Molecular and immune landscape of hepatocellular carcinoma for therapeutic development

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, with an estimated 750,000 deaths in 2022. Recent emergence of molecular targeted agents and immune checkpoint inhibitors and their combination therapies have been transforming HCC care, but their prognostic impact in advanced-stage disease remains unsatisfactory. In addition, their application to early-stage disease is still an unmet need. Omics profiling studies have elucidated recurrent and heterogeneously present molecular aberrations involved in pro-cancer tumor (immune) microenvironment that may guide therapeutic strategies. Recurrent aberrations such somatic mutations in TERT promoter and TP53 have been regarded undruggable, but recent studies have suggested that these may serve as new classes of therapeutic targets. HCC markers such as alpha-fetoprotein, glypican-3, and epithelial cell adhesion molecule have also been explored as therapeutic targets. These molecular features may be utilized as biomarkers to guide the application of new approaches as companion biomarkers to maximize therapeutic benefits in patients who are likely to benefit from the therapies, while minimizing unnecessary harm in patients who will not respond. The explosive number of new agents in the pipelines have posed challenges in their clinical testing. Novel clinical trial designs guided by predictive biomarkers have been proposed to enable their efficient and cost-effective evaluation. These new developments collectively facilitate clinical translation of personalized molecular-targeted therapies in HCC and substantially improve prognosis of HCC patients.

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

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.

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

Table 1.

Ongoing biomarker-guided therapeutic trials in HCC

Clinical context	Phase	Trial type	Agent	Class of agent	Biomarker-based eligibility	NCT number	Last updated
Neo-adjuvant theapy	II	Biomarker enrichment trial	Lenvatinib	Multi-kinase inhibitor	AFP>100 ng/mL, infiltrative/ macrotrabecular pattern	NCT05113186	2024
Adjuvant therapy	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06560827	2024
	II	Biomarker enrichment trial	Sintilimab +/- lenvatinib	Anti-PD-1 +/- multi-kinase inhibitor	VETC positive	NCT06461936	2024
	IV	Biomarker enrichment trial	Anti-PD-1 +/- lenvatinib	Anti-PD-1 +/- multi-kinase inhibitor	VETC positive	NCT06311929	2024
First-line systemic therapy	I	Basket/bucket trial	TSR-022	Anti-TIM-3	TIM-3-positive	NCT02817633	2024
	I/II	Biomarker enrichment trial	DKN-01	DKK1 inhibitor	Glutamine synthetase IHC-positive	NCT03645980	2020
First or second-line systemic therapy	I	Biomarker enrichment trial	AFPc332 T cells	Engineered T cells	AFP≥100 ng/mL	NCT03132792	2024
	I	Biomarker enrichment trial	CT0180 cells	Engineered T cells	GPC3 IHC-positive	NCT04756648	2023
	I	Basket/bucket trial	BOXR1030 T cells	Engineered T cells	GPC3 IHC-positive	NCT05120271	2024
	I/II	Basket/bucket trial	CAR-T/TCR-T cells	Engineered T cells	DR5 or EGFRvIII IHC-positive	NCT03941626	2021
	II	Basket/bucket trial	32 molecular-targeted agents	Molecular-targeted agents	Positive targets by tumor DNA-seq, IHC	NCT02465060	2024
	II	Basket/bucket trial	Spartalizumab	Anti-PD-1	PD-1 mRNA, high expression	NCT04802876	2023
	II	Basket/bucket trial	Ad-p53 + approved ICI	Viral gene therapy + ICI	p53 wild type or IHC-negative	NCT03544723	2020
Second-line systemic therapy	I	Biomarker enrichment trial	C-TCR055	Engineered T cells	AFP IHC-positive	NCT04368182	2020
	I	Biomarker enrichment trial	TC-CAR031	Engineered T cells	GPC3 IHC-positive	NCT05155189	2024
	I	Basket/bucket trial	EpCAM CAR-T cells	Engineered T cells	EpCAM-positive	NCT05028933	2024
	I	Basket/bucket trial	AFP CAR-T cells	Engineered T cells	AFP IHC-positive or AFP≥400 ng/mL	NCT06515314	2024
	I	Basket/bucket trial	p53 MVA vaccine + pembrolizumab	MVA vaccine + anti-PD-1	p53 IHC-positive or gene mutation	NCT02432963	2024
	I	Basket/bucket trial	HER2 CAR-macrophages	Engineered macrophages	HER2-positive	NCT04660929	2024
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05003895	2024
	I	Biomarker enrichment trial	GPC3 CAR-T cells + fludarabine + cytoxan	Engineered T cells + STAT1 inhibitor + alkylating agent	GPC3 IHC-positive	NCT05103631	2023
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06461624	2024
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06144385	2024
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05620706	2022
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05926726	2023
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05344664	2022
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05783570	2024
	I	Biomarker enrichment trial	GPC3 CAR-NK cells	Engineered NK cells	GPC3-positive	NCT05845502	2024
	I/II	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06084884	2024
	I/II	Biomarker enrichment trial	B7H3 CAR-T cells	Engineered T cells	B7H3-positive	NCT05323201	2024
	I/II	Biomarker enrichment trial	ECT204 T cells	Engineered T cells	GPC3 IHC-positive	NCT04864054	2024
	I/II	Biomarker enrichment trial	HBV-TCR T cells (LioCyx-M) +/- lenvatinib	Engineered T cells +/- multi-kinase inhibitor	HLA class I profile matching	NCT05195294	2022
	II	Biomarker enrichment trial	PD-0332991	CDK4/6 inhibitor	RB-positive	NCT01356628	2023
	II	Biomarker enrichment trial	Futibatinib + pembrolizumab	FGFR inhibitor + anti-PD-1	FGF19 mRNA or IHC-positive	NCT04828486	2024
Third-line systemic therapy	I/II	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06590246	2024
Not defined	I	Basket/bucket trial	IL-15 and IL-21 armored GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06198296	2024
	I	Biomarker enrichment trial	GPC3/TGF-β CAR-T cells	Engineered T cells	GPC3 WB or IHC-positive	NCT03198546	2024
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05070156	2023
	I	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT06478693	2024
	I/II	Basket/bucket trial	HMBD-001	Anti-HER3	HER3-positive	NCT05057013	2023
	I/II	Biomarker enrichment trial	GPC3 CAR-T cells	Engineered T cells	GPC3 IHC-positive	NCT05652920	2024
	I/II	Biomarker enrichment trial	RZ-001 + valganciclovir	Trans-splicing ribozyme + antiviral	hTERT-positive	NCT05595473	2022

ClinicalTrials.gov accessed in October 2024.

HCC, hepatocellular carcinoma; NCT, National Clinical Trial; AFP, alpha-fetoprotein; GPC3, glypican-3; CAR, chimeric antigen receptor; IHC, immunohistochemistry; PD-1, programmed death-1; VETC, vessels encapsulating tumor clusters; TIM-3, T cell immunoglobulin and mucin containing protein-3; DKK1, dickkopf-1; TCR, T cell receptor; DR5, death receptor 5; EGFR, epidermal growth factor receptor; DNA-seq, DNA sequencing; Ad, adenoviral; C-TCR, AFP specific T cell receptor transduced T cells; ICI, immune checkpoint inhibitor; EpCAM, epithelial cell adhesion molecule; MVA, modified vaccinia virus ankara; HER, human epidermal growth factor receptor; STAT1, signal transducer and activator of transcription 1; NK, natural killer; HBV, hepatitis B virus; HLA, human leukocyte antigen; CDK, cyclin-dependent kinase; RB, retinoblastoma; FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; IL, interleukin; TGF-β, transforming growth factor beta; WB, western blotting; hTERT, human telomerase reverse transcriptase.

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).

Figure 2.

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

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.