**2. Vascular endothelial growth factor (VEGF) receptor signalling**

defined, such as hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcohol

The main reasons for the high rate of mortality are lack of diagnostic methods and limited treatment options for patients with advanced HCC. Surveillance programmes to identify patients with early HCC, such as by ultrasound sound screening and by serum alpha fetoprotein (AFP) levels, are not well implemented. Additionally, AFP levels are also dysregulated in benign liver diseases [3]. Some of the treatment options for HCC patients include surgical resection, liver transplantation (LTx), radiofrequency ablation (RFA), transarterial chemoembolization (TACE) and sorafenib. Surgery has a 5-year survival rate of 70% but unfortunately at the time of diagnosis, only 10–30% of patients are suitable for this option. The biggest risk post-surgery is that of recurrence. The 5-year recurrence rate in patients with early HCC is about 68% after surgery [4]. LTx is suggested to HCC patients with tumours within the Milan criteria (a single lesion ≤5 cm, or up to three lesions ≤3 cm each) and is associated with a 5-year overall survival (OS) rate of 75%. However, the limitation of LTx is shortage of organ donation [5]. RFA is another option for patients with early HCC (<3 cm) and its survival benefits are comparable to those with surgical resection. However, high costs of RFA and complications involving peritoneal bleeding hinder its use [6]. TACE is the standard of care for intermediate HCC with preserved liver function, and with no signs of macrovascular invasion or extrahepatic spread. It has reported a median survival of 34 months and a survival benefit at 1, 3, 5 and 7 year as 82%, 47%, 26% and 16%, respectively. However, TACE is a heterogeneous operating technique with variation in efficacy depending on the choice of chemotherapeutic agents used [7]. For advanced HCC patients, sorafenib is the only drug approved by the

**Trial name and no. Drug Target Ref** SHARP, NCT00105443 Sorafenib *versus* placebo VEGFR, PDGFR, Raf [8] Asia Pacific, NCT00492752 Sorafenib *versus* placebo VEGFR, PDGFR, Raf [15] STORM, NCT00692770 Sorafenib *versus* placebo VEGFR, PDGFR, Raf [17] REACH, NCT01140347 Ramucirumab *versus* placebo VEGFR-2 [23] --, NCT00699374 Sunitinib *versus* sorafenib VEGFR, PDGFR [26] LIGHT, NCT01009593 Linifanib *versus* sorafenib VEGFR and PDGFR [28] RESOURCE, NCT01774344 Regorafenib *versus* placebo VEGFR1-3, PDGFR-β, and FGFR-1 [30] EVOLVE-1, NCT01035229 Everolimus *versus* placebo mTOR [39] SILVER, NCT00355862 Sirolimus *versus* placebo mTOR [43] METIV-HCC, NCT01755767 Tivantinib *versus* placebo c-MET [47] JET-HCC, NCT02029157 Tivantinib *versus* placebo c-MET [47] CELESTIAL, NCT01908426 Cabozantinib *versus* placebo c-MET and VEGFR, PDGFR [48]


**Table 1.** Phase III clinical trials of molecular targets in HCC.

consumption, and non-alcoholic steatohepatitis (NASH) [2].

174 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

Angiogenesis is a critical step for tumour growth and metastasis. With HCC being a highly vascular tumour, controlling tumour-associated angiogenesis offers a promising approach to inhibiting tumour progression.

VEGF is the most well documented growth factor in angiogenesis. It exerts its effect by binding to its receptors, VEGF receptor 1 (VEGF-R1), VEGF-R2, and VEGF-R3, present on endothelial cells. VEGF secreted by tumour cells bind to its receptors and results in activation of signal transduction pathways promoting cell migration, proliferation, and survival of cancer cells leading to angiogenesis. VEGF overexpression is possibly induced by the hypoxic tumour environment, activation by epidermal growth factor (EGF) receptor (EGFR) and cyloonxygenase-2 signalling. Increased levels of VEGF, VEGFRs have been reported in HCC cell lines, tissue and serum of HCC patients. High levels of VEGF in HCC patients has been associated to poor OS and disease-free survival [9], vascular invasion [10] and portal vein emboli [11]. Additionally, Guo et al. [12] and colleagues reported poor prognosis for HCC patients with increased serum VEGF following TACE. VEGF is also more commonly expressed in HCV-associated HCC than in HBV-associated HCC providing clinical implications for different population of HCCs.

Other growth factors stimulating angiogenesis include fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR). Overexpression of either of these growth factors has also been associated to poor survival. There are four types of FGFRs (FGFR1, 2, 3, 4) and the PDGFR consist of PDGFRα and PDGFRβ [13, 14].

Sorafenib is currently the only drug approved for treatment of advanced HCC patients who cannot undergo TACE treatment. It is an orally active anti-angiogenic multi-kinase inhibitor. Several clinical studies have reported promising results. In the randomised phase III SHARP trial (ClinicalTrials.gov number NCT00105443), 400 mg of sorafenib twice daily, significantly increased the OS of advanced HCC patients (7.9 months *versus* 10.7 months) and the time to progression (TTP) (2.8 months *versus* 5.5 months) compared to the placebo group [8]. Similarly in another phase III Asia Pacific trial (NCT00492752), sorafenib increased the OS and TTP from 4.2 months to 6.5 months and from 1.4 months to 2.8 months, respectively [15]. The difference in the OS and TTP results in both studies could be due to patient HCC aetiology. The Asia Pacific trail had more HBV-associated HCC compared to the SHARP trial (73% *versus* 12%).

The use of sorafenib as an adjuvant after surgery or TACE remains doubtful. In a small retrospective study with 36 HCC patients, 12 patients received sorafenib post-surgery and the remaining 24 patients had surgery only. The group of patients who received sorafenib postsurgery had a significantly longer OS (37 months *versus* 30 months) and TTP (29 months *versus* 22 months) [16]. However, in the phase III placebo-controlled study (STORM, NCT00692770), which recruited 1602 patients from 28 countries, sorafenib as an adjuvant treatment after surgery/local ablation, did not affect time to recurrence or OS [17]. Similar findings were reported in the SPACE trial (NCT00855218). In this phase II trial Lencioni et al. [18] tested the efficacy of doxorubicin-eluting beads (DEB)-TACE plus sorafenib *versus* sorafenib in patients with intermediate HCC. The authors did not report a significant improvement in TTP following addition of sorafenib to DEB-TACE.

Linifanib is a multikinase inhibitor targeting VEGFR and PDGFR. In a phase II trial involving 44 HCC patients with unresectable or metastatic HCC, linifanib yielded a median OS of 9.7 months (compared to 10.4 months in patients with Child-Pugh class A hepatic function) [26]. In an open-label phase III trial (LIGHT, NCT01009593), Cainap et al. [27] compared linifanib with sorafenib treatment in advanced HCC. Both drugs had similar OS with 9.1 months for linifanib and 9.8 months for sorafenib. The median TTP was found to be 5.4 months and 4.0 months for linifanib and sorafenib respectively. However, linifanib caused more adverse side effects than sorafenib, implying sorafenib could be more safe

Emerging Targeted Therapies for Treatment of Hepatocellular Carcinoma (HCC)

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Regorafenib is a novel diphenylurea multikinase inhibiting VEGFR1-3, PDGFR-β, and FGFR-1. It has been approved for treatment of metastatic colorectal cancer after failure of oxaliplatin and irinotecan-based systemic chemotherapy and has also been approved for treatment of metastatic gastrointestinal stroma tumours after failure of imatinib and sunitinib. This year regorafenib was approved by the FDA as a second-line treatment for HCC. HCC patients not responding to sorafenib now have an option of FDA-approved regorafenib as a second line of treatment. This makes regorafenib the first FDA approved drug for treatment of liver cancer in almost a decade. In a small phase II study involving 36 advanced HCC patients who had progressed following sorafenib, regorafenib at 160 mg once daily in cycles of 3 weeks yielded a median TTP of 4.3 months median OS of 13.8 months. The side effects of regorafenib appeared similar to that of sorafenib such as fatigue, diarrhoea, hypertension and, hand–foot skin reaction [28]. A phase III trial of regorafenib (RESOURCE, NCT01774344) involving 573 patients from 21 countries evaluated the efficacy and safety of regorafenib in HCC patients and observed disease progression after systemic first-line treatment with sorafenib. Regorafenib treatment resulted in a survival benefit of 2.8 months compared to placebo (10.6 months *versus* 7.8 months). The median PSF for patients taking regorafenib was 3.1 months compared to 1.5 months for patients taking placebo. The overall response rate was 11% compared to 4% of patients taking placebo [29]. Following these promising results from the RESOURCE trail, regorafenib was approved by the FDA in April 2017 for the treatment of HCC patients who

The mitogen-activated protein kinase (MAPK) cascade consists of serine/threonine kinases, which converts extracellular molecules such as growth factors, hormones, and differentiation factors, into intracellular signals for regulating several cellular processes including proliferation, apoptosis and migration. The four core proteins kinases of this pathway include, Ras, Raf, MEK and ERK. The pathway is activated by binding of ligand to receptor tyrosine kinases (RTK). In the nucleus, phosphorylation of these four protein kinases regulates gene transcription. Around 58% of HCC cases have activated MAPK pathway with Ras, MEK, ERK and MAPK up regulated in 33%, 40%, 50% and 50% of HCC patients, respectively [30]. This pathway has also been shown to be activated by hepatitis virus infections. Dysregulation of this pathway by hepatitis B virus X protein has contributed to loss of function of the tumour

than linifanib.

have previously been treated with sorafenib.

**3. RAF/MEK/ERK pathway**

Bevacizumab is an anti-VEGF monoclonal antibody that has demonstrated improved efficacy in patients with unresectable HCC. Treatment with bevacizumab at 5–10 mg/kg produced partial response (PR) in 14% and disease control rates (DCR) in 56% of patients. A phase II trial of bevacizumab with capecitabine and oxaliplatin (chemotherapeutic drugs) also showed encouraging results with a median progression-free survival (PFS) of 6.8 months, and a median OS of 9.8 months. Twenty three patients had stable disease with overall 77.5% disease control rate and eight patients produced (PR) [19]. Hsu et al. [20] investigated the combination of bevacizumab plus capecitabine in a phase II study yielding median PFS and OS of 2.7 and 5.9 months, respectively.

Ramucirumab is another example of monoclonal antibody targeting VEGFR-2. The above mentioned bevacizumab targets the proangiogenic factor VEGF while ramucirumab blocks the receptor. In a phase II study involving advanced HCC patients, ramucirumab monotherapy yielded a disease control rate (DCR) of 50%, PFS of 4.0 months and OS of 12 months [21]. The promising results from this study lead to a phase III trial (REACH, NCT01140347) of ramucirumab monotherapy in advanced HCC patients (post-sorafenib). Although ramucirumab did not improve OS, interestingly in a sub-group of HCC patients with AFP base line levels ≥400 ng/mL, ramucirumab significantly enhanced the OS [22].

Sunitinib is an orally administered multi-kinase inhibitor with activity against various kinases including VEGFR and PDGFR. It has been approved for treatment of renal cell carcinoma (RCC), and imatinib-resistant gastrointestinal stromal tumours (GIST). However, it is not considered for HCC patients due to its high toxicity. In two phase II studies of sunitinib, 50 mg daily of sunitinib orally, 4 weeks on and 2 weeks off, both Barone et al. [23] and Faivre et al. [24] reported high toxicity. Barone et al. [23] observed treatment-related deaths in 18% of patients and with PR in 12% of patients. Median TTP was 2.8 months and median OS was 5.8 months. Faivre et al. [24] reported 10% deaths related to treatment and 80% patients experienced grade 3/4 adverse effects because of which the study could not proceed to the second phase and was terminated [23, 24]. Similarly, a phase III trial (NCT00699374) comparing sunitinib to sorafenib was discontinued. Patients were administered 37.5 mg of sunitinib once daily or 400 mg of sorafenib twice a day but a majority of patients experienced adverse effects such as thrombocytopenia and neutropenia. Additionally, sunitinib did not show a better OS than sorafenib [25].

Linifanib is a multikinase inhibitor targeting VEGFR and PDGFR. In a phase II trial involving 44 HCC patients with unresectable or metastatic HCC, linifanib yielded a median OS of 9.7 months (compared to 10.4 months in patients with Child-Pugh class A hepatic function) [26]. In an open-label phase III trial (LIGHT, NCT01009593), Cainap et al. [27] compared linifanib with sorafenib treatment in advanced HCC. Both drugs had similar OS with 9.1 months for linifanib and 9.8 months for sorafenib. The median TTP was found to be 5.4 months and 4.0 months for linifanib and sorafenib respectively. However, linifanib caused more adverse side effects than sorafenib, implying sorafenib could be more safe than linifanib.

Regorafenib is a novel diphenylurea multikinase inhibiting VEGFR1-3, PDGFR-β, and FGFR-1. It has been approved for treatment of metastatic colorectal cancer after failure of oxaliplatin and irinotecan-based systemic chemotherapy and has also been approved for treatment of metastatic gastrointestinal stroma tumours after failure of imatinib and sunitinib. This year regorafenib was approved by the FDA as a second-line treatment for HCC. HCC patients not responding to sorafenib now have an option of FDA-approved regorafenib as a second line of treatment. This makes regorafenib the first FDA approved drug for treatment of liver cancer in almost a decade. In a small phase II study involving 36 advanced HCC patients who had progressed following sorafenib, regorafenib at 160 mg once daily in cycles of 3 weeks yielded a median TTP of 4.3 months median OS of 13.8 months. The side effects of regorafenib appeared similar to that of sorafenib such as fatigue, diarrhoea, hypertension and, hand–foot skin reaction [28]. A phase III trial of regorafenib (RESOURCE, NCT01774344) involving 573 patients from 21 countries evaluated the efficacy and safety of regorafenib in HCC patients and observed disease progression after systemic first-line treatment with sorafenib. Regorafenib treatment resulted in a survival benefit of 2.8 months compared to placebo (10.6 months *versus* 7.8 months). The median PSF for patients taking regorafenib was 3.1 months compared to 1.5 months for patients taking placebo. The overall response rate was 11% compared to 4% of patients taking placebo [29]. Following these promising results from the RESOURCE trail, regorafenib was approved by the FDA in April 2017 for the treatment of HCC patients who have previously been treated with sorafenib.
