ALDH1 has been reported to regulate CSCs in breast cancer by promoting the metabolism of retinoid [94]. RA binds to RARs and RXRs and activates the expression of genes associated with differentiation, cell cycle arrest, and morphological variation [95]. Increasing RAR and RXR levels creates a positive feedback loop for retinoid signaling. RA formation by the oxidation of all-*trans*-retinal and 9-*cis*-retinal in retinoid signaling is closely associated with the function of SCs and CSCs [96].

#### **6.2. ALDH1 in acetaldehyde metabolism**

Alcohol dehydrogenase catalase and cytochrome P4502E1 metabolize ethanol to acetaldehyde. Acetaldehyde produces ROS, which suppress DNA repair and methylation and form DNA and protein adducts, thereby promoting carcinogenesis and tumor growth [97, 98]. ALDH1A1 and ALDH2 primarily metabolize acetaldehyde to acetate. ALDH activity maintains a low ROS level and inhibits CSC apoptosis [99]. Reactive aldehydes' metabolism and the ROS level are closely related to the characteristics of CSCs and cancer development. However, the relationship between ALDH and ROS in the functions of SCs and CSCs is still unclear.

#### **6.3. ALDH1 in HCC**

ALDH1 expression evaluated by immunohistochemistry is heterogenous and is present in the normal liver tissue, especially in hepatocytes [100]. However, ALDH bright cells, including ALDH1 isoforms, evaluated using the Aldefluor assay, have been reported to be a marker of liver progenitor cells in the normal liver tissue [101] and of CSCs in HCC [102]. Interestingly, ALDH bright cells are attributed to ALDH1 activity. Thus, ALDH1 expression in immunohistochemistry is considered to be slightly different from ALDH bright cells in HCC [93].

ALDH1 expression is associated with a favorable outcome for HCC patients [100, 103]. Furthermore, putative CSC markers such as CD24, CD13, CD90, EpCAM, BMI1, and CD133 were not colocalized with ALDH1-expressing cells in HCC [100]. Consequently, immunohistochemistry with an ALDH1 antibody shows differentiated cells that look like mature hepatocytes but not CSCs.

Taken together, these findings suggest that increased ALDH1 expression is associated with a factor indicative of a well-differentiated morphology and favorable prognosis in HCC. Furthermore, ALDH1-expressing cells may serve as a useful differentiation biological marker for HCC rather than as a CSC marker.

#### **6.4. ALDH1 in CCA**

**6.1. ALDH1 in retinoid signaling**

38 Updates in Liver Cancer

function of SCs and CSCs [96].

**6.3. ALDH1 in HCC**

tocytes but not CSCs.

for HCC rather than as a CSC marker.

**6.2. ALDH1 in acetaldehyde metabolism**

Retinoid signaling has important roles in SCs and CSCs [92]. In retinoid signaling, retinol dehydrogenases oxidize the retinol absorbed by cells to retinal [93]. Retinal is then oxidized to RA in a reaction catalyzed by ALDH1 family members such as ALDH1A1, ALDH1A2, and ALDH1A3. The metabolized product RA includes all-*trans* RA, 9-*cis* RA, and 13-*cis* RA. RA enters the nucleus and induces the transcription of downstream genes through the activation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Finally, increased ALDH1 contributes to not only RA synthesis but also cellular protection against cytotoxic drugs.

ALDH1 has been reported to regulate CSCs in breast cancer by promoting the metabolism of retinoid [94]. RA binds to RARs and RXRs and activates the expression of genes associated with differentiation, cell cycle arrest, and morphological variation [95]. Increasing RAR and RXR levels creates a positive feedback loop for retinoid signaling. RA formation by the oxidation of all-*trans*-retinal and 9-*cis*-retinal in retinoid signaling is closely associated with the

Alcohol dehydrogenase catalase and cytochrome P4502E1 metabolize ethanol to acetaldehyde. Acetaldehyde produces ROS, which suppress DNA repair and methylation and form DNA and protein adducts, thereby promoting carcinogenesis and tumor growth [97, 98]. ALDH1A1 and ALDH2 primarily metabolize acetaldehyde to acetate. ALDH activity maintains a low ROS level and inhibits CSC apoptosis [99]. Reactive aldehydes' metabolism and the ROS level are closely related to the characteristics of CSCs and cancer development. However, the relationship between ALDH and ROS in the functions of SCs and CSCs is still unclear.

ALDH1 expression evaluated by immunohistochemistry is heterogenous and is present in the normal liver tissue, especially in hepatocytes [100]. However, ALDH bright cells, including ALDH1 isoforms, evaluated using the Aldefluor assay, have been reported to be a marker of liver progenitor cells in the normal liver tissue [101] and of CSCs in HCC [102]. Interestingly, ALDH bright cells are attributed to ALDH1 activity. Thus, ALDH1 expression in immunohistochemistry is considered to be slightly different from ALDH bright cells in HCC [93].

ALDH1 expression is associated with a favorable outcome for HCC patients [100, 103]. Furthermore, putative CSC markers such as CD24, CD13, CD90, EpCAM, BMI1, and CD133 were not colocalized with ALDH1-expressing cells in HCC [100]. Consequently, immunohistochemistry with an ALDH1 antibody shows differentiated cells that look like mature hepa-

Taken together, these findings suggest that increased ALDH1 expression is associated with a factor indicative of a well-differentiated morphology and favorable prognosis in HCC. Furthermore, ALDH1-expressing cells may serve as a useful differentiation biological marker Shuang et al. [104] demonstrated that ALDH1 is a valuable marker of CSCs in CCA. Further, patients with high ALDH1 expression had a poor prognosis in the cases of both intrahepatic and extrahepatic CCA. ALDH1 and CD133 are two other molecular markers of putative CSCs in extrahepatic CCA [105]. ALDH1 has been reported to play a crucial role in the identification of CSCs and/or tumor-initiating cells in various types of cancers [106]. In breast cancer, ALDH1+ seems to be a more significant predictive marker than other markers for the identification of breast CSCs. However, the identification of putative CSCs using a single marker such as ALDH1 is controversial. Nevertheless, ALDH1 has been shown to be a very important molecular marker for CSCs. To clarify the correlation among ALDH1 and other putative CSC markers, i.e., CD133, CD24, CD44, and EpCAM, and to identify cells with multiple CSC phenotypes might improve the selection of CSCs, and further studies are needed in this regard.

Recently, HCC and CCA have been shown to share the same origin. Hepatic progenitor cells can differentiate into hepatocytes and cholangiocytes and give rise to HCC as well as CCA [107]. ALDH1 expression has been reported to be specific to the liver CSCs' population [102] and can be assessed to reliably identify CCA cells with stem-like properties. With respect to other ALDH isoforms, only one study has described that ALDH1A3 was a poor prognostic factor and a good biomarker of gemcitabine resistance in intrahepatic CCA [108].

### **7. Conclusions**

CSCs represent key cell populations among the heterogenous malignant cells of liver cancers, and their biological characteristics highlight them as a major target for cancer research. In particular, they provide reliable biomarkers for prognosis, such as ALDH1. Discovery of the mechanisms and molecules associated with CSCs offers great potential to accelerate the development of novel therapeutic options and improve the treatment outcome and quality of life of patients with liver cancers.

## **Conflicting interest**

The authors declare no conflicts of interest.

## **Acknowledgements**

We thank all the members of our laboratory and our collaborators for their research work and helpful discussions. This work was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (#15K11289 and #26430111).

## **Author details**

Hiroyuki Tomita1,\*, Tomohiro Kanayama1 , Ayumi Niwa1 , Kei Noguchi1 , Kazuhisa Ishida1 , Masayuki Niwa2 and Akira Hara1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Tumor Pathology, Gifu University Graduate School of Medicine, Gifu, Japan

2 Medical Science Division, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu, Japan

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Masayuki Niwa2

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**Chapter 4 Provisional chapter**

#### **Lipid Metabolism in Liver Cancer Lipid Metabolism in Liver Cancer**

Guo-Dong Lu and Shing Chuan Hooi Guo-Dong Lu and Shing Chuan Hooi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64993

#### **Abstract**

Hepatocellular carcinoma (HCC) represents 90% cases of liver cancer that is the second leading cause of cancer death in the world. With the pandemic of obesity and other metabolic syndromes in both adults and children, the incidences of fatty liver diseases and the derived HCC are on their upward track. Emerging metabolomic studies have revealed the perturbation of lipid profiles and other metabolites in fatty liver diseases and HCC. Two common metabolic features including enforced fatty acid oxidation and glycolysis could distinguish HCC from healthy liver and chronic non-tumor liver diseases. The potential translational impacts of fatty acid oxidation are gaining great interests, because many recent investigations have demonstrated that tumor cells were dependent on fatty acid oxidation for cell survival and tumor growth. Blockage of fatty acid oxidation could sensitize to metabolic stress-induced cell death and tumor growth inhibition. Thus, lipid catabolism, in terms of fatty oxidation, is tuned for tumor maintenance but vulnerable to pharmacological disruption. The therapeutic potentials of blocking fatty acid oxidation are yet to be further carefully examined.

**Keywords:** liver cancer, lipid metabolism, metabolomics, fatty liver diseases, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cirrhosis

### **1. Introduction**

Livercanceristhesecondleadingcauseofcancerdeathworldwide[1].Hepatocellularcarcinoma (HCC) represents 90% of primary liver cancer. The incidence of HCC has been successfully improved in China and Southeast Asia, owing to several decades' endeavor in controlling viral hepatitis B and environmental toxicants, for example, aflatoxin in contaminated food and microcystin in pond water [1–3]. By contrast, HCC incidence has been increased in the United States and other Western countries in the last three decades. The fast-growing fraction of the

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cases was reported to result from chronic fatty liver diseases: nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) [4].

Epidemiologic studies lend the credence to the importance of NAFLD as the most common cause of liver disease in the world [4]. The prevalence of NAFLD can reach as high as 25–45% [4–6], when image scanning is applied into diagnostic decision. This heavy disease burden of NAFLD may mirror the epidemic of obesity, diabetes, and other metabolic syndromes, which are still on their upward trend in the world [7]. These metabolic disorders are well characterized with the disruption of glucose and lipid homeostasis, accumulated deposition of systemic and hepatic fat, and insulin resistance. As a consequence, these metabolic alterations may predispose liver to chronic inflammation and fibrogenesis, and finally cancerous transformation into HCC, no matter whether liver cirrhosis ensues. Furthermore, the coexistence of NAFLD with viral hepatitis and environmental toxicants may drive the disease progression in a more complicated manner.

The emerging metabolomic technologies enable hepatologists and biologists to have a panorama view of a highly complex and dynamic flux of small metabolites in liver diseases [8]. This methodology includes high-throughput analytical mass spectrometry, nuclear magnetic resonance spectroscopy, and multivariate data analysis, permitting unbiased comparison of "global" profiles of hundreds to thousands of metabolites between samples from two or more liver disease status. As same as the other omics technologies, the metabolomic investigations have provided new insights into liver disease mechanisms and identified novel biomarkers involved in liver diseases and oncogenesis. Recent findings, comparing different phases of liver diseases from healthy liver to NAFLD/NASH, cirrhosis, and eventually HCC, revealed that both Warburg shift (from mitochondrial oxidative phosphorylation to enforced cytosolic glycolysis) and the up-regulation of lipid catabolism occurred as early as in NAFLD/NASH, and throughout the whole oncogenic processes [9].

Different from the well-studied lipogenesis and fat deposition in the liver, the emerging roles of lipid catabolism in cancer maintenance and progression have been unveiled recently [10]. Mechanically, lipid catabolism in terms of fatty acid oxidation and its upstream autophagy pathway may promote cancer cell survival and tumor growth, especially during stringent nutrient deprivation. The aforementioned nutrient deprivation and the metabolic stress ensued usually occur during rapid solid tumor development and during clinical embolization intervention. These recent findings brought to light the translational significance of lipid catabolism in cancer therapeutics.

### **2. Lipid metabolism in liver physiology: a brief introduction**

Liver is the central organ for lipid metabolism and fat deposition in the body [11]. Through well-tuned coordination with adipose, muscle, and other tissues, liver plays an essential role in the maintenance of lipid homeostasis and energy balance. Whenever an excess of calories is ingested, fatty acids are synthesized primarily in the liver, and to a lesser extent in the adipose tissue [12]. Dietary carbohydrate, which is digested into two-carbon units acetyl-CoA, is the major source of carbon for the synthesis of fatty acids. The process is termed as lipogenesis. An excess of dietary protein also can promote lipogenesis through conversion to acetyl-CoA and other intermediates of tricarboxylic acid cycle (also known as citric acid cycle). After elongation and desaturation, three fatty acids join together by one glycerol molecule to form triacylglycerols. Subsequently, triacylglycerols are packaged into very long low-density lipoprotein (VLDL) particles with cholesterol, phospholipids, and proteins. VLDL particles are then released into blood and then transported to major organs including adipose tissues for storage in the form of triacylglycerols, and muscle for energy metabolism. The aforementioned process is briefly summarized in **Figure 1**.

cases was reported to result from chronic fatty liver diseases: nonalcoholic fatty liver disease

Epidemiologic studies lend the credence to the importance of NAFLD as the most common cause of liver disease in the world [4]. The prevalence of NAFLD can reach as high as 25–45% [4–6], when image scanning is applied into diagnostic decision. This heavy disease burden of NAFLD may mirror the epidemic of obesity, diabetes, and other metabolic syndromes, which are still on their upward trend in the world [7]. These metabolic disorders are well characterized with the disruption of glucose and lipid homeostasis, accumulated deposition of systemic and hepatic fat, and insulin resistance. As a consequence, these metabolic alterations may predispose liver to chronic inflammation and fibrogenesis, and finally cancerous transformation into HCC, no matter whether liver cirrhosis ensues. Furthermore, the coexistence of NAFLD with viral hepatitis and environmental toxicants may drive the disease progression

The emerging metabolomic technologies enable hepatologists and biologists to have a panorama view of a highly complex and dynamic flux of small metabolites in liver diseases [8]. This methodology includes high-throughput analytical mass spectrometry, nuclear magnetic resonance spectroscopy, and multivariate data analysis, permitting unbiased comparison of "global" profiles of hundreds to thousands of metabolites between samples from two or more liver disease status. As same as the other omics technologies, the metabolomic investigations have provided new insights into liver disease mechanisms and identified novel biomarkers involved in liver diseases and oncogenesis. Recent findings, comparing different phases of liver diseases from healthy liver to NAFLD/NASH, cirrhosis, and eventually HCC, revealed that both Warburg shift (from mitochondrial oxidative phosphorylation to enforced cytosolic glycolysis) and the up-regulation of lipid catabolism occurred as early as in NAFLD/NASH,

Different from the well-studied lipogenesis and fat deposition in the liver, the emerging roles of lipid catabolism in cancer maintenance and progression have been unveiled recently [10]. Mechanically, lipid catabolism in terms of fatty acid oxidation and its upstream autophagy pathway may promote cancer cell survival and tumor growth, especially during stringent nutrient deprivation. The aforementioned nutrient deprivation and the metabolic stress ensued usually occur during rapid solid tumor development and during clinical embolization intervention. These recent findings brought to light the translational significance of lipid

Liver is the central organ for lipid metabolism and fat deposition in the body [11]. Through well-tuned coordination with adipose, muscle, and other tissues, liver plays an essential role in the maintenance of lipid homeostasis and energy balance. Whenever an excess of calories is ingested, fatty acids are synthesized primarily in the liver, and to a lesser extent in the adipose tissue [12]. Dietary carbohydrate, which is digested into two-carbon units acetyl-CoA, is the

**2. Lipid metabolism in liver physiology: a brief introduction**

(NAFLD) and nonalcoholic steatohepatitis (NASH) [4].

and throughout the whole oncogenic processes [9].

catabolism in cancer therapeutics.

in a more complicated manner.

50 Updates in Liver Cancer

**Figure 1.** Schematic diagram of lipogenesis in the liver. Fatty acids are synthesized from glucose and excess protein in the liver after meals. After conversion into triacylglycerol, fatty acids are packaged in VLDL particles and then transported to adipose for storage and muscle for energy metabolism. TCA cycle: tricarboxylic acid cycle; TG: triacylglycerol; VLDL: very long low-density lipoprotein.

When the serum level of glucose runs low before next meal or during short-term fasting, the body can mobilize energy deposits sequentially from glycogen to triacylglycerols. This process is tightly regulated by hormones, in a coordination of decrease in insulin and increase in glucagon. The hormone-sensitive lipase breaks down triacylglycerols into glycerol and fatty acids, and the latter are released into the blood. The fatty acids are then transported to muscle and other tissues to meet immediate energy demands, where fatty acids are oxidized to CO2 and water to produce energy. During extended fasting, the body mainly mobilizes lipids through fatty acid beta-oxidation, and even digests unnecessary protein and organelles through a catabolic process called autophagy.

Therefore, lipid metabolism is important for normal liver physiology. Triacylglycerols are the predominant form to store fat energy, mainly in adipose tissue and also in liver. However, unhealthy lifestyles of nutrition overload and physical inactivity may tilt the balance of lipid homeostasis and disrupt the body sensitivity to insulin [7]. In the long run, fat accumulates systemically in the adipose tissues and locally in the liver, contributing to inflammation and fibrogenesis, and consequently obesity and fatty liver diseases [12].

## **3. A heavy burden of fatty liver diseases for liver carcinogenesis**

NAFLD is a well-recognized common liver disease in the world [4, 13, 14]. Most individuals with NAFLD are restricted to benign and dormant liver steatosis. Up to 25–30% NAFLD patients progress to NASH [14], an aggressive form of NAFLD with combined complications of steatosis, inflammation, and fibrosis. NAFLD is a growing etiological cause of HCC, especially in those regions with low incidence of viral hepatitis, for example, USA, UK, and other Western countries [13, 14]. The incidence of HCC has tripled from 1.5 to 4.9 per 100,000 individuals in the past three decades in the United States alone [15]. It has been suggested that NAFLD will become the leading cause of HCC in the coming decade [16, 17], due to not only the epidemics of obesity and type 2 diabetes but the successful control of risk factors including hepatitis B virus (HBV) by at-birth vaccination, hepatitis C virus (HCV) by novel antiviral treatments, aflatoxin contamination by food hygiene, and microcystin in pond water by the change of drinking water source.

NAFLD coincides with or occurs on the basis of preexisting metabolic conditions, accounting for up to 90% of obese patients [18, 19] and up to 70% of type 2 diabetic patients [20, 21], which were confirmed by large cohort studies based on examinations using ultrasound examination and liver biopsy. The high prevalence of NAFLD (25–45% for all ages and 10–20% for children) is not surprising [14, 22], if the pandemic background of overweight and obesity in the world is considered. The proportion of adults with overweight or obesity, whose BMI (body mass index) was more than 25 kg/m2 , increased between 1980 and 2013 from 28.8 to 36.9% in male and from 29.8 to 38.0% in female globally [7]. Note that there were 23.8% boys (younger than 20) and 22.6% girls suffering from overweight or obesity in 2013. In China, the biggest developing country in the world, the prevalence of adult overweight (30.1%, BMI between 25 and 30) and obesity (11.9%, BMI of >30) in 2012 was catching up with those of Western countries. Thus, obesity has become and will remain a major public health challenge in both developed and developing countries. Undoubtedly, the prevalence of NAFLD is also on its upward track in the foreseeable future.

Meta-analysis of large prospective population-based cohorts demonstrated that overweight or obese persons had relatively 17 or 89% higher risks, respectively, to develop HCC, compared to their normal-weight peers [23]. The risk in male was much higher than that in female. According to a large Swedish cohort study, obese males had 3.1-fold higher risk than the normal-weight control [24]. Another US study reported a 4.5-fold increase in HCC risks in overweight and obese males [25]. Similar trends have been found in diabetic patients. El-Serag and colleagues reported that the risk of males with type 2 diabetes to develop HCC was 2.5 times of those without, according to a systematic review and meta-analysis of case-control and cohort studies [26]. Although yet unproven in large cross-section populations, some casecontrol studies have shown that the active treatment of obesity (by Statins) and/or diabetes (by Metformin) may be beneficial for HCC reduction with odds ratio of 0.74 (95% confidence interval (CI): 0.64–0.87) and 0.38 (95% CI: 0.24–0.59), respectively [27, 28].

NAFLD may coexist with other chronic liver disease and synergistically promote liver oncogenesis [13, 14]. A large Taiwan cohort study observed that obesity and hepatitis C combined had a higher HCC risk than obesity alone or obesity and hepatitis B combined (odds ratio 4.13 vs. 2.36 and vs. 1.36, respectively) [29]. Furthermore, according to another study based on 23,712 Taiwan residents, obesity and alcohol use had a synergistic effect in HCC incidence with an unadjusted odds ratio of 7.19 (95% CI: 3.69–14.00), compared to obesity alone (odds ratio 1.47, 95% CI: 0.95–2.30) and alcohol use alone (odds ratio 2.56, 95% CI: 1.96–3.35) [30]. After multivariate adjustment, the combined effect was still significant with an odds ratio of 3.82 (95% CI: 1.94–7.52). NAFLD may also contribute to cryptogenic cirrhosis, which represents 30–40% of HCC cases in developed countries [31]. The relationship between NAFLD and cryptogenic HCC could only be verified through medical history taking. Cryptogenic HCC may account for more advanced stage HCC through a complicated process from initial NAFLD/NASH-based hepatosteatosis to subsequent extensive lipid catabolism [32–34], so that the original steatosis was not observable at diagnosis. Although cirrhosis usually precedes HCC, increasing studies showed that NAFLD might induce HCC independent of cirrhosis [34– 36]. It has been reported that NAFLD may account for 59% of non-cirrhotic HCC, compared to diabetes (36%) and chronic hepatitis C (22%), according to a recent study based on US healthcare claims database [17].

**3. A heavy burden of fatty liver diseases for liver carcinogenesis**

change of drinking water source.

52 Updates in Liver Cancer

index) was more than 25 kg/m2

upward track in the foreseeable future.

NAFLD is a well-recognized common liver disease in the world [4, 13, 14]. Most individuals with NAFLD are restricted to benign and dormant liver steatosis. Up to 25–30% NAFLD patients progress to NASH [14], an aggressive form of NAFLD with combined complications of steatosis, inflammation, and fibrosis. NAFLD is a growing etiological cause of HCC, especially in those regions with low incidence of viral hepatitis, for example, USA, UK, and other Western countries [13, 14]. The incidence of HCC has tripled from 1.5 to 4.9 per 100,000 individuals in the past three decades in the United States alone [15]. It has been suggested that NAFLD will become the leading cause of HCC in the coming decade [16, 17], due to not only the epidemics of obesity and type 2 diabetes but the successful control of risk factors including hepatitis B virus (HBV) by at-birth vaccination, hepatitis C virus (HCV) by novel antiviral treatments, aflatoxin contamination by food hygiene, and microcystin in pond water by the

NAFLD coincides with or occurs on the basis of preexisting metabolic conditions, accounting for up to 90% of obese patients [18, 19] and up to 70% of type 2 diabetic patients [20, 21], which were confirmed by large cohort studies based on examinations using ultrasound examination and liver biopsy. The high prevalence of NAFLD (25–45% for all ages and 10–20% for children) is not surprising [14, 22], if the pandemic background of overweight and obesity in the world is considered. The proportion of adults with overweight or obesity, whose BMI (body mass

and from 29.8 to 38.0% in female globally [7]. Note that there were 23.8% boys (younger than 20) and 22.6% girls suffering from overweight or obesity in 2013. In China, the biggest developing country in the world, the prevalence of adult overweight (30.1%, BMI between 25 and 30) and obesity (11.9%, BMI of >30) in 2012 was catching up with those of Western countries. Thus, obesity has become and will remain a major public health challenge in both developed and developing countries. Undoubtedly, the prevalence of NAFLD is also on its

Meta-analysis of large prospective population-based cohorts demonstrated that overweight or obese persons had relatively 17 or 89% higher risks, respectively, to develop HCC, compared to their normal-weight peers [23]. The risk in male was much higher than that in female. According to a large Swedish cohort study, obese males had 3.1-fold higher risk than the normal-weight control [24]. Another US study reported a 4.5-fold increase in HCC risks in overweight and obese males [25]. Similar trends have been found in diabetic patients. El-Serag and colleagues reported that the risk of males with type 2 diabetes to develop HCC was 2.5 times of those without, according to a systematic review and meta-analysis of case-control and cohort studies [26]. Although yet unproven in large cross-section populations, some casecontrol studies have shown that the active treatment of obesity (by Statins) and/or diabetes (by Metformin) may be beneficial for HCC reduction with odds ratio of 0.74 (95% confidence

NAFLD may coexist with other chronic liver disease and synergistically promote liver oncogenesis [13, 14]. A large Taiwan cohort study observed that obesity and hepatitis C

interval (CI): 0.64–0.87) and 0.38 (95% CI: 0.24–0.59), respectively [27, 28].

, increased between 1980 and 2013 from 28.8 to 36.9% in male

It is worth noting that the risk of NAFLD to develop HCC may not be as high as those of hepatic virus and aflatoxin. The relative risk of NAFLD/NASH alone in the absence of cirrhosis for HCC mortality was found to be as weak as 0–3% for a follow-up period up to 20 years [37]. The cumulative incidence for NAFLD with cirrhosis increased to a range between 2.4 and 11.3%, which was yet comparatively lower than that of hepatitis C with cirrhosis (17–30% for a 5-year cumulative incidence) [34, 35, 38]. By contrast, the relative risks for HBV and HCV to develop HCC are 15–20 folds, according to large case-control and cross-sectional studies [3, 39, 40]. A nested case-control study of 18,000 male residents in Shanghai, China, found that exposure to HBV alone caused an increased HCC risk by 7.3, exposure to aflatoxin alone 3.4, and exposure to both remarkably 59.4 [41]. The public health impact of NAFLD on HCC development, however, could not be overlooked, if considering the higher prevalence of NAFLD than that of viral hepatitis (20–40% for NAFLD vs. 6.3% for viral hepatitis in the Western countries, and 15–30% for NAFLD vs. 11–14% for viral hepatitis in China) [3, 4, 13, 40].

The underlying molecular mechanisms involving how NAFLD promote HCC are yet unclear. Several hypotheses have been suggested. First, chronic inflammation, the increased release of adipokine, and insulin resistance may affect cell proliferation and responsiveness [42, 43]. Second, enhanced lipogenesis and fat deposition may induce extensive lipotoxicity, oxidative stress, and subsequent DNA damages [44, 45]. Third, oncogenic insulin-like growth factor (IGF)-1/PI3K/mTOR, tumor necrosis factor (TNF)-alpha/mitogen-activated protein kinase (MAPK), and/or interleukin (IL)-6/STAT3 pathways were actively involved in HCC [46, 47]. Fourth, hepatosteatosis may influence the hepatic stellate cells and alter microenvironment, causing irreversible fibrosis and cirrhosis [48]. Lastly, the alteration of gut microbiota may influence HCC through bacterial metabolites [49].

Taken together, the growing contribution of NAFLD to HCC development is acknowledged globally. Although the risk of NAFLD alone is comparatively low, the public health impact of NAFLD and its synergistic effects with other chronic liver diseases on HCC development may pose a huge threat in the coming decades.

## **4. Lipid metabolism in liver oncogenesis: insights from metabolomic studies**

Emerging metabolomic technologies represent a powerful platform to dissect global metabolite profiles in an unbiased manner and to discover novel biomarkers and pathways in liver oncogenesis [8]. This high-throughput strategy may complement the other omics technologies (genomics, proteomics, and others) to improve diagnosis, prognostication, and tumor therapy in HCC. Recent metabolomic investigations have shed light on the importance of lipid metabolism on the liver oncogenic processes.

Dr. Beyoglu and Idle in the University of Bern, Switzerland [9], recently well summarized the metabolomic findings conducted in chronic liver diseases and HCC, and proposed a threestage biochemical progression from healthy liver to carcinoma through intermediate phases of chronic liver diseases including NAFLD/NASH and cirrhosis, according to the alterations of major metabolites and the involved metabolic pathways. A common alteration of "core metabolic phenotype [9]" was found between liver diseases and healthy liver. Deregulation of bile acid and phospholipid occurred in the early phase of NAFLD/NASH, and maintained in cirrhosis and HCC. In HCC, Warburg effect (enforced cytosolic glycolysis over mitochondrial oxygen respiration) and induction of lipid catabolism were commonly observed phenotypes, and could be detected as early as in a few NAFLD/NASH cases. The NAFLD-derived HCC also demonstrated the up-regulation of metabolites from triglycerides that were originally stored in adipose. These alterations observed in HCC (also termed as metabolic reprogramming), as summarized in **Figure 2**, were regarded as one of common hallmarks of tumor [50].

**Figure 2.** The metabolomic alterations during liver oncogenesis. Liver oncogenesis progresses from healthy liver to HCC through several intermediate chronic liver diseases including NAFLD/NASH, cirrhosis, and viral hepatitis. The percentages of early-stage or intermediate liver diseases progress to the next advanced stage were shown as indicated. The major metabolomic alterations were summarized.

Metabolomic investigations of NAFLD utilized serum and tissue specimens from animal models and human. As a manifestation of steatosis, the lipogenesis (triacylglycerols, diacylglycerol, and phospholipids) [51–53] and bile acid biosynthesis (cholesterol esters, choline, and bile acids) [54, 55] pathways were found to be up-regulated. The hepatic lipids were found to be rearranged and repartitioned from adipose to liver, rather than *de novo* lipogenesis and deposition in the liver, possibly through increased turnover of phosphatidylcholine and phosphatidylethanolamine [9]. This conclusion was supported by a mouse starvation experiment that TG(44:2) and TG(48:3), the most abundant triacylglycerols in adipose, were significantly deposited in the liver after 24-h starvation [52]. Similar increase in glycolysis was also demonstrated by the common elevation of lactate and reduction of glucose in mouse obesity models and human NAFLD specimens [56, 57], which is consistent with the inductive role of insulin resistance on pyruvate kinase M2 [58]. On the other hand, the sustained hyperactivation of hepatic lipid metabolism subsequently aroused extensive oxidative stress and competitively repressed antioxidant biochemical species such as glutathione and cysteineglutathione disulfide [54, 56]. As an advanced stage of NAFLD, NASH has a similar demonstration of increased lipogenesis (triacylglycerols) and bile acid biosynthesis [53, 56], but differs in the decreased lysophosphatidylcholine [59]. The decrease of lysophosphatidylcholine and increase of bile acids were indicative of inflammation in NASH.

NAFLD and its synergistic effects with other chronic liver diseases on HCC development may

Emerging metabolomic technologies represent a powerful platform to dissect global metabolite profiles in an unbiased manner and to discover novel biomarkers and pathways in liver oncogenesis [8]. This high-throughput strategy may complement the other omics technologies (genomics, proteomics, and others) to improve diagnosis, prognostication, and tumor therapy in HCC. Recent metabolomic investigations have shed light on the importance of lipid

Dr. Beyoglu and Idle in the University of Bern, Switzerland [9], recently well summarized the metabolomic findings conducted in chronic liver diseases and HCC, and proposed a threestage biochemical progression from healthy liver to carcinoma through intermediate phases of chronic liver diseases including NAFLD/NASH and cirrhosis, according to the alterations of major metabolites and the involved metabolic pathways. A common alteration of "core metabolic phenotype [9]" was found between liver diseases and healthy liver. Deregulation of bile acid and phospholipid occurred in the early phase of NAFLD/NASH, and maintained in cirrhosis and HCC. In HCC, Warburg effect (enforced cytosolic glycolysis over mitochondrial oxygen respiration) and induction of lipid catabolism were commonly observed phenotypes, and could be detected as early as in a few NAFLD/NASH cases. The NAFLD-derived HCC also demonstrated the up-regulation of metabolites from triglycerides that were originally stored in adipose. These alterations observed in HCC (also termed as metabolic reprogramming), as summarized in **Figure 2**, were regarded as one of common hallmarks of tumor [50].

**Figure 2.** The metabolomic alterations during liver oncogenesis. Liver oncogenesis progresses from healthy liver to HCC through several intermediate chronic liver diseases including NAFLD/NASH, cirrhosis, and viral hepatitis. The percentages of early-stage or intermediate liver diseases progress to the next advanced stage were shown as indicated.

**4. Lipid metabolism in liver oncogenesis: insights from metabolomic**

pose a huge threat in the coming decades.

metabolism on the liver oncogenic processes.

The major metabolomic alterations were summarized.

**studies**

54 Updates in Liver Cancer

Cirrhosis is characterized by extensive liver fibrosis/regeneration and liver dysfunction, attributable directly to liver cell damages and indirectly to portal hypertension. It has been estimated that 10–20% of NASH cases eventually progressed to cirrhosis over a follow-up period of 10 years [4]. Metabolomic studies examined the alterations of metabolite profiles comparing serum or biopsies from healthy liver and from cirrhotic liver in human, but the results varied possibly due to the different etiologies. As same as NASH, the cirrhotic liver had attenuated lysophosphatidylcholine [60]. But cirrhosis differed from NASH in liver dysfunctions of several metabolic pathways. First, amino acids metabolism was impaired, as exemplified by the increase of serum nonessential amino acids and aromatic amino acids but the decrease of essential amino acids particularly branched-chain amino acids valine and isoleucine [61, 62]. Second, ammonium detoxication was found to be reduced, as evidenced by the increase of glutamate but the decrease of glutamine and glucose [63]. Therefore, these metabolic dysfunctions of amino acid metabolism and ammonium detoxification may collectively mirror the pathological damages in cirrhosis.

The metabolic reprogramming in HCC has been investigated by a greatest number of metabolomic studies. Accumulated data suggested that glycolysis and beta-oxidation were commonly elevated in HCC. On one hand, the enforced glycolysis over mitochondrial oxygen respiration (also called Warburg effect), although comparatively lower in reaction rates (fourfold increase) than the other types of tumor, was a common metabolic phenotype in tumor. This phonotype was evidenced by the following metabolic changes including the decrease in glucose, citrate, and glycerol 3-phosphate but the increase in lactic acid and pyruvate [64–69]. On the other hand, the common induction of beta-oxidation was exemplified by the elevation of 2-oxoglutarate and reduction of free fatty acids, carnitine and carnitine esters [61, 64, 66, 68, 70–73]. This theme will be further explored in the next section. As same as the intermediate liver diseases, HCC maintained the aforementioned "core metabolic phenotype" of bile acid and phospholipid perturbation [66, 71, 73–75]. Some findings also demonstrated similar liver impairments of ammonium detoxication and amino acid metabolism in HCC as in cirrhosis [66, 72, 73, 76].

Other chronic liver diseases, for example, alcoholic liver disease and viral hepatitis, share some similar metabolic alterations to NASH, such as increased lipogenesis and reduction in lysophosphatidylcholine [77, 78]. Metabolomic profiling of a hepatitis B virus X (HBx) transgenic mouse model confirmed the involvement of lipid metabolism (triacylglycerol, cholesterol, saturated, and monounsaturated fatty acids) in HBV-induced liver oncogenesis [79]. This similarity indicated that chronic liver diseases may share some common biochemical and mechanical pathways particularly lipid metabolism, in spite of the differences in etiologies.

A recent nested case-control study from a large European prospective cohort compared serums collected before diagnosis in 114 HCC with 222 matched controls and carefully controlled possible confounders such as tobacco usage, alcohol consumption, and so on [80]. Sixteen metabolites involved in amino acid metabolism, choline metabolism, polyunsaturated lipid metabolism, and ammonium detoxication were selected for potential biomarkers. These metabolomic changes are actually reflective of the underlying precancer liver dysfunctions before HCC occurrence (as discussed above). And these data are pivotal for understanding the process of HCC oncogenesis. The results also demonstrated that the pre-diagnostic metabolic profiles between HCC cases and respective control were different, which was dependent on hepatitis infection status, liver function, and the length of time from blood collection to HCC diagnosis.

Many recent investigations applied metabolomics technologies in biomarker discovery to advance HCC diagnosis. One Chinese study using a panel of metabolic markers (formate, phytosphingosine, and 3*α*,6*α*,7*α*,12*α*-tetrahydroxy-5*β*-cholan-24-oleic acid) achieved high accuracy (area under curve (AUC): 0.995–1.000, sensitivity: 100%, and specificity: 94.7–100%) in diagnosing HCC patients with low alpha-fetoprotein (a commonly accepted but mediocre biomarker to differentiate HCC from non-tumor liver at a cut-off value of 20 ng/ml) [73]. Another Chinese study, which used tryptophan, glutamine, and 2-hydroxybutyric acid as biomarkers in 183 human serum, also achieved high accuracy (AUC: 0.969–0.990) in both training and validation dataset [69]. Furthermore, the aforementioned European study [80] also demonstrated better diagnostic performance of metabolites than alpha-fetoprotein and liver enzymes.

Taken together, metabolomic investigations have suggested many useful biomarkers and potential biochemical pathways in chronic liver diseases and HCC. Some of them such as bile acid and lysophosphatidylcholine may belong to the "core metabolic phenotype" of background chronic liver diseases and are not necessarily indicative of the tumor status of HCC. Some other alterations, for example, those of ammonium detoxication or amino acid metabolism, may suggest the impaired liver functions. In HCC, there are common findings of enforced glycolysis and enhanced lipid catabolism. These two changes are consistent with the high demand of energy and intermediate metabolites in HCC cells for protein and membrane synthesis during fast tumor growth.

## **5. Lipid catabolism in liver cancer: a potential therapeutic target**

as the intermediate liver diseases, HCC maintained the aforementioned "core metabolic phenotype" of bile acid and phospholipid perturbation [66, 71, 73–75]. Some findings also demonstrated similar liver impairments of ammonium detoxication and amino acid metabo-

Other chronic liver diseases, for example, alcoholic liver disease and viral hepatitis, share some similar metabolic alterations to NASH, such as increased lipogenesis and reduction in lysophosphatidylcholine [77, 78]. Metabolomic profiling of a hepatitis B virus X (HBx) transgenic mouse model confirmed the involvement of lipid metabolism (triacylglycerol, cholesterol, saturated, and monounsaturated fatty acids) in HBV-induced liver oncogenesis [79]. This similarity indicated that chronic liver diseases may share some common biochemical and mechanical pathways particularly lipid metabolism, in spite of the differences in

A recent nested case-control study from a large European prospective cohort compared serums collected before diagnosis in 114 HCC with 222 matched controls and carefully controlled possible confounders such as tobacco usage, alcohol consumption, and so on [80]. Sixteen metabolites involved in amino acid metabolism, choline metabolism, polyunsaturated lipid metabolism, and ammonium detoxication were selected for potential biomarkers. These metabolomic changes are actually reflective of the underlying precancer liver dysfunctions before HCC occurrence (as discussed above). And these data are pivotal for understanding the process of HCC oncogenesis. The results also demonstrated that the pre-diagnostic metabolic profiles between HCC cases and respective control were different, which was dependent on hepatitis infection status, liver function, and the length of time from blood collection to HCC

Many recent investigations applied metabolomics technologies in biomarker discovery to advance HCC diagnosis. One Chinese study using a panel of metabolic markers (formate, phytosphingosine, and 3*α*,6*α*,7*α*,12*α*-tetrahydroxy-5*β*-cholan-24-oleic acid) achieved high accuracy (area under curve (AUC): 0.995–1.000, sensitivity: 100%, and specificity: 94.7–100%) in diagnosing HCC patients with low alpha-fetoprotein (a commonly accepted but mediocre biomarker to differentiate HCC from non-tumor liver at a cut-off value of 20 ng/ml) [73]. Another Chinese study, which used tryptophan, glutamine, and 2-hydroxybutyric acid as biomarkers in 183 human serum, also achieved high accuracy (AUC: 0.969–0.990) in both training and validation dataset [69]. Furthermore, the aforementioned European study [80] also demonstrated better diagnostic performance of metabolites than alpha-fetoprotein and

Taken together, metabolomic investigations have suggested many useful biomarkers and potential biochemical pathways in chronic liver diseases and HCC. Some of them such as bile acid and lysophosphatidylcholine may belong to the "core metabolic phenotype" of background chronic liver diseases and are not necessarily indicative of the tumor status of HCC. Some other alterations, for example, those of ammonium detoxication or amino acid metabolism, may suggest the impaired liver functions. In HCC, there are common findings of enforced glycolysis and enhanced lipid catabolism. These two changes are consistent with the high

lism in HCC as in cirrhosis [66, 72, 73, 76].

etiologies.

56 Updates in Liver Cancer

diagnosis.

liver enzymes.

As same as the enforced glycolysis, hyperactivation of lipid catabolism is commonly observed in HCC and other types of cancer. In tumor cells, lipid catabolism and glycolysis may share a few common oncogenic functions. First, both of them are important energy resources physically and pathologically. Second, their metabolisms support cell survival and tumor growth during stringent metabolic stress. Third, they can donor essential metabolic intermediates for protein and membrane anabolic biosynthesis in the fast growing tumor.

The body stores two types of major energy resources: triacylglycerol and glycogen. After a meal, the liver synthesizes both triacylglycerol and glycogen. Comparatively, triacylglycerol can provide six times as much ATP as glycogen, so that triacylglycerol is the preferred resource for storage. Lipid catabolism in terms of fatty acid oxidation is physically performed in heart and skeletal muscle, the highest energy-demanding tissues in the body. But the high activity of fatty acid oxidation in HCC and other types of cancer tissues, as revealed above by metabolomic studies, may provide excess ATP generation when needed.

During fast proliferation, tumor cells, especially those in the core of solid tumor nodules, may experience limited nutrient and oxygen availability because of insufficient tumor neoangiogenesis. This type of nutrient deprivation and the metabolic stress ensued may drive the tumor cells to programmed cell death. However, active fatty acid oxidation in the resistant HCC cells can protect cells with necessary ATP generation even if the exogenous glucose is unavailable and the endogenous glycogen is depleted. The author recently confirmed this hypothesis by comparing the starvation-sensitive with -resistant HCC cells [81]. The results demonstrated that the sensitive HCC cells, lack of an important transcription factor C/EBPalpha, could not initiate fatty acid oxidation and died within 12 h during enforced glucose deprivation *in vitro*. By contrast, the resistant C/EBPalpha-expressing HCC cells could activate autophagymediated lipid catabolism and survive as long as 8 days. But blockage of autophagy and the downstream fatty acid beta-oxidation by either pharmacological inhibitors or genetic shRNA intervention could significantly abolish the protective effect. Furthermore, this phenotype could be reproduced *in vivo* in a mice xenograft experiment that the C/EBPalpha-silenced HCC cells failed to develop tumor nodules due to extensive necrosis within a few days after inoculation of the tumor cells into the flank of mice. More importantly, we observed an inverse association of the expression level of C/EBPalpha with tumor necrosis in human HCC tissues. The higher C/EBPalpha expressed in human HCC, the less tumor necrosis observed. Consistently, these results shed the light on the importance of fatty acid oxidation on cell survival and tumor maintenance.

The involvement of fatty acid oxidation in cell survival was also observed during cell detachment from tumor matrix. Cells derived from solid tumor are dependent on fatty acid oxidation to survive when the cell experiences loss of attachment (LOA) to the extracellular matrix [82]. Otherwise, the cell will die in anoikis, a specific form of apoptotic cell death induced by inadequate cell-matrix interactions. Accumulating data have shown that antioxidants and oncogenes (e.g., promyelocytic leukemic protein, carnitine palmitoyltransferase isoform 1C) could activate fatty acid oxidation to support cell survival during LOA and other types of metabolic stresses [82–84]. Promyelocytic leukemic protein was found to be overexpressed in a subset of aggressive breast cancer [83], while the brain-isoform carnitine palmitoyltransferase 1C was abnormally up-regulated in human lung cancer [84]. With the aid of fatty acid oxidation, the survived cancer cells could migrate to distant locations and settle down in possible metastatic sites. Second, during metabolic stress, fatty acid oxidation could sustain NADPH level and consequently counteract harmful oxidative stress. In glioma cells, inhibition of fatty acid oxidation caused significant reduction of NADPH, accumulation of redox oxygen species, and eventually cell death [85]. The involved mechanisms of NADPH production by fatty acid oxidation were later found to be mediated by LKB1-AMPK pathway [86, 87]. Lastly, one study demonstrated that leukemia progenitor cells required fatty acid oxidation to maintain stem cell property [88].

The translational impact of fatty acid oxidation on cell survival and tumor growth has been tested recently. Dr. Samuduio and colleagues initially showed that pharmacological inhibitors (etomoxir and ranolazine) of fatty acid beta-oxidation could sensitize leukemia cells to chemotherapeutic drugs-induced cell death *in vitro* [89]. Later, the antitumor effects of a novel inhibitor ST1326 were confirmed in patients-derived leukemia primary cells *in vitro* [90] and in an *in vivo* mice model of Burkitt's lymphoma [91]. Furthermore, the synergistic effects of fatty acid oxidation inhibitors (etomoxir or CVT-4325) were reproduced in L-asparaginasetreated childhood acute lymphoblastic leukemia cells [92] and in dexamethasone-treated lymphocytic leukemia cells [93], respectively. Recently, etomoxir was applied to MYCoverexpressing triple-negative breast cancer [94]. The results showed that the inhibition of fatty acid oxidation significantly abolished tumor growth in both an MYC-driven transgenic mice model of breast cancer and patient-derived xenografts.

Nutrient deprivation may occur not only in pathological solid tumor growth but during clinical intervention. Embolization treatment, especially transarterial chemoembolization, is applied to intermediate-stage HCC (BCLC B), which may account for 20% HCC patients [95]. By blocking the main arterial of blood supply to tumor nodules, this clinical intervention aims to starve and suffocate tumor cells. Whether fatty acid oxidation helps HCC cells escape cell death induced by embolization and whether this survival advantage contributes to HCC recurrence are yet to be determined. It will be also interesting to learn whether the addition of pharmacological inhibitors of fatty acid beta-oxidation is beneficial for the efficacy and safety of transarterial chemoembolization therapy.

Taken together, lipid catabolism (especially fatty acid oxidation) is important for tumor cell survival and tumor growth during nutrient deprivation-induced metabolic stress. Several investigations have confirmed the antitumor effects of the inhibitors of fatty acid oxidation *in vitro* and *in vivo*. The therapeutic potentials of this target need to be further explored.

## **6. Conclusion and perspective**

Otherwise, the cell will die in anoikis, a specific form of apoptotic cell death induced by inadequate cell-matrix interactions. Accumulating data have shown that antioxidants and oncogenes (e.g., promyelocytic leukemic protein, carnitine palmitoyltransferase isoform 1C) could activate fatty acid oxidation to support cell survival during LOA and other types of metabolic stresses [82–84]. Promyelocytic leukemic protein was found to be overexpressed in a subset of aggressive breast cancer [83], while the brain-isoform carnitine palmitoyltransferase 1C was abnormally up-regulated in human lung cancer [84]. With the aid of fatty acid oxidation, the survived cancer cells could migrate to distant locations and settle down in possible metastatic sites. Second, during metabolic stress, fatty acid oxidation could sustain NADPH level and consequently counteract harmful oxidative stress. In glioma cells, inhibition of fatty acid oxidation caused significant reduction of NADPH, accumulation of redox oxygen species, and eventually cell death [85]. The involved mechanisms of NADPH production by fatty acid oxidation were later found to be mediated by LKB1-AMPK pathway [86, 87]. Lastly, one study demonstrated that leukemia progenitor cells required fatty acid oxidation to

The translational impact of fatty acid oxidation on cell survival and tumor growth has been tested recently. Dr. Samuduio and colleagues initially showed that pharmacological inhibitors (etomoxir and ranolazine) of fatty acid beta-oxidation could sensitize leukemia cells to chemotherapeutic drugs-induced cell death *in vitro* [89]. Later, the antitumor effects of a novel inhibitor ST1326 were confirmed in patients-derived leukemia primary cells *in vitro* [90] and in an *in vivo* mice model of Burkitt's lymphoma [91]. Furthermore, the synergistic effects of fatty acid oxidation inhibitors (etomoxir or CVT-4325) were reproduced in L-asparaginasetreated childhood acute lymphoblastic leukemia cells [92] and in dexamethasone-treated lymphocytic leukemia cells [93], respectively. Recently, etomoxir was applied to MYCoverexpressing triple-negative breast cancer [94]. The results showed that the inhibition of fatty acid oxidation significantly abolished tumor growth in both an MYC-driven transgenic

Nutrient deprivation may occur not only in pathological solid tumor growth but during clinical intervention. Embolization treatment, especially transarterial chemoembolization, is applied to intermediate-stage HCC (BCLC B), which may account for 20% HCC patients [95]. By blocking the main arterial of blood supply to tumor nodules, this clinical intervention aims to starve and suffocate tumor cells. Whether fatty acid oxidation helps HCC cells escape cell death induced by embolization and whether this survival advantage contributes to HCC recurrence are yet to be determined. It will be also interesting to learn whether the addition of pharmacological inhibitors of fatty acid beta-oxidation is beneficial for the efficacy and safety of

Taken together, lipid catabolism (especially fatty acid oxidation) is important for tumor cell survival and tumor growth during nutrient deprivation-induced metabolic stress. Several investigations have confirmed the antitumor effects of the inhibitors of fatty acid oxidation *in*

*vitro* and *in vivo*. The therapeutic potentials of this target need to be further explored.

maintain stem cell property [88].

58 Updates in Liver Cancer

mice model of breast cancer and patient-derived xenografts.

transarterial chemoembolization therapy.

Lipid metabolisms are essential for both healthy and diseased liver. It is apparent that with the pandemic of obesity, diabetes, and other metabolic syndromes, the incidence of NAFLD-, NASH-, and NAFLD-derived HCC will be on the upward trend in the foreseeable future. The *de novo* lipogenesis induced by nutrition overload dominates in the liver. Excess lipids deposit in the adipose and liver, causing lipotoxicity, oxidative stress, and inflammatory reactions. The metabolomic investigations have analyzed the perturbed metabolite profiles in the fatty liver diseases and identified several novel biomarkers and biochemical pathways. At this precancer stage, enhancement of lipid catabolism, together with dietary control, will be beneficial for the health. This goal can be achieved by regular physical exercises, medical intervention (e.g., administration of Statins or Metformin), and other preventive countermeasures. It has been reported that the consumption of coffee or tea could reduce HCC risks, according to large epidemiological studies [96]. Mechanically, the main components of coffee (caffeine) and tea (epigallocatechin-3-gallate) were found to promote autophagy and fatty acid oxidation [97, 98].

The induction of fatty acid beta-oxidation in HCC may reflect the high-energy demand for rapid tumor growth and cell survival. Accumulating metabolomic studies have confirmed this change. The oncogenic drivers for the metabolic reprogramming from the original net lipid gain in fatty liver diseases to the increased lipid loss in HCC, however, are yet to be unveiled. The translational impacts and therapeutic potentials of fatty acid oxidation are gaining growing interests. Oriented inhibition of fatty acid oxidation in the tumor cells may be useful for cancer treatment, especially if applied with the strictly localized application of transarterial chemoembolization. However, this hypothesis has not been carefully tested. It is of particular importance to examine the safety of systemic administration of fatty acid oxidation inhibitors in the non-tumor tissues.

## **Author details**

Guo-Dong Lu1,2\* and Shing Chuan Hooi3

\*Address all correspondence to<EMAIL_ADDRESS>
1 School of Public Health, Guangxi Medical University, Nanning, Guangxi Province, China

2 The Key Laboratory of High-Incidence Diseases Prevention and Control, Guangxi Medical University, Nanning, Guangxi Province, China

3 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

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#### **Hepatitis B Virus–Related Hepatocellular Carcinoma: Carcinogenesis, Prevention, and Treatment Hepatitis B Virus–Related Hepatocellular Carcinoma: Carcinogenesis, Prevention, and Treatment**

Bolin Niu and Hie-Won Hann Bolin Niu and Hie-Won Hann

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65424

#### **Abstract**

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the second leading cause of cancer death. Hepatitis B virus (HBV) infection is one of the major risk factors for the development of HCC in the world. Most of the burden of disease (85%) is observed in the HBV endemic regions. Chronic infection with HBV predisposes patients with or without cirrhosis to HCC. Patients with high HBV DNA levels are at an increased risk for HCC. Studies have shown that the suppression of HBV with anti-viral therapy (nucleos(t)ide analogs) (NAs) decreases the incidence of HCC but does not eliminate the risk entirely. Chronic viral suppression alone is not sufficient treatment to prevent HCC development. Therefore, along with NAs, treatment may need to include targeting the cccDNA and inhibiting the viral entry into the newly formed hepatocytes and T-cell vaccine which specifically targets HBV and enhancing innate immunity with Toll-like receptor agonist. With all of these working together, we may achieve the goal of HBV cure.

**Keywords:** HBV, HCC, nucleos(t)ide analogs, antiviral treatment, prevention of HCC, HBV cure, hepatocarcinogenesis

## **1. Hepatitis B virus (HBV)**

#### **1.1. The discovery of HBV**

Following the "icteric epidemic" in the 1880s, viral hepatitis was recognized as infectious in nature [1]. The discovery of HBV did not occur until 1965 when Baruch Blumberg, an American physician and geneticist, found a unique antigen in the serum of Australian aborigines (Australia Antigen, AuAg) that reacted with the serum of hemophiliacs [2, 3]. This

antigen AuAg is now recognized as the hepatitis B surface antigen (HBsAg). Later, the link between viral hepatitis and this newly discovered antigen was firmly established when a technician in Blumberg's laboratory developed acute hepatitis [4]. Blumberg was awarded the Nobel Prize in Medicine in 1976 for his discovery of HBV.

Dane et al. identified the entire viral particle using electron microscopy in the 1970s [5]. Subsequently, in 1971, Blumberg and Millman developed a blood test to start screening blood donations for HBV [6]. In 1980, the FDA approved the first commercially available HBV vaccine once the genome of HBV was sequenced [7, 8]. While this first generation vaccine is no longer available in the United States, a recombinant HBV vaccine has been in use since 1986. A strong association between HBV and hepatocellular carcinoma (HCC) was described by Beasley et al. in their landmark study of 22,707 men in Taiwan [9]. Therefore, this vaccine has been designated by the World Health Organization as a bonafide "cancer vaccine".

#### **1.2. HBV epidemiology**

From a global view, a recent meta-analysis shows that the worldwide HBsAg prevalence is 3.61% [10]. There are over 248 million people currently living with chronic hepatitis B (CHB). Africa has the highest endemicity, with an HBsAg prevalence of 8.83%. However, the country with the largest number of people living with CHB is China with 95 million people, with an HBsAg prevalence of 5.49%. India and Nigeria have the second and third highest population of HBsAg (+) individuals, respectively, at 17 and 15 million people.

Chronic hepatitis B is a major risk factor for the development of hepatocellular carcinoma. A study in New York City found that Korean males had the highest rate of liver cancer–related mortality compared with all racial/ethnic groups. In fact, liver cancer was the second and third cause of cancer-related deaths in NYC Chinese and Korean men, respectively [11]. The Asian American Hepatitis B Program (AAHBP), a large community-based program in New York City, has found 13.3% HBsAg positivity among over 4000 newly screened individuals born in Asia [12].

## **2. HBV carcinogenesis**

#### **2.1. Risk of HCC from HBV infection**

In a landmark paper in 1981, Beasley et al. established the association between HBV and HCC in 22,000 HBsAg (+) Taiwanese men. Compared to uninfected controls, their relative risk for HCC was found to be 63 [9]. Since then, co-infection with HCV [13], family history of HCC [14], alcohol intake [15], HBV genotype C greater than B [16, 17], and core promoter mutations [18, 19] have all been identified as risk factors for HCC development.

In highly endemic areas, HBV transmission is nearly all from mother to newborn and as many as 90% of infected babies develop chronic infections [20]. This differs from areas that have a low prevalence of HBV, where transmission is horizontal through sexual and parenteral routes in adulthood. More than 90% of these cases of acute HBV infection resolve spontaneously and do not lead to chronic infections. Longer periods of chronic HBV infection contribute to a higher risk for HCC; therefore, endemic areas have a higher incidence of HCC.

Approximately 25% of those chronically infected people with HBV will develop HCC [21]. In addition to the earlier report by Beasley et al. [9], Franceschi et al. also reported a 30-fold increased risk of HCC in chronic HBV carriers [22]. A systematic review estimated the incidence rates of HCC in subjects with chronic HBV infection in East Asian countries to be 0.2 per 100 person-years in inactive carriers (HBsAg-positive but with normal levels of ALT), 0.6 person-years for those with chronic HBV infection without cirrhosis, and 3.7 person-years for those with compensated cirrhosis [23]. HBV can cause HCC in the absence of cirrhosis though 70–90% of HBV-related HCC occur in patients with cirrhosis [24].

The risk of HCC is increased in patients with higher levels of HBV replication. One large study followed 11,893 Taiwanese men for a mean of 8.5 years to evaluate the effect of HBV replication on the risk of HCC. The incidence rate of HCC was 1169 per 100,000 person-years among men who were positive for both HBsAg and HBeAg, 324 per 100,000 person-years for those who were only HBsAg-positive, and 39 per 100,000 person-years for those who were HBsAgnegative [25]. The relative risks of HCC among men who were positive for both HBsAg and HBeAg were increased 60-fold compared to 10-fold among those who were only HBsAg positive [25]. Another prospective study from Taiwan reported that in a cohort of 3653 HBsAgpositive participants, the incidence of cirrhosis and HCC increased in proportion to the HBV DNA level, from <300 copies/mL at 0.74% incidence to ≥1,000,000 copies/mL at 13.50% incidence over 13 years of follow up [26]. Furthermore, inactive carriers of HBV (HBeAg negative, HBV DNA <10,000 copies/mL, normal liver enzyme levels, no cirrhosis) are still at a 5-fold greater risk for HCC than HBsAg-negative controls [27].

### **2.2. Entry of HBV DNA into host cells**

Hepatitis B virus is an enveloped DNA virus belonging to the *Hepadnaviridae* family. HBV contains a partially double-stranded circular DNA genome (rcDNA) [28]. HBV recognizes highly sulfated heparin sulfate proteoglycans (HSPGs) on the surface of liver cells, allowing the virus to be highly hepatotropic [29]. When HBsAg binds a liver-specific receptor named sodium taurocholate cotransporting polypeptide (NTCP or SLC10A1) during an infection, the virus gains entry into its host cell [30].

Upon entering the human hepatocyte, rcDNA becomes a covalently closed circular DNA (cccDNA) in the nucleus. This cccDNA functions as a template for transcription of all four viral mRNAs, which then translate all seven HBV proteins [28]. The largest viral mRNA transcript encodes the viral polymerase and is a template for DNA [31]. Current HBV antiviral medications thwart this step of the viral replication [32].

#### **2.3. HBV X protein**

antigen AuAg is now recognized as the hepatitis B surface antigen (HBsAg). Later, the link between viral hepatitis and this newly discovered antigen was firmly established when a technician in Blumberg's laboratory developed acute hepatitis [4]. Blumberg was awarded the

Dane et al. identified the entire viral particle using electron microscopy in the 1970s [5]. Subsequently, in 1971, Blumberg and Millman developed a blood test to start screening blood donations for HBV [6]. In 1980, the FDA approved the first commercially available HBV vaccine once the genome of HBV was sequenced [7, 8]. While this first generation vaccine is no longer available in the United States, a recombinant HBV vaccine has been in use since 1986. A strong association between HBV and hepatocellular carcinoma (HCC) was described by Beasley et al. in their landmark study of 22,707 men in Taiwan [9]. Therefore, this vaccine has been desig-

From a global view, a recent meta-analysis shows that the worldwide HBsAg prevalence is 3.61% [10]. There are over 248 million people currently living with chronic hepatitis B (CHB). Africa has the highest endemicity, with an HBsAg prevalence of 8.83%. However, the country with the largest number of people living with CHB is China with 95 million people, with an HBsAg prevalence of 5.49%. India and Nigeria have the second and third highest population

Chronic hepatitis B is a major risk factor for the development of hepatocellular carcinoma. A study in New York City found that Korean males had the highest rate of liver cancer–related mortality compared with all racial/ethnic groups. In fact, liver cancer was the second and third cause of cancer-related deaths in NYC Chinese and Korean men, respectively [11]. The Asian American Hepatitis B Program (AAHBP), a large community-based program in New York City, has found 13.3% HBsAg positivity among over 4000 newly screened individuals born in

In a landmark paper in 1981, Beasley et al. established the association between HBV and HCC in 22,000 HBsAg (+) Taiwanese men. Compared to uninfected controls, their relative risk for HCC was found to be 63 [9]. Since then, co-infection with HCV [13], family history of HCC [14], alcohol intake [15], HBV genotype C greater than B [16, 17], and core promoter mutations [18,

In highly endemic areas, HBV transmission is nearly all from mother to newborn and as many as 90% of infected babies develop chronic infections [20]. This differs from areas that have a low prevalence of HBV, where transmission is horizontal through sexual and parenteral routes in adulthood. More than 90% of these cases of acute HBV infection resolve spontaneously and

Nobel Prize in Medicine in 1976 for his discovery of HBV.

**1.2. HBV epidemiology**

70 Updates in Liver Cancer

Asia [12].

**2. HBV carcinogenesis**

**2.1. Risk of HCC from HBV infection**

nated by the World Health Organization as a bonafide "cancer vaccine".

of HBsAg (+) individuals, respectively, at 17 and 15 million people.

19] have all been identified as risk factors for HCC development.

The HBV X protein (HBx) is a 154 amino acid polypeptide with a mass of 17 kDa. Its role in the development of HCC is critical. HBx regulates cellular transcription, protein degradation, and cellular proliferation and apoptosis. HBx acts on cellular promoters by protein-protein interactions instead of binding directly to DNA. HBx can downregulate Wnt/β-catenin expression and suppress cell growth by not only repressing cell proliferation but also triggering cell apoptosis [33]. HBx protein also interacts with the tumor suppressor adenomatous polyposis coli to activate Wnt/β-catenin signaling, which upregulates the epithelial cell adhesion molecule in HCC cells to promote tumor initiation [34, 35]. Therefore, HBx activation of Wnt/β-catenin may directly promote the transformation of hepatocytes into cancer initiating cells [36]. Overall, the seemingly contradictory roles of HBx in regulating apoptosis demonstrate the complexity of hepatocarcinogenesis.

There are numerous ways in which HBx may induce anti-apoptotic effects. The most salient is its ability to inhibit p-53-mediated apoptosis. HBx may increase the expression of telomerase reverse transcriptase and telomerase activity, thus prolonging the lifespan of hepatocytes and leading to malignant transformation [36]. In addition, carboxyl-terminal (C-terminal) truncated HBx protein loses its proapoptotic properties and may enhance the protein's ability to transform oncogenes [36].

#### **2.4. Integration of HBV DNA into host DNA**

HBx truncation occurs with HBV integration into host DNA. The 3′-end of HBx is the preferred region of HBV genome involved in integration. When HBV integrates, the 3′-end of HBx is often deleted. Therefore, HBV integration is an important step in HCC development [37]. The C-terminal region produced by HBx truncation also contributes to HCC development. The Cterminal region has been suggested to be required for ROS production and 8-oxoguanine formation, biomarkers of oxidative stress [38]. The 24 amino acids truncated at the C-terminal end play a role in increasing cell invasiveness and metastasis in HCC through activation of MMP10 by C-Jun signaling [39]. Lastly, C-terminal truncated HBx has been reported to directly regulate miRNA transcription and promote hepatocellular proliferation [40].

## **3. Natural history**

HBV carriers often are asymptomatic without significant liver injury because HBV replication in itself is not directly cytotoxic to hepatocytes [21, 41]. Hepatocellular injury occurs largely from host immune responses, both through major-histocompatibility-complex (MHC) class IIrestricted, CD4+ helper T cells and MHC class I-restricted, CD8+ cytotoxic T lymphocytes [21, 42]. Four distinct phases comprise the natural history of HBV infection.

#### **3.1. Acute "immune tolerant" phase**

In the acute phase of infection with HBV, the "immune tolerant" phase is HBeAg (+) with high viral loads, normal serum alanine aminotransferase (ALT), and near normal liver histology [43]. When HBV is acquired in adulthood, this phase is very short [44]; however, perinatal and early childhood infection lead to a long "immune-tolerant" phase [45, 46]. The risk of progression to chronic carrier state differs greatly between those infected perinatally (90%) and as an adult (<1%) [44, 47, 48]. At the current time, antiviral treatment is not recommended during the immune-tolerant phase but rather for the immune clearance phase. Interestingly, some recent reports have shown evidence of immune reactivity during the immune-tolerant stage [49–51]. As was presented by Zoulim and Mason, there is an argument to consider earlier treatment of CHB in order to prevent HCC [52].

#### **3.2. "Immune clearance" phase**

interactions instead of binding directly to DNA. HBx can downregulate Wnt/β-catenin expression and suppress cell growth by not only repressing cell proliferation but also triggering cell apoptosis [33]. HBx protein also interacts with the tumor suppressor adenomatous polyposis coli to activate Wnt/β-catenin signaling, which upregulates the epithelial cell adhesion molecule in HCC cells to promote tumor initiation [34, 35]. Therefore, HBx activation of Wnt/β-catenin may directly promote the transformation of hepatocytes into cancer initiating cells [36]. Overall, the seemingly contradictory roles of HBx in regulating apoptosis

There are numerous ways in which HBx may induce anti-apoptotic effects. The most salient is its ability to inhibit p-53-mediated apoptosis. HBx may increase the expression of telomerase reverse transcriptase and telomerase activity, thus prolonging the lifespan of hepatocytes and leading to malignant transformation [36]. In addition, carboxyl-terminal (C-terminal) truncated HBx protein loses its proapoptotic properties and may enhance the protein's ability to

HBx truncation occurs with HBV integration into host DNA. The 3′-end of HBx is the preferred region of HBV genome involved in integration. When HBV integrates, the 3′-end of HBx is often deleted. Therefore, HBV integration is an important step in HCC development [37]. The C-terminal region produced by HBx truncation also contributes to HCC development. The Cterminal region has been suggested to be required for ROS production and 8-oxoguanine formation, biomarkers of oxidative stress [38]. The 24 amino acids truncated at the C-terminal end play a role in increasing cell invasiveness and metastasis in HCC through activation of MMP10 by C-Jun signaling [39]. Lastly, C-terminal truncated HBx has been reported to directly

HBV carriers often are asymptomatic without significant liver injury because HBV replication in itself is not directly cytotoxic to hepatocytes [21, 41]. Hepatocellular injury occurs largely from host immune responses, both through major-histocompatibility-complex (MHC) class IIrestricted, CD4+ helper T cells and MHC class I-restricted, CD8+ cytotoxic T lymphocytes [21,

In the acute phase of infection with HBV, the "immune tolerant" phase is HBeAg (+) with high viral loads, normal serum alanine aminotransferase (ALT), and near normal liver histology [43]. When HBV is acquired in adulthood, this phase is very short [44]; however, perinatal and early childhood infection lead to a long "immune-tolerant" phase [45, 46]. The risk of progression to chronic carrier state differs greatly between those infected perinatally (90%) and as an adult (<1%) [44, 47, 48]. At the current time, antiviral treatment is not recommended

regulate miRNA transcription and promote hepatocellular proliferation [40].

42]. Four distinct phases comprise the natural history of HBV infection.

demonstrate the complexity of hepatocarcinogenesis.

**2.4. Integration of HBV DNA into host DNA**

transform oncogenes [36].

72 Updates in Liver Cancer

**3. Natural history**

**3.1. Acute "immune tolerant" phase**

The "immune clearance" phase, developing during adolescence, is characterized by high viral load, HBeAg (+), and elevated ALT. Antiviral therapy is usually recommended during this phase. The salient feature of this phase is elevated ALT levels, which is a result of T-cell immune-mediated lysis of hepatocytes [53, 54]. The frequency of flares and duration of this phase are correlated with the risk of cirrhosis and HCC [55, 56]. High ALT level is a marker of vigorous host immune response, which is correlated with spontaneous HBeAg seroconversion. HBeAg seroconversion to anti-HBe is a pertinent outcome of this phase [57, 58].

#### **3.3. "Inactive carrier" phase**

Following HBeAg seroconversion, an "inactive HBsAg carrier" phase begins. It is marked by HBeAg (−), anti-HBe (+), normal ALT, and low or undetectable viral load [59]. Liver biopsy at this time would show mild hepatitis, minimal fibrosis, but cirrhosis may also be seen in patients who have experienced severe liver injury in the previous "immune clearance" phase [60]. Antiviral therapy is not indicated in this phase, but patients do need regular screening for HCC given the persistent risk while remaining positive for HBsAg and anti-HBc (IgG). Spontaneous seroclearance of HBsAg at a yearly incidence of 0.7–2.4% may happen after patients become HBeAg (−) [57, 61]. This phase may persist indefinitely.

#### **3.4. "Reactivation/HBeAg-negative chronic hepatitis" phase**

The last phase in the natural history of HBV infection is more recently recognized. The "reactivation of HBV replication/HBeAg-negative chronic hepatitis B" stage, also known as "e-CHB", is marked by HBeAg (−), anti-HBe (+), detectable viral load, elevated ALT, and continued necroinflammation on histology [62]. Patients may enter the "e-CHB" phase after some years in the "inactive carrier" phase or directly progress from HBeAg (+) chronic hepatitis to HBeAg (−) chronic hepatitis [63]. Many mutations in the viral core promoter and pre-core regions inhibit the synthesis of HBeAg without affecting HBV replication. Nucleotide 1896 is one of the most studied mutations associated with e-CHB in the pre-core region [64].

## **4. Prevention of HCC**

#### **4.1. Results of vaccination**

Taiwan, a country with a high prevalence of chronic HBV, instituted a nationwide HBV vaccination program in 1984 for all citizens ranging from neonates to adults. A landmark paper published in the *New England Journal of Medicine* in 1997 reports the effect of vaccination on childhood HCC in Taiwan [65]. In 1984, the prevalence of seropositivity of HBsAg was 10.6% in six-year olds. Ten years after launching the vaccination campaign, this prevalence was reduced to <1% in six-year olds by 1994. The incidence of HCC in children ages 6–9 significantly declined from 0.52 per 100,000 for those born between 1974 and 1984 to 0.13 for those born between 1984 and 1986.

Given the availability of national health records, Taiwan is a country with tremendous potential for public health and epidemiological investigations. A newer study from the same group published recently re-examines the effect of HBV vaccination by comparing the rate of HCC in different time periods [66]. Between 1983 and 2011, 1509 patients were diagnosed with HCC. 1343 were born before and 166 were born after the HBV vaccination program began. The relative risk for HCC in patients 6–9 years old, 10–14 years old, 15–19 years old, and 20–26 years old who were vaccinated vs. unvaccinated were 0.26, 0.34, 0.37, and 0.42, respectively. Out of the 166 cases of HCC that occurred after HBV vaccination began in Taiwan, the two strongest risk factors were transmission of HBV from highly infectious mothers and incomplete immunization.

At this point, 180 countries have introduced infant HBV vaccination, and the global HBV vaccination coverage rate for the third dose is about 78% [67]. HBV vaccines are usually administered in three doses, with the second dose given one month after the first dose and the third dose given six months after the first dose. The dose recommended for adults is 10–20 μg and for infants and children 5–10 μg. With regard to the immunogenicity of HBV vaccines, over 90% of infants, children, and adolescents have protective serum anti-HBs antibody concentrations (>10 mIU/mL) after the vaccine series has been completed. However, host factors such as age older than 30, obesity, immunosuppression, and smoking have been linked to inadequate immunogenicity to the HBV vaccine.

Vaccination is most important for infants, particularly those born to HBsAg (+) mothers. In addition, the WHO recommends high-risk groups should also be vaccinated as well, including [1] people who frequently require blood transfusions, such as dialysis patients and recipients of solid organ transplantations [2]; people interned in prisons [3]; IV drug users [4]; household and sexual contacts of people with chronic HBV infection [5]; people with multiple sexual partners, health-care workers, and others who are exposed to blood or blood products through work [6]; and travelers who have not completed their HBV vaccine series. Although post-vaccination testing for immunity is not generally recommended, it has been the practice at our institution to conduct post-vaccination test to confirm the presence of anti-HBs at the protective level (>10 IU). For those who fail to produce antibody, it is important to rule out the occult HBV infection not uncommonly seen among the family members of HBV patients.

#### **4.2. Surveillance for HCC**

It is generally recommended to perform HCC surveillance in those with CHB and especially if the patient has cirrhosis. CHB is an independent risk factor for the development of HCC, which can occur even without cirrhosis. The surveillance method includes imaging, whether triple-phase CT or MRI with contrast, should occur every 6 months. The evidence for serological testing for alpha fetal protein (AFP) in surveillance for HCC is unclear; however, at our institution it is obtained at 6-month intervals with imaging.

childhood HCC in Taiwan [65]. In 1984, the prevalence of seropositivity of HBsAg was 10.6% in six-year olds. Ten years after launching the vaccination campaign, this prevalence was reduced to <1% in six-year olds by 1994. The incidence of HCC in children ages 6–9 significantly declined from 0.52 per 100,000 for those born between 1974 and 1984 to 0.13 for those born

Given the availability of national health records, Taiwan is a country with tremendous potential for public health and epidemiological investigations. A newer study from the same group published recently re-examines the effect of HBV vaccination by comparing the rate of HCC in different time periods [66]. Between 1983 and 2011, 1509 patients were diagnosed with HCC. 1343 were born before and 166 were born after the HBV vaccination program began. The relative risk for HCC in patients 6–9 years old, 10–14 years old, 15–19 years old, and 20–26 years old who were vaccinated vs. unvaccinated were 0.26, 0.34, 0.37, and 0.42, respectively. Out of the 166 cases of HCC that occurred after HBV vaccination began in Taiwan, the two strongest risk factors were transmission of HBV from highly infectious mothers and incom-

At this point, 180 countries have introduced infant HBV vaccination, and the global HBV vaccination coverage rate for the third dose is about 78% [67]. HBV vaccines are usually administered in three doses, with the second dose given one month after the first dose and the third dose given six months after the first dose. The dose recommended for adults is 10–20 μg and for infants and children 5–10 μg. With regard to the immunogenicity of HBV vaccines, over 90% of infants, children, and adolescents have protective serum anti-HBs antibody concentrations (>10 mIU/mL) after the vaccine series has been completed. However, host factors such as age older than 30, obesity, immunosuppression, and smoking have been linked

Vaccination is most important for infants, particularly those born to HBsAg (+) mothers. In addition, the WHO recommends high-risk groups should also be vaccinated as well, including [1] people who frequently require blood transfusions, such as dialysis patients and recipients of solid organ transplantations [2]; people interned in prisons [3]; IV drug users [4]; household and sexual contacts of people with chronic HBV infection [5]; people with multiple sexual partners, health-care workers, and others who are exposed to blood or blood products through work [6]; and travelers who have not completed their HBV vaccine series. Although post-vaccination testing for immunity is not generally recommended, it has been the practice at our institution to conduct post-vaccination test to confirm the presence of anti-HBs at the protective level (>10 IU). For those who fail to produce antibody, it is important to rule out the occult HBV infection not uncommonly seen among the family members of HBV patients.

It is generally recommended to perform HCC surveillance in those with CHB and especially if the patient has cirrhosis. CHB is an independent risk factor for the development of HCC, which can occur even without cirrhosis. The surveillance method includes imaging, whether triple-phase CT or MRI with contrast, should occur every 6 months. The evidence for

between 1984 and 1986.

74 Updates in Liver Cancer

plete immunization.

**4.2. Surveillance for HCC**

to inadequate immunogenicity to the HBV vaccine.

A recent study shows the importance of HCC surveillance even in those with seroclearance of HBsAg [68]. In a retrospective analysis of 829 patients (mean age: 52.3 years; 575 males; 98 with cirrhosis) after HBsAg seroclearance, the estimated annual incidence of HCC was 2.85% and 0.29% in patients with and without cirrhosis, respectively. In non-cirrhotic patients, the annual rate of HCC was higher in males than females (0.40% vs. 0%, respectively). The study concludes that HCC surveillance should be considered for cirrhotic patients and non-cirrhotic male patients over age 50, even after HBsAg seroclearance, especially those infected with HBV genotype C.

#### **4.3. Prevention of recurrent HCC post-resection, transplantation, and local tumor ablation**

Antiviral therapy after tumor resection aims to improve prognosis by suppressing viral replication. Recent evidence indicates high serum HBV DNA levels, either preoperatively or postoperatively, is associated with a higher risk of HCC recurrence [69]. Furthermore, the incidence of HCC recurrence was significantly higher in patients who experienced acute postoperative exacerbations of hepatitis with high-serum concentrations of HBV DNA and sustained HBsAg expression postoperatively [70]. Antiviral therapy has been shown to induce the remission of active hepatitis, maintain liver function, and increase the likelihood of successful treatment for HCC recurrence even if recurrence developed after curative resection [71]. In addition, high levels of HBV DNA are significantly associated with shorter survival times, with the cause of death being HCC recurrence [71]. A recent meta-analysis shows that antiviral therapy with nucleos(t)ide analogs (NAs) reduces HCC-related mortality and HCC recurrence postoperatively, and improves overall survival in patients with HBV-related HCC [72].

There is a lack of evidence to guide the management of HBV after liver transplantation for HBV-related HCC; however, lifelong antivirals are used in most centers. In the case of transplantation for HBV cirrhosis, recurrent HBV may lead to graft loss and poor posttransplant survival. There is a direct relationship between the HBV VL at time of transplantation and the rate of HBV recurrence [73]. Since the study by Samuel et al. [74], hepatitis B immune globulin (HBIG) has been use as prophylaxis against HBV recurrence after liver transplantation for HBV cirrhosis.

We have reported favorable effects of antiviral therapy on the survival of HCC patients following local tumor ablation through interventional radiology [75]. We included 25 patients, who met criteria with a single HCC ≤ 7 cm and underwent tumor ablation with curative intent. Sixteen patients (diagnosed 1999 and after) received antiviral therapy and nine patients (diagnosed before 1999) did not. While there was no difference in their median tumor size and AFP, the survival was significantly different (*p* < 0.001). The median survival of the untreated was 16 months while that of the treated was 80 months. Fourteen of 16 treated patients are alive to date with two longest survivors alive for ≥151 months. Overall, there is evidence for lifelong antiviral therapy for patients with HCC treated with resection, transplantation, or local regional therapy.

## **5. Current treatment of hepatitis B**

Since the advent of antiviral drugs, survival of patients with HBV has been remarkably improved. Current treatments for hepatitis B include nucleos(t)ide analogs (lamivudine, adefovir, entecavir, telbivudine, and tenofovir) and an interferon [pegylated-interferon alpha-2a (peg-IFN α-2a)] (**Table 1**) [4, 76]. The ultimate goal in the treatment of chronic hepatitis B is to prevent the development of HCC.

**Table 1.** Current approved drugs for treatment of HBV.

#### **5.1. Pegylated-interferon alpha-2a**

Pegylated-interferon alpha-2a (peg-IFN α-2a) has replaced interferon alpha-2b due to better pharmacokinetic properties, weekly injection schedule, and similar efficacy. Its major mechanism of action is in immune modulation with a weak antiviral effect [77]. Peg-IFN α-2a has the highest rate of sustained response after 1 year of therapy, with a 27% rate of HBeAg seroconversion and 25% rate of loss of HBV DNA after 48 weeks of treatment [78, 79]. After 18 months of follow up, 4–6% of patients showed serum positivity for anti-HBs and had loss of HBsAg [78, 79]. Even after the end of treatment, 12–65% of patients had seroclearance of HBsAg within 5 years of losing HBeAg [80, 81]. A study of 542 patients, who received the medication for 48 weeks, shows patients with the best response include genotype A with HBV DNA <9 log10 copies/mL or ALT ≥ 2×ULN, or genotype B and C with ALT ≥ 2×ULN and low HBV DNA (<9 log10 copies/mL) [84]. Remission long after discontinuing therapy was associated with an early virological response, defined as suppressing levels to below 105 copies/mL within the first 2 weeks of therapy or >2 log10 decrease in serum HBV DNA [82, 83].

Peg-IFN α-2a only makes up about 10% of all hepatitis B prescriptions in the United States due to its substantial side effect profile and need for administration by injection [85].

#### **5.2. Lamivudine**

**5. Current treatment of hepatitis B**

B is to prevent the development of HCC.

Hepsera Oral

Lamivudine Epivir Oral

Entecavir Baraclude Oral

Telbivudine Tyzeka Oral

Tenofovir Viread Oral

Adapted from Halegoua-De Marzio and Hann [4].

**5.1. Pegylated-interferon alpha-2a**

**Table 1.** Current approved drugs for treatment of HBV.

Pegasys Finite duration of treatment

Least expensive

Low resistance

Low resistance

Potent viral suppression Safe with negligible side effects

Potent viral suppression Effective and safe in pregnancy

Potent viral suppression Safe with negligible side effects No known resistance so far Effective and safe in pregnancy

No known resistance

Durable response post-treatment

Safe with negligible side effects Effective and safe in pregnancy

Pegylated interferon-2a

76 Updates in Liver Cancer

Adefovir dipivoxil

Since the advent of antiviral drugs, survival of patients with HBV has been remarkably improved. Current treatments for hepatitis B include nucleos(t)ide analogs (lamivudine, adefovir, entecavir, telbivudine, and tenofovir) and an interferon [pegylated-interferon alpha-2a (peg-IFN α-2a)] (**Table 1**) [4, 76]. The ultimate goal in the treatment of chronic hepatitis

**Name Trade name Strengths Weaknesses Approved**

Pegylated-interferon alpha-2a (peg-IFN α-2a) has replaced interferon alpha-2b due to better pharmacokinetic properties, weekly injection schedule, and similar efficacy. Its major mechanism of action is in immune modulation with a weak antiviral effect [77]. Peg-IFN α-2a has the highest rate of sustained response after 1 year of therapy, with a 27% rate of HBeAg

Needle injection High cost

65–70% fail to respond Significant side effects

Long-term treatment is necessary High incidence of resistance

Long-term treatment is necessary Long-term treatment for renal toxicity Less potent than other treatments

Long-term treatment is necessary

Long-term treatment is necessary High incidence of resistance

Long-term treatment is necessary 2008

High cost

1991 2005

1998

2002

2005

2006

Approved by the Food and Drug Administration (FDA), lamivudine is a nucleoside analog reverse transcriptase inhibitor. Due to availability of other oral antivirals that have higher genetic barriers to resistance, lamivudine is not commonly used today. The most common reasons for its use currently are during pregnancy in HBsAg (+) women to perinatal transmission and during chemotherapy and immunosuppression to prevent reactivation of HBV in HBsAg (+) patients.

With 12 months of treatment, lamivudine is associated with 16–18% rate of HBeAg seroconversion [86]. In HBeAg (+) patients, the rate of HBeAg seroconversion increases with the duration of treatment, from 17% at 1 year to 27% at 2 years to 47% at 4 years [87]. Therapy for 1 year also results in 60–70% HBV DNA suppression in HBeAg (−) patients with chronic hepatitis B [88].

Lamivudine has been shown to decrease the rate of fibrosis and the incidence of HCC [89]. A study of 651 Asian patients with advanced fibrosis was stopped prematurely at 32 months because a significantly lower proportion of the lamivudine-treated group reached the primary endpoint of development of hepatic decompensation, HCC, or death from liver disease compared to placebo (7.8% vs. 17.7%) [89]. Lamivudine-treated patients have been observed to have a significant reduction in the incidence of HCC [90]. Reversal of fibrosis was significantly more likely to be seen on histology after 52 weeks of treatment with lamivudine than placebo [87].

Despite these positive attributes, there is a decrease in lamivudine usage due to its resistance profile. A large-scale safety study showed resistance rates of 23% at one year and 67% at five years of therapy in HBeAg (+) patients [91]. In a small study at our institution, lower resistance rate of 3% at one year and 10% at two years was found when 150 mg dose of lamivudine was used [92]. Pretreatment HBV DNA level is the most important factor for lamivudine resistance. Tenofovir has been shown to have stronger antiviral effect than adefovir against lamivudineresistant HBV [93]. Furthermore, tenofovir monotherapy has been shown to be superior to adefovir and lamivudine combination therapy in lamivudine-resistant HBV [94].

#### **5.3. Adefovir dipivoxil**

Approved in 2002 by the FDA, adefovir dipivoxil is a nucleotide analog reverse transcriptase inhibitor. Treatment with adefovir for one year in HBeAg (+) patients leads to a 12% HBeAg seroconversion and 53% histological improvement [89, 95, 96]. Furthermore, HBeAg seroconversion is sustained in 91% of patients [97]. Development of resistance is associated with persistent viremia after 48 weeks of therapy. Rates of adefovir resistance at 1, 2, 4, and 5 years of therapy have been reported at 0%, 3%, 18%, and 29%, respectively [98]. Nevertheless, adefovir use is declining with the arrival of newer medications.

#### **5.4. Entecavir**

Approved by the FDA in 2005 for the treatment of CHB, entecavir is a nucleoside analog that inhibits HBV polymerase. It is administered as an oral dose of 0.5 mg/day, resulting in superior reduction of HBV DNA levels compared to lamivudine (6.98 log10 copies/mL vs. 5.4 log10 copies/mL) [99]. In a phase three clinical trial entecavir to lamivudine, those who received 52 weeks of entecavir achieved better virological response with HBV DNA < 400 copies/mL (67% entecavir vs. 36% lamivudine), normalization of ALT (78% vs. 70%), and histological improvement (72% vs. 62%) [99]. While entecavir is superior to lamivudine in HBeAg (−) patients, it does require indefinite treatment to maintain viral suppression and prevent relapse [100, 101]. After 6 years of therapy, 96% of HBeAg (+) CHB patients had histological improvement and 88% showed improved fibrosis scores even in cirrhosis [102]. Continuous entecavir treatment for up to 5 years in HBeAg (+) patients has been able to maintain HBV DNA suppression <300 copies/mL in 94% of patients [103].

In comparison to adefovir, entecavir has been shown to achieve viral suppression more rapidly within 14 days of initiating therapy [98]. Entecavir also has a higher rate of HBV clearance (58% vs. 19%) and ALT normalization (76% vs. 63%) when compared with adefovir after 48 weeks of treatment. No significant difference was observed in the rate of HBeAg loss or HBeAg seroconversion [104].

The incidence of HCC has been shown to decrease in entecavir-treated patients compared to non-treated. The five-year cumulative HCC incidence was 3.7% and 13.7% for entecavir-treated and control groups, respectively [105]. HBsAg loss has been associated with entecavir treatment [106, 107]. Compared to lamivudine treatment in HBeAg (+) patients, 96 weeks of entecavir treatment resulted in HBsAg loss in 5% of patients and 3% of lamivudine-treated patients [108]. Unlike HBeAg (+) patients, HBeAg (−) patients show no significant HBsAg loss on entecavir [109].

The biggest advantage of entecavir is its high genetic barrier and low resistance profile. The cumulative incidence of resistance after 6 years of entecavir in nucleoside-naïve patients is low at 1.2%. However, in lamivudine-refractory patients, the rate of resistance to entecavir is 57% at 6 years [110].

#### **5.5. Telbivudine**

**5.3. Adefovir dipivoxil**

78 Updates in Liver Cancer

**5.4. Entecavir**

copies/mL in 94% of patients [103].

seroconversion [104].

on entecavir [109].

at 6 years [110].

Approved in 2002 by the FDA, adefovir dipivoxil is a nucleotide analog reverse transcriptase inhibitor. Treatment with adefovir for one year in HBeAg (+) patients leads to a 12% HBeAg seroconversion and 53% histological improvement [89, 95, 96]. Furthermore, HBeAg seroconversion is sustained in 91% of patients [97]. Development of resistance is associated with persistent viremia after 48 weeks of therapy. Rates of adefovir resistance at 1, 2, 4, and 5 years of therapy have been reported at 0%, 3%, 18%, and 29%, respectively [98]. Nevertheless,

Approved by the FDA in 2005 for the treatment of CHB, entecavir is a nucleoside analog that inhibits HBV polymerase. It is administered as an oral dose of 0.5 mg/day, resulting in superior reduction of HBV DNA levels compared to lamivudine (6.98 log10 copies/mL vs. 5.4 log10 copies/mL) [99]. In a phase three clinical trial entecavir to lamivudine, those who received 52 weeks of entecavir achieved better virological response with HBV DNA < 400 copies/mL (67% entecavir vs. 36% lamivudine), normalization of ALT (78% vs. 70%), and histological improvement (72% vs. 62%) [99]. While entecavir is superior to lamivudine in HBeAg (−) patients, it does require indefinite treatment to maintain viral suppression and prevent relapse [100, 101]. After 6 years of therapy, 96% of HBeAg (+) CHB patients had histological improvement and 88% showed improved fibrosis scores even in cirrhosis [102]. Continuous entecavir treatment for up to 5 years in HBeAg (+) patients has been able to maintain HBV DNA suppression <300

In comparison to adefovir, entecavir has been shown to achieve viral suppression more rapidly within 14 days of initiating therapy [98]. Entecavir also has a higher rate of HBV clearance (58% vs. 19%) and ALT normalization (76% vs. 63%) when compared with adefovir after 48 weeks of treatment. No significant difference was observed in the rate of HBeAg loss or HBeAg

The incidence of HCC has been shown to decrease in entecavir-treated patients compared to non-treated. The five-year cumulative HCC incidence was 3.7% and 13.7% for entecavir-treated and control groups, respectively [105]. HBsAg loss has been associated with entecavir treatment [106, 107]. Compared to lamivudine treatment in HBeAg (+) patients, 96 weeks of entecavir treatment resulted in HBsAg loss in 5% of patients and 3% of lamivudine-treated patients [108]. Unlike HBeAg (+) patients, HBeAg (−) patients show no significant HBsAg loss

The biggest advantage of entecavir is its high genetic barrier and low resistance profile. The cumulative incidence of resistance after 6 years of entecavir in nucleoside-naïve patients is low at 1.2%. However, in lamivudine-refractory patients, the rate of resistance to entecavir is 57%

adefovir use is declining with the arrival of newer medications.

Telbivudine is an L-nucleoside that is structurally related to lamivudine; it was approved by the FDA in 2006. It specifically inhibits HBV viral DNA synthesis, and it has been shown to be superior to lamivudine in both HBeAg (+) and HBeAg (−) patients with CHB. The seroconversion of HBeAg with telbivudine was found to be 22% and 30% at 1 and 2 years, respectively, in patients who are HBeAg (+) [111, 112]. In these patients, suppression of HBV DNA < 300 copies/mL was 60% and 56% at 1 and 2 years, respectively [105, 106]. Furthermore, recent evidence shows telbivudine has renoprotective effects, both in preventing adefovir-induced nephrotoxicity and improving renal function in liver transplant patients [113–116].

However, resistance to telbivudine has been reported at 21.6% and 8.6% after 2 years of therapy in HBeAg (+) and HBeAg (−) patients [117]. Predictive factors for response to telbivudine include ALT > 2×ULN at baseline or HBV DNA < 9 log10 copies/mL in HBeAg (+) patients [118, 119]. Telbivudine treatment has good therapeutic result in patients with low baseline HBV DNA and negative HBV DNA at week 24 [118].

Telbivudine is a pregnancy category B medication. A study of 186 pregnant Asian women with HBV DNA > 6,000,000 copies/mL, half received telbivudine from second trimester of pregnancy until 4 weeks postpartum and all infants received hepatitis B immune globulin (HBIG) within 24 h of birth, telbivudine treatment showed better outcomes compared with the control group with more women achieving undetectable HBV DNA (30% vs. 0%) [120]. Importantly, no infants born to women in the treatment group were HBsAg (+) compared to 8.7% in the control group.

#### **5.6. Tenofovir**