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

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 prognostic value; iv) in HCC staging.

*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 differentiated tumors (Miyaaki et al., 2007).

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

New Molecular Biomarkers Candidates for the Development of Multiparametric

**material mainly analyzed**

Serum

Tissue/Serum

Tissue

Tissue/Serum

**HCC biomarker Biological** 

(AFP) Serum

prothrombin (DCP) Serum

Glypican-3 Tissue/Serum

Growth Factor (VEGF) Tissue/Serum

Factor β1 (TGF-β1) Tissue/Serum

Factor (HGF) Serum

p53 antibodies Serum

Survivine Tissue

Alpha-fetoprotein

*Lens culinaris*  agglutinin-reactive AFP (AFP-L3)

Des-c-carboxy

Squamous cell carcinoma antigen

Vascular Endothelial

Transforming Growth

Hepatocyte Growth

Epidermal Growth Factor Receptor (EGFR)

Nerve Growth Factor (NGF) and its highaffinity receptor

trkANGF

(SCCA)

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 77

Early diagnosis; HCC staging; monitoring the response to treatment during the followup of patients with HCC

Early diagnosis and prognosis; progression from moderately differentiated to poorly differentiated tumors

Early diagnosis and prognosis (more specific than AFP); related to early portal vein invasion and metastasis

Early diagnosis; detection of micro-metastasis in tissues; large-scale screening of serum

HCC early diagnosis; useful for discriminating malignant from benign hepatic lesions

HCC prognosis; predictive of invasion and metastatization

HCC progression; prognostic factor for reduced survival in

HCC prognosis; predictive of HCC recurrence and metastasis after hepatic

HCC prognosis; predictive of reduced survival of HCC

HCC prognosis (poor differentiation); associated with a poor prognosis of HCC

HCC prognosis; poor prognosis following resection of HCC; associated with reduced disease-free survival.

HCC prognosis and progression; predictive of progression of liver fibrosis

towards HCC

Table 1. List of the main biomarkers useful for HCC diagnosis/prognosis

Golgi protein-73 Serum HCC early diagnosis Malaguarnera et al., 2010

in patients at risk

of HCC cells

resection

patients

patients

patients with HCC

**Main use/s References**

Terentiev & Moldogazieva, 2006; Mizejewski, 2002; Malaguarnera et al., 2010; Marrero et al., 2010

Oka et al., 2001; Sato et al., 1993; Malaguarnera et al., 2010; Marrero et al., 2004

Malaguarnera et al., 2010

Malaguarnera et al., 2010

Suzuki et al., 1996; Mise et al., 1996; Li et al., 1999; Qin &Tang, 2004; Mann et al., 2007; Malaguarnera et al.,

Ito et al., 1990; Grizzi et al., 2007; Mann et al., 2007; Okumoto et al., 2004; Tsai et

Breuhan et al., 2006; Yamagamim et al., 2002; Wu et al., 2006; Malaguarnera et

Daveau et al., 2003; Ito et al., 2001; Mann et al., 2007

Malaguarnera et al., 2010

Fields et al., 2004; Mann et

Malaguarnera et al., 2010

2010

al., 1997

al., 2010

al., 2007

Rasi et al, 2007;

Grizzi et al., 2007; Malaguarnera et al., 2010; Marrero et al., 2004

associated to an increase of DCP serum levels. Apart its diagnostic significance, increased DCP levels may also have a prognostic value, being often related to early portal vein invasion and metastatization by cancer cells.

#### **2.2 Some growth factors used as diagnostic/prognostic aid**

Vascular Endothelial Growth Factor (VEGF), plays an crucial role in angiogenesis and is highly expressed in various human cancers (Brown et al., 1993; Mattern et al., 1996; Toi et al., 1994), including HCC (Mise et al., 1996; Suzuki et al., 1996). Specifically, VEGF levels are higher in HCC patients than in patients suffering from chronic hepatitis, and its expression is more elevated in advanced HCC as compared to early HCC. High serum VEGF levels are associated with tumors with portal vein emboli, poor-encapsulated tumors, microscopic vein invasion, and recurrence in HCC patients (Li et al., 1999). It is considered as a possible marker for predicting invasion and metastatization of HCC, and in general, of tumor aggressiveness.

Transforming Growth Factor β1 (TGF-β1), is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is an important mediator of control of liver cell proliferation and replication. In normal liver tissues, TGF-β1 is produced by nonparenchymal cells (Kupffer cells, storing cells, and endothelial cells), but not by hepatocytes. Conversely, transcription of TGF-β1 gene is activated in human HCC tissues and is higher in patients with advancing histological aggressiveness (Ito et al., 1990). Moreover, TGF-β1 serum levels are reported to be increased in HCC patients (Grizzi et al., 2007). TGF-β1 has been proposed as a possible prognostic factor for reduced survival in patients with HCC (Mann et al., 2007; Okumoto et al., 2004; Tsai et al., 1997)

Hepatocyte growth factor (HGF) is a cytokine with a wide range of effects, including liver regeneration for protection and/or repair of different organs, including kidney, lung, and cardiovascular system (Birchmeier et al., 1998). It promotes proliferation in normal hepatocyte and in hepatocellular carcinoma cells (Breuhan et al., 2006) through expression of its high-affinity tyrosine kinase receptor (Met/*HGF*-R). HGF is detected in the serum from patients suffering from hepatic chronic disease and its serum values seems to be correlated with a worsening of liver disease (Breuhan et al., 2006). Increased HGF serum levels in cirrhotic patients is an indicator of HCC development (Yamagamim et al., 2002). It is considered a prognostic marker since elevated HGF serum levels, are predictive of HCC recurrence and metastasis after hepatic resection (Wu et al., 2006).

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases, EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). These receptors bind ligands of the EGF family, including EGF, TGF-α and heparin-binding EGF. EGFR has been found to be overexpressed in poorly differentiated HCC and primarily in patients with early tumor recurrence (Daveau et al., 2003; Ito et al., 2001) EGFR tissue overexpression is also correlated with high proliferating activity, advanced stage, the presence of intrahepatic metastasis and poor disease-free survival following resection (Ito et al., 2001). EGFR strongly reflects the biological aggressiveness of HCC and might be considered a possible prognostic factor of reduced survival of HCC patients.

associated to an increase of DCP serum levels. Apart its diagnostic significance, increased DCP levels may also have a prognostic value, being often related to early portal vein

Vascular Endothelial Growth Factor (VEGF), plays an crucial role in angiogenesis and is highly expressed in various human cancers (Brown et al., 1993; Mattern et al., 1996; Toi et al., 1994), including HCC (Mise et al., 1996; Suzuki et al., 1996). Specifically, VEGF levels are higher in HCC patients than in patients suffering from chronic hepatitis, and its expression is more elevated in advanced HCC as compared to early HCC. High serum VEGF levels are associated with tumors with portal vein emboli, poor-encapsulated tumors, microscopic vein invasion, and recurrence in HCC patients (Li et al., 1999). It is considered as a possible marker for predicting invasion and metastatization of HCC, and

Transforming Growth Factor β1 (TGF-β1), is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is an important mediator of control of liver cell proliferation and replication. In normal liver tissues, TGF-β1 is produced by nonparenchymal cells (Kupffer cells, storing cells, and endothelial cells), but not by hepatocytes. Conversely, transcription of TGF-β1 gene is activated in human HCC tissues and is higher in patients with advancing histological aggressiveness (Ito et al., 1990). Moreover, TGF-β1 serum levels are reported to be increased in HCC patients (Grizzi et al., 2007). TGF-β1 has been proposed as a possible prognostic factor for reduced survival in patients with HCC

Hepatocyte growth factor (HGF) is a cytokine with a wide range of effects, including liver regeneration for protection and/or repair of different organs, including kidney, lung, and cardiovascular system (Birchmeier et al., 1998). It promotes proliferation in normal hepatocyte and in hepatocellular carcinoma cells (Breuhan et al., 2006) through expression of its high-affinity tyrosine kinase receptor (Met/*HGF*-R). HGF is detected in the serum from patients suffering from hepatic chronic disease and its serum values seems to be correlated with a worsening of liver disease (Breuhan et al., 2006). Increased HGF serum levels in cirrhotic patients is an indicator of HCC development (Yamagamim et al., 2002). It is considered a prognostic marker since elevated HGF serum levels, are predictive of HCC

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases, EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). These receptors bind ligands of the EGF family, including EGF, TGF-α and heparin-binding EGF. EGFR has been found to be overexpressed in poorly differentiated HCC and primarily in patients with early tumor recurrence (Daveau et al., 2003; Ito et al., 2001) EGFR tissue overexpression is also correlated with high proliferating activity, advanced stage, the presence of intrahepatic metastasis and poor disease-free survival following resection (Ito et al., 2001). EGFR strongly reflects the biological aggressiveness of HCC and might be considered a possible prognostic factor of

invasion and metastatization by cancer cells.

in general, of tumor aggressiveness.

reduced survival of HCC patients.

**2.2 Some growth factors used as diagnostic/prognostic aid** 

(Mann et al., 2007; Okumoto et al., 2004; Tsai et al., 1997)

recurrence and metastasis after hepatic resection (Wu et al., 2006).


Table 1. List of the main biomarkers useful for HCC diagnosis/prognosis

New Molecular Biomarkers Candidates for the Development of Multiparametric

an attractive target for a new therapeutic approach.

legend). hep: hepatocytes.

trkA

trkA

trkA

**– –** 

**+\* ±** 

**++\* ++**

**Health** NGF

**HCC** NGF

**CIRR** NGF

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 79

possible use of NGF, not only as a marker of progression and transformation, but also as

Fig. 1. NGF distribution (red hue) in tissues from healthy donors (**A**) and from patients suffering from HCC (**B**). Green hue represents the auto-fluorescence used to visualize liver tissue morphology. **A1** and **B1**: Images of H&E stained sections close to that used for immunohistochemistry. Differently coloured arrows indicate the different cell types (see

> **– –**

**± ±** 

**++\*\* ++\*\*** 

IF: immunofluorescence labelling; IG: immunogold labelling; nd: not determined. \*Immunoreaction mainly localized on cytoplasmic vesicles and endoplasmic reticulum. \*\*Immunoreaction mainly localized in the portion of cells near the ductal lumen.

**Marker Hep Bec Ec Ssc Lymph Kpf**

nd nd

nd nd

> **+ +**

**–** 

**±** 

**+**  nd

**+**  nd

> **+ +**

**–**

**+** 

**± +** 

**– –** 

**+ ±**

**++ +** 

Table 2. Expression of NGF and trkANGF in liver cell types from healthy donors and from patients with cirrhosis and HCC. Hep: Hepatocyte; Bec, Biliary epithelial cells; Ec, Endothelial cells; Ssc, Spindle-shaped cells; Lymph, Lymphocytes; Kpf, Kupffer cells.

#### **2.3 The Nerve Growth Factor (NGF): A new candidate proposed as potential histological/serum marker for HCC diagnosis and prognosis**

In the last years, some new candidates have been proposed as potential biological markers of HCC insurgence, recurrence and metastasis, that could be therefore useful for early diagnosis of this malignancy and improve patient's prognosis. In particular we focus on our recently published data that suggested an involvement of Nerve Growth Factor (NGF) in liver tissue remodelling processes and HCC progression, describing the correlation between NGF tissue distribution and serum levels in patients suffering from cirrhosis and/or HCC (Rasi et al, 2007).

NGF is a prototypical member of neurotrophin family essential for survival, differentiation, and maintenance of neuronal cells in the central and peripheral nervous system (Levi-Montalcini, 1987). In recent years, many findings have indicated that NGF could also have a role outside the central and peripheral nervous system. In particular, it may be involved in lung and skin tissue repair (Micera et al., 2001) as well as in allergic inflammation and fibrosis (Micera et al., 2003). Increased levels of circulating NGF were observed in several autoimmune, chronic inflammatory and fibrotic disorders (Aloe & Tuveri, 1997; Bonini et al., 1999). Numerous data also indicate that NGF is involved in tumor growth, invasion and metastasis (Bold et al., 1995; Descamps et al., 1998; Djakiew et al., 1991; Koizumi et al., 1998; McGregor et al., 1999; Oelmann et al., 1995; Pflug et al., 1992; Revoltella & Butler, 1980; Sortino et al., 2000).The NGF effects are mediated by two types of receptor: the high-affinity receptor trkANGF, specific for NGF, and the low-affinity glycoprotein receptor p75NTR, also binding other neurotrophins (Meakin & Shooter, 1992). Most of the biological activities elicited by NGF are mediated by binding to the trkANGF receptor (Sofroniew et al., 2001).

In the 2007 (Rasi et al., 2007), we provided immunohistochemical evidence that NGF and its high-affinity receptor trkANGF are over expressed in patients suffering from HCC (**Fig. 1**) and to a greater extent from HCC with cirrhosis (**Fig. 2B, C**). Specifically, in HCC tissues NGF was detectable in a high number of cells (**Table 2**), at different levels of intensities depending on the patient, but never in normal liver tissue. Interestingly NGF and trkANGF were negative in liver specimens from patients with cirrhosis undergoing transplantation (Child-C) but without HCC (**Fig. 2A**), while they were markedly positive in patients with cirrhosis that had evolved into HCC, already at early staging (Child- Pugh A, **Fig. 2B**).

Transmission electron microscopy, after immunogold labeling, showed that in hepatocytes of HCC tissue and, at higher extent, of cirrhotic tissue from the same liver, NGF mainly localized on cytoplasmic vesicles, free in the cytoplasm and along endoplasmic reticulum (**Fig. 3**), indicating that it might be actively produced by the hepatocytes constituting the cirrhotic/HCC tissues. The evidence that hepatocytes in HCC and cirrhotic tissues from the same liver produce NGF and express its receptor suggested that NGF may act by both autocrine and paracrine mechanisms, as a messenger molecule in the cross-talk between different cell types. Moreover, in sera obtained from patients with documented cirrhosis, HCC, or both, circulating NGF levels elevated 25-fold over the normal (range 73-520pg/ml, compared to a mean of 20pg/ml in healthy donors) were recorded (**Fig. 4**). These elevated circulating NGF levels, as well as the tissue distribution of NGF and its receptor strongly support a correlation between NGF activity and the progression of liver fibrosis towards HCC. This open up an interesting perspective for the

In the last years, some new candidates have been proposed as potential biological markers of HCC insurgence, recurrence and metastasis, that could be therefore useful for early diagnosis of this malignancy and improve patient's prognosis. In particular we focus on our recently published data that suggested an involvement of Nerve Growth Factor (NGF) in liver tissue remodelling processes and HCC progression, describing the correlation between NGF tissue distribution and serum levels in patients suffering from cirrhosis and/or HCC

NGF is a prototypical member of neurotrophin family essential for survival, differentiation, and maintenance of neuronal cells in the central and peripheral nervous system (Levi-Montalcini, 1987). In recent years, many findings have indicated that NGF could also have a role outside the central and peripheral nervous system. In particular, it may be involved in lung and skin tissue repair (Micera et al., 2001) as well as in allergic inflammation and fibrosis (Micera et al., 2003). Increased levels of circulating NGF were observed in several autoimmune, chronic inflammatory and fibrotic disorders (Aloe & Tuveri, 1997; Bonini et al., 1999). Numerous data also indicate that NGF is involved in tumor growth, invasion and metastasis (Bold et al., 1995; Descamps et al., 1998; Djakiew et al., 1991; Koizumi et al., 1998; McGregor et al., 1999; Oelmann et al., 1995; Pflug et al., 1992; Revoltella & Butler, 1980; Sortino et al., 2000).The NGF effects are mediated by two types of receptor: the high-affinity receptor trkANGF, specific for NGF, and the low-affinity glycoprotein receptor p75NTR, also binding other neurotrophins (Meakin & Shooter, 1992). Most of the biological activities elicited by NGF are mediated by binding to the trkANGF receptor (Sofroniew et al., 2001).

In the 2007 (Rasi et al., 2007), we provided immunohistochemical evidence that NGF and its high-affinity receptor trkANGF are over expressed in patients suffering from HCC (**Fig. 1**) and to a greater extent from HCC with cirrhosis (**Fig. 2B, C**). Specifically, in HCC tissues NGF was detectable in a high number of cells (**Table 2**), at different levels of intensities depending on the patient, but never in normal liver tissue. Interestingly NGF and trkANGF were negative in liver specimens from patients with cirrhosis undergoing transplantation (Child-C) but without HCC (**Fig. 2A**), while they were markedly positive in patients with cirrhosis that had evolved into HCC, already at early staging (Child- Pugh A, **Fig. 2B**).

Transmission electron microscopy, after immunogold labeling, showed that in hepatocytes of HCC tissue and, at higher extent, of cirrhotic tissue from the same liver, NGF mainly localized on cytoplasmic vesicles, free in the cytoplasm and along endoplasmic reticulum (**Fig. 3**), indicating that it might be actively produced by the hepatocytes constituting the cirrhotic/HCC tissues. The evidence that hepatocytes in HCC and cirrhotic tissues from the same liver produce NGF and express its receptor suggested that NGF may act by both autocrine and paracrine mechanisms, as a messenger molecule in the cross-talk between different cell types. Moreover, in sera obtained from patients with documented cirrhosis, HCC, or both, circulating NGF levels elevated 25-fold over the normal (range 73-520pg/ml, compared to a mean of 20pg/ml in healthy donors) were recorded (**Fig. 4**). These elevated circulating NGF levels, as well as the tissue distribution of NGF and its receptor strongly support a correlation between NGF activity and the progression of liver fibrosis towards HCC. This open up an interesting perspective for the

**2.3 The Nerve Growth Factor (NGF): A new candidate proposed as potential** 

**histological/serum marker for HCC diagnosis and prognosis** 

(Rasi et al, 2007).

possible use of NGF, not only as a marker of progression and transformation, but also as an attractive target for a new therapeutic approach.

Fig. 1. NGF distribution (red hue) in tissues from healthy donors (**A**) and from patients suffering from HCC (**B**). Green hue represents the auto-fluorescence used to visualize liver tissue morphology. **A1** and **B1**: Images of H&E stained sections close to that used for immunohistochemistry. Differently coloured arrows indicate the different cell types (see legend). hep: hepatocytes.


IF: immunofluorescence labelling; IG: immunogold labelling; nd: not determined.

\*Immunoreaction mainly localized on cytoplasmic vesicles and endoplasmic reticulum. \*\*Immunoreaction mainly localized in the portion of cells near the ductal lumen.

Table 2. Expression of NGF and trkANGF in liver cell types from healthy donors and from patients with cirrhosis and HCC. Hep: Hepatocyte; Bec, Biliary epithelial cells; Ec, Endothelial cells; Ssc, Spindle-shaped cells; Lymph, Lymphocytes; Kpf, Kupffer cells.

New Molecular Biomarkers Candidates for the Development of Multiparametric

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 81

Fig. 3. NGF distribution in cirrhotic tissue from patient with HCC by immunogold labelling. Transmission electron micrographs of hepatocytes showing positive immunogold reaction on cytoplasmic vesicles (black arrows), free in the cytoplasm (double pointed arrows) and along endoplasmic reticulum (white arrows). hep: hepatocytes; ER: endoplasmic reticulum.

**Normal Cirrhosis HBV Cirrhosis HCV Cirrhosis**

Fig. 4. Bar diagram illustrating the circulating NGF levels, determined by ELISA test , in patients with documented cirrhosis/HCC. NGF amounts, reported with regard to the etiology, is calculated either as total mean values SD (all patients examined) or as mean values for Child-Pugh class A (score = 5-6), for Child-Pugh class B (score = 7-9) and for Child-Pugh class C (score = 10-15). As a control, mean value SD of circulating NGF levels

**Total Child-Pugh A Child-Pugh B Child-Pugh C**

**HCV/alcohol** 

**Cirrhosis alcohol** 

**Cirrhosis-HCC** 

Scale bars = **A**: 2*μm*; **B**: 100*nm*.

**0**

from some healthy individuals is also reported

**100**

**200**

**300**

**NGF (pg/ml)**

**400**

**500**

**600**

Fig. 2. NGF and trkANGF distribution in liver specimens. **A**: tissues obtained before transplantation from patients with cirrhosis but without HCC (Child-Pugh C)**. B:** tissue from patient also suffering from cirrhosis with HCC. Red hue represents the NGF or trkA immunostaining; green hue represents the auto-fluorescence used to visualize liver tissue morphology. **C**: Images of H&E stained sections close to that used for immunohistochemistry. Differently coloured arrows indicate the different cell types (see legend). hep: hepatocytes; bec: biliary epithelial cells; ssc: spindle-shaped cells

Fig. 2. NGF and trkANGF distribution in liver specimens. **A**: tissues obtained before

patient also suffering from cirrhosis with HCC. Red hue represents the NGF or trkA immunostaining; green hue represents the auto-fluorescence used to visualize liver tissue

immunohistochemistry. Differently coloured arrows indicate the different cell types (see

morphology. **C**: Images of H&E stained sections close to that used for

legend). hep: hepatocytes; bec: biliary epithelial cells; ssc: spindle-shaped cells

transplantation from patients with cirrhosis but without HCC (Child-Pugh C)**. B:** tissue from

Fig. 3. NGF distribution in cirrhotic tissue from patient with HCC by immunogold labelling. Transmission electron micrographs of hepatocytes showing positive immunogold reaction on cytoplasmic vesicles (black arrows), free in the cytoplasm (double pointed arrows) and along endoplasmic reticulum (white arrows). hep: hepatocytes; ER: endoplasmic reticulum. Scale bars = **A**: 2*μm*; **B**: 100*nm*.

Fig. 4. Bar diagram illustrating the circulating NGF levels, determined by ELISA test , in patients with documented cirrhosis/HCC. NGF amounts, reported with regard to the etiology, is calculated either as total mean values SD (all patients examined) or as mean values for Child-Pugh class A (score = 5-6), for Child-Pugh class B (score = 7-9) and for Child-Pugh class C (score = 10-15). As a control, mean value SD of circulating NGF levels from some healthy individuals is also reported

New Molecular Biomarkers Candidates for the Development of Multiparametric

Fig. 5. Cellular signaling pathways implicated in the pathogenesis of HCC and therapeutics targeting molecular components of these pathway, useful for HCC treatment. EGF, epidermal growth factor; EGFR, EGF receptor; IGFR, insulin-like growth factor receptor; PDGFR, platelet-derived growth factor receptor; WNT, family of secreted glycoproteins that act as ligands of the Frizzled receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Details and main function/s of AKT, BAD, c-JUN, c-MYC, DSH, ERK 1/2, FOXO, GSK-3β, GBP, MEK 1/2, mTOR, PI3K, PTEN, p53, RAF, RAS, β-catenin, are reported in **Table 3**. Adapted from Whittaker

signaling pathway (Gonsalves et al., 2011; Luu et al., 2004; Moon et al., 2004).

Besides the mentioned pathways, directly or indirectly involved in the angiogenic signaling, in the last years numerous studies demonstrated that the WNT/β-catenin pathway is actively involved in initiation and progression of several kinds of human cancers, including HCC (De La et al., 1998; Polakis, 1999; Waltzer & Bienz, 1999) and growing attention has been given to new anti-tumor therapeutic approaches targeting components of this

et al., 2010

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 83
