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This study investigated hepatitis B virus (HBV) single-nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety-three HCC patients and 108 non-HCC patients were enrolled for HBV genome-wide next-generation sequencing (NGS) analysis. A systematic literature review and a meta-analysis were performed to validate NGS-defined HCC-associated SNVs and deletions. The experimental results identified 60 NGS-defined HCC-associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n = 24) or genotype C (n = 34), except for nt53C, which was present in both genotypes. The pathogenic frequencies of these HCC-associated SNVs showed a distinct U-shaped distribution pattern. According to the meta-analysis and literature review, 167 HBV variants from 109 publications were categorized into four levels (A-D) of supporting evidence that they are associated with HCC. The proportion of NGS-defined HCC-associated SNVs among these HBV variants declined significantly from 75% of 12 HCC-associated variants by meta-analysis (Level A) to 0% of 10 HCC-unassociated variants by meta-analysis (Level D) (P < 0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P = 0.030) and C (P = 0.049). For genotype C, preS deletions involving a specific fragment (nt2977-3013) were significantly associated with HCC (HCC versus non-HCC, 6/34 versus 0/32, P = 0.025). Meta-analysis of preS deletions showed significant association with HCC (summary odds ratio 3.0; 95% confidence interval 2.3-3.9). Transfection of Huh7 cells showed that all of the five novel NGS-defined HCC-associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including endoplasmic reticulum-stress and DNA repair systems, as shown by microarray, real-time polymerase chain reaction and western blot analysis. Their carcinogenic mechanisms are worthy of further research. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Summary Background Deregulation of mammalian target of rapamycin (mTOR) signalling is common in human hepatocellular carcinoma (HCC). Aim To determine the maximum tolerated dose (MTD) of the oral mTOR inhibitor everolimus in advanced HCC patients. Methods Patients with locally advanced or metastatic HCC (Child-Pugh class A or B) were enrolled in an open-label phase 1 study and randomly assigned to daily (2.5–10 mg) or weekly (20–70 mg) everolimus in a standard 3 + 3 dose-escalation design. MTD was based on the rate of dose-limiting toxicities (DLTs). Secondary endpoints included safety, pharmacokinetics and tumour response. In a post hoc analysis, serum hepatitis B virus (HBV) DNA levels were quantified. Results Thirty-nine patients were enrolled. DLTs occurred in five of 21 patients in the daily and two of 19 patients in the weekly cohort. Daily and weekly MTDs were 7.5 mg and 70 mg respectively. Grade 3/4 adverse events with a ≥10% incidence were thrombocytopenia, hypophosphataemia and alanine transaminase (ALT) elevation. In four hepatitis B surface antigen (HBsAg)-seropositive patients, grade 3/4 ALT elevations were accompanied by significant (>1 log) increases in serum HBV levels. The incidence of hepatitis flare (defined as ALT increase >100 IU/mL from baseline) in HBsAg-seropositive patients with and without detectable serum HBV DNA before treatment was 46.2% and 7.1% respectively (P

Strategies to increase antitumor efficacy of oncolytic adenoviruses are actively investigated. We have previously shown that E1B-55 kDa-deleted adenovirus, designated Ad5WS1, has therapeutic potential for treating hepatocellular carcinoma (HCC). To achieve HCC-restricted replication of oncolytic adenovirus, we generated Ad5WS2, an E1B-55 kDa-deleted adenovirus with its E1A gene driven by the liver-specific transthyretin promoter. Our results showed that Ad5WS2 could replicate within tumor cells where the transthyretin gene was expressed. Mouse transthyretin promoter was active in murine and human HCC cells, but relatively quiescent in cells of non-liver origin. Ad5WS2 caused severe cytolytic effect on HCC cells, but was much attenuated in non-HCC cells. Peritoneal administration of Ad5WS2 into mice bearing liver tumors grown in ascites resulted in enhanced survival. In an orthotopic HCC model, Ad5WS2, when systemically administered, exerted higher antitumor effects than Ad5WS1. Lack of viral replication in normal organs and minimal hepatic toxicity was noted after Ad5WS2 treatment. Furthermore, the antitumor effect of Ad5WS2 could be enhanced when combined with chemotherapeutic agent cisplatin in the ascites tumor model. These results suggest that E1B-55 kDa-deleted adenovirus driven by the transthyretin promoter may be a safer and more efficacious oncolytic agent for the treatment of primary and metastatic HCC.

Background: The availability of pigs homozygous for α1,3-galactosyltransferase gene-knockout (GT-KO) has enabled study of the incidence and cytotoxicity of primate antibodies directed to antigens other than Galα1,3Gal (Gal), termed non-Gal antigens. Methods: Sera from 27 healthy humans and 31 patients awaiting renal allotransplantation, who were either unsensitized [panel reactive antibodies (PRA) 70%), were tested by flow cytometry for binding of immunoglobulin M (IgM) and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs. Complement-dependent cytotoxicity to WT and GT-KO PBMC was also measured. Results: IgM and IgG from all 27 (100%) healthy human sera bound to WT PBMC, while 78% and 63% of these sera had IgM and IgG that bound to GT-KO PBMC, respectively. Mean binding to WT PBMC was significantly greater than GT-KO PBMC. Whereas 100% of sera were cytotoxic to WT PBMC, only 61% were cytotoxic to GT-KO PBMC, and the extent of lysis was significantly less. Neither mean binding of IgM and IgG nor cytotoxicity of unsensitized and allosensitized sera to WT and GT-KO PBMC was significantly different to that of healthy sera. Conclusions: More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.