November 11, 2013

Systemic Therapies in Hepatocellular Carcinoma

Future Oncology

Present and Future

Gaetano Bertino, Isidoro Di Carlo, Annalisa Ardiri, Giuseppe Stefano Calvagno, Shirin Demma, Giulia Malaguarnera, Nicoletta Bertino, Mariano Malaguarnera, Adriana Toro, Michele Malaguarnera

Future Oncol. 2013;9(10):1533-1548.

Abstract and Introduction

Abstract

Hepatocellular carcinoma (HCC) is now the third leading cause of cancer deathsworldwide and is generally presented at an advanced stage, limiting patients' quality of life. The conventional cytotoxic systemic therapy has proved to be ineffective in HCC, since its induction several decades ago. Today it is possible to use our knowledge of molecular hepatocarcinogenesis to provide a targeted therapy. Sorafenib has demonstrated large improvements in overall survival in HCC. This review describes the molecular mechanisms and potential therapeutic targets, focusing on sorafenib, sunitinib, tivantinib, antiangiogenic agents, and current and future immunotherapies. Thus, it will be necessary in the future to classify HCCs into subgroups according to their genomic and proteomic profiling. The identification of key molecules/receptors/signaling pathways and the assessment of their relevance as potential targets will be the main future challenge potentially influencing response to therapy. Defining molecular targeted agents that are effective for a specific HCC subgroup will hopefully lead to personalized therapy.

Introduction

Over the past 15 years, the incidence of hepatocellular carcinoma (HCC) has more than doubled. Every year there are 500,000 new cases in the Asia–Pacific region, often due to chronic hepatitis B virus (HBV) infection.[1] More than 60% of the total number of HCC cases occur in China alone, and an estimated 360,000 patients residing in Far East countries, including China, Japan, Korea and Taiwan, die from this disease each year. In Japan hepatitis C virus (HCV)-related HCC represents 70% of all cases.[2] In addition, in the USA and Europe, an increased incidence of HCV has led to an increased incidence of HCC.[3] A relevant risk factor for the high incidence of nonalcoholic fatty liver disease is obesity and diabetes, which can promote the development of liver cancer.[4] This involves a poor diagnosis and a low level of survival (5-year survival rate: less than 5%) in patients with advanced HCC at diagnosis. For a correct and effective treatment strategy in patients with cirrhosis, it is necessary to perform a liver ultrasound twice a year. Recently, the role of AFP serum levels has been discussed to be less useful than previously assumed. Furthermore, in addition to AFP there are other biomakers but we know that none of them are optimal.[5–7] For some years there have been reports that some cancer biomarkers, such as DCP and GPC-3, have mitogenic and migratory activities in the angiogenesis of HCC and are contributing factors to tumor growth. The role of these tumor markers is important in hepatocarcinogenesis and represents a great opportunity in HCC treatment.[8,9] Only 30–40% of HCC patients at initial diagnosis are early stage (0 or A) according to the Barcelona Clinic Liver Cancer (BCLC) classification, which defines patients who may be treated with local ablation (particularly radiofrequency), resection or orthotopic liver transplantation.[10]Orthotopic liver transplantation provides 5-year survival rates of 70%, whereas radiofrequency, transcatheter arterial chemoembolization (TACE) or resection show lower rates (50–60%) due to the high rate of tumor recurrence (up to 70% within 5 years).[11] For patients in the intermediate stage (asymptomatic HCC multifocal without vascular localization or metastasis; BCLC stage B), TACE is considered the standard of care, achieving partial response (PR) in 20–50% of patients and an expansion of median survival for up to 20 months throughout the development of new vector systems (polymers) and more accurate patient selection methods.[12] In the last decade, no effective conventional cytotoxic systemic therapy was available,[13,14] which has contributed to the dismal prognosis in patients with HCC.[10] However, a better knowledge of molecular hepatocarcinogenesis today provides the opportunity for targeted therapy.[15] A significant improvement in overall survival (OS) for patients with advanced HCC is due to the oral multikinase inhibitor sorafenib (Nexavar®; Onyx Pharmaceuticals, CA, USA), the first effective remedy after years of limited options, as the positive data from SHARP confirms.[16] On the basis of the SHARP data, the EMA and the US FDA granted the approval for sorafenib to be used as a first-line therapy in advanced HCC in October 2007. In the future it will be used as a systemic therapy. Nexavar has been used largely to improve OS in treated HCC, but recently it has been assessed that in patients with a PR after 3 months of treatment, this drug does not seem surfficient to ensure a significant chance of survival and prognosis. In the near future, we must understand the processes governing neoangiogenesis, which are the molecular alterations in the mitotic phase, to develop more effective targeted therapies to improve OS.[17,18] Many new molecules have been tested, some with antiangiogenic activity, and others targeting the EGFR in mTOR or HGF/c-Met. In the near future, scientific research will need to commit to develop other molecularly targeted agents in HCC: this is not just about the antiangiogenic therapy but other molecularly targeted agents. Another form of strategy based on investigation is about combining targeted agents with chemotherapy. Probably, the strategy to achieve the best results will be the combination of different molecularly targeted agents able to cope with the greatest number of 'molecular errors'.

Conventional Cytotoxic Chemotherapy for Advanced HCC

The management of patients with advanced HCC was previously characterized by limited therapeutic options because of the lack of efficacy of conventional cytotoxic chemotherapy. In fact, HCC is highly refractory to cytotoxic chemotherapy and, until now, no conventional systemic chemotherapy has provided response rates >25% and prolonged survival in patients with advanced HCC.[19]

Patients with HCC have been observed to need high rates of chemotherapy sessions due to tumor drug resistance mechanisms. The intrinsic drug resistance of tumor cells is mediated by enhanced cellular drug efflux mechanisms in association with an increase in a drug transporter family (ATP-binding cassette proteins containing MDR1 and P-gp).[20]Furthermore, resistance is also determined by p53 mutations and overexpression of DNA topoisomerase IIa.[21] In addition, we must consider that the liver cirrhosis and hepatic dysfunction complicate administration of systemic therapy due to pharmacokinetic properties.[22–24] Altogether, no systemic therapy could be considered as a standard of care for patients with advanced HCC in the pre-era of targeted therapy.[25]

Hepatocarcinogenesis

Hepatocarcinogenesis is known to be a highly complex multistep process and nearly every pathway involved in carcinogenesis is altered to some degree in HCC.[17]Therefore, no single dominant or pathognomonic molecular mechanism exists in HCC.

Hepatocarcinogenesis is considered to be a process originating from hepatic stem cells (however, the role of liver stem cells as HCC cells of origin is under debate)[19] or mature hepatocytes and evolving from chronic liver disease driven by oxidative stress, chronic inflammation and cell death followed by unrestricted proliferation/restricted regeneration, and permanent liver remodeling. Chronic liver injury caused by HBV, HCV, chronic alcohol consumption, nonalcoholic steatohepatitis, hereditary hemochromatosis, primary biliary cirrhosis and α-1 antitrypsin deficiency leads to permanent hepatocyte damage followed by a massive compensatory cell proliferation and regeneration in response to cytokine stimulation. Finally, fibrosis and cirrhosis develop in this setting of permanent liver remodeling, particularly driven by the synthesis of extracellular matrix components from hepatic stellate cells. In this carcinogenic environment, the development of hyperplastic and dysplastic nodules as preneoplastic conditions is only a question of time. However, the suspected sequential accumulation of molecular events at different stages of liver disease (normal liver tissue, chronic hepatitis, cirrhosis, hyperplastic and dysplastic nodules and cancer) is only partially understood. The molecular pathogenesis of HCC involves different genetic/epigenetic aberrations and alterations in multiple signaling pathways leading to the known heterogeneity of the disease in terms of its biologic and clinical behavior. Current evidence indicates that in hepatocarcinogenesis, two main mechanisms seem to be involved: cirrhosis and the association with hepatic regeneration after chronic liver damage caused by several factors (hepatitis infection, toxins or metabolic impairments), and the collection of DNA mutations with impairment of the celluar oncogenesis–oncosuppression equilibrium leading to the development of neoplastic cells. Several important cellular signaling pathways have been observed to be part of the oncogenetic involvement in HCC. The knowledge of these signaling cascades opens multiple ways to better focus therapeutic interventions, aiming to reverse, delay or prevent tumorigenesis. The major signaling pathways in HCC are the RAF/MEK/ERK, PI3K/AKT/mTOR, WNT/β-catenin, IGF, HGF/c-MET and growth factor-regulated angiogenic signaling as shown in Figures 1 & 2.

812561-fig1

Figure 1.

Cellular signaling pathways implicated in the pathogenesis of hepatocellular carcinoma.
HCC: Hepatocellular carcinoma.

812561-fig3

Figure 2. The major signaling pathways and the cascade of angiogenic growth factors in hepatocellular carcinoma.
HCC: Hepatocellular carcinoma

DCP is a novel type of VEGF, having mitogenic and migratory activities in the angiogenesis of HCC. DCP is an abnormal prothrombin induced by the absence of vitamin K2, which is increased in the serum of patients with HCC. In hepatoma cells, genetic alterations, the inability of membrane receptors to uptake labeled low-density lipoprotein, cytoskeletal changes and hepatocyte cytoplasmic transfers involved in vitamin K metabolism could play an important role in producing detectable DCP serum levels. DCP is not exclusively a diagnostic or prognostic biomarker for HCC, but is also a novel type of VEGF, with mitogenic and migratory roles in the angiogenesis of HCC. In HCC patients, DCP production is independent of vitamin K deficiency, although pharmacological doses of vitamin K can transiently suppress DCP production in some tumors. In an in vitro model, DCP production was observed in HepG2 cells and inhibited by introducing additional vitamin K2 into the treated cells. In addition to the decrease in DCP production, there has been a reduction of the growth and invasiveness of carcinoma cells. Therefore, administration of vitamin K2, associated with therapies of proven efficacy, could be considered to be a promising option for the treatment of HCC.[5,40] GPC-3 is a heparan sulfate proteoglycan. Recent studies have shown that GPC3 levels are increased in HCC patients. It is thought that GPC3 stimulates the growth of HCC cells by upregulating autocrine/paracrine canonical Wnt signaling. GPCs stimulate both the canonical and noncanonical pathways and regulate migration, adhesion and actin cytoskeleton organization in tumor cells through Wnt signaling modulation.[5] The principal factors in the development of HCC and in hepatocarcinogenesis are the heparan sulfate chains of GPC-3 combined with other heparin-binding growth factors. Therefore, GPC-3 could be an interesting molecular therapy.[41] In addition, the tyrosine kinase inhibitor of IGF-1R (NUP-AEW541) expressing HCC cell line (PLC/PRF/5) has interestingly been recently reported on.[42] A new cancer therapy could be a combination of the anti-GPC-3 antibody and molecular therapy targeting GPC-3 related molecules, such as FGFR.

Molecular Targeted Therapy

The knowledge of molecular hepatocarcinogenesis broadened the horizon for patients with advanced HCC. During the last few years, several molecular targeted agents have been evaluated in clinical trials in advanced HCC (Figure 3). Despite the only modest objective response rates according to the Response Evaluation Criteria in Solid Tumors (RECIST),[43] several studies showed encouraging results in terms of prolongation of the time to progression (TTP), disease stabilization (DS) and survival.

812561-fig2

Figure 3.

Molecular sites of action of active biochemical agents in hepatocellular carcinoma treatment
↓: Decreased; HCC: Hepatocellular carcinoma

Sorafenib

Sorafenib (BAY43–9006) is currently the only approved systemic treatment for HCC. It has been approved for the therapy of asymptomatic HCC patients with well-preserved liver function who are not candidates for potentially curative treatments, such as surgical resection or liver transplantation, and it is the first FDA-approved systemic therapy for patients with advanced HCC. In clinical practice, the failure of locoregional therapy, such as TACE, led to the use of sorafenib. Its efficacy and safety in HCC patients was demonstrated by the SHARP trial in western patients.

Sorafenib is an multikinase inhibitor showing anti-tumoral properties in cell proliferation and angiogenesis, the serine/threonine kinases Raf-1/B-Raf, and the tyrosine kinases of VEGFR-2/-3 and PDGFR-β are its molecular targets.[44] Patients with higher phosphorylated ERK staining in tumor biopsies had a significantly longer TTP (178 vs 46 days), suggesting Raf inhibition as an important mechanism of action for sorafenib, and phosphorylated ERK as a potential marker of response as demonstrated in the results of Phase II studies. These data have not been confirmed in more recents studies.[45,46]

This oral multikinase inhibitor has been shown to block neoangiogenesis and to induce apoptosis in many tumor cell lines, although the mechanisms are not completely understood.[47] Two important suggested mechanisms are the inhibition of the eIF4E factor, which, when phosphorylated, mediates the upregulation of some oncogenic proteins, and the downregulation of an antiapoptotic protein, Mcl-1. The proapoptotic activity of sorafenib can be enhanced by adding other chemotherapeutics (e.g., gemcitabine) or nonchemotherapeutic agents (e.g., sirolimus, an inhibitor of the mTOR pathway), which inhibit signal transduction pathways,[48] showing good opportunities for combination therapy.[49]Insufficient data are available demonstrating the combinated treatment of sorafenib with those agents, and further studies are needed. The efficacy, safety profile and influencing clinical factors of sorafenib are analyzed by Xie et al.[50]

Studies on Treatment With Sorafenib Alone

There are seven trials that studied sorafenib alone, including a total of 1072 patients, divided in two Phase I trials,[51,52] three Phase II trials[53–55] and two Phase III randomized, placebo-controlled, clinical trials.[16,46] All the trials showed a disease control rate (DCR) range from 26 to 82%.[51] Five of the trials supplied short-term data about OS and TTP,[16,46,51,53,55]respectively, with an interval ranging from 5[55] to 15.6[51]months for OS and from 3[55] to 5.5[16] months for TTP. Major adverse events (AEs; grade 3 or 4), such as hand–foot syndrome (HFS), diarrhea and fatigue, were described in all trials but with a wide variation in frequency. HFS ranged from 3[16] to 27%,[51] diarrhea from 0[51] to 82%[52] and fatigue from 0[31] to 91%.[51]

The two highest quality reports (multicenter, Phase III, double-blind, placebo-controlled trials; we randomly assigned patients with advanced HCC who had not received previous systemic treatment to these trials) were the SHARP and the Asia–Pacific trials,[16,46] which recruited patients with Child's A cirrhosis (95 and 97%, respectively), even if HBV infection frequency was much lower in the Asia–Pacific trial (71 vs 19%). These were the data provided by the SHARP trial in terms of OS and TTP: OS and TTP values were 10.7 and 5.5 months in the sorafenib group, and 7.9 and 2.8 months in the placebo group, respectively, with p < 0.05 in both groups.[16]

The data provided by the Asia–Pacific trial were 6.5 vs 4.2 months for OS and 2.8 vs 1.4 months for TTP, respectively, for the sorafenib group versus the placebo group.[16,56]

Considering the six studies that provided data about Child–Pugh score,[16,46,51–53,55] they have shown that the majority of patients had Child's A cirrhosis. Child's B cirrhosis ranged from 3[46] to 52%,[51] and only one study had included Child's C cirrhosis with two patients.[55] HFS is more frequent in patients with Child's B cirrhosis, with an incidence of 27%[31] in the seven trials. At the same time, in the Phase II trial by Abou-Alfa et al., there was a relatively low frequency of HFS (5%) among a population in which 28% had Child's B cirrhosis.[53]

Thus, it seems that HBV infection could adversely affect the response to sorafenib. In fact, in the Hong Kong trial, most patients with HBV infection (90%) also had the lowest OS (5 months) and DCR (26%).[55]

The Japanese trial, instead, studied mostly HCV patients (74%), demonstrating the longest OS (15.6 months) and the highest DCR (82%).[51] Three (43%) of the seven studies on sorafenib alone showed an OS equal to 10 months. In the six trials that provided data about the TTP, none reported a TTP >6 months. Three of them reported a DCR >60%.

The seven studies show that treatment with sorafenib alone produces statistically significant but modest results clinically, and demonstrates improvements in OS, TTP and DCR in patient populations including men less than 70 years of age with advanced stage HCC and Chid's A cirrhosis.[16,46,51–55] Insufficient data are still available on the effects of sorafenib in women, older patients (>70 years) and late stage cirrhotic patients. Sorafenib treatment alone seems to have more efficacy in HCV-infected patients than in HBV-infected patients. Moreover, sorafenib's side effects are frequent, mostly in advanced cirrhosis patients.

In a study by Di Costanzo et al., a single center's experience in treating 116 patients with advanced HCC with sorafenib was reported on.[57] The study evaluated the response of HCC to and safety of sorafenib. The efficacy of sorafenib was evaluated considering the time to radiographical progression, DCR, OS and survival by grade of radiological response. The TTP was measured every 3 months with the modified RECIST (mRECIST) for HCC. With regards to the appearance of AEs, patients were monitored every month using the common toxicity criteria version 3.0. The 3-month overall DCR was 71%: stable disease was observed in 37%, PR in 31% and complete response in 2–5% of patients. The 3-month radiological response is in relation to OS. In the study the authors emphasize that it is very important to evaluate tumor response after 3 months of therapy. In fact the therapeutic response correlates with the prediction of survival. The OS rate at 12 months increased from 10% in complete responder patients to 100% in patients with disease progression. In both the SHARP trial and Asia–Pacific study, 71 and 54% of patients achieved stable disease, while only 2 and 3.3% achieved a PR, respectively, and no patients with a complete response were observed.[16,58,59] The authors explain that the difference is due to the different criteria used to establish the response (RECIST was conventional in the SHARP and Asia–Pacific trials, whereas mRECIST was used in the authors studies) and early termination of the study may have underestimated the SHARP response assessment. These facts can also explain the most significant radiological TTP in this study compared with the SHARP study (12 vs 5.5 months). The AEs were moderate (grade 1 or 2), and symptom treatment and dose adjustments were usually enough to manage AEs.[46,57,60]

Xie et al. demonstrated that sorafenib treatment is significant from the statistical point of view, but clinically only modest improvements in OS, TTP and DCR have been demonstrated.[50] HCV patients seem to respond better. Moreover, sorafenib used in advanced cirrhosis and in combination with fluorouracil drugs is accompanied by a higher frequency of HFS. It is still not clear if sorafenib treatment alone is less effective than sorafenib used in combination with other drugs.

Studies on Sorafenib Combined With Other Treatments

Fourteen studies investigating sorafenib in combination with other treatments included a total of 470 patients, with a similar patient populations as the sorafenib-only studies, but with a smaller number of patients.

These studies collected better OS, TTP and DCR results than those observed in the sorafenib-only trials, even though a direct comparison between all these studies seems difficult to assess. Sorafenib in combination with other agents does not show excessive toxicity, but the HFS seems to be much more frequent, especially sorafenib in combination with fluorouracil and derivates.[61–67] Furthermore, addition of erlotinib to sorafenib did not provide additional benefit to patients with unresectable liver cancer versus sorafenib alone in the Phase III trial.[68]

Sunitinib

The most common cause of death in HCC is due to tumor recurrence and metastasis. Clinical outcomes have not met expectations, despite positive results obtained in preclinical studies with VEGF inhibitors. Sorafenib is the only drug that has been shown to improve survival significantly in HCC patients. Recently, a VEGF inhibitor called sunitinib has shown survival benefits in HCC patients but not in the HCV patients.

Sunitinib malate (SU11248, Sutent®; Pfizer, NY, USA) is an oral multikinase inhibitor that targets several tyrosine kinases receptors, such as VEGF-1/2 and PDGFR-α/β,[69] and is implicated in HCC proliferation and angiogenesis. In addition, it was defined as an inhibitor of c-kit, Fit-3 and RET.[69]

Sunitinib was approved for treatment of gastrointestinal stromal tumors after progression or intolerance to imatinitib mesylate.

It has already demonstrated preliminary anti-tumoral activity and an acceptable safety profile in different Phase II trials for patients with advanced HCC.[70–72] However, despite the other tumor types, sunitinib seems to have more toxic side effects in HCC. Due to more treatment-related toxicities when using the 50-mg dose, in most planned trials, a 37.5-mg dose has been used.

In any case, sorafenib therapeutic effects are transient so additional treatments are warranted.

In the Huynh et al. study (xenograft models), the authors wanted to compare the effectiveness of sunitinib relative to sorafenib. Both are strong inhibitors of tyrosine kinases proteins, which are involved in tumor growth, angiogenesis and metastasis, reporting suppressed tumor growth, angiogenesis, cell proliferation and induced apoptosis in both HCC models, orthotopic and ectopic, for both drugs.[73]

However, the anti-tumoral effectiveness of sorafenib was greater when administered at the dose of 50 mg/kg than sunitinib at a dose of 40 mg/kg. Futhermore, sorafenib inhibited p-eIF4E Ser209 and p-p38 Thr180/Tyr182, and reduced survivin expression. This was not seen with sunitinib. In addition, the anti-tumoral properties and the proapoptotic effects of sorafenib (upregulation of fast migrating Bin and ASK1, plus downregulation of survivin) was greater than sunitinib. These data seem to confirm the apparently higher efficacy of sorafenib in anti-tumoral activity, if it were compared with sunitinib.

Huynh concludes that the anti-tumoral effect of sunitinib is inferior to sorafenib, in both ectopic and orthotopic models of human HCC. However, these observations should be verified in humans. Concomitant liver function, liver disease and the local liver environment have a huge impact on treatment outcomes.[73]

Sunitinib antiproliferative action on HCC cell lines, either in vitro, or in xenograft and orthotopic models was studied by Bagi et al. in order to evaluate the effect of local liver vasculature on drug efficacy.[74]The in vitro studies used human cancer cell lines Huh7.5, Hep3B and SK-Hep-1, whereas, in in vivostudies, mice carried Huh7.5 cells either in the subcutaneous or intrahepatic compartment. Drug exposure and treatment regimens were the same in both tumors. In the Huh7.5 cell lines sunitinib can mildly inhibit proliferation. It can also promote the expression of p53 in p53-wildtype cell line SK-hep-1 and can increase the expression of elements of the cell cycle (S-phase and sub-G1) in the Hep3B cell line. HCC tumors manifest their heterogeneity in a different response to sunitinib in HCC cell lines, which probably explains the discrepancy between preclinical and clinical results. Comparing sunitinib effects on the models, the in vivo results show that it is much less effective against intrahepatic tumors compared with xenograft models. Sunitinib affects large, solid, intrahepatic tumors, as shown by histological data, but unopposed local growth of the small turmors and the development of distant micrometastases seems to be a problem with these types of VEGF inhibitors. There is no doubt that both xenograft and orthotopic models are limited, but they can add value in understanding tumor biology to plan treatment paradigms for these types of patients.

Orthotopic models are particularly useful in enhancing our knowledge of the role of organ vasculature in local metastasis development and tumor resistance to antiangiogenic treatments.

Thus, recently, sunitinib efficacy/safety assessment studies have been suspended due to an unfavorable risk–benefit relationship of its administration (SUN 1170 Phase III open-label study), in comparison to sorafenib.[67]

Linifanib

Linifanib (ABT-869) is a multitargeted tyrosine kinase inhibitor that inhibits multiple members of the VEGFR and PDGFR families.[75] In a xenograft model of HCC, ABT-869 significantly reduced tumor burden. Interim Phase II results in patients with advanced HCC showed a median TTP of 3.7 months with ABT-869 treatment and a safety profile consistent with angiogenesis inhibition. In the Toh et al. study, the authors demonstrate that linifanb as a single agent was found to be clinically active in patients with advanced HCC, with an acceptable safety profile.[75]

Tivantinib

Tivantinib (ARQ 197) is a new oral selective MET inhibitor that acts by blocking growth and inducing apoptosis in human tumor cell lines that express MET. MET is a tyrosine kinase receptor involved in tumor development and metastatic progression, which is encoded by a MET proto-oncogene. When binding to HGF, MET activates the RAS–MAPK and PI3K–AKT signaling pathways.[76,77] Tivantinib anti-tumor activity was demonstrated in murine xenograft models and its efficacy was confirmed in a panel of HCC cell lines.[78] The inhibitory effect on MET activity and downstream pathways was also demonstrated in tumor biopsy samples gained before and after tivantinib treatment.[79] Ultimately, preclinical studies on tivantinib combination with sorafenib have shown good therapeutic opportunities for the additive and synergistic activity of these drugs, as freshly confirmed by a Phase I clinical trial in HCC.[80]

A Phase II trial in patients who were surgically and pharmacologically untreatable with previous therapeutic options was performed by Santoro et al.[81] For this multicenter, randomized, placebo-controlled, double-blind, Phase II study,[201] patients with advanced HCC and Child–Pugh A cirrhosis who were untreatable with other options were enrolled. We randomly allocated patients 2:1 to receive tivantinib (360 mg twice a day) or placebo until progression of the disease. An amendment of tivantinib dose (from 360 to 240 mg) was necessary for the high incidence of grade 3 or worse neutropenia during the treatment. Randomization was carried out centrally, stratified by Eastern Cooperative Oncology Group performance status and vascular invasion, and the primary end point was TTP, according to an independent radiological review in the intention-to-treat population. MET expression on tumor samples was estimated using immunohistochemistry (high expression was regarded as grade ≥2 in ≥50% of tumor cells). Tivantinib was received by 71 patients, of which 38 received a dose of 360 mg twice daily and 33 received 240 mg twice daily. Placebo was received by 36 patients. At the time of analysis, 46 (65%) patients in the tivantinib group and 26 (72%) in the placebo group demonstrated disease progression. Patients treated with tivantinib had a longer TTP (1.6 months [95% CI: 1.4–2.8]) than placebo patients (1.4 months [95% CI: 1.4–1.5]; hazard ratio: 0.64, 90% CI: 0.43–0.94; p = 0.04). Patients with a high MET expression had a longer TTP if treated with tivantinib instead of placebo (2.7 months [95% CI: 1.4–8.5] for 22 MET-high patients on tivantinib vs 1.4 months [95% CI: 1.4–1.6] for 15 MET-high patients on placebo). Neutropenia and anemia were observed with more frequency in the tivantinib group with the following rates: 14 versus 0% (placebo group) and 8 versus 0% (placebo group), respectively. Furthermore, the administration of a higher dosage (360 vs 240 mg) of tivantinib seems to lead to higher rates of neutropenia and anemia (21 vs 6%, respectively) and to higher death rates for severe neutropenia.

In conclusion, tivantinib may provide an option for second-line treatment in patients affected by advanced HCC with well-compensated cirrhosis, especially if they have MET-high tumors. MET can represent an important prognostic and predictive biomarker in this type of patient. Further studies of tivantinib in a biomarker-selected patient population are warranted.[81]

The Trojan and Zeuzem paper covers the preclinical data, the Phase I studies of monotherapy or in combination with sorafenib, and a Phase II study of second-line systemic treatment in patients with advanced HCC.[82] The tivantinib safety profile and MET expression diagnostic role are mentioned. Tivantinib was shown to improve progression-free survival (PFS) and OS in the highly expressed MET group versus placebo, as a second choice treatment (Phase II study) while MET overexpression was considered a negative prognostic factor. Tivantinib in combination with sorafenib seems to be a promising therapy. AEs are represented by hematological toxicity, asthenia and loss of appetite. Tivainib safety was examinated in the Santoro et al. Phase Ib study[83] of a population of patients with Child's A or B cirrhosis, previously treated for HCC. They were administered a twice-daily dose of 360 mg, until toxicity effects or disease progression were observed. Common drug-related AEs detected in 21 HCC patients were neutropenia, anemia, asthenia, leukopenia, anorexia, diarrhea and fatigue. Drug-related decline of liver function or performance status was not observed except for a Child's B cirrhosis patient that experienced a bilirubin increase. Four patients had serious AEs, one of them suffering a neutropenia-related death. Compared with the previous study, there were more hematologic toxicities but those were promptly treated. In nine out of 16 evaluable patients (56%), the best response was stable disease (median: 5.3 months). Median TTP was 3.3 months. In conclusion, tivantinib demonstrated a manageable safety profile and preliminary anti-tumor activity in patients with HCC and Child's A or B cirrhosis.

Antiangiogenic Agents

Bevacizumab

Bevacizumab (Avastin®; Genentech, CA, USA), a recombinant, humanized monoclonal antibody that targets VEGF, is one of the central drugs of colorectal tumor treatment.[84] In addition to inhibiting tumor growth, growth factor release and metastasis, it can enhance chemotherapeutic agent delivery by normalizing tumor vasculature.[85] Many Phase I–II studies in advanced HCC examined bevacizumab either as a single agent[86] or combined with conventional chemotherapy, such as gemcitabine/oxaliplatin[87] or with capecitabine and oxaliplatin.[88,89] As a single agent it reached objective response rates in up to 13% of patients.[86] In combination with gemcitabine/oxaliplatin it showed a response rate of 20%, with an additional 27% of patients having DS.[87] Good results were also achieved with capecitabine and oxaliplatin (PR: 11%; DS: 78%).[88,89]

A recent study by Kaseb et al. evaluated the use of bevacizumab in combination with erlotinib in patients with advanced HCC and achieved encouraging results.[90] However, apart from more contraindications (cardiovascular and renal impairment) compared with other targeted agents, the risk of hemorrhagic and thromboembolic events needs further evaluation.[86,90] In addition, due to the small sample size, the nonrandomized nature and the single-arm setting in these studies, the relative contribution from the chemotherapy regimen remains unknown and no recommendation for routine clinical use of bevacizumab can be recommended.

Fang and collegues have performed a systematic review of the efficacy and safety of bevacizumab for the treatment of advanced HCC,[91] reporting approximately eight trials involving 300 patients. One trial observed bevacizumab as a monotherapy and seven trials observed it in combination with several others agents, such as erlotinib, capecitabine, capecitabine plus oxaliplatin and gemcitabine plus oxaliplatin. PFS and OS reported in those studies were assessed with averages of between 5.3 and 9.0 months and 5.9 and 13.7 months, respectively. The control of the disease appeared to be significant in five out of eight trials, ranging from 51.1 to 76.9%. The response (complete or partial) rates ranged from 0 to 23.7%. Increased aspartate transaminase/alanine transaminase serum levels were frequently observed (13%), as well as fatigue (12%), hypertension (10%), diarrhea (8%) and neutropenia (5%). Esophageal varices bleeding was observed in 30% of patients.[86,87,92–96]

Bevacizumab can represent a workable treatment option for advanced HCC. Its efficacy appears to positively compare with that of sorafenib. In six of the eight Phase II trials on bevacizumab, it has been reported how the median OS rates are similar or greater than those reported in the SHARP and Asia–Pacific trials.[16,46,97] Interestingly, none of the patients involved in the Yau et al. trial responded to treatment.[95] All patients had very advanced HCC and were refractory to sorafenib. The lack of response seemed to be due to increased proliferation and invasion in the absence of VEGF-related angiogenesis.[95] The lack of efficacy of bevacizumab was not completely surprising, considering its pharmacodynamics.

This suggests that these types of patients cannot receive any benefit from treatment with bevacizumab/erlotinib if they are refractory to sorafenib but it is important to consider that this trial included a very small number of patients (n = 10) so the data are unreliable. Moreover, the Kaseb et al.study suggests that patients previously treated with sorafenib (n = 7) can respond to a treatment combination of bevacizumab and erlotinib (as indicated by PFS and OS).[90] There was no uniformity among the studies regarding doses, coadministered treatment, number of treatment cycles or line of treatment. In four of the eight studies, bevacizumab was somministrated in combination with erlotinib, an EGFR tyrosine kinase inhibitor.[90,94–96]

The dual targeting of VEGF and EGFR was considered a logical treatment approach. Many data from Phase II studies have suggested that erlotinib may facilitate disease control. However, any meaningful comparison among treatment options is not possible for the nonhomogenenous population of patients in the trials involving bevacizumab/erlotinib combination therapy.

There were no grade 1 or 2 unexpected AEs related to bevacizumab in all of the Phase II studies.

Bevacizumab was usually well tolerated, as these findings suggest. In particular, the most common cause of gastrointestinal bleeding was esophageal varices, suggesting that patients with this comorbidity may not be suitable for this kind of treatment. In fact, Thomas et al. excluded patients with esophageal varices from treatment.[94] Studies are necessary to further characterize the efficacy and safety profile of bevacizumab, to determine which patients are most likely to benefit from treatment and how bevacizumab can be optimally incorporated into HCC treatment strategies.[91]

Brivanib

Brivanib, a selective dual inhibitor of FGF and VEGF signaling, has recently been shown to have activity as a first-line treatment for patients with advanced HCC.[97] In the Phase II open-label study by Finn et al., brivanib was assessed as a second-line therapy in patients with advanced HCC who had not been successfully treated with prior antiangiogenic treatment.[67,98] In this study, brivanib was administered orally at a dose of 800 mg once daily. The primary objectives were tumor response rate, time to response, duration of response, PFS, OS, DCR, TTP, and safety and tolerability. Forty six patients were treated. The best responses to treatment with brivanib (n = 46 patients) using modified WHO criteria were PRs for two patients (4.3%), stable disease for 19 patients (41.3%) and progressive disease for 19 patients (41.3%). The tumor response rate was 4.3%; the DCR was 45.7%. Median OS was 9.79 months. Median TTP, as assessed by study investigators following second-line treatment with brivanib, was 2.7 months. The most common AEs were fatigue, decreased appetite, nausea, diarrhea and hypertension. The authors conclude that brivanib had a manageable safety profile and is one of the first agents to show promising anti-tumor activity in advanced HCC patients treated with prior sorafenib. Nevertheless, recent data showed that patients administrated with brivanib did not reach the primary end point (OS) both in first- and second-line therapy.[98]

Ramucirumab

The monoclonal antibody ramucirumab is a specific inhibitor of VEGFR-2. A Phase II study of 42 patients with advanced HCC and primarily well-preserved liver function (75% Child–Pugh A status) showed that first-line ramucirumab monotherapy produced a DCR of 50% and a median PFS of 4.3 months.[99] This positive study prompted the initiation of the Phase III REACH trial in HCC, which compares ramucirumab/supportive care with placebo/supportive care for second-line treatment after sorafenib (Table 1). REACH Phase III trial enrollment has been completed but no results are available yet. It will be very interesting to see the results and the final end points.

Table 1.  Molecular targeted therapy, phase and state of trials.

Drugs in HCC treatment Mechanism of action Target Phase Trials (n) State of trials
Multikinase inhibitors
Sorafenib TKI VEGFR-2/-3, PDGFR-β, Raf-1, B-Raf, Flt-3, c-KIT, RET I, I–II, II, III, IV 65 Closed
Sunitinib TKI VEGFR-1/-2, PDGFR-α and -β, Flt-3, c-KIT, RET II, III 6 Closed
Linifanib TKI VEGFR, PDGFR family II, III 2 Ongoing
Cabozantinib TKI VEGFR2 I, II 2 Ongoing
MET inhibitors
Tivantinib Inhibits growth, induces apoptosis in c-MET-positive HCC c-MET/HGF I, II 3 Ongoing
Cabozantinib Inhibits growth, induces apoptosis in c-MET-positive HCC c-MET I, II 2 Ongoing
Antiangiogenic agents
Bevacizumab MAB VEGF I, I–II, II 20 Closed
Brivanib MAB FGF, VEGF I, II, III 6 Ongoing
Ramucirumab MAB VEGFR-2 III 1 Ongoing

HCC: Hepatocellular carcinoma; MAB: Monoclonal antibody; TKI: Tyrosine kinase inhibitor.

Cabozantinib

Cabozantinib acts as a dual c-Met/VEGFR2 inhibitor, inhibiting the tyrosine kinase activity of RET, MET, VEGFR-1, -2 and -3, KIT, TRKB, FLT-3, AXL and TIE-2. The Verslype et al. study also demonstrated early evidence of anti-tumor activity in a randomized discontinuation Phase II study. Interestingly, the clinical benefits were observed regardless of whether patients had received prior sorafenib treatment. Cabozantinib is undergoing additional evaluation in HCC to better assess its efficacy and safety profile in several ongoing clinical trials.[100] Phase III studies should evaluate the effectiveness of cabozantinib versus placebo, and OS in patients with advanced HCC who have already been treated. OS was the primary end point, and the objective response rate and PFS for RECIST 1.1 were the secondary end points. Additional end points were: safety and tolerability of cabozantinib; pharmacokinetics (PK); change from baseline tumor biomarker levels in the serum; and health-related quality of life as assessed by the EuroQol Health questionnaire.

Immunotherapy of HCC

The human immune system against tumors is mainly dependent on cellular immunity. Patients with HCC are found to have a functional deficiency in a variety of immunocytes.[101] Thus, cellular immunotherapy would improve the immune state and has potential in enhancing the therapeutic outcome for HCC patients. Current attempts at harnessing the immune system to eliminate tumors has been focusing on vaccination, such as a dendritic cell (DC) vaccine, to increase the frequency of tumor-specific cytotoxic T lymphocytes and adoptive transfer of effector T cells to promote tumor regression.[102] However, despite considerable success in preclinical studies, the outcome of immunotherapy is often disappointing when translated to clinical trials, which is at least in part due to the complexity of the immune escape mechanism of tumor cells. Cancer cells not only protect themselves from recognition by the immune system through downregulation of MHC molecules and stimulation of molecules on the cell surface, but they also secrete cytokines such as IL-10 and TGF-β, to alter the tumor microenvironment to diminish the effectiveness of anti-tumor responses.[103] The cancer uses most of these interactions to shield itself from an effective anti-tumor immune response, which needs to be overcome by tumor immunotherapy strategies. On the other hand, evidence shows that the reduction of tumor burden (e.g., with radiofrequency ablation) enhances the effect of immunotherapy. Therefore, the ideal timing of applying cellular immunotherapy is also critical. The cytokine and/or chemokine milieu might also enhance the antineoplastic function and promote the recruitment of circulating immune cells to the tumor site.[104]

An innate system of pattern recognition and immediate response allows the host to have a series of mechanisms that provide rapid action against tumors. A more evolved, adaptive immune response allows them to react to more variable threats that trigger an initial weak response, followed by a stronger long-term memory response.

Immunosuppression Induced by Chronic HBV & HCV Infection

Chronic HBV or HCV infections are well known to induce a chronic proinflammatory hepatic and systemic state associated with immunosuppressive and immunomodulatory effects.[27,28,105–107]

HCC & Immune Escape

In addition, it should be noted that there are many mechanisms of HCC immune escape. The peripheral blood of patients with HCC shows impaired IL-12 production and reduced allostimulatory activity.[108] An impairment of natural killer cell production and activity has also been described in HCC patients.[109]

Current & Future Immunotherapy of HCC

Immune-based therapy can represent an improvement in outcomes for patients with HCC,[102] as many clinical trials demonstrate. In a historical study, 150 patients were randomized to receive either IL-2 and anti-CD3-activated PBMC, or observation after curative resection.[110] The results were encouraging, both with respect to time to relapse and disease-free survival (p = 0.09). A trial testing the administration of APC in HCC patients who received pulsed DC with autologous tumor lysate[111] showed an increase of 1-year survival (63 vs 10%; p = 0.038). Most recently, murine models have demonstrated that immunotherapy and DC in combination with IL-12 in an adjuvant setting activates T and natural killer cells and reduces HCC recurrence.[112–114]

DCs could be used as a potential cellular adjuvant for the production of specific tumor vaccines. Recently, El Ansary and colleagues' study[114] evaluated the safety and efficacy of the autologous pulsed DC vaccine in advanced HCC patients in comparison with supportive treatment. Thirty patients with advanced HCC who were not suitable for radical or locoregional therapies were enrolled. Patients were divided into two groups, group I, consisting of 15 patients, received vaccination with mature autologous DCs pulsed ex vivo with a liver tumor cell line lysate. Group II (control group; n = 15) received supportive treatment. To generate DCs, 100 and 4 ml of venous blood were obtained from each patient. DCs were identified by CD80, CD83, CD86 and HLA-DR expressions using flow cytometry. Follow-up at 3 and 6 months postinjection by clinical, radiological and laboratory assessment was carried out. Improvements in OS were observed. Partial radiological response was obtained in two patients (13.3%), stable course in nine patients (60%) and four patients (26.7%) showed progressive disease (died at 4 months postinjection). Both CD8+ T cells and serum IFN-γ were elevated after DC injection. The authors conclude that autologous DC vaccination in advanced HCC patients is safe and well tolerated. Ex vivotreatment with CTLA-4 blocking antibodies of T-cell CD8+, isolated from patients affected by HCC, showed an expanded antigen-specific T-cell repertoire, alluding that ipilimumab may possess a therapeutic potential in treating hepatocarcinoma.[115] A therapeutic advantage, regarding refractory solid tumors, can be obtained by an antibody-mediated block of PD-1, meanwhile the inhibition of Tim-3 signaling has been demonstrated to restore anti-tumor T-cell action in preclinical models.[116] Another approach has been described to overcome cancer-mediated immunosoppression, involving the reactivation of hyporesponsive tumor-specific T cells by supplying T-cell growth factors (IL-15 and IL-7) or costimulatory agonists (anti-4-1BB and anti-OX40).[117] Other treatment options regarding tumor homing and penetration of T-effector cells are being evaluated because of the correlation between T-cell infiltration of hepatocarcinoma lesions and OS. Strategies are divided into two big groups: restoring the tumor vascularity, and upregulation of chemokines and molecules of adhesion. Monoclonal antibodies against VEGF and its receptors, such as sorafenib or bevacizumab, appear to have a restricted therapeutic effect in clinical trials.[16,50] In fact a hallmark of new vessel formation in HCC is their structural and functional abnormality; this leads to an abnormal tumor microenvironment characterized by low oxygen tension and low therapeutic agent levels.[118] Preclinical data sustain the idea that angiogenesis and tumor vascularity still represent a potential target that, through the generation of long-lived antivascular T-cell responses via VEGFR2 vaccine, can be suppressed via a T-cell dependent process.[114] Proinflammatory chemokines demonstrated their importance in HCC-specific T-cell immunity, such as IFN-γ- inducible chemokines CXCL9/Mig and CXCL10/IP-10, high levels of which correlated with the presence of CD8+ T cell in hepatocarcinoma. It is still unknown if this pattern of chemokine expression is correlated with a positive prognosis, as has been seen in patients with cervical/uterine tumors. Agents that can induce the expression of chemokines and adhesion molecules by vascular activation represent another promising approach.[119]

Conclusion

Cytotoxic chemotherapy, hormonal agents and immunotherapy have been tested in HCC with marginal efficacy to date. Recent insights into the molecular pathogenesis of HCC have identified several aberrant signaling pathways that have served as targets for novel therapeutic agents. Several pathways are now implicated in hepatocarcinogenesis, and agents that target these pathways continue to be developed. Thus, in the future, new therapeutic options will be represented by a blend of immunotherapy-like vaccines and T-cell modulators, supplemented by molecularly targeted inhibitors of tumor signaling pathways.[120,121]

Future Perspective

Molecular alterations may differ depending on the underlying risk factors and etiologies, potentially influencing patient responses to therapy. Thus, it will be necessary in the future to classify HCCs into subgroups according to their genomic and proteomic profiling. The identification of the key molecules/receptors/signaling pathways and the assessment of their relevance as potential targets will be the main future challenge. Defining molecular targeted agents effective for a specific subgroup will hopefully lead to personalized therapy.

References

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