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.

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 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).

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

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

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

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.