**4.2** γ**-Glutamyl transferase (GGT)**

γ-Glutamyl transferase (GGT) is a membrane-binding enzyme, which appears in the development of liver cells during pregnancy, its concentration is high throughout pregnancy and decreases immediately after birth. The total GGT concentration increased in chronic liver diseases, HCC, and some extra-liver cancer diseases [63]. A study by Cui et al. on 90 patients with cirrhosis and 120 patients with HCC showed that the sensitivity of HS-GGT was 74%, irrespective of size, and 43.8% for small tumors (<3 cm) [64] (**Table 1**). The diagnostic value improves when combined with other biomarkers such as AFP, PIVKA II, or AFP-L3. This is a promising sign in the detection of small cancers and can be used in combination with AFP and AFP-L3.

## **4.3 Matrix metalloproteinases (MMPs)**

Matrix metalloproteinase (MMP) is an enzyme belonging to the endopeptidase group, which helps regenerate tissue in various pathogenetic processes including tumor progression, and wound healing [65]. Kuo et al. showed that only cases of HBsAg-positive have high levels of MMP-2 expression [66], but the relationship between other markers of HBV and MMP was not clarified. Positive cases with HBeAg showed a high tendency for portal vein thrombosis along with high manifestations of MMP-7 and MMP-9. MMPs have a synergistic effect on HCC generation, proliferation and invasion, through ways that the study did not elucidate [67]. A significantly higher MMP-9/MMP-2 ratio was found in patients with advanced HCC compared to patients at an early stage [68]. The mRNA of MMP-14, MMP-15 and MMP-2 are highly expressed in most HCC cells suggesting an important role of MMPs in the growth, invasion, and metastasis of tumor cells. Selective inhibitors for these MMPs promise to be an effective mean of preventing the growth and metastasis of HCC [69].

### **4.4 Glutamine synthetase (GS)**

Glutamine synthetase (GS) is an enzyme involved in catalyzing the synthesis of glutamine from glutamate and ammonia, it plays an important role in the function of ammonia metabolism and nitrogen balance of the liver [70]. Research by Haupt et al. demonstrated that GSmRNA increased its tissue and protein expression in the serum of HCC patients [71]. In addition, Osada et al. reported increasing GS expression correlated with cancer progression, suggesting GS can play a role in promoting HCC metastases [72].

### **4.5 Alpha L fucosidase (AFU)**

Alpha L fucosidase (AFU) is a glycosidase responsible for hydrolysing fucoseglycoside bonds of glycoprotein and glycolipids and is found in all mammalian cell

lysosomes and is involved in the degenerative reaction of a series of fucoglycocontaining fucoglyco complexes [73]. Serum AFU levels are constantly elevated in cirrhotic patients who tend to progress to HCC. Deugnier et al. found that serum AFU had greater sensitivity and specificity than AFP and that it can be considered a marker for HCC diagnosis. However, the cause of this increased serum AFU activity is still unknown. The most likely explanation is that increased serum AFU activity is a result of an increase in tumor protein synthesis that increases fucoses [74]. Measuring the activity of serum AFU regularly during follow-up of cirrhotic patients provides very useful clinical data in monitoring cirrhosis progression to HCC. Although an increased serum AFU activity was not correlated with tumor size and was common in cases of early HCC, the HCC tumor would appear within a few years in 82% of patients with liver fibrosis if serum AFU activity exceeds 700 nmol/ ml/hour. Serum AFU activity increased in 85% of patients at least six months before HCC was detected by a diagnostic imaging method [75]. AFU activity was significantly increased in HCC patients compared with patients with other liver diseases or other cancers. AFU sensitivity is 81.5% and its specificity is 85.4% in HCC diagnosis [76] indicating a promising specific marker for HCC diagnosis.
