Immunotherapy has been investigated in patients with hepatocellular carcinoma (HCC) for decades. However, initial clinical trials that tested various immune-based therapies showed little clinical efficacy in HCC, despite eliciting tumor-specific immune responses. In recent years, knowledge of HCC-specific tumor-associated antigens (TAAs) and the development of immune checkpoint blockade therapy has offered a positive outlook for patients with HCC, according to a review by Gaetano Bertino, MD, with the University of Catania in Catania, Italy.

Immunotherapies offer great promise, as the search for a second-line therapy continues. Currently, sorafenib, an oral kinase inhibitor, is the only US Food and Drug Administration (FDA)-approved agent available to treat metastatic HCC. No suitable second-line therapies are available for patients who relapse or who are refractory to sorafenib treatment.

Additionally, the recently published results from a double-blind, phase III study indicate that sorafenib is not an effective therapeutic strategy in the adjuvant setting following surgical resection or ablation for patients with HCC, further limiting options.

Given that most patients with HCC do not receive a diagnosis until the disease is at an advanced stage, and only liver resection and transplantation are considered curative, there is an urgent clinical need to improve the therapy landscape. Although the inherent immune tolerance associated with the liver makes immune-based strategies challenging, the immune system can be utilized in multiple ways. For example, the immune response can be enhanced indirectly with either tumor vaccines or immunomodulators, or directly by introducing effective immune cells with adoptive immunotherapy.

describe 7 TAAs that are recognized by the adaptive immune system, elicit a T-cell response, and show HCC specificity. The TAAs described are alpha-fetoprotein (AFP), glypican-3 (GPC3), NY-ESO-1, SSX-2, melanoma antigen gene-A (MAGE-A), telomerase reverse transcriptase (TERT), and HCCassociated antigen-519/targeting protein for Xklp-2 (HCA519/TPX2). These antigens may provide suitable targets for vaccine-based immunotherapies for patients with HCC (

Table. Targetable TAAs in Hepatocellular Carcinoma

10 to 15 AFP-derived epitopes in development

Multiple epitopes but very little activity

When considering the choice of peptide for an immune-based vaccine, several factors must be evaluated. Weak immune responses from native TAAs might be limiting; Hong and colleagues

used an in silicoguided method for optimizing epitopes and engineered a highly immunogenic AFP. An additional method for amplifying immune response is the use of fusion peptides. One example of such a peptide is the full-length hepatitis B virus (HBV) core protein fused with epitopes from HBx, AFP, and MAGE-3.

Some have argued that the use of HBV antigens alone does not make a sufficient target for an HCC vaccine therapy, as they have not been shown to be expressed stably on HBV-associated HCC cells, and therefore pose some additional risk in cases of liver-sparing treatment.

As such, the fusion peptide approach may pose a suitable comparable strategy. Alternatively, strategies that combine the use of vaccine-activated, TAA-specific T cells with a blockade against inhibitory receptors or immunoregulatory cell types may also provide effective antitumor suppression in HCC.

Despite the heightened antitumor immune response that may be mounted against HCC-specific antigens, overexpression of co-inhibitory signals, such as programmed cell death-1 (PD-1) or cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) checkpoint molecules, may dampen the antitumor response. As such, a focused research effort has been placed on developing clinically effective therapies that target and modulate the immune checkpoint blockade. Two monoclonal antibodies (mAbs) that are currently in clinical development and have shown some clinical efficacy in HCC are tremelimumab and nivolumab.

The anti-CTLA-4 antibody, tremelimumab, prevents the binding between CTLA-4 and its ligands on antigen-presenting cells, and subsequently inhibits the suppression of T-cell activation. In a phase I pilot study of 18 patients with HCC, the safety and feasibility of 2 dose levels of tremelimumab were evaluated when used in combination with either transcatheter arterial chemoembolization (TACE) or radiofrequency ablation (RFA), which have shown poor clinical efficacy on their own.

The results were recently presented at the 2015 American Society of Clinical Oncology (ASCO) Annual Meeting. The most common adverse event (AE) associated with tremelimumab was pruritus. Of those patients evaluable for a response, 40% showed a partial response (PR) and all showed immune cell infiltration with possible reductions in HCV viral load. The median progression-free survival (PFS) time was 7.4 months.

Ongoing phase I and phase I/II studies of tremelimumab in combination with other therapeutic approaches are currently under exploration. In a phase I study currently recruiting participants, tremelimumab will be combined with chemoembolization or ablation in patients with advanced HCC who have not responded to other treatments.

The primary outcome of the study will be the safety and feasibility of treatments.

In a phase I/II study, tremelimumab will be studied alone or in combination with MEDI4736, an anti-programmed cell death ligand-1 (PD-L1) antibody.

The primary outcomes will be determining the number of patients reporting AEs and the number of patients experiencing dose-limiting toxicities. A similar phase I study performed in patients with advanced non-small cell lung cancer (NSCLC) showed tolerable safety with high levels of clinical activity in patients with PD-L1-positive and PD-L1-negative tumors.

A phase III study is currently under way.

Tremelimumab has already been granted an orphan drug designation by the FDA for the treatment of malignant melanoma.

Nivolumab, an anti-PD-1 mAb, blocks the interaction between PD-1 and its ligand, PD-L1, enhancing the antitumor immune response. PD-L1 is highly expressed in many tumor types and is associated with a poor prognosis because of its immunosuppressive properties. Early clinical trials of nivolumab in patients with HCC are promising. In a phase I/II study presented at the 2015 ASCO Annual Meeting, the safety and preliminary efficacy of nivolumab was evaluated in 41 patients with advanced HCC.

In this dose escalation study, patients received nivolumab intravenously for up to 2 years. The most common drug-related AEs were increases in the levels of aspartate aminotransferase, alanine aminotransferase, lipase, and amylase. Grade 3/4 AEs occurred in 17% of patients. Of those patients with an evaluable response, 5% had a complete response (CR; response duration, 14-17 months) and 18% had a PR (response duration, 1-8 months). The overall survival (OS) at 6 months was 72%.

The results of the phase I/II trial are encouraging for patients with advanced HCC, who currently have very limited therapeutic options. Treatment responses with nivolumab were durable, and the results compared favorably with existing treatment options. The safety profile of nivolumab in this study was consistent with that previously reported with nivolumab in other types of tumors.

Nivolumab is currently approved for use in patients with unresectable or metastatic melanoma and metastatic squamous NSCLC.

Trials of nivolumab in HCC are ongoing. An expansion, dose-escalating, phase I trial will determine the safety of nivolumab in patients with HCC from 3 different cohorts: uninfected HCC subjects, HBV-infected patients, and hepatitis C virus (HCV)-infected subjects.

The primary outcomes of the study will determine the incidence of worst AEs and hematology and serum chemistry abnormalities, with secondary outcomes evaluating the objective response and disease control rates.

Adoptive Immunotherapy and Immunomodulation

Other immune-based therapeutic approaches still under investigation include the use of immunomodulators and adoptive immunotherapy. Interferon (IFN), a cytokine well known for its immunomodulatory properties, is known to induce HCC apoptosis and autophagy.

As such, IFN-alpha therapy is currently being evaluated in ongoing clinical trials. In one multicenter study currently recruiting participants, IFN-alpha will be analyzed as an adjuvant therapy after surgical resection in patients with HCC who express low levels of miR-26.

A phase II study will compare the combination of IFN-alpha plus fluorouracil (5-FU) versus cisplatin plus 5-FU in patients with HCC to determine OS and PFS rates.

Adoptive immunotherapy strategies also are being investigated as potential alternatives for patients who are refractory to conventional therapies. Notably, strategies using cytokine-induced killer (CIK) cells are furthest along in clinical development in studies of patients with HCC. These cells have some distinct advantages over other immune cell types, including strong cytolytic activity against tumor cells of both autologous and allogeneic origins, minimal toxicity, and high proliferation rate. At this time, there are numerous phase III trials exploring CIK cells in patients with radical resection of HCC.

Overall, numerous translational studies and early-stage clinical trials have shown that immunotherapy is safe and well tolerated by patients with a variety of cancers. Clinical studies of immune-based therapies, such as peptide-based vaccines and immune checkpoint blockade therapy, are in their infancy in HCC, but offer hope to patients with advanced disease. Although more randomized, controlled trials in HCC patients are needed, the incorporation of immunotherapy with standard antitumor approaches likely will provide patients with effective alternatives. The future outlook for immunotherapy in HCC is bright.

Bertino G, Demma S, Ardiri A, et al. The immune system in hepatocellular carcinoma and potential new immunotherapeutic strategies.

. 2015(2015). Article ID 731469. doi:10.1155/2015/731469.

Nobuoka D, Yoshikawa T, Sawada Y, et al. Peptide vaccines for hepatocellular carcinoma.

Bruix J, Takayama T, Mazzaferro V, et al; STORM investigators. Adjuvant sorafenib for hepatocellular carcinoma after resection or ablation (STORM): a phase 3, randomised, double-blind, placebo-controlled trial [published online September 8, 2015].

. pii: S1470-2045(15)00198-9. doi: 10.1016/S1470-2045(15)00198-9.

Miamen AG, Dong H, Roberts LR. Immunotherapeutic approaches to hepatocellular carcinoma treatment.

Butterfield LH, Ribas A, Meng WS, et al. T-Cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.

Komori H, Nakatsura T, Senju S, et al. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma.

Korangy F, Ormandy LA, Bleck JS, et al. Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-1 in hepatocellular carcinoma.

Liang J, Ding T, Guo Z-W, et al. Expression pattern of tumour-associated antigens in hepatocellular carcinoma: association with immune infiltration and disease progression.

Zerbini A, Pilli M, Soliani P, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma.

Mizukoshi E, Nakamoto Y, Marukawa Y, et al. Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma.

Aref AM, Hoa NT, Ge L, et al. HCA519/TPX2: a potential T-cell tumor-associated antigen for human hepatocellular carcinoma.

Hong Y, Peng Y, Guo ZS, et al. Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.

Articles

Clinical Articles

In recent years, knowledge of hepatocellular carcinoma (HCC)-specific tumor-associated antigens and the development of immune checkpoint blockade therapy has offered a positive outlook for patients with HCC.

Novel Immunotherapies on the Horizon in HCC

Hepatocellular carcinoma (HCC) has traditionally been an aggressive malignancy, with diagnosis taking place at later stages of disease and a poor prognosis in most patients. However, recent advances in molecular diagnostics and treatment show promise in changing the HCC clinical landscape.

Researchers have made particularly intriguing discoveries in the area of microRNAs (miRNAs), the small, noncoding RNAs associated with regulatory functions in gene expression. miRNAs associated with HCC can be used as biomarkers for the purposes of diagnosis and prognosis, and certain miRNAs have also been used as therapeutic targets.

As a part of the molecular pathology of cancer, many genes are dysregulated, including genes that express miRNAs. Chromosomal rearrangements, methylation of promoters, and transcriptional induction are all capable of dysregulating miRNA expression.

In many cases, the miRNAs that are dysregulated are associated with oncogenes or tumor-suppressor genes, thereby contributing to a pro-oncogenic signaling milieu within the cell.

In the specific case of HCC, a number of miRNAs have been shown to be differentially regulated in the serum, plasma, and cancer cells of an affected individual. As such, many of these miRNAs show promise as biomarkers for the purpose of molecular diagnostics in the context of both HCC diagnosis and prognosis.

early detection of HCC at a stage when the cancer is still surgically resectable (ie, in a single nodule smaller than 2 cm) provides the best odds of patient survival. Detecting HCC at such an early clinical stage is difficult using traditional diagnostic methods; thus, molecular diagnostics that make use of biomarkers represent a large unmet need in HCC care.

Although some molecular markers of HCC have already been described, such as serum &alpha;-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), and Dickkopf-1 (DKK1), these markers are neither specific for HCC nor present in all early-stage hepatocellular cancers.

As such, researchers have sought to identify novel biomarkers with enhanced specificity for HCC, whose presence can be detected reliably at the extreme early stages of HCC tumor progression. In addition, researchers have particularly sought out circulating biomarkers for HCC, as these markers can be detected noninvasively.

Whereas differentially regulated miRNAs have been detected noninvasively in serum, plasma, and urine derived from patients with HCC, the specificity and sensitivity of detecting these miRNAs has varied depending on the type of sample. Samples derived from patient serum have thus far demonstrated the best sensitivity and selectivity profile, suggesting that serum-derived miRNAs may be the most useful in HCC early diagnosis.

Fortunately, two miRNAsmiR-21 and miR-199a&mdash;have shown particular promise in early diagnosis of HCC. The expression of miRNA-21 is associated with reduced expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in HCC cells, whereas the expression of miRNA-199a is associated with suppression of pro-oncogenic MAPK signaling. Amr et al

found that miR-21 was significantly unregulated in the serum of patients with HCC, while miR-199a was significantly downregulated in patient serum. These investigators found that expression of miRNA-21 and downregulation of miRNA-199a discriminated patients with HCC from patients with chronic hepatitis (

Notably, in this study, miR-21 upregulation was able to predict HCC with a sensitivity of 100% and specificity of 81.2%, whereas miR-199a downregulation predicted HCC with a sensitivity of 54.5% and specificity of 100%. Clinical use of miRNA-21 and miRNA-199 expression to diagnose HCC will require that standardized threshold expression values be set.

Although miRNA-21 and miRNA-199 are useful in the early detection of HCC, these miRNAs are potentially less useful in monitoring the progression of HCC. Instead, another miRNA, miRNA-335, has been shown to be associated with HCC prognosis and treatment response. Cui et al

determined that serum levels of miR-335 are associated with response to transarterial chemoembolization (TACE) in HCC, and that miR-335 expression tracked overall HCC prognosis. These investigators found that miR-355 levels were significantly lower in patients with HCC than in patients with hepatitis or in healthy controls.

Low miR-335 expression levels were associated with HCC clinical features associated with tumor progression, including vascular invasion, cirrhosis, and larger tumor size; low miR-335 expression also correlated with poorer prognosis and shorter overall survival. Notably, TACE response rate was elevated in those patients expressing relatively higher levels of miR-335. Taken together, these results suggest that miR-335 can serve as a molecular marker for aggressive HCC disease with poor prognosis. However, standardized thresholds for miR-335 expression have yet to be determined.

Other miRNAs that may be useful in detecting HCC include miR-101, which is downregulated in HCC patient serum, and miR-375, which is upregulated in patient serum.

The combination of upregulated miR-23b, miR-423, miR-375, miR-23a, and miR-342-3p in patient serum is also strongly associated with HCC, as is the combination of miR-10a and miR-125b.2 These miRNAs also have been variously associated with tumor size, invasive potential, grade, migration, and recurrence.

Aside from their diagnostic and prognostic potential, miRNAs serve as valuable therapeutic targets. Indeed, many miRNAs are intimately involved in regulating the expression of genes that contribute to oncogenesis, tumor progression, and loss of tumor suppressor capabilities within the cell. One of the more promising miRNA therapeutic targets is miR-221, an oncogenic miRNA that is involved in growth, cell-cycle progression, cell proliferation, tumor invasion, and cell survival.

designed a &ldquo;microRNA sponge&rdquo; that inhibits miR-221 activity.

Their miR-221 sponge consists of adenovirus and adenovirus-associated viral vectors that drive expression of miR-221 binding sites, thereby sequestering miR-221 via competitive inhibition. In HCC cell culture, these miR-221 sponges were able to significantly reduce endogenous levels of miR-221, commensurate with a rise in expression of the cell cycle regulatory CDKN1B gene, a known target of miR-221. Moshiri and colleagues also noted that miR-221 sponge expression in HCC cells had a pro-apoptotic effect. Although preliminary, these results with miR-221 sponges are promising and warrant further testing of safety and efficacy in treating HCC.

Another promising miRNA target is miR-122. In the liver, miR-122 plays a role in lipid metabolism and is highly expressed. In some instances, miR-122 is capable of acting as a tumor suppressor; miR-122 levels are reduced in HCC cells, and expression of miR-122 is capable of reversing the malignant phenotype of these cells.

However, miR-122 also plays an important role in the development of HCC from hepatitis C virus (HCV) infection.

As an important host factor for hepatitis C virus replication, miR-122 strongly promotes the accumulation of HCV RNA. Thus, miR-122 is an attractive therapeutic target in HCC that is associated with HCV infection.

An anti-miR that targets miR-122, miravirsen, is capable of producing prolonged decreases in HCV RNA levels in patients with chronic hepatitis C, in a dose-dependent manner. Miravirsen is a locked nucleic acid anti-miR, composed of a DNA and phosphorothioate backbone that stably and specifically binds to its target miRNA, preventing it from exerting any further regulatory effects.

evaluated the virological response rate to the more traditional anti-HCV therapeutics peginterferon and ribavirin in patients taking miravirsen, assessing the long-term safety of miravirsen treatment in these patients. In their retrospective follow-up study, van der Ree and colleagues observed sustained virological response in 7 of 12 patients with HCV genotype 1 who received miravirsen followed by peginterferon and ribavirin. Among 27 patients who received miravirsen, there were no long-term safety issues or adverse events (AEs) over 35 months of follow-up. Thus, anti-miR-122 therapy for HCV infection achieved via administration of miravirsen appears to be safe and effective; however, larger-scale clinical trials are necessary before any definitive conclusions can be drawn.

Currently, the US Food and Drug Administration (FDA) requests a 5-year follow-up period for studies of anti-miR-122 therapies in humans because of the theoretical risk that HCC might be induced by lowered miR-122 expression. Moving beyond simple targeting of miRNA, some novel therapeutics seek to directly modulate miRNA expression. Xiao et al

have described a small-molecule modulator of miR-34a that is capable of inhibiting HCC growth. In a variety of cancer types, miR-34a serves as a tumor suppressor, inducing apoptosis and senescence and inhibiting cell proliferation. In HCC cells, miR-34a is typically severely downregulated or silenced completely.

performed a screen of small molecules and identified a compound named Rubone (2&rsquo;-hydroxy-2,4,4&rsquo;,5,6&rsquo;-pentamethoxychalcone) that rescued expression of miR-34a in HCC cell culture. These researchers determined that Rubone did not inhibit growth in nontransformed human hepatocytes, suggesting that Rubone can restore the tumor suppressor function of miR-34a in cancerous cells without damaging healthy tissue.

The prospects of Rubone are particularly exciting given that Rubone showed similar antitumor activity to sorafenib, the frontline anti-HCC therapeutic, with seemingly less potential for systemic toxicity. Notably, Rubone was effective in cells that expressed mutant or wild-type p53, but failed to restore miR-34a expression in cells that were p53-deficient. Xiao et al

therefore hypothesized that the pro-miR-34a activity demonstrated by Rubone is p53-dependent, and that Rubone achieves its effects on miR-34a expression by enhancing the DNA-binding or cofactor-recruiting function of p53 protein as it binds to the miR-34a promoter.

Taken together, the results described by Xiao et al

demonstrate that small molecules capable of modulating miRNA expression can serve as HCC therapeutic agents; however, these researchers performed their studies using only HCC cell culture and mouse xenograft models. These preclinical results warrant further investigation of the safety and efficacy of Rubone in human subjects. Although many applications for miRNA in HCC have been described in the scientific literature, much of the existing work has been conducted at the level of cell culture or xenograft models. The true clinical implications of miRNA are not yet fully understood.

A recent review described many of the potential applications of miRNA in clinical practice, along with the inevitable limitations of miRNA technology.

For example, one major limitation of using serum miRNA levels to diagnose HCC is that standardization of serum miRNA assays is currently lacking. The interpretability of serum miRNA assays is hampered by spurious signal from the miRNAs of contaminating blood cells and difficulties in normalizing expression data between studies due to differing sample collection methodologies. Thus, more and better standardized studies are warranted before many serum miRNAs can be taken into clinical practice as truly useful biomarkers.

Other limitations of miRNA technology that concern treatment of HCC have to do with the somewhat uncertain safety profile of modulating miRNA expression. Although preclinical trials have demonstrated that modulating miRNA expression does not have deleterious effects on nontransformed cells in culture, the effects of modulating miRNA expression in a whole person remain somewhat unclear, and safety studies looking at systemic toxicity have, in large part, yet to be conducted.

Because miRNA are usually pleiotropic, influencing many diverse phenotypes at once, modulating their expression can have unexpected effects on cellular physiology in systems entirely unrelated to HCC, and such effects must be carefully monitored before miRNA therapies can be used in the clinic. Along similar lines, the delivery mechanisms used to introduce miRNA and anti-miRNA into cells may have toxicity issues in a whole organism that are unapparent in cell culture and xenograft-based studies.

For example, many miRNA targeting strategies make use of viral vectors, which may have immunostimulatory and pro-inflammatory effects when used in a whole person, thereby inducing serious AEs. Nonetheless, whereas the full implications of miRNA technology for clinical practice have yet to be worked out, studies and clinical trials in human patients are currently ongoing.

One such study is a phase I trial sponsored by Mirna Therapeutics to evaluate the safety of a liposomal formulation of miR-34, known as MRX34.

The study investigators are evaluating MRX34 in patients with unresectable primary HCC, as well as in patients with other advanced metastatic cancers. Based on the hopeful results obtained with the miR-34aenhancing agent, Rubone, results from this human trial are highly anticipated by the scientific community.

Preliminary findings of the MRX34 trial, reported in April 2015 at the Annual Meeting of the American Association for Cancer Research, demonstrated that MRX34 had a reasonable safety profile in 23 patients with HCC. Adverse events associated with MRX34 treatment were chiefly attributable to infusion reactions, and included fever, chills, and back pain, as well as gastrointestinal symptoms. The patients with HCC treated with MRX34 experienced a dose-dependent repression of oncogenes known to be miR-34 targets in circulating blood cells, which suggests that MRX34 may be efficacious in the treatment of various cancer types.

Although fraught with challenges, miRNA technology offers many opportunities in HCC diagnosis, prognosis, and treatment. For a condition that is frequently diagnosed very late and with few treatment options, miRNA provides a promising avenue of study. Circulating miRNA biomarkers can allow for noninvasive diagnosis much earlier than other methods, and therapeutics that modulate expression of key oncogenic and tumor-suppressive miRNAs can complement the current unsatisfactory standard of care, namely resection and sorafenib treatment.

Although few in number presently, future clinical trials studying safety and efficacy of miRNA technologies in the management of HCC surely are forthcoming.

Ghidini M, Braconi C. Non-coding RNAs in primary liver cancer.

. 2015;2:36. doi:10.3389/fmed.2015.00036.

He S, Zhang D-C, Wei C. MicroRNAs as biomarkers for hepatocellular carcinoma diagnosis and prognosis [published online March 3, 2015].

. 2015;39(4):426-434. doi:10.1016/j.clinre.2015.01.006.

Amr KS, Ezzat WM, Elhosary YA, et al. The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma.

Cui L, Hu Y, Bai B, Zhang S. Serum miR-335 level is associated with the treatment response to trans-arterial chemoembolization and prognosis in patients with hepatocellular carcinoma [published online August 20, 2015].

. 2015;37(1):276-283. doi:10.1159/000430352.

Moshiri F, Callegari E, D'Abundo L, et al. Inhibiting the oncogenic mir-221 by microRNA sponge: toward microRNA-based therapeutics for hepatocellular carcinoma.

van der Ree MH, van der Meer AJ, de Bruijne J, et al. Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients [published online September 8, 2014].

. 2014;111:53-59. doi:10.1016/j.antiviral.2014.08.015.

Xiao Z, Li CH, Chan SL, et al. A small-molecule modulator of the tumor-suppressor miR34a inhibits the growth of hepatocellular carcinoma [published online September 12, 2014].

. 2014;74(21):6236-6247. doi:10.1158/0008-5472.CAN-14-0855.

Clinicaltrials.gov. A Multicenter Phase I Study of MRX34, MicroRNA miR-RX34 Liposomal Injection. https://clinicaltrials.gov/ct2/show/ NCT01829971?term=miR34&rank=1. Accessed September 17, 2015.

Hepatocellular carcinoma has traditionally been an aggressive malignancy, with diagnosis taking place at later stages of disease and a poor prognosis in most patients. However, recent advances in molecular diagnostics and treatment show promise in changing the HCC clinical landscape.

Role of MicroRNAs Evolving in the Diagnosis and Treatment of HCC

To date, no established regimens exist for the treatment of patients with advanced hepatocellular carcinoma (HCC) after failure of first-line treatment with the multikinase inhibitor sorafenib. Outcomes in the setting of sorafenib resistance or intolerance are poor, with a median expected overall survival (OS) for the placebo arms of second-line trials in the range of 7 to 8 months.

A recent study evaluating survival among 260 patients with HCC permanently discontinuing sorafenib reported median OS durations of 4.1 months overall, 4.6 months in patients with progression, and 1.8 months in patients with decompensated disease.

The search for an active second-line regimen in advanced HCC has been marked by multiple disillusions: Within the past few years, 3 candidate agents failed to meet primary endpoints in large phase III trials, including brivanib (BRISK-PS study), everolimus (EVOLVE-1 study), and ramucirumab (REACH study).

Brivanib, a dual inhibitor of the vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR)mediated pathways, was the first agent evaluated in second-line therapy for HCC. A phase II study reported promising antitumor activity and a median OS of 9.79 months among patients with HCC resistant to antiangiogenic regimens, suggesting that FGF targeting may overcome resistance to antiangiogenic therapy.

However, the subsequent phase III BRISK-PS study involving 395 patients with HCC who progressed during or after sorafenib treatment or were intolerant to the drug failed to demonstrate a survival benefit versus placebo.

Compared with placebo, brivanib was associated with significant improvement in objective response rate (ORR; 10% vs 2% with placebo;

= .003) and other secondary endpoints, including time to progression (TTP; 4.2 vs 2.7 months;

However, brivanib did not meet the prespecified primary endpoint of OS improvement (9.4 months vs 8.2 months with placebo; hazard ratio [HR], 0.89;

=.3307). The minimum HR of 0.78 to demonstrate positivity was also not met after adjustment for prognostic factors (adjusted HR, 0.81). Treatment-related toxicities were substantial, with a discontinuation rate secondary to adverse events (AEs) of 23%.

Despite preliminary antitumor activity observed in phase I/II trials, the mTOR inhibitor everolimus failed to demonstrate clinical benefit in the phase III EVOLVE-1 study.

The survival of patients with HCC progression on or after sorafenib allocated to everolimus (n=362) or placebo (n=184) did not differ (median OS, 7.6 vs 7.3 months; HR, 1.05;

=.68); everolimus also did not significantly affect secondary endpoints including TTP (HR, 0.93).

of ramucirumab, a fully human IgG1 monoclonal antibody (mAb) targeting VEGFR-2, failed to detect a significant overall OS benefit versus placebo (median OS, 9.2 months vs 7.6 months; HR, 0.87;

=.14). Ramucirumab was associated with significant improvement in progression-free survival (PFS; 2.8 months vs 2.1 months; HR, 0.63;

Despite overall disappointing results, subgroup analysis of this trial revealed that patients with elevated baseline levels of serum alpha-fetoprotein (AFP), a marker of poor prognosis, benefited from ramucirumab, a finding that is being further investigated in an ongoing phase III trial.

Proposed reasons to explain the recent trial failures in second-line therapies include bias between treatment arms caused by inadequate patient stratification. According to David Bolos, MD, and Richard S. Finn, MD, from the David Geffen School of Medicine, University of California, Los Angeles, &ldquo;strata used in the front-line setting may not be applicable in the second-line setting.&rdquo;

In particular, separate consideration of 2 key prognostic factors for survival, extrahepatic spread and vascular invasion, has been proposed future trials.

Other relevant factors to explain the recent phase III trial failures include insufficient exploration of liver toxicities in phase II trials that may have led to more discontinuations than anticipated; misinterpretation of the predictive value of the phase II trial endpoint TTP on OS; and lack of analysis of biomarkers that might predict response to treatment.

Consequently, the design of ongoing trials has been reconsidered and adapted to include more thorough exploration of toxicity and pharmacokinetics of novel agents in dose-escalation studies, as well as approaches for marker-guided patient selection.

Identifying Genetic Signature  Personalized Therapy for HCC

The molecular heterogeneity of HCC, which may contain as many as 30 to 40 different mutations per tumor, including 5 to 8 mutations in putative oncogenic driver genes, renders the identification of key oncogenic pathways that would guide selection of targeted treatment challenging.

Encouraging findings for a potential enrichment strategy based on signaling pathway activation were made in a randomized phase II trial with the small-molecule MET inhibitor tivantinib: Patients with tumors expressing high levels of MET had a survival benefit, and tumor MET expression appeared both predictive and prognostic.

The MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) are implied in carcinogenesis and metastatic progression in HCC; 25% to 87% of HCC overexpresses MET. High-MET expression is particularly frequent in advanced tumors with vascular invasion.

In the phase II trial, 107 patients with advanced HCC with progression on or intolerance to sorafenib were randomly allocated at a 2:1 ratio to tivantinib (n=71; 39 with initial starting dose of 360 mg twice daily, amended to 240 mg twice daily due the incidence of severe neutropenia) or placebo (n=36).

In the overall study population, tivantinib was associated with significantly longer TTP (1.6 vs 1.4 months; HR, 0.64; 90% CI, 0.43-0.94

Tumor MET expression was assessed by immunohistochemistry in 77 patients (72%), revealing high-MET tumors in 37 patients (48% of the study population). Patients with high-MET tumors had significant benefits with tivantinib treatment, resulting in prolongation of median TTP (2.7 months vs 1.4 months with placebo; HR, 0.43;

=.03) and of median OS (7.2 months vs 3.8 months with placebo; HR, 0.38;

Tumor MET status appeared predictive for response to treatment: Patients with low-MET tumors did not benefit from tivantinib treatment (median TTP, 1.5 months vs 1.4 months with placebo; HR, 0.96;

=.92; median OS, 5.0 months vs 9.0 months with placebo; HR, 1.33;

Outcomes from the phase II trial also suggest MET expression as a prognostic factor for survival in pretreated patients with HCC. Among patients receiving placebo, patients with high-MET tumors had a significantly shorter median OS than patients with low-MET tumors (3.8 months for high-MET vs 9.0 months for low-MET; HR, 2.94;

Tivantinib as second-line treatment in HCC is currently being investigated in a randomized phase III study in a selected population of patients with high-MET advanced HCC after sorafenib failure (METIV-HCC trial; ClinicalTrials.gov identifier NCT01755767). The primary endpoint is OS, and secondary endpoints include progression-free survival (PFS) and safety; the planned enrollment is 346 patients.

Promising outcomes in the second-line treatment of advanced HCC have also been observed with cabozantinib (XL-184), a small-molecule inhibitor of MET, RET, and VEGFR. A phase II study including 41 patients with advanced HCC reported clinical activity, with a 12-week DCR of 68%, median PFS of 4.4 months, and median OS of 15.1 months.

Based on these results, a randomized phase III trial (CELESTIAL; ClinicalTrials.gov identifier NCT01908426) has been initiated to evaluate cabozantinib versus placebo in patients with advanced HCC that progressed following sorafenib. With a targeted recruitment of 760 subjects, the study projects 90% power to detect 31.6% increase in the primary endpoint, OS (HR, 0.76).

Biomarker-Driven and Novel Multi-Pathway Strategies

A different biomarker-driven approach has emerged from outcomes of the phase III REACH trial

evaluating ramucirumab. Although this study failed to detect a significant OS benefit in the overall study population, a prespecified subgroup of patients with elevated baseline AFP >400 ng/mL had a 20% ORR and significant improvement in survival after receiving ramucirumab (median OS of 7.8 months vs 4.2 months with placebo; HR, 0.67;

The molecular mechanisms for this correlation remain unclear, but according to the authors, high &alpha;-fetoprotein expression may be associated &ldquo;with a particular subtype of hepatocellular carcinoma with increased angiogenesis and possibly enhanced sensitivity to VEGFR-2 inhibition.&rdquo;

Based on these findings, the phase III REACH-2 trial (ClinicalTrials.gov identifier NCT02435433) has been initiated to assess ramucirumab in a second-line setting in patients with elevated AFP. In a post-hoc analysis of REACH, ramucirumab treatment led to a greater reduction in the risk of death in patients with progressively higher baseline AFP values.

The multistep oncogenic transformation underlying HCC involves alterations in several signaling cascades, resulting in molecularly heterogeneous tumors. Presumptive key carcinogenic signaling pathways include the Wnt/&beta;-catenin pathway, chromatin remodeling, oxidative stress, and signaling involving EGF, PDGF, FGF, VEGF, and IGF, and intracellular mediators such as RAS/RAF/MAPK and PI3K/AKT. The simultaneous inhibition of multiple pathways is, therefore, an appealing concept that is currently being explored using the multikinase inhibitor regorafenib.

Structurally related to sorafenib, regorafenib is a potent inhibitor of kinases involved in angiogenic, oncogenic, and stromal/tumor environment-related signaling cascades, including VEGFR1-3 and TIE2, c-kit, Ret, BRAF, and PDGFR, among others.

Data from a single-arm phase II study have shown an acceptable safety profile and evidence of antitumor activity of regorafenib in patients with advanced HCC with progression on sorafenib.

Disease control was achieved in 26 of 36 patients (72%), median TTP was 4.3 months, and median OS was 13.8 months.

Based on these outcomes, the phase III RESORCE trial (Regorafenib after Sorafenib in Patients with Hepatocellular Carcinoma; ClinicalTrials.gov identifier NCT01774344) has been initiated to compare the efficacy and tolerability of regorafenib versus placebo in patients with advanced HCC that has progressed on sorafenib. The primary endpoint is OS; secondary endpoints include TTP, PFS, objective tumor response, DCR, and safety.

Cancer immunotherapy has emerged as a highly effective therapeutic approach in multiple solid malignancies, and recent evidence suggests promise in the treatment of advanced HCC.

Data from an ongoing phase I/II trial of nivolumab, a fully human mAb directed against the immune checkpoint receptor programmed cell death protein-1 (PD-1) indicate safety and efficacy in patients with advanced HCC.

Both the PD-1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA- 4) inhibitory immune checkpoints normally temper T-cell responses to avoid damage to self; however, overexpression of these receptors and their ligands in cancers mediates escape from an antitumor response.

No established regimens exist for the treatment of patients with advanced hepatocellular carcinoma after failure of first-line treatment with the multikinase inhibitor sorafenib. Outcomes in the setting of sorafenib resistance or intolerance are poor, with a median expected overall survival for the placebo arms of second-line trials in the range of 7 to 8 months.

Second-Line Treatments for Advanced Hepatocellular Carcinoma 

Treatment with tivantinib was highly effective in patients with hepatocellular carcinoma (HCC) with high MET expression, suggesting a prognostic and predictive biomarker of response, according to findings from a phase II study presented at the 2015 International Liver Cancer Association Annual Conference.

In the analysis, which was conducted on data from the ARQ-197-215 trial, the risk of death was reduced by 62% with tivantinib compared with placebo in patients with MET-high HCC (HR, 0.38; 95% CI, 0.18-0.81). Conversely, in MET-low tumors, placebo outperformed tivantinib (HR, 1.33; 95% CI, 0.58-3.03). In this analysis, immunohistochemistry (IHC) was an effective test for MET expression, if strict testing criteria were followed.

The ongoing phase III METIV-HCC study is currently exploring tivantinib in patients with MET-high inoperable HCC who have received prior treatment with sorafenib. The study remains open for recruitment, with the goal of randomizing 346 patients to tivantinib or placebo in a 2:1 ratio. By the end of 2015, the company developing the drug, ArQule anticipates that 300 patients will have enrolled (NCT01755767).

"The biomarker data from this trial demonstrates that patients with MET-high tumors are more likely to benefit from tivantinib therapy," Brian Schwartz, MD, head of research and development at ArQule, said in a statement. "On the basis of the results from the phase II trial and in partnership with Daiichi Sankyo, we are conducting the pivotal phase III METIV-HCC trial that is enrolling MET-high HCC patients as determined by a required companion diagnostic test."

In the phase II study that was the basis for the biomarker analysis, 107 patients were randomized to tivantinib (n = 71) or placebo (n = 36). This analysis did not restrict enrollment to MET-high individuals but did assess tumor samples for MET expression, with a high expression level defined as &ge;2+ in &ge;50% of tumor cells.

In the full population of the study, the median time to progression in patients treated with tivantinib was 6.9 weeks compared with 6.0 weeks with placebo (HR, 0.64;

= .04). The median progression-free survival was 1.7 months for the tivantinib group versus 1.5 months for the placebo arm (HR, 0.67;

In the biomarker analysis, 77 samples were available for analysis. MET was overexpression more commonly in patients who received prior sorafenib. In this group, MET-high status was seen in 82% of patients compared with 40% for samples taken prior to the administration of sorafenib. Additionally, following sorafenib treatment, just 3 patients (18%) were MET-low.

As a prognostic factor, MET overexpression was associated with significantly worse outcomes. For patients treated with placebo, those with MET-low expression had a median overall survival (OS) of 9 months compared with 3.8 months in the MET-high group (HR, 0.34; 95% CI, 0.13-0.86;

A significant interaction was seen between tivantinib and tumor MET levels as a predictive factor for OS (

= .039). The median OS in the placebo arm for patients with MET-low status was 9 months versus 7.2 months in the MET-high tivantinib group (HR, 0.72; 95 % CI, 0.30-1.70;

In the presentation, lead investigator Lorenza Rimassa, MD, deputy director of the Medical Oncology Unit at Humanitas Cancer Center in Milan, Italy, noted that tumor MET status was the only prognostic and predictive factor out of the biomarkers analyzed.

"Overall, the biomarker data from this trial support the use of tivantinib in MET-high patients only," she said during her presentation of the results. "The ongoing phase III METIV-HCC trial will validate the role of biomarkers in HCC."

Rimassa L, Abbadessa G, Personeni N, et al. Tivantinib in Pretreated Hepatocellular Carcinoma (HCC): Tumor and Plasma Biomarker Analysis from the Randomized Controlled Phase 2 Trial (RCT) ARQ 197-215. Presented at: International Liver Cancer Association 9th Annual Conference; September 4-6; Paris, France. Abstract O-029.

Treatment with tivantinib has proven highly effective in patients with hepatocellular carcinoma and with high MET expression, suggesting a prognostic and predictive biomarker of response.


Tivantinib Biomarker Analysis Validates Phase III Design

The presence of circulating tumor DNA in serum could predict microscopic vascular invasion of the portal vein and extrahepatic metastasis following surgery for patients with hepatocellular carcinoma (HCC), according to a study published in

Cellular and Molecular Gastroenterology and Hepatology (CMGH)

In the analysis, whole-genome sequencing was performed on pre- and post-surgical serum samples from 46 patients who were undergoing HCC resection. Various mutations were detected in all patients, with circulating tumor DNA found in 15.2% of samples. Overall, circulating DNA was associated with a larger tumor size and increased risk of tumor recurrence within 2 years. Moreover, circulating tumor DNA levels were found to increase with disease progression.

"We uncovered that circulating tumor DNA levels accurately reflect cancer progression and therapy effects on hepatocellular carcinoma," said lead study author Atsushi Ono, PhD, from Hiroshima University. "With further research, the identification of genome profiles through circulating tumor DNA analysis may guide individualized management of hepatocellular carcinoma."

In the study, circulating tumor DNA was detected in 100 &mu;L of serum in 7 of the 46 patients before surgery. When compared with the negative group, those with detectable circulating tumor DNA had a statistically significant increase in the cumulative incidence of recurrence (

= .0386). In a multivariate analysis, circulating tumor DNA was found to be an independent predictor of microscopic vascular invasion of the portal vein (odd ratios, 6.10; 95% CI, 1.11-33.33;

"Circulating tumor DNA analysis represents a potential paradigm shift in personalized medicine for liver cancer," Jerrold R. Turner, MD, PhD, editor-in-chief,

, said in a statement. "We look forward to seeing future research build on this encouraging data to ultimately improve the effectiveness of therapy against advanced hepatocellular carcinoma."

Ono A, Fujimoto A, Yamamoto Y, et al. Circulating Tumor DNA Analysis for Liver Cancers and Its Usefulness as a Liquid Biopsy, Summary.

Cellular and Molecular Gastroenterology and Hepatology.

The presence of circulating tumor DNA in serum could predict microscopic vascular invasion of the portal vein and extrahepatic metastasis following surgery for patients with hepatocellular carcinoma.

Monitoring Circulating Tumor DNA Could Improve Outcomes in HCC

The FDA has granted the A3AR agonist CF102 a fast track designation as a second-line treatment for patients with hepatocellular carcinoma (HCC), according to a statement from the drug's developer, Can-Fite BioPharma.

The safety and efficacy of CF102 has been assessed in an open-label phase I/II trial for patients with advanced unresectable HCC. The median overall survival (OS) was 7.8 months with CF102 in the full population. In those with CP class B tumors, the median OS was 8.1 months. At a 4-month analysis, stable disease by RECIST criteria was seen in 22% of patients. Liver function was maintained over a 6-month period.

"We are very pleased that the FDA recognizes the potential for CF102 to treat HCC patients who have tried, and not been responsive to Nexavar, the only FDA approved drug currently on the market for this indication," Pnina Fishman, PhD, chief executive officer and co-founder of Can-Fite, said in a statement. "We consider Fast Track designation to be a major catalyst for our CF102 development program and we believe it could shorten our time to market for CF102, thereby making a considerable difference for patients."

CF102 selectively binds to A3AR on the surface of cells, causing deregulation of the Wnt and NF-&kappa;B signal transduction pathways. A3AR, which plays a role in cellular proliferation, is highly expression on the surfaces of various solid tumors, specifically HCC.

The phase I/II study examined the pharmacokinetic (PK) behavior of CF102 at varying doses. Additionally, A3AR was examined as a potential predictive biomarker of response.

The study enrolled 18 patients, 28% with CP class B tumors and 67% who had received prior sorafenib. PK data demonstrated good oral bioavailable, and there were no serious adverse events or dose-limiting toxicities.

The study found a correlation between expression of A3AR and OS, warranting the initiation of further studies for a blood-based biomarker test kit. In March 18, 2015, the company announced that it had completed the development of a commercial predictive blood test for A3AR that could return results within a few hours.

&ldquo;The completion of the development of our commercial A3AR biomarker test kit comes at a very important time since we plan to use the test kit in our upcoming advanced clinical studies,&rdquo; said Fishman. &ldquo;We believe the test kit will create efficiencies in our trials in patient enrollment and monitoring."

CF102 received an orphan drug designation as a second-line treatment for HCC in February 2012. The fast track and orphan drug designations allow Can-Fite to complete a rolling submission of data to the FDA for a new drug application.

A phase II study exploring CF102 is currently being conducted in the United States, Europe, and Israel. In this 78-patient randomized study, the agent will be compared with placebo in those with CP class B cirrhosis following progression on sorafenib. The primary endpoint of the study is OS (NCT02128958).

In addition to HCC, Can-Fite plans a phase III study for CF102 in patients with rheumatoid arthritis. Additionally, the company is planning studies for patients with advanced psoriasis. Outside of these indications, the agent has shown promise in proof-of-principle studies for patients with colon cancer, prostate cancer, and melanoma.

Stemmer SM, Benjaminov O, Medalia G, et al. CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.

The FDA has granted the A3AR agonist CF102 a fast track designation as a second-line treatment for patients with hepatocellular carcinoma. The drug is also planned to be tested in patients with rheumatoid arthritis and psoriasis. 

October 2015 | HCC Monitor

Novel Immunotherapies on the Horizon in HCC

Role of MicroRNAs Evolving in the Diagnosis and Treatment of HCC

Second-Line Treatments for Advanced Hepatocellular Carcinoma

Tivantinib Biomarker Analysis Validates Phase III Design

Monitoring Circulating Tumor DNA Could Improve Outcomes in HCC

CF102 Receives Fast Track Designation for HCC