**Meet the editor**

Dr. Georgios Tsoulfas received his medical degree from Brown University School of Medicine and completed his general surgery residency at the University of Iowa Hospitals and Clinics, as well as a transplant research fellowship at the Thomas E. Starzl Transplant Institute at the University of Pittsburgh. He then completed a 2-year transplantation surgery fellowship at the Massachusetts

General Hospital, Harvard Medical School and then joined the Division of Solid Organ Transplantation and Hepatobiliary Surgery at the University of Rochester Medical Center as an Assistant Professor of Surgery. He has currently moved back to Greece, where he is an Associate Professor of Surgery at the Aristotle University of Thessaloniki. He has published over 70 papers in peer-reviewed journals and in PubMed, as well as 17 book chapters. He is a reviewer for 15 international journals and on the editorial board of several others. He is currently the Chairman of the Education, Quality, and Communications Subcommittee of the IRC of the American College of Surgeons and the World President of the International College of Surgeons.

## Contents

#### **Preface XI**


Kazuyuki Suzuki, Ryujin Endo and Akinobu Kato


## Preface

Chapter 8 **Portal Vein Thrombosis in Patients with Liver Cirrhosis 155** Anca Trifan, Carol Stanciu and Irina Girleanu

Chapter 9 **Hemodynamic Optimization Strategies in Anesthesia Care for**

Chapter 10 **Management of Hepatocellular Carcinoma in the Setting of**

Chapter 11 **Impact of Glyoxalase-I (Glo-I) and Advanced Glycation End Products (AGEs) in Chronic Liver Disease 211**

Chapter 12 **Regenerative Medicine in Liver Cirrhosis: Promises and**

Chapter 13 **The Promising Role of Anti-Fibrotic Agent Halofuginone in**

Alexander Giakoustidis and Dimitrios E. Giakoustidis

Alexander A. Vitin, Dana Tomescu and Leonard Azamfirei

Asima Tayyeb, Fareeha Azam, Rabia Nisar, Rabia Nawaz, Uzma

**Liver Transplantation 173**

**Liver Cirrhosis 197**

**VI** Contents

Marcus Hollenbach

Qaisar and Gibran Ali

Berna Karakoyun

**Liver Fibrosis/Cirrhosis 257**

**Pitfalls 233**

Liver cirrhosis represents one of the major challenges for most physicians and surgeons giv‐ en the critical role of the liver, the increasing incidence of cirrhosis and its threatening na‐ ture. The problem that cirrhosis represents on a global scale makes it necessary to obtain a better understanding of the mechanisms involved in its development, identify novel and less invasive methods of diagnosis, and most importantly, select the best practices for the management of cirrhosis and its significant manifestations and complications. One of the major challenges in cirrhosis is the evolution of hepatocellular carcinoma, which creates added pressure regarding timely diagnosis and management.

This book with the contribution of an excellent group of world authorities in this field pro‐ vides a valuable update in the most current methods used for the diagnosis and management of cirrhosis, as well as being able to identify and address many of the remaining significant challenges. In addition to the knowledge shared, the authors provide their personal clinical experience making this book an extremely useful tool for every practicing hepatologist, gas‐ troenterologist and liver surgeon. The chapters that appear in the book start with those focus‐ ing on diagnostic issues and molecular pathways, followed by those addressing the significant complications of cirrhosis, including ascites, encephalopathy, bleeding, portal vein thrombo‐ sis, and the presence of hepatocellular carcinoma. The issue of hepatocellular carcinoma is addressed in a separate chapter with a special focus on the different management strategies and treatment modalities. Finally, the last chapters offer a glimpse into the future by discus‐ sing regenerative medicine practices and novel anti-fibrotic agents.

Overall, this book represents a true tour de force of a variety of topics having to do with liver cirrhosis, in an effort to present the many different challenges that we may face when trying to deal with this disease. This is true whether it has to do with making the correct diagnosis, evaluating the extent of the cirrhosis and the level of hepatic failure, identifying and managing the significant complications that can occur, and treating the resultant liver insufficiency or failure, as well as the possibility of hepatocellular carcinoma. It is essential to note that this book is not addressed at a single specialty, but rather the goal is to stress the fact that in order to manage liver cirrhosis successfully the cooperation of several different medical and surgical specialties is necessary.

> **Georgios Tsoulfas, MD, PhD, FICS, FACS** Associate Professor of Surgery Aristotle University of Thessaloniki Thessaloniki, Greece

## **Non-Alcoholic Steatohepatitis, Liver Cirrhosis and Hepatocellular Carcinoma: The Molecular Pathways**

Dzeina Mezale, Ilze Strumfa, Andrejs Vanags, Matiss Mezals, Ilze Fridrihsone, Boriss Strumfs and Dainis Balodis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68771

#### **Abstract**

Non-alcoholic steatohepatitis (NASH) is growing into global problem, mainly due to NASH-induced cirrhosis and hepatocellular carcinoma (HCC), that can develop either subsequently to cirrhosis or preceding it. In addition, NASH-induced cirrhosis constitutes a significant fraction of cases diagnosed as cryptogenic cirrhosis. Thus, there is a need for deeper understanding of the molecular basis, leading to liver steatosis, then—to the associated inflammation seen in NASH, loss of liver architecture and cirrhosis, followed or paralleled by carcinogenesis and HCC. Insulin resistance, increased hepatic iron level, and certain cytokines, including TNF-α and IL-6 derived from extrahepatic adipose tissues, can trigger the chain of events. The imbalance between leptin and adiponectin is important as well. These markers remain important during the whole course from NASH through liver cirrhosis to HCC. The molecular pathogenesis substantiates treatment: hypertriglyceridemia can be lowered by low calorie diet; mTOR complex can become inhibited by physical activity and metformin; cholesterol synthesis, RAF/ MAPK1/ERK and p21 pathway by statins; inflammation by pentoxyfillin, and kinases (in HCC) by sorafenib. Bidirectional regulation of telomere attrition, senescence and p21 pathway, restoration of wild-type p53 activity and regulation of miRNA network represent attractive future treatment options. Focusing on relevant molecular pathways allows deeper understanding of NASH pathogenesis, leading to identification of predictive markers and treatment targets.

**Keywords:** non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver cirrhosis, cryptogenic cirrhosis, hepatocellular carcinoma

© 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.

### **1. Introduction**

Non-alcoholic fatty liver disease (NAFLD) is a clinical and pathological entity with features that resemble alcohol-induced liver steatosis, but, by the definition, it occurs in patients with little or no history of alcohol consumption. NAFLD is subdivided into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). It encompasses a histological spectrum that ranges from fat accumulation in hepatocytes without concomitant inflammation or fibrosis (simple hepatic steatosis, NAFL) to hepatic steatosis with a necroinflammatory component (inflammation-induced apoptosis in hepatocytes) that may or may not have associated fibrosis. The latter condition, referred to as non-alcoholic steatohepatitis (NASH), can lead to NASH-induced liver cirrhosis (**Figure 1**). In addition, NASH is now recognised as the main cause of cryptogenic cirrhosis [1], as sequential association has been demonstrated in up to 75% of cryptogenic cirrhosis cases (see also Section 3 for detailed discussion of the relationships between NASH and cryptogenic cirrhosis). Liver cirrhosis may further lead to hepatocellular carcinoma (HCC), the most common primary liver cancer known for its poor clinical outcome and limited therapeutic options. Although previously it was considered that risk of HCC is limited to cirrhotic patients [2], a significant fraction of NASH-associated HCC develops in liver showing none or mild fibrosis. The association between NAFLD/NASH and increased HCC risk is supported by strong epidemiologic evidence.

In the year 2010, the annual incidence of HCC in the population of the USA was at least 6 per 100,000. The mortality rate was almost identical to the incidence underscoring the serious prognosis [3]. Patients with NAFLD/NASH are subjected to an increased lifetime risk of HCC. In a 16-year follow-up study, the standardised incidence ratio of HCC in patients with NAFLD/NASH was 4.4 [4]. In a recent global meta-analysis, the HCC incidence among NAFLD patients reached 0.44 (range, 0.29–0.66) per 1000 person-years [5]. The HCC-related mortality rates among NAFLD patients range from 0.25 to 2.3% over 8.3 and 13.7 years of follow-up, respectively [5, 6]. NAFLD/NASH-associated HCC is believed to be the leading cause of obesity-related cancer deaths in middle-aged men in the USA [4]. Consistently, the proportion of HCC related to NAFLD/NASH is increasing worldwide and is reported to range between 4 and 22% in Western countries [7]. Although the exact burden of HCC associated with NAFLD/NASH still remains uncertain, it seems evident that NAFLD and NASH will become the most common causative/risk factors for HCC, surpassing viral or alcohol-related cirrhosis in the future [7]. In the USA, the number of NAFLD-associated HCC cases is annually growing (2004–2009) for 9% [8], while decreased burden of viral hepatitis-induced HCC might be expected due to the achievements in antiviral treatment targeting hepatitis C virus [9].

NAFLD is the major hepatic manifestation of obesity and associated metabolic conditions. The epidemiology of NAFLD mirrors the recent spread of obesity and diabetes. With increasing prevalence of these conditions, NAFLD has become the most common liver disorder in USA [10] and other Western industrialised countries, facing high occurrence of the major risk factors for NAFLD, namely, central obesity, type 2 diabetes mellitus, dyslipidemia and metabolic syndrome [11]. In a recent meta-analysis of 86 studies, comprising 8,515,431 persons from 22 countries, the global prevalence of NAFLD was 25.24% (95% confidence interval [CI], 22.10–28.65) showing the highest occurrence in the Middle East and South America and the lowest in Africa [5]. Non-Alcoholic Steatohepatitis, Liver Cirrhosis and Hepatocellular Carcinoma: The Molecular Pathways http://dx.doi.org/10.5772/intechopen.68771 3

**1. Introduction**

2 Liver Cirrhosis - Update and Current Challenges

Non-alcoholic fatty liver disease (NAFLD) is a clinical and pathological entity with features that resemble alcohol-induced liver steatosis, but, by the definition, it occurs in patients with little or no history of alcohol consumption. NAFLD is subdivided into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). It encompasses a histological spectrum that ranges from fat accumulation in hepatocytes without concomitant inflammation or fibrosis (simple hepatic steatosis, NAFL) to hepatic steatosis with a necroinflammatory component (inflammation-induced apoptosis in hepatocytes) that may or may not have associated fibrosis. The latter condition, referred to as non-alcoholic steatohepatitis (NASH), can lead to NASH-induced liver cirrhosis (**Figure 1**). In addition, NASH is now recognised as the main cause of cryptogenic cirrhosis [1], as sequential association has been demonstrated in up to 75% of cryptogenic cirrhosis cases (see also Section 3 for detailed discussion of the relationships between NASH and cryptogenic cirrhosis). Liver cirrhosis may further lead to hepatocellular carcinoma (HCC), the most common primary liver cancer known for its poor clinical outcome and limited therapeutic options. Although previously it was considered that risk of HCC is limited to cirrhotic patients [2], a significant fraction of NASH-associated HCC develops in liver showing none or mild fibrosis. The association between NAFLD/NASH and

In the year 2010, the annual incidence of HCC in the population of the USA was at least 6 per 100,000. The mortality rate was almost identical to the incidence underscoring the serious prognosis [3]. Patients with NAFLD/NASH are subjected to an increased lifetime risk of HCC. In a 16-year follow-up study, the standardised incidence ratio of HCC in patients with NAFLD/NASH was 4.4 [4]. In a recent global meta-analysis, the HCC incidence among NAFLD patients reached 0.44 (range, 0.29–0.66) per 1000 person-years [5]. The HCC-related mortality rates among NAFLD patients range from 0.25 to 2.3% over 8.3 and 13.7 years of follow-up, respectively [5, 6]. NAFLD/NASH-associated HCC is believed to be the leading cause of obesity-related cancer deaths in middle-aged men in the USA [4]. Consistently, the proportion of HCC related to NAFLD/NASH is increasing worldwide and is reported to range between 4 and 22% in Western countries [7]. Although the exact burden of HCC associated with NAFLD/NASH still remains uncertain, it seems evident that NAFLD and NASH will become the most common causative/risk factors for HCC, surpassing viral or alcohol-related cirrhosis in the future [7]. In the USA, the number of NAFLD-associated HCC cases is annually growing (2004–2009) for 9% [8], while decreased burden of viral hepatitis-induced HCC might be expected due to the achievements in antiviral treatment targeting hepatitis C virus [9].

NAFLD is the major hepatic manifestation of obesity and associated metabolic conditions. The epidemiology of NAFLD mirrors the recent spread of obesity and diabetes. With increasing prevalence of these conditions, NAFLD has become the most common liver disorder in USA [10] and other Western industrialised countries, facing high occurrence of the major risk factors for NAFLD, namely, central obesity, type 2 diabetes mellitus, dyslipidemia and metabolic syndrome [11]. In a recent meta-analysis of 86 studies, comprising 8,515,431 persons from 22 countries, the global prevalence of NAFLD was 25.24% (95% confidence interval [CI], 22.10–28.65) showing the highest occurrence in the Middle East and South America and the lowest in Africa [5].

increased HCC risk is supported by strong epidemiologic evidence.

**Figure 1.** Progression of NAFLD. Abbreviations: IL, interleukin; TNF, tumour necrosis factor; ROS, reactive oxygen species; Fe, accumulation of iron compounds.

Thus, 90% of patients suffering from morbid obesity (defined as having body mass index 40 kg/m2 or higher) and 74% patients affected by diabetes mellitus develop NAFLD. In addition, NAFLD has been observed even in non-obese, non-diabetic patients who have increased insulin levels in blood and resistance to insulin action. Consequently, NAFLD affects up to 20–30% of adults in Europe and 46% in the USA: a tremendously high prevalence for a condition that can cause any significant complications [9, 10].

Most patients are diagnosed with NAFLD in their 40s or 50s. Studies vary in regard to the gender distribution of NAFLD, with some suggesting that it is more common in women and others suggesting more frequent occurrence in men [11, 12].

Since 1998, non-alcoholic fatty liver disease has been considered a condition with a "two-hit" course of pathogenesis, first proposed by Day and James [13], describing the role of lipid peroxidation in liver injury. The "first hit" is the development of hepatic steatosis. It was suggested that hepatic triglyceride accumulation increased the susceptibility of the liver to the "second injury hit" by inflammatory cytokines and/or adipokines, mitochondrial dysfunction and elevated oxidative stress that together promote steatohepatitis and fibrosis [14]. Alternatively, many factors may act simultaneously leading to the development of NAFLD: this hypothesis corresponds to the multihit model proposed by Tilg and Moschen [15].

Experimental and population studies have shown the links between NAFLD/NASH and development of HCC. However, the mechanisms by which NASH progresses to HCC are only beginning to be elucidated [14]. NASH is the most rapidly growing risk for liver transplantation because of HCC. Wong et al. in their study included 61,868 patients over the period 2002–2012 and found that the proportion of NASH-related HCC increased from 8.3 to 13.5%, an increase of near 63% [16].

This increase is alarming as HCC already is the fifth most frequently diagnosed cancer and the second leading oncologic death cause worldwide [17], with increasing incidence and mortality rates in Europe [18]. Thus it is crucial to analyse molecular pathways involved in NASH-induced cirrhosis and HCC carcinogenesis. Focusing on the molecular events involved in pathogenetic chain of events from NASH to liver cirrhosis and HCC would provide not only better theoretical understanding of liver diseases preceding and following cirrhosis but would also allow to recognise predictive markers and treatment targets before HCC development.

## **2. Common pathogenetic mechanisms of NAFLD**

Hepatic steatosis or excessive triglyceride accumulation in the liver is a prerequisite to the histological diagnosis of NAFLD. Several mechanisms may lead to steatosis, including (1) increased fat supply because of high-fat diet or excess lipolysis in adipose tissues, which increase free fatty acid (FFA) level; (2) decreased fat export in the form of very low density lipoprotein-triglyceride complex, secondary to either reduced synthesis of the relevant proteins or compromised excretion; (3) decreased or impaired β-oxidation of FFA to adenosine triphosphate and (4) increased hepatic synthesis of fatty acids through *de novo* lipogenesis [1, 19]. Free fatty acid delivery to the liver accounts for almost two-thirds of its lipid accumulation. *De novo* lipogenesis therefore only contributes to the accumulation of hepatic fat in case of NAFLD [15].

The molecular mechanisms responsible for the accumulation of fat in the liver are complex (**Figure 2**). Certain inflammatory cytokines, particularly those derived from extrahepatic Non-Alcoholic Steatohepatitis, Liver Cirrhosis and Hepatocellular Carcinoma: The Molecular Pathways http://dx.doi.org/10.5772/intechopen.68771 5

**Figure 2.** Pathogenesis of liver steatosis. Abbreviations: FFA, free fatty acids; TG, triglycerides; IL, interleukin; TNF, tumour necrosis factor; CCL2, CC motif chemokine ligand 2; VLDL, very low density lipoproteins.

adipose tissues, can trigger this process. Insulin resistance appears to be at the centre for the massive metabolic dysregulations that initiate and aggravate hepatic steatosis. At a certain point, the simple steatosis transforms to steatohepatitis in about 20–30% of NAFLD patients [19]. A major feature in the transition from NAFLD to NASH is the appearance of hepatic inflammation [14]. This breakthrough-like process is mediated by the interplay of multiple hit factors and is orchestrated by rich network of miRNAs [20]. Currently, a number of common pathogenetic mechanisms have been proposed and characterised for the transition from simple steatosis to NASH [19]. A summary of these mechanisms is shown in **Figure 3**.

#### **2.1. Inflammation in peripheral adipose tissue**

Most patients are diagnosed with NAFLD in their 40s or 50s. Studies vary in regard to the gender distribution of NAFLD, with some suggesting that it is more common in women and

Since 1998, non-alcoholic fatty liver disease has been considered a condition with a "two-hit" course of pathogenesis, first proposed by Day and James [13], describing the role of lipid peroxidation in liver injury. The "first hit" is the development of hepatic steatosis. It was suggested that hepatic triglyceride accumulation increased the susceptibility of the liver to the "second injury hit" by inflammatory cytokines and/or adipokines, mitochondrial dysfunction and elevated oxidative stress that together promote steatohepatitis and fibrosis [14]. Alternatively, many factors may act simultaneously leading to the development of NAFLD: this hypothesis corresponds to the multihit model proposed by Tilg and Moschen [15].

Experimental and population studies have shown the links between NAFLD/NASH and development of HCC. However, the mechanisms by which NASH progresses to HCC are only beginning to be elucidated [14]. NASH is the most rapidly growing risk for liver transplantation because of HCC. Wong et al. in their study included 61,868 patients over the period 2002–2012 and found that the proportion of NASH-related HCC increased from 8.3 to 13.5%,

This increase is alarming as HCC already is the fifth most frequently diagnosed cancer and the second leading oncologic death cause worldwide [17], with increasing incidence and mortality rates in Europe [18]. Thus it is crucial to analyse molecular pathways involved in NASH-induced cirrhosis and HCC carcinogenesis. Focusing on the molecular events involved in pathogenetic chain of events from NASH to liver cirrhosis and HCC would provide not only better theoretical understanding of liver diseases preceding and following cirrhosis but would also allow to recognise predictive markers and treatment targets before

Hepatic steatosis or excessive triglyceride accumulation in the liver is a prerequisite to the histological diagnosis of NAFLD. Several mechanisms may lead to steatosis, including (1) increased fat supply because of high-fat diet or excess lipolysis in adipose tissues, which increase free fatty acid (FFA) level; (2) decreased fat export in the form of very low density lipoprotein-triglyceride complex, secondary to either reduced synthesis of the relevant proteins or compromised excretion; (3) decreased or impaired β-oxidation of FFA to adenosine triphosphate and (4) increased hepatic synthesis of fatty acids through *de novo* lipogenesis [1, 19]. Free fatty acid delivery to the liver accounts for almost two-thirds of its lipid accumulation. *De novo* lipogenesis therefore only contributes to the accumulation of hepatic fat in case

The molecular mechanisms responsible for the accumulation of fat in the liver are complex (**Figure 2**). Certain inflammatory cytokines, particularly those derived from extrahepatic

others suggesting more frequent occurrence in men [11, 12].

**2. Common pathogenetic mechanisms of NAFLD**

an increase of near 63% [16].

4 Liver Cirrhosis - Update and Current Challenges

HCC development.

of NAFLD [15].

Hypoxia and death of rapidly expanding adipocytes are considered important initiating factors of adipose tissue inflammation in obesity [19]. During inflammation, typical cytokines like tumour necrosis factor (TNF)-α, interleukin (IL)-6 and CC motif chemokine ligand 2 (CCL2) are secreted by inflammatory cells infiltrating adipose tissue [21]. TNF-α was the first pro-inflammatory cytokine detected in adipose tissue. TNF-α and IL-6 are involved in the regulation of insulin resistance [19]. TNF-α and IL-6 induce insulin resistance in adipocytes, stimulating triglyceride lipolysis and fatty acid release into the circulation. CCL2 recruits macrophages to the adipose tissue, resulting in even higher local cytokine production and perpetuating the inflammatory cycle [19]. In the liver, increased expression of hepatic IL-6 correlates with higher degree of insulin resistance in patients with suspected NAFLD [1].

At the same time, extrahepatic adipocytes are compromised in their natural ability to secrete adiponectin, an anti-inflammatory adipokine that facilitates the normal partitioning of lipid

**Figure 3.** Pathogenesis of non-alcoholic steatohepatitis. Abbreviations: CYPE1, cytochrome CYP2E1; ROS, reactive oxygen species; Fe, iron; NF-κB, nuclear factor kappaB; IL, interleukin; TNF, tumour necrosis factor; HSC, hepatic stellate cells.

to adipocytes for storage [19]. Adiponectin is a hormone secreted exclusively by adipose tissue. It has beneficial effects on lipid metabolism. In the liver, adiponectin is considered to have insulin-sensitising, anti-fibrogenic and anti-inflammatory properties by acting on hepatocytes, liver stellate cells and hepatic macrophages (Kupffer cells), respectively. Adiponectin suppresses the transportation of free fatty acids to the liver as well as gluconeogenesis and *de novo* synthesis of fats but enhances oxidisation of FFAs [21]. The adiponectin-induced suppression of aldehyde oxidase and transforming growth factor has net anti-fibrotic effect [21], while decreased release of pro-inflammatory cytokines including TNF-α reduces inflammation [1]. Decreased levels of adiponectin result in loss of these protective metabolic, antifibrotic and anti-inflammatory effects.

Together, these abnormalities accentuate fat loss from adipocytes and promote ectopic fat accumulation [19].

#### **2.2. Insulin resistance**

Obesity and type 2 diabetes mellitus, both conditions associated with peripheral insulin resistance, are frequently diagnosed in patients affected by non-alcoholic fatty liver disease [12]. Evaluating patients suffering from diabetes mellitus, NAFLD was found in 74% of them in North American study, 70% in Italian population and 35–56% in Eastern countries. In Mexico, prevalence of NASH in diabetics was 18.5%. The prevalence of NAFLD in obese patients is 57–90% in Western and 10–80% in Eastern populations. NASH is present in 15–20% patients affected by obesity. The frequency of NASH is higher in those undergoing bariatric surgery and can reach 48–60% in USA men, 20–31% in USA females and up to 80% in Taiwan patients [9, 10, 12].

Insulin resistance has also been observed in NASH patients who are not obese and those who have normal glucose tolerance [1]; however, not all people with NAFLD have increased insulin resistance. NAFLD also cannot be considered as a cause for insulin resistance but rather as a consequence [19].

Resistance to the action of insulin results in important metabolic changes, including the turnover of lipids. It is characterised not only by increased circulating insulin levels but also by increased hepatic gluconeogenesis, impaired glucose uptake by muscle, enhanced peripheral lipolysis, increased triglyceride synthesis and increased hepatic uptake of fatty acids, as well as increased release of inflammatory cytokines from peripheral adipose tissues, which are the key factors promoting accumulation of liver fat and progression of hepatic steatosis [1, 19].

#### **2.3. Lipotoxicity**

**Figure 3.** Pathogenesis of non-alcoholic steatohepatitis. Abbreviations: CYPE1, cytochrome CYP2E1; ROS, reactive oxygen species; Fe, iron; NF-κB, nuclear factor kappaB; IL, interleukin; TNF, tumour necrosis factor; HSC, hepatic

stellate cells.

6 Liver Cirrhosis - Update and Current Challenges

The term "lipotoxicity" describes the deleterious effects of excess FFA and ectopic fat accumulation resulting in organ dysfunction and/or cellular death. In obesity, excessive food intake combined with high FFA output from insulin-resistant adipose tissue surpasses the storage and oxidative capacity of tissues such as skeletal muscle, liver, or pancreatic β-cells [22]. Long-chain saturated fatty acids, as well as free cholesterol derived from *de novo* synthesis can be harmful to hepatocytes. Free cholesterol accumulation leads to liver injury through the activation of intracellular signalling pathways in Kupffer cells, liver stellate cells, and hepatocytes [19], ultimately promoting inflammation and fibrosis [23]. FFAs are redirected into noxious pathways of nonoxidative metabolism with intracellular accumulation of toxic metabolites. It is not TG accumulation *per se* that is uniquely hazardous, but rather the lipidderived metabolites that trigger the development of reactive oxygen species (ROS) and activation of inflammatory pathways [22], including up-regulation of nuclear factor kappaB, production of TNF-α and IL-6 [24], and the subsequent inflammatory reaction in the liver [1].

#### **2.4. Oxidative stress**

In the context of increased supply of fatty acids to hepatocytes, oxidative stress can occur. It is attributable to the raised levels of reactive oxygen/nitrogen species and lipid peroxidation that are generated during free fatty acid metabolism in microsomes, peroxisomes, and mitochondria [19]. NAFLD and NASH-induced oxidative stress is partly regulated through cytochrome P450 2E1 (CYP2E1) as it metabolises C10–C20 fatty acids [14] that in turn produce hepatotoxic free oxygen radical species [1]. Peroxidation of plasma and intracellular membranes may cause direct cell necrosis/apoptosis and development of megamitochondria, while ROS-induced expression of Fas-ligand on hepatocytes may induce fratricidal cell death [19]. Recent studies support the idea that oxidative stress may be a primary cause of liver fat accumulation and subsequent liver injury [25], as well as ROS may play a part in fibrosis development. Lipid peroxidation and free oxygen radical species can also deplete antioxidant stores such as glutathione, vitamin E, beta-carotene, and vitamin C, rendering the liver susceptible to oxidative injury [1].

#### **2.5. Increased hepatic iron concentration**

The degree of liver fibrosis in nonalcoholic steatohepatitis shows correlation with the concentration of iron compounds in the hepatocytes. The underlying mechanism might involve the ferric-to-ferrous reduction (switch of trivalent Fe(III) to divalent Fe(II) compounds), resulting in simultaneous production of free oxygen radicals [1]. In addition, sinusoidal iron accumulation might also have a pathogenetic role in the progression of chronic liver diseases and development of hepatocellular carcinoma [26]. However, at least in Eastern populations, disturbances of iron metabolism are rarely observed in NAFLD patients [12]. In patients without iron overload, increased ferritin level in the blood may still be associated with insulin resistance and fatty liver [27].

#### **2.6. MicroRNAs in NAFLD**

MicroRNAs are small molecules of non-coding RNA that act as large-scale molecular switches. The pathogenetic chain of events in the transition to NAFL, NASH, and liver cirrhosis is richly regulated by miRNA network: it has been estimated that approximately 54 miRNAs regulate 107 genes involved in the development of NAFLD. The up-regulation of miR-26b and down-regulation of miR-26a decrease insulin sensitivity, while lower levels of miR-451 are associated with pro-inflammatory background. The up-regulation of miR-155 and miR-107 promotes fat accumulation in liver cells. Enhanced fibrosis is mediated by miR-21. Assessing patients with NAFLD-associated liver fibrosis, at least 9 miRNAs are expressed in modified levels, including higher expression of miR-31, miR-182, miR-183, miR-224, and miR-150 as well as down-regulated levels of miR-17, miR-378i, miR-219a, and miR-590. In the progression of liver fibrosis, the normally high levels of miR-22 and miR-125b are suppressed. The miR-29 family showing anti-fibrotic action in many organs is also suppressed [20].

## **3. NASH-induced liver cirrhosis**

the activation of intracellular signalling pathways in Kupffer cells, liver stellate cells, and hepatocytes [19], ultimately promoting inflammation and fibrosis [23]. FFAs are redirected into noxious pathways of nonoxidative metabolism with intracellular accumulation of toxic metabolites. It is not TG accumulation *per se* that is uniquely hazardous, but rather the lipidderived metabolites that trigger the development of reactive oxygen species (ROS) and activation of inflammatory pathways [22], including up-regulation of nuclear factor kappaB, production of TNF-α and IL-6 [24], and the subsequent inflammatory reaction in the liver [1].

In the context of increased supply of fatty acids to hepatocytes, oxidative stress can occur. It is attributable to the raised levels of reactive oxygen/nitrogen species and lipid peroxidation that are generated during free fatty acid metabolism in microsomes, peroxisomes, and mitochondria [19]. NAFLD and NASH-induced oxidative stress is partly regulated through cytochrome P450 2E1 (CYP2E1) as it metabolises C10–C20 fatty acids [14] that in turn produce hepatotoxic free oxygen radical species [1]. Peroxidation of plasma and intracellular membranes may cause direct cell necrosis/apoptosis and development of megamitochondria, while ROS-induced expression of Fas-ligand on hepatocytes may induce fratricidal cell death [19]. Recent studies support the idea that oxidative stress may be a primary cause of liver fat accumulation and subsequent liver injury [25], as well as ROS may play a part in fibrosis development. Lipid peroxidation and free oxygen radical species can also deplete antioxidant stores such as glutathione, vitamin E, beta-carotene, and vitamin C, rendering the liver sus-

The degree of liver fibrosis in nonalcoholic steatohepatitis shows correlation with the concentration of iron compounds in the hepatocytes. The underlying mechanism might involve the ferric-to-ferrous reduction (switch of trivalent Fe(III) to divalent Fe(II) compounds), resulting in simultaneous production of free oxygen radicals [1]. In addition, sinusoidal iron accumulation might also have a pathogenetic role in the progression of chronic liver diseases and development of hepatocellular carcinoma [26]. However, at least in Eastern populations, disturbances of iron metabolism are rarely observed in NAFLD patients [12]. In patients without iron overload, increased ferritin level in the blood may still be associated with insulin resis-

MicroRNAs are small molecules of non-coding RNA that act as large-scale molecular switches. The pathogenetic chain of events in the transition to NAFL, NASH, and liver cirrhosis is richly regulated by miRNA network: it has been estimated that approximately 54 miRNAs regulate 107 genes involved in the development of NAFLD. The up-regulation of miR-26b and down-regulation of miR-26a decrease insulin sensitivity, while lower levels of miR-451 are associated with pro-inflammatory background. The up-regulation of miR-155 and miR-107 promotes fat accumulation in liver cells. Enhanced fibrosis is mediated by miR-21. Assessing

**2.4. Oxidative stress**

8 Liver Cirrhosis - Update and Current Challenges

ceptible to oxidative injury [1].

tance and fatty liver [27].

**2.6. MicroRNAs in NAFLD**

**2.5. Increased hepatic iron concentration**

Liver cirrhosis develops (**Table 1**) when simple steatosis progresses to steatohepatitis and then fibrosis [11]. The composition of the hepatic fibrosis is similar regardless of the cause of injury as it follows the paradigm for wound healing in other tissues, including skin, lung and kidney. Fibrosis occurs first in regions of most severe injury over several months to years of ongoing tissue damage [23, 28, 29].


**Table 1.** The key structures in the development of liver cirrhosis.

Cryptogenic cirrhosis is the end stage of a chronic liver disease in which the underlying aetiology remains unknown after extensive clinical, serological and pathological evaluation [30, 31]. In different studies, 3–30% of liver cirrhosis cases have been attributed to the cryptogenic group [9]. Naturally, occasionally the diagnosis of cryptogenic cirrhosis is issued just due to lack of information despite the definition demanding complete investigation. Studying explanted livers of cirrhotic patients undergoing liver transplantation and having preoperative diagnosis of cryptogenic cirrhosis, specific cause was identified in 28.6% of cases. The relevant diagnoses included autoimmune hepatitis, sarcoidosis, primary biliary cirrhosis, sclerosing cholangitis, congenital hepatic fibrosis and Wilson's disease [32]. Other data/investigational methods can yield significant information as well. For instance, a significant fraction of cases initially diagnosed as cryptogenic liver cirrhosis can be associated with occult hepatitis B infection [33].

Recent evidence suggests that cryptogenic cirrhosis is strongly associated with development of HCC, while in a varying percentage (6.9–50%) of HCC, the underlying aetiology of liver disease cannot be determined. In a retrospective study of 641 HCC patients, cryptogenic cirrhosis was found in 44 (6.9%) cases, characterised also by more frequent occurrence of obesity and diabetes mellitus than in patients having history of chronic viral hepatitis and alcohol abuse. Considering the known association between obesity, diabetes and NASH, it was hypothesised that NASH is the precursor of cryptogenic cirrhosis and hepatocellular carcinoma [34].

At present, there is strong evidence that cryptogenic cirrhosis represents the end state of NASH at least in a fraction of patients. First, the progression of fibrosis in NASH is associated with gradual loss of fat vacuoles. Thus, the specific morphological changes would be burned out when the cirrhosis develops. Second, patients diagnosed with cryptogenic cirrhosis have high prevalence of metabolic changes as type 2 diabetes mellitus, obesity, or history of those disorders. If the history of preceding diabetes mellitus or obesity or liver biopsy revealing NAFLD is considered as the diagnostic criteria, 30–75% of cryptogenic cirrhosis cases can be retrospectively associated with NASH [9]. Third, due to growing awareness of the entity of NASH-induced cirrhosis, direct evidence has been brought by data obtained in explanted livers. Cases that were clinically diagnosed as cryptogenic cirrhosis were reclassified as NAFLD (either cirrhosis or pre-cirrhotic stage) in 78.6% of cases [12, 35, 36].

In comparison with liver cirrhosis due to other aetiologies, NASH-induced cirrhosis is diagnosed in older patients. Higher cardiovascular mortality is observed, in addition to the classic complications of liver cirrhosis attributable to portal hypertension and oesophageal variceal bleeding, infections and renal failure [9].

In a population-based, large study, carried out in the United Kingdom, the following distribution of cirrhosis by the cause was found (in patients, diagnosed in 1987–2006): alcoholinduced, 56.1%; cryptogenic, 20.8%; attributable to viral hepatitis, 12.0%; autoimmune or metabolic (i.e., in this study—haemochromatosis or alpha-1-antitrypsin deficiency), 11.0% [37]. In a nationwide Danish study regarding 11,605 patients diagnosed with liver cirrhosis in 1977–1989, 61.7% of cases were alcohol-induced, 2.8%—attributable to primary biliary cirrhosis, 14.6%—related to chronic hepatitis (including autoimmune inflammation) and 20.9%—

non-specified [38]. Regarding the cause of cirrhosis in explanted livers, 48.6% were related to chronic viral hepatitis (31.1% to HCV and 15.9% to HBV, 1.6% to HCV and HBV coinfection), 23.1% to alcohol-induced liver damage and 16.7% to NAFLD [36]. The data on explanted livers may not reflect the true incidence of NASH-induced cirrhosis as NAFLD patients are less likely to receive transplant. The probability to receive liver transplant within 1 year is 40.5% in NAFLD, contrasting with 47% for hepatitis C or alcohol-induced cirrhosis. The difference is the result of several factors: contraindications due to morbid obesity, comorbidities, older physiologic age, impaired renal function as well as slower disease progression [9].

Cryptogenic cirrhosis is the end stage of a chronic liver disease in which the underlying aetiology remains unknown after extensive clinical, serological and pathological evaluation [30, 31]. In different studies, 3–30% of liver cirrhosis cases have been attributed to the cryptogenic group [9]. Naturally, occasionally the diagnosis of cryptogenic cirrhosis is issued just due to lack of information despite the definition demanding complete investigation. Studying explanted livers of cirrhotic patients undergoing liver transplantation and having preoperative diagnosis of cryptogenic cirrhosis, specific cause was identified in 28.6% of cases. The relevant diagnoses included autoimmune hepatitis, sarcoidosis, primary biliary cirrhosis, sclerosing cholangitis, congenital hepatic fibrosis and Wilson's disease [32]. Other data/investigational methods can yield significant information as well. For instance, a significant fraction of cases initially diagnosed as cryptogenic liver cirrhosis can be associated with occult

Recent evidence suggests that cryptogenic cirrhosis is strongly associated with development of HCC, while in a varying percentage (6.9–50%) of HCC, the underlying aetiology of liver disease cannot be determined. In a retrospective study of 641 HCC patients, cryptogenic cirrhosis was found in 44 (6.9%) cases, characterised also by more frequent occurrence of obesity and diabetes mellitus than in patients having history of chronic viral hepatitis and alcohol abuse. Considering the known association between obesity, diabetes and NASH, it was hypothesised that NASH is the precursor of cryptogenic cirrhosis and hepatocellular

At present, there is strong evidence that cryptogenic cirrhosis represents the end state of NASH at least in a fraction of patients. First, the progression of fibrosis in NASH is associated with gradual loss of fat vacuoles. Thus, the specific morphological changes would be burned out when the cirrhosis develops. Second, patients diagnosed with cryptogenic cirrhosis have high prevalence of metabolic changes as type 2 diabetes mellitus, obesity, or history of those disorders. If the history of preceding diabetes mellitus or obesity or liver biopsy revealing NAFLD is considered as the diagnostic criteria, 30–75% of cryptogenic cirrhosis cases can be retrospectively associated with NASH [9]. Third, due to growing awareness of the entity of NASH-induced cirrhosis, direct evidence has been brought by data obtained in explanted livers. Cases that were clinically diagnosed as cryptogenic cirrhosis were reclassified as NAFLD

In comparison with liver cirrhosis due to other aetiologies, NASH-induced cirrhosis is diagnosed in older patients. Higher cardiovascular mortality is observed, in addition to the classic complications of liver cirrhosis attributable to portal hypertension and oesophageal variceal

In a population-based, large study, carried out in the United Kingdom, the following distribution of cirrhosis by the cause was found (in patients, diagnosed in 1987–2006): alcoholinduced, 56.1%; cryptogenic, 20.8%; attributable to viral hepatitis, 12.0%; autoimmune or metabolic (i.e., in this study—haemochromatosis or alpha-1-antitrypsin deficiency), 11.0% [37]. In a nationwide Danish study regarding 11,605 patients diagnosed with liver cirrhosis in 1977–1989, 61.7% of cases were alcohol-induced, 2.8%—attributable to primary biliary cirrhosis, 14.6%—related to chronic hepatitis (including autoimmune inflammation) and 20.9%—

(either cirrhosis or pre-cirrhotic stage) in 78.6% of cases [12, 35, 36].

bleeding, infections and renal failure [9].

hepatitis B infection [33].

10 Liver Cirrhosis - Update and Current Challenges

carcinoma [34].

Thus, cryptogenic cirrhosis is a significant burden for health care systems. Patients undergoing liver transplantation for cryptogenic cirrhosis are subjected to higher postoperative mortality, lower cumulative 5- and 10-year survival and higher rate of chronic rejection [32]. NASH is the most rapidly growing indication for simultaneous liver and kidney transplantation. NASH and cryptogenic cirrhosis in patients having body mass index greater than 30 kg/m<sup>2</sup> constituted 6.3% in the years 2002–2003 but 19.2% in the years 2010–2011 [39].

As the liver becomes fibrotic, significant changes occur in the extracellular matrix (ECM) quantitatively and qualitatively. ECM refers to macromolecules that comprise the scaffolding of either normal or fibrotic liver. These include collagens, non-collagen glycoproteins, matrix-bound growth factors, glycosaminoglycans, proteoglycans and matricellular proteins. In case of fibrosis, the total collagen content increases 3- to 10-fold including an increase in fibril-forming collagens (i.e., types I, III and IV) and some non-fibril forming collagens (types IV and VI). Glycoproteins (fibronectin; laminin; secreted protein, acidic and rich in cysteine: SPARC; osteonectin; tenascin, and von Willebrand factor), proteoglycans and glycosaminoglycans (perlecan, decorin, aggrecan, lumican, and fibromodulin) also accumulate in cirrhotic liver. Particularly notable is the shift from heparan sulphate-containing proteoglycans to those containing chondroitin and dermatan sulphates. These processes represent a change in the type of ECM in subendothelial space from the normal low-density basement membranelike matrix to the interstitial type.

The replacement of the low-density matrix with the interstitial type influences the function of hepatocytes, liver stellate cells, and endothelium of blood vessels: the microvilli disappear on the surface of liver parenchymal cells, and endothelium loses fenestrations precluding effective molecule exchange between blood and liver parenchyma. In addition, stellate cells undergo activation [23].

The hepatic stellate cell is the primary source of ECM in normal and fibrotic liver. Hepatic stellate cells, located in subendothelial space of Disse between hepatocytes and sinusoidal endothelial cells, represent one-third of the non-parenchymal population or approximately 15% of the total number of resident cells in normal liver. Stellate cells comprise a heterogeneous group of cells that are functionally and anatomically similar but differ in their expression of cytoskeletal filaments, retinoid content, and potential for activation. Stellate cells with fibrogenic potential are not confined to liver and have been identified in other organs such as the pancreas, where they contribute to desmoplasia in chronic pancreatitis and carcinoma. Hepatic stellate cell activation is the common pathway leading to hepatic fibrosis. During activation, stellate cells undergo a transition from a quiescent vitamin A-rich cell into proliferating, fibrogenic, and contractile myofibroblasts [23], which have strong ability to secrete collagen and migrate to the area of necrosis and inflammation [40]. Proliferation of stellate cells occurs predominantly in regions of greatest injury.

Considering liver fibrosis, the balance between synthesis and degradation of extracellular matrix also is of importance as enhanced destruction of the normal matrix in the space between hepatocytes and endothelial cells leads to accumulation of dense scar tissue. Degradation occurs through the actions of at least four enzymes: matrix metalloproteinase (MMP) 2 and MMP9, which degrade type IV collagen; membrane-type metalloproteinase 1 or 2, which activate latent MMP2 and stromelysin 1, which degrades proteoglycans and glycoproteins and activates latent collagenases. Stellate cells are the principal source of MMP2 and stromelysin. Activation of latent MMP2 may require interaction with hepatocytes. Markedly increased expression of MMP2 is a characteristic of cirrhosis. MMP9 is secreted locally by Kupffer cells. Disruption of the normal liver matrix is also a prerequisite for tumour invasion and stromal desmoplasia.

The cytochrome CYP2E1 may have an important role in the generation of reactive oxygen species that stimulate liver stellate cells. Cultured hepatic stellate cells grown in the presence of CYP2E1-expressing cells increase the production of collagen, an effect prevented by antioxidants or a CYP2E1 inhibitor. These data suggest that the CYP2E1-derived reactive oxygen species are responsible for the increased collagen production. Such findings may help to explain the pathogenesis of liver injury in alcoholic liver disease since CYP2E1 is alcohol inducible. As noted above, reactive oxygen species are generated through lipid peroxidation from hepatocytes, macrophages, stellate cells, and inflammatory cells. In alcoholic or nonalcoholic steatohepatitis, ROS generation in hepatocytes results from induction of cytochrome P450 2E1, leading to pericentral (zone 3) injury. Also, oxidase of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) mediates fibrogenic activation of hepatic stellate cells, as well as of Kupffer cells or resident liver macrophages through generation of oxidative stress. Increasing knowledge about NADPH oxidase isoforms and their cell-specific activities is leading to their emergence as a therapeutic target [23].

Pathology of telomeres and the related molecular events represent another key mechanism that is associated both with induction of liver steatosis and progression of NAFLD [41]. Telomerase mutations can accelerate progression of chronic liver disease to cirrhosis [42]. Missense mutations in telomerase reverse transcriptase hTERT are found more frequently in cirrhosis regardless of aetiology [41]. Thus, missense mutations were observed in 7% of cirrhotic patients in USA [43]. Functional mutations were identified in 3% of German patients affected by cirrhosis [44].

Telomeres are repeated, short DNA sequences (in humans—TTAGGG) located at the chromosome end. These structures prevent chromosomal end-to-end fusion as well as protect the coding DNA from progressive loss at mitosis. During each mitosis, the DNA polymerase complex cannot replicate the terminal 5′ end of the lagging strand. Consequently, the chromosomal end is lost. Due to the presence of telomeres, this loss is limited to telomeres. However, the telomeres shorten in each mitosis. Telomere attrition is especially marked in chronic diseases associated with increased cell loss and proliferation. When they become critically short, cellular ageing *s*. senescence and apoptosis follows. To ensure the unlimited proliferation of cancer, malignant cells maintain telomere length via different mechanisms. The most significant ones include telomerase reverse transcriptase hTERT, its RNA template: telomerase RNA component hTERC, the hTERC-protecting and stabilising dyskerin complex (consisting of four nucleolar proteins) and shelterin complex, including six proteins [41].

NAFLD is characterised by telomere shortening and increased cellular senescence in comparison to healthy controls [45]. The changes in telomeres represent an important mechanism in the transition to liver cirrhosis. However, dual effects are observed. In progressing chronic liver disease, cellular senescence enhances the loss of parenchyma, limiting the replicative potential of hepatocytes. In contrast, in advanced liver damage, the ageing of stellate cells stops the remodelling and thus, the further progression of fibrosis. Still another prognostic aspect can be involved regarding HCC development: senescent stellate cells can promote carcinogenesis by secreting pro-carcinogenic mediators. These changes are described as the senescence-associated secretory program [41]. The extent of fibrosis in NAFLD is associated with p21 protein representing another molecular regulator of cellular senescence [41].

Although shorter telomeres are considered a hallmark of liver cirrhosis regardless of aetiology [41], the telomeres in NAFLD patients are shorter than in those affected by cryptogenic cirrhosis. In NAFLD, telomere length correlates with the level of hTERT mRNA, while hTERT-independent mechanisms already start to operate in cryptogenic cirrhosis [45].

## **4. NASH-induced HCC**

proliferating, fibrogenic, and contractile myofibroblasts [23], which have strong ability to secrete collagen and migrate to the area of necrosis and inflammation [40]. Proliferation of

Considering liver fibrosis, the balance between synthesis and degradation of extracellular matrix also is of importance as enhanced destruction of the normal matrix in the space between hepatocytes and endothelial cells leads to accumulation of dense scar tissue. Degradation occurs through the actions of at least four enzymes: matrix metalloproteinase (MMP) 2 and MMP9, which degrade type IV collagen; membrane-type metalloproteinase 1 or 2, which activate latent MMP2 and stromelysin 1, which degrades proteoglycans and glycoproteins and activates latent collagenases. Stellate cells are the principal source of MMP2 and stromelysin. Activation of latent MMP2 may require interaction with hepatocytes. Markedly increased expression of MMP2 is a characteristic of cirrhosis. MMP9 is secreted locally by Kupffer cells. Disruption of the normal liver matrix is also a prerequisite for tumour invasion and stromal

The cytochrome CYP2E1 may have an important role in the generation of reactive oxygen species that stimulate liver stellate cells. Cultured hepatic stellate cells grown in the presence of CYP2E1-expressing cells increase the production of collagen, an effect prevented by antioxidants or a CYP2E1 inhibitor. These data suggest that the CYP2E1-derived reactive oxygen species are responsible for the increased collagen production. Such findings may help to explain the pathogenesis of liver injury in alcoholic liver disease since CYP2E1 is alcohol inducible. As noted above, reactive oxygen species are generated through lipid peroxidation from hepatocytes, macrophages, stellate cells, and inflammatory cells. In alcoholic or nonalcoholic steatohepatitis, ROS generation in hepatocytes results from induction of cytochrome P450 2E1, leading to pericentral (zone 3) injury. Also, oxidase of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) mediates fibrogenic activation of hepatic stellate cells, as well as of Kupffer cells or resident liver macrophages through generation of oxidative stress. Increasing knowledge about NADPH oxidase isoforms and their cell-specific activities

Pathology of telomeres and the related molecular events represent another key mechanism that is associated both with induction of liver steatosis and progression of NAFLD [41]. Telomerase mutations can accelerate progression of chronic liver disease to cirrhosis [42]. Missense mutations in telomerase reverse transcriptase hTERT are found more frequently in cirrhosis regardless of aetiology [41]. Thus, missense mutations were observed in 7% of cirrhotic patients in USA [43]. Functional mutations were identified in 3% of German patients

Telomeres are repeated, short DNA sequences (in humans—TTAGGG) located at the chromosome end. These structures prevent chromosomal end-to-end fusion as well as protect the coding DNA from progressive loss at mitosis. During each mitosis, the DNA polymerase complex cannot replicate the terminal 5′ end of the lagging strand. Consequently, the chromosomal end is lost. Due to the presence of telomeres, this loss is limited to telomeres. However, the telomeres shorten in each mitosis. Telomere attrition is especially marked in chronic diseases associated with increased cell loss and proliferation. When they become critically short, cellular

stellate cells occurs predominantly in regions of greatest injury.

12 Liver Cirrhosis - Update and Current Challenges

is leading to their emergence as a therapeutic target [23].

desmoplasia.

affected by cirrhosis [44].

Although the association between NAFLD and HCC was first observed more than two decades ago, mostly through NASH-induced cirrhosis [11], the molecular events that link NAFLD and HCC are still incompletely understood. Following the general principles of cancerogenesis, HCC in cirrhotic liver develops by dysplasia—carcinoma pathway: from a dysplastic cirrhotic nodule. The process is slow and can last for several decades [34]. The genetic events that are prerequisite for malignant change develop in the background of increased cellular proliferation. Hypothetically, it is possible that the molecular portrait of HCC in DNA, mRNA, microRNA and protein level is different in accordance to the inciting factor of the underlying liver disease. If this is true, specific molecular targets may exist for the diagnostics, prevention or treatment of NASH-induced HCC or HCC arising in diabetic and/or obese patients [10].

The course of HCC that is associated with cryptogenic cirrhosis differs from HCC developing in other clinical settings [46]. HCC also varies by epigenetic signature in accordance to the cause [47].

The risk of hepatocellular carcinoma differs by the aetiology of cirrhosis. To estimate this, a large population-based study was carried out in the United Kingdom. All patients diagnosed with liver cirrhosis were identified, and the results were compared to national cancer registry identifying those diagnosed with HCC. The 10-year cumulative incidence of HCC was 4% in cirrhosis induced by chronic viral hepatitis, 3.2% in cirrhosis due to autoimmune or metabolic (in this study—haemochromatosis, alpha-1-antitrypsin deficiency) diseases, 1.2% in alcohol-induced cirrhosis and 1.1% in cryptogenic cirrhosis, while the same estimates at 1 year were 1.0, 0.8, 0.3 and 0.3%, respectively. This study has the significant benefit of exploring HCC risk in patients that differ by aetiology of cirrhosis but belong to the same population [37]. Considering patients referred for liver transplantation, the frequency of hepatocellular carcinoma in cryptogenic cirrhosis is lower (8%) than in cirrhosis related to chronic hepatitis B (29%) or C (19%) as reported by Alamo et al. [32]. For the epidemiological estimates of HCC in different liver pathology, see also **Table 2** [37, 38].

The causal distribution of HCC shows geographic variations. Thus, in Canadian patients, 45% of cases were attributable to alcohol-induced cirrhosis, 26% to cryptogenic cirrhosis and 13% to hepatitis C. In patients from Saudi Arabia, 47% of HCC were caused by hepatitis C, 27% by cryptogenic cirrhosis and 21% to hepatitis B [48]. In USA, regarding the HCC cause, 54.9% of cases were induced by HCV, 16.4% by alcohol, 14.1% by NAFLD and 9.5% by HBV [10]. In explanted livers, 81.8% of HCC were associated with viral hepatitis, 9.1% with alcohol-induced liver damage and 9.1% with NAFLD [36].

In the USA, the number of NAFLD-associated HCC cases is annually growing for 9%, if the time span 2004–2009 is evaluated [10]. In Europe, NAFLD-related HCC comprised 35% of all HCC cases in 2010. HCC that is not related to hepatitis B or C is becoming increasingly frequent in Japan as well; however, here, it comprises only 10% of all HCC cases [53]. NASH is responsible for higher percentage of HCC in Western than in Eastern societies [12].

Hepatocellular carcinoma in patients affected by metabolic syndrome has distinct morphology [49]. NAFLD-associated HCC is characterised by larger size [34] and moderate or high differentiation degree [34], showing high differentiation as frequently as in 65% of cases [49]. However, the tumours lack capsule thus confirming the true malignant biological potential [34]. This is an important diagnostic trait considering the association between NAFLD, low-grade HCC [49], and liver adenomatosis [50].

The prognostic estimates are somewhat controversial. The NAFLD-associated hepatocellular carcinomas are diagnosed as more advanced tumours in older patients showing higher cardiovascular morbidity. The patients are less likely to receive liver transplant and have higher


Abbreviations: SIR, standardised incidence ratio; CI, confidence interval.

1 Primary biliary cirrhosis.

2 Including viral and autoimmune causes. 3 Viral hepatitis.

**Table 2.** Epidemiological estimates of hepatocellular carcinoma by the cause of chronic advanced liver pathology.

tumour-specific mortality [10]. HCC associated with cryptogenic cirrhosis is larger than cancers related to HCV even in patients who correspond to Milan criteria [51]. However, after curative treatment, the recurrence risk and mortality are lower for HCC arising in cryptogenic cirrhosis—finding that is in accordance with the grade difference [52].

in alcohol-induced cirrhosis and 1.1% in cryptogenic cirrhosis, while the same estimates at 1 year were 1.0, 0.8, 0.3 and 0.3%, respectively. This study has the significant benefit of exploring HCC risk in patients that differ by aetiology of cirrhosis but belong to the same population [37]. Considering patients referred for liver transplantation, the frequency of hepatocellular carcinoma in cryptogenic cirrhosis is lower (8%) than in cirrhosis related to chronic hepatitis B (29%) or C (19%) as reported by Alamo et al. [32]. For the epidemiological estimates of HCC in

The causal distribution of HCC shows geographic variations. Thus, in Canadian patients, 45% of cases were attributable to alcohol-induced cirrhosis, 26% to cryptogenic cirrhosis and 13% to hepatitis C. In patients from Saudi Arabia, 47% of HCC were caused by hepatitis C, 27% by cryptogenic cirrhosis and 21% to hepatitis B [48]. In USA, regarding the HCC cause, 54.9% of cases were induced by HCV, 16.4% by alcohol, 14.1% by NAFLD and 9.5% by HBV [10]. In explanted livers, 81.8% of HCC were associated with viral hepatitis, 9.1% with alcohol-induced liver damage and

In the USA, the number of NAFLD-associated HCC cases is annually growing for 9%, if the time span 2004–2009 is evaluated [10]. In Europe, NAFLD-related HCC comprised 35% of all HCC cases in 2010. HCC that is not related to hepatitis B or C is becoming increasingly frequent in Japan as well; however, here, it comprises only 10% of all HCC cases [53]. NASH is

Hepatocellular carcinoma in patients affected by metabolic syndrome has distinct morphology [49]. NAFLD-associated HCC is characterised by larger size [34] and moderate or high differentiation degree [34], showing high differentiation as frequently as in 65% of cases [49]. However, the tumours lack capsule thus confirming the true malignant biological potential [34]. This is an important diagnostic trait considering the association between NAFLD, low-grade HCC [49],

The prognostic estimates are somewhat controversial. The NAFLD-associated hepatocellular carcinomas are diagnosed as more advanced tumours in older patients showing higher cardiovascular morbidity. The patients are less likely to receive liver transplant and have higher

**Chronic hepatitis Cryptogenic** 

**cirrhosis**

25.2–67.3 43.4; 30.3–60.4 Sorensen et al.

4.3–13.4 3.1; 1.6–5.9 West et al. [37]

**Reference**

[38]

responsible for higher percentage of HCC in Western than in Eastern societies [12].

**Autoimmune and genetic diseases**

3.2; 2.1–4.8 5.3; 2.6–10.5 7.6;<sup>3</sup>

Abbreviations: SIR, standardised incidence ratio; CI, confidence interval.

12.6–120.2 42.7;<sup>2</sup>

**Table 2.** Epidemiological estimates of hepatocellular carcinoma by the cause of chronic advanced liver pathology.

different liver pathology, see also **Table 2** [37, 38].

14 Liver Cirrhosis - Update and Current Challenges

9.1% with NAFLD [36].

and liver adenomatosis [50].

**Estimate Alcohol-induced** 

Incidence rate per 1000 person years; 95% CI

Viral hepatitis.

Primary biliary cirrhosis.

1

2

3

**cirrhosis**

SIR; 95% CI 70.6; 59.5–83.2 47.0;<sup>1</sup>

Including viral and autoimmune causes.

Although previously it was considered that HCC risk is limited to cirrhotic patients, currently at least 25–30% of NAFLD-related hepatocellular carcinomas develop in the absence of cirrhosis [9]. In Japanese group, 33% of NAFLD-related HCC occurred in the background of none or mild fibrosis contrasting with only 16% in alcohol-induced HCC [53]. According to other researchers, up to 65% of NAFLD-associated HCC evolve in the absence of fibrosis [49]. The proportion of NAFLD-associated HCC developing in non-cirrhotic liver has been variably estimated as 15, 38, or 49% [54–57]. These tumours tend to be larger [57].

The development of HCC in noncirrhotic liver has been associated with malignant transformation in liver cell adenoma [34, 49]. Malignant change in hepatic adenoma correlates with metabolic syndrome [58]. Inflammatory molecular type of liver cell adenoma shows clinical correlation with obesity. The underlying molecular basis could include either activated IL-6 signalling or hyperoestrogenemia associated with obesity. However, a controversy exists here as inflammatory type of liver adenoma is not prone to malignisation [50].

Several pathogenetic ways account for a tumour-promoting environment in obesity and diabetes, allowing to distinguish the pathogenesis of HCC linked to NAFLD from that of viral and other aetiologies.

Obesity has been linked to higher frequency of cancers in a variety of tissues [59, 60] including the liver (**Table 3**). HCC is increasingly diagnosed among obese individuals. In a prospective cohort of the Cancer Prevention Study with more than 900,000 North American subjects, the relative risk of dying from liver cancer among men with a body mass index reaching or exceeding 35 kg/m<sup>2</sup> was remarkably higher (4.5 fold) compared to a reference group with normal body weight. In a large cohort involving 362,552 Swedish men, the relative risk of HCC in individuals with a body mass index reaching or exceeding 30 kg/m<sup>2</sup> was 3.1 fold higher than in controls having normal weight. Studies from other parts of the world indicate that the link between obesity and increased incidence of HCC has been globally recognised [61].

Obesity has a significant tumour-promoting effect regarding HCC. This effect largely depends on the chronic general low-grade inflammatory response it induces, which involves production of TNF-α and IL-6. Both these molecular mediators are tumour-promoting cytokines [62] and major drivers of cell proliferation in NAFLD and NASH [21]. TNF-α and other mediators produced by activated inflammatory macrophages stimulate compensatory hepatocyte proliferation and expand HCC progenitors. TNF-α further reinforces the inflammatory microenvironment and induces expression of chemokines (CCL2, CCL7 and CXCL13) and growth factors/cytokines (IL-1β, IL-6, TNF–α itself and hepatocyte growth factor) both by progenitors of hepatocellular carcinoma and surrounding cells [63]. TNF-α up-regulates the cellular proliferation through the molecular pathways of nuclear factor kappaB, mTOR and wide spectrum of kinases. The proliferative and anti-apoptotic activities of IL-6 are largely mediated through the signal transducer and activator of transcription 3, STAT3 [10]. IL-6 also


**Table 3.** Obesity-related human cancers [60].

contributes to the metabolic background of cancer sustaining insulin resistance that can be improved by systemic neutralization of IL-6 [64].

Another mechanism involved in the progression of NAFLD to HCC in obese individuals is the imbalance between leptin and adiponectin. Particularly, obesity is linked to increased levels of leptin [34]. Apart from its role in obesity-associated insulin resistance and inflammation, leptin is a pro-inflammatory, pro-angiogenic, and pro-fibrogenic cytokine with a growth-promoting effect by activating the Janus kinase/STAT, phosphoinositide 3-kinase (PI3K)/Akt, and extracellular signal-regulated kinase (ERK) signalling pathways [61]. The up-regulation of PI3K/Akt pathway leads to activation of downstream molecular mediator mTOR that is found in 40% of HCC cases. Leptin-induced up-regulation of mTOR also inhibits autophagy—a process that normally would limit oxidative stress by removing damaged mitochondria. Suppression of autophagy, in turn, increases oxidative tissue damage and subsequent inflammation [21]. Since leptin exerts pro-inflammatory and pro-fibrogenic effects by activating Kupffer cells and stellate cells, it has been associated to disease progression in fibrotic NAFLD [10]. Leptin can also promote invasion and migration of hepatocellular carcinoma cells [65].

Adiponectin, another major adipokine with potent anti-inflammatory, antiangiogenic and tumour growth-limiting properties, is suppressed in obesity [15, 24]. Adiponectin activates 5′-adenosine monophosphate–activated protein kinase, which can suppress tumour growth and increase apoptosis by regulating the mTOR and c-Jun N-terminal kinase/caspase 3 pathways. Moreover, adiponectin opposes the effects of leptin by inhibiting activation of Akt and STAT3, as well as by increasing the expression of SOCS3: the suppressor of cytokine signalling 3 [61]. Thus, low adiponectin levels may be insufficient to suppress endotoxin-mediated inflammatory signalling in Kupffer cells and other macrophages, as well as control angiogenesis, a pivotal mechanism of tumour growth [10]. Microarray analysis of tissue adiponectin levels in HCC patients revealed that adiponectin expression was inversely correlated with tumour size, supporting the hypothesis that adiponectin may inhibit proliferation and dedifferentiation [66].

HCC can show marked accumulation of fat within the neoplastic cells (**Figure 4**). In a study by Salomao et al., 36% of patients who developed HCC in the setting of steatohepatitis were diagnosed as having a steatohepatitic variant of HCC as compared to 1.3% of HCC patients without steatohepatitis [67]. Increased intensity of fatty acid synthesis and characteristic pattern of perilipin proteins has been demonstrated in HCC. Regarding gene expression pattern, activated lipogenesis is associated with higher cell proliferation and worse prognosis in HCC [10]. Hypothetically, HCC cells might benefit from the energetic value of fat compounds or use lipids as building blocks of new cells.

Lipotoxicity, defined as the cellular dysfunction caused by ectopic deposition of fat in nonadipose tissues, may contribute to the development of HCC in NAFLD. Activated oxidation of fatty acids generates high burden of free radicals and lipid peroxide compounds that oxidise and damage large molecules and cell organoids, e.g., mitochondria and endoplasmic reticulum. The damaged cells are subjected to apoptosis, leading to higher activity in liver destruction and progression towards cirrhosis that in turn is closely associated with enhanced proliferation and accumulation of genetic damage. Accumulation of fatty acids may interfere with cellular signalling and promote oncogenesis through altered regulation of gene transcription [10]. Oxidative stress can induce mutations in the tumour suppressor gene *TP53* in a pattern observed in HCC [68].

contributes to the metabolic background of cancer sustaining insulin resistance that can be

Another mechanism involved in the progression of NAFLD to HCC in obese individuals is the imbalance between leptin and adiponectin. Particularly, obesity is linked to increased levels of leptin [34]. Apart from its role in obesity-associated insulin resistance and inflammation, leptin is a pro-inflammatory, pro-angiogenic, and pro-fibrogenic cytokine with a growth-promoting effect by activating the Janus kinase/STAT, phosphoinositide 3-kinase (PI3K)/Akt, and extracellular signal-regulated kinase (ERK) signalling pathways [61]. The up-regulation of PI3K/Akt pathway leads to activation of downstream molecular mediator mTOR that is found in 40% of HCC cases. Leptin-induced up-regulation of mTOR also inhibits autophagy—a process that normally would limit oxidative stress by removing damaged mitochondria. Suppression of autophagy, in turn, increases oxidative tissue damage and subsequent inflammation [21]. Since leptin exerts pro-inflammatory and pro-fibrogenic effects by activating Kupffer cells and stellate cells, it has been associated to disease progression in fibrotic NAFLD [10]. Leptin can also promote invasion and migration of hepatocellular car-

Adiponectin, another major adipokine with potent anti-inflammatory, antiangiogenic and tumour growth-limiting properties, is suppressed in obesity [15, 24]. Adiponectin activates 5′-adenosine monophosphate–activated protein kinase, which can suppress tumour growth and increase apoptosis by regulating the mTOR and c-Jun N-terminal kinase/caspase 3 pathways. Moreover, adiponectin opposes the effects of leptin by inhibiting activation of Akt and STAT3, as well as by increasing the expression of SOCS3: the suppressor of cytokine signalling 3 [61].

improved by systemic neutralization of IL-6 [64].

**Table 3.** Obesity-related human cancers [60].

**Location Level of evidence**

Liver cancer Highly suggestive

Colonic cancer in females Suggestive Ovarian cancer Suggestive Prostate cancer Suggestive Thyroid cancer Suggestive Melanoma in males Weak

Oesophageal adenocarcinoma Strong Colorectal cancer in males Strong Pancreatic cancer Strong Breast cancer Strong Endometrial cancer Strong Renal cancer Strong Multiple myeloma Strong

16 Liver Cirrhosis - Update and Current Challenges

cinoma cells [65].

Adipose tissue expansion, release of pro-inflammatory cytokines, and lipotoxicity collectively promote systemic and hepatic insulin resistance, resulting in hyperinsulinemia [34]. The risk of HCC in patients affected by diabetes mellitus is 2.31 [57]. Insulin resistance and hyperinsulinemia are the most common metabolic features of NAFLD, which correlate with impaired hepatic clearance of insulin and have been linked to tumour development [69]. Deregulated metabolic effects of insulin result in excessive activation of proliferative signalling cascades.

**Figure 4.** Hepatocellular carcinoma showing nuclear atypia and presence of fat in tumour cells. Haematoxylin-eosin stain, original magnification 100× and 400×.

Hyperinsulinemia causes reduced hepatic synthesis of insulin-like growth factor (IGF) binding protein-1 and increased bioavailability of IGF1, which further promotes cellular proliferation and inhibits apoptosis [10, 34]. It has been shown recently that elevated fasting insulin, which is inversely related to insulin sensitivity, is an independent risk factor for HCC. Baseline serum levels of C-peptide have also been found to be associated with a higher risk of HCC in the general population independently of obesity and other established liver cancer risk factors [69]. Loss of heterozygosity for IGF2 has been observed in over 60% of HCC cases. This likely coincides with IGF2 overexpression, found in HCC, which has been associated to reduced apoptosis and increased cellular proliferation [68].

The importance of insulin resistance is illustrated by the observations that obesity and type 2 diabetes mellitus comprise increased HCC risk even regardless of the presence or cause of liver cirrhosis [9].

A number of studies have demonstrated a critical role for phosphatase and tensin homolog (PTEN) in the progression of NASH to tumour. *PTEN* deletion results in PKB/Akt activation, promoting proliferation and reducing apoptosis. Insulin-like growth factor 2 mRNA binding protein p62 was reported to be a possible upstream regulator of PTEN. Aberrant microRNAs contribute to carcinogenesis. MiR-21 was found to be another upstream regulator of PTEN participating in NASH-associated cancer induction [10, 14, 70].

The oral iron test has revealed increased absorption of iron compounds in patients affected by NASH [71]. In turn, increased amount of iron in liver tissues is associated with increased risk of HCC in patients affected by NASH-related liver cirrhosis [72]. As the reductive conversion of Fe(III) to Fe(II) necessitates increased oxidation of other compounds, oxidative DNA damage can develop and lead to the malignancy [34, 73]. Iron overload also is known to enhance insulin resistance [74] and to act in concert with other factors damaging liver. The significance of iron overload in hepatic carcinogenesis is shown in several models. The risk of HCC is increased in hereditary haemochromatosis, characterised by excessive iron accumulation in the body and caused by excessive absorption because of homozygous C282Y mutation in *HFE* gene. Almost 8–10% of patients with hereditary haemochromatosis develop HCC. Increased relative risk of HCC (10×) has also been demonstrated in association with long-lasting excess dietary iron intake [37, 74, 75]. Thus, there is significant evidence of the carcinogenic action of iron overload, and evidence of iron accumulation in NAFLD and especially NASH that allows drawing conclusion that iron metabolites are contributing to the development of NASH-related HCC.

The expression profile of *Wnt* signalling genes in NASH strongly suggests inhibition of Wnt pathway. IHC staining of β-catenin shows predominately membrane staining with loss of nuclear staining indicating that β-catenin is not active in NASH. In contrast, 20–90% of HCC cases exhibit active Wnt pathway [76]. Thus, the long-lasting conversion of NASH into HCC hypothetically involves up-regulation of Wnt pathway either by activators or loss of inhibitors [77].

Hepatocyte apoptosis is a prominent feature of NASH (**Figure 5**). The executing mechanism of apoptosis includes activation of characteristic lytic enzymes—the caspases. In an apoptotic

hepatocyte, activated caspase-3 is splitting various cell structures, including cytokeratin (CK) 18—the intermediary filament that represents the specific cytoskeleton protein of hepatocytes. Consequently, blood tests can reveal increased concentration of CK18 fragments [70]. In liver tissues, CK8 and CK18-containing Mallory bodies are evident by light microscopy as large, brightly eosinophilic inclusions in liver cell cytoplasm. Although Mallory hyaline is the hallmark of alcohol-induced hepatitis, its development can also be induced by diet rich in saturated fatty acids. The molecular pathways associated with Mallory body development include IL-6, protein p62 that binds ubiquitin in cell cytoplasm, and reduced concentration of HSP72 that prevents protein misfolding. The presence of CK18 in Mallory bodies correlates with plasma CK18 levels [78]. In a longitudinal paired liver biopsy study, the change of CK18 correlated with disease progression. Patients with increased NAFLD activity score 3 years after initial evaluation had greater increase of plasma CK18 compared with those who had stable or decreased activity score [79]. El-Zefzafy et al. proved that CK18 was a sensitive indicator of the severity of liver disease and also could predict the development of HCC. In their study, the sensitivity and specificity of serum CK18 were 95 and 96.7%, respectively, with a cut-off value of 534.5 U/L for HCC diagnosis [80].

Hyperinsulinemia causes reduced hepatic synthesis of insulin-like growth factor (IGF) binding protein-1 and increased bioavailability of IGF1, which further promotes cellular proliferation and inhibits apoptosis [10, 34]. It has been shown recently that elevated fasting insulin, which is inversely related to insulin sensitivity, is an independent risk factor for HCC. Baseline serum levels of C-peptide have also been found to be associated with a higher risk of HCC in the general population independently of obesity and other established liver cancer risk factors [69]. Loss of heterozygosity for IGF2 has been observed in over 60% of HCC cases. This likely coincides with IGF2 overexpression, found in HCC, which has been associated to

The importance of insulin resistance is illustrated by the observations that obesity and type 2 diabetes mellitus comprise increased HCC risk even regardless of the presence or cause of

A number of studies have demonstrated a critical role for phosphatase and tensin homolog (PTEN) in the progression of NASH to tumour. *PTEN* deletion results in PKB/Akt activation, promoting proliferation and reducing apoptosis. Insulin-like growth factor 2 mRNA binding protein p62 was reported to be a possible upstream regulator of PTEN. Aberrant microRNAs contribute to carcinogenesis. MiR-21 was found to be another upstream regulator of PTEN

The oral iron test has revealed increased absorption of iron compounds in patients affected by NASH [71]. In turn, increased amount of iron in liver tissues is associated with increased risk of HCC in patients affected by NASH-related liver cirrhosis [72]. As the reductive conversion of Fe(III) to Fe(II) necessitates increased oxidation of other compounds, oxidative DNA damage can develop and lead to the malignancy [34, 73]. Iron overload also is known to enhance insulin resistance [74] and to act in concert with other factors damaging liver. The significance of iron overload in hepatic carcinogenesis is shown in several models. The risk of HCC is increased in hereditary haemochromatosis, characterised by excessive iron accumulation in the body and caused by excessive absorption because of homozygous C282Y mutation in *HFE* gene. Almost 8–10% of patients with hereditary haemochromatosis develop HCC. Increased relative risk of HCC (10×) has also been demonstrated in association with long-lasting excess dietary iron intake [37, 74, 75]. Thus, there is significant evidence of the carcinogenic action of iron overload, and evidence of iron accumulation in NAFLD and especially NASH that allows drawing conclusion that iron metabolites are contributing to the development of

The expression profile of *Wnt* signalling genes in NASH strongly suggests inhibition of Wnt pathway. IHC staining of β-catenin shows predominately membrane staining with loss of nuclear staining indicating that β-catenin is not active in NASH. In contrast, 20–90% of HCC cases exhibit active Wnt pathway [76]. Thus, the long-lasting conversion of NASH into HCC hypothetically involves up-regulation of Wnt pathway either by activators or loss of

Hepatocyte apoptosis is a prominent feature of NASH (**Figure 5**). The executing mechanism of apoptosis includes activation of characteristic lytic enzymes—the caspases. In an apoptotic

reduced apoptosis and increased cellular proliferation [68].

participating in NASH-associated cancer induction [10, 14, 70].

liver cirrhosis [9].

18 Liver Cirrhosis - Update and Current Challenges

NASH-related HCC.

inhibitors [77].

In a study by Salomao et al., devoted to HCC in NASH, immunohistochemically there was diffuse loss of cytoplasmic CK8/18 and an increased number of activated hepatic stellate cells within the steatohepatitic HCC, identical to the pattern seen in the surrounding non-neoplastic liver [67, 81].

**Figure 5.** Apoptotic bodies (arrows) in non-alcoholic steatohepatitis. Haematoxylin-eosin stain, original magnification 400×.

The HCC development shows complex associations with telomere shortening. The senescence-associated secretory program of liver stellate cells promotes carcinogenesis. The telomere shortening induces also genomic instability thus facilitating HCC development [41]. Indeed, HCC is characterised by significantly shorter telomeres in comparison to adjacent tissues [82]. However, cancer cells still maintain unlimited proliferation. Evidently, hepatocellular carcinoma cells develop compensatory mechanisms either for telomere extension or for cellular proliferation despite telomere shortening. The elongation of telomeres again can be ensured via diverse mechanisms, including hTERT or alternative lengthening of telomeres via telomerase-independent mechanism seen in 7% of HCC cases [41].

Over the progression of HCC, the telomere length changes in contrary direction. Early liver carcinogenesis is associated with telomere shortening, while disease progression is associated with telomere extension, cell immortalisation and reactivation of telomerase [83]. Longer telomeres in HCC are associated with higher stage (regional or distant spread *versus* localised tumour) and grade (III–IV *versus* lower grade) as well as with worse survival [83, 84]. Telomerase promotes HCC development via several pathways, not limited to maintenance of telomeres and thus cellular proliferation. In addition, hTERT can act as a transcription factor in the Wnt molecular cascade [41]. Experimental data by HCC induction in telomerase-deficient mice have shown increased number of early tumours and reduced incidence of high-grade HCC [85].

Interestingly, shorter telomeres are observed more frequently (telomere length ratio between HCC and surrounding tissues lower than the mean, 70.1% *versus* higher, 29.9%) in HCC that is not related to hepatitis B (50.0% *versus* 50.0%) or C (60.0% *versus* 40.0%), or alcohol abuse (50.0% *versus* 50.0%), although the difference does not reach statistical significance [83]. Telomere shortening can be detected in peripheral blood. Notably, this assay can be used to predict HCC persistence (by telomere shortening) in cases attributable to viral hepatitis B or C but not in HCC attributable to non-infectious causes despite comparable size of patient groups [86].

Genetic predisposition has been studied in NAFLD trying to identify those patients that are at particularly increased risk of HCC. The possible candidate genes could be associated with telomere length and mechanisms involved in preserving telomeres [42]. About 10% of patients affected both by HCC and NASH have germline mutations in *hTERT* in comparison to complete absence of such mutations in NASH patients having cirrhosis and healthy controls [41]. In addition, *PNPLA3* polymorphisms have been studied in NAFLD patients, finding twice increased risk of HCC in association with rs738409 C>G. The proposed mechanism involves retinol metabolism in hepatic stellate cells [34].

The interaction of these pathogenetic mechanisms and genetic predisposition finally results in the increased incidence of HCC in NAFLD that reaches 76–201 per 100,000 contrasting with the incidence of 4.9–16 per 100,000 of the general population [57].

## **5. Potential treatment strategies**

As no specific treatment is approved for NAFLD, lifestyle interventions play the leading role in NAFLD management. Weight loss due to low calorie diet in combination with physical activities is the main therapeutic approach in overweight patients with NAFLD. As hypertriglyceridemia is a frequent and promoting feature of NAFLD [87] reduction of the triglyceride level must be among therapeutic goals. In severe hypertriglyceridemia, total fat consumption should be limited to less than 30 g/day, and carbohydrate amount in daily nutrition should be strictly controlled as well [88].

The HCC development shows complex associations with telomere shortening. The senescence-associated secretory program of liver stellate cells promotes carcinogenesis. The telomere shortening induces also genomic instability thus facilitating HCC development [41]. Indeed, HCC is characterised by significantly shorter telomeres in comparison to adjacent tissues [82]. However, cancer cells still maintain unlimited proliferation. Evidently, hepatocellular carcinoma cells develop compensatory mechanisms either for telomere extension or for cellular proliferation despite telomere shortening. The elongation of telomeres again can be ensured via diverse mechanisms, including hTERT or alternative lengthening of telomeres via

Over the progression of HCC, the telomere length changes in contrary direction. Early liver carcinogenesis is associated with telomere shortening, while disease progression is associated with telomere extension, cell immortalisation and reactivation of telomerase [83]. Longer telomeres in HCC are associated with higher stage (regional or distant spread *versus* localised tumour) and grade (III–IV *versus* lower grade) as well as with worse survival [83, 84]. Telomerase promotes HCC development via several pathways, not limited to maintenance of telomeres and thus cellular proliferation. In addition, hTERT can act as a transcription factor in the Wnt molecular cascade [41]. Experimental data by HCC induction in telomerase-deficient mice have shown

Interestingly, shorter telomeres are observed more frequently (telomere length ratio between HCC and surrounding tissues lower than the mean, 70.1% *versus* higher, 29.9%) in HCC that is not related to hepatitis B (50.0% *versus* 50.0%) or C (60.0% *versus* 40.0%), or alcohol abuse (50.0% *versus* 50.0%), although the difference does not reach statistical significance [83]. Telomere shortening can be detected in peripheral blood. Notably, this assay can be used to predict HCC persistence (by telomere shortening) in cases attributable to viral hepatitis B or C but not in HCC attributable to non-infectious causes despite comparable size of patient groups [86].

Genetic predisposition has been studied in NAFLD trying to identify those patients that are at particularly increased risk of HCC. The possible candidate genes could be associated with telomere length and mechanisms involved in preserving telomeres [42]. About 10% of patients affected both by HCC and NASH have germline mutations in *hTERT* in comparison to complete absence of such mutations in NASH patients having cirrhosis and healthy controls [41]. In addition, *PNPLA3* polymorphisms have been studied in NAFLD patients, finding twice increased risk of HCC in association with rs738409 C>G. The proposed mechanism

The interaction of these pathogenetic mechanisms and genetic predisposition finally results in the increased incidence of HCC in NAFLD that reaches 76–201 per 100,000 contrasting

As no specific treatment is approved for NAFLD, lifestyle interventions play the leading role in NAFLD management. Weight loss due to low calorie diet in combination with physical activities is the main therapeutic approach in overweight patients with NAFLD. As hypertriglyceridemia is a frequent and promoting feature of NAFLD [87] reduction of the triglyceride

increased number of early tumours and reduced incidence of high-grade HCC [85].

telomerase-independent mechanism seen in 7% of HCC cases [41].

20 Liver Cirrhosis - Update and Current Challenges

involves retinol metabolism in hepatic stellate cells [34].

**5. Potential treatment strategies**

with the incidence of 4.9–16 per 100,000 of the general population [57].

Physical activity has beneficial effect of reducing triglyceride level, even independently from diet [89]. Thus, at least 30 min of moderate activity most days of the week would be a necessary part of dyslipidemia management [90]. Loss of 5% of body weight decreases hepatic steatosis, but body weight loss of 10% could even improve inflammation and fibrosis in liver [87].

Experimentally investigating hepatocyte-specific PTEN-deficient mouse model, Piguet et al. showed that physical activity could reduce HCC growth in fatty liver. In PTEN-deficient mice, HCC incidence was 71% of exercised mice and 100% of sedentary mice. In addition, liver tumour volume in exercised mice was significantly smaller than that of sedentary mice (444 ± 551 *versus* 945 ± 1007 mm3 ) [91]. The physiological substantiation relies on fact that regular physical activity could inhibit mTOR complex, which is engaged in cell growth and proliferation [92].

Increased hepatic free cholesterol accumulation is typical for NASH. Statins are commonly prescribed to reduce cholesterol synthesis in the liver and thus serum levels of free cholesterol [14]. In a recent European multi-centre cohort study, statin use was associated with protection from steatosis (odds ratio, OR 0.09; 95% CI, 0.01–0.32; p = 0.004), steatohepatitis (OR, 0.25; 95% CI, 0.13–0.47; p <0.001), and fibrosis stage F2–F4 (OR, 0.42; 95% CI, 0.20–0.80; p = 0.017). The protective effect of statins on steatohepatitis was stronger in subjects not carrying the I148M PNPLA3 risk variant (p = 0.02), indicating the role of genetic predisposition [93]. Statins also have been associated with reduced risk (range, 0.46–0.79) of HCC [94].

In a meta-analysis, including 4298 patients with HCC, statin use was associated with a 37% reduction in the risk of hepatocellular carcinoma. The effect was stronger in Asian patients but was also present in Western populations. Moreover, the reduction of cancer risk was independent of statin lipid-lowering effects [95]. Several hypotheses have been proposed, including statin ability to inhibit cell proliferation via inhibition of v-myc avian myelocytomatosis viral oncogene homolog protein phosphorylation which seems to play a role in liver carcinogenesis [96], as well as capacity to inhibit the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which activates multiple proliferative pathways [95]. Simvastatin selectively induces apoptosis in cancer, but not in healthy cells. This proapoptotic effect is maintained via RAF/MAPK1/ ERK and growth-inhibitory action by suppression of angiogenesis and proteasome pathway [95, 96]. However, data about liver carcinogenesis and statin effects remain controversial. In another large meta-analysis, including 86,936 participants, no beneficial effect of statin in terms of incidence or death from cancer was observed. Even more, in 67,258 patients who received statins, 35 new liver cancers and 24 deaths from liver cancer were reported showing no significant difference from control group, comprising 67,279 patients who received placebo, and developed 33 new liver cancer (p = 0.93) cases leading to 24 deaths (p = 1.00) as analysed by Carrat [97].

Metformin, a widely prescribed drug for treating type 2 diabetes mellitus, is one of the most extensively recognised metabolic modulators which decreases aminotransferase levels and hepatic insulin resistance. It has no beneficial effects on NAFLD histology but still retains an important anti-cancer action [87, 98]. The hypothetic antitumor mechanisms of metformin are believed to be (1) inhibition of mTOR, (2) weight loss and (3) suppressed production of ROS and the associated DNA damage, in combination with (4) reduction of hyperinsulinemia, which is known to lead to cell proliferation [99]. In meta-analysis comprising 105,495 patients with type 2 diabetes, Zhang et al. showed that metformin was associated with an estimated 70% reduction in the risk of developing HCC [98]. The risk reduction in metformin users is significant, regarding both incidence (78%) and mortality (77%) from HCC [100].

The mammalian target of rapamycin (mTOR) promotes growth in a majority of liver cancers, including hepatocellular carcinoma. It participates in the formation of two protein complexes—mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin and has ability to activate downstream targets which regulate cellular growth and metabolism. Prolonged mTORC1 activation is related to liver steatosis and insulin resistance in obese patients [14, 101]. Due to the ability suppress mTORC1, rapamycin and its analogues Everolimus and Temsirolimus have been tested to treat HCC. Unfortunately, results have not been promising. In a phase 3 study of patients with advanced HCC, Everolimus increased the frequency of hepatic injury and showed no improvements regarding survival [14]. After 2 weeks with rapamycin treatment, the lipid droplets in the liver decreased, as well as ROS burden. However, rapamycin treatment promoted liver damage with augmented IL-6 and decreased anti-inflammatory IL-10 production, leading to increased hepatic inflammation and hepatocyte necrosis [101].

Inflammation promotes development of complications in patients with cirrhosis contributing to mortality and to liver insufficiency mediated by pro-inflammatory cytokines. The most recognisable pro-inflammatory cytokine associated with liver damage in case of NAFLD is TNF-α that can be inhibited by pentoxifylline. Lebrec et al. performed randomised, placebo controlled, double-blind trial assessing pentoxifyline effect in 335 patients with cirrhosis. Although pentoxifylline had no effect on short-term mortality, it significantly (p = 0.04) prolonged the complication-free time span [102].

Knowing the important role of NADPH oxidases (NOXs) and production of ROS in liver fibrosis, different strategies to prevent the oxidative damage have been developed [23]. In hepatocytes, NOX4 mediates suppressor effects on TGF-β and can inhibit hepatocyte growth and liver carcinogenesis. In turn, dual NOX4/NOX1 pharmacological inhibitor GKT137831 could decrease both the apparition of fibrogenic markers as well as hepatocyte apoptosis *in vivo* [103].

Currently, multikinase inhibitor sorafenib is the only pharmacological agent that prolongs survival of HCC patients, although the median survival is improved only by 12 weeks [14]. It acts against Raf-1 and B-raf, vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor receptor kinases [104]. Sorafenib as well as VEGF inhibitors have radiosensitizing effect. However, combined regimens including sorafenib and liver stereotactic radiation or whole liver radiotherapy are characterized by poor tolerability [104]. Various beneficial effects of sorafenib have been reported in liver cirrhosis. As epithelial-mesenchymal transition and TGF-β play crucial roles in liver fibrosis, Ma et al. proved that sorafenib had ability to strikingly suppress TGF-β1 induced epithelial-mesenchymal transition, as well as apoptosis in hepatic stellate cells, in dose-dependent manner [105].

Several treatment strategies might involve the telomere and telomerase complex. In cancer, telomerase inhibitors might arrest tumour growth, prevent further malignisation in surrounding cirrhotic nodules and/or enhance HCC chemosensitivity. In early liver disease, telomerase activation might prevent tissue loss if the etiologic factor cannot be removed. This could be reached via transplantation of liver cells engineered for hTERT expression, direct supply of hTERT to the patient's cells or by small molecules enhancing telomerase activity. However, side effects and enhanced cancer risk must be considered and prevented [41]. The treatment modulating cellular senescence and proliferation control may also target p21 [106–108] and p53 [109] pathways.

The p21 protein, a strong and universal inhibitor of cyclin-dependant kinases, is an important regulator of cell proliferation, apoptosis and senescence [107, 108]. Based on its intracellular location and the molecular background, it can have dual activity. Intranuclear p21 acts as tumour suppressor, as it binds cyclin-dependant kinases and thus suppresses cellular proliferation. Cytoplasmic p21 prevents apoptosis by binding caspases and promotes proliferation and migration of p53-deficient cells. The p21 pathway is also closely associated with senescence. Few small molecular inhibitors of p21 are known, including LLW10, butyrolactone and UC2288. In addition, sorafenib also exhibits anti-p21 activity. LLW10 binds to p21 and induces proteosomal degradation via ubiquitination. Despite the reliable mechanism, the high concentration that is necessary for sufficient activity as well as the instability of LLW10 prevents it from being clinically useful drug. Butyrolactone also induces proteosomal degradation of p21. UC2288 decreases p21 concentration via suppressed transcription and modified posttranscriptional modulation [107]. In turn, upregulation of p21 can be achieved via statins or by anticancer agents including histone deacetylase inhibitors [106]. Induction of senescence would be desirable if the tumour is already present while suppressed senescence might prevent or slow down the development of liver cirrhosis. As was noted, it is possible to modulate p21 level in both directions. However, the net effects must be carefully considered and studied experimentally, knowing the bidirectional activity of p21.

p21 is also an effector of p53-mediated responses in cells maintaining functional p53. In p53-deficient cell, it manifests carcinogenic effects. Thus, restoration of wild-type p53 could be attractive, either in combination with p21-targeted treatment or with other oncological approach. In liver cancer, restoration of p53 activity has resulted in senescence and increased immune response. The therapeutic approaches could include (1) restoration of wild type function to mutant p53 by low molecular weight compounds PRIMA 1 or PRIMA-1MET. The last one has progressed to phase II clinical trials; (2) stabilising p53 due to blocked interaction with MDM2 or MDM4 by nutlins, representing low molecular weight molecules, or by stapled peptides; (3) gene therapy using viral vectors that has already been tested in HCC; (4) induction of synthetic lethality [109].

## **6. Conclusions**

important anti-cancer action [87, 98]. The hypothetic antitumor mechanisms of metformin are believed to be (1) inhibition of mTOR, (2) weight loss and (3) suppressed production of ROS and the associated DNA damage, in combination with (4) reduction of hyperinsulinemia, which is known to lead to cell proliferation [99]. In meta-analysis comprising 105,495 patients with type 2 diabetes, Zhang et al. showed that metformin was associated with an estimated 70% reduction in the risk of developing HCC [98]. The risk reduction in metformin users is

The mammalian target of rapamycin (mTOR) promotes growth in a majority of liver cancers, including hepatocellular carcinoma. It participates in the formation of two protein complexes—mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin and has ability to activate downstream targets which regulate cellular growth and metabolism. Prolonged mTORC1 activation is related to liver steatosis and insulin resistance in obese patients [14, 101]. Due to the ability suppress mTORC1, rapamycin and its analogues Everolimus and Temsirolimus have been tested to treat HCC. Unfortunately, results have not been promising. In a phase 3 study of patients with advanced HCC, Everolimus increased the frequency of hepatic injury and showed no improvements regarding survival [14]. After 2 weeks with rapamycin treatment, the lipid droplets in the liver decreased, as well as ROS burden. However, rapamycin treatment promoted liver damage with augmented IL-6 and decreased anti-inflammatory IL-10 production, leading to increased hepatic inflammation

Inflammation promotes development of complications in patients with cirrhosis contributing to mortality and to liver insufficiency mediated by pro-inflammatory cytokines. The most recognisable pro-inflammatory cytokine associated with liver damage in case of NAFLD is TNF-α that can be inhibited by pentoxifylline. Lebrec et al. performed randomised, placebo controlled, double-blind trial assessing pentoxifyline effect in 335 patients with cirrhosis. Although pentoxifylline had no effect on short-term mortality, it significantly (p = 0.04) pro-

Knowing the important role of NADPH oxidases (NOXs) and production of ROS in liver fibrosis, different strategies to prevent the oxidative damage have been developed [23]. In hepatocytes, NOX4 mediates suppressor effects on TGF-β and can inhibit hepatocyte growth and liver carcinogenesis. In turn, dual NOX4/NOX1 pharmacological inhibitor GKT137831 could decrease both the apparition of fibrogenic markers as well as hepatocyte apoptosis *in vivo* [103].

Currently, multikinase inhibitor sorafenib is the only pharmacological agent that prolongs survival of HCC patients, although the median survival is improved only by 12 weeks [14]. It acts against Raf-1 and B-raf, vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor receptor kinases [104]. Sorafenib as well as VEGF inhibitors have radiosensitizing effect. However, combined regimens including sorafenib and liver stereotactic radiation or whole liver radiotherapy are characterized by poor tolerability [104]. Various beneficial effects of sorafenib have been reported in liver cirrhosis. As epithelial-mesenchymal transition and TGF-β play crucial roles in liver fibrosis, Ma et al. proved that sorafenib had ability to strikingly suppress TGF-β1 induced epithelial-mesenchymal transition, as well as

apoptosis in hepatic stellate cells, in dose-dependent manner [105].

significant, regarding both incidence (78%) and mortality (77%) from HCC [100].

and hepatocyte necrosis [101].

22 Liver Cirrhosis - Update and Current Challenges

longed the complication-free time span [102].

Non-alcoholic steatohepatitis is recognised as the cause of NASH-induced cirrhosis. It has also been associated with a significant fraction of cases previously diagnosed as cryptogenic cirrhosis. Liver cirrhosis can become further complicated by hepatocellular carcinoma, the most frequent primary liver tumour known for serious prognosis and limited treatment options. In addition, the development of HCC in NAFLD patients can precede cirrhosis in a significant fraction of cases. NAFLD is the major hepatic manifestation of obesity and associated metabolic diseases, such as diabetes mellitus. With increasing prevalence of these conditions, NAFLD has become the most common liver disorder worldwide. It affects around 25% of general population and 90% of patients suffering from morbid obesity, i.e., having body mass index equal or greater than 40 kg/m<sup>2</sup> .

The mechanisms of liver steatosis include up-regulation of inflammatory cytokines, as TNF-α, IL-6 and CCL2, released from extrahepatic adipose tissues due to prolonged low-grade inflammation triggered by hypoxia-induced death of fast-growing fat cells. Insulin resistance further contributes to NAFLD and can be aggravated by the pro-inflammatory cytokine background. Free fatty acids and cholesterol cause lipotoxicity due to released reactive oxygen species as well as toxic metabolites generated by non-oxidative biochemical pathways. Decreased level of adiponectin, exaggerated oxidative stress and hepatic iron accumulation also are among the mechanisms of NAFLD.

In the pathogenesis of NAFLD, 20–30% of patients, initially affected by simple liver steatosis, develop hepatic inflammation and thus correspond to the diagnostic criteria of NASH. These cases are at risk to progress to liver cirrhosis and hepatocellular carcinoma. The standardised incidence ratio of HCC in NASH patients reaches 4.4. Regarding the epidemiological profile of hepatocellular carcinoma, the proportion of NASH-related cases is growing and has increased from 8.3 to 13.5% in the time period 2002–2012.

Obesity has been linked to higher frequency of cancers in different organs including the liver. The relative risk of HCC-attributable death in obese patients (body mass index equal or greater than 35 kg/m2 ) can be as high as 4.5. The underlying mechanisms of carcinogenesis include chronic general low-grade inflammation characterised by elevated levels of TNF-α and IL-6, both of which are tumour-promoting cytokines and major drivers of cell proliferation in NAFLD and NASH. The increased levels of leptin and suppressed production of adiponectin represent another mechanism involved in the progression of NAFLD to HCC in obese individuals. Leptin is a pro-inflammatory, pro-angiogenic and pro-fibrogenic cytokine with a growth-promoting effect. Adiponectin has anti-inflammatory, antiangiogenic and tumour growth-limiting properties. Insulin resistance and hyperinsulinemia lead to excessive cell proliferation. Iron compound deposition has also been related to HCC development in NAFLD-related cirrhosis, possibly due to oxidative DNA damage. Thus, the same molecular pathways that induced NAFLD continue to be active until the development of HCC. These mechanisms are supplemented by critical genetic events including *PTEN* deletion, switch from inactivated to upregulated Wnt pathway and typical mutation pattern in *TP53*. Certain microRNAs, including miR-21, act as molecular switches.

Pathogenetically related molecular markers, e.g., cytokeratin 18, can serve as predictive tests to detect increased risk of HCC.

The molecular pathogenesis of NAFLD is closely related to the selection of treatment targets. NAFLD patients can benefit from low calorie diet, reducing hypertriglyceridemia and potentially reversing steatosis and even fibrosis; physical activity inhibiting mTOR complex; statins influencing cholesterol synthesis, RAF/MAPK1/ERK and p21 pathway; metformin acting through suppression of mTOR and ROS; pentoxyfillin lowering production of pro-inflammatory cytokines. Multikinase inhibitor sorafenib is indicated in HCC patients. Bidirectional regulation of telomere attrition, senescence, and p21 pathway could be at least theoretically considered in the future. Restoration of wild-type p53 activity becomes possible. The regulation of miRNA machinery also represents a highly attractive future treatment option.

Thus, NAFLD is gaining increasing importance in nowadays medicine as a frequent condition that can lead to such grave complications as liver cirrhosis and hepatocellular carcinoma. Awareness of the molecular profile is helpful to identify the treatment targets and predictive markers.

## **Acknowledgements**

cirrhosis. Liver cirrhosis can become further complicated by hepatocellular carcinoma, the most frequent primary liver tumour known for serious prognosis and limited treatment options. In addition, the development of HCC in NAFLD patients can precede cirrhosis in a significant fraction of cases. NAFLD is the major hepatic manifestation of obesity and associated metabolic diseases, such as diabetes mellitus. With increasing prevalence of these conditions, NAFLD has become the most common liver disorder worldwide. It affects around 25% of general population and 90% of patients suffering from morbid obesity, i.e., having body

.

The mechanisms of liver steatosis include up-regulation of inflammatory cytokines, as TNF-α, IL-6 and CCL2, released from extrahepatic adipose tissues due to prolonged low-grade inflammation triggered by hypoxia-induced death of fast-growing fat cells. Insulin resistance further contributes to NAFLD and can be aggravated by the pro-inflammatory cytokine background. Free fatty acids and cholesterol cause lipotoxicity due to released reactive oxygen species as well as toxic metabolites generated by non-oxidative biochemical pathways. Decreased level of adiponectin, exaggerated oxidative stress and hepatic iron accumulation

In the pathogenesis of NAFLD, 20–30% of patients, initially affected by simple liver steatosis, develop hepatic inflammation and thus correspond to the diagnostic criteria of NASH. These cases are at risk to progress to liver cirrhosis and hepatocellular carcinoma. The standardised incidence ratio of HCC in NASH patients reaches 4.4. Regarding the epidemiological profile of hepatocellular carcinoma, the proportion of NASH-related cases is growing and has

Obesity has been linked to higher frequency of cancers in different organs including the liver. The relative risk of HCC-attributable death in obese patients (body mass index equal

include chronic general low-grade inflammation characterised by elevated levels of TNF-α and IL-6, both of which are tumour-promoting cytokines and major drivers of cell proliferation in NAFLD and NASH. The increased levels of leptin and suppressed production of adiponectin represent another mechanism involved in the progression of NAFLD to HCC in obese individuals. Leptin is a pro-inflammatory, pro-angiogenic and pro-fibrogenic cytokine with a growth-promoting effect. Adiponectin has anti-inflammatory, antiangiogenic and tumour growth-limiting properties. Insulin resistance and hyperinsulinemia lead to excessive cell proliferation. Iron compound deposition has also been related to HCC development in NAFLD-related cirrhosis, possibly due to oxidative DNA damage. Thus, the same molecular pathways that induced NAFLD continue to be active until the development of HCC. These mechanisms are supplemented by critical genetic events including *PTEN* deletion, switch from inactivated to upregulated Wnt pathway and typical mutation pattern in *TP53*. Certain

Pathogenetically related molecular markers, e.g., cytokeratin 18, can serve as predictive tests

The molecular pathogenesis of NAFLD is closely related to the selection of treatment targets. NAFLD patients can benefit from low calorie diet, reducing hypertriglyceridemia and potentially reversing steatosis and even fibrosis; physical activity inhibiting mTOR complex;

) can be as high as 4.5. The underlying mechanisms of carcinogenesis

mass index equal or greater than 40 kg/m<sup>2</sup>

24 Liver Cirrhosis - Update and Current Challenges

also are among the mechanisms of NAFLD.

or greater than 35 kg/m2

to detect increased risk of HCC.

increased from 8.3 to 13.5% in the time period 2002–2012.

microRNAs, including miR-21, act as molecular switches.

The team of authors gratefully acknowledges artist Ms. Sandra Ozolina for professional preparation of illustrations.

## **Author details**

Dzeina Mezale<sup>1</sup> \*, Ilze Strumfa1 , Andrejs Vanags<sup>2</sup> , Matiss Mezals3 , Ilze Fridrihsone1 , Boriss Strumfs<sup>4</sup> and Dainis Balodis<sup>1</sup>


## **References**


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## **Noninvasive Diagnostic and Prognostic Assessment Tools for Liver Fibrosis and Cirrhosis in Patients with Chronic Liver Disease**

Ju‐Seop Kang and Min‐Ho Lee

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68317

#### Abstract

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34 Liver Cirrhosis - Update and Current Challenges

Liver fibrosis, that is, excessive accumulation of extracellular matrix protein, occurs and is the wound-healing response and common final pathway of various chronic liver diseases. Advanced hepatic fibrosis caused by chronic liver inflammation eventually progresses to cirrhosis, and prognosis and management of chronic liver diseases depend on the fibrotic severities. Therefore, the early and precise evaluation of severity and status of liver fibrosis provides useful information for diagnosis as well as treatment planning and treatment efficacy and prognosis. Although invasive liver biopsy is the gold standard to assess the nature and severity of hepatic fibrosis, it has several recognized limitations including sampling error and inter-observer variability in interpretation and staging. Furthermore, the dynamic process of fibrosis resulting from progression and regression is difficult to capture with biopsy alone. Therefore, alternative, simple, reliable, and noninvasive direct and indirect serum markers able to predict the presence of significant fibrosis or cirrhosis in patients with chronic liver disease with considerable accuracy were needed. The hepatology experts are actively researching noninvasive methods of fibrosis quantification. The aims of this chapter were to review the nature and limitations of the several noninvasive methods for the assessment of presence and severity of liver fibrosis in patients with chronic liver disease.

Keywords: noninvasive method, biomarker, stage of liver fibrosis, cirrhosis, chronic liver disease

### 1. Introduction

Liver fibrosis and cirrhosis are an important and growing global health problem. Patients with non-cirrhotic chronic liver disease may have an increased mortality rate compared to controls [1]. However, mortality and morbidity rates increase exponentially once cirrhosis

develops. Prognosis and management of chronic liver diseases greatly depend on the amount and progression of liver fibrosis. Therefore, the ability to reliably rule out cirrhosis may be considered an important characteristic of any test designed to assess liver fibrosis [2]. The diagnosis of cirrhosis also portends an increased risk of liver-related morbidity [3] as well as mortality [4]. Liver-related mortality and decompensation are expected to continue to increase over the next decade, due to the projected increase in the number of patients with advanced liver fibrosis in the population [5]. Therefore, the accurate and timely evaluation of liver fibrosis is a key step to manage a chronic liver disease and to assess its prognosis and in need of close monitoring, management of complications, and underlying liver disease in patients with advanced stages [6]. For many years, liver biopsy has been considered the "gold standard" for evaluation of liver fibrosis [7]. Pathologists have proposed robust scoring system for staging liver fibrosis such as the semi-quantitative Metavir score (F0: no fibrosis, F1: portal fibrosis, F2: bridging fibrosis, F3; bridging fibrosis, and marked, F4: cirrhosis) [8] and the modified Ishak score, an expansion of Metavir score [9]. In addition, computer-aided morphometric measurement of collagen-proportional area, a partly automated technique, provides an accurate and linear evaluation of the amount of fibrosis [10]. However, liver biopsy is an invasive procedure with rare but potentially life-threatening complications and prone to sampling errors. Also, liver biopsy gives a snapshot and not an insight into the dynamic changes during the process of fibrogenesis (progression, atatic, or regression). Therefore, liver biopsy has some limitations as follows. First, biopsy is an invasive technique, which has associated morbidity; pain occurs in 20% of patients and major complications such as bleeding or hemobilia in 0.5% [11]. The bleeding rate (0.5%) has not changed significantly in recent years, according to a large multicenter study [12]. The primary factor that appeared to contribute to bleeding risk was platelet count rather than qualitative factors such as operator experience, needle size, or the use of ultrasound to localize the site. Second, the small size of the biopsy makes it prone to sampling variability [13]. Third, the interpretation of the histologic changes can be problematic with inter- and intra-observer variation [14]. These limitations as well as the availability of powerful viral diagnostic tools and new antiviral drugs have rapidly decreased the use of liver biopsy in viral hepatitis and led to the development of noninvasive techniques for the assessment of liver fibrosis. On the other hand, at least some correlation between biopsy stage and outcomes has begun to emerge. In the NIH-HALT C cohort, a correlation was found between the Ishak fibrosis stage and clinical outcomes, the need of liver transplantation, and liver-related deaths in patients with chronic HCV. However, even in this study, up to 25% of the liver biopsy samples were fragmented, which significantly diminished the ability to draw correlations between biopsy findings and clinical outcomes [15]. While some of these methodologies are now generally applied in patients for a top priority of evaluation, biopsy exists within the clinical technique of hepatologists for estimating the causes of complicated diseases or when there are unconformities between clinical characteristics and extents of fibrosis evaluated by noninvasive methodologies [16]. The dynamic process of fibrosis should be best measured as a continuous variable and classical histological staging systems do not permit this [17]. Since liver biopsy is an invasive procedure, cost-intensive, mostly uncomfortable for the patients, and sometimes prone to complication, alternative, simple noninvasive tests have been developed to reliably assess the stage of liver fibrosis. Ongoing efforts include serum markers and imaging based on ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). The goal is to develop tests with high specificity and sensitivity to estimate liver fibrosis and predict outcomes [18]. Ideally, noninvasive methodologies of liver fibrosis should be liver specific, easy to perform, reliable, and inexpensive. In addition, it should be accurate not only for the staging of fibrosis but also for the monitoring of disease progression and antiviral therapy efficacy [19]. Scientific attention is currently focused on new antifibrotic therapies, aiming at fibrosis reversibility and cirrhosis regression [20]. It is therefore important, now more than ever, to ensure accurate and prompt assessment of hepatic fibrosis in therapeutic trials of chronic liver disease. Consequently, the demand for noninvasive method substitutes to estimate hepatic fibrosis is a main trial that has provoked research and induced the improvement of noninvasive serological markers of hepatic fibrosis. Several noninvasive serological markers have been described to forecast the existence of significant fibrosis or cirrhosis in patients with chronic hepatic disease with good accuracy. However, most of these markers require complicated calculations, and manipulation in various clinical situations is difficult and inconvenient [21]. Recently, transient elastography (TE, FibroScan) has been introduced as a novel, rapid, noninvasive, and reproducible method to measure liver stiffness [22]. In several studies [22, 23], liver stiffness measurement (LSM) using M probe of FibroScan accurately predicted hepatic fibrosis and cirrhosis in patients with chronic liver disease.

This chapter focuses and provides comparison of invasive and noninvasive methods for assessing the severity of liver fibrosis and aims to provide update on noninvasive diagnostic and prognostic assessment tools for liver fibrosis and cirrhosis in patients with chronic liver disease.

## 2. Mechanism of liver fibrosis

develops. Prognosis and management of chronic liver diseases greatly depend on the amount and progression of liver fibrosis. Therefore, the ability to reliably rule out cirrhosis may be considered an important characteristic of any test designed to assess liver fibrosis [2]. The diagnosis of cirrhosis also portends an increased risk of liver-related morbidity [3] as well as mortality [4]. Liver-related mortality and decompensation are expected to continue to increase over the next decade, due to the projected increase in the number of patients with advanced liver fibrosis in the population [5]. Therefore, the accurate and timely evaluation of liver fibrosis is a key step to manage a chronic liver disease and to assess its prognosis and in need of close monitoring, management of complications, and underlying liver disease in patients with advanced stages [6]. For many years, liver biopsy has been considered the "gold standard" for evaluation of liver fibrosis [7]. Pathologists have proposed robust scoring system for staging liver fibrosis such as the semi-quantitative Metavir score (F0: no fibrosis, F1: portal fibrosis, F2: bridging fibrosis, F3; bridging fibrosis, and marked, F4: cirrhosis) [8] and the modified Ishak score, an expansion of Metavir score [9]. In addition, computer-aided morphometric measurement of collagen-proportional area, a partly automated technique, provides an accurate and linear evaluation of the amount of fibrosis [10]. However, liver biopsy is an invasive procedure with rare but potentially life-threatening complications and prone to sampling errors. Also, liver biopsy gives a snapshot and not an insight into the dynamic changes during the process of fibrogenesis (progression, atatic, or regression). Therefore, liver biopsy has some limitations as follows. First, biopsy is an invasive technique, which has associated morbidity; pain occurs in 20% of patients and major complications such as bleeding or hemobilia in 0.5% [11]. The bleeding rate (0.5%) has not changed significantly in recent years, according to a large multicenter study [12]. The primary factor that appeared to contribute to bleeding risk was platelet count rather than qualitative factors such as operator experience, needle size, or the use of ultrasound to localize the site. Second, the small size of the biopsy makes it prone to sampling variability [13]. Third, the interpretation of the histologic changes can be problematic with inter- and intra-observer variation [14]. These limitations as well as the availability of powerful viral diagnostic tools and new antiviral drugs have rapidly decreased the use of liver biopsy in viral hepatitis and led to the development of noninvasive techniques for the assessment of liver fibrosis. On the other hand, at least some correlation between biopsy stage and outcomes has begun to emerge. In the NIH-HALT C cohort, a correlation was found between the Ishak fibrosis stage and clinical outcomes, the need of liver transplantation, and liver-related deaths in patients with chronic HCV. However, even in this study, up to 25% of the liver biopsy samples were fragmented, which significantly diminished the ability to draw correlations between biopsy findings and clinical outcomes [15]. While some of these methodologies are now generally applied in patients for a top priority of evaluation, biopsy exists within the clinical technique of hepatologists for estimating the causes of complicated diseases or when there are unconformities between clinical characteristics and extents of fibrosis evaluated by noninvasive methodologies [16]. The dynamic process of fibrosis should be best measured as a continuous variable and classical histological staging systems do not permit this [17]. Since liver biopsy is an invasive procedure, cost-intensive, mostly uncomfortable for the patients, and sometimes prone to complication, alternative, simple noninvasive tests have been developed to reliably assess the stage of liver fibrosis. Ongoing efforts include serum markers and imaging based on ultrasound, computed

36 Liver Cirrhosis - Update and Current Challenges

Liver fibrosis is the result of the continuous wound-healing process of the liver to repeated damage [24]. After acute liver injury (e.g., viral hepatitis), parenchymal cells regenerate and replace the necrotic or apoptotic cells. The process is associated with a hepatic inflammatory response and a limited deposition of extracellular matrix (ECM) in the hepatic parenchyma. If the liver injury persists, then eventually the liver regeneration fails, and hepatocytes are substituted with abundant ECM, including fibrillar collagen [25]. This process results in cirrhosis, which can have a bad outcome and high mortality. Progression to this end stage is typically variable but slow, developing over 20–40 years in patients with chronic liver damage; the speed is dependent on both genetic and environmental factors [26]. Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, alcohol, and autoimmune, drug-induced, cholestatic and metabolic diseases [18, 26–28]. Deposition of excess ECM is rich in fibril-forming collagens [29], which change the normal structure of the liver resulting in pathophysiologic damage to the organ [30]. Liver fibrosis is beneficial at first because it can encapsulate the injury and is considered a reversible process at this stage [31]. In normal liver, ECM is highly dynamic substratum with a precisely regulated balance between synthesis and degradation. Normally, the hepatic ECM comprises less than 3% of the relative area on a liver tissue section and approximately 0.5% of the total wet weight of liver [32]. It is also a component of Glisson's capsule, portal tracts, central veins, and the subendothelial space of Disse. The most important structural ECM components in liver are collagen, proteoglycans, laminin, fibronectin, and matricellular proteins. The hepatic parenchyma is composed of hepatocytes, endothelial cells, and other resident cells, including hepatic stellate cell (HSCs) and Kupffer cells (KCs). The sinusoid is the hepatic microvascular unit that has an endothelial lining distinguished by fenestration of pores and is separated from the hepatocytes by the space of Disse, where HSCs reside. This space contains a low-density basal membrane-like matrix that is essential for maintaining the differentiated function of parenchymal cell yet is sufficiently porous to enable metabolic exchange between the bloodstream and hepatocytes [26]. During chronic liver injury, however, ECM production exceeds ECM degradation, and liver fibrosis develops as results of the progressive thickening of fibrotic septae and chemical cross-linking of collagen. Moreover, these changes in ECM composition directly stimulate fibrogenesis (Figure 1) [33]. After liver injury, disruption of this matrix and replacement by fibrillar collagens I and III and fibronectin have occurred [34, 35]. Fibrosis is characterized histologically and biochemically by a several-fold elevation in the total ECM content of the liver [25].

Accumulation of ECM in the space of Disse leads to loss of the normal fenestrating structures that are characteristic of the endothelial lining, which causes the impairment of the normal bidirectional metabolic exchange between portal blood and hepatocytes. This process is sinusoidal remodeling, termed capillarization of the sinusoid [38]. All major constituents of normal ECM are represented, to some extent, in the newly formed matrix during the fibrogenic process. As in normal ECM, collagen (especially types I and III) and elastin are most abundant proteins, but glycoproteins (fibronectin and laminin) and pure carbohydrates are also present. When compared to normal matrix, scar tissue produced in liver fibrosis has a significantly higher percentage of type I collagen [39]. ECM deposition occurs as a result of an imbalance between excessive ECM production and less degradation. In the normal liver, matrix metalloproteinases (MMPs) have a well-described ECM-degrading function. The activity of MMPs, however, is suppressed in the setting of liver injury as a result of overexpression of tissue inhibitor of metalloproteinase (TIMPs) by the activated HSCs [25]. TIMPs are key regulators of MMPs, by blocking their collagenolytic activity. In addition, TIMP-1 is antiapoptotic toward HSCs, in part through the induction of Bcl-2, thus promoting the survival of fibrogenic cells [40]. This balance between MMPs and TIMPs is crucial for ECM homeostasis [41]. In human liver, the degree of TIPM-1 expression correlates with the extent of liver fibrosis [42]. In order to preserve matrix homeostasis, ECM also contains MMPs, MMP-1, MMP-8, and MMP-13 that degrade the fibrillary collagen types I and III predominating in fibrosis, while MMP-2 and MMP-9 degrade collagen types IV as well as denatured fibrillary collagens. HSCs are the key source of both MMPs in liver. Although the increase of MMP production should control the excessive increase of the ECM, it can also promote injury. Early increases in MMP, particularly MMP-2, degrade normal matrix and recruit cells that amplify fibrosis [43, 44]. In addition, there is also enhanced secretion of TIMP-1 and -2 by HSCs during progressive tissue injury and cellular activation. Different populations of cells play roles in fibrogenesis, but the activation of HSCs is an essential factor in fibrinogenesis [45]. The mechanism of liver fibrosis is thought to be associated with the hepatic damage of various etiologic

Noninvasive Diagnostic and Prognostic Assessment Tools for Liver Fibrosis and Cirrhosis in Patients with… http://dx.doi.org/10.5772/intechopen.68317 39

also a component of Glisson's capsule, portal tracts, central veins, and the subendothelial space of Disse. The most important structural ECM components in liver are collagen, proteoglycans, laminin, fibronectin, and matricellular proteins. The hepatic parenchyma is composed of hepatocytes, endothelial cells, and other resident cells, including hepatic stellate cell (HSCs) and Kupffer cells (KCs). The sinusoid is the hepatic microvascular unit that has an endothelial lining distinguished by fenestration of pores and is separated from the hepatocytes by the space of Disse, where HSCs reside. This space contains a low-density basal membrane-like matrix that is essential for maintaining the differentiated function of parenchymal cell yet is sufficiently porous to enable metabolic exchange between the bloodstream and hepatocytes [26]. During chronic liver injury, however, ECM production exceeds ECM degradation, and liver fibrosis develops as results of the progressive thickening of fibrotic septae and chemical cross-linking of collagen. Moreover, these changes in ECM composition directly stimulate fibrogenesis (Figure 1) [33]. After liver injury, disruption of this matrix and replacement by fibrillar collagens I and III and fibronectin have occurred [34, 35]. Fibrosis is characterized histologically and biochemically by a several-fold elevation in the total ECM content of

Accumulation of ECM in the space of Disse leads to loss of the normal fenestrating structures that are characteristic of the endothelial lining, which causes the impairment of the normal bidirectional metabolic exchange between portal blood and hepatocytes. This process is sinusoidal remodeling, termed capillarization of the sinusoid [38]. All major constituents of normal ECM are represented, to some extent, in the newly formed matrix during the fibrogenic process. As in normal ECM, collagen (especially types I and III) and elastin are most abundant proteins, but glycoproteins (fibronectin and laminin) and pure carbohydrates are also present. When compared to normal matrix, scar tissue produced in liver fibrosis has a significantly higher percentage of type I collagen [39]. ECM deposition occurs as a result of an imbalance between excessive ECM production and less degradation. In the normal liver, matrix metalloproteinases (MMPs) have a well-described ECM-degrading function. The activity of MMPs, however, is suppressed in the setting of liver injury as a result of overexpression of tissue inhibitor of metalloproteinase (TIMPs) by the activated HSCs [25]. TIMPs are key regulators of MMPs, by blocking their collagenolytic activity. In addition, TIMP-1 is antiapoptotic toward HSCs, in part through the induction of Bcl-2, thus promoting the survival of fibrogenic cells [40]. This balance between MMPs and TIMPs is crucial for ECM homeostasis [41]. In human liver, the degree of TIPM-1 expression correlates with the extent of liver fibrosis [42]. In order to preserve matrix homeostasis, ECM also contains MMPs, MMP-1, MMP-8, and MMP-13 that degrade the fibrillary collagen types I and III predominating in fibrosis, while MMP-2 and MMP-9 degrade collagen types IV as well as denatured fibrillary collagens. HSCs are the key source of both MMPs in liver. Although the increase of MMP production should control the excessive increase of the ECM, it can also promote injury. Early increases in MMP, particularly MMP-2, degrade normal matrix and recruit cells that amplify fibrosis [43, 44]. In addition, there is also enhanced secretion of TIMP-1 and -2 by HSCs during progressive tissue injury and cellular activation. Different populations of cells play roles in fibrogenesis, but the activation of HSCs is an essential factor in fibrinogenesis [45]. The mechanism of liver fibrosis is thought to be associated with the hepatic damage of various etiologic

the liver [25].

38 Liver Cirrhosis - Update and Current Challenges

Figure 1. Schematic illustration of fibrosis progression and regression (modified from Refs. [18, 36, 37]).

factors followed by the activation of HSCs within the liver that develop into liver myofibroblasts (LMFs) [46]. LMFs include a heterogeneous population of highly proliferative cells that accumulate at injury sites and promote ECM accumulation [47]. The pool of LMF originates mainly from liver mesenchymal cells, namely HSCs [48]. Although HSCs are the primary source of LMFs in liver fibrosis, extrahepatic precursors such as bone marrow-derived mesenchymal cells and portal fibroblasts contribute in ECM production [49, 50]. HSCs are resident peri-sinusoidal cells in the subendothelial space of Disse between hepatocytes and sinusoidal endothelial cells. The main cells affected by liver fibrosis are the HSCs and fibroblasts, which are activated by soluble mediators produced by activated KCs or inflammatory cells in the course of chronic liver disease [51]. ECM may thereby regulate cellular activity and availability of growth factors. For instance, decorin and biglycan, two ECM components, bind transforming growth factor-β (TGF-β), fibronectin and laminin bind tumor necrosis factor-α (TNF-α), and collagen binds platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and interleukin-2 (IL-2). The binding of survival factors to the ECM may prevent apoptosis of hepatocyte in the pathologic condition and also prevent growth factor degradation [33]. In liver tissues, HSCs store retinoids such as vitamin A and produce glial fibrillary acidic protein (GFAP), the so-called fat-storing cells or vitamin A-rich cells [52]. Following liver injury, HSCs become activated, which leads to the conversion of a quiescent to activated HSCs that has lost vitamin A droplets, leading to increased proliferation and contraction and the release of proinflammatory, profibrogenic, and promitogenic cytokines. These activated HSCs are capable of enhanced migration and deposition of ECM components [46, 53]. The activation of HSCs can be divided into two stages: initiation and perpetuation [46]. In the first or initiation phase, HSCs undergo the initial changes toward a myofibroblast-like cell differentiation and become more responsive to proliferative and fibrogenic cytokines by up-regulation of membrane receptors [54]. This stage also called a "pre-inflammatory" stage refers to early changes in gene expression that result primarily from paracrine stimuli derived from damaged resident liver cells (sinusoidal endothelial cells, KCs, and hepatocytes) and platelets. KCs engagement drives release cytokines (especially TGF-β) and ROS signaling [55]. Endothelial cells participate in the conversion of latent TGF-β into active form and produce fibronectin, which also provokes early HSC activation. In addition, PDGF, TGF-β, and endothelial growth factor (EGF) which is potent activators of HSCs [56]. Persistence of these stimuli accompanying sustained injury leads to a perpetuation stage regulated by autocrine and paracrine stimuli. Perpetuation stage involves at least seven distinct changes in HSC behavior, including proliferation, chemotaxis, fibrogenesis, contractility, altered matrix degradation, retinoid loss, and inflammatory signaling [57]. Therefore, a critical event in liver fibrogenesis is that the ECM is a dynamic structure, and even advanced fibrosis may be reversible [58, 59]. Multiple interactions between the ECM, HSCs, endothelial cells, and immune cells have been identified. The central event in liver fibrogenesis appears to be the activation of HSCs, which is a complex process [59]. Different patterns of fibrosis progression have been described on the basis of their etiology, region of injury (e.g., portal or central), the source of fibrogenic cells involved, and the predominant fibrogenic mechanisms [60]. For example, chronic viral hepatitis B and C are major causes of bridging fibrosis, resulting in the formation of portal-central fibrotic septa. Perisinusoidal or pericellular fibrosis is typically found in alcohol-related disorders and nonalcoholic fatty liver disease (NAFLD). Progression of hepatic pathology with sustained fibrogenesis leads to cirrhosis, which is not merely the end-stage accumulation of scar, but rather is characterized by a destruction of the hepatic parenchyma and vascular architecture. The main pathological characteristic of cirrhosis is the formation of nodules of regenerative parenchyma enclosed by fibrotic septa, which may contain terminal hepatic venules and portal tracts when the nodules are especially large (i.e., macronodular cirrhosis). Portosystemic shunts and venous occlusion often occur, leading to impairment in liver function and the development of portal hypertension. The formation of vascularized fibrous septa that link portal tracts and central veins is stimulated by angiogenesis and contributes to portosystemic shunting that bypasses the liver parenchyma [61].

#### 3. Liver biopsy: pros and cons, and limitations

Liver biopsy is usually known as the most specific test to evaluate the feature and severity of liver pathology and can be useful in monitoring the efficacy of various treatments. There are currently several techniques available for obtaining liver tissue and each of these has pros and cons [7]. The size of the biopsy specimen, which varies between 10 and 30 mm in length and between 1.2 and 2 mm in diameter, represents only 1:50,000 of the total mass of liver [62]. Therefore, in disease affecting the liver in a diverse way, the histologic findings of biopsy specimen may not be representative of the pathologic process. However, most cases of chronic liver disease causing fibrosis, such as viral and autoimmune hepatitis, as well as nonalcoholic steatohepatitis (NASH), affect the liver in a relatively uniform pattern [63]. Then the extent, to which the biopsy will be representative, will depend greatly on the size of the specimen obtained. The number of portal triads present in the specimen is important; most hepatopathologists are satisfied with a biopsy specimen containing at least 6–8 portal triads. The indications of liver biopsy are outlined in Table 1 [7].

◈ Diagnosis, grading, and staging of chronic hepatitis C or chronic hepatitis B.

◈ Diagnosis of heavy metal storage disorders (e.g., hemochromatosis, Wilson's disease)

◈ Evaluation of the cholestatic liver disease, primary biliary cirrhosis, and primary sclerosing cholangitis

◈ Evaluation of abnormal results of biochemical tests of the liver in association with serological workup that is negative or inconclusive

◈ Use of hepatotoxic regimens (e.g., methotrexate therapy for psoriasis): monitoring

◈ Diagnosis of liver mass (e.g., cancer or unexplained lesions)

◈ Liver donor status before transplantation

phase, HSCs undergo the initial changes toward a myofibroblast-like cell differentiation and become more responsive to proliferative and fibrogenic cytokines by up-regulation of membrane receptors [54]. This stage also called a "pre-inflammatory" stage refers to early changes in gene expression that result primarily from paracrine stimuli derived from damaged resident liver cells (sinusoidal endothelial cells, KCs, and hepatocytes) and platelets. KCs engagement drives release cytokines (especially TGF-β) and ROS signaling [55]. Endothelial cells participate in the conversion of latent TGF-β into active form and produce fibronectin, which also provokes early HSC activation. In addition, PDGF, TGF-β, and endothelial growth factor (EGF) which is potent activators of HSCs [56]. Persistence of these stimuli accompanying sustained injury leads to a perpetuation stage regulated by autocrine and paracrine stimuli. Perpetuation stage involves at least seven distinct changes in HSC behavior, including proliferation, chemotaxis, fibrogenesis, contractility, altered matrix degradation, retinoid loss, and inflammatory signaling [57]. Therefore, a critical event in liver fibrogenesis is that the ECM is a dynamic structure, and even advanced fibrosis may be reversible [58, 59]. Multiple interactions between the ECM, HSCs, endothelial cells, and immune cells have been identified. The central event in liver fibrogenesis appears to be the activation of HSCs, which is a complex process [59]. Different patterns of fibrosis progression have been described on the basis of their etiology, region of injury (e.g., portal or central), the source of fibrogenic cells involved, and the predominant fibrogenic mechanisms [60]. For example, chronic viral hepatitis B and C are major causes of bridging fibrosis, resulting in the formation of portal-central fibrotic septa. Perisinusoidal or pericellular fibrosis is typically found in alcohol-related disorders and nonalcoholic fatty liver disease (NAFLD). Progression of hepatic pathology with sustained fibrogenesis leads to cirrhosis, which is not merely the end-stage accumulation of scar, but rather is characterized by a destruction of the hepatic parenchyma and vascular architecture. The main pathological characteristic of cirrhosis is the formation of nodules of regenerative parenchyma enclosed by fibrotic septa, which may contain terminal hepatic venules and portal tracts when the nodules are especially large (i.e., macronodular cirrhosis). Portosystemic shunts and venous occlusion often occur, leading to impairment in liver function and the development of portal hypertension. The formation of vascularized fibrous septa that link portal tracts and central veins is stimulated by angiogenesis and contributes to portosystemic

shunting that bypasses the liver parenchyma [61].

40 Liver Cirrhosis - Update and Current Challenges

3. Liver biopsy: pros and cons, and limitations

Liver biopsy is usually known as the most specific test to evaluate the feature and severity of liver pathology and can be useful in monitoring the efficacy of various treatments. There are currently several techniques available for obtaining liver tissue and each of these has pros and cons [7]. The size of the biopsy specimen, which varies between 10 and 30 mm in length and between 1.2 and 2 mm in diameter, represents only 1:50,000 of the total mass of liver [62]. Therefore, in disease affecting the liver in a diverse way, the histologic findings of biopsy specimen may not be representative of the pathologic process. However, most cases of chronic liver disease causing fibrosis, such as viral and autoimmune hepatitis, as well as nonalcoholic steatohepatitis (NASH), affect the liver in a relatively uniform pattern [63]. Then the extent, to ◈ Evaluation of systemic illness (e.g., fever of unknown origin, inflammatory or granulomatous disorders)

◈ Hepatosplenomegaly of unknown cause: diagnosis

Table 1. Indication for liver biopsy (modified from Ref. [7]).

Even for patients where serological tests point to a specific liver disease, a liver biopsy can provide valuable information regarding staging, prognosis, and management. There are bad interrelationships between clinical characteristics or status of serum liver enzymes and hepatic histopathologic findings, but also patients with healthy status of liver enzymes may be diagnosed to have clinically advanced fibrosis or cirrhosis on histopathologic findings [64]. If the patient has minor-state illness and is infected with genotype 1a or 1b of the hepatitis C virus, a medical judgment may be made to delay treatment. If the patients have the above degree of moderate disease, treatment will be commonly suggested. If the patients have a virological reaction and acceptable adverse reactions with treatment, continued therapy would be firmly encouraged. The cirrhotic findings on hepatic histopathology will indicate the need for extra tests, such as upper endoscopic procedure to rule out esophageal varices and monitoring for hepatoma with continuing assessment of serum α-fetoprotein and hepatic sonography [7]. In alcoholic liver disease (ALD), the grade of the clinical symptoms and the severity of serum liver enzymes elevation correlate poorly with the degree of liver pathology, particularly in patients who continue to consume alcohol. The long-term prognosis depends upon the extent of liver damage [65]. In patients with ALD as well as NASH, liver biopsy may demonstrate hepatic fatty infiltration, ballooning degeneration of hepatocyte, Mallory's bodies, and hepatonecrosis, regardless of clinically severe fibrosis or cirrhosis [7]. In primary biliary cirrhosis (PBC), sequential liver biopsies may assist one to investigate the natural history, track the responses of therapy, or identify a recurrence of the disease after liver transplantation [66, 67]. Liver biopsy allows a precise evaluation in approximately 90% of patients with obscure disorders revealed on liver function tests [68]. The explanation of diverse courses that appear in a transplanted liver including immune reaction, systemic or infectious complications, drug

<sup>◈</sup> Diagnosis, grading, and staging of alcoholic liver disease, nonalcoholic steatohepatitis (NASH), or autoimmune hepatitis

toxic reaction, and the recurrence of primary disease necessitates a liver histological examination [69]. Liver biopsy can also provide the diagnosis of systemic diseases that can influence the liver, such as sarcoidosis, lymphoma, acquired immune deficiency syndrome (AIDS), and amyloidosis. The histopathological examination of the biopsy material is a subjective process; therefore, diagnostic reproducibility at the 100% level is practically impossible. Intra-observer and inter-observer agreement studies suggest that biopsy specimen size and observer experience (specialization, duration of practice, and academic practice) are important factors in reducing the variation of assessment [8, 70]. Most studies of specimen adequacy have focused on chronic hepatitis because it represents the most common indication for liver biopsy [71]. At present, the most common suggestions for the precise assessment of the degree of fibrosis in chronic hepatic diseases are that the size of biopsy tissue materials must be at a minimum of 20 mm in size and 1.4 mm in radius and must be retained at a minimum of 11 intact portal tracts [72]. In addition, the type of biopsy needle is important, as suction needles tend to miss the fibrous tissue of the septa, as opposed to cutting needles, thus providing the wrong impression regarding the degree of fibrosis and the presence or absence of cirrhosis [73]. Finally, it should be kept in mind that biopsy specimens obtained from subcapsular locations generally contain more fibrous tissue than deeper specimens taken perpendicular to the hepatic surface. For many years, liver biopsy has been considered the gold standard for the staging liver fibrosis [7]. For instance, in patients with chronic HCV, precise definition of the liver fibrosis stage is the important parameter to assess the risk of disease progression and to decide the need for immediate antiviral therapy [74]. Several standardized semi-quantitative scoring systems have been proposed for the staging histological activity index (HAI) proposed in 1981 by Knodell [75] and, more recently, the Ishak score [9] and the Metavir system [76] (Table 2). All of these scoring systems have some limitations, being not linear and prone to intra- and inter-observer variation and to sampling variability [77].

The Knodell score is a composite score that is based on histological assessment of periportal and/or bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation, and fibrosis. The score ranges from 0 to 22, with higher scores representing more advanced disease [75]. Knodell score is frequently used in trials of treatments for chronic hepatitis, particularly HCV. The score is used to assure that baseline histologic features in treatment groups are equally matched and to assess histologic changes after therapy. A limitation of the Knodell score is that it combines inflammation and fibrosis to arrive at one composite score, so it is relatively insensitive to changes in fibrosis. This is important because it is fibrosis, and not inflammation per se, that leads to many of the sequelae of chronic liver disease. In addition, patients may have the same Knodell score despite having markedly different degree of fibrosis. Also, the Knodell score is associated with high inter- and intra-observer variability. The Metavir system is a semi-quantitative classification that consists of four intensity degrees of an activity score (A0–A3) and a five-point scale of fibrosis (F0–F4) [8, 76]. In contrast to the Knodell score, the Metavir system was specifically designed and validated for patients with HCV [76]. The inter- and intra-observer reliabilities of the activity and fibrosis scores of the Metavir system are similar to the Knodell score. The Ishak score is a modification of the Knodell score that includes six stages of fibrosis [9]. This permits documentation of small changes in fibrosis compared with the standard Knodell score, which has only four stages.


toxic reaction, and the recurrence of primary disease necessitates a liver histological examination [69]. Liver biopsy can also provide the diagnosis of systemic diseases that can influence the liver, such as sarcoidosis, lymphoma, acquired immune deficiency syndrome (AIDS), and amyloidosis. The histopathological examination of the biopsy material is a subjective process; therefore, diagnostic reproducibility at the 100% level is practically impossible. Intra-observer and inter-observer agreement studies suggest that biopsy specimen size and observer experience (specialization, duration of practice, and academic practice) are important factors in reducing the variation of assessment [8, 70]. Most studies of specimen adequacy have focused on chronic hepatitis because it represents the most common indication for liver biopsy [71]. At present, the most common suggestions for the precise assessment of the degree of fibrosis in chronic hepatic diseases are that the size of biopsy tissue materials must be at a minimum of 20 mm in size and 1.4 mm in radius and must be retained at a minimum of 11 intact portal tracts [72]. In addition, the type of biopsy needle is important, as suction needles tend to miss the fibrous tissue of the septa, as opposed to cutting needles, thus providing the wrong impression regarding the degree of fibrosis and the presence or absence of cirrhosis [73]. Finally, it should be kept in mind that biopsy specimens obtained from subcapsular locations generally contain more fibrous tissue than deeper specimens taken perpendicular to the hepatic surface. For many years, liver biopsy has been considered the gold standard for the staging liver fibrosis [7]. For instance, in patients with chronic HCV, precise definition of the liver fibrosis stage is the important parameter to assess the risk of disease progression and to decide the need for immediate antiviral therapy [74]. Several standardized semi-quantitative scoring systems have been proposed for the staging histological activity index (HAI) proposed in 1981 by Knodell [75] and, more recently, the Ishak score [9] and the Metavir system [76] (Table 2). All of these scoring systems have some limitations, being not linear and prone to

42 Liver Cirrhosis - Update and Current Challenges

intra- and inter-observer variation and to sampling variability [77].

The Knodell score is a composite score that is based on histological assessment of periportal and/or bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation, and fibrosis. The score ranges from 0 to 22, with higher scores representing more advanced disease [75]. Knodell score is frequently used in trials of treatments for chronic hepatitis, particularly HCV. The score is used to assure that baseline histologic features in treatment groups are equally matched and to assess histologic changes after therapy. A limitation of the Knodell score is that it combines inflammation and fibrosis to arrive at one composite score, so it is relatively insensitive to changes in fibrosis. This is important because it is fibrosis, and not inflammation per se, that leads to many of the sequelae of chronic liver disease. In addition, patients may have the same Knodell score despite having markedly different degree of fibrosis. Also, the Knodell score is associated with high inter- and intra-observer variability. The Metavir system is a semi-quantitative classification that consists of four intensity degrees of an activity score (A0–A3) and a five-point scale of fibrosis (F0–F4) [8, 76]. In contrast to the Knodell score, the Metavir system was specifically designed and validated for patients with HCV [76]. The inter- and intra-observer reliabilities of the activity and fibrosis scores of the Metavir system are similar to the Knodell score. The Ishak score is a modification of the Knodell score that includes six stages of fibrosis [9]. This permits documentation of small changes in fibrosis compared with the standard Knodell score, which has only four stages. Table 2. Comparison between three scoring systems for liver fibrosis in chronic viral hepatitis (modified from Refs. [19, 78–80]).

This staging system has become widely used in clinical trials because of its ability to detect mild changes in fibrosis [81]. The Scheuer system is a simple scoring system that separates necrotic inflammation from fibrosis [78]. Histologic findings of portal inflammation, interface hepatitis, and lobular inflammation are each assigned a score of 0–4. A separate score (0–4) is assigned to the stage of fibrosis. Batts-Ludwig system is also known as the modified Scheuer system [79]. This system is applicable to both chronic viral hepatitis and autoimmune hepatitis and is more useful for assessing an individual patient's liver biopsy for clinical care than therapeutic trials. In addition, disease-specific scoring systems are also available, including scoring systems for nonalcoholic fatty liver disease (NAFLD), ALD, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). However, it is not common to encounter two or more concurrent diseases in a liver biopsy specimen, no scoring systems are available that specifically address these situations [81]. Absolute contraindication to liver biopsy includes patient's inability to remain still and to maintain brief expiration for the procedure, suspected vascular lesion (e.g., hemangioma), bleeding tendency (e.g., INR >1.2 despite receiving vitamin K, bleeding time >10 min), and severe thrombocytopenia (<50,000/mL). Relative contraindications include profound anemia, peritonitis, marked ascites, high-grade biliary obstruction, and a subphrenic or right pleural infection or effusion. Nonetheless, percutaneous liver biopsy is sufficiently safe to be performed on an outpatient setting [82]. Despite liver biopsy being the standard test for an appropriate assessment of patients with chronic liver diseases, there are several limitations of this including variable quality of liver biopsy specimens of <20 mm in length which may be difficult to interpret. Therefore, larger caliber needles may yield better than fine-needle biopsies [71]. Because of fluctuating disease activity, histologic changes obtained at a single point in time may not reflect overall disease activity, which may vary. On the other hand, one would often want to be aware of the progression of liver disease in order to assess therapy response. Limitations entailed by repeat liver biopsy as regards potential patient's risks demand the development of new methods for liver fibrosis evaluation. The features and limitations of liver biopsy are summarized in Table 3. On all these grounds, noninvasive diagnostic tests (serum markers and imaging modalities) have been developed of late mainly to assess liver fibrosis severity. The following pages attempt to describe available information on the better-known serum markers as well as imaging techniques.


Table 3. Pros and cons of liver biopsy and noninvasive methods for the evaluation of liver fibrosis in chronic liver disease (modified from Refs. [83, 84]).

## 4. Noninvasive assessment of liver fibrosis in patients with chronic liver disease

Liver biopsy remains the "gold standard" of assessing hepatic fibrosis. However, it has limitations, such as high cost, invasiveness, associated risk for complications, and sampling or observer variability. Therefore, liver biopsy has recently been challenged by the development of novel noninvasive modalities, including serum direct and/or indirect markers of hepatic fibrosis, noninvasive modalities of predicting fibrosis and imaging techniques, including TE (FibroScan), ultrasonography (US), computed tomography (CT), magnetic resonance imaging (MRI), and thallium 201 per rectal scintigraphy (TI-201 test). As well as TE [85, 86], TI-201 test is a relatively new technique for assessment of liver fibrosis or cirrhosis [87–91]. However, the cost of the equipment may limit the use of TE in some institutions with limited resources. In the past decade, several noninvasive methods for assessing hepatic fibrosis have been published, resulting in more noninvasive tests than histologic scoring systems. The noninvasive tests were introduced to estimate the likelihood of advanced liver fibrosis in patients with chronic viral liver disease at presentation, and on follow-up to assess fibrosis regression in post-treatment period [92]. These tests were later applied in ALD [93, 94] and NAFLD [95, 96]. Our previous studies on the clinical value of the TI-201 test in chronic liver disease may be useful in differentiating chronic hepatitis from cirrhosis and prediction of its prognosis for the management of disease [90, 91, 97, 98]. The first important clinical topic in the assessment of new diagnostic methodologies for evaluation of liver fibrosis is its validation against the present clinical gold standard, liver biopsy, to calculate sensitivity, specificity, and negative- (NPV) and positive-predictive values (PPV). The standard statement of the efficiency of modalities is to examine the area under the receiver operator characteristic curve (AUROC), which plots the sensitivity over 1-specificity using liver biopsy as the reference [17]. The AUROC

## ◈ Liver specific

◈ Levels not influenced by alterations in liver, renal, or reticuloendothelial function

◈ Measurement of one or more of the following processes: Stage of fibrosis, imbalance of activity of ECM (fibrogenesis vs. degradation)

◈ Easy to perform

4. Noninvasive assessment of liver fibrosis in patients with chronic liver

Table 3. Pros and cons of liver biopsy and noninvasive methods for the evaluation of liver fibrosis in chronic liver disease

Liver biopsy Noninvasive methods

• Noninvasive

tions

stages • False-positive values

stages

• Not quantitative

14–33% of cases)

• No complications and no contraindica-

• Accurate assessment of cirrhosis and

• Less accurate for intermediate fibrosis

• Scores may change in different disease

• Unsuitable for diagnosing liver disease

• "Grey zone" (intermediate results in

• Inter-laboratory reproducibility • High applicability and wide availability

for repeated assays • Reasonable cost

minimal/no fibrosis

• Direct observation and quantitative assessment of fibrosis,

• Accurately assessing progression of liver disease or the

• Sampling variability/evaluation of a tiny part of the

• Risk of complications, rare major complications,

Pros • Gold standard to assess fibrosis

44 Liver Cirrhosis - Update and Current Challenges

effect of therapy

whole organ (1:50,000) • Intra- and inter-observer variability • Unsuitable for repeated assays

morbidity and mortality

Cons • Invasive

• High cost

(modified from Refs. [83, 84]).

inflammation, and steatosis • Different stage by different scoring systems • Diagnosing different forms of liver disease

Liver biopsy remains the "gold standard" of assessing hepatic fibrosis. However, it has limitations, such as high cost, invasiveness, associated risk for complications, and sampling or observer variability. Therefore, liver biopsy has recently been challenged by the development of novel noninvasive modalities, including serum direct and/or indirect markers of hepatic fibrosis, noninvasive modalities of predicting fibrosis and imaging techniques, including TE (FibroScan), ultrasonography (US), computed tomography (CT), magnetic resonance imaging (MRI), and thallium 201 per rectal scintigraphy (TI-201 test). As well as TE [85, 86], TI-201 test is a relatively new technique for assessment of liver fibrosis or cirrhosis [87–91]. However, the cost of the equipment may limit the use of TE in some institutions with limited resources. In the past decade, several noninvasive methods for assessing hepatic fibrosis have been published, resulting in more noninvasive tests than histologic scoring systems. The noninvasive tests were introduced to estimate the likelihood of advanced liver fibrosis in patients with chronic viral liver disease at presentation, and on follow-up to assess fibrosis regression in post-treatment period [92]. These tests were later applied in ALD [93, 94] and NAFLD [95, 96]. Our previous studies on the clinical value of the TI-201 test in chronic liver disease may be useful in differentiating chronic hepatitis from cirrhosis and prediction of its prognosis for the management of disease [90, 91, 97, 98]. The first important clinical topic in the assessment of new diagnostic methodologies for evaluation of liver fibrosis is its validation against the present clinical gold standard, liver biopsy, to calculate sensitivity, specificity, and negative- (NPV) and positive-predictive values (PPV). The standard statement of the efficiency of modalities is to examine the area under the receiver operator characteristic curve (AUROC), which plots the sensitivity over 1-specificity using liver biopsy as the reference [17]. The AUROC

disease

Table 4. Features of an ideal marker of liver fibrosis (modified from Ref. [108]).

indicates the probability that a test will correctly rank two randomized patient groups, one with a liver biopsy considered "normal group" and the other "diseased group" [99, 100]. Because liver biopsy itself is not a perfect gold standard, a perfect test will never reach maximal value (1.0) [17]. According to a range of accuracies of the biopsy and a range of prevalence of significant disease (that influence the AUROC), an AUROC of >0.90 in the most favorable scenario cannot be achieved when assessing the so-called "significant fibrosis" even for a perfect marker [99, 101]. This is important for several reasons. First, studies have already shown that these maximal AUROC values have been reached for surrogate markers, especially when assessing cirrhosis versus non-cirrhosis, suggesting that these surrogate markers may be at least as good as liver biopsy in the diagnosis of cirrhosis [102]. Second, some reports suggest that a definitive method for assessing the performance of surrogate markers would employ a clinical end point rather than biopsy as gold standard [101]. The AUROC values may also depend on the biopsy tissue size and fragmentation [103] as well as the incidence of each stage of fibrosis within the studied population (e.g., the spectrum bias) [104]. Indeed, if extreme stages of fibrosis (F0 and F4) are overestimated in a population, the sensitivity and specificity achieved will automatically be higher than in a population that included only patients with near stages of fibrosis (F1 and F2). Several strategies of prohibiting the "spectrum bias" have been suggested including the realignment of AUROC by the DANA method that define advanced (F2–F4) and non-advanced fibrosis (F0–F1) [104] or the Obuchowski measure that is multinomial version of the AUROC [105, 106]. Today, noninvasive methods are widely available. Their most advantages are the absence of contraindication and dangerous complications for the patients, and their reproducibility [107]. In contrast to liver biopsy, many noninvasive methods can effectively evaluate the extent of fibrosis in the whole organ and not only in a part of it. Their potential ability to identify and differentiate between advanced fibrosis stages, the high specificity and sensitivity to diagnose cirrhosis, and their easy application makes them a useful tool in daily clinical practice. Many liver fibrosis experts would therefore consider noninvasive fibrosis tests with an AUROC of 0.85–0.90 to be as good as liver biopsy for diagnosis and staging for liver fibrosis [108]. The role of noninvasive diagnostic tests becomes more significant because their diagnostic accuracy can be increased if they are combined, that is, a serological panel may be used in conjunction with an imaging technique [90, 99, 109]. Features of ideal noninvasive markers of liver fibrosis are summarized in Table 4.

#### 4.1. Serological markers of liver fibrosis

The clinical need for good noninvasive markers of fibrosis is underlined by the marked increase in the number of reports in this area in recent years. A large number of the serological markers of liver fibrosis have been assessed for the noninvasive evaluation of liver fibrosis and are broadly categorized into two groups (direct and indirect) [107]. First, we will refer to direct markers of fibrosis that are thought to directly reflect ECM turnover. Fields in which these methods may have clinical or investigational values involve both the noninvasive method for staging of liver fibrosis but they may also be useful for monitoring the behavior of fibrogenesis and ECM metabolism. Therefore, such assays may be valuable in forecasting fibrotic disease deterioration as well as the efficiency of treatment. Second, there are those that reflect changes in hepatic function but do not directly reflect ECM turnover, for instance, platelet count, coagulation studies, and evaluation of liver enzymes, the so-called indirect markers of liver fibrosis. Researches and developments of these markers have largely focused on the diagnosis of cirrhosis, but more recent researches have emphasized the availability of these markers to assess patients with more advanced fibrosis and hence may be valuable in guiding treatment decisions and prediction of complications of liver cirrhosis [90, 108].

#### 4.1.1. Direct markers of liver fibrosis

Direct markers of liver fibrosis include serum markers, which have been shown to be, or are thought to be, directly involved in the deposition or degradation of ECM. The best-validated marker is hyaluronic acid (HA), a glycosaminoglycan synthesized by HSCs [110]. HA levels correlate with fibrosis in ALD [111] and chronic viral hepatitis [112, 113] and a highly negative score may be used in clinical practice as a reliable index for exclusion of liver fibrosis. Aminoterminal propeptide of type III collagen is a marker associated with collagen deposition and its level is increased in acute and chronic hepatic diseases [114]. TIMPs (TIMP-1 and -2), on the other hand, are associated with the procedure of collagen degradation, which is progressive to fibrosis consequence [114]. The direct markers include several cytokines and markers of matrix turnover (Table 5). The circulating retention times of these molecules are short, so levels may reflect the behavior of ECM turnover. Since ECM turnover is related to both new ECM accumulation and degradation and rebuilding of formed ECM, circulating levels probably exhibit both the activity of the fibrogenesis and the total amount of ECM rebuilding [108]. This phenomenon is identified by at least three properties. First, circulating amounts of these markers are often most increased in situations with rapidly processing fibrosis (e.g., advanced ALD or more active viral hepatitis) and may be high ahead of the significant accumulation of ECM [113, 115]. Second, circulating ECM levels tend toward a decrease in reaction to therapy of the underlying illness, often before any perceptible decrease in the stage of fibrosis [116]. Third, in chronic liver diseases, elevations of several, but not all of these markers associate independently with the stage of fibrosis, rather than with either serological or histopathological findings of inflammatory reaction [112, 117, 118]. In some studies, however, levels of these markers correlated more strongly with the degree of histopathological inflammation or serum liver enzymes [119]. The observation that markers of ECM metabolism are increased in parallel with markers of liver inflammation and necrosis may reflect the importance of these processes in up-regulating fibrogenesis. Direct markers of fibrosis can also be categorized according to their molecular structures. These include (a) collagens: procollagen I and III, propeptides released into the circulation during matrix accumulation and rebuilding. Type IV collagen, which is secreted during interstitial filament metabolism, reflects matrix depletion and rebuilding; (b) glycoproteins and polysaccharides including HA [120], laminin [121], tenascin, and Noninvasive Diagnostic and Prognostic Assessment Tools for Liver Fibrosis and Cirrhosis in Patients with… http://dx.doi.org/10.5772/intechopen.68317 47


Table 5. Direct markers of ECM turnover (deposition vs. degradation) (modified from Ref. [108]).

YKL-40 [122]; and (c) collagenase and their inhibitors, include the MMPs and TIMPs, and cytokines involved in liver fibrosis, the best studies of these is TGF-β. Others, including PDGFs and the antifibrotic cytokine IL-10, have been less well evaluated [108]. The greatest clinical utility of HA may be its ability to exclude patients with significant fibrosis and cirrhosis [112].

#### 4.1.2. Indirect marker and combined panels of liver fibrosis

are broadly categorized into two groups (direct and indirect) [107]. First, we will refer to direct markers of fibrosis that are thought to directly reflect ECM turnover. Fields in which these methods may have clinical or investigational values involve both the noninvasive method for staging of liver fibrosis but they may also be useful for monitoring the behavior of fibrogenesis and ECM metabolism. Therefore, such assays may be valuable in forecasting fibrotic disease deterioration as well as the efficiency of treatment. Second, there are those that reflect changes in hepatic function but do not directly reflect ECM turnover, for instance, platelet count, coagulation studies, and evaluation of liver enzymes, the so-called indirect markers of liver fibrosis. Researches and developments of these markers have largely focused on the diagnosis of cirrhosis, but more recent researches have emphasized the availability of these markers to assess patients with more advanced fibrosis and hence may be valuable in guiding treatment

Direct markers of liver fibrosis include serum markers, which have been shown to be, or are thought to be, directly involved in the deposition or degradation of ECM. The best-validated marker is hyaluronic acid (HA), a glycosaminoglycan synthesized by HSCs [110]. HA levels correlate with fibrosis in ALD [111] and chronic viral hepatitis [112, 113] and a highly negative score may be used in clinical practice as a reliable index for exclusion of liver fibrosis. Aminoterminal propeptide of type III collagen is a marker associated with collagen deposition and its level is increased in acute and chronic hepatic diseases [114]. TIMPs (TIMP-1 and -2), on the other hand, are associated with the procedure of collagen degradation, which is progressive to fibrosis consequence [114]. The direct markers include several cytokines and markers of matrix turnover (Table 5). The circulating retention times of these molecules are short, so levels may reflect the behavior of ECM turnover. Since ECM turnover is related to both new ECM accumulation and degradation and rebuilding of formed ECM, circulating levels probably exhibit both the activity of the fibrogenesis and the total amount of ECM rebuilding [108]. This phenomenon is identified by at least three properties. First, circulating amounts of these markers are often most increased in situations with rapidly processing fibrosis (e.g., advanced ALD or more active viral hepatitis) and may be high ahead of the significant accumulation of ECM [113, 115]. Second, circulating ECM levels tend toward a decrease in reaction to therapy of the underlying illness, often before any perceptible decrease in the stage of fibrosis [116]. Third, in chronic liver diseases, elevations of several, but not all of these markers associate independently with the stage of fibrosis, rather than with either serological or histopathological findings of inflammatory reaction [112, 117, 118]. In some studies, however, levels of these markers correlated more strongly with the degree of histopathological inflammation or serum liver enzymes [119]. The observation that markers of ECM metabolism are increased in parallel with markers of liver inflammation and necrosis may reflect the importance of these processes in up-regulating fibrogenesis. Direct markers of fibrosis can also be categorized according to their molecular structures. These include (a) collagens: procollagen I and III, propeptides released into the circulation during matrix accumulation and rebuilding. Type IV collagen, which is secreted during interstitial filament metabolism, reflects matrix depletion and rebuilding; (b) glycoproteins and polysaccharides including HA [120], laminin [121], tenascin, and

decisions and prediction of complications of liver cirrhosis [90, 108].

4.1.1. Direct markers of liver fibrosis

46 Liver Cirrhosis - Update and Current Challenges

Indirect markers of fibrosis are simple routine blood tests reflecting alterations in liver function but not directly representing ECM homeostasis. These biomarkers include indices related to portal hypertension (platelet count and spleen size), liver synthetic parameters (i.e., albumin), liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) [123], AST/ALT ratio [124, 125], bilirubin, prothrombin index (PT) [126], γ-glutamyl transferase (γ-GT), and apolipoprotein A1(apo-A1). They can be used in combination to produce sophisticated serological panels such as PGA index (prothrombin time, γ-GT, and apo-A1) [127–129] and APRI (AST to platelet ratio index) [130, 131]. PGA is one of the first biological indexes used for the noninvasive detection of cirrhosis in ALD patients [127]. APRI is based on serum AST level and platelet [131]. It is calculated as (AST/upper limit of normal\*) 100/platelet count and has been extensively studied in patients with HCV or ALD (\*adjusted according to the reference values of each laboratory) [107, 132]. PGA index was subsequently modified to the PGAA index by the addition of α2-macroglobulin which resulted in some improvement in its performance (PGAA) [128]. Analysis of studies of indirect markers of fibrosis reveals several features, which are applicable to routine clinical practice. First, in viral and NAFLD, an AST/ ALT ratio of greater than 1 is frequently associated with progressive liver fibrosis or cirrhosis [133–135]. Second, both components of the PGA index such as γ-GT and thrombin index are markers of advanced liver fibrosis and can be used to discern patients with more advanced liver fibrosis. Indeed, the prothrombin index has been carried out alike or better than specific other markers of liver fibrosis [117, 136]. It should be emphasized that these markers represent liver dysfunction or structure rather than the disturbance of normal ECM metabolism (Table 6) [108].

#### 4.1.3. Indices/algorithms combining indirect and direct markers of liver fibrosis

The limitations of each marker to assess liver fibrosis have led to the development of more sophisticated algorithms or indices combining the results of panels of markers that substantially improved diagnostic accuracy in noninvasive evaluation of liver fibrosis (Table 7).


Abbreviation: TIMP, tissue inhibitors of metalloproteinases; MMP, matrix metalloproteinase; ALT, alanine aminotransferase; HA, hyaluronic acid; APRI, aspartate aminotransferase/platelet ratio index; PIIINP, Procollagen III amino terminal; γ-GT, γ-glutamyl transpeptidase.

Table 6. Serum noninvasive marker of liver fibrosis (modified from Refs. [19, 84]).



Abbreviation: AP, alkaline phosphatase; NPV, negative-predictive value; PPV, positive-predictive value, CHC, BMI, body mass index; H/L ratio, heart/liver uptake ratio; γ-GT, γ-glutamyl transpeptidase.

Table 7. Combined scores/algorithms for evaluation of liver fibrosis.

Scores/algorithms Description

γ-GT, γ-glutamyl transpeptidase.

Fibrosis Probability Index (FPI)

Goteborg University Cirrhosis

Index (GUCI) [153]

[152]

FibroTest [137] • Most validated algorithm and consider patient age and gender

gender.

Table 6. Serum noninvasive marker of liver fibrosis (modified from Refs. [19, 84]).

Direct serum markers Indirect serum markers/combined panels

◈ HA ◈ Liver enzymes (ALT, AST)

◈ Laminin ◈ AST/ALT ◈ YKL-40 ◈ <sup>γ</sup>-GT

48 Liver Cirrhosis - Update and Current Challenges

◈ Procollagen type III ◈ Platelet count ◈ PIIINP ◈ Albumin ◈ MMP-1 and -2 ◈ Bilirubin ◈ TIPMs ◈ PGA ◈ TGF-<sup>β</sup> ◈ APRI

Fibrospect [148] • 3 parameters: serum HA, TIMP-1, α2-macroglobulin

BUN

Hepascore [147] • Four parameters: bilirubin, γ-GT, HA, TIMP-1, α2-macroglobulin), age, and

Abbreviation: TIMP, tissue inhibitors of metalloproteinases; MMP, matrix metalloproteinase; ALT, alanine aminotransferase; HA, hyaluronic acid; APRI, aspartate aminotransferase/platelet ratio index; PIIINP, Procollagen III amino terminal;

• Moderate or severe fibrosis versus no fibrosis Fibrometer [149] • Six parameters: platelet count, prothrombin time, AST, α2-macroglobulin, HA,

ViraHep C model [150] • Probability <sup>¼</sup> 1/(exp[�y]) <sup>þ</sup> 1, <sup>y</sup> ¼ �5.17 <sup>þ</sup> 0.2xrace <sup>þ</sup> age(years) <sup>þ</sup> 1.19 � ln (AST, IU/L) –1.76�ln(platelet, 10<sup>3</sup>

Glycocirrhotest [151] • Detection of compensated cirrhosis with 100% specificity and 75% sensitivity.

Forns score [154] • 7.811–3.131�ln(PT) <sup>þ</sup> 0.781�ln(γ-GT) <sup>þ</sup> 3.467�ln(age) – 0.014� (cholesterol)

in chronic hepatitis C

87% at a score of ≥0.8.

stage)

10<sup>9</sup> /L)

• Prediction of severe fibrosis in chronic viral hepatitis

independent predictors of significant fibrosis.

and useful guide to make decision for need of biopsy.

• Five parameters: apo-A1, α2-macroglobulin, γ-GT, total bilirubin, haptoglubin.

• Dependent on race (AA, African American <sup>¼</sup> 0, CA, Caucasian American <sup>¼</sup> 1)

• Multivariate logistic regression analysis identified age, AST, total cholesterol level, insulin resistance (by homeostasis model), and past alcohol intake as

• 96% sensitivity and NPV 93% at a score of ≥0.2 versus 94% specificity and PPV

• Probability of significant liver fibrosis in patients with chronic HCV infection

• Multivariate logistic regression analysis between fibrosis stage (ref. as Ishak

• GUCI formula: normalized AST � prothrombin-INR � 100/platelet count(�

• 80% sensitivity and 78% specificity for cirrhosis with NPV 97% and PPV 31%

• Validation in patients with CHC as well as nonviral chronic hepatitis

• Follow-up of chronic liver diseases patients without repeated biopsy

/mL) þ 1.38�ln(AP, IU/L) for severe fibrosis

• Prediction with AUROC 0.81 in significant fibrosis and 0.88 for cirrhosis

In most studies, indices have been validated against the current clinical gold standard, liver biopsy, using as expression of their effectiveness the AUROC with optimal value being as close as possible [99]. The first proposed index was based on a parented mathematical formula combining five variables (total bilirubin, γ-GT, haptoglobin, α2-macroglobulin, and apo-A1) [137] and the results of this test were ranged from 0 to 1.0, using Fibrotest as a reference. In the initial report, a very low score (<0.1) allowed the exclusion of significant fibrosis with a 100% negative-predictive value (NPV), whereas a moderate high score (>0.6) allowed the diagnosis of significant fibrosis with a 90% positive-predictive value (PPV), using liver biopsy as a reference. Overall, liver biopsy could have been avoided in 46% of the patients from that study. Fibrotest has been primarily used for patients with chronic viral hepatitis and is now extensively evaluated in the patients with chronic hepatitis C [109, 138, 139] but also in other cases, such as hepatitis B [140, 141], HCV and HIV coinfection [142], NAFLD [143], ALD [93], and renal-transplanted patients with chronic HCV [144]. The recent meta-analysis that pooled 7985 subjects (with analysis of individual data in 3282) with both Fibrotest and biopsy (HCV, 4600; HBV, 1580; NAFLD, 267; ALD, 524; mixed form, 1014) and the mean standardized AUROC for diagnosing significant fibrosis was 0.84 (95% confidence interval: 0.83–0.89), without differences between causes of liver disease. Therefore, Fibrotest have been used as an alternative to liver biopsy for the first-line assessment of fibrosis and common chronic liver diseases, namely HCV, HBV, NAFLD, and ALD [145]. One of the important issues of these algorithms is that in individual patients they cannot reliably differentiate the intermediate stages of fibrosis. Finally, in patients with chronic HCV, the application of these algorithms or indices can confirm or exclude fibrosis in less than 40% of patients [146].

#### 4.1.4. Combination of indices and algorithms for assessment of liver fibrosis

In order to increase diagnostic accuracy, new approaches using stepwise algorithms combining continually different indices have been proposed in patients with chronic hepatitis C [161] and B [140]. For instance, one group was able to identify significant fibrosis with high diagnostic outcome above 94% diagnostic accuracy by APRI as screening procedure, followed by Fibrotest in APRI non-classified cases and prohibiting liver biopsy to patients classified F0–F1 by noninvasive procedures. Cirrhosis could also have been recognized with 95% diagnostic accuracy applying a similar algorithm by the combination of APRI and Fibrotest (Figure 2). On the whole, liver biopsy could have been prevented in approximately 50 and 80% of patients for the diagnosis of severe fibrosis and cirrhosis in patients with chronic hepatitis C, respectively. Other groups have proposed alternative algorithms combining Fibrotest and APRI either with [162] or without Forns index [146]. Otherwise, high diagnostic accuracy for the evaluation of significant fibrosis and cirrhosis has been reported for the combination of Fibrotest with Fibroscan that is based on the assessment of hepatic stiffness by TE [22, 109]. However, this method requires the availability of complex equipment, with limited access and costs that most likely exceed those of their more simple and accessible algorithms [161].

Several noninvasive markers of liver fibrosis have been represented but their application in substitute for liver biopsy may still remain controversy and is not generally acceptable due to still insufficient diagnostic performance. In fact, some of these methodologies such as APRI and Forns index remain in many cases unclassified group and all of them are not over 80–85% diagnostic performance [131, 139, 163, 164]. As a consequence, many patients still need to have a liver biopsy taken, and in those classified without liver biopsy, misdiagnosis is expected to occur in at least 15–20%, a figure that is considered inadequate by many clinicians [165, 166]. Most of them, such as APRI and Forns index, are not able to identify individual stages of fibrosis. APRI cannot be completely standardized due to the variability of measurement and normal ranges of AST in different laboratories [167]. Since the diagnostic performance of described noninvasive markers is variable depending on the stage of fibrosis and other patients' characteristics, they can be used to reduce rather than completely substitute the need for liver biopsy. Even though many studied have been shown that Fibrotest had the best performance when compared to other noninvasive methods, none of the investigated noninvasive markers of liver fibrosis has adequate accuracy for universal use instead of liver biopsy [140]. And, one of the major critical points of the clinical application of serum markers and indices of liver fibrosis is that they are not regularly useful in most clinical situation. Another clinical point of these markers is that they are liver nonspecific and may be influenced by changes of their level; for example, HA levels increase after the meal [168] or in senile Noninvasive Diagnostic and Prognostic Assessment Tools for Liver Fibrosis and Cirrhosis in Patients with… http://dx.doi.org/10.5772/intechopen.68317 51

diagnosing significant fibrosis was 0.84 (95% confidence interval: 0.83–0.89), without differences between causes of liver disease. Therefore, Fibrotest have been used as an alternative to liver biopsy for the first-line assessment of fibrosis and common chronic liver diseases, namely HCV, HBV, NAFLD, and ALD [145]. One of the important issues of these algorithms is that in individual patients they cannot reliably differentiate the intermediate stages of fibrosis. Finally, in patients with chronic HCV, the application of these algorithms or indices can confirm or

In order to increase diagnostic accuracy, new approaches using stepwise algorithms combining continually different indices have been proposed in patients with chronic hepatitis C [161] and B [140]. For instance, one group was able to identify significant fibrosis with high diagnostic outcome above 94% diagnostic accuracy by APRI as screening procedure, followed by Fibrotest in APRI non-classified cases and prohibiting liver biopsy to patients classified F0–F1 by noninvasive procedures. Cirrhosis could also have been recognized with 95% diagnostic accuracy applying a similar algorithm by the combination of APRI and Fibrotest (Figure 2). On the whole, liver biopsy could have been prevented in approximately 50 and 80% of patients for the diagnosis of severe fibrosis and cirrhosis in patients with chronic hepatitis C, respectively. Other groups have proposed alternative algorithms combining Fibrotest and APRI either with [162] or without Forns index [146]. Otherwise, high diagnostic accuracy for the evaluation of significant fibrosis and cirrhosis has been reported for the combination of Fibrotest with Fibroscan that is based on the assessment of hepatic stiffness by TE [22, 109]. However, this method requires the availability of complex equipment, with limited access and costs that most

Several noninvasive markers of liver fibrosis have been represented but their application in substitute for liver biopsy may still remain controversy and is not generally acceptable due to still insufficient diagnostic performance. In fact, some of these methodologies such as APRI and Forns index remain in many cases unclassified group and all of them are not over 80–85% diagnostic performance [131, 139, 163, 164]. As a consequence, many patients still need to have a liver biopsy taken, and in those classified without liver biopsy, misdiagnosis is expected to occur in at least 15–20%, a figure that is considered inadequate by many clinicians [165, 166]. Most of them, such as APRI and Forns index, are not able to identify individual stages of fibrosis. APRI cannot be completely standardized due to the variability of measurement and normal ranges of AST in different laboratories [167]. Since the diagnostic performance of described noninvasive markers is variable depending on the stage of fibrosis and other patients' characteristics, they can be used to reduce rather than completely substitute the need for liver biopsy. Even though many studied have been shown that Fibrotest had the best performance when compared to other noninvasive methods, none of the investigated noninvasive markers of liver fibrosis has adequate accuracy for universal use instead of liver biopsy [140]. And, one of the major critical points of the clinical application of serum markers and indices of liver fibrosis is that they are not regularly useful in most clinical situation. Another clinical point of these markers is that they are liver nonspecific and may be influenced by changes of their level; for example, HA levels increase after the meal [168] or in senile

exclude fibrosis in less than 40% of patients [146].

50 Liver Cirrhosis - Update and Current Challenges

4.1.4. Combination of indices and algorithms for assessment of liver fibrosis

likely exceed those of their more simple and accessible algorithms [161].

Figure 2. Proposed best algorithm for the detection of significant fibrosis and cirrhosis using APRI and Fibrotest in patients with chronic hepatitis C and B with diagnostic accuracy (modified from Refs. [90, 159]).

patients with chronic inflammatory states such as rheumatoid arthritis [169]. Also, the repeatability of assessments of several biomarkers included in direct serum markers, such as AST levels or platelet count, is doubtful [170]. The effect of serum lipid levels caused by anticholesteremic agents on the Forns index was taken into consideration. Finally, when applying Fibrotest in clinical situation, the evaluation should consider each of the five markers individually in order to escape false-positive outcomes related to hemolysis (low hepatoglobin level), Gilbert syndrome (high bilirubin level), or false-negative outcomes related to inflammatory reactions [171]. However, a panel that combines proteins and proteinases of the ECM has been proposed and the results are promising [155]. The combined use of some of these markers with the aim of reducing rather than completely abolishing liver biopsy may represent a rational and more convincing approach [172]. In a large-scale multicenter study, the diagnostic accuracy of a stepwise combination of two well-studied noninvasive markers of fibrosis (APRI and Fibrotest) was followed by liver biopsy in only a subset of cases [171]. This approach, called SAFE (sequential algorithm for fibrosis evaluation) biopsy, has been built up with double goal of detecting both severe fibrosis and cirrhosis and has here been confirmed to assure >90% diagnostic accuracy in comparison with respect to liver biopsy as the gold standard with <2% underestimation of the stage of liver disease as derived from NPV. The SAFE biopsy may be particularly useful for screening HCV-infected patients in whom an immediate approach with liver biopsy is particularly problematic or questionable [173]. Using two algorithms (Fibrotest and APRI), liver biopsy could be avoided in 50% of cases for the diagnosis of significant fibrosis and in 70% of cases for the identification of cirrhosis [174].

#### 4.2. Imaging modalities and combinations with other markers for the diagnosis of liver fibrosis

#### 4.2.1. Transient elastography (TE)

Liver fibrosis can be staged using one-dimensional ultrasound TE (Fibroscan) [22], which is the most widely used imaging method for noninvasive and rapid measurement of hepatic tissue stiffness. Many studies have evaluated the diagnostic accuracy of TE for diagnosing cirrhosis with specificity and sensitivity approaching 90%. The accuracy for liver fibrosis detection is lower, with sensitivity and specificity approaching 70–80% [102, 175, 176]. Because both adipose tissue and the presence of fluid may influence the velocity of shear wave [107], obesity, ascites, acute inflammation, liver congestion, and elevated portal vein pressure may reduce TE accuracy. Furthermore, a falsely increased liver stiffness, due to postprandial increase in portal vein pressure, has been observed [177, 178]. Comparison of TE with biopsy results has provided that cut-off values can be demonstrated to differentiate mild and moderate fibrosis from advanced fibrosis and cirrhosis, with validation tests showing variable performance and with greatest statistical significance being ensured in the distinction of cirrhosis from mild fibrosis (AUROC F ¼ 4 (0.94), sensitivity F ≥ 2 (85%), specificity F ≥ 2 (91%)) [179, 180]. Investigations have applied various best stiffness cut-off values, making comparison between researches. Generally, advanced fibrosis is more likely with higher cut-off values (Table 8) [181, 182]. The optimal cut-off value is 14.6 kPa for the detection of cirrhosis, but a cut-off value of 10.0 and 14.1 kPa was adequate to achieve 95% sensitivity and specificity in their HCV patients with cirrhosis [183]. Otherwise, the performance of TE was low for discriminating mild from significant liver fibrosis [184] and Spearman's correlation coefficient between the elasticity scores using real-time TE and histopathological fibrosis stage was low at 0.48 [185]. However, TE was more useful for the identification of advanced fibrosis and their necroinflammatory activity influences TE measurements in patients without cirrhosis [186] and might be overestimated liver fibrosis when ALT is elevated [187]. Some reports were shown that good correlation between TE and fibrosis exists, but data on TE in an Asian cohort show only 8% of patients having limited HCV [188]. Liver stiffness measurement (LSM) by TE is a reliable predictor of liver fibrosis in Indian patients with chronic hepatitis C and B. LSM is superior to APRI for noninvasive diagnosis of hepatic fibrosis and cirrhosis, and high bilirubin (10.5 mg/dL) and Ishak HAI grade (>11) were independent predictors of discordance between liver biopsy and LSM [189]. Liver stiffness has also been revealed to have good correlation with steatosis, necrotic inflammatory activity and hepatic iron accumulation as well as fibrosis [190]. TE is restrictive, however, by its impossibility to perform in patients with ascites and patients with narrow intercostal spaces or morbid obesity. Advantages of TE include a short procedure time (<5 min), immediate results, and the ability to perform the test at the bedside or in an outpatient clinic.

Noninvasive Diagnostic and Prognostic Assessment Tools for Liver Fibrosis and Cirrhosis in Patients with… http://dx.doi.org/10.5772/intechopen.68317 53


Abbreviations: AUROC, area under the receiver operator characteristic curve; CC, correctly classified: true positive and negative; HBV, chronic hepatitis B; HCV, chronic hepatitis C; SE, sensitivity; SP, specificity.

Table 8. Diagnostic performance of TE for significant fibrosis (F ≥ 2) and cirrhosis (F4) in patients with Hepatitis B or C (modified from Refs. [191]).

#### 4.2.2. Magnetic resonance elastography (MRE)

SAFE biopsy may be particularly useful for screening HCV-infected patients in whom an immediate approach with liver biopsy is particularly problematic or questionable [173]. Using two algorithms (Fibrotest and APRI), liver biopsy could be avoided in 50% of cases for the diagnosis of significant fibrosis and in 70% of cases for the identification of cirrhosis [174].

Liver fibrosis can be staged using one-dimensional ultrasound TE (Fibroscan) [22], which is the most widely used imaging method for noninvasive and rapid measurement of hepatic tissue stiffness. Many studies have evaluated the diagnostic accuracy of TE for diagnosing cirrhosis with specificity and sensitivity approaching 90%. The accuracy for liver fibrosis detection is lower, with sensitivity and specificity approaching 70–80% [102, 175, 176]. Because both adipose tissue and the presence of fluid may influence the velocity of shear wave [107], obesity, ascites, acute inflammation, liver congestion, and elevated portal vein pressure may reduce TE accuracy. Furthermore, a falsely increased liver stiffness, due to postprandial increase in portal vein pressure, has been observed [177, 178]. Comparison of TE with biopsy results has provided that cut-off values can be demonstrated to differentiate mild and moderate fibrosis from advanced fibrosis and cirrhosis, with validation tests showing variable performance and with greatest statistical significance being ensured in the distinction of cirrhosis from mild fibrosis (AUROC F ¼ 4 (0.94), sensitivity F ≥ 2 (85%), specificity F ≥ 2 (91%)) [179, 180]. Investigations have applied various best stiffness cut-off values, making comparison between researches. Generally, advanced fibrosis is more likely with higher cut-off values (Table 8) [181, 182]. The optimal cut-off value is 14.6 kPa for the detection of cirrhosis, but a cut-off value of 10.0 and 14.1 kPa was adequate to achieve 95% sensitivity and specificity in their HCV patients with cirrhosis [183]. Otherwise, the performance of TE was low for discriminating mild from significant liver fibrosis [184] and Spearman's correlation coefficient between the elasticity scores using real-time TE and histopathological fibrosis stage was low at 0.48 [185]. However, TE was more useful for the identification of advanced fibrosis and their necroinflammatory activity influences TE measurements in patients without cirrhosis [186] and might be overestimated liver fibrosis when ALT is elevated [187]. Some reports were shown that good correlation between TE and fibrosis exists, but data on TE in an Asian cohort show only 8% of patients having limited HCV [188]. Liver stiffness measurement (LSM) by TE is a reliable predictor of liver fibrosis in Indian patients with chronic hepatitis C and B. LSM is superior to APRI for noninvasive diagnosis of hepatic fibrosis and cirrhosis, and high bilirubin (10.5 mg/dL) and Ishak HAI grade (>11) were independent predictors of discordance between liver biopsy and LSM [189]. Liver stiffness has also been revealed to have good correlation with steatosis, necrotic inflammatory activity and hepatic iron accumulation as well as fibrosis [190]. TE is restrictive, however, by its impossibility to perform in patients with ascites and patients with narrow intercostal spaces or morbid obesity. Advantages of TE include a short procedure time (<5 min), immediate results, and the ability to perform the test at the bedside or in an outpatient clinic.

4.2. Imaging modalities and combinations with other markers for the

diagnosis of liver fibrosis

4.2.1. Transient elastography (TE)

52 Liver Cirrhosis - Update and Current Challenges

MRE is a noninvasive method of measuring the viscoelastic properties of the liver and evaluate liver stiffness by measuring the propagation of mechanical waves [194]. MRE indicated that patients with hepatic fibrosis have higher LSM than normal volunteers [195] and that those with mild fibrosis were able to be distinguished from those with moderate or advanced fibrosis, with a mean hepatic shear elasticity being 2.24, 2.56, and 4.68 kPa in patients with F0–F1, F2–F3, and F4 fibrosis, respectively [196]. MRE is superior to TE because of its ability to scan the whole organ and its application in patients with ascites or obesity. MRE was accurate in liver fibrosis staging and superior to biochemical testing with APRIs in patients with chronic HBV and HCV infection [197, 198]. These findings suggest that noninvasive MRE potentially has a role in determining the treatment and the prognosis of patients with chronic liver disease because it enables substantial and advanced fibrosis to be readily diagnosed. More particularly, MRE might be useful in the selection of patients with liver fibrosis who should either be treated (score of ≥F2) or undergo surveillance for portal hypertension and hepatocellular carcinoma (score of ≥F3) [197]. Antiviral treatment should be considered in patients with liver stiffness values of ≥2.8 kPa [199]. The main drawbacks are the high cost and complexity of the method that is too procrastinating for daily clinical practice. MRE values may be affected by the increased portal vein pressure following a meal similar to TE [200].

#### 4.2.3. Acoustic radiation force impulses (ARFI)

ARFI use conventional hepatic ultrasonography to assess liver stiffness [199, 201]. ARFI uses short duration of acoustic pulses that produce mechanical excitation. The speed of the produced waves correlates directly with the extent of liver fibrosis and results are expressed in m/s. For fibrosis quantification, the "Virtual Touch (VT) tissue quantification" application was used, allowing for the measurement of SWV (shear wave velocity, m/s) within the interest area chosen by the examiner, according to principles. The higher the tissue stiffness shows, the higher the SWV produces [202]. The theoretical advantage of ARFI as compared to TE is its implementation on an ultrasound device, via additional software imaging control and detection algorithms, thus allowing the visualization of B-mode, color Doppler mode, and ARFI images with same equipment [201]. Advantages of this technology include the ability to select the area to be assessed, avoiding large vessels or ribs [107] and the fact that steatosis does not influence the accuracy of the procedure. Otherwise, ARFI and TE are influenced by high ALT levels. In European patients with chronic hepatitis B and C, ALT values between 1.1x and 5xULN had only limited influence on ARFI values. The best cut-off values for predicting significant fibrosis and cirrhosis were similar in patients with moderately elevated ALT levels [203].

#### 4.2.4. Real-time sonography-based elastography (RTE)

RTE is a new method for the measurement of tissue elasticity different from TE. The echo signals are captured in the real time, while the probe slightly compresses or relaxes the body through freehand operation. Many clinical researches indicated that RTE could allow a high accuracy on the differential diagnosis of superficial focal pathological lesion such as mammary gland tumors, thyroid tumors, and prostate tumors [204, 205]. This method estimates the velocity of a shear wave through the liver using US and results are expressed in kPa. The diagnostic accuracies expressed as AUROC were 0.75 for the diagnosis of significant fibrosis (F ≥ 2), 0.73 for severe fibrosis (F ≥ 3), and 0.69 for cirrhosis. For a combined elasticitylaboratory scores (platelet count and γ-GT), AUROCs were 0.93, 0.95, and 0.91, respectively. Therefore, RTE is a new and promising sonography-based noninvasive method for the assessment of liver fibrosis in patients with chronic viral hepatitis [185].

#### 4.2.5. 2D-Shear wave elastography (2D-SWE)

2D-SWE combines ultrasound images with radiation force induced into the liver. 2D-SWE can measure shear waves propagation in real time [16]. Advantages of 2D-SWE (m/s or kPa) include good applicability and adjustable region of interest depending on the operator [84]. Its failure rate is significantly lower than that of TE [206–208], particularly in patients with ascites [207, 208], but not in obese patients when the XL probe is used for TE (10.4 vs. 2.6%, respectively) [209]. In a pilot study in 121 patients with chronic hepatitis C (Metavir, 41% F0/ F1, 27% F2, 12% F3, and 20% F4), AUROCs of 2D-AWE for the diagnosis of significant fibrosis and cirrhosis were 0.92 and 0.98, respectively [206]. Sensitivities and specificities were 85 and 92% for the diagnosis of significant fibrosis using a cut-off of 7.1 kPa, and 97 and 93% for the diagnosis of liver cirrhosis using a cut-off of 10.1 kPa. Therefore, 2D-SWE is a promising technique that is currently under investigation. It seems to be at least equivalent to TE and pSWE/ARFI for noninvasive staging of liver fibrosis in viral hepatitis [16].

#### 4.2.6. Sonography-based imaging

practice. MRE values may be affected by the increased portal vein pressure following a meal

ARFI use conventional hepatic ultrasonography to assess liver stiffness [199, 201]. ARFI uses short duration of acoustic pulses that produce mechanical excitation. The speed of the produced waves correlates directly with the extent of liver fibrosis and results are expressed in m/s. For fibrosis quantification, the "Virtual Touch (VT) tissue quantification" application was used, allowing for the measurement of SWV (shear wave velocity, m/s) within the interest area chosen by the examiner, according to principles. The higher the tissue stiffness shows, the higher the SWV produces [202]. The theoretical advantage of ARFI as compared to TE is its implementation on an ultrasound device, via additional software imaging control and detection algorithms, thus allowing the visualization of B-mode, color Doppler mode, and ARFI images with same equipment [201]. Advantages of this technology include the ability to select the area to be assessed, avoiding large vessels or ribs [107] and the fact that steatosis does not influence the accuracy of the procedure. Otherwise, ARFI and TE are influenced by high ALT levels. In European patients with chronic hepatitis B and C, ALT values between 1.1x and 5xULN had only limited influence on ARFI values. The best cut-off values for predicting significant fibrosis and cirrhosis were similar in patients with moder-

RTE is a new method for the measurement of tissue elasticity different from TE. The echo signals are captured in the real time, while the probe slightly compresses or relaxes the body through freehand operation. Many clinical researches indicated that RTE could allow a high accuracy on the differential diagnosis of superficial focal pathological lesion such as mammary gland tumors, thyroid tumors, and prostate tumors [204, 205]. This method estimates the velocity of a shear wave through the liver using US and results are expressed in kPa. The diagnostic accuracies expressed as AUROC were 0.75 for the diagnosis of significant fibrosis (F ≥ 2), 0.73 for severe fibrosis (F ≥ 3), and 0.69 for cirrhosis. For a combined elasticitylaboratory scores (platelet count and γ-GT), AUROCs were 0.93, 0.95, and 0.91, respectively. Therefore, RTE is a new and promising sonography-based noninvasive method for the

2D-SWE combines ultrasound images with radiation force induced into the liver. 2D-SWE can measure shear waves propagation in real time [16]. Advantages of 2D-SWE (m/s or kPa) include good applicability and adjustable region of interest depending on the operator [84]. Its failure rate is significantly lower than that of TE [206–208], particularly in patients with ascites [207, 208], but not in obese patients when the XL probe is used for TE (10.4 vs. 2.6%, respectively) [209]. In a pilot study in 121 patients with chronic hepatitis C (Metavir, 41% F0/ F1, 27% F2, 12% F3, and 20% F4), AUROCs of 2D-AWE for the diagnosis of significant fibrosis

assessment of liver fibrosis in patients with chronic viral hepatitis [185].

similar to TE [200].

4.2.3. Acoustic radiation force impulses (ARFI)

54 Liver Cirrhosis - Update and Current Challenges

ately elevated ALT levels [203].

4.2.4. Real-time sonography-based elastography (RTE)

4.2.5. 2D-Shear wave elastography (2D-SWE)

US imaging has been used to noninvasively evaluate the severity of liver fibrosis in patients with chronic HCV. Results vary with some studies showing associations between US score and diagnosis of cirrhosis with various sensitivities (87.5–100%) and specificities (81.5–93.5%) [210, 211]. The application of US to assess liver fibrosis was used by calculating a fibrosis extraction ratio (FER) (fiber volume/total volume), which was able to distinguish F0/F1 from ≥F2 fibrosis with a sensitivity of 55% in the HCV cohort [212]. In sonography, contrastenhanced sonography is based on intravenous injection of specifically sized microbubbles, transferred with a shell of protein or biopolymers that facilitate their sonographic imaging [213]. Some report studied the hepatic vein transit time (HVTT) for grading liver disease using a microsound microbubble contrast agent as a tracer. This study also applied Doppler sonography to make a decision for several indices to assess portal vein congestive index, but found that there was no significance. HVTT was significantly shorter in cirrhotic patients than in non-cirrhotic patients (p < 0.001) and distinguished between these patients with high accuracy [214]. Therefore, unenhanced Doppler ultrasound is not reliable in the discrimination of varying degrees of fibrosis, but that results can be improved with additional measurement such as heart pulsation at the liver surface and portal venous flow measurements. Color Doppler is a noninvasive method for assessing portal hemodynamics. In the study for portal hemodynamics by color Doppler and gastric mucosal blood flow (GMBF) by laser Doppler velocimetry in patients with cirrhosis, portal venous blood flow (PVBF), portal flow velocity (PFV), and GMBF were all significantly slower in cirrhotic patients and PVBF and PFV were lower in Child's class B/C than in class A [215]. A statistically significant difference has been shown in all US markers between patients with and without cirrhosis, but sensitivity and specificity were significantly increased when evaluation of the transmission of heart pulses on the liver surface area included as part of the US test a sensitivity of 85 versus 55% and a specificity of 93 versus 86%, respectively [216].

#### 4.2.7. Per rectum TI-201 scintigraphy (TI-201 test)

A complete understanding of the hepatic disease requires the evaluation of portal circulation, which allows for more appropriate treatment and follow-up of patients. During the last six decades or more, several clinical reports have investigated portal circulation by radioactive tracers [87, 88]. These reports have been established that TI-201 test allows us to understand the portal circulation, and a new method using TI-201 distribution patterns seems to be useful in evaluating the portosystemic shunt (heart/liver uptake ratio, H/L ratio), which can develop to varying extents in liver cirrhosis and positive correlation to portal pressure in patients with chronic hepatitis [87–89, 217]. Our previous studies on the clinical value of H/L ratio in chronic liver disease may be useful in differentiating chronic hepatitis from cirrhosis and the prediction of its prognosis for the management of disease [97]. Noninvasive test such as maximal removal rate of indocyanine green and H/L ratio, as well as ALT/AST ratio, prothrombin time, and platelet count, may be used to evaluate the progression of chronic liver disease without liver biopsy [98] as well as progression of variceal bleeding without an endoscopy in biopsy-proven patients with cirrhosis [218]. However, because most serum markers except H/L ratio may be changeable by medical treatment of chronic liver disease, serum markers are not suitable for monitoring long-term outcomes of patients with cirrhosis. On assessing the predictive values of H/L ratio for decompensation during the follow-up period of 45.5 months in 107 patients [90], the last visiting value of H/L ratio provided a strongly reliable predictor of decompensation with an odds ratio estimate of 14.4, an AUROC of 0.825, a cut-off of 0.4, a sensitivity of 73.1%, and a specificity of 71.6% (Figure 3).

Figure 3. I. Typical scintigrams after administration per rectum of 18.5 MBq 201TI and H/L ratio versus time profiles in a decompensated patient (A, B) and compensated patient (C, D) at the first and last visits. On the left I (AYD) are scintigrams after administration per rectum of 18.5 MBq 201TI in each patient (ROI g1, liver area vs. ROI g2, heart area) and on the right I (AYD) are time-activity curves for the H/L ratio in each patient. ROI, regions of interest. II. Mean H/L ratio of the first visit when the patient is diagnosed with cirrhosis and the last visit before the development of decompensation in patients with liver cirrhosis. III. ROC curve and cut-off point of last visit H/L ratio (Q0.4) (permission from Ref. [90]).

### 5. Conclusion and perspectives

There is an urgent need to pursue the development of noninvasive tests in addition to a liver biopsy for the staging of fibrosis. The area of liver fibrosis and cirrhosis has been extensively studied during the few decades. As a result of growing understanding of liver injury and fibrosis, a number of noninvasive tests for fibrosis that are accurate and replace liver biopsy are being used to develop, commercialized, and are being used more and more in practices. The current serum tests are a start and may have utility in identifying patients with minimal fibrosis who do not require a liver biopsy. Because of the conditional relationship with biopsy, the development of serum markers will always have obvious limitations. The use of noninvasive tools varies widely depending on practice setting and the individual physician's management style. However, as with many new diagnostic methodologies, such tests are being adopted and marketed while the evidence of their general usefulness in various clinical settings remains incomplete. For instance, there is no solid evidence that the currently available tests for liver fibrosis have the precision necessary for tracing disease progression in real time or patient's response to therapy. Before such tests are accepted, their superiority to routine laboratory studies should be demonstrated. Although invasive liver biopsy is still the gold standard to assess the nature and severity of hepatic fibrosis, it has several recognized limitations including sampling error and inter-observer variability in interpretation and staging. Furthermore, the dynamic process of fibrosis resulting from progression and regression is difficult to capture with biopsy alone. Therefore, alternative, simple, reliable, and noninvasive direct and indirect serum markers able to predict the presence of significant fibrosis or cirrhosis in patients with chronic liver disease with considerable accuracy were needed. The hepatology experts are actively researching noninvasive methods of fibrosis quantification. This chapter reviewed the nature and limitations of the several noninvasive methods for the assessment of the presence and severity of liver fibrosis in patients with chronic liver disease.

## Author details

of its prognosis for the management of disease [97]. Noninvasive test such as maximal removal rate of indocyanine green and H/L ratio, as well as ALT/AST ratio, prothrombin time, and platelet count, may be used to evaluate the progression of chronic liver disease without liver biopsy [98] as well as progression of variceal bleeding without an endoscopy in biopsy-proven patients with cirrhosis [218]. However, because most serum markers except H/L ratio may be changeable by medical treatment of chronic liver disease, serum markers are not suitable for monitoring long-term outcomes of patients with cirrhosis. On assessing the predictive values of H/L ratio for decompensation during the follow-up period of 45.5 months in 107 patients [90], the last visiting value of H/L ratio provided a strongly reliable predictor of decompensation with an odds ratio estimate of 14.4, an AUROC of 0.825, a cut-off of 0.4, a

There is an urgent need to pursue the development of noninvasive tests in addition to a liver biopsy for the staging of fibrosis. The area of liver fibrosis and cirrhosis has been extensively

Figure 3. I. Typical scintigrams after administration per rectum of 18.5 MBq 201TI and H/L ratio versus time profiles in a decompensated patient (A, B) and compensated patient (C, D) at the first and last visits. On the left I (AYD) are scintigrams after administration per rectum of 18.5 MBq 201TI in each patient (ROI g1, liver area vs. ROI g2, heart area) and on the right I (AYD) are time-activity curves for the H/L ratio in each patient. ROI, regions of interest. II. Mean H/L ratio of the first visit when the patient is diagnosed with cirrhosis and the last visit before the development of decompensation in patients with liver cirrhosis. III. ROC curve and cut-off point of last visit H/L ratio (Q0.4) (permission from Ref.

sensitivity of 73.1%, and a specificity of 71.6% (Figure 3).

56 Liver Cirrhosis - Update and Current Challenges

5. Conclusion and perspectives

[90]).

Ju-Seop Kang<sup>1</sup> \* and Min-Ho Lee<sup>2</sup>

\*Address all correspondence to: jskang@hanyang.ac.kr

1 Department of Pharmacology, College of Medicine, Hanyang University, Seoul, South Korea

2 Department of Internal Medicine, College of Medicine, Ministry of Food and Drug Safety, Hanyang University, Seoul, South Korea

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