Therapeutic options in hepatocellular carcinoma: a comprehensive review

Hepatocellular carcinoma (HCC) is a chronic liver disease that is highly fatal if not detected and treated early. The incidence and death rate of HCC have been increasing in recent decades despite the measures taken for preventive screening and effective diagnostic and treatment strategies. The pathophysiology of HCC is multifactorial and highly complex owing to its molecular and immune heterogeneity, and thus the gap in knowledge still precludes making choices between viable therapeutic options and also the development of effective regimens. The treatment of HCC demands multidisciplinary approaches and primarily depends on tumor stage, hepatic functional reserve, and response to treatment by patients. Although curative treatments are limited but critical in the early stages of cancer, there are numerous palliative treatments available for patients with intermediate and advanced-stage HCC. In recent times, the use of combination therapy has succeeded over the use of monotherapy in the treatment of HCC by achieving effective tumor suppression, increasing survival rate, decreasing toxicity, and also aiding in overcoming drug resistance. This work focuses on reviewing the current and emerging treatment strategies for HCC.


Introduction
Liver cancer is the third leading cause of cancer-related mortality worldwide, accounting for 830,180 deaths and 905,677 new cases in 2020 [1].Hepatocellular carcinoma accounts for 80% of primary liver cancers and has become a serious public health issue with its increase in both incidence and mortality rates [2].There are numerous etiological risk factors for HCC including, viral infections (Hepatitis B virus and Hepatitis C virus), diabetes, obesity, fatty liver disease, alcohol consumption, tobacco smoking, aflatoxins, genetic factors and environmental toxins that act as carcinogens [3].HBV is the leading cause of HCC and mortality worldwide (33%), followed by alcohol (30%), HCV (21%), and other factors (16%) [4].The geographical distribution of HCC incidence is attributed to the variation in the prevalence of these varied risk factors.In developing countries such as Asia and Africa, 70% of HCC is attributed to HBV infection, the condition in which the double-stranded circular DNA of the virus gets incorporated into the human genome, causing p53 inactivation, oxidative stress, and inflammation leading to hepatocarcinogenesis [5].Since a decade, there has been a 9% increase in HCC cases attributed to nonalcoholic fatty liver disease in Europe and the USA, compared to HCV cirrhosis, which previously accounted for 60% of HCC cases.Unlike HBV, the HCV protein modulates a wide range of cellular activities in HCV-induced HCC, including transcriptional regulation, epigenetic alterations, and cytokine modulation, all of which contribute to chronic liver injury, cirrhosis, and HCC development [6].The pathological state characterized by insulin resistance, hyperglycemia, and inflammation in obesity and diabetes leads to increased endoplasmic reticulum (ER) stress and oxidative stress, changes in the gut microbiota, and dysregulated adipokines, all of which contribute to the pathogenesis of HCC [7].Furthermore, the contribution of alcohol to the HCC burden varies across countries (Europe: 16-78%, America: 17-52%, Oceania: 15-37%, and lowest in Asia: 0-41%) [8].In Africa and China, aflatoxin exposure and p53 mutations have been tightly linked, contributing to 40% of HCC, with comparatively negligible cases in North America and Europe [9].Thus, the prolonged period of exposure to etiologies results in hepatic injury due to inflammation followed by hepatocyte necrosis, regeneration, and transition to fibrosis, cirrhosis, and thereby the development of HCC [10,11].Because of the complicated pathophysiology of HCC, which involves changes in numerous signaling pathways, treating HCC requires interdisciplinary approaches, which makes the procedure difficult [12].
The treatment options for HCC are mainly based on the tumor stage, and this classification varies across the globe.The currently used classifications to determine the treatment strategies include the Barcelona Clinic Liver Cancer (BCLC) classification, the Hong Kong Liver Cancer (HKLC) score, the Japan Integrated Staging (JIS) score, and the Chinese Liver Cancer (CNLC) classification (Table 1).The other scoring systems that are used to predict survival are the Okuda score, the Cancer of the Liver Italian Program score, the Tumor-Node Metastasis (TNM) score, the Chinese University Prognostic Index (CUPI) score, the Tokyo score, the Bilirubin-albumin-AFPL3-AFP-DCP (BALAD) score, the Advanced Liver Cancer Prognostic System (ALPCS), and the Taipei Integrated Score System (TIS) [13].However, with the heterogeneity associated with HCC, other factors such as the severity of liver dysfunction, patient performance, availability of resources, and expertise play a critical role in the choice of therapy [14].While surgical resection, liver transplantation, and ablation therapies continue to be effective in the early stages of HCC, embolization and radiation therapy are used in the intermediate stages.Other treatment options such as chemotherapy, molecular targeted therapy, immunotherapy, and gene therapy are used to treat advanced stages of HCC [15] (Fig. 1A and B).Although all these treatment options are found to be effective in treating HCC, in recent times, combination therapy has been used for its broad-spectrum activity and efficacy.The use of combination therapy for unresectable HCC has also gained importance, and the use of different agents in combination may be potentially effective against HCC by inhibiting tumor growth and increasing survival [16].Undoubtedly, improved treatments may result in a better prognosis.In this review, we will cover the different treatment options for HCC and shed light on the combination therapies that are currently available and under investigation for the treatment of advanced HCC.

Surgery
Curative treatments are first-line treatments for patients with HCC at early stages, defined by a single liver tumor (< 2 cm) or three nodules (≤ 3 cm).Patients at this early-stage benefit from surgery that involves either surgical resection or liver transplantation [17].Partial liver resection would be successful in patients (15-20%) with good liver function having a single tumor without vascular invasion.Since most patients with HCC also have cirrhosis, utmost care is taken in assessing the liver function based on the Child-Turcotte-Pugh (CTP) score and the Model for End-Stage Liver Disease (MELD) system [18].Iwao Ikai et al. have reported the cumulative survival rates at 1, 5, and 10 years in HCC patients with cirrhosis (n = 6785) treated with hepatic resection between 1988 and 1999 to be 85.2%, 45%, and 20.7%, respectively.Patients undergoing surgical resection for HCC at an early stage (≤ 2 cm tumor size) may have a 5-year survival rate of up to 67.4% [19].It is interesting to note that the reports of Western and Asian studies have similar results in terms of survival rates after curative surgical resection of HCC [20].A recent study by Wee et al. showed that surgical resection treatment can also be taken into consideration for BCLC system patients who are in the intermediate stage and fall outside of BCLC A. This is because BCLC B, those who meet the "Up-To-7" criteria and its subgroups have 5-year overall survival rates that are higher than 50% [21].However, Roayaie et al. discovered that as a result of the processes that underpin the growth of intra-and extrahepatic recurrence, up to 70% of patients experience a disease recurrence in the first five years after a margin-free surgical resection [22].Undoubtedly, HCC patients in the early stages may benefit from surgical resection and achieve survival benefits with advanced minimally invasive surgical technologies and peri-operative management.
Liver transplantation remains an outstanding treatment option as it obviates HCC and resolves the underlying hepatic inefficiency, resulting in the long-term survival of the patients.The stage of HCC is very critical when opting for liver transplantation as extrahepatic metastasis and vascular invasion may make the therapy futile [2].The Milan criteria have been devised to specify the anatomic extent of the illness that HCC patients should be assessed for eligibility for liver transplantation for patients with a singular lesion of ≤ 5 cm or up to three lesions of ≤ 3 cm in the maximum diameter and without vascular invasion [23].Despite the limitations of not taking tumor biology into account, the Milan Criteria (MiC) proposed by Mazzaferro and colleagues remain the gold standard for the selection criteria of HCC patients for liver transplantation as it indicates a high post-transplant survival rate compared to others [23,24].The number of liver transplants varies worldwide, and as of 2018, America has the highest rate, followed by Europe, the Western Pacific, South East Asia, the Eastern Mediterranean, and Africa [25].According to the organ procurement and transplantation network (OPTN), 8896 liver transplants were performed in the USA in 2019.Of those, about 1200 liver transplants were done in people with HCC [26].Although liver transplantation offers a potential therapy for HCC when opted at early stages, it remains a major challenge due to the shortage of organ donors, as well as unprecedented clinical outcomes and costs associated with the procedure.

Ablation
This curative treatment option is employed in HCC patients with poor health or reduced liver function despite the detection at an early stage.Of note, tumor location and size represent a crucial factor in choosing ablation instead of surgery.Although this minimally invasive technique does not remove tumors, it causes complete remission given that tumors are small, usually less than 5 cm in diameter.Ablation is used as a treatment option in liver metastasis to reduce the risk of cancer recurrence [27].Additionally, ablation can also be repeated in the case of tumor recurrence or applied in combination with surgery.Several types of ablation techniques have been developed to destroy small tumors, including the application of chemical agents or energy sources.In the chemical ablation process, either ethanol (PEI, percutaneous ethanol injection) or acetic acid (PAI, percutaneous acetic acid injection) is injected directly into the tumor to destroy the cancer cells.Energy-based ablation includes thermal ablation (radiofrequency ablation, microwave ablation, laser photocoagulation), cryoablation, high-intensity focused ultrasound, or irreversible electroporation that causes the cancer cells to die [28].A study of a randomized controlled trial involving 187 HCC patients has demonstrated that radiofrequency thermal ablation therapy is most effective and provides higher overall survival than percutaneous ethanol and acetic acid injection in treating early-stage HCC [29].On the other hand, studies by Miura et al. suggest that hepatic resection provides better overall survival compared to ablation, but this conclusion seems irrational as the study captures all types of ablation that might have influenced the outcome [30].Hence, it is evident that multiple factors, including patient profile, tumor characteristics, and technical expertise, have an impact on treatment allocation [18].Albeit possessing a safety profile and good efficacy, the 5-year tumor recurrence rate after resection and after radiofrequency ablation is found to be more than 50% and up to 80%, respectively [31], thus emphasizing the need for alternative therapeutic options for the treatment of HCC.

Embolization therapy
HCC patients who have a tumor burden but are confined to the liver, meaning unresectable cancer that has not spread, are considered to have intermediate-stage HCC and chemoembolization therapy is of great benefit to these patients to ensure preserve their liver function [32].The embolization procedure involves blockage of the hepatic artery, causing a reduction in the blood supply to the tumor, and is also termed transcatheter therapy or transcatheter liver-directed intra-arterial therapy [33].This technique exploits the distinct characteristic of blood supply to the cancer cells in the liver that is usually fed by the hepatic artery versus the portal vein that supplies blood to the normal hepatic cells, thus leaving the healthy cells unharmed [34].The types of embolization therapy vary with the methodology of how the hepatic artery has been blocked and include trans-arterial embolization (TAE-agents such as gelatin sponge, starch microspheres, polyvinyl alcohol beads, or collagen particles are injected into the catheter inserted into the hepatic artery), trans-arterial chemoembolization (TACE-giving chemotherapy drugs via the catheter into the hepatic artery), drug-eluting bead trans-arterial chemoembolization (DEB-TACE-tiny beads containing chemotherapy drugs are injected into the catheter), and trans-arterial radioembolization (TARE-small beads having radioisotope injected into the hepatic artery), all of which aims in inducing vascular occlusion [35][36][37][38][39].A prospective randomized study by Lammer J et al., involving 212 patients from 19 centers in 5 countries, has shown that DEB doxorubicin-TACE is more effective than doxorubicin-TACE [40].Above all, the studies carried out by two independent groups in 2002 provided strong evidence that chemoembolization therapy significantly improved the overall survival rate of the patients, underscoring the role of arterially directed therapies in the treatment of HCC [41,42].

Radiation therapy
With the impact of rapidly evolving technologies, radiotherapy has advanced over the past decades and is considered a treatment option for candidates ineligible for other local therapeutic options with unresectable HCC [43].Hence, as this technique employs high-energy rays to kill cancer cells, it is very crucial to avoid damaging normal liver cells during the treatment.Conformal radiation therapy is employed to treat focal HCC as it delivers tumoricidal doses with low rates of toxicity.Stereotactic body radiation therapy (SBRT) has been used for patients with Chinese liver cancer (CNLC) stages Ia and Ib HCC.For patients with CNLC stages IIa, IIb, and IIIa HCC, TACE in combination with external beam radiation therapy (EBRT) prolongs survival.In addition, EBRT has been employed to treat lymph node metastasis [44], bone metastasis [45], and soft tissue metastasis [46] in patients with HCC.External radiotherapy can also serve as a bridge treatment before liver transplantation for HCC.Radiation therapy has also been shown to be effective in the treatment of HCC with portal venous thrombosis.Thus, all stages of HCC may benefit from radiation therapy, especially liver-confined HCC that is unsuitable for or resistant to other locoregional treatments.Various studies have provided evidence that the use of radiation therapy has resulted in improved outcomes in terms of overall survival and efficacy when compared to other local therapies for HCC [47][48][49].Furthermore, studies by Sapir et al. in 209 patients who underwent TACE (n = 84) and SBRT (n = 125) over a period of 8 years, have demonstrated that SBRT is a safe and effective treatment when compared to TACE for HCC [50].However, it is important to note that tumor volume, liver function, and tumor location all play important roles in determining the radiation dose and its effective delivery without affecting nearby organs.

Chemotherapy
As the treatment regimen depends on the stage of HCC, chemotherapy is an option for intermediate or advancedstage HCC patients, for whom curative treatments will no longer be helpful [51].The reasons that lead to palliative therapies such as chemotherapy, molecular targeted therapy, immunotherapy, and gene therapy include tumor size, metastasis, liver function status, vascular invasion, multifocality, and late diagnosis of HCC [15].In chemotherapy, drugs are used to destroy cancer cells either by injecting them into the vein (intravenous-IV) or taking them by mouth, which possibly reaches all areas of the body via circulation (systemic chemotherapy), or by injecting drugs directly into the hepatic artery so that they reach only the liver, performing site-specific action (regional chemotherapy).Since traditional chemotherapy drugs target cells at different phases of the cell life cycle (Fig. 2), there is a possibility that they may damage not only cancer cells but also healthy cells.Chemotherapeutic drugs are either topoisomerase inhibitors or cell cycle inhibitors that are cytotoxic and possess diverse anticancer mechanisms [52].Several studies have proved that the response rates of traditional chemotherapeutic agents like doxorubicin, oxaliplatin, mitoxantrone, cisplatin, gemcitabine, and 5-fluorouracil are low and show only marginal improvements in survival along with toxicity.Hence, the efficacy of chemotherapy is often limited to a poor prognosis, and it is not an approved treatment for HCC [53][54][55][56].Out of the four large randomized trials reported, two provide evidence that chemotherapy may improve survival [57].However, resistance brought on by signaling pathway activation, hypoxia, and genetic abnormalities reduces the effectiveness of chemotherapy.Additionally, chemotherapy usage may also be severely restricted by concurrent liver cirrhosis, which carries a very high risk of hepatic decompensation and little to no improvement in survival rates [58].

Molecular targeted therapy
Often, molecular targeted therapy is considered under the umbrella of systemic therapy which employs either small molecules or monoclonal antibodies that specifically target key signaling pathways dysregulated in HCC pathogenesis [59,60].In addition to molecular targeted therapy, systemic treatments include immune checkpoint inhibitors or a combination of both and benefits patients with advanced-stage HCC (Stage C of BCLC staging system-refer to Table 1).Multiple molecular signaling pathways are implicated in HCC pathogenesis, which includes growth factor signaling (IGF-insulin-like growth factor, PDGF-plateletderived growth factor, SCF-stem cell factor, FGF-fibroblast growth factor, HGF-hepatocyte growth factor) that activates receptor tyrosine kinase pathways; angiogenesis (VEGF-vascular endothelial growth factor) and cell differentiation (Wnt, Hedgehog, Notch) [61,62].Thus, molecular targeted therapies include signal transduction inhibitors (proliferation, cell survival, migration, and differentiation), apoptosis inducers, and angiogenesis inhibitors [59] (Fig. 3).On account of such complexity in HCC biology, a molecular agent that targets one or more signaling pathways could be a potential candidate to effectively treat HCC patients.
Globally, numerous drugs have undergone clinical trials but have failed at different phases [63].Herein, we discuss only the approved drugs that are used in the treatment of advanced HCC.Llovet J. M. et al., in 2008, got approval from the United States (US) Food and Drug Administration (FDA) for the chemotherapeutic agent called sorafenib, a multi-tyrosine kinase inhibitor (TKI) as a first-line treatment option for HCC, which inhibits tumor progression, tumor angiogenesis, and increases apoptosis in tumor cells [64].A SHARP (Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol) trial sub-analysis by Raoul et al. showed that irrespective of the disease condition such as etiology, liver enzyme levels, α-fetoprotein (AFP) levels, bilirubin levels, tumor size and its stage or any prior therapy doesn't affect the efficacy of sorafenib [65].Along the same lines, a GIDEON (Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib) study conducted by Marrero et al. in 2016 demonstrated that the efficacy and safety of the drug sorafenib had no substantial toxicity in a broader patient population [66].Although the drug showed promising results in restricting HCC progression, it possessed primary and acquired resistance as its limitations.According to RESORCE and CELES-TIAL study investigations, Regorafenib and Cabozantinib are approved for use in patients who are resistant to or intolerant to sorafenib because they both target VEGFRs 1-3 as well as MET and AXL receptor tyrosine kinase (AXL) pathways.Both studies showed the overall survival benefit of the patients was increased to 26 months compared to 19 months in patients who received sorafenib as first-line and placebo as second-line treatment [67,68].After almost a decade of sorafenib failure, in 2018, followed by the published data of the REFLECT trial by Kudo et al., lenvatinib was another drug that was approved for first-line therapy in advanced HCC [69].Recently, in 2020, donofenib, which is also a multi-tyrosine kinase inhibitor, is a very promising The other small molecule tyrosine kinase inhibitors that are used for second-line therapy include regorafenib, brivanib, carbozantinib, pemigatinib, and apatinib [71][72][73][74][75]. Additionally, monoclonal antibodies such as ramucirumab and bevacizumab have been approved for the treatment of HCC because they target VEGF and inhibit the formation of new blood vessels (angiogenesis inhibitors) that promote tumor growth [76,77].

Immunotherapy
Immunotherapeutic strategies have been proven safe and effective in treating advanced HCC and mainly involve four approaches: immune checkpoint inhibitors (ICI), adoptive cell transfer (ACT), vaccines, and immune system modulators [78] (Fig. 4).ICIs are monoclonal antibodies that block the checkpoint proteins from binding to their partner proteins, enabling the T cells to kill tumor cells.To date, there are three inhibitors, namely PD1 (programmed death-1) inhibitor, PD-L1 (programmed death-ligand 1) inhibitor, and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor, which have been evaluated and approved by the  [80,81].Furthermore, a recent multicentric study carried out in China in 2020 assessed the safety and efficacy of camrelizumab, another PD-1 monoclonal antibody, and provided evidence that this can be chosen as a new regimen in pretreated patients with advanced HCC [82].On the other hand, atezolizumab, a monoclonal antibody targeting PD-L1, was also tested in unresectable HCC patients [83].Though several anti-CTLA-4 monoclonal antibodies have been evaluated, ipilimumab is the only drug approved by the FDA in 2020 that is administered in combination with nivolumab for patients with HCC pretreated with sorafenib [84].Besides, monoclonal antibodies that block angiogenesis either by inhibiting VEGFR2 or VEGFA are also used for HCC treatment [85].
Several clinical trials have shown the anti-tumor effects of adoptive cell transfer (ACT), which includes tumorinfiltrating lymphocytes (TILs), cytokine-induced killer cells (CIKs), natural killer cells (NK), and chimeric antigen receptor T cells (CAR-T) cell therapy that enhances the overall immune response in HCC patients [86][87][88].A randomized study by Takayama et al. concluded that adoptive immunotherapy is a safe treatment with a lower postsurgical recurrence rate of HCC [89].Following the first reported study of CAR-MUC1 T cell therapy in 2008 by Wilkie et al., CAR-T immunotherapy has emerged as a novel and effective therapeutic option [90].These receptor proteins are engineered to provide T cells with higher specificity to target and destroy cancer cells.CAR-T cell therapies are widely tested in clinical trials and some of the CAR-T targets of HCC cells are Glypican-3 (GPC-3), Alpha-Fetoprotein (AFP), Cluster of Differentiation 147 (CD147), Mucin-1 (Muc1), Epithelial Cell Adhesion Molecule (EpCAM), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) as these are highly expressed or their levels are high in HCC patients but not in healthy subjects [91].Similarly, some vaccines are being evaluated in phase I/II clinical trials for treating advanced HCC, but unfortunately, these vaccines show a limited degree of efficacy and include AFP-derived peptidase vaccine, GPC3 peptide vaccine, telomerase peptide vaccine, and dendritic cell (DC) vaccines [78].Additionally, some immune-modulating agents enhance the body's immune response and delay tumor progression, showing an effective therapeutic response [92].Cytokines such as interferons (IFNs) and interleukins (ILs) play a major role in immunoregulation, leading to the activation of natural killer (NK) cells, killer T cells, or dendritic cells that trigger an immune response against cancer cells [93,94].Interestingly, Tomov B. et al., demonstrated that treating a patient with metastasized HCC with IL-2, bacillus calmette Guerin (BCG), and melatonin resulted in tumor regression [95].Although the advances in immunotherapy have been a promising therapeutic strategy for the treatment of HCC, immunotherapy resistance has been a significant barrier, and overcoming this by using combination therapy is the need of the hour in treating advanced HCC patients [78,93].

Gene therapy
Resistance to targeted therapies used against HCC has made gene therapy an attractive option in recent times.Gene therapy involves the modification of a gene in patients to treat or cure HCC, either by replacing or inactivating the diseasecausing gene or by introducing a new or modified gene.This includes restoration of tumor suppressor genes, inhibition of oncogenes, gene-directed enzyme/prodrug therapy, and targeted expression of pro-apoptotic genes by in vivo or ex vivo gene therapy [96].Various techniques have been employed in gene therapy, such as the use of viral vectors (adenovirus, vaccinia virus, and lentivirus), nanoparticles, chimeric antigen receptor (CAR)-T cells or clustered regularly interspaced short palindromic repeats/cas associated proteins (CRISPR/Cas9) to treat HCC patients.Notably, China is the country that is very active and leading in gene therapy clinical trials for HCC, and the data suggest that only limited gene therapy techniques have entered clinical trials (thymidine kinase, CAR-T, p53, JX-594, rhAdV, and RNAi) [97].However, ongoing clinical research offers a ray of hope that gene therapy could be an effective therapeutic strategy for HCC.

Combination therapy
This multimodal therapeutic option employs two or more kinds of therapy and is found to be effective in the treatment of advanced HCC when surgery is not an option.The therapies may be used at the same time or in sequence or at different time points that aid in inhibiting tumor growth and metastatic potential and improve survival by targeting multiple oncogenic pathways in a synergistic or additive manner [16].Thus, combination therapy is more advantageous than singleagent treatment or monotherapy as it decreases the likelihood of drug resistance and toxicity.In this review, we will discuss the combination therapies under investigation in phase II clinical trials, with a special focus on phase III clinical trials for HCC and combination therapies that have been approved for the treatment of HCC (Fig. 5).

Ablation in combination with embolization, immunotherapy, or molecular targeted therapy
Numerous meta-analysis and retrospective studies have shown the effectiveness of the combined effect of radiofrequency ablation (RFA) and trans-arterial chemoembolization (TACE) against early and intermediate-stage HCC.Most of these studies have employed TACE followed by RFA [98].Studies by Wang et al. have provided evidence that the combination of TACE and RFA significantly improves the survival rates and is an effective treatment approach providing a better prognosis for HCC patients [99].Indeed, it is shown that TACE diminishes the blood flow and RFA elevates the necrosis level in the tumor cells.In support of this, a study by Liu et al. showed that while there was an 83.2% necrosis rate in tumor cells in patients subjected to TACE plus RFA, there was only a 27.9% tumor necrosis rate in TACE alone treated HCC patients [100].In addition, thermal ablation is also used in place of RFA.The best combination regimen in phase 2 clinical trials is RFA and sorafenib, which is used in first-and second-line therapy and is found to be effective against medium-sized tumors [101].However, the efficacy of RFA plus sorafenib in advanced HCC remains unclear.Of note, the ablation procedure followed by the treatment with tremelimumab (a human monoclonal antibody against CTLA-4, an immune checkpoint inhibitor) has provided promising results in patients with advanced HCC [102] and, albeit the lack of long-term effects, a few studies have demonstrated that combination therapy of radio ablation and immune checkpoint inhibitors has improved the overall survival rate with a reduced recurrence rate [103].

Chemoembolization in combination with immunotherapy or molecular targeted therapy
Trans-arterial radioembolization (TARE) has been extensively used in the treatment of HCC as ionizing radiation has been attributed to inducing cell death via DNA damage of tumor cells [104].On the contrary, studies have demonstrated that the rate of tumor recurrence is high with the upregulation of oncogenic pathways when TARE has been the only treatment administered to HCC patients [105].Thus, as the need for an additional treatment option was very critical, the use of immune checkpoint inhibitors was a game changer in this scenario and showed significant improvement in HCC patients [106].With this advancement, care has been taken to optimize the treatment to maximize the additive or synergistic effect of this combination therapy.Studies by Zhan et al. have provided evidence that the combined therapy of radioembolization and immunotherapy is significantly effective and safe in treating HCC patients [107].A study conducted by Li et al. showed that the combination of TACE and hepatic arterial infusion chemotherapy (HAIC) has a more advantageous progression-free survival (PFS) due to its significantly higher conversion rate compared to conventional TACE (48% vs. 9%, respectively) [108].Additionally, the administration of trans-arterial chemoembolization (TACE) followed by sorafenib treatment is found to be therapeutically effective in treating advanced HCC.A nationwide population-based cohort study by Kok et al. demonstrated that combination therapy of sorafenib with TACE improved the survival rate of advanced HCC patients compared to sorafenib alone [109].In addition to this, the phase II studies conducted by Park et al. and Erhardt et al. have also proved the therapeutic efficacy and safety profile of this combination therapy [110,111].Overall, the trials show that the combination of sorafenib and TACE has superior results in terms of reducing tumor burden and increasing time to progression (TTP) when compared to sorafenib or TACE alone.

Radiation therapy in combination with immunotherapy or molecular targeted therapy
Radiotherapy has been an attractive regimen for the treatment of HCC as the process involves the application of highenergy particles that induce DNA damage, causing tumor cell death [43].However, radiotherapy is also known to potentiate the activation and infiltration of immune cells into the tumor microenvironment and increase the expression of PD-1 on tumor cells, which leads to radiation resistance [103].These sequential molecular events create the need for combining immune checkpoint inhibitors to reduce the antitumor responses.Kim et al. and Yoshimoto et al. found that combining radiotherapy with anti-PD-L1 and anti-CTLA-4 antibodies significantly reduced tumor growth and increased overall survival compared to radiotherapy alone [112,113].Furthermore, Chiang et al. have tested the efficacy of the combined effect of SBRT and nivolumab (PD-1 inhibitor) for the treatment of advanced HCC [114].The other combination under investigation is the application of radiotherapy along with durvalumab (PD-1 inhibitor) and tremelimumab (CTLA-4 inhibitor) for the treatment of advanced HCC.Along with immune modulation, radiotherapy also increases the expression of VEGF, causing vascular abnormalities.Thus, radiotherapy along with an anti-angiogenic agent (sunitinib) that enhances the anti-tumor effect has been considered to be a potential synergistic treatment strategy for advanced HCC [103].

Combination immunotherapy
Immunotherapy is an innovative approach to the treatment of advanced HCC.The regimen used comprises anti-PD1/ anti-PDL1, anti-CTLA4, and anti-VEGF/anti-VEGFR monoclonal antibodies [79].The currently used immunotherapeutic agents include nivolumab (anti-PD1), ipilimumab (anti-CTLA4), durvalumab (anti-PDL1), bevacizumab (anti-VEGFA), tremelimumab (anti-CTLA4), and atezolizumab (anti-PDL1) and are used either individually or in combination with each other.Undoubtedly, combination immunotherapy has been very promising as it involves simultaneous blockade of multiple immune checkpoints compared to single-agent immunotherapy [115].A combination of durvalumab plus bevacizumab and durvalumab and tremelimumab is in a phase 3 clinical study [116,117].The multicenter phase trial study conducted by Simonelli et al. found that the combination of isatuximab plus atezolizumab, an anti-CD3 monoclonal antibody, and an anti-PD-L1 monoclonal antibody, respectively, has a well-tolerated and manageable safety profile in treating advanced solid tumors.When combined with isatuximab, the efficacy of atezolizumab was found to be increased [118].A STRIDE (Single Tremelimumab Regular Interval Durvalumab) study conducted by Ghassan et al. found that the combination of tremelimumab plus durvalumab in 393 patients with unresectable HCC had a significant survival rate of 30.7% compared to durvalumab (24.7%, n = 389) and sorafenib (20.2%, n = 389) alone [119].Studies have proved that the FDA-approved drug; Nivolumab (PD1 inhibitor), used in combination with ipilimumab (CTLA-4 inhibitor), increases overall survival and is used in the second-line treatment of advanced HCC patients previously treated with sorafenib [84].

Combination of molecularly targeted therapy
Hepatocarcinogenesis is a complex biological process involving the activation of multiple signaling pathways, leading to tumor proliferation, angiogenesis, and metastasis.With the advancement in the understanding of tumor biology and molecular mechanisms involved in this process, targeting these signaling pathways to inhibit tumor growth and progression seems like a logical approach.Sorafenib was the first approved molecular target agent to treat advanced HCC [64].Due to tumor heterogeneity, changes in signaling pathways, and therapeutic resistance, mono-targeted therapy appears ineffective, paving the way for the testing of combination therapy regimens to enhance the anti-tumor effect.
To date, several clinical trials have been conducted to target multiple pathways simultaneously using various combinations of regimens.Sorafenib, a multikinase inhibitor, is the most commonly used drug along with either mono-or multi-tyrosine kinase inhibitors or chemotherapeutic agents.Although doxorubicin plus sorafenib has been shown to improve overall survival [120], a recent phase 3 clinical trial study by Abou-Alfa et al. demonstrated that the addition of doxorubicin to sorafenib in the treatment of advanced HCC resulted in no difference in patient survival but rather in increased toxicity [121].Similarly, the SEARCH trial, a multinational, multicentric phase III trial in patients with advanced HCC by Zhu A. et al., also showed that sorafenib plus erlotinib (EGFR inhibitor) did not improve the survival of HCC patients [122].So far, it is promising to note that regorafenib administered to patients with advanced HCC after sorafenib is shown to improve liver function and could be considered as an effective treatment option.Based on the outcome of the RESOURCE trial, regorafenib has been reported to be clinically effective as the second-line treatment for HCC patients after progression on first-line sorafenib [123].

Combination of immunotherapy and molecular targeted therapy
Various combinations have been tried among the immune checkpoint inhibitors and molecular targeted agents to improve the overall survival of HCC patients and reduce toxicity.Furthermore, combination therapy involving immune therapy and molecular targeted therapy is being tested in advanced HCC patients in order to improve treatment efficacy and maintain safety profiles.Indeed, targeting oncogenic signaling pathways along with the tumor immune microenvironment plays a pivotal role in anticancer therapy.These combinations so far mainly involve the combinations of tyrosine kinase inhibitors with either anti-PD1/ anti-PDL1/anti-CTLA4 antibodies or anti-angiogenic agents [124] (Fig. 6).
An interesting study by Shigeta K. et al. has demonstrated that the combination therapy of regorafenib with anti-PD1 antibody in HCC mice has inhibited tumor growth and increased survival by promoting anti-tumor immunity and vascular normalization [125].A remarkable investigation of IMbrave150, a multicenter, international trial, tested the efficacy of combining immunotherapy (atezolizumab-anti-PDL1) with anti-angiogenic therapy (bevacizumab-anti-VEGF) and reported that the dual pathway blockade offers longer progressive-free survival and a 42% reduction in death rate compared to sorafenib treatment alone in HCC patients [126].The FDA has approved this combination therapy for patients with unresectable or metastatic HCC with no prior systemic therapy treatment.The other promising combination was lenvatinib (a multikinase inhibitor) and pembrolizumab (PD1 inhibitor), possessing anti-tumor activity that was approved by the FDA based on the interim data for the first-line treatment of unresectable HCC.Unfortunately, in July 2020, the FDA held off on the approval of this combination therapy and the results of the phase 3 trial are much awaited [127,128].The few other molecular targeted and immune checkpoint regimens that are in phase II and phase I trials include bevacizumab plus erlotinib, atezolizumab plus cabozantinib, and camrelizumab plus apatinib.

Conclusions and future outlook
Understanding the evolution of cancer and discovering an effective treatment for HCC has been a global quest.The complex biology involved in the development and progression of HCC makes it a difficult choice to use monotherapy as the treatment option.Blocking a single pathway may just slow down or stop the growth of cancer cells momentarily, which often results in recurrence.Thus, a better understanding of HCC heterogeneity, genetic and epigenetic alterations, the immune microenvironment of HCC tumors, and the crosstalk of various signal transduction pathways is of paramount importance as all of these factors are responsible for drug resistance and disease recurrence, offering a great impact on the clinical outcomes.A collective effort of hepatologists, oncologists, and basic scientists is the need of the hour to ensure that the prognosis continues to improve.
Regardless of the challenges faced, the use of combination therapy is a powerful tool used to attack cancer cells and has been proven to be an effective and promising therapeutic option for treating advanced HCC.Indeed, understanding the additive or synergistic effects of the drugs and the fundamental molecular mechanisms underlying these processes would lead to the exploration of better combination regimens that may potentially increase the Fig. 6 The two FDA-approved combination regimens used in combating HCC and their mode of action overall survival rate and decrease toxicity in HCC patients.Above all, a paradigm shift in the HCC treatment strategy can happen with multidisciplinary approaches with the application of recent technologies (integrated omics, single-cell genomics, next-generation sequencing, and nanotechnology) and a better comprehension of the complex interactions between tumor stages, the severity of the underlying liver disease, and patient performance status.Furthermore, along with the success in the application of advanced combination therapy, the most awaited outcome is the development of biomarkers to be used in precision medicine for treating advanced HCC to achieve the optimal clinical outcomes.

Fig. 2
Fig. 2 Mechanisms of action of different chemotherapeutic agents used to kill tumor cells in HCC treatment

Fig. 3
Fig. 3 Inhibition of proliferation, survival, and angiogenesis genes by multikinase kinase inhibitors used in the molecular targeted therapy in HCC treatment.VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; FLT-3, fms-like

Fig. 4
Fig. 4 Different types of immunotherapy modalities used in the treatment of HCC.Immune checkpoint inhibitors (monoclonal antibodies), adoptive cell transfer, immune system modulators, and vaccines

Fig. 5
Fig. 5 Status of active combination regimen for HCC therapy.Source: Clinical trials for HCC from clinicaltrials.gov