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

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

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

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

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

levels ≥400 ng/mL, ramucirumab significantly enhanced the OS [22].

addition of sorafenib to DEB-TACE.

176 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

OS of 2.7 and 5.9 months, respectively.

better OS than sorafenib [25].

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 suppressor p53 [31]. HCV infection has led to anti-apoptotic effect also following activation by Ras/Raf/Mek/Erk signalling [32].

everolimus was next investigated in advanced HCC patients who did not respond to sorafenib. In a phase III, randomised, double-blind study (EVOLVE-1, NCT01035229) everolimus did not show improvement in OS (7.6 months with everolimus, 7.3 months with placebo). In a separate phase II study (NCT01005199), patients with advanced HCC were compared to those administered sorafenib alone (800 mg) or with everolimus (5 mg) [38]. The results were not encouraging and combination of sorafenib with everolimus did not improve efficacy compared to sorafenib alone with median PFS (6.6 months *versus* 5.7 months), TTP (7.6 months *versus* 6.3 months), and OS (10 months *versus* 12 months) were similar in the Sorafenib alone group *versus* sorafenib + everolimus, respectively. However, loss of tuberous sclerosis complex 2 (TSC2) in HCC has been reported to be predictive of response to everolimus in HCC patients [39]. Immuohistochemical analysis of HCC samples collected in the EVOLVE-1 clinical trial (NCT01035229) had no detection of TSC2 and longer OS than compared to placebo. A larger study is needed to validate the potential of everolimus before it can be used to stratify

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Another mTOR inhibitor, temsirolimus (taken intravenously) has not improved survival either alone or in combination with either sorafenib [40] or bevacizumab [41]. In a recently concluded phase III study (SILVER, NCT00355862), another mTOR inhibitor, sirolimus, did not improve recurrence-free survival (RFS) or OS beyond 5 years in Ltx recipients with HCC but it did improve RFS and OS within 3–5 years. This may suggest the potential use of siroli-

c-Met is a proto-oncogene that encodes the receptor, MET, for the ligand of hepatocyte growth factor (HGF). MET is a tyrosine kinase receptor regulating metastatic progression. Binding of MET to HGF activates the RAS-MAPK and PI3K-AKT signalling pathways leading to tumour development and metastasis. In HCC, c-MET protein is overexpressed in 70% of HCC and has

Foretinib was the first c-MET inhibitor of broad spectrum, including c-Met and VEGFR, to be tested in clinical trials. In a phase I/II study (NCT00920192) involving patients with advanced HCC, the median TTP was 4.2 months and the OS was 15.7 months. Its toxicity profile was also acceptable with the most adverse events including hypertension and anorexia. Baseline plasma levels of Interleukin 6 (IL6) and Interleukin 8 (IL8) were identified as independent predictors of OS by multivariate analysis. A larger randomised study is needed to warrant

Tivantinib is a selective oral inhibitor of c-MET. In a randomised, placebo controlled phase II study (NCT00988741), advanced HCC patients were administered 240 mg daily resulting in a small improvement in TTP (1.4 months *versus* 1.6 months) compared to the placebo group. Additionally, HCC tumours expressing high levels of c-MET protein, as judged by immunohistochemical analysis, demonstrated an improved OS (7.2 months *versus* 3.8 months) and longer TTP (2.7 months *versus* 1.4 months) compared to placebo. There was no difference

mus for selection of immunosuppression in LTx recipients with HCC [42].

HCC patients for response to everolimus.

**5. c-MET inhibitors**

the effects of foretinib [44].

been associated to poor prognosis [43].

Selumetinib is an oral MEK inhibitor. In a small phase II study (NCT00604721) involving 19 HCC patients with advanced HCC, selumetinib was given at a dose of 100 mg twice per day but the study was terminated at interim analysis because there was no response and the TTP was only 8 weeks. However western blot of biopsy samples taken pre and post treatment showed phosphorylation of MEK1/2, and ERK1/2, suggesting failure of selumetinib was not due to lack of target inhibition [33]. A recent phase I study (NCT01029418) looked into the safety, maximum tolerated dose (MTD), and tolerability of selumetinib in combination with sorafenib in 27 Asian patients with advanced HCC. The MTD of selumetinib was at 75 mg daily with sorafenib at 400 mg twice daily. Common treatment-related adverse events included diarrhoea, rash, and hypertension, fatigue, anorexia and hand-foot and mouth disease. Seven patients had a PR and stable disease for more than 6 months. The OS was 14.4 months. Due to the acceptable adverse events, this combination of selumetinib and sorafenib deserves further evaluation [34].

Another MEK inhibitor, refametinib, was evaluated in a phase II study (NCT01204177) in combination with sorafenib in 95 patients with unresectable HCC. Patients received twicedaily refametinib at 50 mg plus twice-daily sorafenib at 200 mg (morning)/400 mg (evening), with dose escalation to sorafenib 400 mg twice daily after cycle 2. The TTP was 122 days and OS was 290 days. Interestingly, the best responders to the combination treatment were those harbouring Ras mutation. A recently completed proof of concept phase II trial (NCT01915602) of refametinib in combination with sorafenib in Ras mutant HCC has recently been completed with results expected soon. Given that Ras mutations are only observed in 3–5% of HCC patients, this study raises questions about feasibility and costs of screening large cohort of patients to identify a small sub-group with particular mutations.
