INTRODUCTION

Globally, liver cancer, predominantly hepatocellular carcinoma (HCC), ranks as the third most common cause of cancer-related mortality. In 2022, it was responsible for approximately 750,000 fatalities worldwide.^1,2 The emergence of technologies based on next-generation sequencing has significantly enhanced our comprehension of the molecular and immunological characteristics of HCC.³ In addition, the treatment landscape for advanced-stage HCC has undergone drastic changes due to the approval of molecular targeted agents (MTAs) and immune checkpoint inhibitors (ICIs) in the past 5 years.⁴ Although the introduction of these new therapeutic options is promising, their impact on survival rates in advanced-stage HCC remains modest, with median survival still falling short of 2 years. Experimental studies and genomic data are anticipated to provide molecular insights that will soon influence the clinical management of patients with HCC. However, the optimal sequencing of these novel therapies remains uncertain. Recent pre-clinical and clinical investigations have indicated that the response to these molecularly targeted treatments may be affected by clinical factors, such as the etiology of liver disease. Therefore, an in-depth understanding the molecular pathogenesis of HCC in specific clinical scenarios is essential to optimize the application of novel therapies for maximum effectiveness. This review serves as a roadmap for clinical trials and the implementation of new therapeutic approaches by offering a comprehensive overview of the molecular and immunological landscape of HCC.

EMERGING MOLECULAR AND IMMUNE TARGETS IN HCC

Extensive multicenter studies have performed in-depth genomic phenotyping of human HCC tumors and the tumor microenvironment (TME)/tumor immune microenvironment (TIME), identifying key molecular characteristics driving/supporting cancer initiation/progression (Fig. 1A).⁵ A thorough grasp of the intricacies and diversity within the molecular and immune landscape of HCC and its TME/TIME will offer insights for personalized treatment approaches. The variations within and between tumors arise not only from inherent epi/genetic abnormalities in cancer cells but also from shifts in the makeup and stromal components within the TME/TIME. Comprehensive multi-omic examination of whole tissue, individual cells/nuclei, and spatially mapped tissue has uncovered a wide range of molecular aberrations in HCC tumors and their microenvironment.^6-16 Some molecular pathways/hallmarks can be targeted using approved or tested clinical agents, while others, previously considered untreatable, are now being explored as novel therapeutic targets (Fig. 1B). These include frequently mutated regions (e.g., TERT promoter [TERT_p], TP53, CTNNB1). In this context, we discuss current and reported molecular and immune mechanisms that could serve as potential therapeutic strategies.^6-11,17

Telomere maintenance mechanisms (TMM)

The disruption of TMM, including the reactivation of telomerase, is a crucial aspect in achieving replicative immortality, which is considered one of the fundamental characteristics in multiple cancer types including HCC.^18,19 Somatic alterations in the TERT_p are the most common structural changes found in human HCC, occurring in 40-60% of cases, thus, these modifications can take the form of nucleotide substitutions, insertions or deletions, and the integration of viral DN.^{6-11,17,20,21} The occurrence of somatic TERT_p mutations in early-stage HCC, dysplastic nodules of varying grades, and cell-free DNA from cirrhosis patients without clinically detectable HCC indicates their role in the initial stages of HCC formation. This finding also suggests that detecting TERT_p mutations could be valuable for assessing risk and/or identifying HCC in its early phases.^22-27 The reactivation of telomerase was linked to worse recurrence-free survival, indicating its role in disease advancement.^28-30 Conversely, a recent case-control investigation discovered a novel single nucleotide polymorphism in the TERT gene (rs2242652) that appeared to protect against HCC in patients with alcohol-induced cirrhosis.³¹ This suggests that telomere length may be a factor in the molecular heterogeneity of HCC tumors.³² The precise mechanistic involvement of these mutations in HCC initiation and progression remains unclear. Nevertheless, due to the high frequency of TERT_p mutations in HCC and other cancers, TMM has emerged as a potential therapeutic target.³³ For example, pre-clinical HCC models have shown promising anti-HCC effects using antisense oligonucleotides that target TERT transcripts.³² Additionally, a nucleoside analog called 6-thio-dG (THIO) has been found to cause telomeric DNA damage in telomerase-activated HCC cells, stimulate innate and adaptive anti-tumor immunity, and improve the response to ICIs in mice.³⁴ These TMM-directed therapeutic approaches, along with others, may prove valuable in the clinical management of individuals at risk for HCC.

Wnt/beta-catenin

The Wnt signaling is an essential pathway in liver development and homeostasis, regulated by a key transcriptional mediator, beta-catenin encoded by the CTNNB1 gene. CTNNB1 is among the most frequently mutated genes in approximately 20-40% of HCC tumors.³⁵ In contrast to other cancer types where CTNNB1 mutations are associated with induction of oncogenic canonical Wnt signaling, these mutations are accompanied with induction of glutamate-ammonia ligase (also known as glutamine synthase) encoded by GLUL gene, more differentiated histology, immune exclusion, and better prognosis in HCC.^35-37 Of note, canonical Wnt signaling can be induced in HCC tumors without CTNNB1 mutations.^35,38 Wnt/beta-catenin pathway is now actively explored as a therapeutic target in multiple cancer types and non-cancer conditions by utilizing various strategies.^39-41 An ongoing phase I/II trial is evaluating a dickkopf-1 (DKK1) inhibitor in patients whose tumors display positive glutamine synthase immunostaining as a surrogate marker of CTNNB1 mutations (NCT03645980).

TP53

TP53 gene, which encodes the p53 tumor protein, is frequently mutated in advanced-stage HCC and is linked to aflatoxin B1 food contamination.^11,42-48 Loss-of-function mutations in this tumor suppressor gene and its related genes (TP73L, TP63, CDKN2A, MDM2/MDM4) are associated with aggressive cancer characteristics, including stemness features and resistance to therapy.49-53 A study using a rodent model suggested that p53 function may be compromised in early HCC development through CD44/STAT3 signaling, independent of somatic mutations.⁵⁴ Another rodent model demonstrated that TP53 loss works in conjunction with c-MET to promote liver cancer development.⁵⁵ Researchers are actively investigating therapeutic approaches to restore p53 function in pre-clinical studies and early-phase clinical trials. These methods include refolding mutant p53 and inhibiting MDM2, which may soon be applicable to HCC cases with TP53 mutations.^56,57 Additionally, adenoviral delivery of the p53 tumor suppressor protein has been evaluated in solid tumors without dominant-negative TP53 mutations (NCT03544723).

Alpha-fetoprotein (AFP)

While the limited positivity of AFP (around 30%) restricts its use as a tumor detection biomarker, it may still prove valuable as an indicator of specific biological/clinical characteristics and treatment responses. Research has demonstrated a connection between high AFP levels and tumor progression, resistance to cell death, and blood vessel formation. These effects might be countered by medications targeting relevant molecular pathways, although the precise mechanisms of therapeutic response, such as in the case of ramucirumab, are not always well understood. Recent investigations have also indicated that AFP might aid in tumor immune evasion by suppressing dendritic cells, natural killer (NK) cells, and T-cells.⁵⁸ While AFP itself has weak immunogenicity, AFP-targeted immune engineering strategies, including chimeric antigen receptor (CAR) T cell therapy and specific peptide enhanced affinity receptor (SPEAR) T cell therapy, have undergone phase I/II clinical trials.⁵⁹ Peptide vaccines targeting have been developed AFP and other cancer-related proteins and neoantigens, including WT-I, ROBO1, FOXM1, HER3, and mutant TP53.⁶⁰

Glypican-3 (GPC3)

GPC3, a heparan sulfate proteoglycan on cell surfaces, serves as an immunohistochemical indicator for HCC.⁶¹ Epithelial cell adhesion molecule (EpCAM), a marker for hepatic stem/progenitor cells on cell surfaces, is highly expressed in various gastrointestinal cancers, including HCC.^62,63 GPC3 (and EpCAM), like AFP, is expressed in a subset of HCC tumors exhibiting stemness characteristics, poor outcomes, and a strong association with the Hoshida S2 subtype (Fig. 1A).^38,63,64 GPC3 is also an ideal target for immune-engineered approaches, such as CAR T-cell therapy, currently evaluated in multiple ongoing phase I/II trials (Table 1).⁶⁴ Codrituzumab (GC33), a humanized monoclonal antibody targeting human GPC3, did not demonstrate anti-tumor effects in a phase II trial. However, it may be useful as a molecular imaging probe when combined with the positron emission tomography radiotracer, I-124.^65,66 ERY974, a bispecific antibody designed to redirect T cells to GPC3-positive tumor cells by targeting GPC3/CD3, showed general tolerability in patients with advanced solid tumors during a phase I trial.⁶⁷

Suppressive immune checkpoints

ICIs, including those targeting programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways, have become crucial elements in the systemic treatment of cancer, including HCC. These agents work by modulating the interactions between tumor and immune cells and are often used in combination with MTAs or other ICIs. There is still no approved companion biomarker for ICI-based therapy specifically for HCC, but several immune-related features such as PD-L1 expression, tumor mutation burden (TMB), and DNA repair defects (e.g., deficient mismatch repair and high microsatellite instability) have been approved for patient selection in several solid cancers such as non-small cell lung cancer.⁶⁸ In ongoing clinical trials for HCC patients, several molecular features, pembrolizumab (anti-PD-1) plus futibatinib (fibroblast growth factor receptor [FGFR]1-4 inhibitor) have been tested in fibroblast growth factor [FGF]19-positive HCC tumors in phase II trials (NCT04828486). However, despite their importance, reliable biomarkers for predicting patient response to these therapies have not yet been identified.^69-71 Currently, a clinical trial (NCT04802876) is evaluating spartalizumab, an anti-PD-1 antibody, in patients exhibiting elevated PDCD1 mRNA expression (encoding PD-1). Recent clinical studies have also focused on targeting established molecular characteristics of HCC, recruiting patients whose tumors display specific molecular alterations. PD-0332991, an inhibitor of CDK4/6 cell cycle regulators, has been studied in HCC patients with intact retinoblastoma (RB) protein (NCT01356628). In HCC patients treated with atezolizumab (anti-PD-L1) and bevacizumab (anti-vascular endothelial growth factor [VEFG]), an increase in Ki-67/PD-1/CD8-positive T cells and TIGIT-positive T cells at 3 weeks after initiation of the therapy was associated with superior progression-free and overall survival, suggesting their role as non-invasive predictive biomarkers of response.⁷²

Vessels that encapsulate tumor clusters (VETC)

VETC pattern is a pathological finding observed in a certain percentage of HCCs and has been demonstrated to correlate with poor patient prognosis.⁷³ VETC-positive HCCs have also been reported to develop under the influence of angiopoietin 2, and the potential efficacy of tyrosine kinase inhibitors has been investigated.⁷⁴ A study showed that unresectable VETC-positive HCC may benefit from treatment with sorafenib.⁷⁵ A clinical trial is currently in progress to evaluate the treatment of VETC-high expressing HCC with a combination of an ICI and a tyrosine kinase inhibitor (NCT06461936, NCT06311929).

The positive outcomes of these trials could potentially pave the way for the clinical application of personalized, biomarker-guided treatments for HCC across various therapeutic approaches. This advancement may enable a more scientifically rational and effective tumor management strategy while reducing the risk of unnecessary adverse effects in patients who unlikely benefit from these therapies.

ONGOING BIOMARKER-GUIDED THERAPEUTIC CLINICAL TRIALS

Although ongoing and planned clinical trials still tend to enroll all-comer patients, biomarker-driven trials have increased in recent years, coinciding with the active development of MTAs.⁷⁶ Predictive biomarkers, which indicate therapeutic response, play a crucial role in innovative clinical trial methodologies like adaptive designs, facilitating the translation of novel therapeutic agents and strategies into clinical practice.^35,77

Biomarker enrichment trials seek to enhance the detection of therapeutic benefits by focusing on a subset of clinically eligible patients who test positive for a biologically rational target biomarker (Fig. 2). In these trials, only biomarker-positive patients are randomly assigned to either the study drug or placebo arm. These trials aim to demonstrate the superiority of the biomarker enrichment approach over the traditional allcomer strategy. The efficacy of experimental therapies and strategies is evaluated using endpoints relevant to specific clinical contexts, such as anti-tumor response, disease control, survival advantage, and cost-effectiveness of interventions. To efficiently assess the growing number of new MTAs across multiple cancer types and histologies, master protocol designs have been developed.⁷⁸ One such design is the basket/bucket trial, which recruits patients with the same molecular target, regardless of tissue type or histology, potentially including HCC alongside other cancer types. Umbrella trials assess various molecularly targeted therapies based on the presence of corresponding biomarkers. Platform trials employ multi-arm/stage designs with an open-ended timeline to evaluate multiple experimental treatments against a shared control group (Fig. 2).

CONCLUSIONS AND FUTURE PERSPECTIVE

HCC remains the third leading cause of cancer-related mortality, and there are large racial, ethnic, and interindividual disparities in HCC risk and survival.^1,2 Our knowledge of whether molecular and genetic factors contribute to these observed differences is limited by a lack of biological samples. Biospecimen collection is the process of collecting, processing, and storing tissue, blood (e.g., ctDNA), or urine samples for research purposes; biospecimens collected through this process are utilized to elucidate the genetic and molecular factors contributing to HCC and to develop novel biomarker assays of significant importance.^79,80 These studies facilitate the development of companion biomarkers based on test results that examine the expression of target molecules, the presence of genetic mutations, and genetic polymorphisms of drug-metabolizing enzymes to predict the efficacy and safety of specific drugs. Furthermore, this research will lead to the development of innovative biomarker-focused trial designs, which will result in improved patient selection, optimizing the application of novel therapies and maximizing their efficacy.

Article information

Acknowledgement

The figures were created using BioRender.com.

Conflicts of Interest

Yujin Hoshida is shareholder for Alentis Therapeutics and Espervita Therapeutics, advises Helio Genomics, Espertiva Therapeutics, Roche Diagnostics, and Elevar Therapeutics.

Ethics Statement

This review article is fully based on articles which have already been published and did not involve additional patient participants. Therefore, IRB approval is not necessary.

Funding Statement

This study was supported by US National Institutes of Health (CA233794, CA255621, CA282178, CA288375, CA283935), European Commission (ERC-AdG-2020-101021417), Cancer Prevention and Research Institute of Texas (RR180016, RP200554), Uehara Memorial Foundation. The funders had no role in this article.

Data Availability

Not applicable.

Author Contributions

Conceptualization: HS, SM, SP, and YH

Data curation: HS, SM, SP, and YH

Funding acquisition: HS, SM, SP, and YH

Methodology: HS, SM, SP, and YH

Project administration: HS, SM, SP, and YH

Resources: HS, SM, SP, and YH

Supervision: HS, SM, SP, and YH

Visualization: HS, SM, SP, and YH

Writing - original draft: HS, SM, SP, and YH

Writing - review & editing: HS, SM, SP, and YH

Figure 1.

Therapeutic strategies according to molecular and immune landscape of HCC. (A) Molecular subtypes of HCC tumors and associated histological and molecular features. (B) Molecular and cellular targets for approved drugs and therapeutic strategies in development in HCC. CAR-T cells, chimeric antigen receptor T cells; GPC3, glypican-3; AFP, alpha-fetoprotein; EpCAM, epithelial cell adhesion molecule; neoAg, neo antigen; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; PDGFβ, platelet-derived growth factor beta; EGF, epidermal growth factor; HGF, hepatocyte growth factor; APC, antigen presenting cell; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; VEGFR, vascular endothelial growth factor receptor; FGFR, fibroblast growth factor receptor; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; MET, MET proto-oncogene (hepatocyte growth factor receptor); PD-L1, programmed cell death ligand 1; mTOR, mechanistic target of rapamycin; JAK/STAT, Janus kinase/signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase; PI3K/Akt, phosphatidylinositol 3-kinase/protein kinase B; PD-1, programmed cell death protein 1; CDK, cyclin-dependent kinase; DKK1, dickkopf-1; Rb, retinoblastoma; HCC, hepatocellular carcinoma; TGFβ, transforming growth factor beta; TGFβR, transforming growth factor beta receptor; TERT, telomerase reverse transcriptase.

Figure 2.

Study designs for biomarker-guided therapeutic clinical trial in HCC. HCC, hepatocellular carcinoma.

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