Emerging Immunotherapy: Liver Cancer Microenvironment for Treatment

Abstract

Hepatocellular carcinoma (HCC) is a highly prevalent primary liver carcinoma and the main cause of deaths (linked with cancer) across the globe. Despite advancements in prevention strategies, testing, and technological advances in diagnosis and treatment, the occurrence and fatality rate of HCC continue to rise. In recent decades, the approval of immune checkpoint inhibitors (ICIs) has transformed palliative treatment for liver cancer. However, the majority of patients with liver cancer do not respond to these treatments. Herein, we elaborated the microenvironment of the liver cancer and candidate immunotherapies based on activating the antitumor activity of myeloid, NK and T cells, chimeric antigen receptors-T or -NK cells, vaccines, oncolytic viruses, and combination therapies, as well as the challenges and opportunities of immunotherapies in liver cancer. This review also explores the rationale, molecular foundation, and supporting preclinical evidence for immunotherapies in HCC, available clinical evidence, and current immunotherapeutic clinical studies.

1. Introduction

Hepatocellular carcinoma (HCC) is the prevalent primary liver carcinoma and the largest cause of cancer-associated deaths across the globe. HCC accounts for ~90% of cases. Hepatitis B and C virus infections, liver flukes in endemic areas, excessive intake of alcohol, cigarettes, elevated level of body fat, and aflatoxins are all significant risk factors for developing HCC. The tumor burden, liver functioning, comorbidities, and health condition of a patient influence treatment options for HCC. HCC treatments have changed dramatically over the past four decades. Surgical or organ transplantation is the first-line treatment for tumors less than 5 cm in diameter. However, the treatment of large HCCs (those greater than 10 cm) is disputed, with considerable heterogeneity in various treatment regimens in different locations [1]. Additionally, radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) are the local modalities utilized for early and intermediate-stage HCC, accordingly [2, 3]. Systemic treatments for advanced-stage HCC were found controversial prior to 2008 because of their ineffectiveness and poor patient tolerability. Moreover, systemic treatments for liver cancers have made little progress in the last decade.

Immuno-oncology has revolutionized cancer treatment, particularly liver cancer, over the last decade. The antitumor immune response combines innate and adaptive immune system elements [4]. Tumors, on the other hand, can harness this response and use it to evade the immune system in a variety of ways, including maintaining an immunosuppressive milieu or inducing cytotoxic cell malfunctions. An immunosuppressive tumor immune microenvironment (TIME) is marked by the existence of regulatory T cells (Treg), immunosuppressive myeloid cells including tumor-associated macrophages (TAMs), and inhibitory B cells [5]. Immune checkpoint activation, which includes coinhibitory substances, prevents effector cell activation and is crucial for tumor immune evasion [6]. Cancer treatment has been transformed by the development of immune checkpoint inhibitor (ICI)-based therapy, which has led to long-term responses and improved survival in a wide spectrum of cancers. However, ICIs have not been very effective against many solid tumors. Checkpoint proteins expressed by immune cells or tumor cells serve as targets for ICI monoclonal antibodies (mAbs), which elicit a robust immunological response from cytotoxic T lymphocytes (CTLs) [7]. Moreover, ICI therapy has shown to be effective in a subset of patients with a range of cancers, including HCC. Anti-PDL1 antibody atezolizumab and the VEGF neutralizing antibody bevacizumab are now the standard therapies for HCC [8]. Herein, we explored the rationale, molecular basis, and underlying preclinical evidence for immunotherapies in HCC, along with existing immunological clinical research.

2. Overview of immune components

2.1 Immune cells

The liver is a key immunological organ that receives antigen-rich blood from the gut via the portal vein. Cancer immunosurveillance and control rely heavily on the innate and adaptive immune systems. The conflicting actions of antitumor effectors and their suppressors in the TME determine immune activation or evasion, as shown in (Figure 1). Through highly conserved cytokine and chemokine-activated inflammatory reactions, adaptive and innate immune cells patrol the liver sinusoids to eliminate invading pathogens and endotoxins. The uninflamed liver generates a tolerogenic milieu that suppresses both innate and adaptive immunity in order to maintain homeostasis and prevent chronic inflammation and tissue damage. The immune cell composition of the TME has a huge impact on HCC that can affect tumor initiation, progression, and therapeutic response. During hepatocarcinogenesis, several molecular processes cause distinct immune cell subsets to respond in ways that either cause inflammation or limit antitumor immunity. Herein, the immune cell landscape of HCC is discussed, with an emphasis on the role of innate immune cells and innate-like T cells in HCC, which may lead to the development of candidate immunotherapies that target the underlined cells.

Figure 1.

Homeostasis and immune cell composition of the liver.

2.2 Cytokines

HCC’s genesis and progression have been linked to inflammation. TNF-α, a key inflammatory mediator, had previously been identified as a possible therapeutic target in a variety of malignancies. Furthermore, TNF-α, interleukin (IL)-1, and IL-6 levels in HCC patients’ serum were found to be considerably greater than in healthy controls. CXCL5 and CCL15, which are produced by tumor cells, attract immunosuppressive neutrophils and monocytes, respectively, while CXCL13, which is produced by hepatic stellate cells, attracts B cells, which differentiate into protumor IgA-producing plasma cells in the context of NASH-associated HCC. IL-2, IFNγ, CXCL10, and CXCL9, on the other hand, recruit lymphocytes to mount an antitumor immune reaction. As a result, the immunological composition and response are determined by the balance of these stimuli. T helper 17 (Th17) cells generate the cytokine IL-17, which has been linked to the development and progression of inflammatory disorders. These findings have ramifications for patients who are undergoing immunotherapy. Activation of the IFN-γ signaling system predicts a favorable response to ICIs, according to the findings of two trials in patients with HCC. In HCC, the tumor microenvironment (TME) has a significant impact on cytokine production.

2.3 Signaling cascades lead to immune evasion

HCC’s immune microenvironment can be influenced by tumor intrinsic signaling pathways. Wnt ligands produced by HCC cells induce M2 polarization of TAMs, which results in tumor development, metastasis, and immunosuppression in HCC. β-Catenin signaling also inhibits MHC-independent immune responses facilitated by NK cells through decreased expression of NKG2D ligands on HCC cells. By reversing NK cell depletion or TGF- β1 reversing NK cell depletion, blocking the CD96 association restores NK cell immunity to tumors, implying that CD96 may considerably contribute to HCC. MYC may suppress PD-L1 expression in HCC, which suggested that treating HCC with a combination therapy targeting MYC and the PD-L1/PD-1 cascade could be beneficial. HCC is linked to TP53 gene mutations and immune cell heterogeneity, with TP53 mutations reported in roughly 40% of all HCCs. The TP53 mutation-associated immunotype is critical for better clinical outcomes and may have important implications for postoperative tailored follow-up and therapeutic decision-making. Furthermore, ARID1A mutations, which are a common driving factor in HCC, have a considerable contribution to antitumor immunity: they stimulate a positive response by suppressing mismatch repair, leading to an elevated TMB, and also reduce IFNγ signaling by lowering chromatin availability. In co-cultured CD4+ T cells, MDSC suppresses the immune system by generating CD4+ CD25+ Foxp3+ regulatory T cells. The IL6-STAT3-PDL1 signaling cascade is used by HCC-CAFs to control neutrophil survival, stimulation, and function in HCC. Finally, T cell fatigue is caused by the upregulation of immunological checkpoint molecules such as PD-1, PDL1, CTLA4, LAG3, and TIM3 in HCC cells. PDL1 overexpression in Kupffer cells and leukocytes is also caused by prolonged HBV infection, which can boost the protein’s level and enhance immunosuppression in HCCs of this etiology.

2.4 Virus associated with HCC

HCC is the main cause of death in cirrhotic patients, with HCV or HBV infection accounting for the majority of cases, especially in developing countries. HBV is a hepatotropic virus that causes liver inflammation. While another established risk factor for severe liver disease is HCV, a Flaviviridae virus. Long-term infection with HBV or HCV causes an inflammatory reaction in the liver that can develop into cirrhosis and, ultimately, HCC.

HBV and HCV-induced immune responses might be either procarcinogenic or anticarcinogenic. Platelets generate components that induce a necroinflammatory infiltration, i.e., virus-specific CD8 + T cells, which drive HCC development and can be slowed down with antiplatelet medications. An effective HBV-specific T cell response, on the other hand, can aid in the control of HCC cells displaying HBV epitopes, as clinically demonstrated by tumor regressions accomplished with adoptive T cells genetically modified to produce TCRs targeting such epitopes. Despite this, persistent HBV infection causes multiple changes in the hepatic immune infiltrate, which promote a tolerogenic milieu, limiting effective antitumor immunity. B reg cells are a primary source of IL-10, an immunosuppressive cytokine that is increased during HBV flares. Furthermore, HBV-specific T cells are susceptible to BIM-induced apoptotic process and TRAIL + NKG2D + NK cell deletion. Chronic HBV infection also increases the production of inhibitory immunological checkpoint proteins on virus-specific T cells, mainly in the liver, restricting any T cells capable of attacking HCC cells. In patients with HBV-associated HCC, the presence of elevated suppressive PD-1 T reg cells is linked with worse survival outcomes, although CD8+ tissue-resident memory T cells are correlated with a better outcome.

HCV has a single polyprotein genome, which is translated into structural as well as nonstructural proteins. These HCV proteins are targets for the host’s innate and adaptive immune systems. The principal pattern recognition receptors that identify HCV PAMPs are RIG-I-like receptors and Toll-like receptors. The correlation stimulates a cascade of antiviral cytokines, including interferons. Perforin, as well as granzyme B, is secreted by CD8 + T cells and NK cells while interferon-gamma (IFN-γ) secreted by CD8 + T cells and NK cells causes noncytolytic HCV clearance. Moreover, the host-HCV interactions could make developing an HCV vaccine challenging. It is hard to neutralize a virus that typically has so many mutations in its E1 and E2 proteins, and attempts to do so clear the most abundant variants and leave replicative space for the expansion and continued replication of other quasispecies.

Understanding how virus-related HCCs regulate their metabolism could open up new avenues for immunotherapy. By decreasing arginine, granulocytic MDSCs accumulate in HBV-infected livers and can suppress HBV-specific T cells. High expression of the esterification enzyme sterol Oacyltransferase 1 (SOAT1) disrupts lipid homeostasis, promoting proliferative and migratory potential of the tumor cell in a subset of HBV-associated HCCs while reducing the activity of HBV/HCC-specific tumor-infiltrating lymphocytes.

2.5 Nonviral HCC

Hepatic steatosis and chronic necroinflammation in the liver can result from persistent alcohol exposure or high-calorie diets combined with a sedentary lifestyle, leading to HCC, which could be fatal. Moreover, NASH-induced inflammation is more frequently linked with diffuse inflammatory infiltrates.

New developments regarding the cellular and molecular cascades that drive NASH and the NASH-HCC transition have emerged in recent years. It has been revealed that platelets drive NASH and the NASH-HCC transition by fostering the first inflammatory reactions in the context of steatosis. Platelets interact with Kupffer cells and inflammatory monocytes through the platelet-specific glycoprotein Ibα (GPIbα). In rodents, and possibly also in humans, preventive and therapeutic antiplatelet treatment lowers the development of NASH and NASH-associated HCC. The study revealed that the usage of low-dose aspirin is linked with a considerably decreased risk of HCC and liver-associated death. NASH-related liver cancer is caused by a variety of immunological mechanisms. NKT cells mediate lipid uptake via LTβR activation on hepatocytes, and metabolic stimulation of intrahepatic CD8 + T cells and NKT cells, for example, has been demonstrated to induce NASH and HCC via hepatocyte cross talk. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. The metabolic machinery in hepatocytes is downregulated as a result of this cross talk, which involves direct interaction between immune cells and hepatocytes via Fas and release of porforins and granzymes as well as indirect communication via secreted substances (e.g., cytokines, chemokines), resulting in increased metabolic, endoplasmic reticulum, and mitochondrial stress. Surprisingly, human NASH has been found to have a considerable elevation in the number of intrahepatic CD8 + PD1 + T cells. As previously stated, metabolic imbalance triggers autoaggression in these CD8 + PD1 + T cells, culminating in MHC I independent cytotoxicity against hepatocytes and necroinflammation. This autoaggressive behavior of CD8 + PD1 + T cells has implications for patients receiving ICIs for NASH-associated HCC: nonviral HCCs, particularly NASH-associated HCCs, are less susceptible to these drugs than viral HCCs. This discrepancy has been linked to the autoaggressive intratumoral CD8 + PD1 + T cells losing their tumor surveillance function, resulting in a protumorigenic milieu.

Alcohol consumption is responsible for up to 30% of all HCC cases worldwide. The mucosal damage caused by alcohol might result in an impaired intestinal barrier function, enabling toxins of gut-inhabiting bacteria such as endotoxins to enter the systemic circulation and to contribute to liver injury after alcohol consumption. Alcohol increases gut permeability, allowing immunomodulatory microbiota-derived PAMPs such as LPS to enter the liver and decrease hepatic immune reactions, presumably through effects on resident macrophages. NASH is linked to an increase in protumorigenic, immunosuppressive granulocytic MDSCs in the liver, as well as a decrease in T cell migration to the liver. Furthermore, the neutrophils in the liver parenchyma are a hallmark of alcoholic hepatitis, which is thought to influence the hepatic immune landscape.

2.6 The modulatory of the microbiota

Dysbiosis has been seen in various phases of chronic liver injury, including HCC, according to several investigations. The gut microbiota increases HCC development in the setting of chronic liver injury, presumably via microbial metabolites or PAMPs, according to in vivo studies using a mouse model. Through the primary to the secondary conversion of luminal bile acids, the microbiota of the stomach can reduce immunosurveillance and accelerate the progression of HCC. Furthermore, metabolized bile acids (deoxycholic acid, a secondary bile acid) have been demonstrated to cause senescence in hepatic stellate cells, leading to the production of numerous cytokines such as transforming growth factor-β1, angiotensin II, leptin) that enhance the progression of HCC. The relevance of the gut microbiota in influencing systemic immunity, including immunotherapeutic reactions and chemotherapy-induced immunological effects, is now widely recognized. The antigenic epitope tail length tape measure protein 1 (TMP1) in the genome of bacteriophage Enterococcus hirae had high similarity with the proteasome subunit beta type-4 (PSMB4) tumor antigen. They activated CD8+ T cells simultaneously and improved the efficacy of PD-1 blockade therapy. It has been demonstrated that the antigen epitope SVYRYYGL (SVY) expressed in the commensal bacterium Bifidobacterium breve was similar to the tumor-expressed antigen epitope SIYRYYGL (SIY), resulting in SVY-specific T cells recognizing SIY and inhibiting tumor growth. However, further research on the impacts on hepatic immunity is still needed.

2.7 HCC immune classification

Few studies have attempted to classify HCC according to its immunological status. The “Inflammatory” and “Lymphocyte Depleted” clusters were found prominent in HCCs, according to a pancancer analysis based on clustering of immune-associated gene expression profiles. Using a transcriptome deconvolution technique, the first thorough immune categorization of HCC was published in 2017, which found an “Immune” class (which accounts for 25% of HCCs). Immune tumors have an elevated level of immune infiltration, enhanced PD-1/PDL1 signaling, and signature enrichment that mimics the response to ICIs in other solid tumor types.

More recently, a modification of this classification established an “Inflamed” class of HCC, which accounts for about 30–35% of tumors, expanding the previously documented immune class with an additional subset of tumors labeled as an “Immune-like” subclass. This novel subclass is distinguished by the presence of CTNNB1 mutations and significant activation of interferon signaling and immunological activation. T-cell-inflamed tumors are characterized by type I interferon (IFN) activation, immune potentiating chemokines, antigen presentation, cytotoxic effector molecules, and activated CD8+ T cells. The inflamed tumor microenvironment is additionally characterized by IFN-induced inhibitory pathways such as programmed death-ligand 1 (PD-L1) and indoleamine-2, 3 dioxygenase and higher proportions of FOXP3+ regulatory T cells. Patients with HCC have a higher proportion of inflamed tumors, and those patients who responded to anti-PD-1/PDL1 antibodies were shown to be enriched in the inflamed class. In the “Noninflamed” class of HCCs, two subclasses have been evaluated based on the mechanisms of immune escape: (1) an “Intermediate” class with TP53 mutations, elevated levels of chromosomal instability, and frequent deletions in subcytobands harboring genes linked with interferon signaling or antigen presentation; and (2) an “Excluded” class with CTNNB1 mutations and immune desertification features.

3. Landscape of immunotherapy for HCC

3.1 Immunotherapy for early-stage HCC

Early-stage (BCLC 0 and BCLCA) HCC patients can benefit from treatments such as surgery, liver transplantation, and radiofrequency ablation (RFA) [9]. However, recurrence after liver surgery is prevalent and closely linked to poor overall survival, with recurrence rates of up to 50% and 70% following 2 and 5 years, accordingly [1]. Recurrence is linked to the manifestation of thrombocytopenia, cirrhosis, and satellite lesions [10]. Furthermore, various immunological variables were found with relatively poor resection effects. High PD-L1 expression is linked to an elevated level of immunosuppressive molecules including regulatory T-cells (also known as Tregs) and myeloid-derived suppressor cells (MDSCs) as well as the attenuation of cytotoxic elements such as interferonγ [11, 12, 13, 14]. High CD3+ and CD8+ T cell density in the tumor core and margin, as well as related immune scores, which are based on the number of CD3+ and CD8+ lymphocytes in the intratumor (from the tumor core to the periphery), are linked to a much lower probability of HCC recurrence postsurgical treatment [15, 16]. For patients with unresectable HCC or HCC with end-stage liver disease, liver transplantation is an alternate and possibly ideal therapeutic option. However, only a small percentage of HCC patients match the transplant criteria, and the restricted number of transplant centers makes liver transplantation an unrealistic option for many patients due to the chronic shortage of donor organs. Hence, it is needed to combine adjuvant and neoadjuvant immunotherapy to elevate the likelihood of cure post HCC surgery.

3.1.1 Adjuvant treatment

Post resection, numerous adjuvant therapies were undertaken in the hopes of enhancing survival, but none of them was found successful. Systemic treatment has relied heavily on sorafenib over the previous decade, making it the only medicine approved for first-line treatment since 2017. Currently, the approval of lenvatinib has been accepted in the practice of first-line setting [17]. T lymphocytes that have been grown ex vivo with cytokines make up CIK cells. In an open-label, phase-3 trial involving 230 patients (associated with HCC) who experienced surgical resection, patients who got CIK lived 44 months longer than those who received placebo (HR, 0.63; 95% CI 0.43–0.94; p = 0.010). However, despite these recent advancements, no adjuvant therapy has consistently proven effective.

3.1.2 Neoadjuvant therapy

Neoadjuvant therapy is a treatment strategy applied prior to the primary cancer treatment to enhance the rate of success for the primary treatment. In neoadjuvant therapy, ICIs can take advantage of the elevated levels of tumor antigens present in the primary tumor to enhance the proliferative potential of tumor-specific T lymphocyte clones already existing in the TME [18]. Mice administered neoadjuvant Tregs level was reduced due to diphtheria toxin fragments. While anti-CD25 lived considerably longer (250 days) than control mice (100 days) in preclinical models of triple-negative breast cancer [19]. A limited time interval between the first administration of neoadjuvant immunotherapy and primary tumor excision was found essential for maximum efficacy in a second trial, whereas a greater time interval abolished therapeutic efficacy in the neoadjuvant context. The viability of neoadjuvant cabozantinib with nivolumab in HCC was investigated in a single-arm phase 1b study [18]. The trial included 15 unresectable patients, 12 of whom had successful margin-negative resection after neoadjuvant cabozantinib and nivolumab therapy. Furthermore, responders showed enrichment of CD138+ plasma cells and a specific spatial rearrangement of B cells, with B cells in close proximity to other B cells, indicating that this combination modulated the TIME. These findings point to the need for a B-cell orchestration of antitumor immune responses. Immunotherapy is also being researched as a neoadjuvant treatment for liver transplantation. In HCC patients that meet the Milan criteria, the combination of lenvatinib and pembrolizumab is being tested in PLENTY202001 before liver transplantation. The usage of ICIs in the transplant scenario poses considerable safety concerns, as it can result in allograft rejection, which can be deadly [20]. As a result, solid organ patients are routinely excluded from clinical trials employing ICIs. The PLENTY202001 is an outlier in this regard, as it will collect extremely valuable safety data.

3.2 Intermediate HCC immunotherapy

TACE is the gold standard of therapy for intermediate-stage BCLC-HCC (B). This immunotherapy significantly improves OS [21]. In addition, TACE seems to influence the immunological response of tumors [22, 23, 24]. TACE can improve both the antitumor immune response and the pro-inflammatory tumor response by lowering Tregs and fatigued effector T-cells in the tumor core [23]. HCC patients (n = 32) who were not eligible for liver surgical resection or transplantation were evaluated for the efficacy and safety of tremelimumab (anti-CTLA-4) with ablation [25]. Tremelimumab was given to patients every 4 weeks for six doses. They had TACE (subtotal radiofrequency ablation) on day 36. Five of the 19 evaluable patients had an established partial response, and the median OS was 19.4 months. Six-week tumor biopsies revealed an increase in CD8+ T cells in those individuals who had a therapeutic benefit. Hence, the combination of locoregional plus immunotherapy for HCC at the intermediate stage is mechanistically justified. Because it enrolled patients with unresectable HCC. Furthermore, The IMbrave 150 trial revealed information regarding HCC-associated patients at an intermediate stage [8]. The ABC-HCC study proposes a novel type of primary endpoint called time-to-failure of treatment strategy, which assesses the time until the investigator discontinues either treatment strategy (systemic therapy or TACE) due to failure [26].

3.3 Immunotherapy for advanced-stage HCC

Immunotherapy has been shown to be effective in the treatment of advanced HCC. ICIs, particularly those that target PD-1 or PD-L1, are the most commonly used drugs. They’ve been studied in big clinical studies both individually and in combination, and they have now become an important aspect of HCC treatment. Furthermore, new immunotherapies, including adoptive cell therapy with considerable improvement in ICI’s therapeutic efficacy against HCC.

3.3.1 Monotherapies with ICIs

A monoclonal antibody, i.e., nivolumab, targeting PD-1, was originally investigated in HCC in phase I/II CheckMate 040 investigation, which comprised 262 HCC patients with a previous history of sorafenib treatment. Nivolumab had a median response length of 73.87 weeks (95% confidence interval (CI) 6–24) and an ORR of 14 percent by RECIST 1.1 (18% by mRECIST) [27]. The study found a median OS of 15.6 months and a safety profile that was similar to prior nivolumab trials. As a result, the FDA granted nivolumab expedited approval for patients with advanced-stage HCC who had previously received sorafenib treatment. In the CheckMate 459 research, nivolumab was compared with sorafenib as a first-line treatment in patients with advanced HCC who had not previously undergone systemic treatment.

In HCC patients (with a previous history of sorafenib treatment) after intolerable toxicity or rejection of sorafenib, the trial-22 (phase 1/2) found a median OS of 65.6 months (95% CI 7.7–24.6) and a median PFS of 8.69 months (95% CI 1.8–5.4) with an acceptable safety profile for tremelimumab monotherapy. Tremelimumab plus durvalumab (anti-PD-L1), on the other hand, had a better overall benefit–risk ratio [28]. Furthermore, early growth of Ki67+ CD8 + T cells was linked to response to either the single treatment or the combination. This is the final trial that potentially leads to global regulatory clearance for a single drug checkpoint inhibitor. The focus and expectations have shifted to combination therapies in general.

3.3.2 Dual therapeutic strategy using ICIs and anti-VEGF antibodies

Based on the positive results of the IMbrave150 phase-3 trial 8, the combination of a PD1/PD-L1 inhibitor and a VEGF blocker has become a new strategy to treat advanced HCC [8]. The IMbrave150 experiment resulted in the approval of atezolizumab plus bevacizumab as first-line treatment for unresectable HCC in the United States and Europe, replacing the TKIs sorafenib and lenvatinib. Inhibition of PD-L1, which increases the immune response (especially T-effector cells), and inhibition of VEGF, which stimulates T-cell infiltration in the tumor microenvironment and overcomes VEGF-mediated immunosuppression, is thought to have synergistic antitumor efficacy [29].

The ORIENT-32 phase 2/3 trial compared sintilimab (anti-PD1) and IBI305 (a bevacizumab biosimilar) to sorafenib in systemic treatment-naive Chinese patients, similar to the IMbrave150 trial (NCT03794440). In comparison to sorafenib, sintilimab/IBI305 exhibited an elevated median OS and PFS (median OS: not attained vs. 10.4 months; median PFS: 41.2 weeks vs. 12.17 weeks) with acceptable tolerability of [30].

3.3.3 Combination therapies of PD-1 and CTLA-4 inhibitors

Immune checkpoint inhibitor therapy for cancer patients has undoubtedly been a big achievement in oncology in recent years, and it represents a huge stride forward as a novel type of immunotherapy in cancer treatment. The combined ICIs including (anti-CTLA-4, and anti-PD-1/L1) are currently being investigated in advanced HCC. The CheckMate 040 trial, which evaluated nivolumab with ipilimumab in 148 patients with advanced HCC who had developed resistance to sorafenib, yielded the first clinical data [31]. The underlined therapy has been approved by the FDA post successful results of the trials. Moreover, systemic steroids are needed to treat adverse events.

3.3.4 Combination therapies of the checkpoint and multi-kinase inhibitors

Anti-VEGF antibodies can be replaced with ICIs and TKIs to inhibit VEGF. Currently, several such combinations are being investigated. As a secondary outcome measure, cabinozantinib monotherapy is compared with sorafenib. In a phase 1b trial involving 104 patients with unresectable HCC, the combination of lenvatinib with pembrolizumab was found to have potential anticancer efficacy [32].

Finally, camrelizumab (SHR1210, anti-PD-1) and apatinib (rivoceranib, a TKI inhibits VEGFR2) were tested in the clinic. An ORR of 50% was observed in a phase 1 investigation of individuals with advanced HCC [33]. The combination therapies are being evaluated as a first-line treatment for patients with advanced HCC in the phase 3 clinical trial.

3.3.5 Systemic treatment beyond ICIs

The most common method of cancer immunotherapy is the attenuation of the immunological checkpoints PD1/PD-L1 and CTLA-4. LAG-3 is an alternative immunological checkpoint that suppresses T-cell function, indicating T-cell depletion. In phase 2 RELATIVITY-073 trial in advanced, ICI-naive HCC post-progression on prior TKI therapy, relatlimab, an antibody that blocks LAG-3, is being tested in combination with nivolumab. Additionally, a growing number of novel immunotherapeutic methods are being investigated. Where today’s ICIs fail, such interventions could be effective. Adoptive transfer of NK or T cells to increase tumor infiltration, for example, could benefit patients whose tumors aren’t infiltrated by effector immune cells.

Besides classic checkpoint inhibition, the majority of immunotherapies are still in the preclinical or early clinical stages, and these include allogeneic NK cells, CAR-T, as well as oncolytic viruses [34]. CAR-T cells targeting GPC3 are currently being studied in phase 1 trials. Moreover, autologous T cells expressing improved TCRs specific for AFP (AFP c332 T) are being tested in HLA-A2-positive people with advanced HCC in the first phase 1 experiment targeting AFP. Pexastimogene devacirepvec (Pexa-Vec) failed in the TRAVERSE phase 2b trial and the PHOCUS trial as a second-line monotherapy in advanced HCC [35, 36]. Pexa-Vec and nivolumab are currently being tested in a phase 1/2a experiment. Novel immunotherapeutic techniques have the potential to provide immunotherapy benefits to a larger number of patients. However, it is not yet obvious which approaches will enhance or even replace current systemic therapeutic strategies.

4. Other HCC immunotherapies

4.1 Adoptive cell therapy (ACT)

In ACT combined with effector cells, lymphocytes are activated and/or amplified ex vivo before being reintroduced into the patients. Redirected peripheral blood T cells, TILs, CIK cells, NK cells, and lymphokine-activated killer cells (LAKs) are among the cell types that are used in this procedure. T-lymphocytes in the peripheral circulation have been genetically recoded to preferentially target tumor cells. The two basic techniques are chimeric antigen receptors (CARs) and transgenic tumor antigen-specific TCRs. CIK, TIL, and LAK therapies are not affected by the cancer-associated genes. Except in LAK, CIK, and CAR T cells, MHC-specific antitumor activity has been found in TIL and TCR-redirected cells. Usually, adoptive transplant cells are preconditioned with fludarabine and cyclophosphamide to promote lymphodepletion and enhance in vivo growth. Patients receiving LAK, CIK, or TIL treatment are often given IL-2 to assist the transferred cells’ proliferation in vivo.

Because of the technology’s apparent complexity and absence of efficiency, the first attempts to use ACT to treat HCC never got to the clinical stage. The fact that LAK cells poorly develop ex-vivo and have a poor cytolytic impact in melanoma patients serves as an example of the intricacy of this system. HCC recurrence was reduced by using LAK cells as an additive following resection to reduce the risk of HCC recurrence. However, this had not improved survival. However, CIK cells are more cytotoxic and proliferative when compared with LAK cells. NKT cells principally contribute to the antitumor effect of this diverse population. In 2015, a multicenter, randomized phase III trial involving 226 patients demonstrated that combination immunotherapy with CIK cells increased PFS and OS of HCC patients following percutaneous ablation or curative surgical resection relative to the patients who did not receive combination therapy. Considering the underlined outcomes, the majority of institutions do not use ACT as adjuvant therapy, most likely due to a lack of in-house cell therapy facilities. From tumor samples (fresh), TILs were obtained followed by selecting the tumor-reactive growing cells on the basis of their autologous cell recognition. Next, these cells were amplified to produce many active cells. The effectiveness of combined TIL treatment was indicated in a Phase-I study with individuals who had HCC7. TILs were given to 15 of 17 patients, with doses up to 3 × 10 [9] cells administered with the least side effects. The major challenges for clinical usage are obtaining sufficient T-cells that are selective for tumor neoepitopes and shortening the underlined procedure.

NK cells have a wide spectrum of receptors that allow them to identify tumor cells even without prior sensitization or the acquisition of receptor reconfiguration. For clinical usage, their inability to grow in vitro may be overcome. Phase II clinical trials are now using allogeneic NK cells to treat patients who have an increased risk of HCC recurrence following surgery or TACE.

In the treatment of hematologic malignancies (such as leukemia, multiple myeloma, and lymphoma), CAR T cell therapy has shown significant promise; however, its use in solid tumors is still under investigation. CAR T cells express transmembrane, intracellular signaling domains, and antigen-recognition domains that are common features of CAR T cells. One-chain variable fragments produced from the variable heavy and light chains of monoclonal antibodies selective for certain tumor cell targets, which can be tumor antigens, usually make up the extracellular antigen-recognition domains. In HCC, GPC3 has emerged as the most specific and appealing target. The efficacy of orthotopic and patient-derived xenografts has been demonstrated in various animal models. One of the most serious issues with CAR T cells is off-target toxicity. This occurs when the expression of the targeting molecule is in non-tumor tissues.

According to several studies, AFP is often overexpressed in HCC. Due to its intracellular expression and secretion, TCR-based therapy is relatively more effective when compared with CAR-based therapies. In patients associated with HCC, four HLA-A2-restricted AFP epitopes were identified. TP53 hotspot mutations, which are common in HCC and HBV antigens, are two more possible targets for T-cells (TCR-engineered).

4.2 Therapeutic vaccines

The key stimulus for using cancer vaccines is to induce tumor-specific reactions with higher efficacy. The underlined impact can be obtained by de novo priming T-cells against antigens produced by tumor cells that do not generate a spontaneous response and further enhancing the remaining reactions or expanding the repertoire and breadth of tumor-specific responses. Vaccinations were once thought to be a stand-alone treatment. However, it is now obvious that they should be used in combination with ICIs or ACT. Combinations of ICIs could block these variables, making it easier for antitumor lymphocytes to accomplish their activities.

In situ therapeutic vaccines act by activating tumor-infiltrating APCs, which absorb and display endogenous TAAs. Classic tumor vaccines, on the other hand, rely on the exogenous delivery of antigens or antigen-pulsed DCs. Cancer antigens should be immunogenic enough to overcome the tolerance induced by multiple self-molecules appearing on tumor cells, while also conferring selectivity for tumor cells and blocking the response of non-tumor cells. Antigen identity is uncertain in tumor lysate, which comprises self-molecules that could not confirm the accurate view of relevant TAA. Using tumor cell lysates as a treatment for HCC in a number of trials did not lead to consistent results.

TAAs including GPC-3, AFP, and telomerase have been addressed as HCC peptide vaccines (antigens specific). HCC patients have revealed spontaneous T-cell responses to the above antigens, suggesting that they are immunogenic in some way. In the majority of cases, T-cell sensitivity to their corresponding antigen is unclear. Hardly a few telomerase and GPC-3-targeting techniques have progressed to clinical evaluations, and none of them has yielded clinically relevant data that could serve as an active pharmaceutical candidate. One of the problems with the approach of therapeutic vaccination is tumor phenotypic heterogeneity and the real possibility that despite best efforts, some tumor cells will simply not be targeted by the vaccine, even if the vaccine is multivalent.

The identification of real tumor-specific antigens should be used to develop more immunogenic vaccinations. HLA peptidomics approaches are the first option that can be used to identify peptides that utilize peptides and serve as a unique immunological signature that CTLs may identify. On the basis of this method, a vaccine clinical evaluation has completed recruitment in HCC.

The use of neoantigens is a second approach. Their detection is based on a complicated pathway that involves analyzing mutations in tumor cells in comparison with wild-type cells. This method is utilized to investigate mutant gene expression as well as immune-associated factors including epitope process ability and HLA molecule interaction. Furthermore, the method has been used to anticipate significantly immunogenic neoantigens with antitumor potential as vaccines in different tumors, including glioblastoma and melanoma. There is a need for extensive studies to clinically evaluate HCC. Only a limited number of studies have been reported that relate the existence of mutations to specific immune responses. According to the results obtained from our ongoing research, mutations observed in HCC patients may generate peptides with stronger HLA-interacting potential when compared with non-mutated wild-type sequences. This research is still being conducted by our group. It has been shown that these peptides stimulate T-cells to recognize only the mutant sequence and not the wild type in HLA-transgenic mice, implying that the method could possibly be used as a vaccination for HCC.

5. Locoregional therapies

The immune system has developed to deal with microbial infections as well as severe tissue injuries. These situations are recognized by interconnected innate receptors, the function of which determines whether or not an adaptive immune response is produced and progresses. Furthermore, these activities may influence whether tumor antigens trigger immunization, tolerance, or not. As a result, hard efforts have been made over the years in order to develop cancerous tissue that seems like diseased or stressed tissue, which might encourage cytotoxic immunity’s adaptive response. Immune cell death and the use of PAMPs are two important concepts to look into in this context.

Immunogenic cell death (ICD) occurs when cells secrete or synthesize alarmins, a group of proteins that signal and stimulate APCs, i.e., DCs. ICD is linked to ER stress, necroptosis or necrosis, the secretion of mitochondrial and nuclear substances, and the activation of the Type-I IFNs. Moreover, chemo and radiotherapies, as well as other physical stimuli, activate cell and tissue damage and enhance immunity against tumor progression.

The immune system is activated by entities other than viruses, bacteria, and other prokaryotic entities. This reactivity allows drugs such as inflammasome agonists, Toll-like receptor (TLR) agonists, MDA5 or RIGI agonists, and cGAS–STING agonists to be delivered locally, stimulating localized immunity. These substances are usually synthetic analogs or obtained from microbial products, and when administered systemically, cause systemic inflammation and sepsis-like cytokine secretion syndromes. They may be more potent in triggering an immunological response with fewer adverse effects when administered intratumorally. Microorganisms can boost antitumor immunity without using their immune-stimulating substances. Therefore, Bacillus Calmette-Guérin vaccine is used for the local treatment of superficial bladder carcinoma. Oncolytic virotherapy is an effective therapeutic technique against cancer progression. This technique employs viruses that proliferate specifically in cancerous cells in order to kill them, with promising results in HCC. But instead of the cytopathic effects of the viruses, it has become clear that most of the therapeutic benefits of virotherapy come from boosting immune responses that target tumor antigens. Also, spores of anerobic bacteria have been used to amplify immune responses against tumors, since they only germinate in hypoxic environments, which occur in tumor nodules. This is why some bacterial infections trigger immune stimulation that in turn target tumor cells.

Locoregional methods are highly recommended for the treatment of individuals with unresectable HCC. Catheter-based procedures (yttrium-90 irradiation and transarterial chemoembolization) and locoregional ablative treatments, either chemical (percutaneous ethanol injection) or thermal (thermal ablation), are examples of image-guided therapies (laser, microwave, and radiofrequency ablation, and cryoablation) [4]. HCC is favorable for local therapies that can trigger ICD or local delivery of PAMPs due to its easy availability to tumor-affected regions. Notably, multiple locoregional therapies can be used independently or in combination with systemic immunotherapies, to obtain an elevated level of immune activation. Following RFA, immunological responses are activated, and T-cells infiltrate the tumor. In patients with advanced HCC receiving tremelimumab, partial tumor ablation with RFA or TACE achieved a 26% and 89% response rate and disease control rate, respectively. Furthermore, 45% of stabilizations last longer than 26.07 weeks and have an OS of 53.45 weeks [22]. As a result of these encouraging findings, clinical trials involving the use of ICIs, whether alone or combined with other ICIs or bevacizumab, in accordance with chemo, radioembolization, or post-complete percutaneous or surgical ablation, have given considerable support.

6. Reactions and resistivity

It is critical to developing pretreatment baseline levels of T-cell infiltration and stimulation when evaluating response to checkpoint inhibition in different carcinomas. While the influence of CD4+ and CD8 + T-cell infiltration on survival post advanced HCC treatment using second-line PD1 inhibitors has been observed in the form of poor interactions and trends. The underlined results show that the impact is not as significant as originally assumed. According to the immunohistochemistry results, about 20% of advanced HCC tumors express PDL1. Tumor responses were reported in the CheckMate 040 trial independent of PDL1 expression. However, response rates were higher in patients with a minimum of 1% of tumor cells expressing PDL1. PDL1 expression in stromal immune cells as well as in tumor cells was elevated in pembrolizumab-treated patients who obtained objective remission. However, according to the obtained results, the responses were observed even in the absence of expression in both types of cells. In naive patients associated with PDL1-positive HCC, median OS was 70 weeks and 37.3 weeks (HR 0.80), accordingly. In the case of PDL1-negative HCC patients, after sorafenib and nivolumab treatment, the median OS was 72.6 weeks and 66 weeks (HR 0.84), respectively. Furthermore, a trend toward better OS with nivolumab monotherapy was observed in patients with increased tumor infiltration via CD3 + or CD8 + cells. In addition, various signatures of inflammatory genes (such as COX-2) were linked to an elevated response rate and OS. The underlined gene signatures were linked to inflammatory activities, cytolytic genes, IFN-associated genes, exhaustion markers, NK cell markers, and antigen presentation. In this view, these findings reveal an elevated level and activity of T cells and NK cells that utilize cytolysis and IFNγ as their primary antitumor effector mechanisms. In this study, the most complicated transcriptome classification, which includes a huge set of genes, was not found to be predictive of response. However, in the current evaluation, the number of patients for whom small data of RNA sequencing were existing, as large sample data could provide more definitive outcomes (positive or negative). Integrating genomics and transcriptomics into immunotherapy effectiveness will include comprehensive integrative analysis as well as obtaining biopsy samples before and during therapy.

Patients with objective remissions demonstrated potent CD3 + and CD8 + infiltration as compared with non-responders in paired HCC biopsy samples taken prior to and post two doses of tremelimumab. In the case of the combinations, objective HCC remissions were achieved regardless of PDL1 expression in nivolumab + ipilimumab treated cancerous cells. A single biomarker is insufficiently sensitive to provide timely clinical data. However, thorough immunohistological, mutational, and transcriptomic evaluations are required, as integrated multifactorial indices may be able to determine subgroups of individuals who would take advantage of ICI treatment. The significance of matching biopsies prior to and following treatment cannot be emphasized, since diagnostic biopsies for HCC are rarely taken.

The question of whether we are dealing with synergistic effects or just an additive impact arises when evaluating the enhanced combination of ICI therapeutic efficacy. Complementary analysis from major clinical studies is still lacking. Nevertheless, a subgroup analysis has reported a correlation between durvalumab, tremelimumab, or both in combinations. Furthermore, on day 15 after therapy began, an elevated level of proliferating Ki67 + CD8 + T cells among blood mononuclear cells was observed. In particular, in comparison with durvalumab, tremelimumab monotherapies, or the combined effect of a routine low dosage of tremelimumab with a similar dosage of durvalumab, the elevation in the underlined population of peripheral effector T-cells was found optimum for responders to an elevated priming dosage of durvalumab plus tremelimumab. The underlined combined therapies obtained the highest OS. It could be helpful to use biomarkers that are easily available to help design new combinations of therapies and to compare data between combinations. Significant TME changes were found in animal models with VEGFR blockade. The blockage of VEGFR improves PD1 inhibitory activity. According to multiple reported studies on mouse models, combined VEGFR and PD1 inhibition decreases M2-polarized macrophages and T-reg cells, enhances HCC cells and PDL1 expression in TAMs, and enhances normalized vasculature development triggered by CD4 + cells. Notably, effectiveness was achieved with a low dosage of anti-angiogenics (vascular normalizing rather than anti-vascular), offering a promising path of research into minimizing the associated toxicities.

The development of ADAs (anti-drug antibodies) that can affect the elimination of these drugs or neutralize their effectiveness is another viable cause of tumor resistance. The estimated prevalence of ADAs during monotherapies with anti-CTLA4 (ipilimumab), anti-PD1 (cemiplimab, nivolumab, and pembrolizumab), and anti-PDL1 (durvalumab and avelumab) agents is low, ranging from 0 to 12.7% in all tumor types. While no relevant impact of ADAs on efficiency has been noticed for nivoluma. ADAs, on the other hand, were found in up to 36% of NSCLC patients who had received atezolizumab and had a negative impact on systemic exposure to the drug as well as antitumor effectiveness. In the case of HCC, effectiveness (impact on OS) was poorer in the 20% of patients (ADA-positive by week 6) treated with bevacizumab and atezolizumab in a subanalysis of the IMbrave150 study. PD-L1 and CTLA4 are only two molecules that are involved in T cell exhaustion. There are many others on the surface of tumor cells that could compensate. Also, CTLs may have a hard time penetrating solid tumors, especially those that are encased in a fibrous capsule. This may also limit the effects of immunotherapy.

7. Management of immunotherapy toxicities

7.1 Immune-associated side effects

Immune checkpoints or coinhibitory receptors including CTLA-4 and PD-1 control T cell reactions and are efficient therapeutic targets [37, 38]. One of the drawbacks of the underlined advancements in the development of a novel spectrum of immune-related adverse events (irAEs), which are frequently distinct from the conventional toxicities associated with chemotherapy. Due to the rising use of ICIs in oncology, clinicians will progressively encounter both frequent and rare irAEs; hence, it is needed to increase attention regarding the clinical manifestation, evaluation, and managing these toxicities.

Unlike anti-CTLA-4-related adverse events, the risk of irAEs caused by PD-1/PD-L1 inhibition is independent of dosage [39, 40]. The skin and gastrointestinal tract were the most commonly affected organ systems by anti-CTLA4 and PD-1/PD-L1 inhibitors, whereas the liver and the endocrine system were less frequently damaged [39, 40]. However, ipilimumab was found to be linked with a considerably elevated incidence of rash and colitis than anti-PD-1/PD-L1 medicines [41].

7.2 irAEs management

Since irAEs are linked with a large array of aggravating conditions in the HCC, hepatologists face numerous hurdles while detecting and treating them. First, liver cirrhosis causes immunological dysfunction, which worsens over time [42]. Consequently, the immunological homeostasis linked with the liver in these patients is substantially damaged. Second, cirrhosis-related hepatic and extrahepatic consequences may overlap with or intensify symptoms mediated by irAEs [43]. Consequently, prior to ICI therapy, HCC patients should be carefully selected and evaluated [43].

Furthermore, the underlined approaches should be utilized for managing irAEs. First, strict surveillance is required, with weekly clinical controls, based on the intensity of the incidents. This is especially critical in patients with liver cirrhosis because distinguishing between problems (linked with cirrhosis) and irAEs can be difficult, and prematurely terminating a considerable antitumor therapy or initiating steroid therapy in cirrhotic patients might have serious implications [43].

Depending on the nature and severity of irAEs, it may be required to temporarily stop or permanently discontinue ICI therapy. With the exception of PD-1/PD-L1-driven rash, nephritis, adrenal insufficiency, and hypothyroidism, which recover after 1 month of treatment, permanent termination of ICI therapy should be addressed for irAEs of grade ≥ 3 [43]. There is a considerable risk of recurrence of irAEs when ICI medication is restarted after it has been discontinued: Twenty-five percent (22 of 40) of the 93 patients with irAEs of grade ≥ 2 who were treated with anti-PD-1/PD-L1 drugs had a recurrence of irAEs post-termination [44]. While recurrence of irAEs was linked with a more rapid onset of the early irAE, the frequency of the recurring irAEs did not vary [44]. TKIs used for HCC include sorafenib, lenvatinib, regorafenib and cabozantinib, all of which are associated with skin toxicity. However, the type of adverse effect and time course can help distinguish between ICI- and TKI-related events. For TKIs, the onset of rash is usually within weeks of starting and palmar-plantar erythema is the most common AE, reported in 52% and 27% of patients receiving sorafenib and lenvatinib, respectively. 29 This compares with around 2% for PD1 inhibitors. Additionally, the relatively short half-life of TKIs results in rapid resolution of skin toxicity over the course of days, which contrasts with the weeks or months that may be required for ICI-related toxicity to resolve.

Glucocorticoids may be prescribed for irAEs of grade ≥ 2 (0.5–2 mg/kg/day prednisone PO or IV, depending on the kind and intensity of the irAEs). Topical, oral, and intravenous glucocorticoids, as well as oral or topical antihistamines, are used to treat cutaneous irAEs, which range from simple rash or itch to less common but more serious illnesses such as Stevens-Johnson syndrome (SJS) [43]. Stevens-Johnson syndrome (SJS) is a form of severe adverse drug reactions and is characterized by epidermal necrolysis. The disease has the unique expression of blisters on the skin and the affection of mucous membranes in the mouth, nose, eyes, and genitals. SJS is characterized by a large area of skin and mucosal epithelial cell shedding and typical performance on the oropharynx, eyes, urogenitals, and anal mucosa. SJS has less than 10% of body surface area involvement. Steroids should be continued for at least 3 days before being reduced over 1–4 weeks [21, 45]. It should be noted that steroids are only beneficial in non-viral-associated chronic liver disease. If viruses are involved (HBV, HCV), steroids will simply permit virus replication to increase, and when steroids are withdrawn, a severe exacerbation of chronic liver disease may be seen.

Differential diagnosis is required for gastrointestinal irAEs, notably colitis and/or diarrhea, to rule out infectious illnesses and medication adverse effects [43]. For grades 2 and ≥ 3, glucocorticoids should be started, and hospitalization with sigmoidoscopy/colonoscopy should be considered. Moreover, immunosuppressive medication should be added early in the case of glucocorticoid failure [43, 46]. Depending on the steroid reaction and the severity of clinical presentation, discontinuation should be done over 2–8 weeks [21, 45].

Immune-related hepatitis is particularly difficult to diagnose and treat in individuals with HCC who are receiving ICI therapy [43]. However, early contact with an experienced hepatologist is thus strongly advised. Intrahepatic growth of the tumor, HBV/HCV flares, and adverse events linked with hepatotoxic medication, ascites, cholestasis, and CMV reactivation should all be ruled out before a diagnosis of immune-associated hepatitis is made. A liver biopsy should also be conducted prior to the administration of steroids [43].

Pneumonitis is an irAE that can be life-threatening. Hence, a prompt and extensive differential diagnosis should be conducted with suspected pneumonitis, including the exclusion of portopulmonary hypertension, viral etiologies, and hepatopulmonary syndrome [43]. Steroids should be started for grade 2 and discontinued over a period of 4–6 weeks [21]. Post glucocorticoid failure, infliximab (which is inflammatory by inhibiting TNFα) or mycophenolate mofetil (an immunosuppressive compound that inhibits inosine monophosphate dehydrogenase) may be administered [43]. It should be noted that therapeutic approaches involving prolonged immunosuppression increase the risk for selected infectious diseases and tumor types that usually never develop in the presence of intact immune surveillance.

8. Future directions

The licensing of the initial ICIs in advanced HCC and their clinical efficacy have revolutionized the concept of cancer immunotherapy. However, this field still has to address three major questions: Is immunotherapy beneficial in the preclinical stage? If so, it will reduce the risk of disease progression to cancer. In addition to PD-1/PD-L1/CTLA-4 suppression, which immunological treatments have antitumor effects in HCC? Which therapy options exist for patients not responding to the presently approved ICIs? Perhaps immunomodulation with histone deacetylase inhibitors (HDACi) in combination with ICI would permit a sustained antitumor response with reduced risk of adverse effects.

In response to the first question, various clinical studies in the initial and intermediate stages have reported the use of ICIs. It’s uncertain whether checkpoint inhibitor exhibit regimens are a TACE alternative. As an alternative, the RENOTACE and ABC-HCC trials will test atezolizumab with bevacizumab and regorafenib plus nivolumab. Patients above the up-to-seven criteria, i.e., the subgroup with a relatively great tumor burden at an advanced stage, were recruited for RENOTACE, whereas patients with the whole spectrum of intermediate stages of disease were targeted for ABC-HCC. In addition, the initial trials examining ICIs for neoadjuvant approaches are currently in progress. The underlined collection of trials will look at the safety and efficacy of immunotherapy in the initial and intermediate stages from a variety of perspectives, providing high-quality data that will be useful in understanding the contribution of ICIs in the underlined situations.

To address the second question, there are a number of treatments that are currently being evaluated in clinical trials, which include the use of oncolytic viruses, CAR T/NK cells, and LAG-3 checkpoint inhibitors. These treatments might help patients with established ICIs who are unresponsive or have failed to respond to the treatment.

The third question may also be addressed by the above new immunotherapeutic strategies. A subset of patients, particularly those with an immunological desert TME, is likely to take advantage less of immunotherapy. Although immunotherapy has made tremendous advances. It should not be overlooked when considering treatment options for patients who may benefit from existing and future specialized therapies.

Acknowledgments

This manuscript is supported by Natural Science Foundation of Shandong Province (ZR2021MH022).

Competing interests

No competing interests exist among the authors.

Keypoints

Based on the outcomes of recent clinical trials, it appears that a single agent may not be sufficient for treating HCC; consequently, combination therapy characterizes an important area of research for the systemic treatment of advanced HCC. Moreover, ICIs are now being considered as part of HCC treatments, and their use in combination with molecular targeted therapy is shown to be an effective way to boost the immune system’s response. The combined therapy of atezolizumab/bevacizumab is the first-line treatment to receive regulatory approval.

Immune checkpoint inhibitors (ICIs) may potentially be effective at earlier stages of illness. Combination therapies are being investigated in the intermediate stage in combination with or in place of transarterial chemotherapy, which is currently the standard of care. ICIs are also being explored as adjuvant and neoadjuvant therapy in the setting of early and very early surgery or ablation.

New research reveals that the control of the commensal gut microbiome and hepatic antitumor immunity are linked. Moreover, ICIs have been linked to immune-related side effects. In HCC clinical studies, however, the prevalence of grade 3/4 incidents was moderate. Therefore, ICIs are largely considered to be a choice in advanced HCC patients.