Non-alcoholic Steatohepatitis-associated Hepatocellular Carcinoma Research Articles

Spatial proteomics of immune microenvironment in nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

NASH-HCC is inherently resistant to immune checkpoint blockade, but its tumor immune microenvironment is largely unknown. We applied the imaging mass cytometry to construct a spatially resolved single-cell atlas from the formalin-fixed and paraffin-embedded tissue sections from patients with NASH-HCC, virus-HCC (HBV-HCC and HCV-HCC), and healthy donors. Based on 35 biomarkers, over 750,000 individual cells were categorized into 13 distinct cell types, together with the expression of key immune functional markers. Higher infiltration of T cells, myeloid-derived suppressor cell (MDSCs), and tumor-associated macrophages (TAMs) in HCC compared to controls. The distribution of immune cells in NASH-HCC is spatially heterogeneous, enriched at adjacent normal tissues and declined toward tumors. Cell-cell connections analysis revealed the interplay of MDSCs and TAMs with CD8 + T cells in NASH-HCC. In particular, exhausted programmed cell death 1 (PD-1 + )CD8 + T cells connected with programmed cell death-ligand 1 (PD-L1 + )/inducible T cell costimulator (ICOS + ) MDSCs and TAMs in NASH-HCC, but not in viral HCC. In contrast, CD4 + /CD8 + T cells with granzyme B positivity were reduced in NASH-HCC. Tumor cells expressed low PD-L1 and showed few connections with immune cells. Our work provides the first detailed spatial map of single-cell phenotypes and multicellular connections in NASH-HCC. We demonstrate that interactions between MDSCs and TAMs with effector T cells underlie immunosuppression in NASH-HCC and are an actionable target.

Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis

RNA N6-methyladenosine (m6A) reader protein YTHDF1 has been implicated in cancer; however, its role in hepatocellular carcinoma (HCC), especially in non-alcoholic steatohepatitis-associated HCC (NASH-HCC), remains unknown. Here, we investigated the functional role of YTHDF1 in NASH-HCC and its interplay with the tumor immune microenvironment. Hepatocyte-specific Ythdf1-overexpressing mice were subjected to a NASH-HCC-inducing diet. Tumor-infiltrating immune cells were profiled with single-cell RNA-sequencing, flow cytometry, and immunostaining. The molecular target of YTHDF1 was elucidated with RNA-sequencing, m6A-sequencing, YTHDF1 RNA immunoprecipitation-sequencing, proteomics, and ribosome-profiling. Ythdf1 in NASH-HCC models was targeted by lipid nanoparticle (LNP)-encapsulated small-interfering Ythdf1. YTHDF1 is overexpressed in tumor tissues compared to adjacent peri-tumor tissues from patients with NASH-HCC. Liver-specific Ythdf1 overexpression drives tumorigenesis in dietary models of spontaneous NASH-HCC. Single-cell RNA-sequencing and flow cytometry revealed that Ythdf1 induced accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed cytotoxic CD8+ T-cell function. Mechanistically, Ythdf1 expression in NASH-HCC cells induced the secretion of IL-6, which mediated MDSC recruitment and activation, leading to CD8+ T-cell dysfunction. EZH2 mRNA was identified as a key YTHDF1 target. YTHDF1 binds to m6A-modified EZH2 mRNA and promotes EZH2 translation. EZH2 in turn increased expression and secretion of IL-6. Ythdf1 knockout synergized with anti-PD-1 treatment to suppress tumor growth in NASH-HCC allografts. Furthermore, therapeutic targeting of Ythdf1 using LNP-encapsulated small-interfering RNA significantly increased the efficacy of anti-PD-1 blockade in NASH-HCC allografts. We identified that YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8+ T-cell dysfunction. YTHDF1 may be a novel therapeutic target to improve responses to anti-PD-1 immunotherapy in NASH-HCC. YTHDF1, a N6-methyladenosine reader, is upregulated in patients with non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC); however, its role in modulating the tumor immune microenvironment in NASH-HCC remains unclear. Here, we show that Ythdf1 mediates immunosuppression in NASH-HCC and that targeting YTHDF1 in combination with immune checkpoint blockade elicits robust antitumor immune responses. Our findings suggest novel therapeutic targets for potentiating the efficacy of immune checkpoint blockade in NASH-HCC and provide the rationale for developing YTHDF1 inhibitors for the treatment of NASH-HCC.

Differential methylation patterns in lean and obese non-alcoholic steatohepatitis-associated hepatocellular carcinoma

BackgroundNonalcoholic fatty liver disease affects about 24% of the world’s population and may progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). While more common in those that are obese, NASH-HCC can develop in lean individuals. The mechanisms by which HCC develops and the role of epigenetic changes in the context of obesity and normal weight are not well understood.MethodsIn this study, we used previously generated mouse models of lean and obese HCC using a choline deficient/high trans-fat/fructose/cholesterol diet and a choline supplemented/high trans-fat/fructose/cholesterol diet, respectively, to evaluate methylation differences in HCC progression in lean versus obese mice. Differentially methylated regions were determined using reduced representation bisulfite sequencing.ResultsA larger number of differentially methylated regions (DMRs) were seen in NASH-HCC progression in the obese mice compared to the non-obese mice. No overlap existed in the DMRs with the largest methylation differences between the two models. In lean NASH-HCC, methylation differences were seen in genes involved with cancer progression and prognosis (including HCC), such as CHCHD2, FSCN1, and ZDHHC12, and lipid metabolism, including PNPLA6 and LDLRAP1. In obese NASH- HCC, methylation differences were seen in genes known to be associated with HCC, including RNF217, GJA8, PTPRE, PSAPL1, and LRRC8D. Genes involved in Wnt-signaling pathways were enriched in hypomethylated DMRs in the obese NASH-HCC.ConclusionsThese data suggest that differential methylation may play a role in hepatocarcinogenesis in lean versus obese NASH. Hypomethylation of Wnt signaling pathway-related genes in obese mice may drive progression of HCC, while progression of HCC in lean mice may be driven through other signaling pathways, including lipid metabolism.

Emerging Insights on Immunotherapy in Liver Cancer.

Significance: Hepatocellular carcinoma (HCC) is a liver malignancy with high mortality rate, limited treatment options, and poor prognosis. Sorafenib has been the only systemic treatment option for patients with advanced HCC for more than a decade. HCC is a typical inflammation-related tumor with a distinct immunosuppressive microenvironment especially the upregulation of immune checkpoints. Recent Advances: Immunotherapy has shown persistent and powerful efficacy in HCC treatment. Several preclinical and clinical studies have prompted the application of immunotherapy in first-line, second-line, and postline treatment of HCC, which has profoundly shifted the paradigm for advanced HCC treatment in the past few years. Critical Issues and Future Directions: Major unaddressed challenges in HCC immunotherapy include the discovery and validation of biological markers that predict the efficacy, the application of immunotherapy in patients with impaired liver function and nonalcoholic steatohepatitis-associated HCC, and the exploration of immunotherapy combinations with better effectiveness. This review provides the latest advances in the research of immune microenvironment and immunotherapy in HCC. Antioxid. Redox Signal. 37, 1325-1338.

Outcomes of liver transplantation for nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

Nonalcoholic steatohepatitis-associated hepatocellular carcinoma (NASH-HCC) is the second-leading cause of liver transplantation (LT) performed for HCC. Despite this, little is known about the clinical characteristics and outcomes of NASH-HCC. Patients undergoing LT for HCC from 2001 to 2017at a single center were reviewed. Outcomes of NASH-HCC (n=51) were compared to other etiologies of HCC including hepatitis C (HCV) hepatitis B (HBV), and alcoholic liver disease (ALD). Outcomes of NASH-HCC were also compared to HCV in the direct-acting antiviral (DAA) era (2014-2017). The frequency of NASH-HCC as the primary indication for LT in patients with HCC increased significantly during the study period from 4.4% (2001-2008) to 15.6% in 2017. NASH-HCC patients were significantly older (median age 65 vs. 60; P<0.001) with significantly lower alpha-fetoprotein levels (7.5 vs. 26.5, P<0.001) compared to other etiologies. The 1-, 3-, and 5-year overall survival of NASH-HCC was 92%, 86%, and 80%. Overall survival of NASH-HCC was not significantly different compared to HCV, HBV, or ALD. Compared to HCV-HCC in the DAA era (n=99), NASH-HCC had comparable post-LT survival (3-year survival 87% vs. 86%, P=0.870). In this large single-center experience of NASH-HCC, we demonstrate favorable outcomes of NASH-HCC following LT comparable to other common etiologies of HCC.

Hepatocellular carcinoma.

Liver cancer remains a global health challenge, with an estimated incidence of >1 million cases by 2025. Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ~90% of cases. Infection by hepatitis B virus and hepatitis C virus are the main risk factors for HCC development, although non-alcoholic steatohepatitis associated with metabolic syndrome or diabetes mellitus is becoming a more frequent risk factor in the West. Moreover, non-alcoholic steatohepatitis-associated HCC has a unique molecular pathogenesis. Approximately 25% of all HCCs present with potentially actionable mutations, which are yet to be translated into the clinical practice. Diagnosis based upon non-invasive criteria is currently challenged by the need for molecular information that requires tissue or liquid biopsies. The current major advancements have impacted the management of patients with advanced HCC. Six systemic therapies have been approved based on phase III trials (atezolizumab plus bevacizumab, sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab) and three additional therapies have obtained accelerated FDA approval owing to evidence of efficacy. New trials are exploring combination therapies, including checkpoint inhibitors and tyrosine kinase inhibitors or anti-VEGF therapies, or even combinations of two immunotherapy regimens. The outcomes of these trials are expected to change the landscape of HCC management at all evolutionary stages.

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles’ heel?

BackgroundA promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. MethodsCell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. ResultsPARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. ConclusionsThese proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.

Reduction of Polyunsaturated Fatty Acids with Tumor Progression in a Lean Non-Alcoholic Steatohepatitis-Associated Hepatocellular Carcinoma Mouse Model.

Background and Aim: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. While obesity and diabetes are the hallmarks of NAFLD, it also develops in lean individuals in the absence of metabolic syndrome, with a prevalence of 7 percent in the U.S. and 25-30 percent in some Asian countries. NAFLD represents the spectrum of liver disease, starting with excess liver fat accumulation (NAFL) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and ultimately hepatocellular carcinoma (HCC). To date, the pathogenesis of lean NASH-HCC is poorly understood and a mouse model is lacking. We aimed to develop a mouse model of lean NASH-HCC using a choline deficient and high trans-fat/sucrose/cholesterol diet to enable better understanding of its molecular pathogenesis.Methods: C57BL/6N mice were fed this diet starting at 4 weeks of age for 52 weeks and were compared to mice fed an isocaloric low fat control diet for the same duration. C57BL/6N mice were chosen instead of the C57BL/6J mice due to the high susceptibility of C57BL/6J mice to diet-induced obesity. The plasma and tumor fatty acid profile of these mice was also investigated.Results: Nearly 61% of the mice developed lean NASH-HCC. These mice showed reduction of plasma polyunsaturated fatty acids (PUFAs) (linolenic acids (α and γ, ω-3 and ω-6, respectively), eicosapentanoic acid (ω-3), docosahexanoic acid (ω-3), and linoleic acid (ω-6)) and increasing levels over time in mice with pre-malignant lesions.Conclusions: We developed a novel high penetrance diet-induced lean NASH-HCC mouse model. Plasma PUFA levels were reduced with tumor progression in parallel with reduced expression of genes controlling desaturase expression suggesting their potential use as biomarkers for lean NASH-HCC progression as well as chemopreventive molecules.

The role of neutrophil extracellular traps in nonalcoholic steatohepatitis-associated hepatocellular carcinoma

Abstract Introduction Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease characterized by steatosis, hepatocellular injury and chronic inflammation. Our recent findings showed that neutrophil forming neutrophil extracellular traps(NETs) in the NASH liver provide a pro-tumorigenic micro-environment. However, the mechanism has not been investigated. Here we sought that NASH induced-NETs suppress the development of anti-tumor immunity by promoting an immune-suppressive through Programmed eath-ligand 1 (PD-L1) signaling for HCC survival. Methods NASH-HCC mice were created by exposing C57BL/6 mice to streptozotocinand(STZ) and high fat diet. NET blockade was achieved with injection of DNase1, or by using PAD4 KO mice. Results There is increased neutrophil infiltration in the liver in the NASH-HCC mice and a concomitant decline in CD4+ and CD8+ T cells. Cit-H3, a specific NET marker persists in the liver through 20 weeks when HCC growth was visible. NETs blockade suppressed tumor growth. PD-L1 expression and PD-1, CTLA-4, LAG-3, TIM-3, the hallmarks of T cell exhaustion are all remarkably increased in CD8+ T cells from NASH-HCC liver compared with control and blocking NETs reduced their expressions. Furthermore, PD-L1 is expressed in the structure of NETs and neutrophils by exposure to stimuli which induce NET formation. The viability of CD4+ and CD8+ T cells isolated from spleen with the stimulation of pure NETs decreased. Summary The present results indicate that NASH induced NETs are critical to the inflammation driven carcinogenesis of NASH-HCC by promoting PD-1/PD-L-1 inhibitory signals, and demonstrate the therapeutic potential of disrupting NETs for the prevention of NASH-associated HCC.

THU-461-A diet-induced lean non-alcoholic steatohepatitis-associated hepatocellular carcinoma mouse model

THU-461-A diet-induced lean non-alcoholic steatohepatitis-associated hepatocellular carcinoma mouse model

Bile acid metabolism regulated by the gut microbiota promotes non-alcoholic steatohepatitis-associated hepatocellular carcinoma in mice.

Gut microbiota plays a significant role in the development of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH). However, understanding of the precise mechanism of this process remains incomplete. A new class steatohepatitis-inducing high-fat diet (HFD), namely STHD-01, can promote the development of HCC without the administration of chemical carcinogens. Using this diet, we comprehensively analyzed changes in the gut microbiota and its metabolic functions during the development of HCC in NASH. Mice fed the STHD-01 developed NASH within 9 weeks. NASH further progressed into HCC by 41 weeks. Treatment with antibiotics significantly attenuated liver pathology and suppressed tumor development, indicating the critical role of the gut microbiota in tumor development in this model. Accumulation of cholesterol and bile acids in the liver and feces increased after feeding the mice with STHD-01. Treatment with antibiotics did not reverse these phenotypes. In contrast, accumulation of secondary bile acids was dramatically reduced after the treatment with antibiotics, suggesting the critical role of the gut microbiota in the conversion of primary bile acids to secondary bile acids. Secondary bile acids such as deoxycholic acid activated the mTOR, pathway in hepatocytes. Activation of mTOR was observed in the liver of mice fed STHD-01, and the activation was reduced when mice were treated with antibiotics. Collectively, bile acid metabolism by the gut microbiota promotes HCC development in STHD-01-induced NASH.

Nrf2-mediated anti-oxidant effects contribute to suppression of non-alcoholic steatohepatitis-associated hepatocellular carcinoma in murine model.

The exact mechanisms of hepatocellular carcinoma development in non-alcoholic steatohepatitis remain unclear. In this study, we used a new class of high-fat diet, which could induce hepatocellular carcinoma development without the use of general chemical carcinogens or knockout mice. We investigated the correlation between hepatocellular carcinoma and oxidative stress/anti-oxidant effects after depletion of the gut microbiota by treatment with antibiotics. Mice fed with the steatohepatitis-inducing high-fat diet (STHD-01) for 41 weeks developed hepatocellular carcinoma. Antibiotic-treatment in mice fed with STHD-01 significantly depleted the gut microbiota and significantly ameliorated liver injury/histology. The tumor numbers of hepatocellular carcinoma were dramatically decreased by the antibiotics-treatment. We analyzed the factors involved in oxidative stress and anti-oxidant effects. Oxidative stress was elevated in mice fed with STHD-01, whereas some anti-oxidant factors were significantly elevated after antibiotics treatment. These results suggest that the gut microbiota is a key factor in improving oxidative stress induced by STHD-01 feeding.

Abstract 5372: Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Accumulated evidence in the past decade has stablished that NASH, in addition to other well-identified risk factors, is becoming a major cause of HCC in United States. In the present study, we investigated cancer-associated chromatin alterations in the Stelic Animal Model (STAM) of NASH-related HCC. Using Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq), we identified 1666 differentially opened chromatin regions in HCC tumor tissue as compared to normal livers. Annotation of these regions showed that they were associated with 1773 differentially expressed genes, which were enriched in lipid and steroid metabolism, obesity, and cancer. Among the differentially opened chromatin regions, 10% resided within 5 kb from the transcription start site (TSS), while the remaining 90% were located distal to the TSS (&amp;gt; 5 kb). By combining ATAC-seq and transcriptomic profiles, we demonstrated that 182 of the up-regulated genes were associated with open chromatin regions in tumor tissue. Furthermore, 24 of these up-regulated genes, including Apoa4, Nupr1, Anxa2, and Igfbp1, had accessible chromatin regions located proximal to the TSS. Importantly, analysis of histone modifications at the open chromatin regions of the over-expressed cancer-related Apoa4 and Anxa2 genes demonstrated enrichment by H3K4 mono-methylation (H3H4me1) and H3K27 acetylation (H3K27ac), histone marks that associated with the active gene promoters (H3K27ac) and enhancers (H3Kme1 and H3K27ac). In conclusion, these data provide evidence for alterations in the chromatin accessibility in tumor tissue and potential role of open chromatin regions in the over-expression of critical cancer-related genes in HCC. Citation Format: Mekonnen Lemma Dechassa. Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2017-5372

Utilization of animal models to investigate nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum of liver disorders with fat accumulation from simple fatty liver, nonalcoholic steatohepatitis (NASH), fibrosis/cirrhosis and NAFLD/NASH-associated hepatocellular carcinoma (HCC). NASH is a progressive form of NAFLD and requires medical attention. One of 5-10 NASH patients may progress to end-state liver disease (ESLD or cirrhosis) in 5-10 years; meanwhile, life-threatening complications of ESLD and HCC account for major mortality. An increasing burden of NAFLD in clinics, elucidation of its pathogenesis and progression, and assessment of the efficacy of potential therapeutics demand reliable animal models. Most NASH-associated HCC occurs in cirrhotic subjects; however, HCC does appear in NASH patients without cirrhosis. Lipotoxicity, oxidant stress, insulin resistance, endoplasmic reticulum stress, altered adipokine and lymphokine profiles and gut microbiome changes affect NAFLD progression and constitute key pathobiologic interplays. How these factors promote malignant transformation in a microenvironment of steatotic inflammation and fibrosis/cirrhosis, and lead to development of neoplasms is one of critical questions faced in the hepatology field. The present review summarizes the characteristics of emerging rodent NASH-HCC models, and discusses the challenges in utilizing these models to unveil the mysteries of NASH-associated HCC development.

Nonalcoholic steatohepatitis-associated hepatocellular carcinoma: Our case series and literature review

Recently, nonalcoholic steatohepatitis (NASH) has been considered to be another cause of liver cirrhosis and hepatocellular carcinoma (HCC). The natural history and prognosis of NASH are controversial. Accordingly, we assessed the clinicopathological features of NASH-associated HCC in our experience and reviewed the literature of NASH-associated HCC. We experienced 11 patients with NASH-associated HCC (6 male, 5 female; mean age 73.8 +/- 4.9 years) who received curative treatments. Most (91%) patients had been diagnosed with obesity, diabetes, hypertension, or dyslipidemia. Seven patients (64%) also had a non-cirrhotic liver. The recurrence-free survival rates at 1, 3 and 5 years were 72%, 60%, and 60%. We also summarized and reviewed 94 cases of NASH-associated HCC which were reported in the literature (64 male; mean age 66 years). The majority of patients (68%) were obese, 66% of patients had diabetes, and 24% had dyslipidemia. Furthermore, 26% of the HCCs arose from the non-cirrhotic liver. In conclusion, patients with non-cirrhotic NASH may be a high-risk group for HCC, and regular surveillance for HCC is necessary in non-cirrhotic NASH patients as well as cirrhotic patients.

Hepatocellular Carcinoma.

Liver cancer remains a global health challenge, with an estimated incidence of >1 million cases by 2025. Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ~90% of cases. Infection by hepatitis B virus and hepatitis C virus are the main risk factors for HCC development, although non-alcoholic steatohepatitis associated with metabolic syndrome or diabetes mellitus is becoming a more frequent risk factor in the West. Moreover, non-alcoholic steatohepatitis-associated HCC has a unique molecular pathogenesis. Approximately 25% of all HCCs present with potentially actionable mutations, which are yet to be translated into the clinical practice. Diagnosis based upon non-invasive criteria is currently challenged by the need for molecular information that requires tissue or liquid biopsies. The current major advancements have impacted the management of patients with advanced HCC. Six systemic therapies have been approved based on phase III trials (atezolizumab plus bevacizumab, sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab) and three additional therapies have obtained accelerated FDA approval owing to evidence of efficacy. New trials are exploring combination therapies, including checkpoint inhibitors and tyrosine kinase inhibitors or anti-VEGF therapies, or even combinations of two immunotherapy regimens. The outcomes of these trials are expected to change the landscape of HCC management at all evolutionary stages.