**3.5 Osteopontin (OPN)**

Osteopontin (OPN) is known as a conversion protein and is a glycophosphoprotein associated with integrin, which is overexpressed in many types of malignancies such as lung, breast, and colon cancers [41]. OPN usually manifests in biliary epithelial cells, astrocytes and Kupffer cells, but not in liver cells [42]. However, increased serum OPN expression has been reported in patients with HCC, but not in those with cirrhosis, chronic hepatitis, or healthy controls [43, 44]. In a meta-analysis, the sensitivity and specificity of OPN were 86% for all HCC stages [45]. Shang et al. suggested that serum OPN concentrations at the cut-off level of 91 ng/ml were more sensitive than that of AFP (74% versus 53%) in the diagnosis of HCC. Combining two imprints with an OPN cut-off of 156 ng/ml and an AFP cut-off of 20 ng/ml increased sensitivity and specificity (95% and 96%). The sensitivity and specificity of OPN were 75% and 62% for early HCC, which means the sensitivity was higher than that of AFP, but the specificity lower (46% and 93%). When combined with AFP at the cut-off of 91 ng/ml for OPNs, sensitivity increased to 83% and specificity decreased to 63% [45] (**Table 1**). Based on such findings, OPN can be considered an important marker in HCC diagnosis, especially for tumors in the early stages, and when combined with AFP to significantly increase sensitivity. However, studies with larger sample populations are needed to confirm its relevance.

### **3.6 Tumor-associated glycoprotein 72 (TAG-72)**

Tumor-associated glycoprotein 72 (TAG-72) is a macro-molecular glycoprotein complex, which is rarely expressed in normal tissues, but overexpressed in the majority of human adenocarcinomas, including gastric, colon, and pancreatic cancer. TAG-72 expression is significantly increased in HCC tissues compared to normal liver tissues [46], and it is suspected of promoting tumor invasion and metastasis. A correlation between overexpression of TAG-72 and poor survival in patients with HCC has been observed [46]. This makes TAG-72 a potential prognosis marker for HCC, and anti-TAG-72 monoclonal antibody has been used for tumors clinical detection [47].

### **3.7 Zinc-α2-glycoprotein (ZAG)**

Zinc-α2-glycoprotein (ZAG) is a member of the class I major histocompatibility complex (MHC-I) family. It is considered a new adipokine because of its strong amino acid sequence homology with lipid mobilizing factor (LMF). ZAG is downregulated in human obesity [48], but it is upregulated in different cancers such as breast, lung and prostate cancers, making it a potential biomarker for these. The serum proteome of the HCC, liver cirrhosis and healthy adult groups have been analyzed and it was found that the ZAG is overexpressed in the HCC patients suggesting a potential biomarker for the early detection of HCC [49].

#### **3.8 Annexin A2**

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on the surface of endothelial cells and most epithelial cells [50, 51]. Annexin A2 serum concentrations in patients with HCC were often higher than those with benign liver disease, other malignant tumors, or healthy individuals [52–54]. High annexin A2 levels were observed in 83.2% of early-stage HCC and 78.4% of AFP-negative HCC patients [55]. Annexin A2 sensitivity and specificity were respectively measured at 83.2% and 67.5% in the detection of early-stage HCC, while HCC patients with normal AFP levels were 54.7% and 81.3%, respectively. The diagnostic performance of annexin A2 alone (AUC = 79%) was also greater than for AFP alone (AUC = 73%). As expected, the combination of annexin A2 and AFP further improved the overall diagnostic performance with a sensitivity of 87.4% and a specificity of 68.3%. This makes annexin A2 a potential independent biomarker for detecting early-stage HCC in patients with normal serum AFP.

#### **4. Enzymes and isozymes**

#### **4.1 Des-**γ**-carboxyprothrombin (DCP)**

Des-γ-carboxyprothrombin (DCP) or Prothrombin induced by vitamin K absence II (PIVKA II) is a prothrombin molecule which is synthesized in abnormally high amount in HCC. During malignant transformation in liver cells, vitamin K-dependent carboxylase system weakens [56]. In essence, this is a carboxylation defect that leads to increased DCP synthesis [57]. Serum DCP levels in patients with liver cancer have differed from normal individuals [58]. In a comparative study of cases of chronic hepatitis and liver cirrhosis, DCP showed a sensitivity of 72.7% and a specificity of 90.0%, equivalent to AFP [59]. The combination of these two markers improves HCC diagnosis with a sensitivity and a specificity of 74.2% and 87.2%, respectively [60]. Although DCP has proven to have great potential as a biomarker for early diagnosis of HCC, it needs to be verified by further studies, especially in combination with AFP. In a large multicentre study, the sensitivity of DCP was 56% for early HCC diagnosis. Combining DCP with AFP increased the sensitivity from 65–87% 3 months before HCC diagnosis, but the specificity decreased from 84–69% [61].

Although the diagnostic value of DCP has been studied in Asian countries, its assessments in Western countries, especially in Europe, are still limited. A case– control study to evaluate the performance of serum AFP and DCP concentrations for early HCC diagnosis was conducted in France [62]. The cut-off threshold for serum DCP was 42 mAU/ml and 5.5 ng/ml for AFP, resulting in DCP being better than AFP for early diagnosis of HCC with a sensitivity of 77% compared to a 61%

#### *Circulating Biomarkers for Early Diagnosis of Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.98483*

one, and a specificity of 82% compared to a 50% one. The positive forecast value was 76% compared to 51%, and the negative forecast value was 83% compared to 62%. The combination of DCP and AFP improved diagnostic performance. These results further support the value of DCP as a marker for early HCC diagnosis. According to the 2010 recommendations of the Japan Society of Hepatology (JSH), the three biological markers AFP, AFP-L3 and DCP are checked by the state insurance for HCC screening, as a combination of two of the three biomarkers, or all three combined. These three markers help to increase sensitivity without reducing specificity in small liver cancer [63].
