Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells.

There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G1 -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.

High serum growth differentiation factor 15 is a risk factor for the occurrence of hepatocellular carcinoma in chronic hepatitis B patients treated with nucleos(t)ide analogs.

AIM: Patients with chronic hepatitis B (CHB) remain at risk for hepatocellular carcinoma (HCC) even with nucleos(t)ide analog therapy. We evaluated risk factors for HCC development, including serum hepatitis B virus (HBV) RNA, hepatitis B core-related antigen level, and growth differentiation factor 15 (GDF15) level, a predictor of HCC development in patients with chronic hepatitis C. METHODS: We collected clinical data and stored serum from CHB patients without a history of HCC who were receiving nucleos(t)ide analog treatment for more than 1 year and whose HBV DNA level was less than 3.0 log IU/mL. We measured the serum levels of HBV RNA and GDF15. RESULTS: Among 242 CHB patients, 57 had detectable HBV RNA, and GDF15 was quantified in all patients. The median GDF15 level was 0.86 ng/mL. Cox proportional hazards analysis revealed that male sex and higher GDF15, FIB-4 index, alpha-fetoprotein and gamma-glutamyl transpeptidase were independent risk factors for HCC. The presence of HBV RNA above the lower limit of quantification was not a risk factor. When we set cutoff values based on the Youden index, the cumulative incidence of HCC was significantly higher in the male, AFP ≥3.0 ng/mL, gamma-glutamyl transpeptidase ≥22 U/L, FIB-4 index ≥1.93, and GDF-15 ≥1.17 ng/mL groups. In patients with no or more than three of these five risk factors, the 10-year HCC cumulative incidence rates were 0% and 41.0%, respectively. CONCLUSIONS: High serum GDF15 is an independent risk factor for the occurrence of HCC in CHB patients treated with nucleos(t)ide analogs.

Distinct Clinical Factors in Hospitalized Patients with Diverticular Bleeding and Diverticulitis.

BACKGROUND/AIMS: It is unclear why colonic diverticular bleeding and diverticulitis rarely coexist. This study compared the characteristics of these conditions. METHODS: This single-center retrospective study examined 310 consecutive patients hospitalized with an episode of diverticular disease (cases) and outpatients without a diverticular episode (controls) from January 2012 to December 2015. We investigated distinct clinical factors in hospitalized patients with diverticular bleeding and diverticulitis. RESULTS: We identified 183 patients with 263 episodes of diverticular bleeding and 127 patients with 135 episodes of diverticulitis during the study period. Patients with diverticular bleeding were significantly older than those with diverticulitis (median age 76 vs. 56 years) and had more cardiovascular disease, hypertension, diabetes, cerebrovascular disease, chronic kidney disease, lipid disorder, or a poorer performance status. Significantly more diverticular bleeding patients were taking antiplatelet drugs, anticoagulant drugs, proton pump inhibitors, or laxative agents. Multivariate analysis revealed that an age > 65 years (OR 5.42), and antiplatelet agent use (OR 7.29) were more significant risk factors for diverticular bleeding than for diverticulitis. CONCLUSIONS: Elderly people using antiplatelet drugs may be more susceptible to diverticular bleeding than diverticulitis.

Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration.

Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.

Inhibition of nonhomologous end joining-mediated DNA repair enhances anti-HBV CRISPR therapy.

Current anti-hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA) and fail to eliminate HBV. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been reported to directly target cccDNA and exert antiviral effects. In this study, we hypothesized that the inhibition of the DNA repair machinery, which is important for the repair of CRISPR-induced double-strand breaks, may enhance the effect of CRISPR targeting cccDNA, and we investigated the antiviral effect of potential combination therapy. The antiviral effect of CRISPR targeting cccDNA (HBV-CRISPR) was evaluated in HBV-susceptible HepG2-hNTCP-C4 cells expressing Cas9 (HepG2-hNTCP-C4-iCas9) or primary human hepatocytes (PHHs) expressing Cas9. Following HBV infection, HBV-CRISPR reduced cccDNA levels, accompanied by decreases in pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen and hepatitis B e antigen levels in HepG2-hNTCP-C4-iCas9 cells, and PHHs. HBV-CRISPR induced indel formation in cccDNA and up-regulated poly(adenosine diphosphate ribose) polymerase (PARP) activity in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of PARP2-Histone PARylation factor 1 (HPF1) (involved in the initial step of DNA repair) with small interfering RNA (siRNA) targeting either PARP2 or HPF1 increased the reduction in pgRNA and cccDNA by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of DNA Ligase 4 (LIG4) (essential for nonhomologous end joining [NHEJ]) but not breast cancer susceptibility gene (BRCA) (essential for homologous recombination) enhanced the antiviral effect of HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. Finally, the clinically available PARP inhibitor olaparib increased the reductions in pgRNA and cccDNA levels induced by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells and PHHs. Conclusion: The suppression of the NHEJ-mediated DNA repair machinery enhances the effect of CRISPR targeting cccDNA. The combination of CRISPR and olaparib may represent a therapy for HBV elimination.

Clinically diagnosed late-onset fulminant Wilson's disease without cirrhosis: A case report.

A 64-year-old woman was referred to our hospital with jaundice of the bulbar conjunctiva and general fatigue. After admission, she developed hepatic encephalopathy and was diagnosed with fulminant hepatitis based on the American Association for the Study of Liver Disease (AASLD) position paper. Afterwards, additional laboratory findings revealed that serum ceruloplasmin levels were reduced, urinary copper levels were greatly elevated and Wilson's disease (WD)-specific routine tests were positive, but the Kayser-Fleischer ring was not clear. Based on the AASLD practice guidelines for the diagnosis and treatment of WD, the patient was ultimately diagnosed with fulminant WD. Then, administration of penicillamine and zinc acetate was initiated; however, the patient unfortunately died from acute pneumonia on the 28th day of hospitalization. At autopsy, the liver did not show a bridging pattern of fibrosis suggestive of chronic liver injury. Here, we present the case of a patient with clinically diagnosed late-onset fulminant WD without cirrhosis, who had positive disease-specific routine tests.

Nivolumab Induces Sustained Liver Injury in a Patient with Malignant Melanoma.

A 42-year-old man was diagnosed with cStage IIIb malignant melanoma and underwent resection. After interferon-beta therapy, 18-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) showed multiple lung metastases, and he received nivolumab (2 mg/kg) every 3 weeks, resulting in a total of 17 cycles. After treatment, 18F-FDG PET/CT showed a significant decrease in the size of the metastases, but he had a Grade 4 alanine aminotransferase (ALT) elevation. Liver histology revealed drug-induced liver damage. Therefore, we performed steroid half-pulse therapy followed by oral methylprednisolone, but his ALT level did not completely recover to the normal range even after five months. We herein report a case with specific, sustained liver injury induced by nivolumab as an immune-related adverse events.

Number of polyps detected is a useful indicator of quality of clinical colonoscopy.

BACKGROUND AND STUDY AIMS: Adenoma detection rate (ADR) is a well-known quality indicator (QI) for colonoscopy. It is, however, difficult to evaluate ADR during practice. The aim of this study was to investigate the number of endoscopically detected polyps as a QI for colonoscopy. PATIENTS AND METHODS: This was a retrospective single-center cohort study of 5,190 consecutive patients who underwent colonoscopy from January 2015 to May 2016. Among these patients, we ultimately enrolled 1,937 patients for initial colonoscopy. We evaluated QIs including bowel preparation, cecum intubation time, withdrawal time, number of endoscopically detected polyps, ADR and advanced neoplasia detection rate (ANDR). RESULTS: The mean number of endoscopically detected polyps, ADR and ANDR were 1.5 ±â€Š2.3 (95 % confidence interval (CI)1.4 - 1.6), 38.6 % (95 % CI 36.5-40.8), and 18.3 % (95 % CI 16.6 - 20.1), respectively. ADR and ANDR increased with the number of endoscopically detected polyps, but the correlation reached a plateau at five or more polyps. We divided the patients into three groups based on the number of polyps (1 to 2, 3 to 4, and 5 or more). Logistic regression analysis adjusted by age and sex revealed that presence of a large number of polyps was a strong predictor of advanced neoplasia (odds ratio: 3.1 [95 % CI 2.2 - 4.3] for 3 to 4 polyps and 7.9 [95 % CI 5.4 - 11.8] for 5 or more polyps when using the presence of 1 or 2 polyps as a reference). CONCLUSION: The number of endoscopically detected polyps can predict risk of advanced neoplasia and may thus be a new QI for colonoscopy.

The combination of anti-HBc and anti-HBs levels is a useful predictor of the development of chemotherapy-induced reactivation in lymphoma patients with resolved HBV infection.

Fatal chemotherapy-induced hepatitis B virus reactivation (HBV-R) is a well-described serious complication observed in patients with lymphoma and resolved HBV infection. The aim of the present study was to determine the predictive factors of the development of chemotherapy-induced HBV-R. A total of 77 consecutive newly diagnosed patients with lymphoma and resolved HBV infection, who received chemotherapy from 2007 through 2015 were analysed retrospectively. Significant predictive factors associated with HBV-R were identified based on the data from these patients. Ten patients developed HBV-R during and following chemotherapy, and two of these 10 patients developed HBV-associated hepatitis flares. There was a significant negative correlation between anti-hepatitis B core (HBc) titres prior to chemotherapy and time to HBV-R (P=0.016, R=-0.732). Univariate and multivariate logistic regression analyses demonstrated that anti-HBc and anti-hepatitis B surface (HBs) titres at baseline were significant predictive factors for HBV-R. In addition, patients with high anti-HBc titres at baseline (above 10 S/CO) were significantly more likely to experience HBV-R than patients with low anti-HBc and high anti-HBs titres (above 28 mIU/ml), who did not experience complete reactivation (P<0.0001). Furthermore, patients with low anti-HBs titres were significantly more likely to experience HBV-R than those with high anti-HBs titres (P=0.031). All HBV-R episodes among the patients with high anti-HBc titres occurred within 3 months following the initiation of chemotherapy. The combination of anti-HBc and anti-HBs titres, as opposed to either titre alone, at baseline in patients with lymphoma may serve as a surrogate marker for the occurrence of HBV-R under the influence of chemotherapy.

Combination of two-hour post-endoscopic retrograde cholangiopancreatography amylase levels and cannulation times is useful for predicting post-endoscopic retrograde cholangiopancreatography pancreatitis.

AIM: To estimate the efficacy of 2 h post-endoscopic retrograde cholangiopancreatography (ERCP) serum amylase levels and other factors for predicting post-ERCP pancreatitis. METHODS: This was a retrospective, single-center cohort study of consecutive patients who underwent ERCP from January 2010 to December 2013. Serum amylase levels were measured 2 h post-procedure, and patient- and procedure-related pancreatitis (PEP) risk factors were analyzed using a logistic model. RESULTS: A total of 1520 cases (average age 72 ± 12 years, 60% male) were initially enrolled in this study, and 1403 cases (725 patients) were ultimately analyzed after the exclusion of 117 cases. Fifty-five of these cases developed PEP. We established a 2 h serum amylase cutoff level of two times the upper limit of normal for predicting PEP. Multivariate analysis revealed that a cannulation time of more than 13 min [odds ratio (OR) 2.28, 95%CI: 1.132-4.651, P = 0.0210] and 2 h amylase levels greater than the cutoff level (OR = 24.1, 95%CI: 11.56-57.13, P < 0.0001) were significant predictive factors for PEP. Forty-seven of the 55 patients who developed PEP exhibited 2 h amylase levels greater than the cutoff level (85%), and six of the remaining eight patients who developed PEP (75%) required longer cannulation times. Only 2 of the 1403 patients (0.14%) who developed PEP did not exhibit concerning 2 h amylase levels or require longer cannulation times. CONCLUSION: These findings indicate that the combination of 2 h post-ERCP serum amylase levels and cannulation times represents a valuable marker for identifying patients at high risk for PEP.

Frequency of coexistent carcinoma in sessile serrated adenoma/polyps and traditional serrated adenomas removed by endoscopic resection.

BACKGROUND AND STUDY AIMS: Sessile serrated adenoma/polyps (SSA/Ps) have a different potential than traditional adenomatous polyps for developing into malignant colorectal cancer. However, little is known about the coexistent cancer rate. Here, we evaluate the frequency of carcinoma in serrated polyps removed by endoscopic resection (ER). PATIENTS AND METHODS: This was a retrospective single-center cohort study of consecutive patients with colorectal polyps who underwent ER from March 2003 to October 2014. We determined the frequency of serrated polyps among all resected colorectal polyps and analyzed the clinicopathological findings as well as the frequency and characteristics of coexistent carcinoma in the serrated polyps resected by ER based on pathology reports. RESULTS: A total of 21,048 polyps from 15,326 patients were identified, including 15,984 traditional adenomatous polyps (75.9 %), 621 SSA/Ps (3.0 %), 136 traditional serrated adenomas (TSAs) (0.6 %), 1,121 hyperplastic polyps (5.3 %), and 3,186 polyps of other types (15.1 %). The clinical and endoscopic findings of SSA/Ps revealed a male predominance (68.6 %), with 61.7 % of the polyps located in the proximal colon. Males accounted for 77.2 % of all patients with TSAs, and 77.2 % of these polyps were located in the distal colon. The mean sizes of the SSA/Ps and TSAs were 8.8 and 10.7 mm, respectively. Among the SSA/Ps, 8 (1.3 %) cases had coexistent carcinoma, and 1 (0.7 %) patient with TSA showed coexistent carcinoma. In the patients with SSA/Ps, female sex and a tumor size ≥ 10 mm were predictive factors for coexistent carcinoma. CONCLUSIONS: The frequency of SSA/Ps with carcinoma was lower than that for traditional adenoma. Female sex and tumor size ≥ 10 mm were significant predictive factors for coexistent carcinoma.