Molecular Diagnostics in Liver Cancer 17 Anil Kumar Chauhan, Monika Bhardwaj, and Pankaj Kumar Chaturvedi 17.1 Introduction Liver cancer is the third

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1 Molecular Diagnostics in Liver Cancer 17 Anil Kumar Chauhan, Monika Bhardwaj, and Pankaj Kumar Chaturvedi 17.1 Introduction Liver cancer is the third leading cause of cancer- related mortalities in the world [1], with hepatocellular carcinoma (HCC) [1] and intrahepatic cholangiocarcinoma (IHCC) [1] being the common forms of liver cancer. The incidence of liver cancer in men is particularly 2 4 times higher than in women [2]. Most of the liver cancer is diagnosed in well less developed nations primarily in East Asian and African countries [3, 4]. Every year more than 850,000 new cases of liver cancer are reported worldwide [5]. Underlying risks for the HCC occurrence and progression are influenced by etiology, activity, and stage of the underlying liver disease. Major risk factors for the development of HCC are distinct such as, cirrhosis (chronic liver damage caused by inflammation and fibrosis), hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcohol abuse and metabolic syndrome [6]. Other cofactors which are well-characterized contributors to HCC A. K. Chauhan M. Bhardwaj Laboratory of Biochemistry and Cellular Engineering, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea chauhananil48@dgist.ac.kr; monika03@ dgist.ac.kr P. K. Chaturvedi (*) Department of Radiation Oncology, Chungbuk National University Hospital, Cheongju, Republic of Korea pankaj@chungbuk.ac.kr are tobacco smoke inhalation and intake of aflatoxin B1 (a fungal carcinogen present in food supplies associated with mutations in the tumor suppressor gene TP53) [7 13] Risk Factors Associated with Liver Cancer Several epidemiological studies have demonstrated that chronic viral hepatitis, driven by HBV and HCV can lead to cirrhosis and/or HCC. HBV is a double-stranded, circular DNA molecule with eight genotypes (A to H). Among these, genotype C has been associated with the higher risk of HCC than genotypes A, B, and D [14]. There are various routes of the Hepatitis B transmission, for example, contaminated blood transfusions, intravenous injections, sexual contact and from mother to fetus [15]. Besides positive hepatitis B surface antigen (HBsAg), patients with positive hepatitis B core antibody (anti- HBc) who are HBsAg-negative also remain at risk for development of HCC. Antiviral treatment for hepatitis B has been associated with significant reduction of hepatocarcinogenicity of HBV. HCV is a small, single-stranded RNA virus, which exhibits high genetic variability, which exhibits six different genotypes [16]. Around 80% of the patient infected with HCV progress to chronic hepatitis and ~20% among them may have chance of developing cirrhosis [17]. Cirrhotic patients are at the higher risk of Springer Nature Singapore Pte Ltd K. K. Shukla et al. (eds.), Molecular Diagnostics in Cancer Patients, 293

2 294 developing HCC if they get the dual infection of HBV and HCV [18]. Patients who obtained a sustained viral response after treatment of HCV, are at significantly lower risk of HCC with a 54% reduction in mortality [19]. Besides that, alcohol consumption is considered as an important factor for HCC, which accounts for 40 50% of all HCC cases in Europe and the United States. The relation between alcohol and risk of liver cancer correlates with the heavy amount of alcohol consumed over a lifetime. The risk of liver cancer increases two- to four-times among persons drinking more than 60 g/day of ethanol [20] Diabetes and Nonalcoholic Fatty Liver Disease Obesity and diabetes mellitus are other key factors that are involved in the risk of liver cancer. The liver plays an essential role in the glucose metabolism which is directly affected by diabetes mellitus, that can lead to chronic hepatitis, fatty liver, liver failure, and cirrhosis [21, 22]. Diabetes mellitus has been significantly associated with about threefold increased risk of HCC. Pleiotropic effects of hyperinsulinemia play a role in carcinogenesis by regulating the anti-inflammatory cascade and cellular proliferation. Alongside diabetes mellitus, obesity-induced nonalcoholic fatty liver disease (NAFLD), steatosis, and cryptogenic cirrhosis may also lead to the development of HCC [23]. Majority of NAFLD induces HCC cases are observed in men, which may occur in the absence of cirrhosis. NAFLD-related tumors have an elevated level of des-γ-carboxy prothrombin (DCP) compared to α-fetoprotein (AFP) synthesis[elevated α-fetoprotein (AFP) synthesis is a hall mark for liver cancer] which is associated with HCV-related HCC [24, 25] Other Factors Sex may play a role in the development of HCC, which is found more often in males. Higher testosterone levels and intake of anabolic steroids have been linked to advanced hepatic fibrosis, A. K. Chauhan et al. hepatitis B carrier, and chronic hepatitis C infection compared to women [26]. Also, the higher amount of alcohol intake, cigarette smoking, and higher body mass index, makes the males more prone to viral hepatitis infection that increases the incidence of HCC [26]. Moreover, the food contaminated with certain mycotoxin, for example, aflatoxin B1 (AFB1) is also associated with the development of HCC. AFB1 is produced by Aspergillus species (molds) and is typically found ingrains, corn, peanuts, or soybeans stored in warm humid conditions. The duration and dose of AFB1 exposure is directly related to the risk of HCC [27]. Additionally, the synergistic effect of AFB1 on hepatitis B-and C-induced liver cancer tends to be more lethal compared with AFB1 exposure alone. So, the removal of AB1 exposure from environment may reduce the incidence of HCC occurrence [27]. Notwithstanding, some metabolic and genetic diseases like, Hemochromatosis, Wilson s disease, α-1 antitrypsin disease, tyrosinemia, glycogen- storage disease types I and II, and porphyrias are also associated with HCC. Thalassemia (iron overload) on the other hand has also been reported to have higher risk of HCV infection which eventually may contribute to the increased risk of primary liver cancer [28]. In addition, longer duration (>5 years) of exposure to oral contraceptives showed a significant increase in the risk of HCC [29] Cellular Signaling Pathways Involved in Hepatocarcinogenesis Being a complex process associated with genetic and epigenetic changes that are associated with initiation, development, and progression of hepatocarcinogenesis, liver cirrhosis is a predisposing condition for HCC. Emerging incidences of HCC have thrived the researchers with the hope to develop new therapeutic strategies by understanding the molecular, cellular and physiological mechanism of liver cancer. Activation of oncogenes and several other cellular events that

3 17 Molecular Diagnostics in Liver Cancer regulates survival of cancerous cells by suppressing apoptosis and tumor suppressor genes have been well documented. Signal transduction pathways are being studied extensively to identify potential biomarkers and molecular targets of HCC. These pathways are briefly explained below: Wnt/β-Catenin Pathway First identified in Drosophila melanogaster, the Wnt signaling pathway is highly conserved and is deregulated in many cancers, including HCC. Wnt signaling participates in various evolutionary pathways that involve homeostasis, cell proliferation, differentiation, motility, and apoptosis [30]. The activation of Wnt pathway is generally observed after infection of HBV/ HCV and liver cirrhosis leading to the development of HCC. In many cases, mutation of the proto- oncogeneβ- catenin or the inactivation of sliver- specific tumor suppressor gene adenomatous polyposis coli causes the activation of Wnt signaling [31]. Mutation in β-catenin promotes the upregulation of frizzled-7 and dephosphorylation of β-catenin that prevent subsequent degradation. These mutations are related to the patients that were highly exposed to HCV infection and aflatoxin [32]. In addition, mutations in the negative regulators of Wnt pathway; Axin 1 and Axin 2 were also observed in HCC patients [33]. Therefore, targeted inactivation of Wnt/β-Catenin pathway could be a potential therapeutic target for HCC p53 Pathway 295 p53 is considered as guardian of the genome which plays a critical role in cancer. In physiological conditions, p53 is expressed at low levels, which is up-regulated in response to intracellular and extracellular stress signals. In most of the human tumors, tumor suppressor TP53 gene is inactivated by a single point mutation that leads to subsequent defects in cell cycle arrest and apoptosis [34]. DNA-damaging agents and chemotherapeutic drugs activate p53 by phosphorylation of the transactivation domain and acetylation and phosphorylation of basic allosteric control region by ataxia telangiectasia mutated and related kinases [35]. p53 mutations and inactivation have been reported to play a critical role in HCC. In a clinical study of patients with HCC, a point mutation at the third base position of codon 249 has been found. The G T and the G C transversion were consistent with mutations caused by AFB1 in mutagenesis experiments. Moreover, in several other cancer cell lines, a mutational hotspot in codon 259 was also found [36]. The mutations in the codon 249 occur in HBsAg seropositive carriers and the subsequent late events of these mutations are reflected as p53 DNA and protein mutation correlated to tumor stage [37]. Thus, the presence of mutated p53 in plasma of HCC patients serve as a biomarker for AFB1 exposure-related HCC. Association of p53 mutations in hemochromatosis and Wilson disease is a notable effect of oxidative stress in liver carcinogenesis. G:C, T:A transversions at codon 249 as well as to C:T to A:T and C:G to T:A transversions at codon 250 alters the function of p53 gene [38]. Under oxidative stress, an elevated level of inducible nitric oxide synthase (inos) expression is observed which results in the development of cirrhosis and increased risk of HCC prb Pathway The tumor suppressor retinoblastoma protein (prb1) controls cell cycle progression via repression of E2F transcription factor family of proteins and implement a major barrier to cancer development. prb phosphorylation and G1/S cell cycle transition correlate the cyclin-dependent kinases (CDKs) phosphorylation [39]. Pediatric cancer retinoblastoma is caused by mutational inactivation of both Rb1 alleles. Hereditary retinoblastoma has more tumor foci and early onset as it is produced by germline transmission of one mutational inactivated Rb1 allele and loss of the remaining wild-type allele in somatic retinal

4 296 cells. In contrast, the occurrence of sporadic retinoblastoma requires inactivation of both Rb1 alleles in somatic retinal cells, which delays their onset [40]. Previous studies on human tumor showed that DNA viruses, such as human papilloma viruses (HPV) results in parallel loss of prb and lack of functional p53 [41]. Changes in the expression of CDK inhibitors such as p16 INK4A, p21 (WAF1/CIP1), and p27 Kip1 contributes to hepatocarcinogenesis. Inactivation of p16 INK4A and reduced expression of p21 (WAF1/CIP1), which is mainly associated with p53 gene mutation contributes to HCC [42]. The similarity in mechanism of disruption of prb pathway was observed in various other cancers which suggests that prb is an important target in cancer Ras Pathway Small GTP-binding proteins known as human ras proteins (H-Ras, N-Ras, K-ras4A, and K-Ras4B) function as a molecular switch in various cellular physiological processes. Ras proteins interact with Raf-1(downstream serine/threonine kinase), that results in activation of MAPK kinases MEK1 and MEK2, to regulate cell growth, differentiation, proliferation, and apoptosis [43, 44]. Activation of Ras and expression of Ras pathway proteins such as p21 contributes to human HCC [45, 46]. Various chemicals such as N-nitrosomorpholine, bleomycin, 1-nitropyrene, and methyl (acetoxymethyl) nitrosamine causes single point mutation in codon 13 of H-ras, codon 12 of N-ras, and codon 61of K-ras that are commonly observed in HCC [47 53]. Members of the RASSF family of Ras inhibitors such as RASSF1A and NORE1A are inactivated in human HCC, which highlight the role of Ras pathway in liver cancer [54]. Suppression of Ras expression using antisense RNA and inhibition of kinases are successfully implemented in cell line and animal models of liver cancer [55, 56]. A. K. Chauhan et al. The intracellular mitogen-activated protein kinase (MAPK) family consists of five subgroups; extracellular signal-regulated kinase protein homologs 1 and2 (ERK1/2), big MAPK-1 (BMK-1/ERK5), c-jun N-terminal kinase homologs 1, 2, and 3 (JNK1/2/3), stressactivated protein kinase 2 (SAPK-2) homologs α, β, and δ (p38α/β/δ), and ERK6, also known as p38γ [57]. MAPKs executes diverse cellular functions ranging from cell proliferation to adhesion, and their activation depends upon phosphorylation of T and Y residues located in their activation loop [58, 59]. MAPK signaling pathway is being modulated at multiple steps by hepatitis proteins. Sprd protein (Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology- 1 domain), an inhibitor of Ras/Raf-1/ERK pathway is highly deregulated in HCC, that results in enhanced secretion of matrix metalloproteinases 2 and 9 [60]. In human hepatoma Huh-7cells, HCV E2 protein assists in activation of MAPK pathway and promotes cell proliferation [61]. A family of transcription factors known as signal transducers and activators of transcription (STATs) has been reported to be ubiquitously activated in HCC. Activation of STATs has been mediated by a diverse range of cytokines, hormones, and growth factors, but primarily governed by tyrosine phosphorylation by Janus kinases (JAKs) [62, 63]. Activated STATs synthesize suppressor of cytokine signaling (SOCS) proteins, that are part of a negative feedback loop in JAK/STAT pathway. These proteins suppress over activation of cytokine-stimulated cells by binding with phosphorylated JAKs and their receptors, that attenuates cancer progression [63]. Deregulation of two other STAT inhibitors; SH2-containingproteins and inhibitor of activated STATs and inactivation of SSI-1, a JAKbinding protein has been reported in HCC [64]. Therefore, intervention in MAPKs and JAK/ STAT signaling pathway could be a potential therapeutic target for treatment of HCC MAPK and JAK/STAT Pathway Others The molecular dynamics of hepatocarcinogenesis can be influenced by various growth factors

5 17 Molecular Diagnostics in Liver Cancer and other cellular pathways. For instance, several members of heat shock proteins (HSPs) family are key players in the occurrence of HCC. The decrease in serine phosphorylation HSP27 has been reported to be associated with the progression of HCC in clinical samples [65]. In addition, modulating the expression of inflammatory cytokines might play a critical role in monitoring HCC progression. For example, levels of Th2 cytokines are induced and Th1 cytokines decreased in liver metastases. Furthermore, vascular endothelial growth factor and fibroblast growth factor also play important roles in HCC development. The use of inhibitors against epidermal growth factor receptor and transforming growth factor β, helps to prevent the development of HCC in rat model [66, 67]. Induction of apoptosis by using RNA interference against antiapoptotic myeloid cell leukemia-1 protein could be another target for HCC [68]. Alcohol metabolism, cellular transport, and ubiquitins are other additional physiological processes that regulate hepatocarcinogenesis [69] Staging, Diagnosis, and Treatment of Liver Cancer Early diagnosis for liver cancer plays a key role in determining the best treatment method for HCC. To circumvent such cases based on prognosis, patients diagnosed with HCC are categorized into groups. This stratification of patients (Staging system) can guide the clinicians in patient selection, choice of therapy, patient counseling and randomization for research protocols. Staging system helps to determine liver function, tumor stage and physical status of HCC patients [70]. There are two staging system in HCC i.e. clinical and pathological. Clinical staging includes Barcelona Clinic Liver Cancer Staging System (BCLC), Okuda Staging System, and Italian Program of the Liver Cancer score, on the other hand, American Joint Committee on Cancer, Japanese Integrated system, Liver Cancer Study Group of Japan, and Chinese University Prognostic Index are the pathological 297 staging system. Among them, BCLC staging system is widely accepted for clinical trials and it outlines certain criteria to establish new drugs for HCC [71]. In HCC, cirrhotic livers exhibit increased hepatocyte proliferation that results in degenerative nodules. Ultrasonography (US) that detect nodules <1 cm are usually undefined and the patient must have a repeat US in 3 4 months. A radiologic investigation such as contrast- enhanced triple or quadriphasic multiphase computerized tomography (CT) scan for chest, abdomen and pelvis region or magnetic resonance imaging (MRI) must be conducted for patients who are diagnosed with nodules >1 cm [72]. MRI is a preferred diagnostic analyzer over CT scan for the evaluation of HCC due to its higher per-lesion sensitivity [73]. Moreover, US imaging sensitivity is variable which depends on the equipment and its operator. A bone scan is another vital tool to identify HCC staging and secondary diseases. Radiologic analysis helps to document certain tumor characteristics such as size, maximum diameter, and number of lesions, the location of tumors, vascular invasion, and extrahepatic disease [72]. If uncertainty persists in the diagnosis of HCC by radiologic test, a serum alpha(α)- fetoprotein (AFP) level >400 ng/ml can be used as an indicative prognostic value for HCC. The main function of AFP is to maintain the regulation of fatty acids in fetal and proliferating adult liver cells [74]. The main drawback of measuring serum AFP level is that it can be falsely raised in patients who have active hepatitis but no evidence of HCC. Moreover, elevated levels of AFP have been found in certain diseases such as acute hepatitis, cirrhosis, colitis, germ cell tumors, and intrahepatic cholangiocarcinoma [72, 74, 75]. In addition, another serum marker known as lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) expressed as a percentage of AFP has been found to be elevated in patients with HCC. AFP-L3 is associated with a more aggressive tumor, shorter doubling time, infiltrative growth pattern, vascular invasion, and intrahepatic metastasis [76]. Des-gammacarboxy prothrombin (DCP) is another serum marker produced by malignant hepatocytes

6 298 from an acquired posttranslational defect in vitamin K-dependent carboxylase system. DCP levels >125 mau/ml is sensitive and specific for differentiating HCC from chronic hepatitis and cirrhosis [77]. The levels of DCP in blood serum are high in specificity but low at sensitivity [78]. The bitter truth of erroneous diagnosis for HCC that the patients have to pay a heavy price in the form of liver impairment and increased morbidity related to therapy. Unfortunately, there is no treatment available in present times that could improve the survival of patients diagnosed with the late stage of HCC. The best positive outcomes for treatments are achieved when patients are put on proper consistent surveillance for HCC at early stages and best treatment selection from various options. A multidisciplinary team of a hepatologist, a radiologist, a pathologist, a medical oncologist, an interventional radiologist, a transplant surgeon, and a hepatobiliary surgeon put their unique contributions to ensure optimal long-term outcomes for patients with HCC. Several treatment options available for treatment of HCC that can have a positive impact on survival are resection, liver transplantation (surgical approaches), transarterial chemoembolization, transarterial radiation, percutaneous local ablation, microwave ablation (nonsurgical approaches) and systemic therapy (sorafenib, an oral multitargeted tyrosine kinase inhibitor) [72, 79] Molecular Diagnosis of Liver Cancer by Gene Expression Profile A. K. Chauhan et al. More than decades ago, there has been a plethora of studies that documented about gene expression profiles in HCC (survival or recurrence) and other associated human liver diseases. The most promising way of molecular diagnosis in HCC and associated liver disease however is microarray- based gene profiling which usually is carried out by liver biopsy of normal and diseased liver that allows differentiating the gene responsible for HCC [80]. Patients with liver disease can avail the benefit of identification of therapeutic target by gene expression analysis Gene Expression Signature in Normal Liver There are several gene variations that occur naturally because of the sophisticated gene expression patterns in human [81]. However, the liver is a multifunctional and metabolically active organ that encompasses a complex transcriptome profile second after to brain. Nearly, 25 40% of genes are expressed in this organ, whose function is still unknown [82]. Among them, few functional genes have been specifically characterized for liver cells or liver tissues. However, there are certain obstructions faced by clinicians and researchers during the comparative study between healthy and diseased human livers. Firstly, the complexity of transcriptomes can get double or even triple during disease which makes the situation less well understood [83]. Secondly, liver biopsy sample contained stellate cells, Kupffer cells, sinusoidal endothelial cells, blood cells, capillary material, cholangiocytes, and lymphocytes along with hepatocytes which make the liver samples heterogeneous [84]. Thirdly, there are chances of cell contamination during the microdissection of liver tumor specimens, which may partially deviate the expression signature [80, 85]. Fourthly, in vitro gene signature profiling can perturb which might influence the technique used to culture the cell [80, 85]. In normal liver tissue, ~50% of genes have been reported to be similarly expressed in DNA microarray study. Gene-Tag study which utilizes amplified fragment length polymorphism (AFLP) based method of transcript imaging showed the presence and a >2.5-fold change inexpression level for at least one sample among four normal adult liver samples [86] Gene Expression Signature in HCC The BCLC staging system is based on tumor status, liver functional reserve, and health status and

7 17 Molecular Diagnostics in Liver Cancer links staging with treatment strategy [87]. Patients at early, intermediate or terminal stages can survive up to 5 years, months or 3 months respectively [88]. Gene expression profiling of HCC has classified the patients on the basis of stage of the disease, etiological agents, recurrence and survival that helps to deal with HCC patients [89 97]. Gene expression signature profiles for survival face certain limitations, as patients die from liver failure and tumor progression. Use of cancer- related death and selection of the cohort should be considered to minimize this limitation [96]. In a report, it was found that around 406 genes belonging to cell proliferation, apoptosis, ubiquitination, and histone modification were related to patient survival [97]. In another study, a 153-gene molecular signature relevant to metastatic HCC patients survival was generated using a supervised machine-learning algorithm [91]. Several gene profiling studies have been employed to study the recurrence of HCC. Using high-density oligonucleotide microarrays, the 12-gene signature was identified for HCC recurrence in a study. Another PCR-based array study identified 20-gene expression signature on the platform of 3072 genes in HCC patients. On the other hand, metastases in the liver tumor can be predicted by pathological variables such as vascular invasion, poor histological differentiation, and satellites [94]. In patients with HCC, the expression of mir-26a and mir-26b was reduced that assist in activation of inflammatory signaling pathways (nuclear factor κb and interleukin-6). Patients with lower mir-26 also exhibit a better response to interferon therapy, but the overall survival rate is reported to be short [98]. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) identification and counting helps to predict tumor stages, metastases and potential implications of therapeutic choices and clinical outcomes. However, a number of technical and methodologic drawbacks have limited the detection and interpretation of CTCs, due to their rare quantity (> 1/mL) [99]. This limitation can be circumvented by isolating the epithelial tumor cells (ISET) approach on the basis of their size. ISET is a simple and cheap 299 technology that is broadly used in clinical oncology for cytopathological diagnosis of tumor cells and peripheral blood samples [100]. Many governing bodies have suggested a clear distinction between tissue biomarkers and serum biomarkers. However, between tissue and serum, serum biomarkers (AFP, DCP, and AFP-L3) tend to represent false positive results thus are not a confirmatory diagnosis of HCC. On the other hand, tissue markers seem to be reliable as they might differentiate the early HCC from those of the cirrhotic tissue, preneoplastic lesions and another type of neoplasms [101]. A number of research studies on the genomics of HCC have revealed heat shock protein 70 [102], glypican-3 [102], telomerase reverse transcriptase [93], serine/threonine kinase 15 [93], telomerase reverse transcriptase (TERT), topoisomerase 2A (TOP2A), and platelet-derived growth factor (PDGF) receptor alpha [103], and phospholipase A2 [93]as the principle markers for early HCC detection. Antagonist-II (PIVKA-II) is another recent tumor growth protein marker that is brought by the deficiency of vitamin K. Patients who had no more than ten tumors, of at most 5 cm with PIVKA-II <400 mau/ml have a 5-year survival rate of 68.5% [ ]. Diagnosis of systemic inflammation including neutrophil-to- lymphocyte ratio (NLR) elevation is considered as a predictive marker of HCC recurrence [107]. A combination of elevated inflammatory serum marker C-reactive protein (CRP) >10 mg/l and lower albumin levels in HCC patients, represent an invasion of the vascular system, tumor growth, advanced stage and diminished recurrence-free rates of survival [108, 109]. Single-nucleotide polymorphism (SNP) array is a well appreciated and an evolving technology to study the genetic susceptibility to disease. SNP arrays provide a robust platform to define genome scale, somatic genetic changes in cancer. The use of 100K arrays helps in the completion of the large-scale analysis of various cancers [110]. Integrative genomic analysis approach (DNA microarray + SNP arrays) in molecular oncology research will help in identification of several novel oncogene and tumor suppressor genes that

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