Preface

Chapter 8 **Surgical Treatment Strategies and Prognosis of Hepatocellular**

Bertozzi, Claudio Avellini and Giorgio Soardo

Chapter 11 **Sorafenib in the Continuum of Care for Hepatocellular**

Chapter 12 **Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-**

Chapter 13 **Insulin-Like Growth Factor-II: Molecular-Targeted Therapy for**

Chapter 14 **Simultaneous Changes in Expression of Bile Canalicular CD10**

Ahmed O. Kaseb and Laura M. Kulik

**Hepatocellular Carcinoma 239**

**Hepatocellular Carcinoma 255**

Chapter 15 **Hepatocellular Carcinoma, Steroid Hormones and**

González and Francisco J. Vizoso

Osawa and Shun Fujimaki

**Cirrhosis 281** Toshitsugu Nakamura

**Metalloproteases 295**

Chapter 9 **Conditions that Predispose to the Development of HCC: The**

Alessandro Uzzau, Maria Laura Pertoldeo, Vittorio Cherchi, Serena

**Role of Tumor Associated Fibroblasts and of microRNA 173** C. Avellini, D. Cesselli, A.P. Beltrami, M. Orsaria, S. Marzinotto, F.

Valerio Barghini, Debora Donnini, Alessandro Uzzau and Giorgio

**Carcinoma: Challenges in Defining Optimal Practice 217**

**Independent Pathways as Potential Therapeutic Targets in**

Yasunobu Matsuda, Toshifumi Wakai, Masayuki Kubota, Mami

Min Yao, Li Wang, Meijuan Yan, Xiaodi Yan and Dengfu Yao

Noemí Eiró, Belen Fernandez-Garcia, Antonio Altadill, Luis O.

**and Sinusoidal CD105 (Endoglin) in Chronic Hepatitis and Liver**

**Carcinoma 143**

**VI** Contents

Morassi and S. Uzzau

Chapter 10 **Signs and Symptoms 197**

Soardo

Liver cancer is the fifth most frequently diagnosed cancer in men worldwide, and is the sec‐ ond leading cause of cancer-related death. Hepatocellular carcinoma (HCC) is the most com‐ mon type of primary liver cancers, and has unique age, sex, and geographic distributions that are most likely determined by specific etiologic factors. HCC is potentially curable by surgical resection and liver transplantation. However, the majority of patients present with unresectable, advanced-stage disease, which is most commonly accompanied by severe background liver disease. Therefore, management of HCC is complicated by its highly vari‐ able biologic behavior and the frequent coexistence of underlying liver disease in affected patients. Hence, the care of patients with HCC has long been based upon a multidisciplinary approach, which involves experts from multiple specialties collaborating from the earliest stages of treatment planning to ensure personalizing the treatment for each individual pa‐ tient. Thus, our book will describe not just diagnosis and treatment of HCC, but also the entire range of existing and evolving interdisciplinary surgical, local and systemic therapies for this complex disease.

Furthermore, the demonstration of improved patient outcome in clinical trials of targeted agents in this very challenging malignancy has generated renewed enthusiasm in the field which led to a wide expansion of clinical research efforts in all aspects of HCC management. However, clinical development of targeted therapy in HCC suffers from several handicaps including lack of effective ways of choosing patients who are likely to respond, and moni‐ toring biologic activity as measured by plasma and tissue biomarkers or imaging-based bio‐ markers to assess tumor response. Therefore, we have chosen to present some relevant literature reviews and research studies related to recent research advances in HCC.

We are very grateful for the dedication and work of all authors in putting together this inter‐ disciplinary collection of the clinical and research knowledge on the management of HCC, and for their steadfast efforts in bringing this book to life.

We believe that this description of HCC multidisciplinary care will be a valuable resource for physicians and researchers from multiple specialties and in different practice settings and an example for other multidisciplinary endeavors.

#### **Dr. Ahmed O. Kaseb**

Assistant Professor, Department of Gastrointestinal (GI) Medical Oncology, Division of Cancer Medicine, The University of Texas, Anderson Cancer Center, Houston, USA

**Chapter 1**

**Role of Alleles and Genotypes of Polymorphisms of IL-18**

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver which represents a serious public health problem in the world, corresponding to the fifth more frequent malignant neoplasia among men, the eighth among women, and the third cause of cancer death in the world [1,2]. Seventy to ninety percent of HCC cases occur in patients with cirrhosis or with chronic liver disease, with cell injury followed by regeneration mediated by the immune

In Brazil, a national survey to update HCC epidemiology and clinical profile of patients with HCC (29 centers, with 1,405 patients diagnosed with HCC from 2004 to 2009) showed that the median age at diagnosis was 59 years (1–92 years old; 78% male) and 98% of the patients had cirrhosis (1279/1308), with the hepatitis C virus being the main etiology (54%), followed by hepatitis B virus (16%) and alcohol (14%). In Southeastern and Southern Brazil, hepatitis C virus accounted for over 55% of cases. In the Northeast and North, hepatitis C virus accounted for less than 50%, and hepatitis B virus accounted for 22–25% of cases [4]. In Ribeirão Preto, Southeastern Brazil, the clinical characteristics of 130 patients with HCC attended at the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of São Paulo (HCFMRP-USP) was revised. The mean (± SD) age at the time of HCC diagnosis was 55.6±11.2 years, with 81.5% of them being males. Cirrhosis was present in 89.2% of cases, with 53.4% of

> © 2013 Teixeira et al.; licensee InTech. This is an open access article 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.

**(-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and TNF-α**

**(-238 A/G and -308 A/G) and HLA-G Genes in the**

**Susceptibility of Hepatocellular Carcinoma**

Andreza Correa Teixeira,

Eduardo Antonio Donadi

http://dx.doi.org/10.5772/56350

**1. Introduction**

Ana de Lourdes Candolo Martinelli and

Additional information is available at the end of the chapter

response playing an important role in hepatocarcinogenesis [3].

**Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and TNF-α (-238 A/G and -308 A/G) and HLA-G Genes in the Susceptibility of Hepatocellular Carcinoma**

Andreza Correa Teixeira, Ana de Lourdes Candolo Martinelli and Eduardo Antonio Donadi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56350

### **1. Introduction**

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver which represents a serious public health problem in the world, corresponding to the fifth more frequent malignant neoplasia among men, the eighth among women, and the third cause of cancer death in the world [1,2]. Seventy to ninety percent of HCC cases occur in patients with cirrhosis or with chronic liver disease, with cell injury followed by regeneration mediated by the immune response playing an important role in hepatocarcinogenesis [3].

In Brazil, a national survey to update HCC epidemiology and clinical profile of patients with HCC (29 centers, with 1,405 patients diagnosed with HCC from 2004 to 2009) showed that the median age at diagnosis was 59 years (1–92 years old; 78% male) and 98% of the patients had cirrhosis (1279/1308), with the hepatitis C virus being the main etiology (54%), followed by hepatitis B virus (16%) and alcohol (14%). In Southeastern and Southern Brazil, hepatitis C virus accounted for over 55% of cases. In the Northeast and North, hepatitis C virus accounted for less than 50%, and hepatitis B virus accounted for 22–25% of cases [4]. In Ribeirão Preto, Southeastern Brazil, the clinical characteristics of 130 patients with HCC attended at the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of São Paulo (HCFMRP-USP) was revised. The mean (± SD) age at the time of HCC diagnosis was 55.6±11.2 years, with 81.5% of them being males. Cirrhosis was present in 89.2% of cases, with 53.4% of

© 2013 Teixeira et al.; licensee InTech. This is an open access article 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. the patients being ChildPugh A; chronic hepatitis B or C without cirrhosis was detected in 3.2%, nonalcoholic steatohepatitis (NASH) in 3.8%, and a normal liver in 3.8% [5].

14bp DD, and that the associations were stronger in the hepatitis B-positive than the hepatitis B-negative population [13] Zhang et al, examining fifteen single-nucleotide polymorphisms (SNPs) in the non-classical class I alleles, found that the SNPs rs17875380, rs41557518, rs114465251, and rs115492845 were associated with susceptibility to chronic hepatitis B infection or HCC, and HLA-F\*01:04, HLA-G\*01:05N, and HLA-E\*01:01 were associated with

Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

http://dx.doi.org/10.5772/56350

3

In general, the inflammatory response mediated by the immune system is beneficial to the host; however, when tissue homeostasis is chronically affected the interactions between innate and adaptive immune responses may be deregulated, culminating with chronic inflammation, excessive tissue remodeling, loss of tissue architecture, apoptosis/necrosis and oxidative stress which, under certain circumstances, may increase the risk of tumor development [15]. The cytokines are responsible for the regulation of growth differentiation and activation of immune cells. The ability to produce cytokines by an individual is influenced by genetic components that have been attributed to molecular mechanisms, including variations in the transcription,

Over the last years, regarding HCC, few studies evaluating the role of alleles and genotypes of *HLA-G* polymorphism are available in the literature, and included diverse patient popula‐ tions. Moreover, previous studies have concentrated on cytokine promoters, encouraged by the identification of a series of SNPs [17]. However, the relation of these polymorphisms with

Alleles and genotypes of cytokines polymorphisms and 14bp of HLA-G gene in HCC patients.

We evaluated, in a Brazilian cohort, the association of alleles and genotypes of the 14bp insertion/deletion polymorphism of the *HLA-G* gene, and alleles and genotypes of polymor‐ phisms of genes *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) with susceptibility to HCC and with the type of tumor presentation (infiltrative diffuse, multinodular and uninod‐ ular), with nodule size (>10 cm, 5-10 cm and <5 cm), with the Milan criteria (fulfills or does not fulfill), the evaluation of metastasis (present or absent), and the histological classification of

susceptibility to HCC or disease severity has not been clarified.

the tumor (Edmondson-Steiner Classification) [18,19, 20,21].

hepatitis B or hepatitis B with HCC [14].

translation and secretion pathways [16].

Immune response and carcinogenesis

**3. Method and patients**

**2. Problem statement**

**Application area**

**Research course**

The human major histocompatibility complex (MHC) represents a set of genes responsi‐ ble for coding histocompatibility molecules. It is a high density region [3.6 Mb DNA) located on the short arm of chromosome 6, region 6p21.3, which contains more than 200 genes grouped into three classes denoted class I, II and III. Class I genes (classic or class Ia) code for the classic histocompatibility molecules HLA-A, B and C; class II genes code for the histocompatibility molecules HLA-DR, DQ and DP, and class III genes, although includ‐ ed in the MHC, do not code for histocompatibility molecules. Among class I genes, there are also those denoted non-classic or class Ib, which code for the non-classic histocompat‐ ibility molecules HLA-E, F and G [6].

*HLA-G* is a class I non-classic HLA gene consisting of eight exons and seven introns with a stop codon in exon 6 (exon 7 is always absent from mature mRNA and, due to the stop codon in exon 6, exon 8 is not translated), a 5' promoter region and a 3' untranslated region (3'UTR) [7,8]. The characteristics of *HLA-G* are: low polymorphism, alternative splicing in the primary transcript that codes for seven protein isoforms, limited distribution in normal tissues, and immunsuppressive properties [9]. Of the seven proteins coded by the *HLA-G* gene, four are linked to the membrane (HLA-G1 to HLA-G4) and three are soluble (HLA-G5 to HLA-G7). The HLA-G1 and HLA-G5 proteins are the main isoforms described in healthy issues such as trophoblast, thymus, cornea, and erythroid and endothelial precur‐ sors. On the other hand, the expression of *HLA-G* can be induced in pathological situa‐ tions such as autoimmune and inflammatory diseases, viral infections, cancer, and transplantation. *HLA-G* exerts inhibitory activity by binding to inhibitory receptors denoted immunoglobulin-like transcript (ILT)-2 and ILT-4, which are expressed by lymphoid and myeloid cells and by myeloid cells only, respectively, and killer immunoglobulin-like (KIR)2DL4 receptor, present only in natural killer cells [9].

Thus far, 47 alleles have been attributed to the *HLA-G* gene, which code for 15 distinct functional proteins with all isoforms (*HLA-G*\*01:01, \*01:02, \*01:03, \*01:04, \*01:06, \*01:07, \*01:08, \*01:09, \*01:10, \*01:11, \*01:12, \*01:14, \*01:15, \*01:16, and \*01:17) and truncated (G\*01:05N allele) or no proteins at all (G\*01:13N allele). Polymorphic sites are observed in the coding and noncoding regions. The promoter and 3´UTR regions of *HLA-G* are highly polymorphic, with the variations in the 3´UTR being associated with the levels of *HLA-G* expression. Two main polymorphic sites were identified in the 3´UTR: a 14bp insertion/deletion, in which the insertion has been associated with reduced levels of HLA-G, and the presence of guanine at position +3142, which increases the affinity of specific microRNAs for *HLA-G* mRNA, reducing the expression of HLA-G [8,10].

The expression of *HLA-G* is frequently detected in malignant tumors of various origins and in some situations has been significantly correlated with tumor size, degree of invasion, meta‐ stasis, clinical stages of the disease, and mortality [11,12]. Regarding HCC, few studies evaluating the role of alleles and genotypes of *HLA-G* polymorphism are available in the literature, and evolve different population. Jiang et al, studying a Chinese population showed that the heterozygote and the 14bp II homozygote confer a lower risk of HCC compared with 14bp DD, and that the associations were stronger in the hepatitis B-positive than the hepatitis B-negative population [13] Zhang et al, examining fifteen single-nucleotide polymorphisms (SNPs) in the non-classical class I alleles, found that the SNPs rs17875380, rs41557518, rs114465251, and rs115492845 were associated with susceptibility to chronic hepatitis B infection or HCC, and HLA-F\*01:04, HLA-G\*01:05N, and HLA-E\*01:01 were associated with hepatitis B or hepatitis B with HCC [14].

In general, the inflammatory response mediated by the immune system is beneficial to the host; however, when tissue homeostasis is chronically affected the interactions between innate and adaptive immune responses may be deregulated, culminating with chronic inflammation, excessive tissue remodeling, loss of tissue architecture, apoptosis/necrosis and oxidative stress which, under certain circumstances, may increase the risk of tumor development [15]. The cytokines are responsible for the regulation of growth differentiation and activation of immune cells. The ability to produce cytokines by an individual is influenced by genetic components that have been attributed to molecular mechanisms, including variations in the transcription, translation and secretion pathways [16].

### **2. Problem statement**

the patients being ChildPugh A; chronic hepatitis B or C without cirrhosis was detected in

The human major histocompatibility complex (MHC) represents a set of genes responsi‐ ble for coding histocompatibility molecules. It is a high density region [3.6 Mb DNA) located on the short arm of chromosome 6, region 6p21.3, which contains more than 200 genes grouped into three classes denoted class I, II and III. Class I genes (classic or class Ia) code for the classic histocompatibility molecules HLA-A, B and C; class II genes code for the histocompatibility molecules HLA-DR, DQ and DP, and class III genes, although includ‐ ed in the MHC, do not code for histocompatibility molecules. Among class I genes, there are also those denoted non-classic or class Ib, which code for the non-classic histocompat‐

*HLA-G* is a class I non-classic HLA gene consisting of eight exons and seven introns with a stop codon in exon 6 (exon 7 is always absent from mature mRNA and, due to the stop codon in exon 6, exon 8 is not translated), a 5' promoter region and a 3' untranslated region (3'UTR) [7,8]. The characteristics of *HLA-G* are: low polymorphism, alternative splicing in the primary transcript that codes for seven protein isoforms, limited distribution in normal tissues, and immunsuppressive properties [9]. Of the seven proteins coded by the *HLA-G* gene, four are linked to the membrane (HLA-G1 to HLA-G4) and three are soluble (HLA-G5 to HLA-G7). The HLA-G1 and HLA-G5 proteins are the main isoforms described in healthy issues such as trophoblast, thymus, cornea, and erythroid and endothelial precur‐ sors. On the other hand, the expression of *HLA-G* can be induced in pathological situa‐ tions such as autoimmune and inflammatory diseases, viral infections, cancer, and transplantation. *HLA-G* exerts inhibitory activity by binding to inhibitory receptors denoted immunoglobulin-like transcript (ILT)-2 and ILT-4, which are expressed by lymphoid and myeloid cells and by myeloid cells only, respectively, and killer immunoglobulin-like

Thus far, 47 alleles have been attributed to the *HLA-G* gene, which code for 15 distinct functional proteins with all isoforms (*HLA-G*\*01:01, \*01:02, \*01:03, \*01:04, \*01:06, \*01:07, \*01:08, \*01:09, \*01:10, \*01:11, \*01:12, \*01:14, \*01:15, \*01:16, and \*01:17) and truncated (G\*01:05N allele) or no proteins at all (G\*01:13N allele). Polymorphic sites are observed in the coding and noncoding regions. The promoter and 3´UTR regions of *HLA-G* are highly polymorphic, with the variations in the 3´UTR being associated with the levels of *HLA-G* expression. Two main polymorphic sites were identified in the 3´UTR: a 14bp insertion/deletion, in which the insertion has been associated with reduced levels of HLA-G, and the presence of guanine at position +3142, which increases the affinity of specific microRNAs for *HLA-G* mRNA, reducing

The expression of *HLA-G* is frequently detected in malignant tumors of various origins and in some situations has been significantly correlated with tumor size, degree of invasion, meta‐ stasis, clinical stages of the disease, and mortality [11,12]. Regarding HCC, few studies evaluating the role of alleles and genotypes of *HLA-G* polymorphism are available in the literature, and evolve different population. Jiang et al, studying a Chinese population showed that the heterozygote and the 14bp II homozygote confer a lower risk of HCC compared with

3.2%, nonalcoholic steatohepatitis (NASH) in 3.8%, and a normal liver in 3.8% [5].

ibility molecules HLA-E, F and G [6].

2 Hepatocellular Carcinoma - Future Outlook

the expression of HLA-G [8,10].

(KIR)2DL4 receptor, present only in natural killer cells [9].

Over the last years, regarding HCC, few studies evaluating the role of alleles and genotypes of *HLA-G* polymorphism are available in the literature, and included diverse patient popula‐ tions. Moreover, previous studies have concentrated on cytokine promoters, encouraged by the identification of a series of SNPs [17]. However, the relation of these polymorphisms with susceptibility to HCC or disease severity has not been clarified.

#### **Application area**

Immune response and carcinogenesis

#### **Research course**

Alleles and genotypes of cytokines polymorphisms and 14bp of HLA-G gene in HCC patients.

### **3. Method and patients**

We evaluated, in a Brazilian cohort, the association of alleles and genotypes of the 14bp insertion/deletion polymorphism of the *HLA-G* gene, and alleles and genotypes of polymor‐ phisms of genes *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) with susceptibility to HCC and with the type of tumor presentation (infiltrative diffuse, multinodular and uninod‐ ular), with nodule size (>10 cm, 5-10 cm and <5 cm), with the Milan criteria (fulfills or does not fulfill), the evaluation of metastasis (present or absent), and the histological classification of the tumor (Edmondson-Steiner Classification) [18,19, 20,21].

This was a retrospective cross-sectional study conducted on 109 patients (89 men) with mean age 55.8±11.4 years for the 14bp insertion/deletion polymorphism of the *HLA-G* gene, and 112 consecutive patients [Mean (±SD) age was 55.6±11.2 years, and 81.5% were males] for the polymorphisms of genes *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) followed up from 2001 to 2009 at the Focal Hepatic Injuries Outpatient Clinic of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The study was approved by the Research Ethics Committee of HCFMRP-USP. Individuals of both genders who fulfilled the following criteria were included in the study: a) diagnosis of HCC defined by the Barcelona 2000 criteria [22] for the diagnoses performed up to 2006 and according to the recommendations of the American Association for the Study of Liver Disease (AASLD) [23] for the diagnoses performed since 2007; b) with DNA stored in the Biological Sample Bank of the Laboratory of Gastroen‐ terology, Department of Internal Medicine. Exclusion criteria were patients with focal hepatic injuries other than HCC according to the Barcelona 2000 criteria and the recommendations of the AASLD and patients with other concomitant neoplasias.

The evaluation of the severity of HCC was based on: a) tumor presentation (uninodular, multinodular or diffuse infiltrative); b) nodule size (<5 cm, 5-10 cm or >10 cm); c) Milan criteria (fulfills or does not fulfill); d) metastasis (present or absent); e) histological classification according to Edmondson & Steiner, 1954 (grades I, II, III or IV) [24].

A total of 202 healthy individuals (56 females and 146 males) with a mean age (±SD) of 33.3±8.3 years and from the same geographic region as the patients studied were used as controls for the evaluation of the frequency of 14bp, *IL-18, IFN-γ* and *TNF-α* alleles and genotypes.

For the determination of the 14bp insertion/deletion, IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) genotypes, we first extracted genomic DNA from peripheral leukocytes by the salting out technique [25]. The HLA-G 14bp insertion/deletion genotypes at exon 8 of the HLA-G locus was analyzed as described: 200ng of genomic DNA were amplified in a 25mL reaction mixture containing 0.20mM dNTP (Invitrogen, Carlsbad, CA), 0.2mM of each primer, 0.5U Taq DNA polymerase (Invitrogen, Carlsbad, CA), 1.5mM MgCl2 and a 1x PCR buffer (0.2M Tris–HCl, pH 8.5; 0.5M KCl). After an initial denaturation step at 94°C for 5 minutes, samples were submitted to 30 additional cycles at 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute, with a final extension cycle at 72°C for 7 minutes with 5´-TGTGAAA‐ CAGCTGCCCTGTG-3´ as the forward primer and 5´-AAGGAATGCAGTTCAGCATGA-3´ as the reverse primer [26,27]. After DNA amplification by PCR, the reaction products were submitted to 10% polyacrylamide gel electrophoresis under non-denaturing conditions followed by silver impregnation. The presence of 345 bp fragments corresponded to the deletion allele, while the 359 bp fragment corresponded to the 14bp insertion allele.

The alleles and genotypes of *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) polymor‐ phisms were analyzed by the polymerase chain reaction using allele-specific primers (PCR-SSP) (Table 1). For SNP *IL-18* −607, a generic reverse primer 5′-TAACCTCATTCAGG ACTTCC-3′ and two allele-specific forward primers (5′-GT TGCAGAAAGTGTAAAAAT‐ TATTAC-3′ and 5′-GTTGCAG AAAGTGTAAAAATTATTAA-3′) were used to amplify a 196-bp product. For SNP *IL-18* −137, a common reverse primer 5′-AGGAGGGCAAAATG‐ CACTGG-3′ and two allele-specific forward primers (5′-CCCCAACTTTTACGGAAG AAAAG-3′ and 5′-CCCCAACTTTTACGGAAGAAAAC-3′) were used to amplify a 261-

**Table 1.** Primers for the detection of single nucleotide polymorphisms (SNPs) of genes IL-18 (-607 C/A and -137 C/G),

*IL-18*: interleukin-18; IFN-γ: interferon-gamma; TNF-α: tumor necrosis factor-alpha; HGBA.S and HGBA.A: human

**SNP Primer Sequence (5'-3')**

IFN-γ +874 A/T IFN-γ 874

hemoglobin; GH1 and GH2: human growth hormone.

IFN-γ (+874 A/T) and TNF-α (-238 A/G and -308 A/G).

*IL-18* -607 C/A *IL-18* 607.2 TAACCTCATTCAGGACTTCC

*IL-18* -137 C/G *IL-18* 137.2 AGGAGGGCAAAATGCACTGG

IFN-γ 874 T IFN-γ 874 A

TNF-α -238 A/G TNF 238 UP AGGCAATAGGTTTTGAGGGCCAT

TNF-α -308 A/G TNFAA 308.2 CAGCGGAAAACTTCCTTGGT

*IL-18* 607C GTTGCAGAAAGTGTAAAAATTATTAC *IL-18* 607A GTTGCAGAAAGTGTAAAAATTATTAA

Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

http://dx.doi.org/10.5772/56350

5

HGBA.S CGGTATTTGGAGGTCAGCAC HGBA.A CCCACCACCAAGACCTACTT

*IL-18* 137C CCCCAACTTTTACGGAAGAAAAC *IL-18* 137G CCCCAACTTTTACGGAAGAAAAG HGBA.S CGGTATTTGGAGGTCAGCAC HGBA.A CCCACCACCAAGACCTACTT

GH 1 GCCTTCCCAACCATTCCCTTA GH 2 TCACGGATTTCTGTTGTGTTTC

TNFAS238G CCCCATCCTCCCTGCTCC TNFAS238A TCCCCATCCTCCCTGCTCT HGBA.S CGGTATTTGGAGGTCAGCAC HGBA.A CCCACCACCAAGACCTACTT

TNFAS 308G ATAGGTTTTGAGGGGCATGG TNFAS 308A ATAGGTTTTGAGGGGCATGA HGBA.S CGGTATTTGGAGGTCAGCAC HGBA.A CCCACCACCAAGACCTACTT

TCAACAAAGCTGATACTCCA TTCTTACAACACAAAATCAAATCT TTCTTACAACACAAAATCAAATCA This was a retrospective cross-sectional study conducted on 109 patients (89 men) with mean age 55.8±11.4 years for the 14bp insertion/deletion polymorphism of the *HLA-G* gene, and 112 consecutive patients [Mean (±SD) age was 55.6±11.2 years, and 81.5% were males] for the polymorphisms of genes *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) followed up from 2001 to 2009 at the Focal Hepatic Injuries Outpatient Clinic of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The study was approved by the Research Ethics Committee of HCFMRP-USP. Individuals of both genders who fulfilled the following criteria were included in the study: a) diagnosis of HCC defined by the Barcelona 2000 criteria [22] for the diagnoses performed up to 2006 and according to the recommendations of the American Association for the Study of Liver Disease (AASLD) [23] for the diagnoses performed since 2007; b) with DNA stored in the Biological Sample Bank of the Laboratory of Gastroen‐ terology, Department of Internal Medicine. Exclusion criteria were patients with focal hepatic injuries other than HCC according to the Barcelona 2000 criteria and the recommendations of

The evaluation of the severity of HCC was based on: a) tumor presentation (uninodular, multinodular or diffuse infiltrative); b) nodule size (<5 cm, 5-10 cm or >10 cm); c) Milan criteria (fulfills or does not fulfill); d) metastasis (present or absent); e) histological classification

A total of 202 healthy individuals (56 females and 146 males) with a mean age (±SD) of 33.3±8.3 years and from the same geographic region as the patients studied were used as controls for the evaluation of the frequency of 14bp, *IL-18, IFN-γ* and *TNF-α* alleles and genotypes.

For the determination of the 14bp insertion/deletion, IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) genotypes, we first extracted genomic DNA from peripheral leukocytes by the salting out technique [25]. The HLA-G 14bp insertion/deletion genotypes at exon 8 of the HLA-G locus was analyzed as described: 200ng of genomic DNA were amplified in a 25mL reaction mixture containing 0.20mM dNTP (Invitrogen, Carlsbad, CA), 0.2mM of each primer, 0.5U Taq DNA polymerase (Invitrogen, Carlsbad, CA), 1.5mM MgCl2 and a 1x PCR buffer (0.2M Tris–HCl, pH 8.5; 0.5M KCl). After an initial denaturation step at 94°C for 5 minutes, samples were submitted to 30 additional cycles at 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute, with a final extension cycle at 72°C for 7 minutes with 5´-TGTGAAA‐ CAGCTGCCCTGTG-3´ as the forward primer and 5´-AAGGAATGCAGTTCAGCATGA-3´ as the reverse primer [26,27]. After DNA amplification by PCR, the reaction products were submitted to 10% polyacrylamide gel electrophoresis under non-denaturing conditions followed by silver impregnation. The presence of 345 bp fragments corresponded to the

deletion allele, while the 359 bp fragment corresponded to the 14bp insertion allele.

The alleles and genotypes of *IL-18* (-607 C/A; rs1946518 and -137 C/G; rs187238), *IFN-γ* (+874 A/T; rs62559044) and *TNF-α* (-238 A/G; rs361525 and -308 A/G; rs1800629) polymor‐ phisms were analyzed by the polymerase chain reaction using allele-specific primers (PCR-SSP) (Table 1). For SNP *IL-18* −607, a generic reverse primer 5′-TAACCTCATTCAGG

the AASLD and patients with other concomitant neoplasias.

4 Hepatocellular Carcinoma - Future Outlook

according to Edmondson & Steiner, 1954 (grades I, II, III or IV) [24].


*IL-18*: interleukin-18; IFN-γ: interferon-gamma; TNF-α: tumor necrosis factor-alpha; HGBA.S and HGBA.A: human hemoglobin; GH1 and GH2: human growth hormone.

**Table 1.** Primers for the detection of single nucleotide polymorphisms (SNPs) of genes IL-18 (-607 C/A and -137 C/G), IFN-γ (+874 A/T) and TNF-α (-238 A/G and -308 A/G).

ACTTCC-3′ and two allele-specific forward primers (5′-GT TGCAGAAAGTGTAAAAAT‐ TATTAC-3′ and 5′-GTTGCAG AAAGTGTAAAAATTATTAA-3′) were used to amplify a 196-bp product. For SNP *IL-18* −137, a common reverse primer 5′-AGGAGGGCAAAATG‐ CACTGG-3′ and two allele-specific forward primers (5′-CCCCAACTTTTACGGAAG AAAAG-3′ and 5′-CCCCAACTTTTACGGAAGAAAAC-3′) were used to amplify a 261bp product. An internal positive amplification control was performed using the primers 5′- CG GTATTTGGAGGTCAGCAC-3′ and 5′-CCCACCACCAAGA CCTACTT-3′, which are specific for the human hemoglobin genes. The reactions were performed in a final vol‐ ume of 10μL containing 200ng of genomic DNA, 3pmol of each primer (the generic one and a specific one), 2pmol of each control primer, 0.25mM dNTP (Pharmacia Biotech, Paris, France), 1.5mM MgCl2, 0.75U of Taq DNA poly- merase (Invitrogen, Carlsbad, CA), and 1× PCR buffer (0.2 M Tris – HCl, pH 8.5, 0.5 M KCl). The cyclingconditions were 3min at 94°C, followed by seven cycles of 20s at 94°C, 40s at 64°C for *IL18* -607 or 60s at 68°C for *IL18* -137 and 40s at 72°C and 25 cycles of 20s at 94°C, 40s at 57°C for *IL18* -607 or 20s at 62°C for *IL18* -137 and 40s at 72°C, and final stage of 5min at 72°C [9]. For the *TNF-α* (-238 and -308) and *IFN-γ* +874 specific amplification, the primers were identical to those previously described [28,29]. All amplification products were visualized using 10% nondenaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining.

**4.1. 14bp insertion/deletion polymorphism**

results of the statistical analyses.

Tumor presentation

Nodule size

Metastasis

Milan criteria

Edmondson

Genotype frequencies of patient and control groups were in accordance to Hardy-Weinberg Equilibrium. The 14bp\*D allele was more frequent in cases of HCC than in controls (0.6514 vs. 0.5619; *P*=0.0326), conferring an OR=1.46 (95%CI=1.04-2.05). Evaluation of genotype frequency (genotypes 14pb DD, DI and II) did not show significant difference between the groups studied (*P*=0.0871; OR=1.54; 95%CI=0.96-2.48; *P*=0.7182; OR=0.89; 95%CI=0.56-1.44; and *P*=0.1343; OR=0.60; 95%CI=0.32-1.12, respectively). However, the 14bp DD genotype was marginally more frequent among individuals with HCC than among controls, with 10.3% difference (0.4495 vs*.* 0.3465, respectively) (*P*=0.0871; OR=1.54; 95%CI=0.96-2.48). Patients were stratified according to the characteristics of HCC (type of tumor presentation, nodule size, Milan criteria, presence of metastasis and Edmondson-Steiner classification), and no significant differences were detected between groups regarding the 14bp insertion/deletion allele frequencies (14bp\*D and 14bp\*I alleles) or genotype frequencies (14bp DD, DI and II genotypes). Table 2 shows the frequency of the 14bp insertion/deletion polymorphism and Table 3 shows the

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**Samples 14bp alleles 14bp genotypes** I D II DI DD

Groups n(frequency) n(frequency) n(frequency) n(frequency) n(frequency) Control 177 [0.4381] 227 [0.5619] 45 [0.2228] 87 [0.4307] 70 [0.3465] HCC 76 [0.3486] 142 [0.6514] 16 [0.1468] 44 [0.4037] 49 [0.4495]

Diffuse 7 [0.2500] 21 [0.7500] 1 [0.0714] 5 [0.3571] 8 [0.5714] Multinodular 14 [0.3684] 24 [0.6316] 13 [0.1579] 9 [0.4211] 6 [0.4211] Uninodular 55 [0.3667] 95 [0.6333] 12 [0.1600] 31 [0.4133] 32 [0.4767]

>10cm 6 [0.3333] 12 [0.6667] 1 [0.1111] 4 [0.4444] 4 [0.4444] 5-10cm 11 [0.2750] 29 [0.7250] 1 [0.0500] 9 [0.4500] 10 [0.5000] <5cm 51 [0.4113] 73 [0.5887] 13 [0.2097] 25 [0.4032] 24 [0.3871]

Present 4 [0.2857] 10 [0.7143] 0 [0.0000] 4 [0.5714] 3 [0.4286] Absent 63 [0.3539] 115 [0.6461] 13 [0.1461] 37 [0.4157] 39 [0.4382]

Yes 46 [0.3898] 72 [0.6102] 11 [0.1864] 24 [0.4068] 24 [0.4068] No 30 [0.3000] 70 [0.7000] 5 [0.1000] 20 [0.4000] 25 [0.5000]

I-II 24 [0.4615] 28 [0.5385] 5 [0.1923] 14 [0.5385] 7 [0.2692] III-IV 16 [0.5000] 16 [0.5000] 4 [0.2500] 8 [0.5000] 4 [0.2500]

*HLA-G*: H*uman Leukocyte Antigen-G*; bp: base pairs; HCC: hepatocellular carcinoma; I: insertion; D:deletion

**Table 2.** Distribution of the *HLA-G* 14bp insertion/deletion allele and genotype frequencies.

For statistical analysis, the allele and genotype frequencies were calculated by the direct count method in all groups. Adherence of genotypic proportions to Hardy-Weinberg expectations was determined by the exact test of Guo and Thompson [30] using the GENEPOP software v. 4.0.10. The presence of a significant association between polymorphisms of the same gene was evaluated by a likelihood ratio test of probability of linkage disequilibrium using the ARLE‐ QUIN software, v. 3.1 [31]. If a positive association was detected, but the gameteic phase was unknown, the PHASE (v. 2 package) [32] and EM algorithms [33] were used to reconstruct the TNF or IL haplotypes. Allele, genotype and haplotype frequencies were compared by the twotailed Fisher exact test using the GraphPad InStat 3.06 software, which was also used to estimate the odds ratio (OR) and its 95% confidence interval (95%CI). The level of significance was set at P<0.05.

#### **4. Results**

Cirrhosis was observed in 89% of the patients, and major underlying causes included: hepatitis C (35%), alcohol plus hepatitis C (25%), alcohol (18%), hepatitis B (9%), alcohol plus hepatitis B (4%), alcohol plus hepatitis C plus hepatitis B (1%), hereditary hemochro‐ matosis (1%), non-alcoholic steatohepatitis (1%), autoimmune hepatitis (1%) and cryptogen‐ ic cirrhosis (5%). Four percent of the patients had no underlying liver disease, 4% nonalcoholic steatohepatitis and 3% chronic hepatitis without cirrhosis (two hepatitis B and one hepatitis C). Fifty-four percent (59/109) of the patients met the Milan criteria for liver transplantation [34]. Metastasis search was performed in 88% of cases (17% had metasta‐ sis and 93% did not). Tumors <5cm, 5-10cm and >10 cm were found in 57%, 18% and 8% respectively. Diffuse infiltrative HCC totaled 15% of the cases. Histological evaluation of HCC was performed in 39% (42/109) of subjects with 62% presenting Edmondson-Steiner I or II and 38% III or IV.

#### **4.1. 14bp insertion/deletion polymorphism**

bp product. An internal positive amplification control was performed using the primers 5′- CG GTATTTGGAGGTCAGCAC-3′ and 5′-CCCACCACCAAGA CCTACTT-3′, which are specific for the human hemoglobin genes. The reactions were performed in a final vol‐ ume of 10μL containing 200ng of genomic DNA, 3pmol of each primer (the generic one and a specific one), 2pmol of each control primer, 0.25mM dNTP (Pharmacia Biotech, Paris, France), 1.5mM MgCl2, 0.75U of Taq DNA poly- merase (Invitrogen, Carlsbad, CA), and 1× PCR buffer (0.2 M Tris – HCl, pH 8.5, 0.5 M KCl). The cyclingconditions were 3min at 94°C, followed by seven cycles of 20s at 94°C, 40s at 64°C for *IL18* -607 or 60s at 68°C for *IL18* -137 and 40s at 72°C and 25 cycles of 20s at 94°C, 40s at 57°C for *IL18* -607 or 20s at 62°C for *IL18* -137 and 40s at 72°C, and final stage of 5min at 72°C [9]. For the *TNF-α* (-238 and -308) and *IFN-γ* +874 specific amplification, the primers were identical to those previously described [28,29]. All amplification products were visualized using 10% non-

denaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining.

was set at P<0.05.

6 Hepatocellular Carcinoma - Future Outlook

I or II and 38% III or IV.

**4. Results**

For statistical analysis, the allele and genotype frequencies were calculated by the direct count method in all groups. Adherence of genotypic proportions to Hardy-Weinberg expectations was determined by the exact test of Guo and Thompson [30] using the GENEPOP software v. 4.0.10. The presence of a significant association between polymorphisms of the same gene was evaluated by a likelihood ratio test of probability of linkage disequilibrium using the ARLE‐ QUIN software, v. 3.1 [31]. If a positive association was detected, but the gameteic phase was unknown, the PHASE (v. 2 package) [32] and EM algorithms [33] were used to reconstruct the TNF or IL haplotypes. Allele, genotype and haplotype frequencies were compared by the twotailed Fisher exact test using the GraphPad InStat 3.06 software, which was also used to estimate the odds ratio (OR) and its 95% confidence interval (95%CI). The level of significance

Cirrhosis was observed in 89% of the patients, and major underlying causes included: hepatitis C (35%), alcohol plus hepatitis C (25%), alcohol (18%), hepatitis B (9%), alcohol plus hepatitis B (4%), alcohol plus hepatitis C plus hepatitis B (1%), hereditary hemochro‐ matosis (1%), non-alcoholic steatohepatitis (1%), autoimmune hepatitis (1%) and cryptogen‐ ic cirrhosis (5%). Four percent of the patients had no underlying liver disease, 4% nonalcoholic steatohepatitis and 3% chronic hepatitis without cirrhosis (two hepatitis B and one hepatitis C). Fifty-four percent (59/109) of the patients met the Milan criteria for liver transplantation [34]. Metastasis search was performed in 88% of cases (17% had metasta‐ sis and 93% did not). Tumors <5cm, 5-10cm and >10 cm were found in 57%, 18% and 8% respectively. Diffuse infiltrative HCC totaled 15% of the cases. Histological evaluation of HCC was performed in 39% (42/109) of subjects with 62% presenting Edmondson-Steiner Genotype frequencies of patient and control groups were in accordance to Hardy-Weinberg Equilibrium. The 14bp\*D allele was more frequent in cases of HCC than in controls (0.6514 vs. 0.5619; *P*=0.0326), conferring an OR=1.46 (95%CI=1.04-2.05). Evaluation of genotype frequency (genotypes 14pb DD, DI and II) did not show significant difference between the groups studied (*P*=0.0871; OR=1.54; 95%CI=0.96-2.48; *P*=0.7182; OR=0.89; 95%CI=0.56-1.44; and *P*=0.1343; OR=0.60; 95%CI=0.32-1.12, respectively). However, the 14bp DD genotype was marginally more frequent among individuals with HCC than among controls, with 10.3% difference (0.4495 vs*.* 0.3465, respectively) (*P*=0.0871; OR=1.54; 95%CI=0.96-2.48). Patients were stratified according to the characteristics of HCC (type of tumor presentation, nodule size, Milan criteria, presence of metastasis and Edmondson-Steiner classification), and no significant differences were detected between groups regarding the 14bp insertion/deletion allele frequencies (14bp\*D and 14bp\*I alleles) or genotype frequencies (14bp DD, DI and II genotypes). Table 2 shows the frequency of the 14bp insertion/deletion polymorphism and Table 3 shows the results of the statistical analyses.


*HLA-G*: H*uman Leukocyte Antigen-G*; bp: base pairs; HCC: hepatocellular carcinoma; I: insertion; D:deletion

**Table 2.** Distribution of the *HLA-G* 14bp insertion/deletion allele and genotype frequencies.


associated with susceptibility to HCC. The inference of haplotypes was performed for the polymorphisms of *IL-18* and *TNF-α*. Haplotypes -607A/-137G IL-18 and TNF-α -308G/-238A -308A/-238G were more frequent in patients with HCC compared with the control group (P = 0.0180, OR = 1.69, 95% CI 1.10 to 2.59, P = 0.0036, OR = 2.06, 95% CI 1,28-3.31 and P = 0.0480, OR = 1.75; 95% CI = 1.03 to 2.97, respectively). On the other hand, TNF-α -308G/-238G haplotype was more frequent in the group of healthy subjects (P = 0.0001, OR = 0.46, 95% CI 0.32 to 0.68),

Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

Control n (frequency)

G 162 [0.7232] 284 [0.7030] 0.6464 1.10 0.77-1.59

C 132 [0.5893] 275 [0.6807] 0.0235 0.67 0.48-0.94

T 92 [0.4144] 164 [0.4059] 0.8652 1.03 0.74-1.44

G 181 [0.8153] 365 [0.9035] 0.0025 0.47 0.29-0.76

G 192 [0.8649] 372 [0.9208] 0.0351 0.55 0.32-0.93

*IL-18* -137C/G C 62 [0.2768] 120 [0.2970] 0.6464 0.90 0.63-1.30

*IL-18* -607A/C A 92 [0.4107] 129 [0.3193] 0.0235 1.48 1.06-2.08

IFN-γ +874A/T A 130 [0.5856] 240 [0.5941] 0.8652 0.96 0.69-1.35

TNF-α -238A/G A 41 [0.1847] 39 [0.0965] 0.0025 2.12 1.32-3.40

TNF-α -308A/G A 30 [0.1351] 32 [0.0792] 0.0351 1.82 1.07-3.08

**Table 4.** Distribution of the allele frequencies of polymorphisms of the *IL-18* (-607 C/A and -137 C/G), IFN-γ (+874 A/T), and TNF-α (-238 A/G e -308 A/G) genes among patients with hepatocellular carcinoma (HCC) and healthy

When the -607C/A SNP of *IL-18* was evaluated in terms of the different presentations of the HCC, the frequencies of the -607\*C and -607\*A alleles between individuals with multinodular and uninodular HCC, showed that the -607\*C allele confers significant susceptibility to multinodular lesions (*P*=0.0289; OR=2.4; 95%CI = 1.09-5.28). Evaluation of the genotype frequency in SNP -607C/A of *IL-18* revealed that genotype 607CC was significantly more frequent in cases of multinodular HCC compared to uninodular tumors (*P*=0.0284; OR=3.5; 95%CI=1.24-9.86). On the other hand, genotype +874AT was found to be more frequent among patients with infiltrative diffuse HCC compared to uninodular HCC (*P*=0.0443; OR=3.6;

95%CI=1.04-12.47), thus conferring greater susceptibility to the diffuse tumor.

P OR 95%CI

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providing protection against HCC.

controls.

allele HCC

**SNP Allele frequency**

n (frequency)

*HLA-G*: *Human Leukocyte Antigen-G*; bp: base pairs; HCC: hepatocellular carcinoma; I: insertion; D:deletion

a Odds ratio= 1.46 [95% confidence interval: 1.04-2.05]

b Odds ratio= 1.54 [95% confidence interval: 0.96-2.48]

**Table 3.** Probability values obtained by means of two-tailed Fisher exact test in the comparisons of HLA-G 14bp insertion/deletion allele and genotype frequencies between different groups.

#### **4.2. IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) polymorphism**

The genotypification of the polymorphisms of the genes *IL-18* (-607 C/A and -137 C/G), *IFN-γ* (+874 A/T) and *TNF-α* (-238 A/G and -308 A/G) was performed in 112 patients with HCC and in 202 healthy controls. The genotype distribution of the two groups adhered to the theoretical proportions of Hardy-Weinberg equilibrium. Significant associations were detected between HCC and the following alleles (Table 4): *IL-18* -607\*A (*P*=0.0235; OR=1.48; 95%CI=1.06-2.08); *TNF-α* -238\*A (*P*=0.0025; OR=2.12; 95%CI=1.32-3.40), and *TNF-α* -308\*A (*P*=0.0351; OR=1.82; 95%CI=1.07-3.08). When the genotypes were evaluated (Table 5), the following associations with HCC were detected: *IL-18* -607 AA (*P*=0.0048; OR=3.03; 95%CI=1.40-6.55); *TNF-α* -238 GA (*P*=0.0011; OR=2.44; 95%CI=1.45-4.12); and *TNF-α* -308 GA (*P*=0.0031; OR=2.51; 95%CI=1.39-4.51). Alleles and genotypes from *IL-18* -137G/C and *IFNγ* +874T/A were not associated with susceptibility to HCC. The inference of haplotypes was performed for the polymorphisms of *IL-18* and *TNF-α*. Haplotypes -607A/-137G IL-18 and TNF-α -308G/-238A -308A/-238G were more frequent in patients with HCC compared with the control group (P = 0.0180, OR = 1.69, 95% CI 1.10 to 2.59, P = 0.0036, OR = 2.06, 95% CI 1,28-3.31 and P = 0.0480, OR = 1.75; 95% CI = 1.03 to 2.97, respectively). On the other hand, TNF-α -308G/-238G haplotype was more frequent in the group of healthy subjects (P = 0.0001, OR = 0.46, 95% CI 0.32 to 0.68), providing protection against HCC.

**Comparisons 14bp alleles 14bp genotypes** I D II DI DD

HCC *vs.* control 0.0326 0.0326a 0.1343 0.7182 0.0871b

Diffuse *vs.* multinodular 0.4238 0.4238 0.6197 1,0000 0.4905 Diffuse *vs.* uninodular 0.2839 0.2839 0.6830 0.7738 0.3863 Multinodular *vs.* uninodular 1.0000 1.0000 1.0000 1.0000 1.0000

>10 cm *vs.* 5-10 cm 0.7577 0.7577 0.5320 1.0000 1.0000 >10 cm *vs.* <5 cm 0.6132 0.6132 0.6769 1.0000 0.7318 5-10 cm *vs.* <5 cm 0.1372 0.1372 0.1698 0.7963 0.4382

Yes *vs.* no 0.1995 0.1995 0.2794 1.0000 0.3421

Present *vs.* Absent 0.7739 0.7739 0.5883 0.4556 1.0000

I-II *vs.* III-IV 0.8232 0.8232 0.7109 1.0000 1.0000

*HLA-G*: *Human Leukocyte Antigen-G*; bp: base pairs; HCC: hepatocellular carcinoma; I: insertion; D:deletion

**Table 3.** Probability values obtained by means of two-tailed Fisher exact test in the comparisons of HLA-G 14bp

**4.2. IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and**

The genotypification of the polymorphisms of the genes *IL-18* (-607 C/A and -137 C/G), *IFN-γ* (+874 A/T) and *TNF-α* (-238 A/G and -308 A/G) was performed in 112 patients with HCC and in 202 healthy controls. The genotype distribution of the two groups adhered to the theoretical proportions of Hardy-Weinberg equilibrium. Significant associations were detected between HCC and the following alleles (Table 4): *IL-18* -607\*A (*P*=0.0235; OR=1.48; 95%CI=1.06-2.08); *TNF-α* -238\*A (*P*=0.0025; OR=2.12; 95%CI=1.32-3.40), and *TNF-α* -308\*A (*P*=0.0351; OR=1.82; 95%CI=1.07-3.08). When the genotypes were evaluated (Table 5), the following associations with HCC were detected: *IL-18* -607 AA (*P*=0.0048; OR=3.03; 95%CI=1.40-6.55); *TNF-α* -238 GA (*P*=0.0011; OR=2.44; 95%CI=1.45-4.12); and *TNF-α* -308 GA (*P*=0.0031; OR=2.51; 95%CI=1.39-4.51). Alleles and genotypes from *IL-18* -137G/C and *IFNγ* +874T/A were not

Groups

Nodule size

Milan criteria

Metastasis

a

Edmondson-Steiner

 Odds ratio= 1.46 [95% confidence interval: 1.04-2.05] b Odds ratio= 1.54 [95% confidence interval: 0.96-2.48]

insertion/deletion allele and genotype frequencies between different groups.

**TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) polymorphism**

Tumor presentation

8 Hepatocellular Carcinoma - Future Outlook


**Table 4.** Distribution of the allele frequencies of polymorphisms of the *IL-18* (-607 C/A and -137 C/G), IFN-γ (+874 A/T), and TNF-α (-238 A/G e -308 A/G) genes among patients with hepatocellular carcinoma (HCC) and healthy controls.

When the -607C/A SNP of *IL-18* was evaluated in terms of the different presentations of the HCC, the frequencies of the -607\*C and -607\*A alleles between individuals with multinodular and uninodular HCC, showed that the -607\*C allele confers significant susceptibility to multinodular lesions (*P*=0.0289; OR=2.4; 95%CI = 1.09-5.28). Evaluation of the genotype frequency in SNP -607C/A of *IL-18* revealed that genotype 607CC was significantly more frequent in cases of multinodular HCC compared to uninodular tumors (*P*=0.0284; OR=3.5; 95%CI=1.24-9.86). On the other hand, genotype +874AT was found to be more frequent among patients with infiltrative diffuse HCC compared to uninodular HCC (*P*=0.0443; OR=3.6; 95%CI=1.04-12.47), thus conferring greater susceptibility to the diffuse tumor.


95%CI=0.02-1.21). Regarding genes *IFN-γ* and *TNF-α*, the allele and genotype frequencies did

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The 14bp\*D allele in gene *HLA-G* was more frequent in cases of HCC compared to control, with the 14bp DD genotype tending to be more frequent among individuals with HCC. The alleles and genotypes of the 14bp insertion/deletion polymorphism of the *HLA-G* were not

Chen et al., in a study of 150 individuals of the Chinese Han population, showed that genotypes of the 14bp insertion/deletion polymorphism of *HLA-G* were significantly associated with the expression of soluble HLA-G in plasma in this population [35]. These authors detected a dramatically lower expression of soluble HLA-G in plasma in the presence of the 14 bp I/I genotype than in the presence of the 14 bp I/D (*P* = 0.004) or D/D (*P* = 0.003) genotypes. No significant difference in plasma expression of soluble HLA-G was detected between 14 bp I/D

Many mechanisms of tumor escape have been proposed in the literature, some of them local and others systemic. Particularly important among them is the expression of immunomodu‐ latory molecules in the tumoral microbiota, as well as the expression of soluble suppressive factors by the tumoral cells. HLA-G represents one of these immunomodulatory molecules, playing an important role in the mechanisms of immunotolerance by the inhibition of the

The 14bp insertion/deletion polymorphism in exon 8 of *HLA-G* (3´UTR of the transcript) has been associated with the magnitude of protein production through the modulation of the stability of HLA-G mRNA, although the mechanisms involved still need to be elucidated. It has been demonstrated that the *HLA-G* allele containing the insertion polymorphism may suffer an additional splicing stage, so that 92 bp are removed from the primary mRNA. Thus, smaller *HLA-G* transcripts, without the 92 bp, are more stable than the complete mRNA forms. The alternative splicing may also be related to the presence of other polymorphisms in linkage disequilibrium with the 14bp insertion [8,10]. Overall, HLA-G expression may be modulated by many actors including transcription factors (influenced by the 5´ polymorphisms of the promoter region) and the rate of mRNA degradation or translation, highly influenced by polymorphisms observed at 3´UTR [8]. Ferguson et al. recently reported that the risk for invasive cancer of the uterine cervix in a Canadian population was significantly higher in the presence of 14 bp I/I genotype (OR=2.17, 95% CI: 1.10-4.27, P=0.020) as well as homozygous genotypes HLA-G\*01:01:02 (OR=3.52, 95% CI: 1.43-8.61, P=0.006) and \*01:06 (OR=19.1, 95% CI: 2.29-159, P=0.005) [12]. Similarly, Chen et al. studying the relationship between HLA-G gene polymorphism and the susceptibility of esophageal cancer in Kazakh and Han nationality in Xinjiang, found that the risk of developing esophageal cancer was significantly increased in individuals with 14 bp I/I genotype compared with 14 bp D/D genotype (OR=2.69, 95% CI:

The tumoral microbiota or even the cells that underwent mutation per se can induce the expression of HLA-G. Studies have demonstrated the expression of HLA-G in various malignant tumors, although with variations in the percentage of lesions expressing the

activity of NK cells, cytotoxic T lymphocytes and antigen-presenting cells [9,36].

not differ significantly between the groups with and without metastasis.

associated with disease severity.

1.30-5.55, P=0.04) in Kazakh population [11].

and D/D genotypes.

**Table 5.** Distribution of the genotype frequencies of polymorphisms of the *IL-18* (-607 C/A and -137 C/G), IFN-γ (+874 A/T), and TNF-α (-238 A/G and -308 A/G) genes among patients with hepatocellular carcinoma (HCC) and healthy controls.

No significant differences in the allele or genotype frequencies of SNPs of *IL-18, IFN-γ* and *TNF-α* were detected between the various tumor sizes, although the *TNF-α* -238\*A allele was slightly more frequent in tumors larger than 10cm compared to tumors smaller than 5cm (*P*=0.0889; OR=2.82; 95%CI: 0.94-8.41). Similarly, genotype *TNF-α* -238AG indicated a margin‐ ally greater susceptibility to tumors >10cm (*P*=0.0565; OR=4.63; 95%CI=1.05-20.48) than to tumors < 5 cm, whereas genotype *TNF-α* -238GG conferred marginal protection against large lesions (>10 cm vs. <5cm) (*P*=0.0565; OR=0.22; 95%CI=0.05-0.95).

Evaluation of SNP -607C/A of *IL-18* revealed no significant difference in allele and genotype frequencies between patients with HCC with or without metastasis. In contrast, evaluation of SNP -137C/G of *IL-18* revealed that the -137\*C allele was more frequent among individuals with metastasis than among individuals with no meatastasis (*P*=0.0240; OR=4.00; 95%CI=1.32-12.14). The frequencies of genotypes -137CC, CG or GG did not differ significantly between patients with and without metastasis, although genotype -137CC tended to confer greater susceptibility to metastasis (*P*=0.0564; OR=8.80; 95%CI=1.29-60.13) and genotype -137GG presented marginal protection against secondary lesions (*P*=0.0548; OR=0.14; 95%CI=0.02-1.21). Regarding genes *IFN-γ* and *TNF-α*, the allele and genotype frequencies did not differ significantly between the groups with and without metastasis.

**SNP Genotype frequency**

HCC n (frequency)

Control n (frequency)

CC 7 [0.0625] 18 [0.0891] 0.5157 0.68 0.27-0.68 GG 57 [0.5089] 100 [0.4950] 0.9063 1.06 0.67-1.68

AA 18 [0.1607] 12 [0.0594] 0.0048 3.03 1.40-6.55 CC 38 [0.3393] 85 [0.4208] 0.1845 0.71 0.44-1.14

AA 40 [0.3604] 79 [0.3911] 0.6276 0.88 0.54-1.42 TT 21 [0.1892] 41 [0.2030] 0.8823 0.92 0.91-1.65

AA 0 [0.0000] 0 [0.0000] 1.0000 1.81 0.03-92.15 GG 70 [0.6306] 163 [0.8069] 0.0011 0.40 0.24-0.69

AA 0 [0.0000] 3 [0.0149] 0.5548 0.25 0.01-4.99 GG 81 [0.7297] 173 [0.8564] 0.0098 0.45 0.25-0.80

*IL-18* -137C/G CG 48 [0.4286] 84 [0.4185] 0.9051 1.05 0.66-1.68

*IL-18* -607A/C CA 56 [0.5000] 105 [0.5198] 0.8138 0.92 0.58-1.47

IFN-γ +874A/T AT 50 [0.4505] 82 [0.4059] 0.4741 1.20 0.75-1.91

TNF-α -238A/G GA 41 [0.3694] 39 [0.1931] 0.0011 2.44 1.45-4.12

TNF-α -308A/G GA 30 [0.2703] 26 [0.1287] 0.0031 2.51 1.39-4.51

**Table 5.** Distribution of the genotype frequencies of polymorphisms of the *IL-18* (-607 C/A and -137 C/G), IFN-γ (+874 A/T), and TNF-α (-238 A/G and -308 A/G) genes among patients with hepatocellular carcinoma (HCC) and healthy

No significant differences in the allele or genotype frequencies of SNPs of *IL-18, IFN-γ* and *TNF-α* were detected between the various tumor sizes, although the *TNF-α* -238\*A allele was slightly more frequent in tumors larger than 10cm compared to tumors smaller than 5cm (*P*=0.0889; OR=2.82; 95%CI: 0.94-8.41). Similarly, genotype *TNF-α* -238AG indicated a margin‐ ally greater susceptibility to tumors >10cm (*P*=0.0565; OR=4.63; 95%CI=1.05-20.48) than to tumors < 5 cm, whereas genotype *TNF-α* -238GG conferred marginal protection against large

Evaluation of SNP -607C/A of *IL-18* revealed no significant difference in allele and genotype frequencies between patients with HCC with or without metastasis. In contrast, evaluation of SNP -137C/G of *IL-18* revealed that the -137\*C allele was more frequent among individuals with metastasis than among individuals with no meatastasis (*P*=0.0240; OR=4.00; 95%CI=1.32-12.14). The frequencies of genotypes -137CC, CG or GG did not differ significantly between patients with and without metastasis, although genotype -137CC tended to confer greater susceptibility to metastasis (*P*=0.0564; OR=8.80; 95%CI=1.29-60.13) and genotype -137GG presented marginal protection against secondary lesions (*P*=0.0548; OR=0.14;

lesions (>10 cm vs. <5cm) (*P*=0.0565; OR=0.22; 95%CI=0.05-0.95).

P OR 95%CI

genotype

10 Hepatocellular Carcinoma - Future Outlook

controls.

The 14bp\*D allele in gene *HLA-G* was more frequent in cases of HCC compared to control, with the 14bp DD genotype tending to be more frequent among individuals with HCC. The alleles and genotypes of the 14bp insertion/deletion polymorphism of the *HLA-G* were not associated with disease severity.

Chen et al., in a study of 150 individuals of the Chinese Han population, showed that genotypes of the 14bp insertion/deletion polymorphism of *HLA-G* were significantly associated with the expression of soluble HLA-G in plasma in this population [35]. These authors detected a dramatically lower expression of soluble HLA-G in plasma in the presence of the 14 bp I/I genotype than in the presence of the 14 bp I/D (*P* = 0.004) or D/D (*P* = 0.003) genotypes. No significant difference in plasma expression of soluble HLA-G was detected between 14 bp I/D and D/D genotypes.

Many mechanisms of tumor escape have been proposed in the literature, some of them local and others systemic. Particularly important among them is the expression of immunomodu‐ latory molecules in the tumoral microbiota, as well as the expression of soluble suppressive factors by the tumoral cells. HLA-G represents one of these immunomodulatory molecules, playing an important role in the mechanisms of immunotolerance by the inhibition of the activity of NK cells, cytotoxic T lymphocytes and antigen-presenting cells [9,36].

The 14bp insertion/deletion polymorphism in exon 8 of *HLA-G* (3´UTR of the transcript) has been associated with the magnitude of protein production through the modulation of the stability of HLA-G mRNA, although the mechanisms involved still need to be elucidated. It has been demonstrated that the *HLA-G* allele containing the insertion polymorphism may suffer an additional splicing stage, so that 92 bp are removed from the primary mRNA. Thus, smaller *HLA-G* transcripts, without the 92 bp, are more stable than the complete mRNA forms. The alternative splicing may also be related to the presence of other polymorphisms in linkage disequilibrium with the 14bp insertion [8,10]. Overall, HLA-G expression may be modulated by many actors including transcription factors (influenced by the 5´ polymorphisms of the promoter region) and the rate of mRNA degradation or translation, highly influenced by polymorphisms observed at 3´UTR [8]. Ferguson et al. recently reported that the risk for invasive cancer of the uterine cervix in a Canadian population was significantly higher in the presence of 14 bp I/I genotype (OR=2.17, 95% CI: 1.10-4.27, P=0.020) as well as homozygous genotypes HLA-G\*01:01:02 (OR=3.52, 95% CI: 1.43-8.61, P=0.006) and \*01:06 (OR=19.1, 95% CI: 2.29-159, P=0.005) [12]. Similarly, Chen et al. studying the relationship between HLA-G gene polymorphism and the susceptibility of esophageal cancer in Kazakh and Han nationality in Xinjiang, found that the risk of developing esophageal cancer was significantly increased in individuals with 14 bp I/I genotype compared with 14 bp D/D genotype (OR=2.69, 95% CI: 1.30-5.55, P=0.04) in Kazakh population [11].

The tumoral microbiota or even the cells that underwent mutation per se can induce the expression of HLA-G. Studies have demonstrated the expression of HLA-G in various malignant tumors, although with variations in the percentage of lesions expressing the molecule. In studies involving renal cell carcinoma [37], endometrial adenocarcinoma [38] and gastric cancer [39], at least 30% of the tumors exhibited HLA-G expression.

A higher frequency of the *IL-18* -607\*A allele and -607AA genotype was found among HCC patients compared to healthy individuals in the present study. Mi et al. analyzed a pool of studies on the association between polymorphisms of the *IL-18* gene and the risk of cancer (cancer of renal cells, of the ovary and breast, nasopharyngeal an cervical cancer, cancer of the esophagus, prostate, lung and stomach, and colorectal cancer) involving approximately 2137 cases and 3117 controls for the -607C/A variant, and 2372 cases and 3476 controls for the -137G/ C polymorphism, and they observed that the -607\*A and -137\*C alleles were associated with an increased global risk of cancer compared to patients with the wild allele. Analysis of the different ethnic groups showed that these polymorphisms were associated with an increased risk of cancer in Asians but not in Europeans or Africans [44]. Regarding the evaluation of SNP -137C/G, in the present study no significant differences in allele or genotype frequencies were detected between patients with HCC and healthy individuals. However, the *IL18* -137G/-607A haplotype was more frequent among individuals with HCC than among control. These results suggest that, even though no association was detected between SNP -137G/C and HCC, the interaction between the two polymorphisms may have been involved in the susceptibility to HCC. Regarding the polymorphism of the *IFN-γ* gene (+874 A/T), no significant difference in allele or genotype frequencies were observed between patients with HCC and healthy controls. Migita et al. studied 236 Japanese patients with chronic HBV infection who were divided into two groups: with (n=48) and without (n=188) HCC, for the evaluation of the association of polymorphisms of the *TNF-α*, *IFN-γ, TGF-β1, IL-6* and *IL-10* genes with the risk of HCC. When evaluating the SNP +874 of *IFN-γ*, the authors did not detect a statistically significant difference

Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

http://dx.doi.org/10.5772/56350

13

between the two groups, as also observed in the present study [45].

compared with the wild-type *TNF-α* CC [48].

We observed that alleles *TNF-α* -238\*A and -308\*A confer significant susceptibility to HCC, and that genotypes *TNF-α* -238GA and -308GA and haplotypes *TNF-α* -238A/-308G and -238G/-308A are also more frequent among patients with HCC compared to control. Also in agreement with these results, Akkiz et al. when analyzing 110 patients with HCC and 110 healthy controls from the Turkish population, observed that patients with HCC had a higher frequency of genotype *TNF-α* -308GA and a lower frequency of genotype *TNF-α* -308GG compared to control and after logistic regression, genotype -308GA was found to be associated with risk of HCC. In addition, individuals with the -308\*A allele (genotypes -308AA and -308GA) had a 4.75-fold higher chance to develop HCC compared to individuals with the GG genotype [46]. Similarly, Jung et al., studying 227 Korean patients with HCC and 365 controls, detected a higher frequency of allele -238\*A among patients with HCC than among healthy controls [47]. On the other hand, Yang et al., did not find statistical difference in *TNF-α* -308G/ A alleles or genotypes frequencies between Chinese HCC patients (n=772) and healthy controls (n=852), but they observed that *TNF-α* -863AA genotype may increase the risk of HCC

The expression of *TNF-α* is regulated both at the transcriptional and post-transcriptional level and polymorphisms in the promoter region of *TNF-α* have been related to the production of this cytokine [22]. Greater TNF-α production up to five-fold the basal level and induction of mRNA expression have been associated with the *TNF-α* -308\*A allele, with elevated serum TNF levels being observed even in heterozygous patients [49]. Many mechanisms of the

Regarding hepatic diseases, Souto et al., in a study of 74 liver biopsies of individuals with chronic HBV infection and 10 specimens obtained from previously healthy cadaver liver donors, demonstrated that 77% of the samples of chronic HBV hepatitis presented HLA-G expression in the hepatocytes, as opposed to none of the controls [40]. These authors detected a case of HCC, in which HLA-G expression was not detected in the tumor cells but was detected in adjacent non-tumoral hepatic tissue.

Lin et al. evaluated by immunohistochemistry the expression of HLA-G in 219 HCC and adjacent nontumoral tissue samples. The expression of HLA-G was observed in 50.2% of HCC samples vs. 0% of normal corresponding adjacent tissue. Evaluation of HCC stages showed that HLA-G expression was detected in 37.8%, 41.9% and 71.4% of cases in stages I, II and III, respectively. The data reported by these authors revealed that the expression of HLA-G was strongly correlated with advanced HCC stage and that soluble HLA-G was significantly more elevated in the plasma of patients with HCC compared to healthy controls [41]. Similarly, Cai et al. studied the expression of HLA-G by immunohistochemistry in 173 HCC specimens and observed that HLA-G expression was associated with the prognosis of HCC, especially in the early stages of the disease, with higher HLA-G expression being independently associated with shorter overall survival and greater tumor recurrence after surgical resection [42].

Recently, a study involving 267 patients divided as anti-HBs positive healthy individuals (n=50), chronic HBV carriers (n=45), active hepatitis B (n=46), liver cirrhosis (n=46) and earlystage HCC patients (n=80) showed that serum concentrations of soluble human leukocyte antigen-G (sHLA-G) were significantly higher in the active hepatitis B and HCC groups compared to the other groups (P<0.05). Moreover, the concentrations of sHLA-G were higher in the patients with HCC than in those with liver cirrhosis or active hepatitis B, suggesting that serum sHLA-G concentrations may be associated with the different phases of hepatitis B infection. They did not find any association between sHLA-G concentrations and HCC stage, number of tumors, pathologic grade and presence of vascular invasion [43]. Another study, examining fifteen SNPs in the non-classical class I alleles, found that the SNPs rs17875380, rs41557518, rs114465251, and rs115492845 were associated with susceptibility to chronic hepatitis B infection or HCC, and HLA-F\*01:04, HLA-G\*01:05N, and HLA-E\*01:01 were associated with hepatitis B or hepatitis B with HCC, concluding that these polymorphisms may play an important role in immune surveillance of hepatitis B and HCC, possibly leading to immune responses to virus or cancer cells [14].

It has been demonstrated that, in addition to genetic factors, the microbiota, for example, strees inducers, hypoxia and cytokines (interferons, IL-10, TNF-α), influences the expression of HLA-G, so that more studies are needed for a better understanding of the interaction of molecules derived from the tumor and from host factors [9].

In summary, the present findings show that the deletion allele of the *HLA-G* 14bp insertion/ deletion polymorphism was more frequent among patients with tumors than among healthy individuals, a fact that may confer greater susceptibility to HCC.

A higher frequency of the *IL-18* -607\*A allele and -607AA genotype was found among HCC patients compared to healthy individuals in the present study. Mi et al. analyzed a pool of studies on the association between polymorphisms of the *IL-18* gene and the risk of cancer (cancer of renal cells, of the ovary and breast, nasopharyngeal an cervical cancer, cancer of the esophagus, prostate, lung and stomach, and colorectal cancer) involving approximately 2137 cases and 3117 controls for the -607C/A variant, and 2372 cases and 3476 controls for the -137G/ C polymorphism, and they observed that the -607\*A and -137\*C alleles were associated with an increased global risk of cancer compared to patients with the wild allele. Analysis of the different ethnic groups showed that these polymorphisms were associated with an increased risk of cancer in Asians but not in Europeans or Africans [44]. Regarding the evaluation of SNP -137C/G, in the present study no significant differences in allele or genotype frequencies were detected between patients with HCC and healthy individuals. However, the *IL18* -137G/-607A haplotype was more frequent among individuals with HCC than among control. These results suggest that, even though no association was detected between SNP -137G/C and HCC, the interaction between the two polymorphisms may have been involved in the susceptibility to HCC. Regarding the polymorphism of the *IFN-γ* gene (+874 A/T), no significant difference in allele or genotype frequencies were observed between patients with HCC and healthy controls. Migita et al. studied 236 Japanese patients with chronic HBV infection who were divided into two groups: with (n=48) and without (n=188) HCC, for the evaluation of the association of polymorphisms of the *TNF-α*, *IFN-γ, TGF-β1, IL-6* and *IL-10* genes with the risk of HCC. When evaluating the SNP +874 of *IFN-γ*, the authors did not detect a statistically significant difference between the two groups, as also observed in the present study [45].

molecule. In studies involving renal cell carcinoma [37], endometrial adenocarcinoma [38] and

Regarding hepatic diseases, Souto et al., in a study of 74 liver biopsies of individuals with chronic HBV infection and 10 specimens obtained from previously healthy cadaver liver donors, demonstrated that 77% of the samples of chronic HBV hepatitis presented HLA-G expression in the hepatocytes, as opposed to none of the controls [40]. These authors detected a case of HCC, in which HLA-G expression was not detected in the tumor cells but was detected

Lin et al. evaluated by immunohistochemistry the expression of HLA-G in 219 HCC and adjacent nontumoral tissue samples. The expression of HLA-G was observed in 50.2% of HCC samples vs. 0% of normal corresponding adjacent tissue. Evaluation of HCC stages showed that HLA-G expression was detected in 37.8%, 41.9% and 71.4% of cases in stages I, II and III, respectively. The data reported by these authors revealed that the expression of HLA-G was strongly correlated with advanced HCC stage and that soluble HLA-G was significantly more elevated in the plasma of patients with HCC compared to healthy controls [41]. Similarly, Cai et al. studied the expression of HLA-G by immunohistochemistry in 173 HCC specimens and observed that HLA-G expression was associated with the prognosis of HCC, especially in the early stages of the disease, with higher HLA-G expression being independently associated with shorter overall survival and greater tumor recurrence after surgical resection [42].

Recently, a study involving 267 patients divided as anti-HBs positive healthy individuals (n=50), chronic HBV carriers (n=45), active hepatitis B (n=46), liver cirrhosis (n=46) and earlystage HCC patients (n=80) showed that serum concentrations of soluble human leukocyte antigen-G (sHLA-G) were significantly higher in the active hepatitis B and HCC groups compared to the other groups (P<0.05). Moreover, the concentrations of sHLA-G were higher in the patients with HCC than in those with liver cirrhosis or active hepatitis B, suggesting that serum sHLA-G concentrations may be associated with the different phases of hepatitis B infection. They did not find any association between sHLA-G concentrations and HCC stage, number of tumors, pathologic grade and presence of vascular invasion [43]. Another study, examining fifteen SNPs in the non-classical class I alleles, found that the SNPs rs17875380, rs41557518, rs114465251, and rs115492845 were associated with susceptibility to chronic hepatitis B infection or HCC, and HLA-F\*01:04, HLA-G\*01:05N, and HLA-E\*01:01 were associated with hepatitis B or hepatitis B with HCC, concluding that these polymorphisms may play an important role in immune surveillance of hepatitis B and HCC, possibly leading

It has been demonstrated that, in addition to genetic factors, the microbiota, for example, strees inducers, hypoxia and cytokines (interferons, IL-10, TNF-α), influences the expression of HLA-G, so that more studies are needed for a better understanding of the interaction of molecules

In summary, the present findings show that the deletion allele of the *HLA-G* 14bp insertion/ deletion polymorphism was more frequent among patients with tumors than among healthy

gastric cancer [39], at least 30% of the tumors exhibited HLA-G expression.

in adjacent non-tumoral hepatic tissue.

12 Hepatocellular Carcinoma - Future Outlook

to immune responses to virus or cancer cells [14].

derived from the tumor and from host factors [9].

individuals, a fact that may confer greater susceptibility to HCC.

We observed that alleles *TNF-α* -238\*A and -308\*A confer significant susceptibility to HCC, and that genotypes *TNF-α* -238GA and -308GA and haplotypes *TNF-α* -238A/-308G and -238G/-308A are also more frequent among patients with HCC compared to control. Also in agreement with these results, Akkiz et al. when analyzing 110 patients with HCC and 110 healthy controls from the Turkish population, observed that patients with HCC had a higher frequency of genotype *TNF-α* -308GA and a lower frequency of genotype *TNF-α* -308GG compared to control and after logistic regression, genotype -308GA was found to be associated with risk of HCC. In addition, individuals with the -308\*A allele (genotypes -308AA and -308GA) had a 4.75-fold higher chance to develop HCC compared to individuals with the GG genotype [46]. Similarly, Jung et al., studying 227 Korean patients with HCC and 365 controls, detected a higher frequency of allele -238\*A among patients with HCC than among healthy controls [47]. On the other hand, Yang et al., did not find statistical difference in *TNF-α* -308G/ A alleles or genotypes frequencies between Chinese HCC patients (n=772) and healthy controls (n=852), but they observed that *TNF-α* -863AA genotype may increase the risk of HCC compared with the wild-type *TNF-α* CC [48].

The expression of *TNF-α* is regulated both at the transcriptional and post-transcriptional level and polymorphisms in the promoter region of *TNF-α* have been related to the production of this cytokine [22]. Greater TNF-α production up to five-fold the basal level and induction of mRNA expression have been associated with the *TNF-α* -308\*A allele, with elevated serum TNF levels being observed even in heterozygous patients [49]. Many mechanisms of the cancerigenous activity of TNF-α have been suggested, such as induction of pro-malignant chemokines, metalloproteinases, cell adhesion molecules, angiogenic mediators, reactive oxygen intermediates and inflammatory enzymes. Increased TNF-α levels are correlated with hepatic inflammation, fibrosis and tissue damage [46].

**5. Conclusions**

**Author details**

**References**

Eduardo Antonio Donadi1

Our results suggest that the 14bp-deletion allele in *HLA-G* gene is associated with HCC susceptibility in a Brazilian population, and that the alleles *IL-18* -607\*A and *TNF-α* (-238\*A and -308\*A) may confer susceptibility to HCC, whereas IL-18 -607\*C and -137\*C alleles may confer susceptibility to multinodular and diffuse HCC, respectively. Furthermore, deletion/ deletion genotype was marginally associated with greater risk of HCC. More studies in

Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

1 Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto – University of

2 Department of Surgery and Anatomy, Faculty of Medicine of Ribeirão Preto – University

[1] Bosch, F. X, Ribes, J, Díaz, M, & Cléries, R. Primary liver cancer: worldwide incidence

[2] Jemal, A, Siegel, R, Ward, E, Hao, Y, Xu, J, & Thun, M. J. Cancer statistics, 2009. CA: a

[3] Rockey, D. C, & Friedman, S. L. Hepatic fibrosis and cirrhosis. In: Boyer TD, Wright

[4] Carrilho, F. J, Kikuchi, L, Branco, F, Goncalves, C. S, & De Mattos, A. A. Brazilian HCC Study Group. Clinical and epidemiological aspects of hepatocellular carcinoma

[5] Teixeira, A. C, & Mente, E. D. Cantao CAB, Sankarankutty AK, Souza FF, Motta TC, Monsignore L, Elias-Junior J, Muglia VF, Abud DG, Peria FM, Zucoloto S, Castro-Sil‐ va O, Martinelli ALC Clinical Characteristics of 130 Patients With Hepatocellular Carcinoma Followed at a Tertiary Hospital From Brazil. World Journal of Oncology

and

http://dx.doi.org/10.5772/56350

15

different populations are needed to confirm these findings.

Andreza Correa Teixeira1,2\*, Ana de Lourdes Candolo Martinelli1

and trends. Gastroenterology (2004). Supplement 1.

TL, Manns MP. Hepatology. Philadelphia: Elselvier, (2006).

\*Address all correspondence to: andrezacteixeira@gmail.com

São Paulo, Ribeirão Preto, São Paulo, Brazil

of São Paulo, Ribeirão Preto, São Paulo, Brazil

cancer journal for clinicians (2009).

(2012). , 3(4), 165-172.

in Brazil. Clinics (2010). , 65(12), 1285-1290.

Studies evaluating the association of polymorphisms of *IL-18* with the severity of cancer have been reported. Saenz-Lopez et al. investigated whether the presence of SNPs -137G/C and -607A/C of *IL-18* were associated with size, grade, and TNM Classification of 158 patients with renal cell carcinoma. These authors observed that genotype -607CC was significantly associ‐ ated with larger tumor size (*P*=0.001), grade (*P*=0.030), and T (*P*=0.001) and M (*P*=0.012) stage, while genotype -137GG was correlated with larger tumor size (*P*=0.036), grade (*P*=0.017), and stage T (*P*=0.026) [50]. Another authors, studing the association of SNPs -607A/C and -137G/C of *IL-18* with histology of colorectal and gastric cancer (moderately differentiated or undiffer‐ entiated vs. well differentiated), noticed that no difference in genotype frequency was detected, although the combination of genotypes -607AA/-137GC was more frequent among patients with less differentiated tumors [51].

Literature evidence has demonstrated that IL-18 is a pleiotropic cytokine that enhances the Th1 or Th2 immune response according to the medium and to genetics. In the presence of IL-12, IL-18 induces IFN-γ secretion by NK and T cells, activating the Th1 response, important for defense against tumor cells. On the other hand, IL-18 can increase tumor growth via increased stimulation of VEGF and of the immune response, and also stimulate solid tumor metastasis [52]. A possible explanation for this fact is the increased Th1 response in the early stages of the cancer which, however, is replaced with Th2 as tumor malignancy worsens with tumor development. Thus, as the tumor develops, *IL-18* polymorphisms, which induce great IL-18 production, may contribute to the promotion of more advanced tumors due to the activation of angiogenesis, differentiation of tumor cells and regulation of stimulators of cell proliferation [53]. To corroborate these data, Tangkijvanich et al., in a study of 70 patients with HCC and 10 healthy controls, observed that serum IL-18 levels were significantly correlated with the presence of vascular invasion and of more advanced tumors according to the Okuda classification. In addition, the survival of patients with high serum levels of IL-18 was worse. Multivariate analysis showed that serum IL-18 levels proved to be a significant and inde‐ pendent prognostic factor regarding survival [54].

Regarding the +874 A/T polymorphism of the *IFN-γ* gene, study involving 100 patients with chronic HCV infection and different degrees of disease severity (chronic hepatitis, n=42; cirrhosis + HCC, n=58) and 103 healthy controls detected that the TT and AT genotypes were significantly more frequent among patients with cirrhosis and HCC. These genotypes were associated with a 2.5 higher risk of progression to more severe forms of hepatic disease. In addition, the +874\*T allele was approximately twice more frequent among patients in an advanced stage of hepatitis C than among patients with chronic hepatitis, although multivari‐ able analysis did not show that the +874\*T allele was an independent predictive factor of severity [55].

### **5. Conclusions**

cancerigenous activity of TNF-α have been suggested, such as induction of pro-malignant chemokines, metalloproteinases, cell adhesion molecules, angiogenic mediators, reactive oxygen intermediates and inflammatory enzymes. Increased TNF-α levels are correlated with

Studies evaluating the association of polymorphisms of *IL-18* with the severity of cancer have been reported. Saenz-Lopez et al. investigated whether the presence of SNPs -137G/C and -607A/C of *IL-18* were associated with size, grade, and TNM Classification of 158 patients with renal cell carcinoma. These authors observed that genotype -607CC was significantly associ‐ ated with larger tumor size (*P*=0.001), grade (*P*=0.030), and T (*P*=0.001) and M (*P*=0.012) stage, while genotype -137GG was correlated with larger tumor size (*P*=0.036), grade (*P*=0.017), and stage T (*P*=0.026) [50]. Another authors, studing the association of SNPs -607A/C and -137G/C of *IL-18* with histology of colorectal and gastric cancer (moderately differentiated or undiffer‐ entiated vs. well differentiated), noticed that no difference in genotype frequency was detected, although the combination of genotypes -607AA/-137GC was more frequent among patients

Literature evidence has demonstrated that IL-18 is a pleiotropic cytokine that enhances the Th1 or Th2 immune response according to the medium and to genetics. In the presence of IL-12, IL-18 induces IFN-γ secretion by NK and T cells, activating the Th1 response, important for defense against tumor cells. On the other hand, IL-18 can increase tumor growth via increased stimulation of VEGF and of the immune response, and also stimulate solid tumor metastasis [52]. A possible explanation for this fact is the increased Th1 response in the early stages of the cancer which, however, is replaced with Th2 as tumor malignancy worsens with tumor development. Thus, as the tumor develops, *IL-18* polymorphisms, which induce great IL-18 production, may contribute to the promotion of more advanced tumors due to the activation of angiogenesis, differentiation of tumor cells and regulation of stimulators of cell proliferation [53]. To corroborate these data, Tangkijvanich et al., in a study of 70 patients with HCC and 10 healthy controls, observed that serum IL-18 levels were significantly correlated with the presence of vascular invasion and of more advanced tumors according to the Okuda classification. In addition, the survival of patients with high serum levels of IL-18 was worse. Multivariate analysis showed that serum IL-18 levels proved to be a significant and inde‐

Regarding the +874 A/T polymorphism of the *IFN-γ* gene, study involving 100 patients with chronic HCV infection and different degrees of disease severity (chronic hepatitis, n=42; cirrhosis + HCC, n=58) and 103 healthy controls detected that the TT and AT genotypes were significantly more frequent among patients with cirrhosis and HCC. These genotypes were associated with a 2.5 higher risk of progression to more severe forms of hepatic disease. In addition, the +874\*T allele was approximately twice more frequent among patients in an advanced stage of hepatitis C than among patients with chronic hepatitis, although multivari‐ able analysis did not show that the +874\*T allele was an independent predictive factor of

hepatic inflammation, fibrosis and tissue damage [46].

with less differentiated tumors [51].

14 Hepatocellular Carcinoma - Future Outlook

pendent prognostic factor regarding survival [54].

severity [55].

Our results suggest that the 14bp-deletion allele in *HLA-G* gene is associated with HCC susceptibility in a Brazilian population, and that the alleles *IL-18* -607\*A and *TNF-α* (-238\*A and -308\*A) may confer susceptibility to HCC, whereas IL-18 -607\*C and -137\*C alleles may confer susceptibility to multinodular and diffuse HCC, respectively. Furthermore, deletion/ deletion genotype was marginally associated with greater risk of HCC. More studies in different populations are needed to confirm these findings.

### **Author details**

Andreza Correa Teixeira1,2\*, Ana de Lourdes Candolo Martinelli1 and Eduardo Antonio Donadi1

\*Address all correspondence to: andrezacteixeira@gmail.com

1 Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto – University of São Paulo, Ribeirão Preto, São Paulo, Brazil

2 Department of Surgery and Anatomy, Faculty of Medicine of Ribeirão Preto – University of São Paulo, Ribeirão Preto, São Paulo, Brazil

### **References**


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Role of Alleles and Genotypes of Polymorphisms of IL-18 (-607 C/A; and -137 C/G), IFN-γ (+874 A/T) and…

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17

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[21] Teixeira, A. C, Mendes-junior, C. T, & Marano, L. A. Deghaide NHS, Secaf M, Elias-Junior J, Muglia V, Donadi EA, Martinelli ALC. Alleles and genotypes of polymor‐ phisms of IL-18, TNF-α and IFN-γ are associated with a higher risk and severity of hepatocellular carcinoma (HCC) in Brazil Human Immunology (accepted for publi‐

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**Chapter 2**

**Liver Transplantation for Hepatocellular Carcinoma**

The incidence of hepatocellular carcinoma (HCC) has increased exponentially. Liver cancer, primarily HCC, has become the third cause of death from cancer worldwide and the ninth cause of cancer deaths in the U.S.A. According to the center for disease control and prevention statistics, the annual incidence of HCC has increased significantly to an average of 3.0 per 100,000 persons. The annual percentage change in incidence (APC) is 3.5% and the highest APC was found in age group 50-59 years [1]. These statistics should be tampered with caution as only biopsy proven HCC, histology code 8170 and 8175, were counted. This definitely

While HCC may arise in healthy livers, the majority of cases develop in a back ground of chronic liver disease i.e. cirrhosis which makes management even more challenging. Viral hepatitis, both B (HBV) and C (HCV), cause 78% of primary liver cancer worldwide [1]. Other risk factors include alcoholic liver disease [2], fatty liver disease (FLD) and non-alcoholic steatohepatitis (NASH) [3, 4], hemochromatosis [5], primary biliary cirrhosis [6] and primary sclerosing cholangitis [7]. Moreover, liver adenoma carries a 10% risk for malignant transfor‐ mation [8]. Tobacco smoking increases the incidence of HCC [9]. Fungi *Aspergillus flavus* present in poorly stored grains produce Aflatoxin, an environmental hazard for HCC [10]. Hepatic venous occlusive disease has been implicated in HCC development in anecdotal case

> © 2013 Asham et al.; licensee InTech. This is an open access article 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.

Emad H. Asham, Howard Mansour and

Additional information is available at the end of the chapter

Mark Ghobrial

**1. Introduction**

**2. Risk factors**

reports [11].

http://dx.doi.org/10.5772/55374

underestimates the scope of the disease.
