**6. Circulating RNAs**

#### **6.1 AFP mRNA**

AFP mRNA is a highly valuable marker only found in active cancer cells, which might be a sign of tumor metastasis. The non-recurrence time of HCC patients with high AFP mRNA expression after surgery was shorter than the group without this marker expression in liver cells (53% compared to 88% after 1 year; 37% compared to 60% after 2 years) [89]. In the advanced HCC stage, the AFP mRNA expression rate reaches 100%, and also acts as a predictor of recurrence after liver resection. However, the use of this marker in HCC diagnosis remains controversial, possibly due to the fact that it also manifests in many other malignancies and non-cancerous liver diseases [90]. Therefore, it could be used for diagnosis and prognosis when combined with other markers.

#### **6.2 GGT mRNA**

Gamma-glutamyl transferase mRNA (GGT mRNA) can be found in the blood and peripheral liver cells of healthy individuals, as well as in patients with benign liver disease, benign liver tumors or HCC. It has 3 types: A, B and C. Type A dominates in normal liver cases, non-cancerous liver diseases, benign tumors and secondary liver cancers, while type C is produced by the yolk during pregnancy. In contrast, type B predominates in HCC [91–93]. During malignant development, expression of GGT mRNA in liver tissues may change from type A to type B [93]. Patients with HCC and high type B expression will have a worse prognosis, with higher odds of a sooner and more serious relapse [94]. Therefore, hepatocellular expression of type B mRNA may be a valuable marker for HCC patients. As in liver tissues, peripheral blood type B expression has also been reported to be significantly higher in HCC patients than in healthy adults [91].

#### **6.3 MicroRNA (miRNA)**

MicroRNAs are small non-coding RNAs that inhibit or accelerate the translation process by attenuating or increasing the synthesis of target mRNAs or by binding to additional chains in the UTR region (3′-untranslated region). In recent years, the link between miRNA and tumor development has become a controversial issue. About 500 miRNA genes have been identified and contribute to control a number of cellular processes including proliferation, differentiation and apoptosis. In malignancy, the function of miRNA is determined to be carcinogenic and tumor suppressant [95]. miRNA can regulate many genes at the same time, they control the replication process and determine the characteristics of the cell. The variety in this functional role allows miRNA to be utilized as a diagnostic marker for early detection of cancer, risk assessment, prognosis and as a new therapeutic target.

Yamamoto et al. have used a global miRNA expression profile in mouse liver development and thus shown that miR-500 (miRNA) is a potential biomarker for HCC [95]. Their work showed that miR-500 is significantly associated with the regulation of liver development and thus is related to cirrhosis progression. The serum miR-21 levels were a valuable marker in distinguishing patients with HCC from those with chronic hepatitis with the sensitivity and specificity of 61.1% and 83.3%, respectively. Compared to the healthy group, the sensitivity and specificity

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

were 87.3% and 92.0%, respectively. Both values are higher than serum AFP concentrations, which have been confirmed as a very valuable biological marker for HCC [96]. Serum miR-15b and miR-130b concentrations are relevant miRNA markers that are highly expressed in HCC. miR-130b has 87.7% sensitivity and 81.4% specificity. In contrast, while the sensitivity of miR-15b is high at 98.3%, its specificity is low at 15.3%. Because the sensitivity of these two factors is rather high, it can be used as a valuable marker in HCC screening and early diagnosis with low AFP levels [97].

A group of markers including seven miRNAs (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) has been shown to have a great diagnostic performance for HBV related HCC at an early stage [98]. Although its mechanism and signal path are still unknown, the expression of miRNA-29 may increase the susceptibility of cancer cells to apoptosis and reduce the expression of Mcl-1 and Bcl-2. Indeed, it has the ability to inhibit the formation and growth of cancer cells and is a potential marker in HCC prognosis and treatment [99]. MiR-122 is a specific miRNA found only in HCC, which concentration is inversely correlated with cancer growth and likelihood of invasion and metastasis. An analysis of miRNA markers revealed only tumor miR-21 expression and significantly higher serum miR-21 levels in HCC patients compared to those in chronic liver diseases and healthy control groups. Analysis of ROC curve between HCC and control group showed that sensitivity and specificity were 87.3% and 92% respectively, which is higher than that of serum AFP. Therefore, miR-21 is also a promising marker to support early HCC diagnosis [96].

Some of their features and expressions make miRNA particularly attractive as potential biomarkers. First, many miRNAs exhibit high stability and are easily detectable in peripheral blood of HCC patients. Secondly, miRNAs can be identified in urine, which will be a valuable non-invasive biological marker in detecting and managing HCC. The detection of the expression of some miRNAs in the urine (miR-625, miR-532, miR-618, miR-516-5P and miR-650) has been used for early detection of HCC [100]. However, more research is needed regarding miRNA before it can be used to detect HCC at an early stage.

#### **6.4 Long non-coding RNA (lncRNA)**

Like other cancers, HCC is characterized by a gradual accumulation of epigenetic changes. Among these changes, lncRNA has been found to play a significant role in the initiation and progression of HCC. Most lncRNAs express the characteristics of each species and the specific characteristics of the tumor. Increased or decreased expression of lncRNA has been found in cancerous tissues. Meanwhile, some lncRNA are found in urine, blood, and other body fluids. Moreover, the use of lncRNA as a marker for cancer pathology is superior to the coding RNA protein, due to the characteristic expression of lncRNA [101]. The sensitivity and specificity of lncRNA for HCC diagnosis found in some recent studies are quite high, while it has been demonstrated that JPX (just proximal to XIST) can have a sensitivity of up to 100% [102]. The 2-lncRNA signal has a high specificity of 90.62% but a low sensitivity of 60.65%, which could make it a potential marker to confirm an HCC diagnosis [103]. Recent findings suggest that lncRNA may be a potential marker for early diagnosis and monitoring of the risk of malignant progression in patients with chronic and highly specific chronic liver disease. These markers may contribute to the definitive HCC diagnosis without the need for histopathological diagnosis (**Table 2**).

#### *Hepatocellular Carcinoma - Challenges and Opportunities of a Multidisciplinary Approach*


**Abbreviation:** *DANCR, Differentiation Antagonizing Non-protein Coding RNA; MALAT1, metastasis associated lung adenocarcinoma transcript 1; UCA1, urothelial cancer associated 1.*

#### **Table 2.**

*Diagnostic performance of miRNAs and lncRNAs for HCC.*

#### **7. Gene mutations**

#### **7.1 Mutations in** *TP53* **gene**

P53 is an important protein in the P53 signaling pathway and mutation or loss of *TP53* gene function leads to abnormal cell growth [104]. Notably, the mutation rate of *TP53* varies by geographic area, reflecting the etiology and epidemiological changes of HCC [105]. Mutations in the *TP53* gene, commonly found in sub-Saharan Africa and Southeast Asia, has the highest incidence of HBV infection and Aflatoxin B1 exposure. In these areas, the most common mutation is TP53 R249S, which is associated with an exposure factor of Aflatoxin B1 [106].

*TP53* mutation was identified as one of the common molecular alterations in HCC, of which, the *TP53* R249S mutation in exon 7 was found in HCC patients with a high incidence. Studies suggest that the *TP53* R249S mutation may occur relatively early in areas associated with Aflatoxin exposure and chronic HBV infection [107]. The *TP53* R249S mutation was an important factor in the carcinogenesis of HCC in Brazil, where Aflatoxin exposure is high [108]. In contrast, the *TP53* R249S mutation may not play a role in causing HCC in Egypt, where HCV infection is common [109]. These findings suggest that *TP53* mutations are involved in HCC pathogenesis in individuals with chronic HBV infection, especially in those exposed to high Aflatoxin B1.

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

Recent reports have shown that *TP53* mutation can be used as a marker to predict HCC in high-risk groups. TP53 mutation has been shown to be associated with significantly higher relapse rates and lower disease-free survival rates [110]. It is also documented that *TP53* mutation rate is about 30% and is associated with additional survival, non-recurrent survival and disease-free survival in HCC patients, with similar results observed in patients infected with HBV and HCV [111, 112]. However, a recent study showed that the *TP53* mutation was only associated with a shorter survival time only in HBV-related HCC, while the R249S mutation was not related to the survival rate in the European patients with HCV-related HCC [113]. Growing evidence suggests that the stability of the *TP53* mutation in tumors is important for its carcinogenic activities, decreasing the expression of the *TP53* mutation that reduces malignant growth of cancer cells. Therefore, the *TP53* mutation, especially at R249S position, can be considered as one of the early markers for HCC diagnosis and is an attractive therapy for cancer treatment.

#### **7.2 hTERT gene mutation**

The telomerase reverse transcriptase (hTERT) gene encodes an enzyme that maintains the telomeric DNA length and stabilizes the chromosomes [113]. hTERT is a major determinant of telomerase activity, which plays a key role in protecting cells from apoptosis and transforming into cancerous cells [114]. The reactivation of telomerase activity in cancer may be related to changes that occur during cancer development, including mutations and rearrangements of chromosomes [115].

The frequencies of *hTERT* mutations were observed in about 60% of HCC patients [116] but vary by geographical regions being the most common in Europe (59%) and less common in East Asia (20.7%) [117]. These data indicate that *hTERT* mutation is frequently associated with HCV-related HCC. *hTERT*-promoting mutations have been found with 6% of low-grade dysplasia nodules, 19% of advanced dysplasia nodules, 61% of early HCC and 42% of intermediate and advanced HCC [118]. Another study also found *hTERT* mutation in 57% of patients with chronic hepatitis and in 30% of those with early HCC [119]. Therefore, mutations in the *hTERT* promoter occur early in the course of malignant transformation and persists during tumor development. The regulation and expression of *hTERT* play an important role in the initiation and progression of HCC. *hTERT* mutation is one of the earliest gene mutations in cancer development and is also the most common gene mutation in HCC. Therefore, *hTERT* mutation is one of the most important markers in early diagnosis and may be a promising target for HCC treatment.

#### **7.3 Mutations in ARID1A and ARID2 genes**

*ARID1A* and *ARID2* are two genes in the SWI/SNF complex (SWitch/sucrose non-fermentable) involved in chromosomal reconstruction. The mutation rate of the *ARID1A* and *ARID2* genes found in 10% HCC, depending on the cause. *ARID1A* mutation is associated with alcohol consumption while *ARID2* mutation is often associated with HCV infection [120]. Although the role of these mutations remains unknown, studies have shown that *ARID1A* and *ARID2* genes are associated with the growth of cancer cells through affecting several signaling pathways such as PI3K/AKT, betacatenin and p53 mutation [121] and are thus potential markers for early HCC detection.

#### **8. DNA methylation**

In HCC, methylation can occur in two ways: total methylation and partial methylation. Total methylation affects the structural function of the nucleus by promoting chromosome and genome instability, while partial methylation is associated with tumor suppressor genes [122]. Chronic hepatitis virus infections are the cause of DNA methylation aberrations in cancerous tissues. Although several DNA methyltransferase enzymes such as DNMT1, DNMT3A and DNMT3B have been shown to increase their expression in HCC related to hepatitis viruses, their mechanisms remain controversial and unclear [123].

*p16* (CDKN2A), a tumor suppressor gene involved in cell cycle regulation, has been shown to be methylated and is related to clinical parameters in HCC [124]. A study has shown that the methylation levels of *p16* gene increased in tissue samples from cirrhosis to HCC [125]. The methylation level of *p16* gene is also associated with HBV infection, as the level of *p16* methylation is higher in patients with HBV than those without HBV, the *HBx* gene being especially involved in the methylation of the *p16* gene [126, 127]. A study on 64 HCC patients found that 77% of patients had *p16* methylation and that methylation levels were correlated to serum AFP levels [128]. In a meta-analysis on 272 HCC tissue samples, the methylation rate of p16 gene was 58.5%, much higher than those with cirrhosis and chronic hepatitis [129]. Therefore, methylation in the *p16* gene may serve as a promising molecular marker for HCC in patients with HBV infection.

Another potential marker for HCC prognosis is *SOCS1* methylation. *SOCS1* gene plays a role in modulating the JAK/STAT signaling pathway when methylation causes malignant cell proliferation. SOCS1 methylation correlates with tumor size and risk factors for HCC, it is more common in HCV and cirrhotic patients, but less common in HBV-infected groups. A study has shown that the methylation of *SOCS1* gene in peripheral blood accounted for 38% in the HCC group, 20% in the cirrhotic group and 23% in the control group without liver disease. Expression of methylation of *SOCS1* and *RASSF1A* genes in combination with serum AFP increased sensitivity to 86% and specificity to 75% for HCC diagnosis [130]. *SOCS1* methylation is quite common in HCC, and is correlated with a number of clinical parameters and other serum biomarkers like AFP. Therefore, *SOCS1* methylation in combination with serum AFP increases the sensitivity and specificity for early HCC diagnosis.

GSTP1 belongs to the Glutathione S-transferase family, which protects cells against carcinogens, regulates signaling pathways that control cell proliferation and cell death [131]. The methylation in the *GSTP1* gene promoter was observed in prostate cancer, HCC and other malignancies. GSTP1 has been shown to have a high methylation rate in HCC related to HBV or HCV infection. Interestingly, methylation of the *GSTP1* gene in HCC patients was 76.7% and those with high *GSTP1* expression had a shorter survival time [132].

Detecting the methylation status of genes in serum provides a promising method for diagnosis of HCC. A study found aberrant methylation in the *CCND2* gene in 39 out of 70 serum samples of HCC patients and methylation status was associated with a shorter disease-free survival time [133]. Yeo et al. showed that 17 out of 40 (42.5%) plasma samples of HCC patients had methylation in *RASSF1A* gene, and that methylation occurred mainly in patients with tumors ≥4 cm in size [134]. Methylation in *RASSF1A* in the serum of 85 HCC patients and found that 93% had methylation, it is associated with a shorter survival and disease stage [135]. The level of methylation in the *RASSF1A* gene of the HCC group is significantly higher compared to other liver disease groups and thus it is a promising independent marker for early diagnosis and prognosis of HCC [136].

#### **9. Conclusion**

A large number of markers have been studied and clinically applied for early diagnosis and monitoring of HCC treatment, of which serum AFP is a widely used *Circulating Biomarkers for Early Diagnosis of Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.98483*

with a controversial diagnostic threshold. A number of protein markers such as AFP-L3 and DCP are also being applied to support HCC diagnosis with higher sensitivity and specificity compared to AFP. However, the available marker is neither specific for solely HCC diagnosis nor provides great diagnostic performance for HCC and thus a combination of several serum protein markers can improve the early diagnosis rate. With the development of molecular technology, biomarkers based on miRNA and lncRNA expression, gene mutation (*TP53, hTERT, ARID1A and ARID2*) and DNA methylation have a great potential to improve the rate of HCC diagnosis at an early stage, as well as predicting progression, metastasis and tumor recurrence. In addition, with the development of current cell technology, cancer pathways and the expression of genes specific for HCC tumor may be important markers for early detection and new targets for the treatment of HCC.
