Isolation and characterization of cancer-associated fibroblasts in the tumor microenvironment of hepatocellular carcinoma.

BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) play an immunosuppressive role in the tumor microenvironment (TME) of human cancers; however, their characteristics and role in hepatocellular carcinoma (HCC) remain to be elucidated. METHODS: Nine tumor and surrounding liver tissue samples from patients with HCC who underwent surgery were used to isolate patient-derived CAFs. Cell morphology was observed using an optical microscope after culture, and cell phenotypes were evaluated using flow cytometry and immunoblotting. Cytokines secreted by CAFs into culture medium were quantified using a multiplex cytokine assay. RESULTS: CAFs were abundant in the TME of HCC and were adjacent to immune cells. After culture, the CAFs and non-tumor fibroblasts exhibited spindle shapes. We observed a robust expression of alpha-smooth muscle actin and fibroblast activation protein in CAFs, whereas alpha-fetoprotein, epithelial cell adhesion molecule, platelet/endothelial cell adhesion molecule-1, and E-cadherin were not expressed in CAFs. Furthermore, CAFs showed high secretion of various cytokines, namely C-X-C motif chemokine ligand 12, interleukin (IL)-6, IL-8, and C-C motif chemokine ligand 2. CONCLUSIONS: CAFs are abundant in the TME of HCC and play a crucial role in tumor progression. These fibroblasts secrete cytokines that promote tumor growth and metastasis.

A phase 2, open-label, randomized, multiple-dose study evaluating Inarigivir in treatment-naïve patients with chronic hepatitis B.

BACKGROUND/AIMS: Novel agents acting against hepatitis B virus (HBV) are needed to improve HBsAg seroclearance or termed as 'functional cure'. Inarigivir (retinoic acid-inducible gene I agonist) has immunomodulatory and direct antiviral actions against HBV. We aimed to determine the safety and efficacy of Inarigivir for the treatment of HBV infection. PATIENTS/METHODS: 80 treatment-naïve patients were randomized in 4 ascending dose cohorts to receive 12 weeks of Inarigivir 25, 50, 100, 200 mg or placebo in a ratio of 4:1. All patients were then given tenofovir for another 12 weeks. RESULTS: Least squares (LS) mean reductions in HBV DNA from baseline increased with higher doses of Inarigivir (0.6116 in 25 mg and 1.5774 in 200 mg groups vs. 0.0352 in placebo group) (95% CI 0.9518-0.2011 and 1.921-1.1634 respectively). LS mean changes in HBV RNA and HBsAg from baseline ranged from -0.3856 to -0.5794 versus -0.1474 and -0.0956 to -0.1818 versus +0.0026 in Inarigivir-treated versus placebo groups respectively. During the tenofovir-treated period, LS mean reductions in HBsAg in the Inarigivir-treated groups ranged from 0.1709 to 0.3529 versus 0.1984 in the placebo group. Inarigivir-treated groups showed mean reductions in ALT from baseline between 23.3 and 33.8 versus 0.7 U/L in the placebo group. Treatment-emergent adverse events related to Inarigivir and placebo occurred in 4.7% and 6.3% patients respectively. CONCLUSIONS: Twelve-week Inarigivir up to 200 mg dose was associated with a reduction of HBV DNA, HBV RNA and antigen levels. A trend for greater HBsAg reduction was observed in Inarigivir pre-treated patients after switching to tenofovir.

Safety, tolerability and antiviral activity of the antisense oligonucleotide bepirovirsen in patients with chronic hepatitis B: a phase 2 randomized controlled trial.

Chronic infection with hepatitis B virus (HBV) leads to an increased risk of death from cirrhosis and hepatocellular carcinoma. Functional cure rates are low with current treatment options (nucleos(t)ide analogs (NAs) and pegylated interferons). Bepirovirsen is an antisense oligonucleotide targeting all HBV messenger RNAs; in cell culture and animal models, bepirovirsen leads to reductions in HBV-derived RNAs, HBV DNA and viral proteins. This phase 2 double-blinded, randomized, placebo-controlled trial is the first evaluation of the safety and activity of an antisense oligonucleotide targeting HBV RNA in both treatment-naïve and virally suppressed individuals with chronic HBV infection. The primary objective was to assess the safety and tolerability of bepirovirsen in individuals with chronic hepatitis B (CHB) (NCT02981602). The secondary objective was to assess antiviral activity, including the change from baseline to day 29 in serum hepatitis B surface antigen (HBsAg) concentration. Participants with CHB infection ≥6 months and serum HBsAg ≥50 IU ml-1 were enrolled from seven centers across Hong Kong and the Republic of Korea and randomized (3:1 within each dose cohort) to receive bepirovirsen or placebo via subcutaneous injection twice weekly during weeks 1 and 2 (days 1, 4, 8 and 11) and once weekly during weeks 3 and 4 (days 15 and 22). Participants were then followed for 26 weeks. Twenty-four participants were treatment-naïve and seven were receiving stable NA therapy. Treatment-emergent adverse events were mostly mild/moderate (most commonly injection site reactions). Eleven (61.1%) and three (50.0%) treatment-naïve participants experienced one or more treatment-emergent adverse event in the bepirovirsen and placebo groups, respectively. In participants receiving NA therapy, the corresponding numbers were three (60.0%) and one (50.0%). Transient, self-resolving alanine aminotransferase flares (≥2× upper limit of normal) were observed in eight treatment-naïve participants and three participants on stable NA regimens in the bepirovirsen treatment arms. HBsAg reductions were observed and were significant versus placebo for treatment-naïve participants receiving bepirovirsen 300 mg (P = 0.001), but not for the bepirovirsen 150 mg group (P = 0.245) or participants receiving stable NA therapy (P = 0.762). Two participants in each of the 300 mg dose groups achieved HBsAg levels below the lower limit of quantitation by day 29 (n = 3) or day 36 (n = 1). Bepirovirsen had a favorable safety profile. These preliminary observations warrant further investigation of the safety and activity of bepirovirsen in a larger CHB patient population.

Comparative Analysis of Lenvatinib and Hepatic Arterial Infusion Chemotherapy in Unresectable Hepatocellular Carcinoma: A Multi-Center, Propensity Score Study.

The comparative efficacy and safety between lenvatinib and hepatic artery infusion chemotherapy (HAIC) in patients with unresectable hepatocellular carcinoma (HCC) is still unclear. This multicenter historical cohort study enrolled 244 patients who were treated with HAIC (n = 173) or lenvatinib (n = 71) between 2012 and 2020. Propensity score matching (PSM) was performed, and 52 patients were selected per group. Clinical outcomes and safety were compared. Objective response rate (ORR) was not different between the two groups (26.0% vs. 23.1%, p = 0.736). Before PSM, the HAIC group had a higher proportion of Child-Pugh B and portal vein tumor, whereas the lenvatinib group had more patients with extrahepatic metastases, which was adjusted after PSM. There were no differences in progression-free survival (PFS) and overall survival (OS) after PSM (HAIC vs. lenvatinib, median PFS, 3.6 vs. 4.0 months, p = 0.706; median OS 10.8 vs. 7.9 months, p = 0.106). Multivariate Cox-regression showed that alpha-fetoprotein ≤1000 ng/mL was only an associated factor for OS after PSM in all patients (hazard ratio = 0.421, p = 0.011). Subgroup analysis for patients with a high tumor burden beyond the REFLECT eligibility criteria revealed that the HAIC group (n = 29) had a significantly longer OS than did the lenvatinib group (n = 30) (10.0 vs. 5.4 months, p = 0.004). More patients in the HAIC group achieved better liver function than those in the lenvatinib group at the time of best responses. There was no difference in the incidence of grade 3 and 4 adverse events between the two groups. Therefore, lenvatinib is comparable to HAIC in terms of ORR and OS in unresectable HCC meeting REFLECT eligibility criteria.

Distinct Patterns of HBV Integration and TERT Alterations between in Tumor and Non-Tumor Tissue in Patients with Hepatocellular Carcinoma.

Although hepatitis B virus (HBV) integration into the cellular genome is well known in HCC (hepatocellular carcinoma) patients, its biological role still remains uncertain. This study investigated the patterns of HBV integration and correlated them with TERT (telomerase reverse transcriptase) alterations in paired tumor and non-tumor tissues. Compared to those in non-tumors, tumoral integrations occurred less frequently but with higher read counts and were more preferentially observed in genic regions with significant enrichment of integration into promoters. In HBV-related tumors, TERT promoter was identified as the most frequent site (38.5% (10/26)) of HBV integration. TERT promoter mutation was observed only in tumors (24.2% (8/33)), but not in non-tumors. Only 3.00% (34/1133) of HBV integration sites were shared between tumors and non-tumors. Within the HBV genome, HBV breakpoints were distributed preferentially in the 3' end of HBx, with more tumoral integrations detected in the preS/S region. The major genes that were recurrently affected by HBV integration included TERT and MLL4 for tumors and FN1 for non-tumors. Functional enrichment analysis of tumoral genes with integrations showed enrichment of cancer-associated genes. The patterns and functions of HBV integration are distinct between tumors and non-tumors. Tumoral integration is often enriched into both human-virus regions with oncogenic regulatory function. The characteristic genomic features of HBV integration together with TERT alteration may dysregulate the affected gene function, thereby contributing to hepatocarcinogenesis.

Significance of TERT Genetic Alterations and Telomere Length in Hepatocellular Carcinoma.

Telomerase reverse transcriptase (TERT) mutations are reportedly the most frequent somatic genetic alterations in hepatocellular carcinoma (HCC). An integrative analysis of TERT-telomere signaling during hepatocarcinogenesis is lacking. This study aimed to investigate the clinicopathological association and prognostic value of TERT gene alterations and telomere length in HCC patients undergoing hepatectomy as well as transarterial chemotherapy (TACE). TERT promoter mutation, expression, and telomere length were analyzed by Sanger sequencing and real-time PCR in 305 tissue samples. Protein-protein interaction (PPI) analysis was performed to identify a set of genes that physically interact with TERT. The PPI analysis identified eight key TERT-interacting genes, namely CCT5, TUBA1B, mTOR, RPS6KB1, AKT1, WHAZ, YWHAQ, and TERT. Among these, TERT was the most strongly differentially expressed gene. TERT promoter mutations were more frequent, TERT expression was significantly higher, and telomere length was longer in tumors versus non-tumors. TERT promoter mutations were most frequent in HCV-related HCCs and less frequent in HBV-related HCCs. TERT promoter mutations were associated with higher TERT levels and longer telomere length and were an independent predictor of worse overall survival after hepatectomy. TERT expression was positively correlated with tumor differentiation and stage progression, and independently predicted shorter time to progression after TACE. The TERT-telomere network may have a crucial role in the development and progression of HCC. TERT-telomere abnormalities might serve as useful biomarkers for HCC, but the prognostic values may differ with tumor characteristics and treatment.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Hypofractionated radiotheapy using helical tomotherapy for advanced hepatocellular carcinoma with portal vein tumor thrombosis.

BACKGROUND: We want to evaluate the efficacy of helical tomotherapy (HT) for treating advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT). METHODS: We treated 35 patients for unresectable HCC combined with PVTT in whom other treatment modalities were not indicated. The tumor thrombi involved the main trunk of the portal vein in 18 patients (51.4%) and the first or second order branches in 17 patients (48.6%). A median dose of 50 Gy (range: 45-60 Gy) was delivered in 10 fractions. Capecitabine was given concomitantly at a dose of 600 mg/m2 twice daily during radiotherapy. RESULTS: The responses were evaluated via computed tomography. There was a complete response (CR) in 5 patients (14.3%), partial response (PR) in 10 patients (28.6%), stable disease (SD) in 18 patients (51.4%) and progressive disease (PD) in 2 patients (5.7%). The Child-Pugh classification (A vs B) and the Japan integrated staging (JIS) score (2 vs 3) were statistically significant parameters that predicted the response of PVTT (p = 0.010 and p = 0.026, respectively). The median survival, one and two year survival rate of all patients was 12.9 months, 51.4% and 22.2%, respectively. The patients with tumor thrombi in the main portal trunk showed statistically inferior overall survival than patients with tumor thrombi in the portal vein branches (9.8 versus 16.6 months, respectively, p = 0.036). The responders' median survival was 13.9 months, double 6.9 months as the median survival of the non-responders. No radiation induced liver disease or treatment related mortality was not appeared. CONCLUSIONS: Hypofractionated radiotherapy with HT was effective not only for tumor response but also for survival in the advanced HCC patients with PVTT. And stricter patient selection by Child-Pugh classification and JIS score may maximize the potential benefits of this treatment.

Helical tomotherapy with concurrent capecitabine for the treatment of inoperable pancreatic cancer.

BACKGROUND: Helical tomotherapy, an advanced intensity-modulated radiation therapy with integrated CT imaging, permits highly conformal irradiation with sparing of normal tissue. Capecitabine, a pro-drug of 5-FU that induces thymidine phosphorylase can achieve higher levels of intracellular 5-FU when administered concurrently with radiation. We evaluated the feasibility as well as the clinical outcome of concurrent administration of capecitabine with tomotherapy in patients with advanced pancreatic cancer. METHODS: Nineteen patients with advanced pancreatic cancer including primarily unresectable disease and recurrence after curative surgery were included in the study. Two planning target volumes (PTV) were entered: PTV1 is gross tumor volume; and PTV2, the volume of the draining lymph nodes. The total doses to target 1 and target 2 were 55 and 50 Gy, respectively. Capecitabine at 1600 mg/m2/day was administered on each day of irradiation. RESULTS: Twenty six measurable lesions were evaluated. Overall in-field response rate was 42.3%; partial responses were achieved in 53.3% of the pancreatic masses, 28.6% of distant metastatic lesions and 25.0% of regional lymph nodes. The median duration of follow-up after tomotherapy was 6.5 months. None of the lesions showed in-field progression. Treatment was well tolerated with only minor toxicities such as grade 1 nausea (one patient), grade 1 hand-foot syndrome (one patient) and grade 1/2 fatigue (three patients). CONCLUSIONS: Helical tomotherapy with concurrent capecitabine is a feasible option without significant toxicities in patients with advanced pancreatic cancer. We achieved excellent conformal distribution of radiation doses and minimal treatment-related toxicities with promising target volume responses.