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In solid tumors, the exhaustion of natural killer (NK) cells and cytotoxic T cells in the immunosuppressive tumor microenvironment poses challenges for effective tumor control. Conventional humanized mouse models of hepatocellular carcinoma patient-derived xenografts (HCC-PDX) encounter limitations in NK cell infiltration, hindering studies on NK cell immunobiology. Here, we introduce an improved humanized mouse model with restored NK cell reconstitution and infiltration in HCC-PDX, coupled with single-cell RNA sequencing (scRNA-seq) to identify potential anti-HCC treatments. A single administration of adeno-associated virus carrying human interleukin-15 reinstated persistent NK cell reconstitution and infiltration in HCC-PDX in humanized mice. scRNA-seq revealed NK cell and T cell subpopulations with heightened PDCD1 and TIGIT levels. Notably, combination therapy with anti-PD-1 and anti-TIGIT antibodies alleviated HCC burden in humanized mice, demonstrating NK cell-dependent efficacy. Bulk-RNA sequencing analysis also revealed significant alterations in the tumor transcriptome that may contribute to further resistance after combination therapy, warranting further investigations. As an emerging strategy, ongoing clinical trials with anti-PD-1 and anti-TIGIT antibodies provide limited data. The improved humanized mouse HCC-PDX model not only sheds light on the pivotal role of NK cells but also serves as a robust platform for evaluating safety and anti-tumor efficacy of combination therapies and other potential regimens, complementing clinical insights., Competing Interests: Declaration of interests Q.C. is the scientific cofounder of two biotech companies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: Hepatocellular carcinoma (HCC) is a highly fatal cancer characterized by high intra-tumor heterogeneity (ITH). A panoramic understanding of its tumor evolution, in relation to its clinical trajectory, may provide novel prognostic and treatment strategies., Methods: Through the Asia-Pacific Hepatocellular Carcinoma trials group (NCT03267641), we recruited one of the largest prospective cohorts of patients with HCC, with over 600 whole genome and transcriptome samples from 123 treatment-naïve patients., Results: Using a multi-region sampling approach, we revealed seven convergent genetic evolutionary paths governed by the early driver mutations, late copy number variations and viral integrations, which stratify patient clinical trajectories after surgical resection. Furthermore, such evolutionary paths shaped the molecular profiles, leading to distinct transcriptomic subtypes. Most significantly, although we found the coexistence of multiple transcriptomic subtypes within certain tumors, patient prognosis was best predicted by the most aggressive cell fraction of the tumor, rather than by overall degree of transcriptomic ITH level - a phenomenon we termed the 'bad apple' effect. Finally, we found that characteristics throughout early and late tumor evolution provide significant and complementary prognostic power in predicting patient survival., Conclusions: Taken together, our study generated a comprehensive landscape of evolutionary history for HCC and provides a rich multi-omics resource for understanding tumor heterogeneity and clinical trajectories., Impact and Implications: This prospective study, utilizing comprehensive multi-sector, multi-omics sequencing and clinical data from surgically resected hepatocellular carcinoma (HCC), reveals critical insights into the role of tumor evolution and intra-tumor heterogeneity (ITH) in determining the prognosis of HCC. These findings are invaluable for oncology researchers and clinicians, as they underscore the influence of distinct evolutionary paths and the 'bad apple' effect, where the most aggressive tumor fraction dictates disease progression. These insights not only enhance prognostic accuracy post-surgical resection but also pave the way for personalized treatment strategies tailored to specific tumor evolutionary and transcriptomic profiles. The coexistence of multiple subtypes within the same tumor prompts a re-appraisal of the utilities of depending on single samples to represent the entire tumor and suggests the need for clinical molecular imaging. This research thus marks a significant step forward in the clinical understanding and management of HCC, underscoring the importance of integrating tumor evolutionary dynamics and multi-omics biomarkers into therapeutic decision-making., Clinical Trial Number: NCT03267641 (Observational cohort)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a deadly cancer with a high global mortality rate, and the downregulation of GATA binding protein 4 (GATA4) has been implicated in HCC progression. In this study, we investigated the role of GATA4 in shaping the immune landscape of HCC., Methods: HCC tumor samples were classified into "low" or "normal/high" based on GATA4 RNA expression relative to adjacent non-tumor liver tissues. The immune landscapes of GATA4-low and GATA4-normal/high tumors were analyzed using cytometry by time-of-flight, bulk/spatial transcriptomic analyses and validated by multiplex immunofluorescence., Results: GATA4-low tumors displayed enrichment in exhausted programmed cell death protein 1+ T cells, immunosuppressive regulatory T cells, myeloid-derived suppressor cells, and macrophages, highlighting the impact of GATA4 downregulation on immunosuppression. Spatial and bulk transcriptomic analyses revealed a negative correlation between GATA4 and C-C Motif Chemokine Ligand 20 (CCL20) expression in HCC. Overexpressing GATA4 confirmed CCL20 as a downstream target, contributing to an immunosuppressive tumor microenvironment, as evidenced by increased regulatory T cells and myeloid-derived suppressor cells in CCL20-high tumors. Lastly, the reduced expression of GATA4 and higher expression of CCL20 were associated with poorer overall survival in patients with HCC, implicating their roles in tumor progression., Conclusions: Our study reveals that GATA4 downregulation contributes to an immunosuppressive microenvironment, driven by CCL20-mediated enrichment of regulatory T cells and myeloid-derived suppressor cells in HCC. These findings underscore the critical role of GATA4 reduction in promoting immunosuppression and HCC progression., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Objectives: This study aims to explore the cost-effectiveness of atezolizumab plus bevacizumab against sorafenib for first-line treatment of locally advanced or metastatic hepatocellular carcinoma (HCC) in Singapore., Methods: A partitioned survival model was developed from a healthcare system perspective, with a 10-year lifetime horizon. Clinical inputs and utilities were obtained from the IMbrave150 trial. Healthcare resource use costs were obtained from published local sources; drug costs reflected the most recent public hospital selling prices. Outcomes included life years, quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs). Deterministic and probabilistic sensitivity analyses were performed to assess the model's robustness., Results: Atezolizumab plus bevacizumab offered an additional 1.42 life years and 1.09 QALYs, with an additional cost of S$111,847; the ICER was S$102,988/QALY. The World Health Organization considers interventions with ICERs <1 gross domestic product (GDP)/capita to be highly cost-effective. At a willingness-to-pay (WTP) threshold of S$114,165/QALY (Singapore's 2022 GDP/capita), atezolizumab plus bevacizumab is cost-effective compared with sorafenib. The ICER was most sensitive to variations in utilities, but all parameter variations had no significant impact on the model outcomes., Conclusion: At a WTP threshold of Singapore's GDP/capita, atezolizumab plus bevacizumab is cost-effective compared with sorafenib.

Background: Combination therapy with radioembolization (yttrium-90)-resin microspheres) followed by nivolumab has shown a promising response rate of 30.6% in a Phase II trial (CA209-678) for advanced hepatocellular carcinoma (HCC); however, the response mechanisms and relevant biomarkers remain unknown., Methods: By collecting both pretreatment and on-treatment samples, we performed multimodal profiling of tissue and blood samples and investigated molecular changes associated with favorable responses in 33 patients from the trial., Results: We found that higher tumor mutation burden, NCOR1 mutations and higher expression of interferon gamma pathways occurred more frequently in responders. Meanwhile, non-responders tended to be enriched for a novel Asian-specific transcriptomic subtype (Kaya&#95;P2) with a high frequency of chromosome 16 deletions and upregulated cell cycle pathways. Strikingly, unlike other cancer types, we did not observe any association between T-cell populations and treatment response, but tumors from responders had a higher proportion of CXCL9 + /CXCR3 + macrophages. Moreover, biomarkers discovered in previous immunotherapy trials were not predictive in the current cohort, suggesting a distinctive molecular landscape associated with differential responses to the combination therapy., Conclusions: This study unraveled extensive molecular changes underlying distinctive responses to the novel treatment and pinpointed new directions for harnessing combination therapy in patients with advanced HCC., Competing Interests: Competing interests: DT reports receiving research support from Novartis, Sirtex, and Bristol Myers Squibb. DT reports having received honorarium as an advisor for Novartis, Celgene, Sirtex, Merck Sharp & Dohme, Eisai, Ipsen, Bayer, and Bristol Myers Squibb., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8 , PD-L1 , LAG-3 , and STAT1 , was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC., Methods: HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses., Results: Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3 + and LAG-3 + CD8 + cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3 + cell proportion to the total CD8 + cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8 + cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3 + CD8 + cell proportion was significantly associated with responses to ICB regardless of viral status., Conclusion: Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting., Competing Interests: DT is in the advisory board in Novartis, Celgene, Sirtex, MSD, BMS, Eisai, Ipsen, and Bayer; and as research support in Novartis, Sirtex, and BMS. JJL received research funding from Bayer, has participated in the advisory boards for Eisai, Ipsen, and Bayer, and Taiho; and received honorarium from Ipsen and BMS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Cheung, Seah, Fang, Orpilla, Lau, Lim, Lim, Lee, Lim, Lim, Cheng, Toh, Choo, Lee, Lee, Liu, Lim, Tai and Yeong.)

Unfortunately, available liver cancer treatments are associated with modest survival advantage. The biggest factor improving survival is early detection, but the current understanding of early transformation events is limited. Therefore, we set up a model to study these early events and investigated the relationship of premalignant, senescent hepatocytes, a regenerative environment, and the influence of secreted factors on liver tumorigenesis. Oncogene-induced senescence (OIS) was triggered in a subset of mouse hepatocytes, which under normal conditions, are eliminated by immunosurveillance. Inducing liver damage and regeneration was sufficient to trigger immunosurveillance escape of OIS hepatocytes, resulting in premalignant to malignant transformation and hepatocellular tumor development. Trefoil factor 3 (TFF3) was found to be overexpressed in OIS hepatocytes and in hepatocellular carcinoma. TFF3 deficiency strongly attenuated malignant transformation by increasing insulin-like growth factor binding protein 5 (IGFBP5) expression, which consequently dampened IGF receptor signaling. Furthermore, analysis of precancerous liver tissue validated TFF3 as an early liver cancer biomarker. Altogether, these findings provide mechanistic insights into early transformation and immunosurveillance escape in liver cancer, revealing TFF3 and IGFBP5 to be important players with opposite roles in tumorigenesis., Significance: Liver damage induces a compensatory regenerative response that can drive premalignant to malignant transformation of senescent hepatocytes., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background and Aims: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated., Approach and Results: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DR lo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme B hi PD-1 lo CD8 + T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DR lo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2., Conclusions: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies., (© 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Purpose: SORAMIC is a randomized controlled trial in patients with advanced hepatocellular carcinoma (HCC) undergoing sorafenib ± selective internal radiation therapy (SIRT). We investigated the value of extracellular vesicle (EV)-based proteomics for treatment response prediction., Experimental Design: The analysis population comprised 25 patients receiving SIRT+sorafenib and 20 patients receiving sorafenib alone. Patients were classified as responders or nonresponders based on changes in AFP and imaging or overall survival. Proteomic analysis was performed on plasma EVs by LC/MS, followed by bioinformatics analysis. Clinical relevance of candidate EV proteins was validated by survival and receiver-operating characteristic analysis with bootstrap internal sampling validation. Origin of circulating EV was explored by IHC staining of liver and tumor tissues and transcriptomics of blood cells., Results: Proteomic analysis identified 56 and 27 EV proteins that were differentially expressed in plasma EVs between responders and nonresponders receiving SIRT+sorafenib and sorafenib alone, respectively. High EV-GPX3/ACTR3 and low EV-ARHGAP1 were identified as candidate biomarkers at baseline from the 13 responders to SIRT+sorafenib with statistically significant AUC = 1 for all and bootstrap P values 2.23 × 10-5, 2.22 × 10-5, and 2.23 × 10-5, respectively. These patients showed reduced abundance of EV-VPS13A and EV-KALRN 6 to 9 weeks after combined treatment with significant AUC and bootstrap P values. In reverse, low GPX3 and high ARHGAP1 demonstrated better response to sorafenib monotherapy with AUC = 0.9697 and 0.9192 as well as bootstrap P values 8.34 × 10-5 and 7.98 × 10-4, respectively. HCC tumor was the likely origin of circulating EVs., Conclusions: In this exploratory study, EV-based proteomics predicted response to SIRT+sorafenib and sorafenib-only treatment in patients with advanced HCC of metabolic origin., (©2022 The Authors; Published by the American Association for Cancer Research.)

Atezolizumab (anti-programmed death-ligand 1 (PD-L1)) and bevacizumab (anti-vascular endothelial growth factor (VEGF)) combination therapy has become the new standard of care in patients with unresectable hepatocellular carcinoma. However, potential predictive biomarkers and mechanisms of response and resistance remain less well understood. We report integrated molecular analyses of tumor samples from 358 patients with hepatocellular carcinoma (HCC) enrolled in the GO30140 phase 1b or IMbrave150 phase 3 trial and treated with atezolizumab combined with bevacizumab, atezolizumab alone or sorafenib (multikinase inhibitor). Pre-existing immunity (high expression of CD274, T-effector signature and intratumoral CD8 +  T cell density) was associated with better clinical outcomes with the combination. Reduced clinical benefit was associated with high regulatory T cell (Treg) to effector T cell (Teff) ratio and expression of oncofetal genes (GPC3, AFP). Improved outcomes from the combination versus atezolizumab alone were associated with high expression of VEGF Receptor 2 (KDR), Tregs and myeloid inflammation signatures. These findings were further validated by analyses of paired pre- and post-treatment biopsies, in situ analyses and in vivo mouse models. Our study identified key molecular correlates of the combination therapy and highlighted that anti-VEGF might synergize with anti-PD-L1 by targeting angiogenesis, Treg proliferation and myeloid cell inflammation., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Most small molecule anticancer drugs have high lipophilicity and low water solubility, which is often regarded as a key obstacle to their development and clinical applications. A variety of nano-size drug carriers, like liposomes, has been developed for solubilizing these drugs. Naturally secreted by cells, exosomes have good biocompatibility and are considered as "natural liposomes." Exosomes released by mesenchymal stem cells (MSCs) not only have the properties like the ones generated by other cells but may also possess many therapeutic bioactive factors uniquely secreted by stem cells. In the present study, exosomes secreted by murine adipose stem cells (mASCs) were isolated, identified, and characterized. Its potential as drug delivery carrier and its biological effects on hepatoma cells and normal liver cell lines were explored in vitro. The data indicated that mASC exosomes separated by our improved sequential filtration method have particle size distribution in 30-150 nm, positively expresses TSG101, CD63, CD9, GADPH, and negatively expresses calnexin. The exosomes of mASCs obtained by this method could be taken up by cells and inhibit the cell activity of hepatoma cells HepG2, while enhance the normal cell activity of THLE-2. The results suggest that ASC exosomes are ideal potential drug delivery carriers and have the prospect of applications in carcinoma treatments., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease., (© 2022. The Author(s).)

Background and Aims: Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions., Approach and Results: We established cell line-based or patient-derived xenograft-based humanized-immune-system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human-specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real-time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual-combination, and triple-combination treatment using N-(1',2-Dihydroxy-1,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized-immune-system mice with HCC. In particular, intratumor human cluster of differentiation-positive (hCD14 + ) cells could produce IL-33 through damage-associated molecular pattern/Toll-like receptor 4/activator protein 1, which up-regulated IL-6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL-33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti-HCC effect of C188-9, bevacizumab, and pembrolizumab. The results showed that the anti-HCC effect of triple-combination therapy was superior to that of single or dual treatments., Conclusions: Humanized-immune-system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.

Introduction: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality globally. Immune-checkpoint blockade (ICB) is one of the systemic therapy options for HCC. However, response rates remain low, necessitating robust predictive biomarkers. In the present study, we examined the expression of CD38, a molecule involved in the immunosuppressive adenosinergic pathway, on immune cells present in the tumor microenvironment. We then investigated the association between CD38 and ICB treatment outcomes in advanced HCC., Methods: Clinically annotated samples from 49 patients with advanced HCC treated with ICB were analyzed for CD38 expression using immunohistochemistry (IHC), multiplex immunohistochemistry/immunofluorescence (mIHC/IF) and multiplex cytokine analysis., Results: IHC and mIHC/IF analyses revealed that higher intratumoral CD38 + cell proportion was strongly associated with improved response to ICB. The overall response rates to ICB was significantly higher among patients with high proportion of total CD38 + cells compared with patients with low proportion (43.5% vs 3.9%, p=0.019). Higher responses seen among patients with a high intratumoral CD38 + cell proportion translated to a longer median progression-free survival (mPFS, 8.21 months vs 1.64 months, p=0.0065) and median overall survival (mOS, 19.06 months vs 9.59 months, p=0.0295). Patients with high CD38 + CD68 + macrophage density had a better mOS of 34.43 months compared with 9.66 months in patients with low CD38 + CD68 + macrophage density. CD38 hi macrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB., Conclusions: A high proportion of CD38 + cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC., Competing Interests: Competing interests: DT is in the advisory board in MSD for clinical trials, and as research support in BMS. FM received research support from Janssen Pharmaceuticals, Celgene, Tusk Therapeutics and Centrose, and served on advisory boards for Centrose, Tusk Therapeutics, Jenssen, Takeda and Sanofi. JJXL has research funding from Bayer and has participated in advisory boards for Ipsen and Bayer., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aim: Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis., Methods: We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo., Results: The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome., Conclusion: Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC., Lay Summary: Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: CD38 is involved in the adenosine pathway, which represents one of the immunosuppressive mechanisms in cancer. CD38 is broadly expressed across immune cell subsets, including human macrophages differentiated in vitro from monocytes, but expression by tissue-resident macrophages remains to be demonstrated. Methods: Tissue samples were obtained from 66 patients with hepatocellular carcinoma (HCC) from Singapore and analyzed using immunohistochemistry. Tumor-infiltrating leukocytes (TILs) were further examined using DEPArray™, and the phenotype of freshly isolated TILs was determined using flow cytometry. Results: CD38 was frequently co-expressed with the macrophage-specific marker CD68. CD38 + CD68 + macrophage density was associated with improved prognosis after surgery, while total CD68 + macrophage density was associated with poor prognosis. DEPArray™ analysis revealed the presence of large (>10 μm), irregularly shaped CD45 + CD14 + cells that resembled macrophages, with concurrent CD38 + expression. Flow cytometry also revealed that majority of CD14 + HLA-DR + cells expressed CD38. Conclusion: CD38 expression was clearly demonstrated on human macrophages in an in vivo setting. The positive association identified between CD38 + macrophage density and prognosis may have implications for routine diagnostic work.

Background: Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC., Methods: Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed., Results: Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis., Conclusions: Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background and Aims: Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics., Methods: We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays., Results: In-depth interrogation of the immune landscapes showed that regulatory T cells (T REG ) and CD8 + resident memory T cells (T RM ) were enriched in HBV-related HCC, whereas Tim-3 + CD8 + T cells and CD244 + natural killer cells were enriched in non-viral-related HCC. NGS of isolated T REG and T RM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. T REG and T RM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1 + T REG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that T REG and T RM contributed to overall patient survival: T REG were associated with a poor prognosis and T RM were associated with a good prognosis in HCC., Conclusion: We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: As the current therapeutic strategies for human hepatocellular carcinoma (HCC) have been proven to have limited effectiveness, immunotherapy becomes a compelling way to tackle the disease. We aim to provide humanised mouse (humice) models for the understanding of the interaction between human cancer and immune system, particularly for human-specific drug testing., Design: Patient-derived xenograft tumours are established with type I human leucocyte antigen matched human immune system in NOD- scid Il2rg -/- (NSG) mice. The longitudinal changes of the tumour and immune responses as well as the efficacy of immune checkpoint inhibitors are investigated., Results: Similar to the clinical outcomes, the human immune system in our model is educated by the tumour and exhibits exhaustion phenotypes such as a significant declination of leucocyte numbers, upregulation of exhaustion markers and decreased the production of human proinflammatory cytokines. Notably, cytotoxic immune cells decreased more rapidly compared with other cell types. Tumour infiltrated T cells have much higher expression of exhaustion markers and lower cytokine production compared with peripheral T cells. In addition, tumour-associated macrophages and myeloid-derived suppressor cells are found to be highly enriched in the tumour microenvironment. Interestingly, the tumour also changes gene expression profiles in response to immune responses by upregulating immune checkpoint ligands. Most importantly, in contrast to the NSG model, our model demonstrates both therapeutic and side effects of immune checkpoint inhibitors pembrolizumab and ipilimumab., Conclusions: Our work provides a model for immune-oncology study and a useful parallel-to-human platform for anti-HCC drug testing, especially immunotherapy., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The most frequent chromosomal structural loss in hepatocellular carcinoma (HCC) is of the short arm of chromosome 8 (8p). Genes on the remaining homologous chromosome, however, are not recurrently mutated, and the identity of key 8p tumor-suppressor genes (TSG) is unknown. In this work, analysis of minimal commonly deleted 8p segments to identify candidate TSG implicated GATA4, a master transcription factor driver of hepatocyte epithelial lineage fate. In a murine model, liver-conditional deletion of 1 Gata4 allele to model the haploinsufficiency seen in HCC produced enlarged livers with a gene expression profile of persistent precursor proliferation and failed hepatocyte epithelial differentiation. HCC mimicked this gene expression profile, even in cases that were morphologically classified as well differentiated. HCC with intact chromosome 8p also featured GATA4 loss of function via GATA4 germline mutations that abrogated GATA4 interactions with a coactivator, MED12, or by inactivating mutations directly in GATA4 coactivators, including ARID1A. GATA4 reintroduction into GATA4-haploinsufficient HCC cells or ARID1A reintroduction into ARID1A-mutant/GATA4-intact HCC cells activated hundreds of hepatocyte genes and quenched the proliferative precursor program. Thus, disruption of GATA4-mediated transactivation in HCC suppresses hepatocyte epithelial differentiation to sustain replicative precursor phenotype.

Background: The majority of patients with hepatocellular carcinoma (HCC) are inoperable and results with conventional chemotherapy are dismal. Many end up with no treatment options and resort to alternative medicine. The authors report the use of Coriolus versicolor (CV) in advanced HCC patients with poor liver function or who were unfit to receive standard therapy., Methods: Fifteen eligible cases were randomized 2:1 to either CV or placebo. The primary endpoint was the median time to progression (TTP) between both arms. Secondary endpoints include evaluating response rates, toxicity, quality of life (QOL), progression-free survival (PFS), and overall survival (OS). Further correlative studies were performed looking at the effect of CV on the immune system., Results: The median treatment duration was 1.5 cycles and 3 cycles on the placebo and CV arm, respectively. Median TTP was 2.5 (1.4-5.3) months compared to 4.2 (0.4-4.2) months in the CV and placebo arm, respectively, hazard ratio (HR) 0.70 (0.16-3.05 p = 0.634). Median PFS was 2.5 (1.4-5.3) months in the CV and 1.1 (0.4-4.2) months in the placebo arm, HR 0.42 (0.13-1.34, p = 0.144). Median OS was 6.5 (3.3-24.1) and 2.2 (0.8-23.3) months, respectively, HR 0.35 (0.10-1.25, p = 0.105). Social and emotional functioning scores were higher in the CV group compared to placebo group on treatment. CV subjects had less appetite loss and pain symptoms compared to placebo subjects during treatment., Conclusions: There was no difference in TTP with use of CV compared to placebo. CV subjects generally had better QOL on treatment compared to placebo subjects. The utility of this supplement in patients whose primary treatment goal is palliation should be further explored.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8 + T cells (T RM s), resident natural killer cells (NK R s), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3 + CD4 + and PD-1 + CD8 + T cells. Interestingly, Tregs and T RM s isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and T RM s isolated from the NTME. We found PD-1 + T RM s were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8 + T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NK R s via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed., Competing Interests: The authors declare no conflict of interest.

Objective: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis., Design: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study., Results: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells., Conclusions: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The cumulative effects of hepatic injury due to hepatitis B virus (HBV) infections and aflatoxin-B1 (AFB1) exposure are the major risk factors of HCC. Understanding early metabolic changes involving these risk factors in an animal model closely resembling human hepatocellular carcinoma (HCC) is critical for biomarker discovery and disease therapeutics. We have used the hepatitis B surface antigen (HBsAg) transgenic mouse model that mimics HBV carriers with and without AFB1 treatment. We investigated early metabolic changes from preneoplastic state to HCC by non-invasive longitudinal imaging in three HCC groups of mice: HBsAg + AFB1(Gp-I), AFB1 alone (Gp-II), HBsAg alone (Gp-III) and a control group (wild-type untreated; Gp-IV). For the first time, we have identified acylcarnitine signals in vivo in the liver prior to the histological manifestation of the tumors in all three groups. Acylcarnitine concentration increased with increase in tumor growth in all HCC mouse models, indicating elevated metabolic activity and increased cell turnover. This was confirmed in a pilot study using human serum from HCC patients, which revealed a higher concentration of acylcarnitine compared with normal subjects. Translational clinical studies can be designed to detect acylcarnitine in patients with high risk factors for HCC.

Background and Aims: Surgery is the primary curative option in patients with hepatocellular carcinoma (HCC). Current prognostic models for HCC are developed on datasets of primarily patients with advanced cancer, and may be less relevant to resectable HCC. We developed a postoperative nomogram, the Singapore Liver Cancer Recurrence (SLICER) Score, to predict outcomes of HCC patients who have undergone surgical resection., Methods: Records for 544 consecutive patients undergoing first-line curative surgery for HCC in one institution from 1992-2007 were reviewed, with 405 local patients selected for analysis. Freedom from relapse (FFR) was the primary outcome measure. An outcome-blinded modeling strategy including clustering, data reduction and transformation was used. We compared the performance of SLICER in estimating FFR with other HCC prognostic models using concordance-indices and likelihood analysis., Results: A nomogram predicting FFR was developed, incorporating non-neoplastic liver cirrhosis, multifocality, preoperative alpha-fetoprotein level, Child-Pugh score, vascular invasion, tumor size, surgical margin and symptoms at presentation. Our nomogram outperformed other HCC prognostic models in predicting FFR by means of log-likelihood ratio statistics with good calibration demonstrated at 3 and 5 years post-resection and a concordance index of 0.69. Using decision curve analysis, SLICER also demonstrated superior net benefit at higher threshold probabilities., Conclusion: The SLICER score enables well-calibrated individualized predictions of relapse following curative HCC resection, and may represent a novel tool for biomarker research and individual counseling.

Purpose: This open-label phase III trial evaluated efficacy and tolerability of linifanib versus sorafenib in patients with advanced hepatocellular carcinoma (HCC) without prior systemic therapy., Patients and Methods: Patients were randomly assigned in a 1:1 ratio to linifanib 17.5 mg once daily or sorafenib 400 mg twice daily. Patients were stratified by region (Outside Asia, Japan, and rest of Asia), Eastern Cooperative Oncology Group performance score (ECOG PS; 0 or 1), vascular invasion or extrahepatic spread (yes or no), and hepatitis B virus (HBV) infection (yes or no). The primary end point of the study was overall survival (OS). Secondary end points were time to progression (TTP) and objective response rate (ORR) per RECIST v1.1., Results: We randomly assigned 1,035 patients (median age, 60 years; Asian, 66.6%; ECOG PS 0, 65.2%; HBV, 49.1%; vascular invasion or extrahepatic spread, 70.1%). Median OS was 9.1 months on the linifanib arm (95% CI, 8.1 to 10.2) and 9.8 months on the sorafenib arm (95% CI, 8.3 to 11.0; hazard ratio [HR], 1.046; 95% CI, 0.896 to 1.221). For prespecified stratification subgroups, OS HRs ranged from 0.793 to 1.119 and the 95% CI contained 1.0. Median TTP was 5.4 months on the linifanib arm (95% CI, 4.2 to 5.6) and 4.0 months on the sorafenib arm (95% CI, 2.8 to 4.2; HR, 0.759; 95% CI, 0.643 to 0.895; P = .001). Best response rate was 13.0% on the linifanib arm versus 6.9% on the sorafenib arm. Grade 3/4 adverse events (AEs); serious AEs; and AEs leading to discontinuation, dose interruption, and reduction were more frequent with linifanib (all P < .001)., Conclusion: Linifanib and sorafenib had similar OS in advanced HCC. Predefined superiority and noninferiority OS boundaries were not met for linifanib and the study failed to meet the primary end point. TTP and ORR favored linifanib; safety results favored sorafenib., (© 2014 by American Society of Clinical Oncology.)

The natural course of early HCC is unknown, and its progression to intermediate and advanced HCC can be diverse. Some early stage HCC patients enjoy prolonged disease-free survival, whereas others suffer aggressive relapse to stage IV metastatic cancer within a year. Comparative proteomics of HCC tumor tissues was carried out using 2D-DIGE and MALDI-TOF/TOF MS to identify proteins that can distinguish these two groups of stage I HCC patients. Twelve out of 148 differentially regulated protein spots were found to differ by approximately 2-fold for the relapse versus nonrelapse patient tissues. Four proteins, namely, heat shock 70 kDa protein 1, argininosuccinate synthase, isoform 2 of UTP-glucose-1-phosphate uridylyltransferase, and transketolase, were shown to have the potential to differentiate metastatic relapse (MR) from nonrelapse (NR) HCC patients after validation by western blotting and immunohistochemical assays. Subsequent TMA analysis revealed a three marker panel of HSP70, ASS1, and UGP2 to be statistically significant in stratifying the two groups of HCC patients. This combination panel achieved high levels of sensitivity and specificity, which has potential for clinical use in identifying HCC tumors prone to MR. This stratification will allow development of clinical management, including close follow-up and possibly treatment options, in the near future.

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.

The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.

Currently, only one drug, sorafenib, is FDA approved for the treatment of advanced hepatocellular carcinoma (HCC), achieving modest objective response rates while still conferring an overall survival benefit. Unlike other solid tumors, no oncogenic addiction loops have been validated as clinically actionable targets in HCC. Outcomes of HCC could potentially be improved if critical molecular subclasses with distinct therapeutic vulnerabilities can be identified, biomarkers that predict recurrence or progression early can be determined and key epigenetic, genetic or microenvironment drivers that determine best response to a specific targeting treatment can be uncovered. Our group and others have examined the molecular heterogeneity of hepatocellular carcinoma. We have developed a panel of patient derived xenograft models to enable focused pre-clinical drug development of rationally designed therapies in specific molecular subgroups. We observed unique patterns, including synergies, of drug activity across our molecularly diverse HCC xenografts, pointing to specific therapeutic vulnerabilities for individual tumors. These efforts inform clinical trial designs and catalyze therapeutic development. It also argues for efficient strategic allocation of patients into appropriate enriched clinical trials. Here, we will discuss some of the recent important therapeutic studies in advanced HCC and also some of the potential strategies to optimize clinical therapeutic development moving forward., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p<0.001). In conclusion, prolactin and MCP-1 are overexpressed in HCC and are conveniently quantifiable in patients' sera by ELISA. MCP-1 appears to be a promising complementary biomarker for HCC diagnosis and this MCP-1+AFP model should be further evaluated as potential biomarker on a larger scale in patients at-risk of HCC.

Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3'-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3'-end of the HBx is often deleted. HBx-human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.

Background: The efficacy and safety of linifanib (ABT-869), a selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases, were assessed in this phase 2, single-arm, open-label, multicenter trial., Methods: Eligible patients had unresectable or metastatic hepatocellular carcinoma and had received ≤ 1 prior systemic therapy. Patients received oral linifanib at a fasting dose of 0.25 mg/kg,. The primary endpoint was the progression-free rate at 16 weeks. Tumor response was assessed every 8 weeks. Secondary endpoints included the time to disease progression, overall survival, and objective response rate. Safety was also assessed., Results: Of the 44 patients enrolled, the majority were Asian (89%), had received no prior systemic therapy (82%), had Child-Pugh class A hepatic function (86%), and had hepatitis B virus infection (61%). The estimated progression-free rate at 16 weeks was 31.8% (34.2% for patients with Child-Pugh class A hepatic function), the estimated objective response rate was 9.1% (10.5% for patients with Child-Pugh class A hepatic function), the median time to disease progression was 3.7 months (3.7 months for patients with Child-Pugh class A hepatic function), and the median overall survival was 9.7 months (10.4 months for patients with Child-Pugh class A hepatic function). The most common linifanib-related adverse events were diarrhea (55%) and fatigue (52%). The most common linifanib-related grade 3/4 adverse events were hypertension (25%) and fatigue (14%). Serum levels of biomarkers cancer antigen (CA) 125, cytokeratin fragment (CYFRA)21.1, and protein induced by vitamin K absence or antagonist II (PIVKA) demonstrated potential as prognostic indicators of patient response or outcome., Conclusions: Single-agent linifanib was found to be clinically active in patients with advanced hepatocellular carcinoma, with an acceptable safety profile., (Copyright © 2012 American Cancer Society.)

Background: Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC., Methods: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided., Results: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002)., Conclusions: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

MicroRNA-224 (miR-224) is one of the most commonly up-regulated microRNAs in hepatocellular carcinoma (HCC), which affects crucial cellular processes such as apoptosis and cell proliferation. In this study, we aim to elucidate the molecular mechanism that leads to the overexpression of miR-224 in HCC. We examined the transcript expression of miR-224 and neighboring miR-452 and genes on chromosome Xq28 in tumor and paired adjacent nontumorous tissues from 100 patients with HCC and found that miR-224 is coordinately up-regulated with its neighboring microRNA (miRNA) and genes. This coordinated up-regulation of miRNAs and genes at the Xq28 locus can be mimicked in nontransformed immortalized human liver cells by the introduction of histone deacetylase (HDAC) inhibitors, which resulted in a corresponding increase in histone H3 acetylation in this region. This miR-224-residing locus in Xq28 is reciprocally regulated by HDAC1, HDAC3, and histone acetylase protein, E1A binding protein p300 (EP300). Notably, in HCC tumors that significantly overexpress microRNA-224, EP300 is also overexpressed and displays increased binding to the Xq28 locus. In transformed HCC cells, high miR-224 expression can be attenuated through the inhibition of EP300, using either siRNA or the specific drug C646. In summary, overexpression of EP300 may account, in part, for the up-regulation of miR-224 expression in patients with HCC.

Background: Thalidomide has shown modest activity in advanced hepatocellular carcinomas (HCCs). Single-agent capecitabine has also been used in patients with HCC, with objective responses being reported. In our study, we review the use of thalidomide and capecitabine combination in advanced HCC., Methods: From November 2003 and September 2008, 42 patients with advanced HCC who were not eligible for clinical trial or conventional chemotherapy were treated with oral capecitabine (2000 mg/m/d) for 14 days every 3 weeks and oral thalidomide at the doses of 50 to 200 mg/d., Results: Almost 50% of patients had Child-Pugh B or C liver cirrhosis and a history of regional or systemic therapy. Three patients achieved complete responses lasting more than 52 weeks, including 1 patient who achieved pathological complete response and underwent curative resection. There were 3 patients with partial responses and 13 with stable disease. Median overall survival of all 42 patients was 9.9 months. The median progression-free survival was 5.1 months. The presence of ascites, portal vein thrombosis, and poorer Child-Pugh liver cirrhosis status also resulted in significantly poorer survival outcome. Treatment was well tolerated. Fatigue was the most common side effect occurring in 16 (38%) patients, but only 1 patient had grade 3 toxicity and had to stop treatment. Two other patients developed grade 3 palmar-plantar erythrodysesthesia from capecitabine., Conclusions: The combination of thalidomide and capecitabine has activity in advanced HCC and can result in complete pathological response. Treatment is well tolerated even in less-fit patients who have been pretreated and deserve further study.

Objective: Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated., Methods: Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182., Results: The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8(+) T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death., Conclusion: A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.

Background & Aims: Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC., Methods: Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation)., Results: The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8(+) T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation., Conclusions: Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation., (Copyright (c) 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential., Methods: We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry., Results: We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation., Conclusion: Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient-derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient-derived HCC xenografts resulted in a dose-dependent inhibition of tumour growth. RAD001-induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up-regulation of p27(Kip1) and down-regulation of p21(Cip1/Waf1), Cdk-6, Cdk-2, Cdk-4, cdc-25C, cyclin B1 and c-Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.

Dynamic contrast-enhanced (DCE) CT imaging of four patients with hepatocellular carcinoma (HCC) was performed using a dual-phase imaging protocol designed with initial rapid dynamic imaging to capture the initial increase in contrast medium enhancement in order to assess perfusion, followed by a delayed imaging phase with progressively longer intervals to monitor subsequent tissue enhancement behaviour in order to assess tissue permeability. The DCE CT images were analysed using a dual-input two-compartment distributed parameter model to yield separate estimates for blood flow and permeability, as well as fractional intravascular and extravascular volumes. The HCCs and surrounding cirrhotic liver tissues were found to exhibit enhancement curves that can be appropriately described by two distinct compartments separated by a semipermeable barrier. Early contrast arrival was also found for HCC as compared with background liver. These findings are consistent with the current understanding of sinusoidal capillarization and hepatocarcinogenesis.

Background/aims: Hepatocellular carcinoma is a leading cause of global cancer mortality, with standard chemotherapy being minimally effective in prolonging survival. We investigated if combined targeting of vascular endothelial growth factor protein and expression might affect hepatocellular carcinoma growth and angiogenesis., Methods: We treated patient-derived hepatocellular carcinoma xenografts with (i) bevacizumab; (ii) rapamycin; and (iii) bevacizumab plus rapamycin. Western blotting was employed to determine changes in the proteins. Apoptosis, vascular endothelial growth factor expression, microvessel density, and cell proliferation were analyzed by immunohistochemistry., Results: Hepatocellular carcinoma growth was inhibited by bevacizumab plus rapamycin treatment to a significantly greater degree than bevacizumab or rapamycin monotherapy. Reductions in tumor growth by bevacizumab plus rapamycin were associated with inhibition of downstream targets of the mammalian target-of-rapamycin pathway, reductions in vascular endothelial growth factor expression, and tumor microvessel density. Potentially additive effects of bevacizumab plus rapamycin included reductions in vascular endothelial growth factor expression, cyclin D1, and cyclin B1. In an intra-peritoneal model of hepatocellular carcinoma, bevacizumab plus rapamycin potently inhibited both intra-liver and intra-abdominal tumor growth, reduced ascites levels, and significantly prolonged mouse survival., Conclusions: Bevacizumab and rapamycin, which are both clinically approved drugs, may represent a novel molecularly-targeted combination treatment for hepatocellular carcinoma.

Introduction: Advanced hepatocellular carcinoma (HCC) has a dismal prognosis and is notoriously chemo-resistant. We conducted a Phase II prospective study to evaluate the activity and tolerability of gemcitabine and cisplatin in chemo-naïve advanced hepatocellular carcinoma. The trial considered a "no further interest" response rate of 10% and a target response rate of 30%. Utilising a Simon's minimax two-stage design with a type I error of 0.05 and power of 80%, 25 subjects would be required. Fifteen patients would be needed in stage 1 and if fewer than 2 responses were observed, the trial would be stopped and lack of efficacy claimed., Materials and Methods: Patients with advanced HCC, diagnosed based on histology or by World Health Organization (WHO) criteria, were administered gemcitabine 1000 mg/m2 and cisplatin 25 mg/m2 on day 1 and day 8 of a 21-day schedule. Assessment of response based on computer tomography was performed after every 2 cycles of chemotherapy., Results: The trial was stopped early due to a lack of efficacy. A total of 15 patients were accrued. Twelve patients were hepatitis B positive and the other 3 patients were negative for both hepatitis B and C. Only 1 patient had a history of prior heavy alcohol use. Two patients had Child C liver cirrhosis, 5 patients had Child B cirrhosis, and the remaining 8 patients had Child A cirrhosis. This regime was well tolerated and there was only 1 patient who experienced grade IV toxicities. Only 5 of 15 patients experienced grade III toxicities (nausea and emesis, 1 patient; anemia, 1 patient; thrombocytopenia, 1 patient; and neutropaenia, 2 patients). Only 1 patient experienced a partial response to the combination of gemcitabine and cisplatin. A further 3 patients experienced stable disease and 11 patients progressed on chemotherapy. The median time to progression was 6 weeks. The progression-free curve showed a sharp descent in the initial part of the study, suggesting that many patients had disease progression after enrollment. The median overall survival was 18 weeks., Conclusion: The progression-free survival and overall survival in our study were extremely short. Based on the results of our phase 2 study, we are unable to recommend further studies utilising gemcitabine and cisplatin combination in patients with advanced HCC.