Prediction of Therapeutic Response Using Contrast-Enhanced Ultrasound in Japanese Patients Treated with Atezolizumab and Bevacizumab for Unresectable Hepatocellular Carcinoma.

INTRODUCTION: Atezolizumab and bevacizumab (AB) therapy was the first-line treatment for unresectable hepatocellular carcinoma (u-HCC). However, the predictive marker of therapeutic response that can be easily used in clinical practice is still unknown. We prospectively investigated the utility of time-intensity curve (TIC) analysis using contrast-enhanced ultrasound (CEUS) as a predictive indicator of therapeutic response after the start of AB therapy. METHODS: Thirty-five patients who received AB therapy for u-HCC were included in this study. TIC analysis was performed in 28 patients who were able to undergo CEUS before and 3-7 days after administration. We analyzed prognostic factors related to the initial therapeutic response and long progression-free survival (PFS). RESULTS: The initial therapeutic response using dynamic computed tomography or Gd-EOB magnetic resonance imaging at 8-12 weeks after administration was partial response/stable disease/progressive disease (PD) in 14/12/9 cases (40/34/26%). Cases with PD (n = 9) had more cases without decreased blood flow in TIC analysis compared with cases with non-PD (100 vs. 18%, p = 0.001). Cases without decreased blood flow in TIC analysis (n = 10) had more cases with PD compared with cases with decreased blood flow (60 vs. 0%, p = 0.001). PFS in patients without decreased blood flow early after the administration was shorter than that in those with decreased blood flow (9.1 vs. 28 weeks, p = 0.0051). CONCLUSION: Early evaluation by TIC analysis using CEUS may be useful in predicting the therapeutic response in patients treated with AB therapy for u-HCC.

Relationship between Plasma IP-10/CXCL10 Levels and the Initial Therapeutic Response in Patients Treated with Atezolizumab plus Bevacizumab for Unresectable Hepatocellular Carcinoma.

INTRODUCTION: Atezolizumab plus bevacizumab combination therapy (AB) was the first-line treatment for unresectable hepatocellular carcinoma (u-HCC). IFN-γ-induced protein 10 (IP-10/CXCL10) is a chemokine to inhibit HCC proliferation by promoting the migration of cytotoxic T cells. We focused on the relationship between plasma IP-10/CXCL10 levels and the initial therapeutic response in patients receiving AB therapy. METHODS: Forty-six patients receiving AB therapy were enrolled. Plasma IP-10/CXCL10 levels were measured at baseline, 3-7 days, 3 weeks, 6 weeks, and 8-12 weeks after the start of AB therapy. The initial therapeutic response was evaluated at 8-12 weeks. RESULTS: The baseline IP-10/CXCL10 levels of partial response (PR) group was higher than that of stable disease (SD) or progressive disease (PD) group. Patients with the baseline IP-10/CXCL10 of 84 pg/mL or higher were likely to present PR than patients below (71 vs. 35%, p = 0.031), but prediction of PD using the baseline IP-10/CXCL10 levels was difficult. In contrast, IP-10/CXCL10 ratio of the PR group was lower than that of the SD/PD group at 3, 6, and 8-12 weeks. Patients with the 3, 6, and 8-12 weeks IP-10/CXCL10 ratio of 1.3, 0.4, and 0.4 or lower were likely to present PR than patients with ≥1.3, 0.4, and 0.4 (88, 35, 35 vs. 30, 3.8, 0%, p < 0.001, 0.011, 0.002). In other hand, the 3, 6, and 8-12 weeks IP-10/CXCL10 ratio for PD group was higher than that for non-PD group. Patients with the 3, 6, and 8-12 weeks IP-10/CXCL10 ratio of 1.3, 1.7, and 1.9 or higher were likely to present PD than patients below (85, 62, 57 vs. 32, 23, 14%, p = 0.002, 0.034, 0.009). CONCLUSION: High baseline IP-10/CXCL10 levels may be associated with better outcome, and high IP-10/CXCL10 ratio after 3-12 weeks may be associated with worse outcome in u-HCC patients receiving AB therapy.

Impact of Renal Function on the Prognosis of Patients Receiving Atezolizumab/Bevacizumab Combination Therapy and Lenvatinib Monotherapy for Unresectable Hepatocellular Carcinoma.

INTRODUCTION: Several studies have reported kidney injury caused by immune checkpoint inhibitors, and proteinuria caused by vascular endothelial growth factor inhibitors for unresectable hepatocellular carcinoma (u-HCC). We investigated the relationship between renal function and prognosis in patients with u-HCC receiving atezolizumab and bevacizumab (AB) and lenvatinib (LEN) therapy. METHODS: Fifty-one patients who received AB and 50 patients who received LEN therapy were included. We analyzed prognostic factors related to the overall survival (OS), and characteristics related to renal function. RESULTS: In patients with AB therapy, OS was shorter in patients with baseline proteinuria of 1+ or higher, as assessed by urine dipstick test, compared to those with -/± (p = 0.024). There were many cases with two or more drugs with a high risk of renal dysfunction (p = 0.019) in patients with 1+ or higher. Furthermore, OS was shorter in the group with estimated glomerular filtration rate (eGFR) grade deterioration without urinary protein-creatinine ratio (UPCR) of 2 g/g·Cre or higher than in the other groups (p = 0.027). In the group where eGFR worsened without an increase in UPCR, there were many cases with a daily salt intake of 10 g or more (p = 0.027), three or more drugs with a high risk of renal dysfunction (p = 0.021), and a history of arteriosclerosis (p = 0.021). On the other hand, in patients with LEN therapy, OS tends to be shorter in patients with proteinuria of ± or higher, compared to those without (p = 0.074). There were many cases with a daily salt intake of 10 g or more in patients with ± or higher (p = 0.002). CONCLUSION: In patients receiving AB and LEN therapy, baseline proteinuria was associated with OS. Renal function deterioration without proteinuria was associated with a poor prognosis in AB therapy. Excessive salt intake, preexisting atherosclerotic disease, and drug with a high risk of renal dysfunction were risk factors for renal deterioration.

Significance of serum IP-10/CXCL10 measurement in predicting post-direct acting antiviral treatment liver function in patients with HCV-decompensated liver cirrhosis.

BACKGROUND: Recently, with the advent of sofosbuvir/velpatasvir therapy, sustained virological response (SVR) can now be achieved even in patients with decompensated cirrhosis (dLC). However, the prognosis after SVR does not always improve in dLC, and appropriate indicators enabling prediction of prognosis is desired. PATIENTS AND METHODS: Serum IP-10/CXCL10 levels were measured in 47 patients (15 chronic hepatitis [CH], 17 compensated cirrhosis [cLC], and 15 dLC) receiving direct acting antiviral (DAA) therapy, and their changes during the therapy were examined. RESULTS: All the patients achieved SVR. In patients with CH, the average IP-10 level was 367, 102, and 68 pg/ml respectively at baseline, at the end of therapy and at 12 weeks after SVR (SVR12), and was decreased upon DAA therapy (P < 0.001). In patients with cLC, IP-10 was respectively 215, 91, and 77 pg/ml, and was decreased upon DAA therapy (P < 0.001) while it was 283, 131, and 182 pg/ml in patients with dLC and there was no evident decrease (P = 0.55). When patients with dLC were further classified depending on the difference in Child-Pugh (CP) score improvement at SVR12, a significant decrease in IP-10 was observed after treatment in those with improvement (P = 0.023) while a significant increase was observed in those without improvement (P = 0.016). CONCLUSION: While serum IP-10 level was decreased in patients with CH/cLC and dLC with post-SVR-CP improvement following SVR, it was increased in patients with dLC without post-SVR CP improvement. The result indicates that IP-10 dynamics may be useful for predicting liver function after DAA therapy.

Early diagnosis of hepatic inflammation in Japanese nonalcoholic fatty liver disease patients using 3D MR elastography.

BACKGROUND: The damping ratio (DR) and the loss modulus (G″) obtained by 3D MR elastography complex modulus analysis has been reported recently to reflect early intrahepatic inflammation, and is expected to be a noninvasive biomarker of inflammation in nonalcoholic fatty liver disease (NAFLD). However, the role of the DR and the G″ in Japanese NAFLD patients remains unclear. METHODS: We enrolled 39 Japanese patients with NAFLD who underwent liver biopsy and 3D MR elastography within 1 month and analyzed the association between DR, G″, and histological activity. RESULTS: Regarding DR, no evident correlation was observed between the DR and histological activity (p = 0.14) when patients with all fibrosis stages were included. However, when patients were restricted up to stage F2 fibrosis, the association of the DR and inflammation became significant, the DR increasing with the degree of activity (p = 0.02). Among the constituents of fibrosis activity, ballooning correlated with the DR (p < 0.01) while lobular inflammation did not. Regarding G″, it was correlated with histological activity (p < 0.01), ballooning (p < 0.01), and lobular inflammation (p < 0.01) in patients with all fibrosis stages and in patients up to F2 fibrosis (p = 0.03 for activity and p = 0.04 for ballooning). The best cutoff value of DR for hepatitis activity in patients within the F2 stage was 0.094 (area under the receiver operating characteristic curve 0.775, 95% CI: 0.529-1.000) and G″ was 0.402 (area under the receiver operating characteristic curve 0.825, 95% CI: 0.628-1.000). CONCLUSIONS: The DR and G″ reflected the histological activity in Japanese patients with NAFLD during the early stage, indicating these values for noninvasive diagnosis of inflammation in Japanese patients with NAFLD.

Effect of a combination of pemafibrate and a mild low-carbohydrate diet on obese and non-obese patients with metabolic-associated fatty liver disease.

BACKGROUND AND AIM: Recently, pemafibrate and a low-carbohydrate diet (LCD) have each been reported to improve fatty liver disease. However, it is unclear whether their combination improves fatty liver disease and is equally effective in obese and non-obese patients. METHODS: In 38 metabolic-associated fatty liver disease (MAFLD) patients, classified by baseline body mass index (BMI), changes in laboratory values, magnetic resonance elastography (MRE), and magnetic resonance imaging-proton density fat fraction (MRI-PDFF) were studied after 1 year of combined pemafibrate plus mild LCD. RESULTS: The combination treatment resulted in weight loss (P = 0.002), improvement in hepatobiliary enzymes (γ-glutamyl transferase, P = 0.027; aspartate aminotransferase, P < 0.001; alanine transaminase [ALT], P < 0.001), and improvement in liver fibrosis markers (FIB-4 index, P = 0.032; 7 s domain of type IV collagen, P = 0.002; M2BPGi, P < 0.001). Vibration-controlled transient elastography improved from 8.8 to 6.9 kPa (P < 0.001) and MRE improved from 3.1 to 2.8 kPa (P = 0.017) in the liver stiffness. MRI-PDFF improved from 16.6% to 12.3% in liver steatosis (P = 0.007). In patients with a BMI of 25 or higher, improvements of ALT (r = 0.659, P < 0.001) and MRI-PDFF (r = 0.784, P < 0.001) were significantly correlated with weight loss. However, in patients with a BMI below 25, the improvements of ALT or PDFF were not accompanied by weight loss. CONCLUSIONS: Combined treatment with pemafibrate and a low-carbohydrate diet resulted in weight loss and improvements in ALT, MRE, and MRI-PDFF in MAFLD patients. Although such improvements were associated with weight loss in obese patients, the improvements were observed irrespective of weight loss in non-obese patients, indicating this combination can be effective both in obese and non-obese MAFLD patients.

Deep sequencing analysis of serum hepatitis B virus-RNA during nucleot(s)ide analogue therapy.

AIM: Recently, serum hepatitis B virus (HBV)-RNA has been reported to be detectable even when HBV particle production is inhibited by nucleot(s)ide analogues (NAs). However, the dynamics of the HBV-RNA sequence compared with those of HBV-DNA during the emergence of antiviral resistance are yet to be elucidated. METHODS: First, we quantified serum HBV-RNA in 181 infected patients, and its relationships with clinical characteristics as well as HBV markers were investigated. Next, we undertook simultaneous deep sequencing of HBV-RNA/HBV-DNA and their dynamics among four patients receiving NA therapy who were experiencing viral breakthrough. RESULTS: Serum HBV-RNA was detected in 25% (31/123) of cases among patients with HBV without NAs, and the detection rate was significantly high in hepatitis B e antigen-positive cases with high viral activity. In patients with chronic hepatitis, hepatitis B core-related antigen was significantly correlated with serum HBV-RNA irrespective of NA use. In the analysis of the four patients experiencing viral breakthrough, no NA resistance mutation was detected in the serum HBV-RNA immediately before the breakthrough. However, NA-resistant sequences appeared at the rates of 0%, 3%, 14%, and 100%, and the NA-resistant HBV-RNA sequence rate was correlated with the peak HBV-DNA titer multiplied by the HBV-DNA detection duration during the breakthrough (R2 = 0.978) observed before redisappearance of HBV-DNA following the addition of new NA. CONCLUSION: Serum HBV-RNA could reflect the transcriptional activity of covalently closed circular DNA and hepatitis B core-related antigen. The dynamics of HBV-RNA could help understanding of the turnover process of HBV covalently closed circular DNA in the liver.

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing.

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.

Cancer-related genetic changes in multistep hepatocarcinogenesis and their correlation with imaging and histological findings.

AIM: The landscape of cancer-related genetic aberrations in hepatocellular carcinoma (HCC) has gradually become clear through recent next-generation sequencing studies. However, it remains unclear how genetic aberrations correlate with imaging and histological findings. METHODS: Using 117 formalin-fixed paraffin-embedded specimens of primary liver tumors, we undertook targeted next-generation sequencing of 50 cancer-related genes and digital polymerase chain reaction of hTERT. After classifying tumors into several imaging groups by hierarchal clustering with the information from gadoxetic acid enhanced magnetic resonance imaging, contrast-enhanced computed tomography, contrast-enhanced ultrasound, and diffusion-weighted imaging magnetic resonance imaging, the correlation between genetic aberrations and imaging and histology were investigated. RESULTS: Most frequent mutations were hTERT (61.5%), followed by TP53 (42.7%), RB1 (24.8%), and CTNNB1 (18.8%). Liver tumors were classified into six imaging groups/grades, and the prevalence of hTERT mutations tended to increase with the advancement of imaging/histological grades (P = 0.026 and 0.13, respectively), whereas no such tendency was evident for TP53 mutation (P = 0.78 and 1.00, respectively). Focusing on the mutations in each tumor, although the variant frequency (VF) of hTERT did not change (P = 0.36 and 0.14, respectively) in association with imaging/histological grades, TP53 VF increased significantly (P = 0.004 and <0.001, respectively). In multivariate analysis, stage III or IV (hazard ratio, 3.64; P = 0.003), TP53 VF ≥ 50% (hazard ratio, 3.79; P = 0.020) was extracted as an independent risk for recurrence in primary HCC patients. CONCLUSIONS: Increased prevalence of hTERT mutation and increased TP53 mutation VF are characteristic features of HCC progression, diagnosed with imaging/histological studies.

Hepatitis B virus (HBV)-infected patients with low hepatitis B surface antigen and high hepatitis B core-related antigen titers have a high risk of HBV-related hepatocellular carcinoma.

AIM: Although the viral markers hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HbcrAg) could reflect intrahepatic hepatitis B virus (HBV) replication activity and constitute important biomarkers for hepatocellular carcinoma (HCC), the value of using these two markers in combination for assessing HCC risk has not been clarified in detail. METHODS: Four hundred and forty-nine consecutive patients with chronic HBV infection were included in the study and the association of HBsAg and HBcrAg with HCC risk was investigated cross-sectionally, as well as longitudinally. RESULTS: When the high value cut-offs of HBsAg and HBcrAg were defined as 3.0 log IU/mL and 3.0 log U/mL, respectively, patients with a history of HCC were found frequently in the low HBsAg group (P = 0.002) and high HBcrAg group (P < 0.001). When HBsAg and HBcrAg were combined, an HCC history was most frequent in the subset with low HBsAg and high HBcrAg, among the HBeAg-negative patients (odds ratio [OR], 7.83; P < 0.001), irrespective of nucleos(t) ide analogue (NA) therapy (NA: OR, 4.76; P < 0.001; non-NA: OR, 9.60; P < 0.001). In a longitudinal analysis of the subsequent development of HCC, carried out on the 338 patients without an HCC history at enrollment, HCC developed significantly more frequently in the low HBsAg/high HBcrAg group (P = 0.005). CONCLUSIONS: Patients with low HBsAg/high HBcrAg values are at high risk of developing HBV-related HCC, according to this cross-sectional and longitudinal analysis, indicating that the combination of HBsAg and HBcrAg values is an excellent biomarker for assessing HCC risk.

L-carnitine Reduces Muscle Cramps in Patients With Cirrhosis.

We performed a prospective study to evaluate the ability of L-carnitine, which is involved in the ß-oxidation of fatty acids, to reduce muscle cramps in patients with cirrhosis. Consecutive patients with cirrhosis and muscle cramps were given L-carnitine 300 mg, 3 times/day (900 mg/day, n = 19) or 4 times/day (1200 mg/day, n = 23) for 8 weeks. The frequency of muscle cramps was assessed by questionnaires, and the degree of muscle cramping was assessed by using the visual analogue scale (VAS). Muscle cramping was reduced in 88.1% of all subjects at the end of the 8-week study period and disappeared for 28.6% of patients. Overall VAS scores decreased significantly from 69.9 ± 22.5 at baseline to 26.2 ± 29.1 after 8 weeks (P < .0001). The dose of L-carnitine was significantly associated with percentages of patients with reduced muscle cramps after 8 weeks (43.5% in the 1200 mg/day group vs 10.5% in the 900 mg/day group, P = .037) and VAS scores at 8 weeks (9.9 ± 13.5 in the 1200 mg/day group vs 39.6 ± 31.9 in the 900 mg/day group, P = .003). No adverse events were reported. Therefore, L-carnitine appears to be safe and effective for reducing liver cramps in patients with cirrhosis.

Naturally occurring, resistance-associated hepatitis C virus NS5A variants are linked to interleukin-28B genotype and are sensitive to interferon-based therapy.

AIM: The presence of resistance-associated variants (RAV) may attenuate the efficacy of direct-acting antivirals (DAA) in combination therapy for hepatitis C. The aim of this study was to characterize the NS3 and NS5A regions of hepatitis C virus (HCV) in naturally occurring RAV. METHODS: The NS3 and NS5A regions of HCV were amplified by nested polymerase chain reaction and their nucleotide sequences were determined by direct sequencing in 493 genotype 1b patients naive to DAA-based therapies. The effect of baseline RAV on response to pegylated interferon and ribavirin therapy was analyzed in 65 patients after stratification by interleukin (IL)-28B genotype. RESULTS: The incidence of RAV was 7.9% in NS3 (V36I/L, 1.2%; T54S, 2.8%; Q80K/R, 3.0%; A156S, 0.2%; and D168E/T, 2.4%) and 20.2% in NS5A (L31I/M, 2.2%; and Y93H, 19.0%). The incidence in interferon experienced and naive patients was similar. The incidence of Y93H in NS5A was significantly higher in the IL-28B TT genotype (rs8099917) than non-TT (27.1% vs 9.5%, P < 0.001). The virological response to pegylated interferon plus ribavirin therapy was not affected by the presence of RAV in IL-28B TT genotype. CONCLUSION: RAV, especially Y93H in the NS5A region, were highly prevalent in DAA naive patients with genotype 1b HCV in Japan and were linked to IL-28B TT genotype. Interferon-based therapy could be an alternative for patients with RAV because these variants did not attenuate the response to that therapy. The analysis of RAV may impact the selection of the optimal treatment strategy.

Wisteria floribunda agglutinin positive human Mac-2-binding protein as a predictor of hepatocellular carcinoma development in chronic hepatitis C patients.

AIMS: Wisteria floribunda agglutinin (WFA)-positive human Mac-2-binding protein (WFA(+) -M2BP) is a new glycol marker related to liver fibrosis. The aim of the present study was to evaluate WFA(+) -M2BP as a predictor of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C. METHODS: This case-control study included 14 patients with chronic hepatitis C who developed HCC and 52controls, matched for age, gender, and fibrosis stage. WFA(+) -M2BP was measured at biopsy and follow-up. Time zero was set at the date of liver biopsy. RESULTS: WFA(+) -M2BP increased stepwise with progression of liver fibrosis (p < 0.001). Cumulative incidence of HCC development was significantly higher in patients with WFA(+) -M2BP ≥4.2 (p < 0.001) or in those with time-course changes in WFA(+) -M2BP (ΔWFA(+) -M2BP/year) ≥0.3 (p = 0.03). Multivariate analyses demonstrated that WFA(+) -M2BP ≥4.2 [hazard ratio (HR): 4.1, 95% confidence interval (CI): 1.1-15, p = 0.04], ΔWFA(+) -M2BP/year ≥0.3 (HR: 5.5, 95% CI: 1.5-19, p = 0.008), and AFP ≥10 ng/ml (HR: 4.7, 95% CI: 1.1-19, p = 0.03) were independent predictive factors of HCC development. Based on these data, we developed a simple scoring system to predict HCC development using these three factors. Using these scores, patients were classified into four groups; cumulative incidence of HCC development significantly increased with increasing scores (p < 0.001). CONCLUSIONS: WFA(+) -M2BP measurements and time-course changes in WFA(+) -M2BP can be used to identify patients at high risk of HCC development. Real-time monitoring of WFA(+) -M2BP can be a novel predictor of HCC development.

Spontaneous portosystemic shunt diameter predicts liver function after balloon-occluded retrograde transvenous obliteration.

BACKGROUND AND AIM: Recently, balloon-occluded retrograde transvenous obliteration (BRTO), performed for spontaneous portosystemic shunts (SPSS), has been receiving attention as a measure to improve liver function in cirrhotic patients with portal hypertension. However, it is unclear whether SPSS diameter is associated with changes in hepatic venous pressure gradient (HVPG) and liver function after BRTO. METHODS: In 34 cirrhotic patients receiving BRTO for hepatic encephalopathy/gastric varices, the association of SPSS diameter with liver function at baseline and 6 months after BRTO and the accompanying changes in HVPG were investigated. RESULTS: Patients had Child-Pugh (CP) scores of A/B/C (7/19/8), SPSS diameters of ≤10 mm/11-20 mm/<20 mm (8/21/5), and an average observation period of 3.2 (0.3-8.5) years. SPSS diameter was significantly associated with male sex, alcohol use, and values of albumin, prothrombin time (PT%), and NH3 at baseline. Moreover, the SPSS diameter was significantly correlated with the changes in HVPG observed upon BRTO (r = 0.55, P = 0.005), and a large shunt diameter was significantly associated with a greater increase in HVPG. At 6 months, significant improvements in albumin, PT%, bilirubin, and NH3 were observed overall, but the improvement was marked in those with larger shunt diameters if they had CP A/B. CONCLUSION: SPSS diameter was strongly associated with liver function at baseline and after BRTO and also with changes in HVPG, indicating that SPSS diameter is an important predictor of BRTO outcome.

Analysis of direct-acting antiviral-resistant hepatitis C virus haplotype diversity by single-molecule and long-read sequencing.

The method of analyzing individual resistant hepatitis C virus (HCV) by a combination of haplotyping and resistance-associated substitution (RAS) has not been fully elucidated because conventional sequencing has only yielded short and fragmented viral genomes. We performed haplotype analysis of HCV mutations in 12 asunaprevir/daclatasvir treatment-failure cases using the Oxford Nanopore sequencer. This enabled single-molecule long-read sequencing using rolling circle amplification (RCA) for correction of the sequencing error. RCA of the circularized reverse-transcription polymerase chain reaction products successfully produced DNA longer than 30 kilobase pairs (kb) containing multiple tandem repeats of a target 3 kb HCV genome. The long-read sequencing of these RCA products could determine the original sequence of the target single molecule as the consensus nucleotide sequence of the tandem repeats and revealed the presence of multiple viral haplotypes with the combination of various mutations in each host. In addition to already known signature RASs, such as NS3-D168 and NS5A-L31/Y93, there were various RASs specific to a different haplotype after treatment failure. The distribution of viral haplotype changed over time; some haplotypes disappeared without acquiring resistant mutations, and other haplotypes, which were not observed before treatment, appeared after treatment. Conclusion: The combination of various mutations other than the known signature RAS was suggested to influence the kinetics of individual HCV quasispecies in the direct-acting antiviral treatment. HCV haplotype dynamic analysis will provide novel information on the role of HCV diversity within the host, which will be useful for elucidating the pathological mechanism of HCV-related diseases.

Usefulness of Cell-Free Human Telomerase Reverse Transcriptase Mutant DNA Quantification in Blood for Predicting Hepatocellular Carcinoma Treatment Efficacy.

Although the usefulness of liquid biopsy as a biomarker in the treatment of hepatocellular carcinoma (HCC) has been suggested, its usefulness in transcatheter arterial chemoembolization (TACE) or tyrosine kinase inhibitor (TKI) therapies has not been reported in detail. In this study, we investigated the clinical value of a cell-free (cf)DNA quantification system targeting the human telomerase reverse transcriptase (hTERT) promoter mutation in advanced HCC treatment. Plasma from 67 patients with advanced HCC, treated with TACE and TKI, was used for extraction of cfDNA. We defined cfDNA with the hTERT promoter C228T mutation as circulating mutant DNA (mutant DNA) and without the mutation as circulating wild-type DNA (wild-type DNA). We analyzed the changes in mutant and wild-type DNA levels during HCC treatment and examined the relationship between changes in the cfDNA level and the clinical course. Mutant DNA was detected in 73.1% (49/67) of the patients during HCC treatment. In univariate analysis, factors associated with detection of mutant DNA before treatment were the intrahepatic maximum tumor diameter (P = 0.015) and protein induced by vitamin K absence (PIVKAII) (P = 0.006). The degree of mutant DNA change after TACE was significantly correlated with tumor volume (P < 0.001), reflecting the treated tumor volume. Responders with peak cfDNA levels within 1 week of TKI initiation had significantly better progression-free survival than nonresponders (P = 0.004). Conclusion: Changes in blood hTERT promoter mutant DNA levels during TACE or TKI treatment indirectly reflect the amount of HCCs and are useful for predicting long-term treatment responses.

HBV preS deletion mapping using deep sequencing demonstrates a unique association with viral markers.

AIM: Deletions are observed frequently in the preS1/S2 region of hepatitis B virus (HBV) genome, in association with liver disease advancement. However, the most significant preS1/S2 region and its influences on viral markers are unclear. METHODS: The preS1/S2 HBV regions of 90 patients without antiviral therapy were subjected to deep sequencing and deleted regions influencing viral markers were investigated. RESULTS: From the deletion frequency analysis in each patient, deletions were observed most frequently in the preS2 codon 132-141 region. When the patients were divided into three groups (0-0.1%: n = 27, 0.1%-10%: n = 34, 10-100%: n = 29), based on the deletion frequency, FIB-4 (p < 0.01), HBV DNA (p < 0.01), HBcrAg (p < 0.01) and preS1/S2 start codon mutations (p < 0.01, both) were significantly associated with the deletion. When clinical and viral markers were investigated by multivariate analysis for their association with the deletion, FIB-4 (p < 0.05), HBcrAg (p < 0.05), and preS1 start codon mutation (p < 0.01) were extracted as independent variables. When the influence of the preS codon 132-141deletions on HBsAg and HBcrAg, relative to HBV DNA, was investigated, the HBsAg/HBV DNA ratio was lower (0-10% vs. 10%-100%, p<0.05), while the HBcrAg/HBV DNA rati o was higher (0-0.1% vs. 10%-100%, p<0.05) in the presence of the preS codon 132-141deletions. CONCLUSION: The preS codon.132-141 deletions have a significant influence on the clinical characteristics and viral markers, even when present as a minor population. Importantly, the preS codon 132-141 deletions have a clear influence on the viral life cycle and pathogenesis.

Association between alanine aminotransferase elevation and UGT1A1*6 polymorphisms in daclatasvir and asunaprevir combination therapy for chronic hepatitis C.

BACKGROUND: Liver damage presented as alanine aminotransferase (ALT) elevation and high ALT-caused treatment discontinuation occurs with high frequency in Japanese patients receiving daclatasvir plus asunaprevir (DCV/ASV) therapy for hepatitis C virus (HCV) infection, and its mechanism is unknown. METHODS: A total of 247 Japanese patients consisting of two independent cohorts with genotype-1b HCV infection receiving DCV/ASV therapy were included. The association of ALT levels during therapy and single nucleotide polymorphisms (SNP) of five drug-metabolizing enzyme loci selected for their possible influence on NS3/4A and NS5A inhibitors was investigated. RESULTS: Among five SNPs, we found a significant correlation between the presence of the UGT1A1 rs4148323 A allele and ALT elevation (Grade 3 elevation in AA 57%, AG 18%, and GG 4%, P = 8.4E - 06) and drug discontinuation (AA 22%, AG 11%, and GG 2.5%, P = 8.7E - 04), while no association was observed with ALT values at baseline (Grade 3 elevation AA 0%, AG 4%, and GG 2%, P = 0.5). In contrast, patients with risk A allele for drug-induced ALT elevation had a tendency to respond more favorably to treatment (AA 100%, AG 93%, and GG 90%, P = 0.29). CONCLUSIONS: Through the analysis we suggest that the A allele in UGT1A1 rs4148323 (UGT1A1*6), which is highly prevalent in the Japanese population, should be considered a risk for the development of DCV/ASV therapy-induced ALT elevation. Pretreatment SNP testing of UGT1A1*6 might be beneficial for the prediction of liver damage induced by DCV/ASV or even by DCV/ASV plus beclabuvir.

Irregular vascular pattern by contrast-enhanced ultrasonography and high serum Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein level predict poor outcome after successful radiofrequency ablation in patients with early-stage hepatocellular carcinoma.

Radiofrequency ablation (RFA) is considered the most effective treatment for early-stage hepatocellular carcinoma (HCC) patients unsuitable for resection. However, poor outcome after RFA has occasionally been reported worldwide. To predict such an outcome, we investigated imaging findings using contrast-enhanced ultrasonography (CEUS) with Sonazoid and serum tumor markers before RFA. This study included 176 early-stage HCC patients who had initially achieved successful RFA. Patients were examined using CEUS; their levels of alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), and des-gamma-carboxy prothrombin before RFA were measured. Sonazoid provided parenchyma-specific contrast imaging and facilitated tumor vascular architecture imaging through maximum intensity projection (MIP). Kaplan-Meier analysis examined cumulative rates of local tumor progression, intrasubsegmental recurrence, and survival; factors associated with these were determined with Cox proportional hazards analysis. Local tumor progression (n = 15), intrasubsegmental recurrence (n = 46), and death (n = 18) were observed. Irregular pattern in MIP classification and serum AFP-L3 level (>10%) before RFA were identified as independent risk factors for local tumor progression and intrasubsegmental recurrence. These two factors were independently associated with poor survival after RFA (irregular pattern in MIP: hazard ratio, (HR) = 8.26; 95% confidence interval, (CI) = 2.24-30.3; P = 0.002 and AFP-L3 > 10%: HR = 2.94; 95% CI = 1.09-7.94; P = 0.033). Irregular MIP pattern by CEUS and high level of serum AFP-L3 were independent risk factors for poor outcome after successful RFA. The Patients with these findings should be considered as special high-risk group in early-stage HCC.

Urinary excretion of the water channel aquaporin 2 correlated with the pharmacological effect of tolvaptan in cirrhotic patients with ascites.

BACKGROUND: The water channel aquaporin 2 (AQP2) at the apical membrane of renal collecting duct cells mediates water reabsorption. The expression of AQP2 at the apical membrane is tightly regulated by vasopressin and was quantitated by measurement of the urinary form by a recently developed ELISA. Tolvaptan, an antagonist of vasopressin type 2 receptor, inhibits water reabsorption in cirrhosis. The aim of this study was to determine the correlation between the pharmacological effect of tolvaptan and the dynamics of urinary AQP2 levels. METHODS: Tolvaptan was administered to 41 cirrhotic patients with ascites unresponsive to standard diuretic therapy. Urinary excretion of AQP2 and urinary osmolarity were measured at the baseline and at 4, 8, and 24 h after administration of tolvaptan. RESULTS: At the baseline, urinary AQP2/creatinine ratios were significantly higher in cirrhotic patients with ascites than in healthy controls (P < 0.0001). After administration of tolvaptan, urinary AQP2/creatinine ratios decreased by 45.0 % at 4 h and 77.0 % at 8 h. Similarly, urinary osmolarity decreased by 42.0 % at 4 h and 41.5 % at 8 h. Urinary AQP2 levels and urinary osmolarity significantly correlated at the baseline and at all time points after tolvaptan administration. The degree of the decrease in urinary AQP2 levels and degree of the decrease in urinary osmolarity correlated significantly at 4 h (r = 0.452, P = 0.009) and 8 h (r = 0.384, P = 0.030) after tolvaptan administration. CONCLUSIONS: These results indicate that the vasopressin-AQP2 system plays a major role in fluid retention in cirrhosis and that the pharmacological effect of tolvaptan to inhibit water reabsorption can be monitored by measurement of the dynamics of urinary AQP2 levels.