Immunotherapy For Hepatocellular Carcinoma Research Articles

Dual‐Targeted Engineered Bacterial Outer Membrane Vesicles for Hepatocellular Carcinoma Immunotherapy

AbstractCluster of differentiation 47 (CD47) is an important innate immune checkpoint that empowers tumors to send “don't eat me” signals, leading to immune escape. CD47 monoclonal antibodies have achieved remarkable therapeutic success in hematological malignancies. However, their efficacy in solid tumors is limited, mainly because of immunosuppression in the tumor microenvironment. In addition, hematotoxicity severely hinders the clinical translation of CD47 antibodies. Here, a novel genetically programmed nanovesicle, OMV‐CD47/GPC3, is generated for hepatocellular carcinoma (HCC) immunotherapy by fusing anti‐CD47 nanobodies and anti‐GPC3 scFv onto bacterial outer membrane vesicles (OMVs). Following intravenous administration, dual‐targeted OMV‐CD47/GPC3 specifically recognized tumor cells expressing both CD47 and GPC3, thereby actively targeting and accumulating at the orthotopic HCC tumor site without hematotoxicity. OMV‐CD47/GPC3 potently blocked tumor CD47 and promoted macrophage phagocytosis. More importantly, immunogenic OMV‐CD47/GPC3 can effectively remodel the tumor immune microenvironment of orthotopic HCC, especially reprogram tumor‐associated macrophages (TAMs) to the M1‐phenotype and promote dendritic cell maturation, synergistically inducing a robust CD8+ T cell‐mediated anti‐tumor immune response. With the two‐pronged approach of immune checkpoint inhibition and immune activation, engineered OMV‐CD47/GPC3 significantly suppressed orthotopic HCC growth and prevented tumor recurrence and metastasis, thus providing a promising strategy for HCC immunotherapy.

Elucidating the multifaceted role of MGAT1 in hepatocellular carcinoma: integrative single-cell and spatial transcriptomics reveal novel therapeutic insights

BackgroundGlycosyltransferase-associated genes play a crucial role in hepatocellular carcinoma (HCC) pathogenesis. This study investigates their impact on the tumor microenvironment and molecular mechanisms, offering insights into innovative immunotherapeutic strategies for HCC.MethodsWe utilized cutting-edge single-cell and spatial transcriptomics to examine HCC heterogeneity. Four single-cell scoring techniques were employed to evaluate glycosyltransferase genes. Spatial transcriptomic findings were validated, and bulk RNA-seq analysis was conducted to identify prognostic glycosyltransferase-related genes and potential immunotherapeutic targets. MGAT1’s role was further explored through various functional assays.ResultsOur analysis revealed diverse cell subpopulations in HCC with distinct glycosyltransferase gene activities, particularly in macrophages. Key glycosyltransferase genes specific to macrophages were identified. Temporal analysis illustrated macrophage evolution during tumor progression, while spatial transcriptomics highlighted reduced expression of these genes in core tumor macrophages. Integrating scRNA-seq, bulk RNA-seq, and spatial transcriptomics, MGAT1 emerged as a promising therapeutic target, showing significant potential in HCC immunotherapy.ConclusionThis comprehensive study delves into glycosyltransferase-associated genes in HCC, elucidating their critical roles in cellular dynamics and immune cell interactions. Our findings open new avenues for immunotherapeutic interventions and personalized HCC management, pushing the boundaries of HCC immunotherapy.

NUSAP1 Promotes Immunity and Apoptosis by the SHCBP1/JAK2/STAT3 Phosphorylation Pathway to Induce Dendritic Cell Generation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is associated with high morbidity and mortality rates. The aims of this study were to investigate the immune-promoting action of nucleolar and spindle-associated protein 1 (NUSAP1) and identify an immunotherapy target for HCC. The Cancer Genome Atlas (TCGA) was used to analyze interaction molecules and immune correlation. The interaction between NUSAP1 and SHC binding and spindle associated 1 (SHCBP1) was examined. The role of the SHCBP1/Janus kinase 2/signal transducer and activator of transcription 3 (SHCBP1/JAK2/STAT3) pathway in this process was explored. After co-culture with HCC cell lines, the differentiation of peripheral blood mononuclear cells (PBMCs) into dendritic cells (DC) was evaluated by measuring the expression of surface factors CD1a and CD86. Pathological tissues from 50 patients with HCC were collected to validate the results of cell experiments. The expression levels of CD1a and CD86 in tissues were also determined. The results show that NUSAP1 interacted with SHCBP1 and was positively correlated with DC. In HCC cell lines, an interaction was observed between NUSAP1 and SHCBP1. It was verified that NUSAP1 inhibited the JAK2/STAT3 phosphorylation pathway by blocking SHCBP1. After co-culture, the levels of CD1a and CD86 in PBMC were elevated. In the clinical specimens, CD1a and CD86 expression levels were significantly higher in the high-NUSAP1 group versus the low-NUSAP1 group. In Summary, NUSAP1 enhanced immunity by inhibiting the SHCBP1/JAK2/STAT3 phosphorylation pathway and promoted DC generation and HCC apoptosis. NUSAP1 may be a target of immunotherapy for HCC.

Identification and validation of a prognostic model based on four genes related to satellite nodules in hepatocellular carcinoma

Satellite nodules is a key clinical characteristic which has prognostic value of hepatocellular carcinoma (HCC). Currently, there is no gene-level predictive model for Satellite nodules in liver cancer. For the 377 HCC cases collected from the dataset of Cancer Genome Atlas (TCGA), their original pathological data were analyzed to extract information regarding satellite nodules status as well as other relevant pathological data. Then, this study employed statistical modeling for prognostic model establishment in TCGA, and validation in International Cancer Genome Consortium (ICGC) cohorts and GSE76427. Through rigorous statistical analyses, 253 differential satellite nodules-related genes (SNRGs) were identified, and four key genes related to satellite nodules and prognosis were selected to construct a prognostic model. The high-risk group predicted by our model exhibited an unfavorable overall survival (OS) outlook and demonstrated an association with adverse worse clinical characteristics such as larger tumor size, higher alpha-fetoprotein, microvascular invasion and advanced stage. Moreover, the validation of the model's prognostic value in the ICGC and GSE76427 cohorts mirrored that of the TCGA cohort. Besides, the high-risk group also showed higher levels of resting Dendritic cells, M0 macrophages infiltration, alongside decreased levels of CD8+ T cells and γδT cells infiltration. The prognostic model based on SNRGs can reliability predict the OS of HCC and is likely to have predictive value of immunotherapy for HCC.

Mitotic catastrophe heterogeneity: implications for prognosis and immunotherapy in hepatocellular carcinoma.

The mitotic catastrophe (MC) pathway plays an important role in hepatocellular carcinoma (HCC) progression and tumor microenvironment (TME) regulation. However, the mechanisms linking MC heterogeneity to immune evasion and treatment response remain unclear. Based on 94 previously published highly correlated genes for MC, HCC patients' data from the Cancer Genome Atlas (TCGA) and changes in immune signatures and prognostic stratification were studied. Time and spatial-specific differences for MCGs were assessed by single-cell RNA sequencing and spatial transcriptome (ST) analysis. Multiple external databases (GEO, ICGC) were employed to construct an MC-related riskscore model. Identification of two MC-related subtypes in HCC patients from TCGA, with clear differences in immune signatures and prognostic risk stratification. Spatial mapping further associates low MC tumor regions with significant immune escape-related signaling. Nomogram combining MC riskscore and traditional indicators was validated great effect for early prediction of HCC patient outcomes. MC heterogeneity enables immune escape and therapy resistance in HCC. The MC gene signature serves as a reliable prognostic indicator for liver cancer. By revealing clear immune and spatial heterogeneity of HCC, our integrated approach provides contextual therapeutic strategies for optimal clinical decision-making.

Evolution of Systemic Treatment for Hepatocellular Carcinoma: Changing Treatment Strategies and Concepts.

Systemic therapy for hepatocellular carcinoma (HCC) has undergone substantial advancements. With the advent of atezolizumab plus bevacizumab (ATZ/BEV) combination therapy, followed by durvalumab plus tremelimumab, the era of immunotherapy for HCC has commenced. The emergence of systemic treatment with high response rates has led to improvements in overall survival while enabling conversion to radical surgical resection in some patients with HCC. In patients with intermediate-stage HCC, new treatment strategies combining systemic treatment and transcatheter arterial chemoembolization (TACE) are under development in clinical trials. Moreover, the addition of local therapies, such as TACE, to systemic treatment according to the treatment effect could achieve a certain percentage of complete response. In the IMbrave050 trial, the efficacy of ATZ/BEV combination therapy was validated in patients predicted to have a high risk of recurrence, especially in those who had undergone radical surgery or radiofrequency ablation for HCC. Therefore, systemic treatment for HCC is entering a new phase for all disease stages. The objective of this review is to organize the current position of systemic therapy for each HCC stage and discuss the development of new treatment methods and strategies, with a focus on regimens incorporating immune checkpoint inhibitors, along with future prospects.

The Role of CD4+T Cells in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) has become the fourth leading cause of cancer-related deaths worldwide; annually, approximately 830,000 deaths related to liver cancer are diagnosed globally. Since early-stage HCC is clinically asymptomatic, traditional treatment modalities, including surgical ablation, are usually not applicable or result in recurrence. Immunotherapy, particularly immune checkpoint blockade (ICB), provides new hope for cancer therapy; however, immune evasion mechanisms counteract its efficiency. In addition to viral exposure and alcohol addiction, nonalcoholic steatohepatitis (NASH) has become a major cause of HCC. Owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance, NASH-associated HCC patients respond much less efficiently to ICB treatment than do patients with other etiologies. In addition, abnormal inflammation contributes to NASH progression and NASH-HCC transition, as well as to HCC immune evasion. Therefore, uncovering the detailed mechanism governing how NASH-associated immune cells contribute to NASH progression would benefit HCC prevention and improve HCC immunotherapy efficiency. In the following review, we focused our attention on summarizing the current knowledge of the role of CD4+T cells in NASH and HCC progression, and discuss potential therapeutic strategies involving the targeting of CD4+T cells for the treatment of NASH and HCC.

Systemic therapeutic strategies for hepatocellular carcinoma: current status and prospects

Hepatocellular carcinoma (HCC) is a common type of poorly prognosticated malignant tumor. Surgical resection is the preferred treatment method for early-stage HCC. However, at the time of the initial diagnosis, fewer than 30% of patients with liver cancer are suitable for radical therapy. Systemic therapy plays an important role in the treatment process of patients with intermediate- to advanced-stage HCC, as it can effectively extend patients' survival time. With an emphasis on the status and role of systemic therapy for comprehensive management of HCC, this article summarizes the latest progress at home and abroad in the past five years, including first-line combined immunotherapy for advanced-stage HCC, second-line therapy selection, perioperative systemic therapy application, and combined therapy of systemic and local. Currently, the treatment model combined with local therapy has already become a new research hotspot in the treatment of advanced-stage HCC. Nevertheless, in the future, individualized and precise systemic therapeutic strategies will need further exploration.

Tumor-associated macrophages in anti-PD-1/PD-L1 immunotherapy for hepatocellular carcinoma: recent research progress.

Hepatocellular carcinoma (HCC) is one of the cancers that seriously threaten human health. Immunotherapy serves as the mainstay of treatment for HCC patients by targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis. However, the effectiveness of anti-PD-1/PD-L1 treatment is limited when HCC becomes drug-resistant. Tumor-associated macrophages (TAMs) are an important factor in the negative regulation of PD-1 antibody targeted therapy in the tumor microenvironment (TME). Therefore, as an emerging direction in cancer immunotherapy research for the treatment of HCC, it is crucial to elucidate the correlations and mechanisms between TAMs and PD-1/PD-L1-mediated immune tolerance. This paper summarizes the effects of TAMs on the pathogenesis and progression of HCC and their impact on HCC anti-PD-1/PD-L1 immunotherapy, and further explores current potential therapeutic strategies that target TAMs in HCC, including eliminating TAMs in the TME, inhibiting TAMs recruitment to tumors and functionally repolarizing M2-TAMs (tumor-supportive) to M1-TAMs (antitumor type).

FAT10 induces immune suppression by upregulating PD-L1 expression in hepatocellular carcinoma.

The upregulation of programmed death ligand 1 (PD-L1) plays a crucial role in facilitating cancer cells to evade immune surveillance through immunosuppression. However, the precise regulatory mechanisms of PD-L1 in hepatocellular carcinoma (HCC) remain undefined. The correlation between PD-L1 and ubiquitin-like molecules (UBLs) was studied using sequencing data from 20 HCC patients in our center, combined with TCGA data. Specifically, the association between FAT10 and PD-L1 was further validated at both the protein and mRNA levels in HCC tissues from our center. Subsequently, the effect of FAT10 on tumor progression and immune suppression was examined through both in vivo and in vitro experiments. Utilizing sequencing data, qPCR, and Western blotting assays, we confirmed that FAT10 was highly expressed in HCC tissues and positively correlated with PD-L1 expression. Additionally, in vitro experiments demonstrated that the overexpression of FAT10 fostered the proliferation, migration, and invasion of HCC cells. Furthermore, the overexpression of FAT10 in HCC cells led to an increase in PD-L1 expression, resulting in the inhibition of T cell proliferation and the enhancement of HCC cell resistance to T cell-mediated cytotoxicity. Moreover, in vivo experiments utilizing the C57BL/6 mouse model revealed that overexpression of FAT10 effectively suppressed the infiltration of CD8 + GZMB + and CD8 + Ki67 + T cells, as well as reduced serum levels of TNF-α and IFN-γ. Mechanistically, we further identified that FAT10 upregulates PD-L1 expression via activating the PI3K/AKT/mTOR pathway, but not in a ubiquitin-like modification. In conclusion, our findings indicate that FAT10 promotes immune evasion of HCC via upregulating PD-L1 expression, suggesting its potential as a novel target to enhance the efficiency of immunotherapy in HCC.

Pyroptosis-related pattern depicting tumor microenvironment and association with prognosis and efficacy in patients with hepatocellular carcinoma receiving combined molecular targeted therapy and immune checkpoint inhibitors: A multi-omics study.

e16194 Background: Molecular targeted therapy combined with immunotherapy is among the recommended treatment strategies for initially unresectable hepatocellular carcinoma (HCC). Pyroptosis is believed to be closely associated with immune cell infiltration in many tumors. We attempted to utilize a readily testable set of pyroptosis-related genes (PRGs) to develop genetic features predicting the efficacy of targeted combined immunotherapy in HCC, aiming to identify individuals sensitive to treatment. Methods: We conducted bioinformatics analysis on 53 PRGs using TCGA-LIHC and GTEx databases to construct PIs (Pyroptosis-Immune score). External and internal validation were performed using the GEO database and sequencing data from our center. Paired single-cell data were used to validate the predictive efficacy of PIs on the effectiveness of immune treatment. A matched cohort of 32 vs 32 individuals was established based on whether they underwent surgery after targeted combined immunotherapy. Immunohistochemistry staining analysis was conducted on pre-treatment biopsy samples, and protein expression was converted to corresponding gene expression levels. P-PIs (Pathological PIs) were calculated to verify its clinical applicability. The surgical group was assessed based on postoperative pathological efficacy, and the non-surgical group was evaluated for progression-free survival (PFS) through imaging. Results: We identified 6 PRGs closely associated with prognosis, immune cell infiltration, and the tumor microenvironment, constructing PIs. Log-Rank risk regression analysis suggested worse prognosis in the high PIs group (OS, HR = 3.44, 95% CI: 2.33-5.05, p < 0.01; PFS, HR = 2.00, 95% CI: 1.44-2.81, p < 0.01). GSE76427 dataset (n = 80; OS: HR = 1.61, 95% CI: 1.15-2.26, P < 0.01; PFS: HR = 1.44, 95% CI: 1.03-2.02, P = 0.03) and our center's sequencing data (n = 43; OS: HR = 1.53, 95% CI: 1.10-2.14, P = 0.01; PFS: HR = 1.55, 95% CI: 1.08-2.21, P = 0.02) indicated that the PIs could predict the prognosis for HCC. Single-cell sequencing data showed that tumor cells with high PI scores inhibited the function of CD8+ T cells (R = -0.33), while MDSC cells (R = 0.22), and M2 macrophage functions (R = 0.22) were enhanced. The P-PIs of operation group was higher (P = 0.01). The P-PIs in the surgical group was positively correlated with the degree of pathological remission (R = 0.95, P < 0.01). The median PFS in the non-surgical group was 8.5 months, and the P-PIs AUC was 0.76 (95% CI: 0.66-0.83). Conclusions: We developed a PIs to predict efficacy of targeted combined immunotherapy in HCC based on pre-treatment biopsy pathology. The P-PIs could potentially facilitate clinical trials aiming at personalized efficacy prediction for conversion or adjuvant treatment in HCC patients.

Pyroptosis-related pattern depicting tumor microenvironment and association with prognosis and efficacy in patients with hepatocellular carcinoma receiving combined molecular targeted therapy and immune checkpoint inhibitors: A multi-omics study.

e16194 Background: Molecular targeted therapy combined with immunotherapy is among the recommended treatment strategies for initially unresectable hepatocellular carcinoma (HCC). Pyroptosis is believed to be closely associated with immune cell infiltration in many tumors. We attempted to utilize a readily testable set of pyroptosis-related genes (PRGs) to develop genetic features predicting the efficacy of targeted combined immunotherapy in HCC, aiming to identify individuals sensitive to treatment. Methods: We conducted bioinformatics analysis on 53 PRGs using TCGA-LIHC and GTEx databases to construct PIs (Pyroptosis-Immune score). External and internal validation were performed using the GEO database and sequencing data from our center. Paired single-cell data were used to validate the predictive efficacy of PIs on the effectiveness of immune treatment. A matched cohort of 32 vs 32 individuals was established based on whether they underwent surgery after targeted combined immunotherapy. Immunohistochemistry staining analysis was conducted on pre-treatment biopsy samples, and protein expression was converted to corresponding gene expression levels. P-PIs (Pathological PIs) were calculated to verify its clinical applicability. The surgical group was assessed based on postoperative pathological efficacy, and the non-surgical group was evaluated for progression-free survival (PFS) through imaging. Results: We identified 6 PRGs closely associated with prognosis, immune cell infiltration, and the tumor microenvironment, constructing PIs. Log-Rank risk regression analysis suggested worse prognosis in the high PIs group (OS, HR = 3.44, 95% CI: 2.33-5.05, p < 0.01; PFS, HR = 2.00, 95% CI: 1.44-2.81, p < 0.01). GSE76427 dataset (n = 80; OS: HR = 1.61, 95% CI: 1.15-2.26, P < 0.01; PFS: HR = 1.44, 95% CI: 1.03-2.02, P = 0.03) and our center's sequencing data (n = 43; OS: HR = 1.53, 95% CI: 1.10-2.14, P = 0.01; PFS: HR = 1.55, 95% CI: 1.08-2.21, P = 0.02) indicated that the PIs could predict the prognosis for HCC. Single-cell sequencing data showed that tumor cells with high PI scores inhibited the function of CD8+ T cells (R = -0.33), while MDSC cells (R = 0.22), and M2 macrophage functions (R = 0.22) were enhanced. The P-PIs of operation group was higher (P = 0.01). The P-PIs in the surgical group was positively correlated with the degree of pathological remission (R = 0.95, P < 0.01). The median PFS in the non-surgical group was 8.5 months, and the P-PIs AUC was 0.76 (95% CI: 0.66-0.83). Conclusions: We developed a PIs to predict efficacy of targeted combined immunotherapy in HCC based on pre-treatment biopsy pathology. The P-PIs could potentially facilitate clinical trials aiming at personalized efficacy prediction for conversion or adjuvant treatment in HCC patients.

Primary and secondary resistance to immune checkpoint inhibitors in hepatocellular carcinoma.

e16209 Background: Immunotherapy have revolutionized the therapeutic landscape of hepatocellular carcinoma (HCC). However, clinical features affecting immune resistance remain largely unknown. This retrospective cohort study aims to investigate the outcomes and characteristics of pts with resistance to immunotherapy in HCC. Methods: Pts with HCC who received immune checkpoint inhibitors (ICIs) of PD-1/PD-L1 antibodies at Eastern Hepatobiliary Surgery Hospital between 2016 and 2021 were retrospectively enrolled and screened for eligibility. Recommended by the Society for Immunotherapy of Cancer (SITC), primary resistance was defined as when pts were exposed to ICIs therapy for at least 6 weeks and with the best overall response (BOR) of PD or SD for less than 6 months. Secondary resistance was defined as pts having disease progression after the initial objective response (CR/PR) or SD for more than 6 months. The durable response group consists of pts with the durable response (CR, PR, or SD) for more than 6 months without progression at data cutoff. Time to progression (TTP), overall survival (OS) and clinicopathological features of HCC pts were compared between groups. Subsequent management after resistance and post-progression survival (PPS) were also analyzed. Results: Of 496 pts included, 229 (46.2%) and 141 (28.4%) pts developed primary resistance and secondary resistance, and 126 (25.4%) pts achieved a durable response. The median follow-up was 22.8 months. For pts with primary resistance, secondary resistance, and durable response, the median TTP was 2.83 [2.56-3.09] months, 11.93 [10.45-13.40] months, and not reached, respectively. Whereas the median OS was 12.83 [10.36-15.30] months, 31.53 [28.09-34.97] months and not reached, respectively. Multivariate logistic regression revealed that the Child-Pugh score (Child-Pugh B versus A, OR 3.28 [1.51-7.13], p= 0.003), BCLC stage (BCLC B versus A, OR 3.55 [1.70-7.43], p= 0.001; BCLC C versus A, OR 2.88 [1.47-5.65], p= 0.002), and combinational therapies (ICIs+bevacizumab versus monotherapy, OR 0.14 [0.04-0.51], p= 0.003; ICIs plus lenvatinib+monotherapy, OR 0.43 [0.26-0.72], p= 0.001) were independently associated with primary resistance, and only the combination of ICIs plus bevacizumab (ICIs+bevacizumab versus monotherapy, OR 0.10 [0.01-0.52], p= 0.007) was independently associated with secondary resistance. AFP levels and post-progression therapies were independently associated with PPS in pts with primary resistance, while post-progression therapies were independently associated with PPS in pts with secondary resistance. Conclusions: Risk for resistance was significantly lower among pts who received ICIs plus bevacizumab.High AFP levels independently predict the survival of pts after primary resistance to immunotherapy. ICI-based maintenance therapy after resistance may provide a significant survival advantage for HCC pts.

WDR4 as a potential indicator of clinical prognosis and immunotherapy in hepatocellular carcinoma.

e16275 Background: Immunotherapy has garnered increasing attention in hepatocellular carcinoma (HCC) treatment. However, there remains a pressing need for effective immunological predictive biomarkers. In our previous study, utilizing data extracted from The Cancer Genome Atlas (TCGA) database, we identified a positive correlation between WDR4 expression levels in hepatocellular carcinoma and various clinical parameters, encompassing Stage, T-stage, pathological grade, AFP level, and vascular invasion. Additionally, higher WDR4 expression was linked to poorer prognosis. Leveraging The TIMER and GEPIA databases, we further elucidated a significant association between WDR4 expression levels and the extent of immune cell infiltration, particularly macrophages, B lymphocytes, and myeloid suppressor cells, in hepatocellular carcinoma. These findings propose the potential utility of WDR4 as a biomarker for immune infiltration in hepatocellular carcinoma. As noted in the trial NCT03867084, which hypothesized that adjuvant pembrolizumab might surpass placebo efficacy, there still exists a gap in identifying the specific population benefiting from adjuvant immunotherapy. Methods: Clinical and pathological data from patients who underwent single-agent immunotherapy for HCC were additionally collected from Hunan Provincial People's Hospital between January 1, 2019, and January 31, 2024. These patients participated in the clinical trial (NCT03867084) with informed consent. The relationship between WDR4 expression levels, clinical pathological data, and patient prognosis was assessed using the Kruskal-Wallis test and Kaplan-Meier survival curve analysis. Results: WDR4 displayed significant upregulation in HCC tissues compared to para-carcinoma tissues (P < 0.001) and exhibited robust diagnostic potential. WDR4 expression showed significant associations with various immune cells, including macrophages (r = 0.278, P < 0.001). Kaplan-Meier survival analysis indicated that patients with high WDR4 expression had shorter postoperative progression-free survival in the context of immunotherapy. Data from 37 patients who underwent postoperative single-agent immunotherapy for hepatocellular carcinoma (NCT03867084) revealed a significant correlation between WDR4 expression levels and Disease-Free Survival (DFS), with notable statistical significance (Log-rank P < 0.001). Conclusions: WDR4 demonstrates elevated expression levels within HCC tissues and is associated with immune infiltration, establishing it as a prognostic biomarker in HCC. Furthermore, the positive correlation observed between WDR4 and CD68 as well as PD-L1 underscores its potential as a guiding factor in immunotherapeutic approaches for HCC.

TACE-HAIC combined with donafenib and immunotherapy for hepatocellular carcinoma (HCC) with portal vein tumour thrombus (PVTT): A retrospective analysis.

e16174 Background: The probability of Chinese hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) is relatively large and the long-term survival of this part of patients is poor. Systemic therapy, transcatheter arterial chemoembolization (TACE), and hepatic artery infusion chemotherapy are widely used in HCC patients with PVTT. We want to explore the efficacy and safety of TACE plus hepatic arterial infusion chemotherapy combined with Donafenib, anti-PD-1 antibody in uHCC patients with PVTT. Methods: We retrospectively analyzed the patients who were diagnosed as uHCC with PVTT and received the treatment of TACE plus HAIC combined with Donafenib, anti-PD-1 antibody as first-line therapy from Dec 2021 to Jun 2023 in Harbin Medical University Cancer Hospital. The primary endpoint was objective response rate (ORR). Secondary endpoints included disease control rate (DCR), progress free survival (PFS), overall survival (OS) and safety. Results: Of 106 patients included in the analysis, all of them received first-line treatment of TACE (embolization of pirarubicin hydrochloride 40mg, iodized oil 10-20ml with or without gelatin sponge) plus HAIC (oxaliplatin 85 mg/m2 2h, leucovorin 400 mg/m2 1h, fluorouracil bolus 400 mg/m2 in the first 10 minutes, and fluorouracil infusion 2400 mg/m2 for 46h) combined with donafenib 200mg bid, anti-PD-1 antibody Q3W. The median age was 58 years (IQR, 52-64). The study population was predominantly male (79.2%), and 79.2% were HBV-positive. All HCC patients are accompanied by PVTT, most of which are VP3(72.6%).The rate of patients with extrahepatic metastasis were 16.0%. All patients were classified as BCLC stage C and 84.0% were classified as CNLC stage IIIa. As of December 2023, 106 patients had received at least one imaging evaluation, 11 (10.4%), 60 (56.6%) and 28 (26.4%) of them achieved complete response, partial response and stable disease, and the overall objective response rate and disease control rate was 67.0% and 93.4% per modified RECIST criteria respectively. With 16.3 months of median follow-up time, the 12-month PFS rate was 81.3% and the 12-month OS rate was 90.1%. The median PFS and OS were not reached. 103 of all patients had at least one treatment-related AE (TRAE), and treatment-related deaths did not occur in this study. Grade 3 TRAEs occurred in 33 (31.1%) patients, and no grades 4 or 5 were reported. The most common Grade 3 TRAEs included aspartate transaminase increased (18.9%), platelet count decreased (14.2%) and blood bilirubin increased (8.5%). Conclusions: TACE-HAIC combined with Donafenib and immunotherapy are effective and tolerable for uHCC patients with PVTT.

Clinical and molecular mechanisms for predicting the efficacy of immunotherapy for hepatocellular carcinoma based on gut microbiome.

e16222 Background: Hepatocellular carcinoma (HCC) is an aggressive malignant tumor, ranking as the fifth most common cancer and the third leading cause of cancer-related deaths globally, posing a significant global health issue. Increasing evidence suggests that the gut microbiome and their metabolites play a crucial role in the development and therapeutic processes of cancer. Immune checkpoint inhibitors (ICIs) have recently become a treatment option for unresectable advanced HCC. To further guide the selection of immunotherapy for clinical HCC patients, it is necessary to further elucidate the prediction of immunotherapy efficacy for HCC based on the microbiome. Methods: The study enrolled 26 unresectable advanced HCC patients and collected baseline clinical laboratory data. We administered ICIs therapy to HCC patients every three weeks. After 6 months of treatment, patients were divided the responsive group (R group) and the non-responsive group (NR group) based on imaging evaluation according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Prior to the application of anti-tumor treatment, fecal samples were collected from the patients for 16S rRNA microbiome sequencing analysis. Immunofluorescence was used to detect the expression of PD-L1 in tumor tissue, and immunohistochemistry was used to assess changes in lymphocyte subsets CD3+T cells, CD4+T cells, CD8+T cells and Treg cells, as well as the expression of TNF-α, γ-IFN, and GZMB. Additionally, the peripheral blood lymphocyte subsets CD3+T cells, CD4+T cells and CD8+T cells were analyzed by flow cytometry. Results: In our study, R group showed higher taxa richness and more gene counts than those of NR group. Comparing with NR group, the relative abundance of Firmicutes were significantly increased, whereas Proteobacteria and Bacteroidota were significantly decreased in R group at phylum level (all P < 0.05). Our study showed that R group had high level of lymphocyte subsets CD3+T cells and CD8+T cells and low level of CD4+T cells and Treg cells compared to NR group (all P < 0.05). And the expressions of PD-L1, TNF-α,γ-IFN and GZMB were upregulated in R group(all P < 0.05). Conclusions: These findings suggested that the characteristics of gut-microbial diversity and composition at the early treatment period of ICIs therapy in HCC may have distinct implications on drug efficacy and disease prognosis. ICIs therapy reshaped HCC tumor microenvironment in parallel with regulating the gut microbiome and lymphocyte subsets immunity.

Epigenetic silencing of LDHB to promote hepatocellular carcinoma by remodeling the tumor microenvironment.

e16220 Background: Hepatocellular carcinoma (HCC) is a deadly disease with limited therapeutic options. Low expression of the LDHB has been associated with poor prognosis of HCC patients, but the role and underlying mechanism remains unclear. Methods: The HCC dada from The Cancer Genome Atlas (TCGA) and GEO were obtained to analyze the LDHB expression and DNA methylation levels. Next, the correlation between LDHB and Hallmark gene sets was analyzed by GO and KEGG. RNA-seq, Western blotting, Immunohistochemistry, and Bisulfite DNA sequencing were performed to explore the relationship between DNA methylation and LDHB expression. CCK8 and colony formation assay were used to evaluate the effect of overexpression of LDHB on the proliferation of hepatocellular carcinoma cells in vitro. Finally, the role of LDHB in HCC tumor immunity was assessed in multiple mouse models in vivo. Results: Here, we found that down-regulation of LDHB was coupled with the promoter hypermethylation and knocking down the DNMT 3A restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Notably, HCC patients with high LDHB expression responded better to immune checkpoint inhibitors. Finally, we demonstrated that there was no difference in the tumor growth rate between the LDHB-expressing tumors and the control tumors in immunodeficient BALB/c nude mice. However, the LDHB-expressing tumors were significantly inhibited in immunocompetent C57BL/6 mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Moreover, as compared with the LDHB+/+ mice, LDHB-/- mice showed promoted tumor growth in an NRAS/shp53 plasmid-induced primary liver tumor model. Conclusions: Overall, our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and target for HCC immunotherapy.

Metabolomics reveals ascorbic acid inhibits ferroptosis in hepatocytes and boosts the effectiveness of anti-PD1 immunotherapy in hepatocellular carcinoma

BackgroundImmunotherapy combined with molecular targeted therapy is increasingly popular in patients with advanced hepatocellular carcinoma (HCC). However, immune-related adverse events(irAEs) brought on by immunotherapy increase the likelihood of side effects, thus it is important to look into ways to address this issue.MethodsDifferent metabolite patterns were established by analyzing metabolomics data in liver tissue samples from 10 patients(divided into severe and mild liver injury) before and after immuno-targeted therapy. After establishing a subcutaneous tumor model of HCC, the mice were divided into PBS group, ascorbic acid(AA) group, and anti-PD1 + tyrosine kinase inhibitor (TKI) group, anti-PD1 + TKI + AA group. Liver tissue were stained with hematoxylin-eosin staining(HE) and the content of aspartate transaminase (AST) and alanine transaminase(ALT) in blood were determined. The mechanism was confirmed by western blotting, mass cytometry, and other techniques.ResultsThrough metabolomics analysis, AA was significantly reduced in the sample of patients with severe liver injury caused by immuno-targeted therapy compared to patients with mild liver injury. The addition of AA in vivo experiments demonstrated a reduction in liver injury in mice. In the liver tissues of the anti-PD1 + TKI + AA group, the protein expressions of SLC7A11,GPX4 and the level of glutathione(GSH) were found to be higher compared to the anti-PD1 + TKI group. Mass cytometry analysis revealed a significant increase in the CD11b+CD44+ PD-L1+ cell population in the AA group when compared to the PBS group.ConclusionsAA could reduce liver injury by preventing hepatocyte SLC7A11/GPX4 ferroptosis and improve the immunotherapy effect of anti-PD1 by boosting CD11b+CD44+PD-L1+cell population in HCC.

Immune signature and therapeutic approach of natural killer cell in chronic liver disease and hepatocellular carcinoma.

Natural killer (NK) cells are one of the key members of innate immunity that predominantly reside in the liver, potentiating immune responses against viral infections or malignant tumors. It has been reported that changes in cell numbers and function of NK cells are associated with the development and progression of chronic liver diseases (CLDs) including non-alcoholic fatty liver disease, alcoholic liver disease, and chronic viral hepatitis. Also, it is known that the crosstalk between NK cells and hepatic stellate cells plays an important role in liver fibrosis and cirrhosis. In particular, the impaired functions of NK cells observed in CLDs consequently contribute to occurrence and progression of hepatocellular carcinoma (HCC). Chronic infections by hepatitis B or C viruses counteract the anti-tumor immunity of the host by producing the sheddases. Soluble major histocompatibility complex class I polypeptide-related sequence A (sMICA), released from the cell surfaces by sheddases, disrupts the interaction and affects the function of NK cells. Recently, the MICA/B-NK stimulatory receptor NK group 2 member D (NKG2D) axis has been extensively studied in HCC. HCC patients with low membrane-bound MICA or high sMICA concentration have been associated with poor prognosis. Therefore, reversing the sMICA-mediated downregulation of NKG2D has been proposed as an attractive strategy to enhance both innate and adaptive immune responses against HCC. This review aims to summarize recent studies on NK cell immune signatures and its roles in CLD and hepatocellular carcinogenesis and discusses the therapeutic approaches of MICA/B-NKG2D-based or NK cell-based immunotherapy for HCC.

Pumilio RNA binding family member 1 deficiency activates anti-tumor immunity in hepatocellular carcinoma via restraining M2 macrophage polarization

ABSTRACT Pumilio RNA-binding family member 1 (PUM1) has been implicated in both the progression of colorectal cancer and the regulation of inflammation. The role of PUM1 in the polarization of tumor-associated macrophages (TAMs) into the M2 phenotype has not yet been reported in hepatocellular carcinoma. Using the PUM1-knockout mice model, flow cytometry, and IHC, we validated the role of PUM1 in hepatocellular carcinoma (HCC) TAMs. One-way analysis of variance (ANOVA) or student’s t-tests was used to compare the experimental groups. We found that PUM1 inhibited anti-tumor immunity in HCC through TAM-mediated inhibition of CD8+ T cells. We also showed that PUM1 promotes the transformation of TAMs into pro-tumorigenic M2-like phenotypes by activating cAMP signaling pathway. This study emphasized the potential of PUM1 as a target for immunotherapy in HCC through TAMs. The present study revealed the molecular mechanism underlying the pro-tumor role of PUM1 in HCC.