**New Molecular Biomarkers Candidates for the Development of Multiparametric Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy**

Annalucia Serafino and Pasquale Pierimarchi *Institute of Translational Pharmacology, National Research of Council of Italy (CNR), Rome Italy* 

#### **1. Introduction**

72 Hepatocellular Carcinoma – Basic Research

World Health Organization. Mortality database. (2010) www.who.int/whosis/en. Accessed

ISSN 0944-1174

February 2010

hepatitis C. *Journal of Gastroentelorogy,* Vol.45, No.9, (September 2010) pp.960-7.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, showing a rapid progressive clinical course, poor response to pharmacological treatment and a severe prognosis (Colombo, 2003; Sherlock & Dooley, 2002). HCC generally develops from chronic liver injury, which leads to inflammation, hepatocyte regeneration, liver matrix remodeling, fibrosis, and finally, cirrhosis. The main risk factors for HCC are hepatitis B (HBV) or C virus (HCV) infection, alcohol-induced liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), primary biliary cirrhosis and exposure to environmental carcinogens (particularly aflatoxin), and genetic metabolic diseases. (Chuang et al., 2009; Di Bisceglie, 1995; Kato et al, 1994; Malaguarnera et al., 2006; Malaguarnera et al., 2009; Seitz & Becker, 2007; Takano et al., 1995). Obesity has also been identified as an independent risk factor for developing HCC in patients with alcoholic or cryptogenic cirrhosis (Nair et al, 2002). Actually, HCV-related cirrhosis is considered the major risk factor since many HCV chronically infected patients remain asymptomatic for a long period, with liver cirrhosis developing after approximately 30 years (Yano et al., 1996; Poynard et al., 1997). The lack of predictive markers that makes unforeseeable the insurgence of liver cirrhosis in chronic HCV patients may also contribute to HCC late diagnosis, progression and poor prognosis. Currently, alpha-fetoprotein (AFP) is the most common marker for early malignancy used in clinical practice, in combination with hepatic echography, to detect HCC in patients suffering from cirrhosis. Nevertheless, most episodes of AFP elevation were transient and closely correlated with the presence of bridging hepatic necrosis, without subsequent development of HCC (Liaw et al., 1986). Since an early diagnosis of HCC is extremely important in improving the survival of patients, the identification of new and more reliable biological markers of HCC insurgence, recurrence and metastasis is essential for the proper management of this malignancy. Once hepatic cancer develops, one of the main reasons for the high mortality rate in patients with HCC is the lack of effective treatment options, especially for those with advanced disease. Although surgery and percutaneous ablation can achieve long-

New Molecular Biomarkers Candidates for the Development of Multiparametric

**2.1 Main markers used for the screening for early malignancy** 

incorrect diagnosis.

prognostic value; iv) in HCC staging.

differentiated tumors (Miyaaki et al., 2007).

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 75

Growth Factor receptor (EGFR), and numerous other molecules (**Table 1**), are used as diagnostic/prognostic aid for HCC and for staging (Malaguarnera et al., 2010; Mann et al., 2007; Qin & Tang, 2004). Nevertheless, each existing marker alone is poorly specific to predict the disease and most markers are not related to each other. Currently the absolute positive and negative serological and/or immunohistochemical markers for HCC are still lacking, and even those selected for high sensitivity and specificity do not exhibit an universal diagnostic/prognostic value. Therefore, in the last years, a great number of studies has been dedicated to the discovery and validation of more specific biomarkers for HCC, driven by the idea that the simultaneous screening for multiple markers should greatly reduce errors from false-negative results, which significantly contribute to an

Alpha-fetoprotein (AFP) is a 70 kDa glycoprotein that is physiologically synthesized by the embryonic liver cells of the yolk sac and fetal intestinal tract. The AFP is expressed in hepatocytes and endodermal cells of the yolk sac during fetal life and its expression is reduced after birth, with very low levels in adults. The AFP levels rise in hepatocyte regeneration, hepatocarcinogenesis, and embryonic carcinomas. Its biological function in embryo- and carcinogenesis and in adult organisms is still not well identified, but, due to its structural similarity with albumin, a function as a carrier for several ligands, including bilirubin, steroids, fatty acids and various drugs has been proposed (Mizejewski, 2002; Terentiev & Moldogazieva, 2006). Recognized as a tumor-associated fetal protein, AFP has long been considered the 'gold-standard' among tumor markers, and, it has been purified, characterized, cloned and sequenced for use in the clinical diagnostic. It is principally used: i) for the screening and diagnosis of hepatocarcinoma in patients at risk of developing HCC, in combination with hepatic ultrasonography; ii) as a marker of tumor progression in HCC patients with high levels of AFP; iii) for monitoring the response to treatment during the follow-up of HCC patients, with a

*Lens culinaris* agglutinin-reactive AFP (AFP-L3) is one of the AFP isoform which exhibits an elevated affinity for *Lens culinaris* agglutinin (LCA). This AFP isoform, that has α1→6 fucose residues on N-acetylglucosamine at reducing end, seems to be exclusively expressed by cancer cells, and is considered a more specific marker for HCC (Oka et al., 2001; Sato et al., 1993). AFP-L3 should be used as a supplemental test in patients with elevated total AFP. It has been reported as a potential indicator of a poor prognosis, since increasing AFP-L3 levels seem to correlate with progression from moderately differentiated to poorly

Des-c-carboxy prothrombin (DCP) or prothrombin induced by vitamin K absence (PIVKA) is an abnormal prothrombin derived by an acquired defect in the post-translational carboxylation of the prothrombin precursor in HCC cells (Ono et al., 1990). DCP derives by a reduced activity of gamma-glutamyl carboxylase, highly expressed in the liver; this reduced activity is attributed to defective gene expression in HCC patients (Grizzi et al., 2007). DCP is a HCC marker more specific than AFP since other liver diseases are not

term control in some patients with early HCC, recurrence rates are high, approximately 50% at 3 years (Mulcahy, 2005). Furthermore, due to the asymptomatic nature of early HCC, lack of awareness and poorly defined screening strategies, approximately 80% of patients present with advanced or unresectable disease (Thomas & Abbruzzese, 2005). These patients generally have a very poor prognosis and treatments, such as transarterial chemoembolization, intra-arterial or systemic chemotherapy, radiotherapy, immunotherapy or hormonal therapy, are mainly used as palliative, with a 5-year relative survival rate of only 7% (Bosch et al., 2004).

The lack of effective and well-tolerated treatments for advanced HCC highlights the need for innovative approaches for diagnosis, prognosis and therapy for hepatic cancer. In this context, multiparametric platforms allowing simultaneous detection of multiple serological and immunohistochemical markers for HCC insurgence, recurrence and metastasis would represent a high-performance technological tools useful not only for diagnosis and prognosis, but also for improving the clinical management of HCC patients, allowing us, in the near future, to design therapies adapted to the aggressiveness of each individual tumor.

Starting from this background, in this chapter will be collected some of the data existing in literature on the main serological and immunohistochemical biomarkers for HCC diagnosis, prognosis and target therapy, also focusing on new molecules which might be attractive candidates for improvement of the diagnostic/therapeutic approaches. In particular will be covered the following topics : 1) Some new candidates recently proposed as potential biological markers of HCC insurgence, recurrence and metastasis, that could be useful for early diagnosis of this malignancy and improve patient's prognosis; 2) Some signaling pathways which deregulation or constitutive activation have been demonstrated to have a role in HCC insurgence and progression and that could be of interest for therapeutic perspectives, since targeting them may contribute to prevent tumorigenesis or achieve tumor reversion; 3) Molecules over-expressed in late stages of cancer or in the metastatic diseases that should be considered a good targets for therapy and drug delivery.
