Akihiro Nasu

Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing.

Background and Aims The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. Methods Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy. Results Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents. Conclusion Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.

Comparison of percutaneous radiofrequency thermal ablation and surgical resection for small hepatocellular carcinoma

BackgroundThe purpose of this investigation was to compare the outcome of percutaneous radiofrequency thermal ablation therapy (PRFA) with surgical resection (SR) in the treatment of single and small hepatocellular carcinoma (HCC).MethodsWe conducted a retrospective cohort study on 231 treatment naive patients with a single HCC ≤ 3 cm who had received either curative PRFA (162 patients) or curative SR (69 patients). All patients were regularly followed up after treatment at our department with blood and radiologic tests.ResultsThe 1-, 3- and 5-year overall survival rates after PRFA and SR were 95.4%, 79.6% and 63.1%, respectively in the PRFA group and 100%, 81.4% and 74.6%, respectively in the SR group. The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 82.0%, 38.3% and 18.0%, respectively in the PRFA group and 86.0%, 47.2% and 26.0%, respectively in the SR group. In terms of overall survival and recurrence free survival, there were no significant differences between these two groups. In comparison of PRFA group patients with liver cirrhosis (LC) (n = 127) and SR group patients with LC (n = 50) and in comparison of PRFA group patients without LC (n = 35) and SR group patients without LC (n = 19), there were also no significant differences between two groups in terms of overall survival and recurrence free survival. In the multivariate analysis of the risk factors contributing to overall survival, serum albumin level was the sole significant factor. In the multivariate analysis of the risk factors contributing to recurrence free survival, presence of LC was the sole significant factor. The rate of serious adverse events in the SR group was significantly higher than that in the PRFA group (P = 0.023). Hospitalization length in the SR group was significantly longer than in the PRFA group (P = 0.013).ConclusionsPRFA is as effective as SR in the treatment of single and small HCC, and is less invasive than SR. Therefore, PRFA could be a first choice for the treatment of single and small HCC.

Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

Background and Aims Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. Methods To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. Results Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. Conclusion Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

The effect of long-term supplementation with branched-chain amino acid granules in patients with hepatitis C virus-related hepatocellular carcinoma after radiofrequency thermal ablation.

Goals: To elucidate whether long-term supplementation with branched-chain amino acid (BCAA) granules improves overall survival (OS) and recurrence-free survival (RFS) after radiofrequency thermal ablation (RFA) in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC)⩽3 cm in diameter with up to 3 nodules and a serum albumin level before RFA of ⩽3.5 g/dL. Background: Whether BCAA treatment after curative RFA for patients with HCV-related HCC improves OS and RFS remains unclear. Study: We compared the OS rate and the RFS rate between the BCAA group (n=115) and the control group (n=141). We also examined factors contributing to OS and RFS. Results: The 1 and 3 years OS rates after RFA were 94.0% and 70.0%, respectively, in the BCAA group, and 94.0% and 49.8%, respectively, in the control group (P=0.001). The corresponding RFS rates 1 and 3 years after RFA were 61.8% and 28.0%, respectively, in the BCAA group, and 52.0% and 12.0%, respectively, in the control group (P=0.013). In the multivariate analysis, in terms of OS, BCAA treatment, and serum albumin level of ≥3.4 g/dL, and in terms of RFS, age 70 years or older, BCAA treatment, and a serum albumin level of ≥3.4 g/dL were significant independent factors, respectively. Conclusions: BCAA treatment may improve OS and RFS after RFA in patients with HCV-related HCC⩽3 cm in diameter with up to 3 nodules and a serum albumin level before RFA of 3.5 g/dL.

Transcatheter arterial infusion chemotherapy prior to radiofrequency thermal ablation for single hepatocellular carcinoma reduces the risk of intrahepatic distant recurrence

The aim of the present study was to elucidate the effectiveness of transcatheter arterial infusion chemotherapy (TAI) of the whole liver using an epirubicin-mitomycin-lipiodol emulsion, prior to radiofrequency thermal ablation (RFA), in preventing intrahepatic distant recurrence (IDR) from single hepatocellular carcinoma (HCC). Of the 269 consecutive patients who underwent RFA in our institute for single HCC, a total of 182 patients were analyzed in the present study. The primary endpoint was comparison of the post-RFA IDR-free survival rates in patients treated using TAI with an epirubicin-mitomycin-lipiodol emulsion via the proper hepatic artery (TAI-EML) prior to RFA, and patients that received lipiodol infusion-alone prior to RFA. The secondary endpoints were local tumor progression (LTP) and overall survival (OS). Lipiodol infusion-alone prior to RFA was performed in 88 patients and TAI-EML prior to RFA in 94 patients. The mean tumor size was 2.06 cm (range, 0.9-3.2 cm) in the TAI group and 1.97 cm (range, 0.9-3.3 cm) in the lipiodol-alone group, respectively. The cumulative IDR-free survival rates at 1, 2 and 3 years were 74.0, 50.8 and 34.9%, respectively, in the lipiodol-alone group, and 90.8, 74.8 and 70.0%, respectively, in the TAI group (P<0.001). In terms of the OS, there was a significant difference between these two groups (P=0.048), although there was no significant difference in terms of the LTP (P=0.145). We concluded that TAI-EML prior to RFA appears to be useful in reducing post-RFA IDR and may contribute to improved survival rates.

Association of genetic polymorphisms with interferon-induced haematologic adverse effects in chronic hepatitis C patients

Summary.  Interferon (IFN)‐based combination therapy with ribavirin has become the gold standard for the treatment of chronic hepatitis C virus infection. Haematologic toxicities, such as neutropenia, thrombocytopenia, and anaemia, however, frequently cause poor treatment tolerance, resulting in poor therapeutic efficacy. The aim of this study was to identify host genetic polymorphisms associated with the efficacy or haematologic toxicity of IFN‐based combination therapy in chronic hepatitis C patients. We performed comprehensive single nucleotide polymorphism detection in all exonic regions of the 12 genes involved in the IFN signalling pathway in 32 healthy Japanese volunteers. Of 167 identified polymorphisms, 35 were genotyped and tested for an association with the efficacy or toxicity of IFN plus ribavirin therapy in 240 chronic hepatitis C patients. Multiple logistic regression analysis revealed that low viral load, viral genotypes 2 and 3, and a lower degree of liver fibrosis, but none of the genetic polymorphisms, were significantly associated with a sustained virologic response. In contrast to efficacy, multiple linear regression analyses demonstrated that two polymorphisms (IFNAR1 10848‐A/G and STAT2 4757‐G/T) were significantly associated with IFN‐induced neutropenia (P = 0.013 and P = 0.011, respectively). Thrombocytopenia was associated with the IRF7 789‐G/A (P = 0.031). In conclusion, genetic polymorphisms in IFN signalling pathway‐related genes were associated with IFN‐induced neutropenia and thrombocytopenia in chronic hepatitis C patients. In contrast to toxicity, the efficacy of IFN‐based therapy was largely dependent on viral factors and degree of liver fibrosis.

Recent Progress in Radiofrequency Ablation Therapy for Hepatocellular Carcinoma

In order to attain better ablation and more effective management of hepatocellular carcinoma (HCC), new approaches and devices in radiofrequency ablation (RFA) therapy were presented and discussed in a workshop at the 50th Annual Meeting of the Liver Cancer Study Group of Japan. A novel bipolar RFA apparatus was introduced in Japan in January 2013. Hundreds of subjects with HCC were treated with multipolar RFA with varied devices and plans. Among these, no-touch ablation was one of the most useful procedures in the treatment of HCC with the apparatus. In RFA therapy, a few assisting devices and techniques were applied for convenience and improvement of the thermal ablation procedure. Contrast-enhanced ultrasonography and three-dimensional fusion imaging technique using volume data of CT or MRI could improve exact targeting and shorten the treatment time for RFA procedures under ultrasonographic guidance. A more complicated method using a workstation was also reported as being helpful in planning the ablated shape and volume in multineedle RFA. The effective use of sedatives and antianalgesics as well as a novel microwave apparatus with a cooled-tip electrode was also discussed.

Effect of treatment with branched‐chain amino acids during sorafenib therapy for unresectable hepatocellular carcinoma

To examine the effect of branched‐chain amino acid (BCAA) therapy for patients with unresectable hepatocellular carcinoma (HCC) treated with sorafenib.

Dynamics of Defective Hepatitis C Virus Clones in Reinfected Liver Grafts in Liver Transplant Recipients: Ultradeep Sequencing Analysis

ABSTRACT Hepatitis C virus (HCV) reinfects liver allografts in transplant recipients by replicating immediately after transplantation, causing a rapid increase in blood serum HCV RNA levels. We evaluated dynamic changes in the viral genetic complexity after HCV reinfection of the graft liver; we also identified the characteristics of replicating HCV clones using a massively parallel ultradeep sequencing technique to determine the full-genome HCV sequences in the liver and serum specimens of five transplant recipients with genotype 1b HCV infection before and after liver transplantation. The recipients showed extremely high genetic heterogeneity before transplantation, and the HCV population makeup was not significantly different between the liver and blood serum specimens of the individuals. Viral quasispecies complexity in serum was significantly lower after liver transplantation than before it, suggesting that certain HCV clones selectively proliferated after transplantation. Defective HCV clones lacking the structural region of the HCV genome did not increase in number, and full-genome HCV clones selectively increased in number immediately after liver transplantation. A re-increase in the same defective clone existing before transplantation was detected 22 months after transplantation in one patient. Ultradeep sequencing technology revealed that the genetic heterogeneity of HCV was reduced after liver transplantation. Dynamic changes in defective HCV clones after liver transplantation indicate that these clones have important roles in the HCV life cycle.

Comparison of FIB-4 index and aspartate aminotransferase to platelet ratio index on carcinogenesis in chronic hepatitis B treated with entecavir

Aims: We sought to compare the effects of FIB-4 index and aspartate aminotransferase to platelet ratio index (APRI) on hepatocellular carcinoma (HCC) incidence in chronic hepatitis B (CHB) patients undergoing entecavir (ETV) therapy. Patient and methods: A total of 338 nucleosides analogue therapy naïve CHB patients initially treated with ETV were analyzed. The optimal cutoff points in each continuous variable were determined by receiver operating curve (ROC) analysis. The effects of FIB-4 index and APRI on HCC incidence were compared using time-dependent ROC analysis and factors linked to HCC incidence were also examined using univariate and multivariate analyses. Results: There were 215 males and 123 females with the median age of 52 years and the median baseline HBV-DNA level of 6.6 log copies/ml. The median follow-up interval after the initiation of ETV therapy was 4.99 years. During the follow-up period, 33 patients (9.8%) developed HCC. The 3-, 5- 7-year cumulative HCC incidence rates in all cases were 4.4%, 9.2% and 13.5%, respectively. In the multivariate analysis, FIB-4 index revealed to be an independent predictor associated with HCC incidence, while APRI was not. In the time-dependent ROC analyses for all cases and for all subgroups analyses stratified by viral status or cirrhosis status, all area under the ROCs in each time point (2-, 3-, 4-, 5-, 6-, and 7-year) of FIB-4 index were higher than those of APRI. Conclusion: FIB-4 index rather than APRI can be a useful predictor associated with HCC development for CHB patients undergoing ETV therapy.