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BackgroundHepatitis B virus (HBV) quasispecies are crucial in the pathogenesis of chronic liver disease. Next-generation sequencing (NGS) is powerful for identifying viral quasispecies. To improve mapping quality and single nucleotide variant (SNV) calling accuracy in the NGS analysis of HBV, we compared different mapping references, including the sample-specific reference sequence, same genotype sequences and different genotype sequences, according to the sample.MethodsReal Illumina HBV datasets from 86 patients, and simulated datasets from 158 HBV strains in the GenBank database, were used to assess mapping quality. SNV calling accuracy was evaluated using different mapping references to align Real Illumina datasets from a single HBV clone.ResultsUsing the sample-specific reference sequence as a mapping reference produced the largest number of mappable reads and coverages. With a different genotype mapping reference, the consensus sequence derived from the Real Illumina datasets of the single HBV clone showed 21 false SNV callings in polymerase and surface genes, the regions most divergent between the mapping reference and this HBV clone. A ~6 % coverage of most of these false SNVs was yielded even with a same genotype mapping reference, but none with the sample-specific reference sequence.ConclusionsUsing sample-specific reference sequences as a mapping reference in NGS analysis optimized mapping quality and the SNV calling accuracy for HBV quasispecies.Electronic supplementary materialThe online version of this article (doi:10.1007/s12072-015-9645-x) contains supplementary material, which is available to authorized users.

Programmed cell death protein-1 (PD-1) inhibitors have shown promising results for treating advanced hepatocellular carcinoma (HCC). However, the clinical utility of such inhibitors in HCC patients with vascular tumor thrombosis remains unclear. This study investigated PD-1 inhibitor efficacy in advanced HCC with macrovascular invasion in a clinical setting. Among the 110 patients with unresectable HCC treated with PD-1 inhibitors, 34 patients with vascular metastases in the portal vein and inferior vena cava were retrospectively compared with 34 patients without tumor thrombi. The vascular response and its effect on survival were assessed. Predictors of survival were identified using multivariate analysis. Among patients achieving objective response, those with and without thrombi exhibited similar response to immunotherapy and comparable survival. Among the 34 patients with tumor thrombi, including 13 receiving PD-1 inhibitors alone and 21 receiving it in combination with tyrosine kinase inhibitors, the median overall survival was 8.9 months (95% confidence interval 3.2–12.6). The objective response rate of vascular metastasis was 52.9%, and vascular responders had a significantly longer survival than did non-responders (11.1 vs 3.9 months). Failure to obtain a vascular response correlated significantly with increased post-treatment Child–Pugh score or class. Multivariate analysis showed that vascular response was a significant positive factor for longer overall survival. Treatment-related grade 3/4 adverse events occurred in 3 (8.8%) of the patients with tumor thrombi. Immunotherapy with PD-1 inhibitors may be a feasible treatment option for HCC with tumor thrombi owing to the high response rate of tumor thrombi and favorable survival outcomes.

Germinal center kinase-like kinase (GLK) is a key controller of autoimmunity. In this study, we assessed the clinical relevance and tumorigenic effects of GLK in hepatocellular carcinoma (HCC). Using immunohistochemistry, we showed that the GLK proportion score increased in both cancerous and adjacent non-cancerous liver tissue from patients with HCC recurrence. A Kaplan-Meier analysis revealed that patients with a wide distribution of GLK in non-cancerous liver tissue had a higher rate of HCC recurrence than those with very low or no GLK expression. Multivariate Cox regression analyses indicated that a high GLK proportion score in non-cancerous liver tissue was an independent predictor of early HCC recurrence after resection. Lentiviral vector-mediated overexpression of GLK activated the nuclear factor kappa B (NFκB) signaling cascade and accelerated cell cycle progression in primary human hepatocytes, thereby promoting proliferation. An increase in GLK expression coincided with NFκB activation and enhanced expression of proliferating cell nuclear antigen in HCC tissue. Our findings demonstrate a potential hepatocarcinogenic effect of GLK and the feasibility of using GLK to predict early HCC recurrence.