Immunotherapy and liver transplantation for hepatocellular carcinoma: Current and future challenges

Abstract

Despite existing curative options like surgical removal, tissue destruction techniques, and liver transplantation for early-stage hepatocellular carcinoma (HCC), the rising incidence and mortality rates of this global health burden necessitate continuous exploration of novel therapeutic strategies. This review critically assesses the dynamic treatment panorama for HCC, focusing specifically on the burgeoning role of immunotherapy in two key contexts: early-stage HCC and downstaging advanced HCC to facilitate liver transplant candidacy. It delves into the unique immunobiology of the liver and HCC, highlighting tumor-mediated immune evasion mechanisms. Analyzing the diverse immunotherapeutic approaches including checkpoint inhibitors, cytokine modulators, vaccines, oncolytic viruses, antigen-targeting antibodies, and adoptive cell therapy, this review acknowledges the limitations of current diagnostic markers alpha-fetoprotein and glypican-3 and emphasizes the need for novel biomarkers for patient selection and treatment monitoring. Exploring the rationale for neoadjuvant and adjuvant immunotherapy in early-stage HCC, current research is actively exploring the safety and effectiveness of diverse immunotherapeutic approaches through ongoing clinical trials. The review further explores the potential benefits and challenges of combining immunotherapy and liver transplant, highlighting the need for careful patient selection, meticulous monitoring, and novel strategies to mitigate post-transplant complications. Finally, this review delves into the latest findings from the clinical research landscape and future directions in HCC management, paving the way for optimizing treatment strategies and improving long-term survival rates for patients with this challenging malignancy.

Key Words: Hepatocellular carcinoma; Immunotherapy; Liver transplantation; Neoadjuvant and adjuvant immunotherapy; Immune checkpoint inhibitors; Vaccine therapies; Oncolytic viruses; Adoptive cell therapy; Antigen-targeting antibodies; Diagnostic biomarkers

Core Tip: Hepatocellular carcinoma is a prevalent condition nowadays. The increasing number of new cases worldwide requires intensive research and innovative curative options. Cancer immunotherapy constitutes a constantly growing field in terms of disease treatment, as well as potential tumor downstaging with an eye to liver transplantation. There are two approaches regarding immunotherapy for hepatocellular carcinoma: boosting the existing immune response and stimulating a de novo immune response.

INTRODUCTION

Hepatocellular carcinoma (HCC) constitutes 90% of primary liver malignancies and ranks as the fourth most common cause of cancer mortality globally, even with the development of various treatment approaches in recent years. Projections in the United States suggest that HCC will become the third-leading cause of cancer mortality by 2040, surpassing breast cancer with an estimated 41000 annual deaths[1]. The rate of HCC occurrence shows geographic variation, reflecting differences in the prevalence of major risk factors[2]. Chronic infection with hepatitis C virus (HCV) and hepatitis B virus (HBV), particularly when it progresses to cirrhosis, is the leading risk factor for HCC. Additionally, non-alcoholic fatty liver disease, alcohol-related liver damage, and hereditary hemochromatosis also contribute to the development of HCC. Chronic inflammation, ongoing production of harmful reactive oxygen species with insufficient removal, and a multitude of genetic changes, all contribute to hepatocellular stress and dysfunction, irrespective of the initial cause[3]. Therefore, HCC presents a significant global health challenge and an area of continuous research regarding treatment options.

Implementation of effective surveillance programs has resulted in the early detection of HCC in 40%-50% of cases[1]. However, despite diligent surveillance programs, a substantial proportion of HCC diagnoses are still made in the advanced stages[4]. While curative options exist for early-stage HCC, including surgical removal, tissue destruction techniques and liver transplantation, there is a continuous effort to improve long-term outcomes and reduce recurrence rates. Non-curative options have consisted of locoregional therapies in intermediate unresectable disease, and systemic therapies in advanced disease. For patients with intermediate disease, transarterial chemoembolization is the standard of care. Systemic therapies have been driven by the evolution of molecular cancer biology with the advent of sorafenib in 2007, launching a new era into targeted therapies[1]. Since then, other targeted therapies have been shown to improve clinical outcomes in HCC[1]. Traditionally, liver transplant has served as a potentially curative approach for patients with advanced HCC or those with contraindications to other modalities. Considering the frequent association of HCC with cirrhosis, in the absence of liver transplant, the recurrence rate of HCC approaches 70% after resection or radiofrequency ablation in a time period of 3 years[5]. Consequently, limitations due to organ shortages and the risk of post-transplant recurrence necessitate ongoing exploration of novel therapeutic strategies.

Understanding the unique immunobiology of the liver and HCC is key to advancing immunotherapy as treatment. The liver’s immune tolerance and regulatory T cells create a distinct immunological environment, while HCC employs immunosuppressive mechanisms to evade immune attack. Immunotherapy, which enhances the body’s immune system to fight tumors, has transformed oncology and offers potential in early-stage HCC management. This review highlights the evolving treatment landscape for HCC, with an emphasis on immunotherapy and liver transplant. It explores the benefits and limitations of combining these treatments, the role of immune checkpoint inhibitors (ICIs) like programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and emerging approaches such as cytokine-targeting therapies, cancer vaccines, and cell-based therapies. The novel elements of the article include the examination of the importance of biomarkers like alpha-fetoprotein (AFP) and glypican-3 (GPC3) for diagnosis and monitoring, while acknowledging the need for new markers to guide immunotherapy strategies. Additionally, neoadjuvant and adjuvant immunotherapy, given before or after surgery to reduce tumor size and prevent relapse, represent promising strategies for early-stage HCC. However, balancing anti-tumor immunity with post-transplant risks is crucial. Finally, this review underscores the ongoing clinical trials and future directions in the field, aiming to aid in optimizing treatment strategies and improving long-term survival for HCC patients.

LITERATURE REVIEW

Focusing on existing research, this review prioritizes a literature search strategy and data analysis methods used to evaluate the current state of knowledge and future challenges regarding immunotherapy and liver transplantation for HCC.

Information retrieval strategy

To ensure a comprehensive review, relevant studies were identified through a systematic search of the literature on the application of neoadjuvant and adjuvant immunotherapy for HCC, including its use before liver transplantation and hepatectomy. The search for relevant information was conducted through electronic databases such as PubMed (MEDLINE), Embase, ResearchGate, and the Cochrane Library. Additionally, ClinicalTrials.gov was utilized to identify ongoing clinical trials. Selection Criteria included: (1) Liver immunobiology; (2) HCC immunobiology; (3) Liver transplantation; (4) Hepatectomy; (5) Neoadjuvant therapy; (6) Adjuvant therapy; (7) Immunotherapy; (8) HCC; and (9) HCC biomarkers.

Identification of current research trends

To identify current research trends and guide our investigation, we utilized large language models for information retrieval. Specifically, queries were submitted to large language models such as Gemini and ChatGPT on the following topics: (1) The role of neoadjuvant and adjuvant immunotherapy in HCC; (2) The application of neoadjuvant and adjuvant immunotherapy to prevent or treat HCC metastasis; and (3) The immunological background of patients receiving immunotherapy for HCC.

Literature search and selection

The selection process for studies employed pre-established criteria for inclusion and exclusion. The factors considered for inclusion were: (1) Clinical trials enrolling patients with resectable HCC; (2) Patients with inoperable primary or metastatic HCC disease; and (3) Utilization of different immune therapies. The following criteria were used to exclude studies from the analysis: (1) Fewer than 10 participants; (2) Outcomes not aligned with our pre-defined endpoints; (3) Lack of valid data for evaluating neoadjuvant/adjuvant immunotherapy efficacy; and (4) Duplicate publications.

Study selection and data extraction

Researchers independently assessed the titles and abstracts of 152 identified studies based on predetermined inclusion and exclusion criteria. Any disagreements were resolved through discussion between them. Full-text articles were retrieved for all potentially relevant studies (n = 152). A standardized data collection form was used to extract information from the full-text articles. The following data elements were collected for each included study: Lead author, publication year, study design, ClinicalTrials.gov identifier, intervention details, blinding, study approach, study development stage, location of study, article type and key criteria integration details.

Internal validity and risk of bias assessment

A standardized tool recommended for assessing bias in intervention studies (Cochrane Handbook for Systematic Reviews of Interventions, version 5.1.0) was used to evaluate the included studies. This tool examines potential sources of bias such as: (1) Random sequence generation; (2) Allocation concealment; (3) Participant and personnel blinding; (4) Blinding of outcome assessors; (5) Completeness of outcome data; (6) Selective outcome reporting; and (7) Other potential biases. Two independent reviewers assessed each study’s risk of bias. Any discrepancies were resolved through discussion or by a third reviewer’s evaluation.

LIVER - HCC IMMUNOBIOLOGY

Liver immunobiology

Once solely associated with metabolism, nutrient storage, and detoxification, the human liver is now appreciated for its complex immunological roles. It generates acute phase proteins, complement factors, cytokines, and chemokines, and harbors a vast and varied population of resident immune cells[6]. Hepatic blood flow, with 75% originating from the portal vein and 25% from the hepatic artery, renders the liver distinctively susceptible to exposure to gut pathogens. The liver is distinguished by its network of channels, called sinusoids, densely packed with specialized immune cells such as macrophages [Kupffer cells (KCs)], liver sinusoidal endothelial cells (LSECs), natural killer (NK) cells, and innate lymphoid cells[2]. KCs act as the liver’s sanitation crew, engulfing cellular debris and filtering out circulating endotoxins. In some cases, they trigger the release of signaling molecules (cytokines) and attract immune cells to address specific threats[3].

Within the liver, blood returning from the intestines mixes with oxygenated blood from the hepatic artery. This blood then flows through channels called hepatic sinusoids between plates of liver cells before draining into the central veins[6]. Pattern recognition receptors found on hepatocytes and immune cells called KCs express pattern recognition receptors that interact with molecules derived from microbes and damaged tissues carried by blood from the intestines (portal vein). These receptors capture and break down molecules derived from microbes and damaged tissues without triggering a full inflammatory response, functioning as a filter that protects the rest of the body from an overactive immune response. This unique function shapes the specific immune environment within the liver[6].

LSECs constitute roughly half of the liver’s non-epithelial cells and act as crucial immune system partners, functioning as antigen-presenting cells (APCs) alongside KCs and dendritic cells (DCs) within the liver’s reticuloendothelial system. Hepatic stellate cells located in the space of Disse between the parenchymal cells and LSECs contribute to this intricate immune response[2]. In response to liver injury or chronic inflammation, stressed hepatic stellate cells contribute to fibrosis, which can progress to cirrhosis[3]. The healthy liver continuously encounters a barrage of dietary and commensal bacterial products that have the potential to trigger inflammation. If these inflammatory stimuli are not effectively eliminated and inflammation is not resolved, it can result in long-term infections, malfunctions of the immune system attacking the body’s own tissues, or tumor formation[6]. This persistent pathological inflammation disrupts tissue balance, potentially progressing to fibrosis, cirrhosis, and ultimately, liver failure[6].

Due to the constant influx of antigens through the portal vein, the liver walks a tightrope between effectively responding to pathogens and maintaining immune regulation. These antigens include harmless dietary components, bacterial breakdown products, and damaged cells, alongside potentially harmful or toxic elements[6]. The liver’s adaptation to this unique antigenic environment manifests as an inherent immune tolerance, evident in its significantly lower allograft rejection rates compared to other transplanted organs[2].

HCC immunobiology

To escape immune defenses, HCC tumors exploit the liver’s natural state of immune tolerance, fostering a supportive tumor microenvironment[3]. This microenvironment consists of similar immunological components as the healthy liver but exhibits distinct patterns of local recruitment, cellular communication, and immunoanatomy. The transition from a healthy liver to a tumor microenvironment unfolds through ongoing disruptions to the liver’s normal immunological balance. This process notably involves the secretion of immunosuppressive cytokines, aberrant antigen expression, and alterations in local immune cell communication during inflammatory states.

In most instances, the development of HCC is associated with chronic liver disease, notably cirrhosis. The ongoing inflammatory process in cirrhosis triggers damage to hepatocellular DNA, stress within the endoplasmic reticulum, and subsequent cell death (necrosis), leading to the formation of regenerative nodules, dysplastic nodules, and ultimately carcinoma. Infection with HCV or HBV can trigger inflammation that promotes abnormal cell growth. HBV in particular exerts its carcinogenic effects through direct transformation of host cell DNA[2]. Moreover, once HCC develops, tumors often exhibit a significant infiltration of immune cells. Tumor infiltrating lymphocytes (TILs) are a prominent feature of solid tumors, reflecting the host’s attempt to mount an anti-tumor response. However, this immune response can become dysfunctional. An imbalance between CD4⁺ helper T cells and regulatory T cells relative to CD8⁺ cells can lead to immune tolerance and a worse prognosis. Additionally, the innate immune system may be weakened, as shown by decreased NK cell activity in HCC[2]. Nevertheless, TILs often fail to effectively control tumor growth, partly due to the expansion of myeloid-derived suppressor cells and regulatory T cells, which facilitate immune evasion by secreting factors like transforming growth factor β[2]. Additionally, various mechanisms enable tumor cells to evade immune detection. Consequently, the immune environment in HCC is highly suppressive, posing significant challenges for immune-based therapies, despite being a promising avenue for future treatments.

HCC and boosting the existing immune response

HCC staging and treatment options: Currently, the Barcelona Clinic Liver Cancer (BCLC) staging system is a widely used method for the management of HCC globally, enjoying widespread acceptance and adoption within the clinical community. This staging system categorizes patients into different stages considering factors like tumor size, number of lesions, presence of vascular invasion, overall performance status and liver function. For individuals diagnosed with early-stage HCC (classified as BCLC 0 or BCLC A), a range of treatment modalities are available, including surgical resection, ablation techniques such as radiofrequency or microwave ablation, and liver transplant. These interventions are aimed at achieving complete eradication or control of the tumor while preserving liver function to the greatest extent possible. In cases of intermediate-stage HCC (BCLC B), characterized by larger or multifocal tumors without vascular invasion or extrahepatic spread, locoregional therapies are the preferred treatment strategies. Minimally invasive procedures like injecting chemotherapy drugs directly into the tumor-feeding arteries and delivering radioactive Yttrium-90 microspheres via the same route are commonly employed techniques that deliver targeted therapy directly to the tumor site, effectively reducing tumor burden and delaying disease progression[7]. Additionally, systemic therapies, such as molecularly targeted agents or immunotherapy, may be considered in certain cases to complement locoregional treatments and improve overall outcomes. Advanced-stage HCC (BCLC C) presents a formidable challenge in clinical management, often associated with extensive tumor burden, vascular invasion, or extrahepatic spread. In this setting, systemic therapies take precedence as the primary treatment approach. Chemotherapy, molecularly targeted agents targeting specific signaling pathways implicated in HCC pathogenesis, and immunotherapy aimed at enhancing the anti-tumor immune response are among the therapeutic options available. Furthermore, multidisciplinary care involving hepatologists, oncologists, radiologists, and transplant surgeons plays a crucial role in creating treatment plans to individual patient needs and ensuring optimal outcomes. This collaborative approach enables comprehensive evaluation, personalized treatment selection, and ongoing monitoring to address the complex and dynamic nature of HCC.

Advanced stage therapies: The chance of liver transplantation for patients with BCLC stage B, and occasionally even stage C, improves if they can be effectively down-staged through suitable treatments. The BCLC stage of HCC strongly influences prognosis, with median survival times differing considerably between stages. For those who receive a liver transplant at an early stage (BCLC 0 or BCLC A), the median survival time exceeds 10 years, reflecting the effectiveness of transplant in prolonging life[8]. Patients undergoing surgical resection or ablation for early-stage HCC typically have a median survival time of over 6 years, highlighting the benefits of these interventions when the disease is detected early. For individuals diagnosed with intermediate-stage HCC (BCLC B), the median survival time ranges between 26 months to 30 months. This stage often involves more complex treatment strategies, including locoregional therapies like injecting chemotherapy drugs directly into the tumor-feeding arteries and delivering radioactive Yttrium-90 microspheres via the same route, which aim to control tumor development[8]. Individuals diagnosed with HCC at an advanced stage (BCLC stage C) who have preserved liver function, such as those with Child-Pugh A cirrhosis, face a median survival time of approximately 19 months. This stage is generally treated with systemic therapies, which aim to manage the disease and extend survival. Importantly, the advent of immunotherapy has significantly improved survival outcomes for HCC patients across all stages. Compared to historical data, patients now have better prognosis and longer median survival times, underscoring the transformative impact of these newer therapeutic approaches in the management of HCC[9].

Evolution of systemic therapies: Sorafenib and beyond: Before the year 2007, the landscape of systemic therapies for HCC lacked established standards, leaving clinicians with limited options to effectively combat this aggressive disease[9]. During this period, cytotoxic chemotherapy represented the mainstay of treatment, albeit with modest efficacy and notable risks of toxicity, particularly in patients with pre-existing liver dysfunction. However, the field of HCC management underwent a major change with the groundbreaking SHARP trial of 2007. This pivotal clinical study marked a significant advancement by demonstrating that sorafenib, a tyrosine kinase inhibitor (TKI), improved overall survival in patients with advanced HCC. Sorafenib emerged as the first systemic therapy to hold a survival benefit in this patient population, offering new hope and therapeutic options where previously there were few[10].

Lenvatinib and the REFLECT trial: Building upon the success of sorafenib, subsequent research efforts focused on the development of novel therapeutic agents targeting key pathways implicated in HCC pathogenesis and progression. One such agent, lenvatinib, emerged as a promising candidate. Lenvatinib inhibits multiple protein kinases, disrupting various signaling pathways essential for tumor growth and blood vessel formation. These pathways involve receptors for vascular endothelial growth factor (VEGF), fibroblast growth factor, platelet-derived growth factor, specific RET proteins, and KIT proteins[11].

A key clinical trial (REFLECT) compared lenvatinib to sorafenib in advanced HCC patients to assess its effectiveness and safety as a first-line treatment. The trial demonstrated promising results, showing that lenvatinib was as effective as sorafenib in terms of overall survival. This study established lenvatinib as a valuable alternative for treating advanced or unresectable HCC. Based on these compelling clinical results, lenvatinib gained approval from the United States regulatory agency in 2018 as a first-line treatment option for this patient population. This regulatory approval represented a significant milestone in the management of HCC, providing clinicians and patients with an additional therapeutic option that has demonstrated efficacy, tolerability, and the potential to improve outcomes in this challenging disease setting.

Cabozantinib and regorafenib: Second-line treatment options: In subsequent-line treatment scenarios for HCC, significant steps have been made in expanding therapeutic options, with both cabozantinib and regorafenib emerging as approved treatments[12]. These developments have reshaped the management landscape, offering new possibilities for patients who have experienced disease progression or intolerance to prior therapies. Regorafenib’s journey to approval in HCC began with the RESORCE study, a landmark trial that investigated its efficacy in patients with advanced HCC who had previously progressed on sorafenib. In this pivotal trial, which enrolled patients with Child-Pugh A cirrhosis, individuals were randomized in a 2:1 ratio to receive either regorafenib or placebo. The results demonstrated a remarkable improvement in overall survival for patients treated with regorafenib, leading to its regulatory approval in 2017 as a subsequent-line treatment option for HCC[12].

On a similar trajectory, cabozantinib, a multi-kinase TKI with activity against VEGF receptors 1-3, MET, and AXL, entered the spotlight with the CELESTIAL trial. In a key clinical trial, researchers investigated the effectiveness of cabozantinib in patients with advanced HCC whose disease had worsened after receiving one or two prior systemic therapies. The study design involved randomly assigning patients to receive either cabozantinib or a placebo in a 2:1 ratio. The findings demonstrated a statistically significant increase in median overall survival for the group treated with cabozantinib[13]. The approval of cabozantinib in 2019 marked a significant milestone in the management of HCC, offering a much-needed therapeutic option for patients who had previously been treated with sorafenib. This approval underscored the evolving landscape of HCC treatment and the growing arsenal of targeted therapies available to clinicians and patients alike. These developments not only highlight the expanding treatment options in the subsequent-line setting but also underscore the importance of ongoing research and innovation in improving outcomes for patients with advanced HCC. Ongoing advancements in the field offer exciting possibilities for improved survival and quality of life for patients battling this complex disease through the incorporation of new therapeutic agents and strategies.

ICIs: A new hope for advanced HCC: When considering treatment options for advanced HCC, ICI-based therapies have emerged as promising options. These therapies, particularly those targeting PD-1 and PD-L1 with antibody-based drugs, mark a substantial improvement compared to traditional approaches exemplified by sorafenib. Early trials have shown encouraging results, with ICI-based therapies demonstrating progressive improvements in overall survival outcomes[14] (Figure 1).

Figure 1 Immunotherapy landscape of hepatocellular carcinoma. An overview of various immunotherapeutic agents under investigation for the treatment of hepatocellular carcinoma. AFP: Alpha-fetoprotein; CAR-T: Chimeric antigen receptor expressing T cell; CIK: Cytokine-induced killer; CTLA-4: Cytotoxic T-lymphocyte associated antigen 4; DC: Dendritic cell; GPC3: Glypican-3; LAG-3: Lymphocyte activation gene 3; MRP3: Multidrug resistance-associated protein 3; NK: Natural killer; PD-1: Programmed cell death protein 1; PD-L1: Programmed cell death ligand 1; TGF-β: Transforming growth factor-β; Tim-3: T-cell immunoglobulin and mucin-domain containing-3.

Combining ICIs and the future: The development of combination therapies has significantly improved treatment options for advanced HCC. Combining ICIs that target CTLA-4 with PD-1 inhibitors has been approved by regulatory bodies and demonstrates even better overall survival rates compared to using ICIs alone. These combination approaches leverage synergistic effects to enhance anti-tumor immune responses and combat the formidable challenges posed by advanced HCC. However, despite these advancements, a significant proportion of patients do not achieve the desired therapeutic response with ICI-based regimens. This lack of response can be attributed to various factors, including inherent resistance mechanisms within the tumor or the development of acquired resistance over time. The tumor’s ability to create an overwhelming immunosuppressive microenvironment, as mentioned earlier in this article, poses a formidable barrier to the efficacy of ICI-based therapies, limiting their effectiveness in a substantial number of patients.

Moving forward, efforts to overcome resistance and enhance the efficacy of ICI-based therapies are underway. Research initiatives aimed at elucidating the underlying mechanisms of resistance and identifying predictive biomarkers hold promise for optimizing patient selection and improving treatment outcomes. Additionally, novel combination strategies and therapeutic approaches are being explored to address the complex immunological dynamics of advanced HCC and maximize the potential of immunotherapy in this challenging disease setting. The era of immunotherapy in the management of HCC dawned with the publication of a seminal pilot study in 2013. This research stands out as a significant development, demonstrating the safety and potential effectiveness of tremelimumab, a CTLA-4 inhibitor, particularly for patients with HCC caused by HCV cirrhosis[9]. This early evidence laid the foundation for a transformative shift in the therapeutic landscape of HCC.

Over the past 10 years, there has been a revolution in cancer treatment due to the emergence of immunotherapy as a powerful tool in HCC management. Its acceptance and integration into clinical practice have been remarkable, and immunotherapy has become a mainstay treatment for most HCC management strategies[8]. This widespread adoption underscores the profound impact that immunotherapy has had on reshaping treatment options and improving patient survival. Furthermore, the ongoing evolution of systemic treatments for advanced HCC has increasingly centered around the use of ICIs. Note that most investigational treatments currently undergoing evaluation in phase III randomized trials for advanced HCC incorporate ICI as a core component. This reflects a growing recognition of the therapeutic potential of immunotherapy and its pivotal role in advancing the standard of care for HCC[15,16]. However, despite the significant advancements and paradigm shifts brought about by immunotherapy, challenges remain. A key limitation is that only a portion of patients experience a meaningful response to ICI therapy, particularly when administered as monotherapy. This underscores the complex and heterogeneous nature of HCC and highlights the need for continued research to optimize treatment strategies.

The role of biomarkers in immunotherapy for HCC: There is a growing emphasis on the development of biomarkers to personalize therapy and stratify cancer patients for optimal response to immune checkpoint therapy. Studies investigating predictive biomarkers offer the potential to improve patient selection and optimize treatment outcomes. Additionally, the exploration of combination approaches, which leverage synergistic effects between immunotherapy and other treatment modalities such as targeted therapy or locoregional interventions, represents a promising avenue for enhancing therapeutic efficacy in HCC.

Nivolumab and pembrolizumab: Second-line treatments: The emergence of nivolumab and pembrolizumab as second-line agents in HCC treatment has significantly expanded the therapeutic landscape for patients who experience disease progression on sorafenib, the standard first-line therapy. Phase II trials evaluating the efficacy of nivolumab and pembrolizumab in this setting have yielded promising results, demonstrating notable activity and durable responses. These trials have reported response rates ranging from 15% to 20%, with complete response rates of 1% to 5%. Notably, the responses observed with nivolumab and pembrolizumab have shown remarkable durability, suggesting sustained disease control and long-term benefit for select patients[9]. In particular, the CheckMate 040 trial provided compelling evidence for the clinical efficacy of nivolumab in HCC[9]. Among responders to nivolumab, the 2-year survival rate exceeded 80%, highlighting the potential for prolonged survival in this population.

Building upon these encouraging findings, regulatory agencies have granted accelerated approval to both nivolumab and pembrolizumab as second-line treatments for patients with HCC who have experienced disease progression on sorafenib or intolerable toxicity to sorafenib[15,16]. This regulatory approval represents a significant advancement in HCC management, offering clinicians and patients additional therapeutic options to consider in the second-line setting. The success of nivolumab and pembrolizumab in clinical trials underscores the growing importance of immunotherapy in the treatment paradigm for HCC. These agents have emerged as valuable additions to the armamentarium of therapies available for patients with advanced HCC, offering the potential for improved outcomes and prolonged survival.

The encouraging efficacy and low risk of side effects demonstrated by single-agent ICI in the management of HCC have ignited considerable interest in exploring combination strategies to enhance therapeutic outcomes. These combinations, some of which have already transitioned into clinical practice, represent a pivotal advancement in the quest for more effective treatments for HCC. A treatment combination of particular interest is the PD-L1 inhibitor atezolizumab paired with the VEGF inhibitor bevacizumab. This combination is frequently referred to as ‘atezolizumab-bevacizumab (atezo-bev)’[17] and leverages the complementary mechanisms of action of these agents, targeting both the immune checkpoint pathway and the tumor vasculature, to elicit a more robust and sustained anti-tumor response.

IMbrave150 and atezo-bev: The pivotal IMbrave150 clinical trial, which investigated the atezo-bev combination, heralded a new era in HCC treatment upon its publication in 2020. This study established atezo-bev as a more effective treatment than sorafenib, not just in terms of conventional measures of success (e.g., overall survival and progression-free survival), but also in the critical area of health-related quality of life[17]. Notably, these groundbreaking findings emerged after a period of over a decade with discouraging clinical trial results and a lack of powerful treatment options for patients with advanced HCC. Indeed, patients receiving atezo-bev experienced significantly prolonged overall survival and progression-free survival compared to those treated with sorafenib, highlighting the potent anti-tumor activity of this novel combination regimen[18]. Furthermore, patients treated with atezolizumab and bevacizumab experienced a significant improvement in their health-related quality of life. This is demonstrated by the substantially longer time it took for their reported quality of life to worsen compared to those receiving sorafenib[17]. These findings underscore the holistic benefit of atezo-bev in enhancing both survival outcomes and patients’ overall well-being.

The regulatory approval of atezo-bev by the United States Food and Drug Administration in 2020 marked a pivotal milestone, solidifying its position as the preferred first-line therapy for patients with unresectable or advanced HCC who have not received prior treatment. This landmark approval validated the transformative impact of atezo-bev on the treatment landscape of HCC and provided clinicians and patients alike with a powerful new therapeutic option. Following its regulatory approval, real-world evidence further corroborated the efficacy and safety of atezo-bev in diverse patient populations across different regions. A global observational study, encompassing patients from Europe, Asia, and the United States, provided compelling insights into the real-world outcomes of atezo-bev in advanced HCC. Promising results were observed in this study with a median follow-up duration of 10 months. These included a median overall survival of 15.7 months, a median progression-free survival of 6.9 months, and an overall response rate of 30.8%[9]. These real-world data not only reaffirmed the clinical benefit of atezo-bev observed in the IMbrave150 trial but also highlighted its consistent efficacy and tolerability in a broader patient population. Moreover, they underscored the generalizability of the trial findings and the potential of atezo-bev to deliver meaningful outcomes in routine clinical practice.

Overall, the compelling evidence from the IMbrave150 trial and subsequent real-world studies has firmly established atezo-bev as a cornerstone of first-line therapy for unresectable or advanced HCC. By offering significant improvements in survival outcomes, quality of life, and treatment response rates, atezo-bev has reshaped the treatment landscape of HCC and provided hope for patients. The success of the atezo-bev combination in the IMbrave150 trial represents a major breakthrough in HCC management, offering a paradigm-shifting treatment approach for patients previously faced with limited therapeutic options. By harnessing the synergistic effects of immune checkpoint inhibition and VEGF pathway blockade, this combination therapy has redefined the treatment landscape for HCC and provided new hope for patients and clinicians alike. Looking ahead, the success of atezo-bev has catalyzed further exploration of combination therapies and novel treatment modalities in HCC.

The HIMALAYA trial: New advances in combination immunotherapy: The HIMALAYA trial stands as a milestone in the ever-evolving landscape of HCC management, particularly in the realm of immunotherapy. A landmark study evaluated the combined use of two powerful ICIs: the CTLA-4 inhibitor tremelimumab and the PD-1 inhibitor durvalumab. This successful combination led to a significant regulatory approval for first-line treatment in 2022[13].

The HIMALAYA trial investigated the effectiveness of different treatments for patients with unresectable HCC who had not received prior treatment. Patients were randomly assigned to one of three groups: The STRIDE regimen (a single dose of 300 mg tremelimumab combined with 1500 mg durvalumab every 3 weeks), durvalumab monotherapy (1500 mg every 4 weeks), or the standard treatment of sorafenib (400 mg twice per day). Among these arms, patients receiving the STRIDE regimen exhibited a notable improvement in median overall survival compared to those treated with durvalumab alone or sorafenib. Specifically, the median overall survival durations were reported as 16.4 months, 16.56 months, and 13.77 months, respectively, underscoring the robust efficacy of the combination therapy[9]. Moreover, the trial findings unveiled that overall survival with durvalumab monotherapy was non-inferior to sorafenib, as evidenced by a hazard ratio of 0.86 and a non-inferiority margin of 1.08[9]. These findings suggest that durvalumab could be a valuable treatment option on its own for patients with advanced HCC who are unsuitable for combined immunotherapy or anti-angiogenic therapies. This expands the range of treatment possibilities for patients with advanced HCC. Although the treatment demonstrated promising effectiveness, a significant proportion of patients receiving the STRIDE regimen experienced severe (grade 3-4) side effects. This highlights the crucial role of close monitoring and management of adverse events for patients undergoing combination immunotherapy.

Positive results from the HIMALAYA trial led to regulatory approval of the durvalumab-tremelimumab combination as a first-line treatment for advanced HCC, offering a potential path to better patient outcomes. Additionally, the demonstrated non-inferiority of durvalumab monotherapy to sorafenib provides an alternative therapeutic strategy, particularly for patients with contraindications to combination therapy. Overall, the findings from the HIMALAYA trial represent a landmark advancement in HCC therapeutics, paving the way for personalized treatment approaches and enhancing the prospects for patients grappling with this challenging disease. As further research and clinical investigations continue to unfold, the integration of immunotherapy into the treatment paradigm of HCC holds the potential to revolutionize patient care and improve long-term outcomes.

Selecting optimal first-line treatment for HCC: The therapeutic landscape for advanced HCC has undergone a profound transformation with the emergence of several TKIs and ICIs approved for use as both first-line and second-line treatments[9]. However, amidst this plethora of treatment options, the challenge of determining the optimal first-line regimen for individual patients persists, necessitating a comprehensive evaluation of various factors to inform clinical decision-making. Since head-to-head clinical trials directly comparing the effectiveness and tolerability of all approved treatment options for treatment-naive patients have not been conducted, clinicians are confronted with the task of navigating treatment selection based on a nuanced understanding of drug toxicity profiles, patient-specific medical histories, and performance status. This intricate decision-making process underscores the importance of personalized medicine in HCC management, where treatment strategies are tailored to suit the unique characteristics and needs of each patient.

For instance, imagine a patient with a complicated medical background marked by substantial heart-related conditions, a tendency for abnormal bleeding, or a past episode of severe varices that bled. In such cases, the potential risks associated with anti-angiogenic therapies, such as cardiovascular events and bleeding complications, must be carefully weighed against the anticipated benefits of treatment. Alternative therapeutic approaches, such as combination durvalumab and tremelimumab, may offer a safer and more favorable risk-benefit profile in this scenario, thus warranting consideration as a preferred treatment option. Conversely, patients with pre-existing autoimmune diseases pose a distinct clinical challenge, as the use of combination ICI therapy may exacerbate underlying autoimmune conditions and precipitate immune-related adverse events. In such instances, TKIs may emerge as a more prudent treatment choice, offering therapeutic efficacy while minimizing the risk of immune-mediated complications. Ultimately, the selection of the most appropriate first-line regimen for patients with advanced HCC necessitates a holistic evaluation of clinical factors, guided by a multidisciplinary approach that integrates oncological expertise with considerations of patient-specific characteristics and preferences. By engaging in shared decision-making and individualized treatment planning, clinicians can strive to optimize therapeutic outcomes while mitigating potential treatment-related risks, thus enhancing the quality of care and overall patient well-being.

STIMULATING DE NOVO IMMUNE RESPONSE

Vaccine therapies - oncolytic viruses

Therapeutic vaccines utilize various components like peptides, DCs, whole cells, oncolytic viruses (OVs), and DNA that can stimulate specific immune responses against tumor antigens. Research on peptides like AFP, multidrug resistance-associated protein 3 (MRP3), and GPC3 suggests they may be safe and well-tolerated[19] (Figure 1).

AFP: Primarily produced in the yolk sac during embryonic development and the liver, AFP functions as a transporter protein with a molecular weight of approximately 70 kDa. However, in HCC, AFP occupies a multifaceted niche[20]. Traditionally, serum AFP levels have been the basis for HCC diagnosis and monitoring[21]. Elevated serum AFP is frequently observed in HCC patients, particularly those with advanced disease, reflecting the re-expression of this fetal protein during liver malignancy[22]. Although valuable for initial screening and surveillance due to its high sensitivity, AFP lacks specificity, as levels can also increase in benign and malignant conditions other than HCC[23]. Despite limitations as a diagnostic marker, AFP holds promise as a target for immunotherapy[24]. The unique structure of AFP peptides makes them potentially immunogenic. Specific peptide fragments derived from AFP can be processed and presented by APC to cytotoxic T lymphocytes (CTL)[25]. This idea has fueled the development of AFP-based vaccines and adoptive cell therapy (ACT) approaches.

Building upon previously identified human AFP peptide epitopes, researchers developed a replication-deficient human adenovirus expressing AFP as a target for immunotherapy utilizing T cells[26]. A phase I/II clinical trial (NCT00093548) evaluated the safety and immune response of these AFP constructs in two HCC patients who had received prior treatment and whose tumors expressed AFP. The vaccine was found to be well-tolerated with no major side effects. Encouragingly, both patients displayed markers of immune response directed against AFP. The first patient developed AFP-specific CD8+ T cells after 9 months, while the second patient exhibited a strong response encompassing AFP-specific CD8+ and CD4+ T cells along with antibodies that neutralized an AFP replication-deficient adenovirus vector after 18 months[27].

MRP3: MRP3, encoded by the ABCC3 gene, is a fascinating protein with a broader role than its name suggests. Initially identified for its ability to confer multidrug resistance in cancer cells[28], MRP3 belongs to the ATP-binding cassette transporter superfamily and plays a vital role in hepatobiliary excretion[29]. It mediates the efflux of conjugated organic anions like glucuronides, sulfates, and glutathione conjugates of various endogenous and exogenous compounds essential for bile formation and detoxification, including bilirubin glucuronides. Beyond waste removal, MRP3 also contributes to the transport of essential nutrients like folate and thyroid hormones, influencing their bioavailability. Interestingly, recent research suggests that MRP3 plays a role in immune regulation. The presence of MRP3 in immune cells such as macrophages and DCs suggests that it may play a role in regulating the immune response by controlling the release of immune-modulating molecules[19].

HCC immunotherapy research shows promise in targeting MRP3 due to its potential as a tumor antigen and its role in chemotherapy resistance. Studies have shown a significant increase in MRP3 expression in HCC tissue compared to tumor-free controls, highlighting its potential as an immunotherapeutic target[30]. In addition, MRP3-specific CTL showed activity independent of liver function, HCV infection status, AFP levels, and HCC stage, suggesting a broad application in a diverse HCC patient population[31]. Research has identified a critical role for MRP3 in mediating resistance to sorafenib, a first-line treatment for HCC[32]. These results collectively indicate that immunotherapy against MRP3 may be effective in overcoming treatment resistance and improving patient outcomes in HCC.

A phase I clinical trial (UMIN000005678) has already investigated the safety and potential immune response of a vaccine containing a peptide derived from MRP3 (MRP3765) in patients with HCC[30]. The study included 12 patients who were positive for the human leukocyte antigen (HLA-A24) allele, a factor that affects the immune response. In a study of vaccine tolerability, 72.7% of patients mounted an immune response against MRP3. In this group, the median overall survival was 14.0 months (95% confidence interval: 9.6-18.5 months). This finding suggests an improvement in median overall survival compared to previously reported data for HCC patients treated with hepatic arterial infusion chemotherapy without peptide vaccination, where median overall survival ranged from 12.0 months to 12.6 months[30]. These preliminary findings indicate the potential of MRP3765 as a vaccine to stimulate an immune response and potentially improve survival outcomes in HCC patients, which warrants further investigation through larger clinical trials.

GPC3: GPC3 is a heparan sulfate proteoglycan that attaches to the cell membrane via a glycosyl-phosphatidylinositol anchor[33]. Unlike normal adult liver tissue where GPC3 expression is low, HCC tumors often exhibit high levels of GPC3[34]. This overexpression makes GPC3 an attractive target for developing specific diagnostic and therapeutic strategies for HCC[35]. Research suggests that GPC3 can promote HCC growth by activating the canonical Wnt pathway[33]. Because GPC3 can stimulate Wnt signaling even in the absence of serum, it is possible that GPC3 triggers Wnt signaling through autocrine or paracrine mechanisms, at least in cell culture[33]. In addition to their known interaction with Wnts, glypicans have also been linked to enhancing the activity of other growth factors, including Hedgehogs, bone morphogenetic proteins, and fibroblast growth factors[36]. GPC3 immunostaining is a valuable tool for differentiating HCC from other liver lesions[37]; HCC typically exhibits strong GPC3 staining, while benign lesions like liver adenomas or cirrhotic nodules often show minimal or absent GPC3 expression[38]. This distinction can be crucial for guiding patient management decisions.

Vaccines containing tumor antigen genes or peptides can trigger the body’s acquired immune system to target tumor cells. This activation of specific cellular and humoral immunity helps prevent tumor growth, metastasis, and recurrence[35]. There are two primary types of tumor antigens: those unique to cancer cells (tumor-specific antigens) and those found on both cancer and healthy cells [tumor-associated antigens (TAAs)]. Similar to a tumor-specific antigen, GPC3 is found almost exclusively on the surface of HCC cells[35]. GPC3 vaccines exploit the TAT status of GPC3 in HCC[39] and target specific GPC3 peptide sequences derived from the GPC3 molecule[40]. This targeted approach minimizes the risk of off-target immune responses against healthy tissues with minimal GPC3 expression. The primary mechanism of action involves inducing CTL responses against HCC cells presenting the targeted GPC3 peptides[41]. Activated CTLs can then directly recognize and eliminate GPC3-expressing HCC cells, offering a potential therapeutic strategy for HCC[42].

According to Guo et al[35] preclinical studies in mice identified two GPC3-derived peptides (GPC3298-306 and GPC3144-152) able to trigger CTLs expressing CD8 surface markers, leading to an anti-tumor immune response. Encouraged by this research, a preliminary clinical study (UMIN000001395) investigated a GPC3 peptide vaccine in patients with advanced HCC. The vaccine demonstrated good tolerability and successfully stimulated the production of GPC3-specific CTLs in most participants. Notably, 19 patients achieved stable disease (SD) for 2 months, with four experiencing tumor necrosis or regression. Furthermore, a sorafenib-resistant patient exhibited significant tumor necrosis after GPC3 vaccination, with GPC3-specific CTL infiltration localized to the tumor site without affecting healthy liver tissue. These findings suggest the potential of GPC3-based immunotherapy for HCC and warrant further investigation. A separate phase II study investigated the impact of adjuvant GPC3 vaccination in GPC3-positive patients. The study revealed a significant decrease in recurrence rates for those who received the vaccination compared to those who only underwent surgery. After 1 year, the recurrence rate in the vaccination group was 24%, while the surgery-only group experienced a rate of 48% (P = 0.047). This trend persisted for 2 years, with recurrence rates of 52.4% and 61.9% in the vaccination and surgery-only groups, respectively (P = 0.387)[19]. GPC3 detection in tissue samples using immunohistochemistry remains a reliable method for diagnosing HCC. Additionally, research on soluble GPC3 in blood shows promise as a non-invasive diagnostic tool[35].

New York esophageal squamous cell carcinoma-1 and melanoma-associated antigen-A: New York esophageal squamous cell carcinoma-1 (NY-ESO-1) has emerged as a fascinating and potent target for cancer immunotherapy due to its unique properties[43]. Encoded by the CTLA-4 locus on chromosome Xp11.23, NY-ESO-1 is typically silent in healthy adult tissues but aberrantly expressed in various malignancies. This restricted expression pattern minimizes the risk of autoimmune reactions, making it an ideal candidate for antigen-specific immunotherapies[44]. Its expression in tumors triggers a robust immune response with the generation of NY-ESO-1-specific CTLs that recognize and eliminate tumor cells. This strong immunogenicity stems from a unique amino acid sequence absent in normal tissues and efficient presentation by APCs to T lymphocytes. The potent immunogenicity of NY-ESO-1 has fueled the development of various immunotherapeutic strategies[45]. Cancer vaccines containing NY-ESO-1 peptides or whole protein can activate and expand NY-ESO-1-specific CTL populations[46]. ACT involves isolating and expanding NY-ESO-1-specific T cells ex vivo for reinfusion into the patient to target tumors. Additionally, combining NY-ESO-1-based therapies with ICIs can further enhance the immune response by overcoming suppression within the tumor microenvironment[44].

The melanoma-associated antigen (MAGE) family, particularly the MAGE-A subfamily, has emerged as a captivating area of exploration in cancer immunotherapy[47]. Encoded by a cluster of genes on the X chromosome, MAGE-A proteins are typically absent in healthy adult tissues but aberrantly expressed in various malignancies[48]. This restricted expression pattern minimizes the risk of autoimmune reactions, making MAGE-A antigens ideal candidates for targeted immunotherapies[47]. MAGE-A antigens boast potent immunogenicity[49]. Their presence in tumor cells triggers the generation of MAGE-A-specific CTLs that recognize and eliminate tumor cells. This strong immunogenicity stems from unique amino acid sequences absent in most normal tissues and efficient presentation by APCs to T lymphocytes[50]. The potent immunogenicity of MAGE-A antigens has fueled the development of diverse immunotherapeutic strategies. Cancer vaccines containing MAGE-A peptides or whole proteins can activate and expand MAGE-A-specific CTL populations. OVs engineered to target MAGE-A expressing tumors and ACT with expanded MAGE-A-specific T cells represent other promising approaches[51]. Overall, MAGE-A antigens represent a promising avenue for cancer immunotherapy due to their restricted expression pattern and potent immunogenicity[52].

Studies have shown that two testicular cancer antigens, NY-ESO-1 and MAGE-A, hold promise as therapeutic targets for HCC due to their limited expression in healthy tissues[53]. Researchers observed specific immune responses mediated by CD8+ T cells targeting NY-ESO-1 in nearly half (48%) of HCC patients who tested positive for NY-ESO-1 messenger RNA (mRNA) and expressed the HLA-A2 antigen. The presence of these T cell responses was linked to improved patient survival[54]. A study of MAGE-A expression in HCC found a remarkably high prevalence, with 92.3% of tumors expressing at least one MAGE-A gene[55], while another study found that CD8+ T cells from HCC patients positive for MAGE antigens (recognized by MAGE tetramers) were able to recognize specific peptide sequences: MAGE-1 (amino acids 161-169) and MAGE-3 (amino acids 271-279)[56]. While there are established therapeutic options for patients with HCC, MAGE-A antigens offer promise as additional targets for tumor-specific immunotherapy. However, the clinical response to vaccination with either NY-ESO-1 or MAGE-A in this patient population remains unexplored.

DCs: DCs are specialized immune cells that play a critical role in initiating and regulating adaptive immune responses. These versatile cells excel at capturing, processing, and presenting TAAs to the immune system[2]. DCs begin their journey in an immature state, circulating through the bloodstream and peripheral tissues. In these areas, they encounter and capture antigens shed by pathogenic infection or tumors. After antigen uptake, DCs embark on a maturation process marked by significant changes in form and function. After maturing, these DCs migrate to secondary lymphoid organs such as lymph nodes. There, they present processed antigen fragments to CTLs, triggering their activation. This process ignites an immune response specific to the antigen, ultimately eliminating target cells that display the presented antigen. Additionally, mature DCs can augment the cytotoxic function of NK cells. These innate immune effector cells are critical for eliminating pathogen-infected and tumor cells[57]. Following maturation and activation in vitro with a chosen antigen, DCs are reintroduced into the patient. Studies utilizing DC vaccines loaded with tumor cell lysate have demonstrated antitumor effects in mouse models[19]. Concurrently, exosomes derived from DCs are emerging as a novel vaccine platform for cancer immunotherapy. These exosomes have the potential to trigger powerful immune responses that specifically target tumor antigens, while simultaneously promoting a beneficial tumor microenvironment[19].

Multiple clinical trials, both completed and ongoing, have investigated the effectiveness of DC vaccines created using antigen-pulsing methods as a standalone treatment for patients with HCC[58]. One approach involves incubating DCs with frozen total lysates derived from either the patient’s own tumor (autologous) or tumor cell lines from other individuals (allogeneic). An alternative strategy utilizes co-culturing DCs with synthetic molecules or modified proteins that resemble recognized tumor antigens[59]. A third strategy involves introducing genetic material encoding known tumor antigens into DCs. Several methods can be used to introduce the desired genetic material into cells, including transfection with plasmid DNA, viral vector DNA, or mRNA[60]. The fourth approach involves fusing DCs with whole tumor cells using polyethylene glycol. While polyethylene glycol is a common agent for fusing lipid membranes, this method remains under preclinical evaluation.

The safety and feasibility of using mature DC vaccines loaded with patient-derived tumor lysate have been explored in two completed phase I clinical trials for patients with unresectable HCC. Both studies demonstrated a good safety profile with no severe adverse events reported. The first trial enrolled eight patients and observed SD in four patients, with one patient experiencing tumor shrinkage and another showing a decrease in HCC tumor markers[61]. The second trial involved 31 participants, and the partial response (PR) rate was 12.9%, with an additional 54.8% of patients experiencing SD. Importantly, those who received both the initial and booster vaccinations demonstrated a substantially higher 1-year overall survival rate compared to those who only received the initial vaccination (63.3% vs 10.7%)[58]. These findings indicate that DC vaccines loaded with extracts from a patient’s own tumor could be a promising and safe treatment option for patients with HCC who cannot undergo curative therapies. However, larger clinical trials are necessary to definitively determine their effectiveness in broader patient groups.

The safety and effectiveness of mature DCs loaded with extracts derived from the HepG2 human HCC cell line were evaluated in two phase II clinical trials involving patients with advanced HCC. Both trials reported a good safety profile with no serious adverse events. In terms of efficacy, the studies yielded promising yet modest results. The first trial by Palmer et al[62] observed a PR rate of 4% and a SD rate of 24% among 35 patients receiving the DC vaccine. Additionally, a subset of patients with high baseline AFP levels experienced a decrease in AFP following vaccination. The study found that 33% of patients were alive at 6 months, and 11% were alive at 12 months. The second trial by El Ansary et al[63] compared DC vaccination to a supportive care control group. The vaccination group demonstrated a higher response rate, with a PR rate of 13.3% and an SD rate of 60% compared to none and 13.3% in the control group, respectively. Patients receiving DC vaccination also exhibited a significantly longer median overall survival (7 months) compared to the control group (4 months). Overall, these studies indicate that DC vaccines loaded with extracts from unrelated tumor cell lines offer a promising and safe approach for HCC patients who cannot undergo conventional treatments. Nonetheless, the observed clinical improvements are still limited, underscoring the need for further research to enhance the effectiveness of this therapeutic strategy.

Early clinical trials (phase I/II) have investigated the safety and potential of DC vaccines in treating HCC. For instance, Butterfield et al[64] assessed a matured DC vaccine loaded with peptides derived from AFP in HLA-A*0201 positive HCC patients, demonstrating its safety and increased AFP-specific CTL responses in 60% of patients. Maeda et al[65] investigated a DC vaccine transfected with mRNA encoding heat-shock protein 70 (HSP70) in unresectable HCC, finding the vaccine to be safe with a 16.7% complete response rate and 41.7% SD. In another trial, a DC vaccine loaded with recombinant AFP, MAGE-1, and GPC-3 proteins was evaluated for safety in patients with advanced HCC who did not respond to other treatments. A current clinical trial (identified by ChiCTR1900021177) is exploring a vaccine for HCC that uses a patient’s own tumor antigens to activate DCs. These studies collectively suggest that such vaccines are well-tolerated and practical for HCC patients who cannot undergo standard treatments. However, more research is necessary to improve their effectiveness and solidify their role as a dependable treatment option.

Tumor escape mechanisms, including the expression of immune checkpoint molecules that suppress CTL activity, limit the efficacy of cancer immunotherapy[66]. This discovery has spurred the development of ICIs. ICIs are often monoclonal antibodies that function by blocking the interaction between checkpoint molecules on immune cells[67]. The United States Food and Drug Administration has granted approval for the PD-1 ICI nivolumab as a second-line therapy for advanced HCC[15]. Despite the promise of immunotherapy, HCC patients frequently show a limited response to treatment with single ICIs, mirroring observations in other solid tumors[68]. This has led researchers to explore the potential of combining DC vaccines with various anticancer therapies for HCC treatment, as evidenced by numerous completed and ongoing clinical trials. Furthermore, capitalizing on the ability of DCs to stimulate and strengthen the cytotoxic activity of CTLs and NK cells[69], researchers are investigating the use of DC vaccines alongside immunologically active cells or the infusion of DC-activated immune cells in clinical trials for HCC patients.

A clinical study designated NCT01974661 assessed the safety and practicality of combining a mature DC vaccine (ilixadencel) with toll-like receptor 3 and 7/8 stimulators and interferon-gamma (IFN-γ) in patients with advanced HCC[70]. The study demonstrated the safety and tolerability of the vaccine. Notably, one patient experienced a PR, and five achieved SD. The median time to progression for the disease was 5.5 months, and the median overall survival was 7.5 months. Importantly, vaccination resulted in a significant increase (73.3%) in the number of tumor-specific CTLs producing IFN-γ in evaluable patients. These findings suggest that when combined with toll-like receptor agonists, DC vaccines lacking specific tumor antigens could be a safe and practical approach to support standard treatments for HCC patients. However, further studies are needed to definitively assess their efficacy. Researchers conducted a phase I/II clinical trial (JPRN-UMIN000010691) to assess the safety and effectiveness of combining surgical removal of the tumor with a post-operative vaccine made from DCs transfected with HSP70 mRNA in patients with HCC that could be surgically removed[71]. Forty-five patients were divided into two groups receiving either surgery alone (control) or surgery followed by three intradermal DC vaccinations (2 × 10⁶ cells each). The DC vaccination regimen started 5-9 days after surgery and continued at 5-10 weeks and 9-16 weeks intervals. The study found DC vaccination to be safe and well-tolerated. Although overall survival and disease-free survival showed no significant difference between the groups, analysis of subgroups revealed that patients with HSP70-positive HCC who underwent DC vaccination had a considerably longer median overall survival and disease-free survival compared to the control group (P values = 0.003 and 0.090, respectively). These findings suggest a potential benefit of DC vaccination with HSP70-targeted therapy for patients with HSP70-positive HCC following surgical resection.

OVs: Ovs are engineered to target and lyse tumor cells, leading to the release of tumor-derived antigens. These antigens stimulate the immune system to generate a tailored response with CTLs specifically directed against the cancer cells[72]. OVs can trigger an antitumor immune response by inducing the release of danger signals and tumor antigens from lysed cancer cells[73], activating immune effector cells like NK cells and CTLs to attack remaining tumor cells[74]. OVs can damage the tumor vasculature, limiting the supply of nutrients and oxygen to the tumor, further hindering its growth[75].

A cutting-edge approach involves equipping OVs with bispecific T-cell engagers (BiTE) or trispecific T-cell engagers (TriTE) molecules. These BiTEs are engineered proteins that act like bridges. One arm of the BiTE binds to a specific molecule on the surface of T cells, while the other arm binds to a specific antigen on the surface of tumor cells[76]. TriTE molecules are essentially BiTEs with an extra “arm” that can bind to an additional site on T cells[76]. Research suggests that OVs engineered with either BiTEs or TriTEs initiate a two-pronged attack. These OVs not only eliminate infected tumor cells but also trigger a bystander effect, leading to the destruction of uninfected tumor cells, resulting in enhanced anti-tumor activity[75].

A recent surge in research has explored the potential of combining chimeric antigen receptor T cells (CAR-T) cell therapy with OVs for treating solid tumors. Despite the remarkable effectiveness of CAR-T cell therapy in treating blood cancers, its application in solid tumors has been underwhelming[77]. Combining CAR-T cell therapy with OVs has shown promise in improving CAR-T cell infiltration and persistence within solid tumors[78]. CAR-T cell and OV combined therapy demonstrates increased tumor eradication and improved patient survival compared to single-agent therapies. Additionally, this combination approach may offer some benefit in controlling tumor spread[79]. CAR-T cell and OV combined therapy has not been investigated in HCC, presenting a potential avenue for future research.

Several types of viruses are being explored as OVs for early-stage HCC therapy, each offering unique advantages and potential limitations[80]. Adenoviruses are a commonly studied group due to their ability to infect a broad range of cells, including HCC cells. Adenovirus offers promise as a vector for tumor gene therapy due to its advantageous characteristics, such as efficient infection, high carrying capacity, and minimal risk of insertional mutagenesis. Poxviruses, on the other hand, exhibit strong immunogenicity and a potent ability to stimulate the body’s anti-tumor immune response after replicating within tumor tissues[81]. JX-594 is a modified poxvirus and is currently the most promising OV being investigated in clinical trials for HCC[2]. Research is currently ongoing on six poxviruses from four distinct genera as potential OVs for use in HCC. Vaccinia virus (VV), a well-studied member of the orthopoxvirus genus[81], stands out for its inherent ability to target tumors. Notably, several hallmarks of cancer, such as disrupted apoptotic pathways, dysfunctional cell cycle control, and immune evasion, create ideal conditions for successful VV replication within these cells[81]. The RNA virus Newcastle disease virus offers a different approach. Newcastle disease virus demonstrates potent oncolytic activity against HCC cells while exhibiting minimal toxicity to healthy tissues[82]. However, its RNA nature presents challenges in terms of stability and manufacturing compared to DNA-based viruses. Researchers are actively investigating these and other OVs, such as reoviruses and herpes simplex viruses, to identify the most effective and well-tolerated viral platforms for targeted therapy in early-stage HCC. The selection of the optimal OV platform will likely depend on factors like tumor characteristics, desired immune response profile, and ease of delivery within the liver microenvironment.

Initial clinical trials exploring the safety and effectiveness of different OVs in patients with HCC have shown promising results. These trials reported manageable side effects and indications of anti-tumor activity. One such trial (NCT00554372) investigated the feasibility of using JX-594 (Pexa-Vec) in HCC patients through a randomized phase II design. The trial involved injecting two different doses of JX-594, low and high, directly into the tumors of 30 participants. Researchers observed a substantially greater median overall survival in the high-dose group compared to the low-dose group. Specifically, the median overall survival was 14.1 months and 6.7 months, respectively (hazard ratio = 0.39, P = 0.020)[83]. Following treatment, all patients developed a flu-like syndrome with fever, chills, and vomiting, with the severity increasing as dose increased[83].

Antigen targeting antibodies - ACT

The high levels of GPC3 protein in HCC make it an attractive target for antibody-based treatments. The monoclonal antibody GC33 is derived from mice and specifically binds to the C-terminus of GPC3 with strong affinity. This binding triggers the immune system to destroy HCC cells through a process known as antibody-dependent cellular cytotoxicity, resulting in powerful anti-tumor effects in animal models where human tumors are implanted in mice[84]. To develop GC33 for clinical use, scientists created a humanized version by transplanting the antigen-binding regions (CDRs) of the antibody onto a human antibody scaffold, employing a combination of hybrid variable region and two-step design techniques. For improved stability, scientists made further refinements to the humanized GC33 antibody. This involved strategically substituting certain amino acids within the heavy chain’s variable region, as these residues might influence the antibody’s structure[85].

A phase I clinical trial investigated the safety, pharmacokinetics, and potential efficacy of GC33, a humanized anti-GPC3 antibody, in 20 patients with HCC[86]. The study design involved administering escalating doses (2.5-20 mg/kg) of GC33 weekly via intravenous infusion. Notably, 56% of patients exhibited high tumor GPC3 expression as assessed by immunohistochemistry. GC33 demonstrated a favorable safety profile, and pharmacokinetic data were obtained. Interestingly, four patients with high GPC3 expression achieved SD, suggesting potential antitumor activity linked to GPC3 levels. Additionally, patients with high GPC3 tumors exhibited a considerably longer median time to disease progression compared to those with low GPC3 expression. These observations justify further exploration of GC33, especially in patients diagnosed with GPC3-positive HCC. Several other antibodies targeting GPC3 for HCC treatment are also under development, including the human antibodies MDX-1414 and HN3, as well as the humanized mouse antibody YP7[87]. Current preclinical investigations hold significant promise in evaluating GPC3 as a novel strategy for HCC treatment using antibody therapy.

ACT in HCC

ACT utilizes a range of immune cell types, including NK cells, TILs, cytokine-induced killer cells (CIKs), and CAR-T cells. These strategies have demonstrated significant antitumor activity against HCC.

NK cells: Adoptive NK cell-based immunotherapy emerges as a promising avenue for early-stage HCC management, offering a unique advantage over traditional therapies. NK cells are a type of innate lymphoid cell and are critical for the body’s initial defense against tumors[88]. Their inherent cytotoxic activity and ability to recognize and eliminate cancer cells without prior sensitization make them a compelling candidate for immunotherapy in early-stage HCC[89]. NK cells employ a multifaceted arsenal to combat tumors. They directly eliminate cancer cells through cytotoxicity, releasing potent granules containing perforin and granzymes upon target recognition[90]. Equipping NK cells with antibodies specific to tumors empowers them to more effectively identify and destroy cancer cells through a process called antibody-dependent cellular cytotoxicity[91]. NK cells secrete cytokines and chemokines that influence the tumor microenvironment, recruiting other immune cells and promoting anti-tumor immunity[92].

In HCC, despite their intrinsic ability to suppress tumors, NK cells frequently exhibit impaired function[93]. This dysfunction is attributed to several factors, including tumor-mediated suppression, where HCC tumors secrete immunosuppressive factors like transforming growth factor-β and interleukin (IL)-10, inhibiting NK cell activation and cytotoxicity[94]. Tumor cells can downregulate ligands for activating receptors on NK cells, hindering their recognition and elimination[95,96]. Prolonged presence within the tumor microenvironment can cause NK cells to become exhausted, exhibiting diminished cytotoxic activity and cytokine secretion[97].

The exploration of promising clinical trials and strategies for harnessing the potential of NK cells in early-stage HCC treatment identified adoptive NK cell therapy, which involves isolating and expanding functional NK cells from healthy donors or the patient, followed by reintroduction into the patient to enhance their anti-cancer response[98,99]. Autologous therapy involves the extraction and expansion of NK cells from the patient, offering the advantage of perfect compatibility. However, tumor-induced NK cell dysfunction and the need for personalized cell cultures limit its widespread application. In contrast, allogeneic therapy uses NK cells derived from healthy donors, providing readily available ‘ready-made’ cells and bypassing the need for personalized cultures[100]. Compared to allogeneic T-cell therapies, NK cells have a significantly lower risk of graft-vs-host-disease due to their limited capacity for long-term persistence and alloreactivity, making them a safer option for patients[101]. Combining allogeneic NK cell therapy with other modalities, such as ICIs or irreversible electrosporulation, promises enhanced anticancer activity, especially in the context of early-stage disease[98,94]. Irreversible electrosporing can directly damage cancer tissue, further enhancing the efficacy of NK cell therapy[98]. NK cell-based immunotherapy generally shows a well-tolerated safety profile compared to other immunotherapies, making it a potentially valuable option for HCC treatment[102]. Furthermore, NK cells can be modified with CARs, allowing them to recognize and eliminate cancer cells expressing specific cancer antigens[103]. CAR-NK cell therapy capitalizes on the intrinsic anti-tumor properties of NK cells while augmenting their targeting specificity through CARs[104]. These custom-designed proteins function as artificial receptors on the NK cell surface. They consist of three essential parts: A recognition unit that specifically binds to a selected tumor antigen, allowing for precise targeting of cancer cells. A membrane-anchoring unit tethers the receptor to the cell membrane, and a signaling unit triggers NK cell activation upon target antigen binding, ultimately leading to cancer cell destruction[105]. Unlike unmodified NK cells, CAR-NK cells can be directed against specific tumor antigens, potentially overcoming the antigenic heterogeneity often seen in HCC[106]. CAR-NK cells derived from healthy donors can be readily available, creating an “off-the-shelf” product for broader patient accessibility compared to personalized therapy with CAR-T cells[107]. CAR-NKs have a significantly lower risk of graft-vs-host disease due to their limited capacity for long-term persistence and alloreactivity, making them a potentially safer option for patients[106]. However, ongoing efforts are focused on improving CAR design and optimizing NK cell manufacturing processes to enhance potency and feasibility of large-scale clinical application.

Despite the promise, challenges remain. Optimizing NK cell expansion and activation protocols for large-scale clinical application is crucial for cost-effectiveness and wider accessibility[108,109]. Developing strategies to counteract tumor-mediated NK cell suppression and immune evasion is essential for long-term efficacy[94]. Identifying the most responsive patient populations and refining treatment regimens based on individual tumor characteristics are crucial for maximizing therapeutic benefit[89,110]. NK cell-based immunotherapy holds significant promise as a novel and potentially curative approach for early-stage HCC. Ongoing research focused on overcoming current limitations and refining treatment strategies is paving the way for personalized and effective immunotherapies tailored to individual patients. As the field continues to evolve, NK cell-based immunotherapy has the potential to revolutionize the fight against this aggressive disease, offering a targeted and potentially curative option for patients with early-stage HCC.

CAR-T cells: Adoptive CAR-T-cell therapy emerges as a beacon of hope, offering a potentially curative and personalized approach for early-stage HCC. This innovative strategy leverages the inherent anti-tumor potential of T lymphocytes by genetically modifying them with CARs. CARs represent a revolutionary advancement in cellular immunotherapy, acting as the cornerstone of CAR-T cell therapy. Meticulously engineered molecules act as synthetic receptors on the surface of T cells, which are designed to specifically identify and bind to unique antigens displayed on cancer cells. In contrast to the natural T-cell receptor that recognizes small peptide fragments presented by major histocompatibility complexes, CARs demonstrate exceptional specificity by directly targeting TAAs[111,112], achieved through their intricate architecture, comprised of three key domains. The extracellular antigen recognition domain, often derived from an antibody fragment, binds with high affinity to the chosen tumor antigen[113]. The CAR molecule possesses a transmembrane domain that integrates it into the T cell membrane, guaranteeing its stable positioning and optimal functionality[114]. Finally, the intracellular signaling domain, containing elements like CD28 and CD3ζ, serves as the “trigger”, initiating T cell activation upon antigen binding[115].

Engineered with the ability to recognize and destroy cancer cells, CAR-T cells employ various methods to achieve this, including releasing cytotoxic granules and triggering programmed cell death in the target cells[116]. This potent anti-tumor activity holds significant promise for achieving tumor control in early-stage HCC. Their ability to persist within the body for extended periods provides sustained anti-tumor surveillance and potentially reduces the risk of recurrence, a crucial factor in early-stage HCC management[117]. Despite the promising potential of adoptive CAR-T cell therapy, several challenges necessitate careful consideration. Selecting the most suitable tumor antigens for CAR targeting in HCC is crucial to maximize therapeutic efficacy while minimizing the risk of on-target, off-tumor toxicities[118,119]. The complex antigenic landscape of HCC necessitates meticulous selection of specific and highly expressed targets to ensure precise targeting and minimize potential damage to healthy tissues[119]. The inherently immunosuppressive nature of the HCC microenvironment can hinder CAR-T cell function and survival, potentially compromising their therapeutic efficacy. Therefore, developing strategies to overcome this immunosuppression is vital for maximizing the success of CAR-T cell therapy in HCC patients. Optimizing and scaling up manufacturing processes for CAR-T-cells is essential for broader clinical application and ensuring patient accessibility to this potentially curative treatment.

Building on research demonstrating the ability of GPC3 CAR-engineered T cells to eliminate GPC3-positive HCC cells in mouse tumor xenografts and cell cultures[19], several phase I clinical trials are currently underway to assess the safety and effectiveness of GPC3 CAR-T cell therapy for HCC (NCT03980288, NCT04121273, NCT03884751). These trials are evaluating the therapy alone and in combination with other treatment options, including cyclophosphamide and fludarabine (NCT02905188)[19]. A 2000 Lancet study involving 150 patients followed for 4.4 years investigated the use of adoptive immunotherapy compared to no additional treatment after surgery for HCC. The study found that adoptive immunotherapy was a safe and promising approach for reducing HCC recurrence[19]. Based on the findings reported by Kole et al[19] and previously demonstrated by Tomonari et al[32], adoptive immunotherapy significantly reduced the recurrence frequency by 18% compared to the control group. The risk of recurrence was also significantly lower in the immunotherapy group by 41% (95% confidence interval: 12-60, P = 0.01). Adoptive CAR-T cell therapy presents a paradigm shift in the fight against early-stage HCC. Its potential for enhanced specificity, potent cytotoxicity, and long-term persistence offers a promising avenue for achieving superior tumor control and potentially curative outcomes. Despite ongoing efforts to refine this approach, research advancements and clinical trials hold promise for personalized CAR-T cell therapy as a potential long-term disease management strategy for HCC patients.

CIK cells: CIK cells are a diverse group of immune cells grown outside the body (ex vivo) by stimulating them with a cocktail of cytokines, usually including IFN-γ and IL-2[120]. These cells exhibit diverse antitumor activities, including direct cytolysis of tumor cells via mechanisms like perforin-mediated granzyme release and Fas-Fas ligand interaction[121]. Studies indicate that when activated by tumor cells, CIK cells substantially elevate their production of pro-inflammatory cytokines such as tumor necrosis factor-α, IFN-γ, and IL-2. These cytokines are thought to amplify the body’s overall anti-tumor response and promote a type 1 T helper-type immune response[122], enhancing their cytotoxic activity against tumor cells. CIK cells produce various molecules that can hinder new blood vessel formation, a critical step in tumor growth and spread. These factors include angiostatin, endostatin, and tissue inhibitors of metalloproteinases. By inhibiting angiogenesis, CIK cells can limit the supply of oxygen and nutrients to tumors, further hindering their proliferation and survival.

Several clinical trials have investigated the efficacy and safety of CIK cell therapy in early-stage HCC patients, demonstrating promising results. A phase I study demonstrated a favorable safety profile for autologous TIL therapy in HCC patients, with no major complications observed. A study with a 14-month follow-up observed 100% patient survival and 80% with no signs of disease[123]. According to a comprehensive analysis of various research studies, CIK cell therapy has been shown to demonstrably improve overall survival rates, progression-free survival times, objective response rates, and disease control rates in patients with HCC[19]. Several studies, including a phase II trial, a phase III trial, and a retrospective analysis, evaluated the effectiveness of CIK cells as adjuvant therapy after surgery compared to no such treatment[19]. While all studies showed a notable increase in disease-free survival rates with CIK cells, the improvement in overall survival was not statistically significant. The combination of CIK immunotherapy and minimally invasive therapies shows promise as a safe and potentially effective treatment for HCC[124]. This notion is supported, as reported by Jiang et al[123] and is further discussed in the article by Kole et al[19], where the 1-year and 18-month recurrence rates were significantly lower (8.9% and 15.6%, respectively) in the group receiving CIK cell transfusion compared to the control group (30.0% and 40.0%). These data suggest that CIK cell therapy is effective in enhancing immune function of HCC patients and potentially reducing the risk of recurrence.

CIK cell therapy represents a promising immunotherapeutic approach for early-stage HCC patients. Its favorable safety profile, autologous nature, and demonstrated ability to improve recurrence-free survival and potentially overall survival make it a valuable tool in the fight against this aggressive malignancy. Ongoing research focused on optimizing protocols, exploring combination therapies, and potentially targeting specific tumor antigens holds the potential to further refine and maximize the clinical impact of CIK cell therapy in early-stage HCC management.

AFP AND GPC3 AS DIAGNOSTIC MARKERS FOR HCC

Several factors, including patient characteristics, study design, and chosen cut-off value, can influence the effectiveness of AFP analysis in detecting HCC[125]. In a systematic analysis of studies employing an AFP threshold of 20 ng/mL for HCC detection in patients with remission, sensitivity ranged from 41% to 65%, while specificity ranged from 80% to 94%[126]. While lowering the AFP cutoff threshold can increase sensitivity, this comes at the expense of a higher false-positive rate[127]. For instance, raising the cutoff from 20 ng/mL to 50 ng/mL significantly improves specificity (96%) and positive predictive value (75%), but sensitivity drops to 47%[127]. Additionally, AFP levels correlate with tumor size, with larger tumors generally having higher AFP values. This further reduces sensitivity for smaller HCCs, dropping from 52% for tumors exceeding 3 cm to 25% for those below 3 cm[128].

AFP serves as a valuable tool for monitoring response to treatment in AFP-producing HCC, but its effectiveness is limited for HCC patients without pre-treatment AFP elevation[129]. Additionally, approximately one-third of HCC patients have normal or low AFP levels[130]. Patients with lower AFP levels typically present with smaller tumors, reduced risk of recurrence, and better survival outcomes compared to those with elevated AFP[131]. Studies show that 58% of HCC patients fall below a 100 ng/mL AFP cutoff, with lower rates observed in larger tumors[132]. Furthermore, HCC with typical AFP levels exhibit a lower risk of developing portal vein thrombosis[132]. The binding affinity to lens culinaris agglutinin (LCA) separates AFP into three distinct bands on western blots. AFP-L1, which does not bind to LCA, is associated with chronic hepatitis inflammation. AFP-L2, with intermediate LCA binding, is detectable in the serum of pregnant mothers. Finally, AFP-L3, which reacts with LCA, indicates a higher likelihood of HCC[133].

Early detection of HCC is possible by measuring AFP-L3[134], which may also be informative in differentiating the histological type of tumors[129]. In HCC patients with low total AFP (≤ 200 ng/mL), AFP-L3 exceeding a 35% increase over total AFP can indicate HCC with 100% specificity[135]. While a large multicenter study reported a modest sensitivity of 37% for HCC regardless of stage[136], other studies suggest a much higher sensitivity, particularly for larger tumors[137]. In a study by Ibrahim et al[137], AFP-L3 achieved 100% diagnostic accuracy for HCC when using a cutoff > 12.3 ng/mL in patients with underlying chronic hepatitis and HCC compared to healthy controls and those with chronically active hepatitis. Notably, most HCC patients in this study had larger tumors (> 5 cm), suggesting that further evaluation of its efficacy in detecting smaller tumors is necessary. A separate investigation explored the potential of AFP-L3 as a diagnostic tool for HCC. The study involved 20 healthy controls, 20 individuals with chronic liver disease, and 40 patients with HCC caused by HCV. Notably, a high proportion (65%) of the HCC cases were in advanced stages. Utilizing a cut-off value of 23 ng/mL, AFP-L3 demonstrated promising results, achieving a sensitivity of 97.5% and a specificity of 100% in distinguishing HCC patients from healthy individuals and those with chronic liver disease[137]. Researchers assessed the effectiveness of AFP, AFP-L3, PIVKA-II, and GPC-3 in diagnosing HCC within a patient group. This group comprised 52 individuals with cirrhosis and 101 patients confirmed with HCC. The study revealed that with a threshold exceeding 13.5 ng/mL, AFP-L3 demonstrated the greatest ability to detect HCC compared to the other biomarkers examined. However, the accuracy of detection can be influenced by the chosen cut-off value, along with tumor size and stage[138]. In addition, studies have shown that persistently elevated AFP-L3 levels following HCC treatment correlate with poorer patient prognoses[129]. These findings suggest that AFP-L3 may be a valuable tool not just for HCC diagnosis but also for predicting patient outcomes.

A new iteration of the AFP-L3 test, called highly sensitive AFP-L3 (hs-AFP-L3), shows promising diagnostic accuracy for βHCC. Studies report a sensitivity of 84.9% and a specificity of 88.6% for hs-AFP-L3 in differentiating patients with HCC from those with chronic hepatitis or cirrhosis[139]. These findings suggest that hs-AFP-L3 could be a valuable tool for HCC diagnosis, offering sensitivity comparable to AFP-L3 while potentially improving with specificity. While AFP and the AFP-L3 subtype show potential for detecting HCC, their limitations have prompted research into combination approaches. De-γ-carboxyprothrombin (DCP), also known as PIVKA-II, is emerging as a promising adjunctive biomarker. In contrast to healthy individuals, HCC patients exhibit increased levels of PIVKA-II due to abnormal protein production within the tumor. This highlights the potential of combining AFP/AFP-L3 with PIVKA-II for improved HCC diagnostic performance[140]. AFP and its subtype AFP-L3 show promise for HCC detection, but limitations exist. Combining them with DCP significantly improves diagnostic accuracy, achieving a pooled sensitivity and specificity of 88% and 79%, respectively[141]. This approach has been successfully implemented in Japan’s HCC surveillance programs since 2002, leading to earlier detection and improved prognosis[142]. Scoring systems like GALAD (incorporating AFP, AFP-L3, DCP, age, and sex) further enhance accuracy (area under the curve = 0.976)[143]. While AFP combined with GP73 also shows promise (sensitivity 84%, specificity 92%, area under receiver operating characteristic curve = 0.93)[40], the combined use of AFP, AFP-L3, and DCP shows promise as a particularly effective approach for diagnosing HCC.

GPC3 emerged as a promising biomarker for HCC through pioneering research. In a study of 113 patients with primary HCC, GPC3 mRNA expression was significantly higher (71.7%) compared to serum AFP levels (51.3%)[144]. Additionally, scientists used microarrays containing 23040 genes to identify GPC3 as a specifically overexpressed gene in HCC[145]. Examination of gene expression patterns in 20 HCC samples, along with their corresponding healthy liver tissues and a variety of normal human tissues, revealed that GPC3 is specifically overexpressed in HCC.

Studies employing a mouse monoclonal antibody (1G12) specific to a C-terminal GPC3 peptide corroborated the elevated expression of GPC3 in HCC patients[146]. Immunohistochemical analysis revealed that GPC3 was present at high levels in 72% of HCC samples. These findings suggest that GPC3 expression may be a valuable additional tool in histopathological diagnosis, potentially aiding in differentiating distinguishing HCC from other liver lesions like cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules[147]. Researchers have explored its potential as a biomarker in blood using techniques like enzyme-linked immunosorbent assays and radioimmunoassays[148]. Combining the analysis of various blood markers like AFP and PIVKA-II could potentially improve the accuracy of HCC diagnosis. Notably, GPC3, a cell surface protein, can be released into the bloodstream after being cleaved by the Notum enzyme[149]. The N-terminal fragment of GPC3 shows potential as a biomarker for early-stage HCC detection[150]. Several investigations have evaluated the diagnostic accuracy of GPC3. Qiao et al[151] reported a sensitivity of 51.5% and a specificity of 92.8% for GPC3 in the diagnosis of HCC compared to other markers such as AFP and human cervical cancer oncogene. In addition, combining GPC3 with other markers significantly increased sensitivity. A meta-analysis by Xu et al[152] comparing GPC3 and AFP as serum markers revealed similar sensitivities (59.2% vs 51.9%) but lower specificity for GPC3 (84.8% vs 94%). While these results indicate GPC3’s promise as a diagnostic marker for HCC in blood, further investigations are necessary to fully understand its behavior in serum, including how it is released and how long it remains stable. Future studies should investigate the utility of GPC3 in combination with other markers or as part of a scoring system to improve diagnostic accuracy for HCC.

CONCLUSION

Immunotherapy is emerging as a transformative option for the treatment of HCC, offering a complex, multifaceted approach that leverages ICIs, CAR T-cell therapy, TILs, cancer vaccines, and therapeutic antibodies to target specific immune responses. The current work emphasizes the groundbreaking potential of combining immunotherapy with liver transplant to reshape the treatment paradigm for early-stage HCC, addressing both tumor downsizing pre-transplant and relapse prevention post-transplant. By exploring innovative strategies like checkpoint inhibitors and OVs, this review underscores the need for new biomarkers beyond AFP and GPC3 to optimize patient selection and treatment monitoring. The integration of neoadjuvant and adjuvant immunotherapy in HCC management holds immense promise for improving survival and reducing recurrence. However, challenges remain in balancing anti-tumor immunity with the risks of immunosuppression, especially in liver transplant settings. Ongoing research must focus on refining these therapies, maximizing efficacy, minimizing toxicity, and exploring synergistic combinations with surgical and targeted treatments to revolutionize HCC care.