Clinical risk factors for portal hypertension-related complications in systemic therapy for hepatocellular carcinoma.

BACKGROUND: During systemic therapy, the management of portal hypertension (PH)-related complications is vital. This study aimed to clarify factors associated with the incidence and exacerbation of PH-related complications, including the usefulness of contrast-enhanced computed tomography (CECT) in the management of PH-related complications during systemic therapy. METHODS: A total of 669 patients who received systemic therapy as first-line treatment (443 patients for sorafenib, 131 for lenvatinib, and 90 for atezolizumab/bevacizumab [ATZ/BEV]) were enrolled in this retrospective study. Additionally, the lower esophageal intramural vessel diameters (EIV) on CECT and endoscopic findings in 358 patients were compared. RESULTS: The cutoff values of the EIV diameter on CECT were 3.1 mm for small, 5.1 mm for medium, and 7.6 mm for large varices, demonstrating high concordance with the endoscopic findings. esophageal varices (EV) bleeding predictors include EIV ≥ 3.1 mm and portal vein tumor thrombosis (PVTT). In patients without EV before systemic therapy, factors associated with EV exacerbation after 3 months were EIV ≥ 1.9 mm and ATZ/BEV use. Predictors of hepatic encephalopathy (HE) include the ammonia level or portosystemic shunt diameter ≥ 6.8 mm. The incidence of HE within 2 weeks was significantly higher (18%) in patients with an ammonia level ≥ 73 µmol/L and a portosystemic shunt ≥ 6.8 mm. The exacerbating factors for ascites after 3 months were PVTT and low albumin levels. CONCLUSIONS: Careful management is warranted for patients with risk factors for exacerbation of PH-related complications; moreover, the effective use of CECT is clinically important.

A Prospective Study Exploring the Safety and Efficacy of Lenvatinib for Patients with Advanced Hepatocellular Carcinoma and High Tumor Burden: The LAUNCH Study.

PURPOSE: This study aimed to investigate the safety and efficacy of lenvatinib in real-world settings, including patients excluded from the REFLECT trial, a phase III trial that compared lenvatinib with sorafenib. PATIENTS AND METHODS: This multicenter, nonrandomized, open-label prospective study was conducted at 10 medical facilities in Japan (jRCTs031190017). Eligible patients had advanced hepatocellular carcinoma (HCC) and were suitable for lenvatinib therapy. The study included patients with high tumor burden (with >50% intrahepatic tumor volume, main portal vein invasion, or bile duct invasion), Child-Pugh B status, and receiving lenvatinib as second-line therapy following atezolizumab plus bevacizumab. RESULTS: From December 2019 to September 2021, 59 patients were analyzed (47 and 12 patients with Child-Pugh A and B, respectively). In patients with Child-Pugh A, the frequency of aspartate aminotransferase elevation was high (72.7%) in the high-burden group. No other significant ad verse events (AE) were observed even in second-line treatment. However, patients with Child-Pugh B had high incidence of grade ≥3 AE (100.0%) and high discontinuation rates caused by AE (33.3%) compared with patients with Child-Pugh A (80.9% and 17.0%, respectively). Median progression-free survival was 6.4 and 2.5 months and median overall survival was 19.7 and 4.1 months in Child-Pugh A and B, respectively. Lenvatinib plasma concentration was higher in patients with Child-Pugh B on days 8 and 15 and correlated with dose modifications and lower relative dose intensity. CONCLUSIONS: Lenvatinib is safe and effective for advanced HCC in patients with Child-Pugh A, even with high tumor burden. However, it carries a higher risk of AE and may not provide adequate efficacy for patients with Child-Pugh B status.

Potential of circulating receptor-interacting protein kinase 3 levels as a marker of acute liver injury.

The pathogenesis of acute liver failure (ALF) involves cell death. Necroptosis is a newly suggested programmed cell death, and receptor-interacting protein kinase 3 (RIPK3) has been reported as a marker for necroptosis. However, there are few reports on necroptosis in ALF. Therefore, we evaluated the role of cell death markers such as cytokeratin (CK) 18, cleaved CK (cCK) 18, and RIPK3 in ALF, as well as cytokines and hepatocyte growth factor (HGF). Seventy-one hospitalized patients with acute liver injury (38 nonsevere hepatitis [non-SH]/22 severe hepatitis [SH]/11 ALF) were studied. No significant difference was found for cytokines, but a substantial increase in HGF levels was found following the severity of hepatitis. The non-SH group had lower levels of CK18 and cCK18 than the SH/ALF group. RIPK3 was significantly lower in the non-SH/SH group than in the ALF group. HGF, RIPK3, and albumin levels were found to be important predictive variables. The present study suggests that cCK18, CK18, and RIPK3 are associated with the severity of hepatitis. RIPK3 and other markers related cell death may be useful for understanding the pathogenesis of ALF and as a prognostic marker of acute liver injury.

Estimation of the effect of atezolizumab plus bevacizumab on pulmonary arterial hypertension using computed tomography in HCC patients.

The effect of the combination of atezolizumab and bevacizumab (Atez/Bev) for hepatocellular carcinoma (HCC) on pulmonary arterial hypertension (PAH) is unknown. Estimation of PAH by using computed tomography (CT) has recently been proposed. Thus, we aimed to estimate the effect of Atez/Bev on PAH using CT. Altogether, 113 patients who received Atez/Bev for HCC were enrolled. Probable PAH was defined as the diameter of the main pulmonary artery (mPA-D) ≥ 33 mm, whereas suspicious PAH was defined as mPA-D ≥ 29 mm or mPA-D/the diameter of the ascending aorta (aAo-D) ≥ 1.0. Before treatment, probable/suspicious PAH were diagnosed in 7 (6.7%)/22 (21.0%) patients, respectively. mPA-D and mPA-D/aAo-D significantly increased after induction of Atez/Bev. The increment of mPA-D was correlated with the occurrence of post-treatment respiratory/heart failure. In analysis of 55 patients who underwent CT at 3 months after the last dose of Atez/Bev, mPA-D and mPA-D/aAo-D significantly decreased. However, in the group with continuous treatment of other molecular-targeted drugs after Atez/Bev, mPA-D and mPA-D/aAo-D showed no significant change. In conclusion, PAH may not be a rare complication in patients with HCC and should be managed carefully because of the possible negative effect of Atez/Bev on PAH.

Cabozantinib for Advanced Hepatocellular Carcinoma in the Latest Real-World Practice: A Multicenter Retrospective Analysis.

BACKGROUND: Cabozantinib was found to be effective as a second- or third-line treatment after sorafenib in patients with advanced hepatocellular carcinoma (HCC) in the phase 3 CELESTIAL trial. So far, as immunotherapy has substituted molecular target agents as the primary systemic therapy for advanced HCC, cabozantinib is extensively used in the latest real-world clinical practice in a greatly different position than that shown by the CELESTIAL trial. In the current analysis, we examined the safety and effectiveness of cabozantinib administration in real-life settings for patients with advanced HCC. METHODS: We retrospectively obtained data from patients with advanced HCC who received cabozantinib in three institutions in Japan between 14 September 2018 and 30 November 2021. RESULTS: During the study period, 23 patients with advanced HCC received cabozantinib. Our cohort included 21.7% of patients with Child-Pugh class B, and 52.2% of patients in fourth line or later. The median progression-free survival of patients given cabozantinib was 3.7 months. Regarding patients with Child-Pugh class B or administration in fourth line or later, the discontinuation rate due to adverse events in patients who initialized at 40 or 20 mg was lower than those who initialized at 60 mg (42.9% versus 75.0%). Patients who were able to continue treatment with cabozantinib for more than 3 months were more likely to undergo dose reduction than those who did not (85.7% versus 25.0%). CONCLUSIONS: Cabozantinib has recently been administered to a diverse range of patients, including those who were not enrolled in the CELESTIAL trial. Deliberate dose reduction could potentially offer clinical benefits to patients with impaired liver function. Furthermore, managing adverse events by reducing the dose could play a crucial role in extending the duration of treatment with cabozantinib. The preprint version of this work is available on https://www.researchsquare.com/article/rs-2655181/v1 .

Hyperprogressive disease during atezolizumab plus bevacizumab treatment in patients with advanced hepatocellular carcinoma from Japanese real-world practice.

BACKGROUND: Hyperprogressive disease (HPD) is a phenomenon with greatly accelerated tumor growth and clinical deterioration rates compared to pre-therapy, in patients treated with immune checkpoint inhibitors (ICI). The aim of this study is to clarify the reality of HPD in patients with advanced hepatocellular carcinoma (HCC) who were treated with atezolizumab plus bevacizumab (Atez/Bev) using tumor dynamics. METHODS: Medical records of consecutive patients with advanced HCC who were treated with Atez/Bev were retrospectively reviewed. HPD was defined as a more than two- or fourfold increase in tumor growth rate (TGR) or tumor growth kinetics rate (TGKR) before and after treatment. Overall survival (OS) and baseline characteristics with or without HPD were analyzed. RESULTS: A total of 85 patients were included in the analysis. When HPD was defined as a twofold of TGR or TGKR, 8 patients (8/85, 9.4%) had HPD and 11 had PD without HPD. A total of 5 patients (5/85, 5.9%) were diagnosed with HPD and 14 with PD without HPD when HPD was defined as a fourfold of TGR or TGKR. No significant difference was observed in the baseline characteristics between HPD and non-HPD. CONCLUSION: The prevalence of HPD in patients with advanced HCC treated with Atez/Bev was lower than those treated with nivolumab monotherapy. The HPD mechanism in ICI combined with antibodies targeting vascular endothelial growth factor (VEGF) remains to be elucidated.

Alteration of the tumor microenvironment by pharmacological inhibition of EZH2 in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive component 2 is overexpressed in a variety of cancers and recognized as a therapeutic target molecule. However, EZH2 possesses immunomodulatory functions in the tumor microenvironment (TME). The impact of EZH2 on TME of hepatocellular carcinoma (HCC) using immunocompetent mouse model was evaluated in the present study. UNC1999, an EZH2 inhibitor, impaired growth of the murine HCC cells (H22 cells) and induced apoptosis in a dose-dependent manner. Although UNC1999 significantly inhibited the growth of H22 cells-derived and Hepa1-6 cells-derived tumors in nonobese diabetic/severe combined immunodeficiency mice, its antitumor effect was diminished in allogenic BALB/c and C57BL/6 mice. Flow cytometric analyses of TME cells in BALB/c mice demonstrated a significant decrease in the number of interferon­Î³+ CD8+ T cells and regulatory T cells and a significant increase in the number of myeloid-derived suppressor cells (MDSCs). Administration of Gr-1 neutralizing antibody concomitant with UNC1999 restored antitumor effect accompanied by an increase in the number of CD8+ T cells followed by a decrease in the number of MDSCs. Chemokine antibody array demonstrated an enhanced expression of chemokines responsible for MDSCs recruitment such as C5a, CCL8, and CCL9. In conclusion, the study results demonstrated that EZH2 inhibitor contributed to attenuation of tumor immunity caused by TME arrangement. Combination therapy with EZH2 inhibitors and agents that reduce MDSCs might represent a novel therapeutic strategy for HCC.

Use of ramucirumab for various treatment lines in real-world practice of patients with advanced hepatocellular carcinoma.

PURPOSE: Ramucirumab was shown to be effective as a second-line treatment after sorafenib in patients with advanced hepatocellular carcinoma (HCC) with alpha-fetoprotein levels > 400 ng/mL in a worldwide phase 3 trial. Ramucirumab is used in patients pretreated with various systemic therapies in clinical practice. We retrospectively examined the treatment outcomes of ramucirumab administered to advanced HCC patients after diverse systemic therapies. METHODS: Data were collected from patients with advanced HCC who received ramucirumab at three institutions in Japan. Radiological assessments were determined according to both Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 and modified RECIST and the Common Terminology Criteria for Adverse Events version 5.0 was used to assess adverse events. RESULTS: A total of 37 patients treated with ramucirumab between June 2019 and March 2021 were included in the study. Ramucirumab was administered as second, third, fourth, and fifth-line treatment in 13 (35.1%), 14 (37.8%), eight (21.6%), and two (5.4%) patients, respectively. Most patients (29.7%) who received ramucirumab as a second-line therapy were pretreated with lenvatinib. We found grade 3 or higher adverse events only in seven patients and no significant changes in the albumin-bilirubin score during ramucirumab treatment in the present cohort. The median progression-free survival of patients treated with ramucirumab was 2.7 months (95% confidence interval, 1.6-7.3). CONCLUSION: Although ramucirumab is used for various lines of treatment other than second-line immediately after sorafenib, its safety and effectiveness were not significantly different from the findings of the REACH-2 trial.

Successful Identification of a Novel Therapeutic Compound for Hepatocellular Carcinoma Through Screening of ADAM9 Inhibitors.

BACKGROUND/AIM: MHC-class I-related chain A (MICA) functions as a ligand for natural killer group D, an activating receptor on natural killer (NK) cells, and its expression correlates with the carcinogenesis and progression of hepatocellular carcinoma (HCC). Although membranous MICA (mMICA) activates NK cells, soluble forms of MICA (sMICA), shed by cleaving enzymes, such as A disintegrin and metalloprotease (ADAM) 9, suppress NK cells. Therefore, the prevention of MICA shedding through the inhibition of ADAM9 has the potential to activate cancer immunity. Although we have discovered several ADAM inhibitors, many did not sufficiently activate NK cells without being cytotoxic, and, thus, new ADAM9 inhibitor candidates are needed. MATERIALS AND METHODS: To identify possible compounds for drug development, chemical library screening (a total of 741 compounds) was conducted using a fluorescence assay. Compounds with reduced fluorescence intensity were used as hit compounds in a subsequent analysis. Their impact on sMICA and mMICA in HCC cell lines was assessed using ELISA and flow cytometry, respectively. The cytotoxicity of NK cells was also evaluated by co-culturing NK cells with HCC cells. RESULTS: CCL347, a symmetrical compound with five benzene rings, was identified as a hit compound. CCL347 significantly reduced sMICA levels in the culture medium supernatant with negligible cytotoxicity. Although mMICA was also reduced, CCL347 successfully enhanced NK cell cytotoxicity in co-cultures of NK cells and HCC cells. CONCLUSION: CCL347 has potential as a novel therapeutic drug for HCC.

Clinical effects and emerging issues of atezolizumab plus bevacizumab in patients with advanced hepatocellular carcinoma from Japanese real-world practice.

BACKGROUND: Although the efficacy of atezolizumab has been demonstrated in randomized controlled trials, its long-term efficacy and association with adverse events in real-world practice are unknown. This study was designed to shed light on these issues. METHODS: In this multicenter retrospective study, data were collected from patients with advanced hepatocellular carcinoma treated with atezolizumab plus bevacizumab in seven institutions in Japan. The authors focused on the efficacy and adverse events related to vascular endothelial growth factor (VEGF) inhibition. RESULTS: A total of 123 patients were enrolled in this study. The median progression-free survival (PFS) for the first-line treatment group was 8.0 months (95% confidence interval [CI], 6.1-9.9), whereas the median PFS for the second- or later-line treatment group was 4.1 months (95% CI, 2.6-5.7), which was significantly worse than that of the first-line treatment group (p = .005). Twenty-seven patients had interrupted bevacizumab treatment. Proteinuria accounted for the largest proportion of bevacizumab treatment interruptions. The cumulative incidence rate of bevacizumab interruption due to anti-VEGF-related adverse events was significantly higher in patients with hypertension and/or diabetes mellitus than in those without (p = .026). The landmark analysis showed that patients experienced bevacizumab interruption by 24 weeks from treatment initiation had poorer PFS than those who did not (p = .013). CONCLUSIONS: The PFS of atezolizumab plus bevacizumab as first-line treatment mostly replicates that of a global phase 3 trial. Interrupted bevacizumab treatment was more common in patients with hypertension and/or diabetes mellitus, which may be associated with worsening long-term PFS. PLAIN LANGUAGE SUMMARY: Atezolizumab plus bevacizumab has been the standard front line systemic therapy for advanced hepatocellular carcinoma. With the growing incidence of fatty liver due to metabolic syndrome as a background liver disease for hepatocellular carcinoma, the rate of comorbid hypertension and diabetes mellitus has been increasing accordingly. The present study demonstrated the cumulative incidence rate of bevacizumab interruption due to anti-VEGF-related adverse events was significantly higher in patients with hypertension and/or diabetes mellitus. The landmark analysis clarified that interruption of bevacizumab might be a risk of impaired efficacy of atezolizumab plus bevacizumab over the long term in patients with advanced hepatocellular carcinoma.

Miglustat, a glucosylceramide synthase inhibitor, mitigates liver fibrosis through TGF-ß/Smad pathway suppression in hepatic stellate cells.

Transforming growth factor (TGF)-ß/Smad pathway is implicated in the pathogenesis of liver fibrosis, a condition characterized by excessive deposition of extracellular matrix (ECM) proteins such as collagen in response to chronic inflammation. It has been reported that ceramide regulates collagen production through TGF-ß/Smad pathway activation. In this study, we examined whether miglustat, an inhibitor of glucosylceramide synthase, can suppress liver fibrosis by reducing TGF-ß/Smad pathway activity. Human hepatic stellate cells (HHSteCs) were cultured with TGF-ß and multiple miglustat concentrations to examine dose-dependent effects on the expression levels of ECM-related genes and Smad proteins. To evaluate the efficacy of miglustat for fibrosis mitigation, C57BL/6 mice were treated with carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis, followed by combined CCl4 plus miglustat for a further 2 weeks. To examine if miglustat can also prevent fibrosis, mice were treated with CCl4 for 2 weeks, followed by CCl4 plus miglustat for 2 weeks. Miglustat dose-dependently downregulated expression of α-smooth muscle actin and ECM components in TGF-ß-treated HHSteCs. Both phosphorylation and nuclear translocation of Smad2 and Smad3 were also suppressed by miglustat treatment. Sirius-Red staining and hydroxyproline assays of model mouse liver samples revealed that miglustat reduced fibrosis, an effect accompanied by decreased expression of ECM. Our findings suggest that miglustat can both prevent and reverse liver fibrosis by inhibiting TGF-ß/Smad pathway.

The efficacy of contrast-enhanced computed tomography on the management of gastroesophageal varices in patients with hepatocellular carcinoma.

The screening of gastroesophageal varices (GEV) is critical in hepatocellular carcinoma (HCC) management. Contrast-enhanced computed tomography (CECT) is often performed in patients with HCC. Therefore, this study aimed to examine the use of CECT in screening for GEV and predicting GEV bleeding. This retrospective study enrolled 312 consecutive patients who are initially diagnosed with HCC, measured the lower esophageal (EIV) and fundal intramural vessel (FIV) diameter on CECT, examined the changes after 1, 2, and 3 years, and verified the relationship with GEV bleeding. The EIV and FIV diameter on CECT correlates well with endoscopic variceal classification. EIV significantly worsened after 2 and 3 years. FIV showed worsening at both 1, 2, and 3 years. Cumulative GEV bleeding rates were 3.7% at 1 year and 6.2% at 3 years. The multivariate analysis revealed that EIV, FIV, and portal vein tumor thrombus were associated with GEV bleeding. Furthermore, EIV deterioration at 1, 2, and 3 years correlated with GEV bleeding. In conclusion, CECT is useful in variceal management during the longitudinal clinical course of HCC, and has the potential to decrease screening endoscopy. With deterioration in EIV, treatments should be considered due to a high-risk GEV bleeding.

Antiviral Compounds Screening Targeting HBx Protein of the Hepatitis B Virus.

A functional cure of hepatitis B virus (HBV) infection or HB antigen loss is rarely achieved by nucleos(t)ide analogs which target viral polymerase. HBx protein is a regulatory protein associated with HBV replication. We thought to identify antiviral compounds targeting HBx protein by analyzing HBx binding activity. Recombinant GST-tagged HBx protein was applied on an FDA-approved drug library chip including 1018 compounds to determine binding affinity by surface plasmon resonance imaging (SPRi) using a PlexArray HT system. GST protein alone was used for control experiments. Candidate compounds were tested for anti-HBV activity as well as cell viability using HepG2.2.15.7 cells and HBV-infected human hepatocytes. Of the 1018 compounds screened, 24 compounds showed binding to HBx protein. Of the top 6 compounds with high affinity to HBx protein, tranilast was found to inhibit HBV replication without affecting cell viability using HepG2.2.15.7 cells. Tranilast also inhibited HBV infection using cultured human hepatocytes. Tranilast reduced HB antigen level dose-dependently. Overall, theSPRi screening assay identified novel drug candidates targeting HBx protein. Tranilast and its related compounds warrant further investigation for the treatment of HBV infection.

Carbon-ion radiotherapy versus radiofrequency ablation as initial treatment for early-stage hepatocellular carcinoma.

AIM: Carbon-ion radiotherapy (C-ion RT) has shown potential as a curative treatment for patients with hepatocellular carcinoma (HCC). However, no reports have compared the effectiveness of C-ion RT and radiofrequency ablation (RFA). This study aimed to compare clinical outcomes between C-ion RT and RFA for patients with early-stage HCC. METHODS: Medical records of consecutive patients with HCC (single lesion ≤5 cm or two to three lesions ≤3 cm) who received either C-ion RT or RFA as initial treatment were retrospectively reviewed. Propensity score matching (PSM) was used to adjust for clinical factors between both groups. RESULTS: A total of 560 patients were included, among whom 69 and 491 received C-ion RT and RFA, respectively. After PSM (C-ion RT, 54 patients; RFA, 95 patients), both groups were well balanced. Carbon-ion radiotherapy had significantly lower cumulative intrasubsegmental recurrence rate after PSM compared to RFA (p = 0.004) (2-year, 12.6% vs. 31.7%; 5-year, 15.5% vs. 49.6%, respectively). However, no significant difference in cumulative local recurrence rate, stage progression-free survival, or overall survival (OS) was observed between both groups. In the RFA group, 6 of 491 patients (1.2%) showed grade 3 adverse events, whereas no grade 3 or higher adverse events were observed in the C-ion RT group. CONCLUSION: Carbon-ion radiotherapy provided a lower cumulative intrasubsegmental recurrence rate, but a comparable cumulative local recurrence rate, stage progression-free survival, and OS compared to RFA. Thus, C-ion RT appears to be one of the effective treatment options for early-stage HCC when RFA is deemed not indicated.

The RNA-Binding Protein ELAVL1 Regulates Hepatitis B Virus Replication and Growth of Hepatocellular Carcinoma Cells.

Previous RNA immunoprecipitation followed by proteomic approaches successfully demonstrated that Embryonic Lethal, Abnormal Vision, Drosophila-Like 1 (ELAVL1) interacts with hepatitis B virus (HBV)-derived RNAs. Although ELAVL family proteins stabilize AU-rich element (ARE)-containing mRNAs, their role in HBV transcription remains unclear. This study conducted loss-of-function assays of ELAVL1 for inducible HBV-replicating HepAD38 cells and HBx-overexpressed HepG2 cells. In addition, clinicopathological analyses in primary hepatocellular carcinoma (HCC) surgical samples were also conducted. Lentivirus-mediated short hairpin RNA knockdown of ELAVL1 resulted in a decrease in both viral RNA transcription and production of viral proteins, including HBs and HBx, probably due to RNA stabilization by ELAVL1. Cell growth of HepAD38 cells was more significantly impaired in ELAVL1-knockdown than those in the control group, with or without HBV replication, indicating that ELAVL1 is involved in proliferation by factors other than HBV-derived RNAs. Immunohistochemical analyses of 77 paired HCC surgical specimens demonstrated that diffuse ELAVL1 expression was detected more frequently in HCC tissues (61.0%) than in non-tumor tissues (27.3%). In addition, the abundant expression of ELAVL1 tended to affect postoperative recurrence in HBV-related HCC patients. In conclusion, ELAVL1 contributes not only to HBV replication but also to HCC cell growth. It may be a potent therapeutic target for HBV-related HCC treatment.

A diet-induced murine model for non-alcoholic fatty liver disease with obesity and insulin resistance that rapidly develops steatohepatitis and fibrosis.

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease worldwide. Patients with NAFLD often suffer steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The presence of visceral obesity or type 2 diabetes mellitus (T2DM) is a major risk factor and potential therapeutic target for NAFLD. The establishment of animal models with these metabolic comorbidities and with the rapid progression of the disease is needed for developing treatments for NAFLD but remains to be archived. In the present study, KK-Ay mice, widely used as T2DM models, or C57BL6 mice were fed a high-fat, high-fructose, and high-cholesterol diet supplemented with cholic acid (NAFLD diet). The KK-Ay mice fed a NAFLD diet exhibited remarkable obesity and insulin resistance. A prominent accumulation of triglycerides and cholesterol in the liver was observed at 4 weeks. These mice developed steatohepatitis at 4 weeks and fibrosis at 12 weeks. In contrast, C57BL6 mice fed a NAFLD diet remained lean, although they still developed steatohepatitis and fibrosis. In summary, we established a diet-induced murine NAFLD model with the rapid development of steatohepatitis and fibrosis, bearing obesity and insulin resistance. This model could be useful as preclinical models for drug development of NAFLD.

Effect of Atezolizumab plus Bevacizumab in Patients with Hepatocellular Carcinoma Harboring CTNNB1 Mutation in Early Clinical Experience.

Atezolizumab plus bevacizumab (ATZ/BV) treatment is a combined immunotherapy consisting of immune checkpoint inhibitor (ICI) and anti-vascular endothelial growth factor monoclonal antibody, which has brought a major paradigm shift in the treatment of unresectable hepatocellular carcinoma (HCC). Gain-of-function mutation of CTNNB1 contributes to resistance of ICI monotherapy through the framework of non-T-cell-inflamed tumor microenvironment. However, whether CTNNB1 mutation renders resistance to ATZ/BV similar to ICI monotherapy remains to be elucidated. In this study, a liquid biopsy sample in plasma of 33 patients with HCC treated with ATZ/BV was subjected to droplet digital PCR for detecting hotspot mutations at the exon 3 of CTNNB1 locus. A total of eight patients (24.2%) exhibited at least one CTNNB1 mutation. The objective response rate (ORR) in patients with wild-type (WT) and mutant (MT) CTNNB1 was 8.0% and 12.5%, respectively, and the disease control rate (DCR) was 68.0% and 87.5%, respectively. No significant difference in both ORR and DCR has been observed between the two groups. The median progression-free survival in patients with WT and MT CTNNB1 was 6.6 and 7.6 months, respectively (not statistically significant). Similarly, no significant difference in overall survival has been observed between patients with WT and MT CTNNB1 (13.6 vs. 12.3 months). In conclusion, the treatment effect of ATZ/BV in patients with HCC with MT CTNNB1 was comparable to those patients with WT CTNNB1. These results implicate that BV added to ATZ might improve immunosuppressive tumor microenvironment caused by CTNNB1 mutation.

Liver biopsy technique in the era of genomic cancer therapies: a single-center retrospective analysis.

BACKGROUND: With the evolution of personalized medicine in the field of oncology, which includes optimal treatment selection using next-generation sequencing-based companion diagnostic systems and tumor-agnostic treatments according to common biomarkers, a liver tumor biopsy technique that can obtain a sufficient specimen volume must be established. The current study aimed to evaluate the safety and availability of a liver tumor biopsy technique with multiple puncture sites made using a coaxial introducer needle and embolization with gelatin sponge particles. METHODS: Patients with primary or metastatic liver cancer who underwent liver tumor biopsies with puncture tract embolization using gelatin sponge (Spongel®) from October 2019 to September 2020 were included in the study. The complication and diagnostic rates were evaluated, and whether the specimen volume was sufficient for Foundation® CDx was investigated. RESULTS: In total, 96 patients were enrolled in this analysis. The median total number of puncture times per patient was 3 (range 1-8). The pathological diagnostic rate was 79.2%. Using the FoundationOne® CDx, specimens with a sufficient volume required for genomic medicine were collected in 84.9% of patients. The incidence rate of bleeding was 4.2% (n = 4), and only one patient presented with major bleeding requiring transfusion. CONCLUSIONS: Liver biopsy with puncture tract embolization using a gelatin sponge may be safe and effective for collecting specimens with a volume sufficient for modern cancer treatments.

Durvalumab with or without tremelimumab combined with particle therapy for advanced hepatocellular carcinoma with macrovascular invasion: protocol for the DEPARTURE phase Ib trial.

INTRODUCTION: Advanced hepatocellular carcinoma (HCC) with macrovascular invasion (MVI) has the worst prognosis among all phenotypes. This trial aims to evaluate whether treatment with durvalumab, alone or in combination with tremelimumab, plus particle therapy is a safe and synergistically effective treatment in patients with advanced HCC and MVI. METHODS AND ANALYSIS: This phase Ib, multicentre (two sites in Japan), open-label, single-arm, investigator-initiated clinical trial will assess durvalumab monotherapy in combination with particle therapy (cohort A) and that of durvalumab plus tremelimumab in combination with particle therapy (cohort B) for patients with advanced HCC with MVI. Cohort A will receive 1500 mg durvalumab every 4 weeks. Cohort B will receive 1500 mg durvalumab every 4 weeks in principle and 300 mg tremelimumab only on day 1 of the first cycle. Carbon-ion radiotherapy will be administered after day 8 of the first cycle. The primary endpoints are rates of any and severe adverse events, including dose-limiting toxicities (DLTs); secondary endpoints are overall survival, 6-month survival, objective response, 6-month progression-free survival and time to progression. Patients are initially enrolled into cohort A. If cohort A treatment is confirmed to be tolerated (ie, no DLT in three patients or one DLT in six patients), the trial proceeds to enrol more patients into cohort B. Similarly, if cohort B treatment is confirmed to be tolerated (ie, no DLT in three patients or one DLT in six patients), a total of 15 patients will be enrolled into cohort B. ETHICS AND DISSEMINATION: This study was approved by the ethics committees of the two participating institutions (Chiba University Hospital and National Institutes for Quantum (approval number: 2020040) and Radiological Science and Technology, QST Hospital (approval number: C20-001)). Participants will be required to provide written informed consent. Trial results will be reported in a peer-reviewed journal publication. TRIAL REGISTRATION NUMBER: jRCT2031210046.