Best Practice & Research Clinical Gastroenterology

Vol. 19, No. 1, pp. 9199, 2005

doi:10.1016/j.bpg.2004.10.003
available online at http://www.sciencedirect.com

5
Serological markers of liver cancer
Man-Fung Yuen*

MD

Associate Professor

Ching-Lung Lai MD
Professor
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China

Serological markers for hepatocellular carcinoma (HCC) are important for early diagnosis, as well
as monitoring of tumour aggressiveness, treatment responsiveness, recurrence and survival. The
three most common markers are total alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive
AFP (AFP-L3) and protein induced by vitamin K absence or antagonist-II (PIVKA-II). Total AFP has
the sensitivity of 60% and specificity of 90% for the detection of HCC. Increase in the percentage
of AFP-L3 over the total AFP (O10%) is very specific for small HCC. PIVKA-II is also more
specific than total AFP in detecting HCC. AFP-L3 and PIVKA-II levels correlate with tumour
aggressiveness and prognosis. All three markers are useful for monitoring treatment
responsiveness and tumour recurrence. Since the levels of the three markers are independent
of each other, combination of measurement of two or three markers will increase the sensitivity
and diagnostic accuracy. Some novel markers including glypican-3 are being extensively studied.
Key words: hepatocellular carcinoma; serological marker; alpha-fetoprotein; des-gammacarboxyprothrombin; glypican-3.

Being the fourth most common cancer in the world, hepatocellular carcinoma (HCC)
remains an important disease.1 It is responsible for around 250 000 deaths every year.2
Advances in the diagnosis and management of HCC have significant impact on patients
who are at risk of development of HCC. Serological markers specific for HCC play
important roles in this disease in the following aspects (1) screening of HCC in
susceptible individuals, specifically patients with chronic hepatitis B or C infection
to increase the chance of receiving curative treatment and to improve survival;
(2) association with the stage of the disease, to provide prognostic information;
(3) monitoring the treatment response; and (4) monitoring for relapse after curative
treatment. Ideally, serological markers for HCC should possess the following
* Corresponding author. Tel.: C852 28554252; Fax: C852 28162863.
E-mail addresses: yuenmf@netvigator.com (M.-F. Yuen), hrmelcl@hkucc.hku.hk (C.-L. Lai).
1521-6918/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

92 M.-F. Yuen and C.-L. Lai

characteristics (1) high sensitivity and specificity for the diagnosis of HCC; (2) assays
that are easy to perform and (3) assays that are comparatively cheap.
To date, there are three common serological markers for HCC namely, total alphafetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3) and protein induced
by vitamin K absence or antagonist-II (PIVKA-II) which is also known as des-g-carboxy
prothrombin (DCP).

TOTAL ALPHA-FETOPROTEIN
AFP was first described as a marker for HCC by Abelev in the 1960s.3 It is a
glycoprotein with molecular weight of around 70 kDa. The molecule contains an
asparagine-binding double-chain complex sugar. AFP gene is highly expressed in
hepatocytes and endodermal cells of the yolk sac during foetal development. The
expression is repressed after birth. Pathological elevation of AFP is seen in hepatocyte
regeneration, hepatocarcinogenesis and embryonic carcinomas. The AFP level in the
serum is usually measured by immunoassays. The serum AFP levels of healthy subjects
should normally be less than 10 ng/ml. In the attempt to define the cut-off level for
diagnosing HCC, one needs to consider that the higher the chosen AFP level is, the
higher will be the specificity and the lower the sensitivity. AFP level O500 ng/ml was
previously taken as a diagnostic level for HCC. This chosen level is, however, not
satisfactory because as high as 80% of patients with early HCC have the AFP levels
lower than 500 ng/ml.47 On the other hand, patients with chronic hepatitis B or C with
exacerbations and regenerations sometimes have AFP levels more than 500 ng/ml.
Studies in the European population show that the sensitivity and specificity of using
20 ng/ml as a cut-off level are 5569% and 8392%, respectively.8,9 A review by Gupta et
al, also shows consistent findings of sensitivity and specificity are 4165% and 8094% in
patients with chronic hepatitis C.10 According to another study conducted by Trevisani
et al in Italy, AFP level of 16 ng/ml is found to have the best discriminating power.11 They
suggest adopting the value of 20 ng/ml as the cut-off level because the sensitivity and
specificity do not differ much from the sensitivity and specificity when 16 ng/ml is used
as cut-off level. With 20 ng/ml as the cut-off level, the sensitivity and specificity are 60
and 90.6%, respectively. Assuming the prevalence of HCC is 5%, the negative predictive
value is 97.7%, but the positive predictive value is 25% only. According to a study in
Chinese patients with chronic hepatitis B, of the 44 patients with elevated AFP (O
20 ng/ml) out of 290 patients, only six (14%) had HCC.12 The remaining 38 patients had
elevated AFP due to hepatitis B exacerbations (nZ18) and unknown causes (nZ20).
This study illustrates the very low positive predictive value when an AFP level of just
above normal is chosen as cut-off level.
In spite of the pitfalls of using AFP as a marker of HCC, it is commonly used as a one
of the screening tools in combination with ultrasonography.13,14 Though some clinicians
find AFP to be of no use for screening15, Tremolda et al, demonstrate that the
sensitivity can reach up to 100% by combining AFP measurement and ultrasonography.16 To improve the sensitivity of AFP for the detection of HCC, different isoforms
of the AFP have been studied and this will be discussed in the Section 2.
To determine whether AFP level correlates with the disease stage of HCC, studies
have been conducted to examine the relationship between AFP levels with tumour
sizes, histological grades, intrahepatic metastases and portal vein thromboses.1720

Liver cancer markers 93

These studies show consistently that AFP levels do not correlate any of these
parameters.
However, AFP serves as an excellent marker for monitoring tumour progression in
patients with AFP-producing HCC. Complete clearance of the tumour after treatment
is almost certain if the pretreatment elevated AFP levels drop to and stay at normal
levels during subsequent follow-ups. For palliative treatment, e.g. transarterial
chemoembolisation, reduction of AFP levels usually signifies favourable responses to
the treatment. AFP is also an excellent marker for detection of new occurrences of
HCC after treatment, though there is a possibility that the new HCC may be of the
non-AFP secreting variety.

LENS CULINARIS AGGLUTININ-REACTIVE AFP (AFP-L3)

The sensitivity of AFP can be improved by measuring the isoforms of the AFP. The
molecule of AFP is heterogeneous in structure regarding the carbohydrate sidechains.21,22 The asparagine at position 232 from the N-terminus of AFP which binds
with the sugar moiety has a distinct affinity to lectin. Heterogeneity in the affinity for
lectins allows for the separation of AFP into different isoforms. Examples are
Concanavalin A (Con A) and Lens culinaris agglutinin (LCA) as measured by
immunoblotting following affinity electrophoresis. LCA-reactive AFP (also named
AFP-L3) is very specific for HCC whereas Con A non-reactive AFP is more specific for
yolk sac tumours and other gastrointestinal tumours.2329 The percentage of AFP-L3
over the total AFP levels is used as a specific index for HCC.2325 The cut-off
percentage is taken as 10%, above which HCC is very likely even when imaging
modalities fail to identify the tumours.30 Separation of different AFP glycoforms can also
be performed by using isoelectric focusing technique.3134 It separates the AFP into
different bands in which band II is found to be relatively specific for HCC. Patients with
positive results for HCC-specific AFP isoform either detected by assays dependent on
the affinity to lectins or by isoelectric focusing, should undergo continuous and
frequent follow-ups for the earliest possible detection of HCC by imaging if HCC is not
detected by imaging modalities initially.
Unlike total AFP levels, AFP-L3 correlates with the staging of HCC. According to a
study conducted by Kumada et al, in small HCC (less than 2 cm), AFP-L3 is associated
with poorly differentiated and multifocal HCC.28 In addition, small hypervascular HCC
has a significantly higher AFP-L3 level compared to that of iso- or hypovascular HCC.
The tumour doubling time is shorter in patients with high levels of AFP-L3 compared to
those with low levels of AFP-L3. AFP-L3 also serves as a marker for clearance of HCC
after treatment. Failure to decrease to the normal level indicates residual disease.
Recurrence of HCC is very likely if AFP-L3 levels increase to O10% of the total AFP or
rise again after returning to the normal levels with treatment. It has been found that
relapse with multifocal HCC as well as tumour invasion to portal vein is more frequent
in patients with elevated AFP-L3 compared to those without re-elevation of AFP-L3
after treatment.26 More importantly, patients with higher levels of AFP-L3 have a
poorer survival compared to those with normal AFP-L3.29
Therefore, AFP-L3 as a marker for HCC is superior to the total AFP not only in the
accuracy of diagnosing HCC, but also in the correlation with the stage and the
prognosis of the disease. However, the assay for AFP-L3 is not available in most nonresearch laboratories.

94 M.-F. Yuen and C.-L. Lai

PROTEIN INDUCED BY VITAMIN K ABSENCE OR ANTAGONIST-II

(PIVKA-II)
Production of PIVKA-II is the result of an acquired defect in the post-translational
carboxylation of the prothrombin precursor in tumour cells.35 The diminished gammacarboxylase activity is due to defective gene expression observed in HCC.36 This
results in the production of abnormal prothrombin without carboxylation of the 10
glutamic-acid residues in the N-terminus. PIVKA-II is, therefore, also known as des-gcarboxy prothrombin (DCP) because of the lack of g-carboxylation of the glutamine
residues. PIVKA-II has no biological activities such as coagulation activity.
PIVKA-II was first described as a marker of HCC by Liebman et al in 1984.37 They
observed that PIVKA-II was elevated with a relatively high incidence in patients with
HCC. In addition, PIVKA-II was found to be very specific for HCC. Serum PIVKA-II
level is usually measured by enzyme immunoassays. For the initial versions of the assay,
the lower limit of detection is 0.0625 AU/ml (AU, arbitrary unit; 1 AU is equivalent to
1 mg/ml of prothrombin). The cut-off level is therefore set at 0.1 AU/ml (100 mAU/ml).
Although PIVKA-II has a high specificity for HCC, it is not as sensitive as AFP. PIVKA-II
level is found to be elevated in around 5060% of patients with HCC and only in
1530% of patients with HCC !3 cm.38,39 The assay performance has since been
improved at several centers.4044 A PIVKA-II level of 40 mAU/ml is now commonly
adopted as a cut-off level. This cut-off level improves the rate of early detection of small
HCC. According to several recent studies, PIVKA-II measurement for HCC has a
sensitivity of 4862% and specificity of 8198%.4552 These figures are comparable to
those of AFP in whom the sensitivity is 4054% and the specificity is 8897% according
to these studies. The accuracy for differentiating HCC from other liver diseases by
using PIVKA-II is 5984%.
Similar to total AFP, PIVKA-II is also an excellent marker for monitoring the
treatment efficacy, indication of complete clearance of HCC after curative treatment,
and recurrence of HCC in the future.
However, there are several differences between PIVKA-II and total AFP. Firstly,
PIVKA-II levels and total AFP levels do not correlate either positively or negatively with
each other. Secondly PIVKA-II compared to total AFP, is more specific as a marker for
HCC because other liver diseases rarely give rise to elevated PIVKA-II. However, the
accuracy of PIVKA-II may be diminished in prolonged obstructive jaundice, intrahepatic
cholestasis with vitamin K deficiency, and intake of warfarin and some antibiotics.
Thirdly, the serum half-life of PIVKA-II of around 4072 hours is much shorter than that
of AFP of around 57 days. Therefore, PIVKA-II reflects the therapeutic efficacy on
HCC in a timelier manner. Finally, unlike total AFP, PIVKA-II is found to correlate with
the stages of the HCC as well as survival. It has been found that PIVKA-II-positive
patients have a higher rate of intrahepatic metastasis, portal vein tumour invasion,
hepatic vein tumour thrombosis, capsular infiltration.1720,37,53 Therefore, it is not
surprising to see that PIVKA-II-positive patients have a poorer survival compared to
PIVKA-II-negative patients.54
Since PIVKA-II and total AFP levels are independent of each other in the setting of
HCC and neither one is ideal as a marker for HCC, combination of these two markers
is a logical approach to determine whether it will increase the accuracy of diagnosing
HCC. According to one study using the analysis of areas under the curve, conducted by
Marrero et al, combination of PIVKA-II and AFP is not superior to PIVKA-II alone in
differentiating HCC from other liver diseases.55 However, Grazi et al find that

Liver cancer markers 95

combination is better. According to their study, the sensitivity, specificity and diagnostic
accuracy for PIVKA-II alone and total AFP alone are 53.3, 88.1, 71.1% and 54.9, 97.4,
76.6%, respectively.46 These figures become 74.2, 87.2 and 80.9%, respectively,
by combining PIVKA-II and total AFP. Other studies also find that combination of total
AFP and PIVKA-II increases the sensitivity as well as the specificity for diagnosing
HCC.39,49,54 It has also been shown that combination of PIVKA-II and AFP-L3 is more
effective for the early detection of HCC.56

GLYPICAN-3
Glypican-3 (GPC3) is one of the members of heparan sulfate proteoglycans.5759 It
binds to the cell membrane through the glycosyl-phosphatidylinositol anchors. GPC3 is
able to interact with various growth factors that either stimulates or inhibits the growth
activity. GPC3 gene mutation is responsible for the SimpsonGolabiBehmel syndrome
that is characterised by pre- and postnatal overgrowth.60 Though GPC3 can induce
apoptosis in the breast and the ovary, it is an oncofoetal protein in the liver and the
colon.61 The action of GPC3 is thought to be tissue-specific. Recently, GPC3 mRNA
expression has been demonstrated to be increased in HCC by several studies.6264
Sung et al have shown that GPC3 is secreted in the culture media from HCC cell lines.65
GPC3 protein measured by enzyme linked immunosorbent assays (ELISA) is detectable
in around 4053% of HCC patients whereas it is not detectable in the serum of healthy
individuals.64,66 According to Capurro et al, only one out of 20 patients with hepatitisrelated cirrhosis has detectable GPC3 in the serum.66 In another study by Nakatsura et
al, none of 13 patients with liver cirrhosis, 34 patients with chronic hepatitis and 60
healthy individual has detectable GPC3. The specificity of GPC3 in this study was thus
100%.64 There is no correlation between GPC3 and total AFP levels in patients with
HCC. Combination of GPC3 and total AFP also increases the sensitivity without
affecting the specificity.66 However, more studies are required to determine the
usefulness of this new marker in HCC.

OTHER NOVEL MARKERS

There are other serological markers which have been studied for HCC. Elevated serum
anti-p53 has been observed in around 2025% of patients with HCC in two previous
studies.9,67 Recently, according to the study conducted by Attallah et al, anti-p53 is
found to be positive in 75% of patients with HCC.68 However, positivity rates are also
very high for other gastrointestinal malignancies including cholangiocarcinoma (100%),
pancreatic carcinoma (75%), colonic carcinoma (70%), carcinoma of esophagus (60%)
and gastric carcinoma (35%).
Glycylproline dipeptidyl aminopeptidase (GPDA) level, first used as a serological
marker by Kojima et al in 198769, is recently re-examined by Ni et al who identify that
the isoenzyme GDFA-F is consistently positive in patients with HCC.70 They suggest
that this marker is particularly useful for diagnosis of HCC in patients with non-AFPproducing HCC.
As an important growth factor for angiogenesis, serum vascular endothelial growth
factor (VEGF) is associated with invasiveness and metastasis of HCC.71 This marker has
a predictive value on the behavior of the HCC.

96 M.-F. Yuen and C.-L. Lai

CONCLUSIONS
Though none of the above markers fulfils the ideal criteria as serological markers for
HCC, reasonable levels of sensitivity and specificity are already achieved. Concomitant
ultrasonographic examination of the liver will further increase the sensitivity and
specificity for the detection of HCC. However, high accuracy for diagnosing small and
early staged HCC which may be undetectable by imaging technique is of particularly
importance. The detection rate may be improved by measuring these available markers
in combination as they are independent of each other. These markers are also excellent
for monitoring tumour behaviour, disease stage, treatment responsiveness, tumour
recurrence and prognosis.

Research agenda
investigate the best combination(s) of available serological markers to further
improve diagnostic accuracy, particularly in differentiating small hepatocellular
carcinoma from hepatitis reactivation and other hepatic conditions
investigate the other novel serological markers with high sensitivity and
specificity

Practice points
serological markers for hepatocellular carcinoma are important for early
diagnosis, monitoring of tumour aggressiveness, treatment responsiveness,
recurrence and survival
total alpha-fetoprotein (AFP) is the most commonly used serological marker,
Lens culinaris agglutinin-reactive AFP (AFP-L3) and protein induced by vitamin
K absence or antagonist-II (PIVKA-II) are two other serological markers that
are useful, though much less commonly tested
AFP-L3 and PIVKA-II are associated with the stage and the prognosis of the
disease
combination of two or three markers increases the sensitivity and diagnostic
accuracy for hepatocellular carcinoma
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