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disease. The authors highlighted the superiority of ANXA2 over follistatin but given the low number of
patient samples, more comprehensive studies are required to draw a conclusion.

BIOMARKER COMBINATIONS FOR HCC DIAGNOSIS
In addition to individual biomarkers and those combined with AFP, biomarker combinations for the
diagnosis of early HCC have also been extensively studied. Cytokeratin-1 (CK-1) and nuclear matrix
protein-52 (NMP-52) elevated in sera of patients with HCC. Combination of CK-1, NMP-52 and AFP showed
an AUC of 0.9 for identifying HCC with 80% sensitivity and 92% specificity. More interestingly, this triple
combination could differentiate HCC from liver fibrosis with an AUC of 0.94 with 80% sensitivity and 92%
[54]
specificity . Epithelial membrane antigen and fibronectin, of which the serum levels were increased in
HCC, when in conjunction with total bilirubin and APF, could identify HCC from cirrhosis with an AUC
[55]
of 0.92 with 89% sensitivity and 85% specificity . Combined use of plasma protein with immune cells in
HCC diagnosis has been published as well. A combination of plasma Dickkopf-1, Tie2-expressing monocytes
[56]
and AFP yielded an AUC of 0.833 for HCC diagnosis . The clinical utility of these biomarker combinations
undoubtedly requires further validation in independent cohorts.

Diagnostic biomarkers with therapeutic potential
[14]
Although early diagnosis of HCC should translate into better overall survival, Chen et al. did not find
this link in their study, citing lack of effective treatment as the main reason. Biomarkers that can serve as
both a diagnostic tool and a therapeutic target would, undoubtedly, be more beneficial to the patients, as the
diagnostic results can immediately assist physicians in planning treatment regimen. A number of promising
biomarkers of this type are discussed below.

GPC3
GPC3 is a member of heparin sulfate proteoglycan family, which is bound to the cell membrane by a
[57]
glycosyl-phosphatidylinositol (GPI) anchor . A total of 6 glypicans have been identified to date, namely
[58]
GPC1 to GPC6, and they are predominantly expressed during development . The amino acid sequence
homologies amongst glypicans are low but the location of 14 cysteine residues are conserved, indicating
that they may share similar high-dimension structures. The location of the heparin sulfate insertion sites of
the glypicans appears to be restricted to the C terminus, putting the heparin sulfate chains near to the cell
[58]
membrane . GPC3 is a 580 amino acid protein encoded by the GPC3 gene located on human chromosome
X (Xq26). Despite being a cell membrane protein, GPC3 is cleaved by the Notum lipase at the GPI anchor
[59]
and released into the serum , making it easy for clinical detection. GPC3 is frequently upregulated in HCC
[60]
and melanoma . By fixed tissue staining, GPC3 expression was detected in up to 72% of samples from
[59]
patients with HCC, but not in healthy subjects or patients with benign liver diseases . The mRNA level of
GPC3 was also upregulated in HCC [61,62] . Moreover, at least three independent groups reported significant
elevation of serum GPC3 in HCC but not hepatitis [63-65] . Taken together, the data strongly suggestGPC3 to be
an attractive serum and histochemical biomarker for HCC.

GPC3 can stimulate Wnt signaling through canonical and non-canonical pathways, which are initiated by
[66]
Wnt ligands and Frizzled receptors . Given that Wnt proteins bind to heparin sulfate, it was suggested
that GPC3 acts as a facilitator of the interaction between Wnt ligands and Frizzled receptors [66,67] . GPC3
may promote tumourigenesis by facilitating canonical Wnt signal activation, which is frequently observed
in HCC [65,68,69] . In contrast, GPC3 expression is downregulated in breast and ovarian cancers, suggesting
that the functions of GPC3 may be tissue-specific [70-72] . Indeed, GPC3 is found to be a negative regulator
of Hedgehog signaling pathway. Downregulation of GPC3 causes hyperactive Hedgehog signaling, which
[67]
[73]
promotes ovarian and breast cancer progression . Filmus and Capurro proposed that GPC3 may exert
different functions depending on cell types. In tissues that proliferate mainly via Hedgehog signaling,
overexpression of GPC3 has an inhibitory effect on proliferation whereas in tissues where canonical Wnt
signaling exerts a dominant influence, upregulation of GPC3 promotes cell proliferation.