marker would not be useful for this
purpose. Lowering the threshold identifies a larger number of cases, improving
sensitivity at the price of an increased false positive rate. In otherwords, the higher
the threshold, the lower the number of detected cases obtained (sensitivity
decreases).

A different study by Gambarin-Gelman et al. (34) also used an AFP diagnostic

threshold of 20 ng/mL, and obtained a sensitivity of 58% and a specificity of 91%.
In exchange, PPV increased from 58 to 75% at higher levels of AFP (50 ng/mL),
although it must be underscored that in the latter case, sensitivity dropped from 58
to 47%.
It reported by Nguyen et al. (35), who had specificity (97.3%) at the same AFP
threshold of 100 ng/mL and had to increase it to 200 ng/mL or higher to obtain a
specificity of 100.
2.6 Glutamine Synthetase as Biomarker of Hepatocelullar Carcinoma
Glutamine synthetase (GS) is a member of an enzyme family that catalyzes the
synthesis of glutamine from glutamate and ammonia. It plays an important role in
ammonia detoxification, nitrogen balance and pH regulation in the liver (36). GS
is present predominantly in the brain, kidneys, and liver. (8) From the research
that conducted by Christa et al. (37) it demonstrated that levels of GS mRNA, GS
protein, and GS activity are upregulated in human HCCs. Kuo (38) described
earlier an equal presence of total GS mRNA in total liver mRNA late in fetal life
and in adult life.
Glutamine synthetase (GS) has long been known to be expressed exclusively in
pericentral hepatocytes most proximal to the central veins of liver lobuli. This
enzyme as well as its peculiar distribution complementary to the periportal
compartment for ureogenesis plays an important role in nitrogen metabolism,
particularly in homeostasis of blood levels of ammonium ions and glutamine.
Despite this fact and intensive studies in vivo and in vitro, many aspects of the

regulation of its activity on the protein and on the genetic level remained
enigmatic. Recent experimental advances using transgenic mice and new analytic
tools have revealed the fundamental role of morphogens such as wingless-type
MMTV integration site family member signals (Wnt), -catenin, and adenomatous
polyposis coli in the regulation of this particular enzyme. (39)

Fig. 1. Schematical drawing of a pericentral GS-positive hepatocyte illustrating

major metabolic steps in glutamine synthesis. Obviously, these cells are equipped
with efficient molecular machinery for efficient scavenging of ammonium ions
and ammonia and conversion into glutamine that is excreted from these cells via
the SNAT3 transporter. (39)

By using immunohistochemistry (IHC) of HCC tissue, Di Tommaso et al. (40)

further identified the overexpression of GS in HCC and proposed GS as an useful
biomarker for early diagnosis and prognosis in HCC patients. The level of GS in
stage I and stage II HCC found to be significantly lower than in stage III and stage
IV HCC. This observation indicates that GS correlates with HCC progression and
thus could be a reliable prognostic marker for HCC.
Medina MA et al. (41) noticed that the serum concentration of glutamine is
decreased in advanced cancers, especially in the parenchyma of cirrhotic liver in
HCC patients. Glutamine is the end product of GS activity, which is the main
energy source of tumor cells (42, 43, 44). It is reasonable to hypothesize that
increased GS might be the result of glutamine deficiency. (45)

From the study that conducted by Jiang Long et al showed that GS expression is
increased in HCC and CHB stage 14 patients. The study revealed the important
phenomenon that strong and diffuse cytoplasmic immunoreactivity frequently
occurred not only in tumor tissue, but also in non-malignant tissues with
necroinflammation and regeneration. This indicates that GS is associated with
necroinflammation and regeneration. It suggest that GS also plays a potential role
in the pathogenesis of chronic inflammation and subsequent malignant
transformation, probably through its role in protein metabolism secondary to bcatenin mutation in the liver. The study confirmed the findings that serum GS
level is increased in HCC and CHB (stage 14) patients, so serum GS level may
be recommended as a diagnostic marker for liver diseases, and it is a serum
marker for diagnosis of HCC, especially for those patients with borderline AFP
value (100\AFP B 200 mg/ml). (22)

2.6.1

Serum Levels Of Glutamine Synthetase In Hepatocelullar Carcinoma

Jiang Long et al from his research investigated whether GS is detectable in the

sera of HCC patients. From their statistical analysis using the Mann- Whitney U
test showed that the distribution of serologic levels of GS in HCC patients was
significantly higher than in those with cirrhosis or healthy donors, and the latter
two groups were significantly different. On the other hand, if a cutoff value of 200
ng/ml was used for AFP, GS was elevated in mostly HCC patients. Thus, the
simultaneous use of the two markers significantly increased the sensitivity of
diagnosis, especially for patients with low AFP levels ( 200 ng/ml). (14)

Another research that conducted by Jiang et all also showed that the distribution
of serological levels of GS in HCC, CHB (stage 13), and CHB (stage 4) patients
is significantly higher than in normal patients. In the HCC group, elevated GS
values also known that has correlate with AFP values. From their result it also
showed that GS was more effective than AFP in differentiating between HCC
versus healthy controls. (22)

3.

CONCLUSION

Glumatate synthetase (GS) as a diagnostic marker has been shown to have several
advantages compared with diagnostic markers that have been used for HCC,
Alpha Feto Protein. However, the period of examination for diagnosis using the
GS is still need to be investigated further, aimed to determine when is the ideal
time for a person with a high risk of HCC after the first examination of serum. In
addition, further clinical examination should still be conducted to determine the
evidence of the use of GS to the diagnosis of Hepatocellular carcinoma.
Since hepatocellular carcinoma is a complex disease with a lot of mechanisms of
pathogenesis, the use of single biomarker will probably have a lot of obstacles,
such as in some of the researches that had been reviewed in this literature.
Therefore, the use of GS in combination with other biomarkers may help improve
the function of GS for diagnosis, staging, and prognosis. As described previously,
GS do not only have the possibility as a diagnostic tool, but also has potential to
be a prognostic tool, which both are extremely important for the future
management of patients with high risk of hepatocellular carcinoma.
Further research is highly recommended to make the glutamate synthetase as
diagnostic tools for early hepatocellular carcinoma become the first-line choice in
diagnosing HCC patients.

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