Machine learning for individualized prediction of hepatocellular carcinoma development after the eradication of hepatitis C virus with antivirals.

BACKGROUND AND AIMS: Accurate risk stratification for hepatocellular carcinoma (HCC) after achieving a sustained viral response (SVR) is necessary for optimal surveillance. We aimed to develop and validate a machine learning (ML) model to predict the risk of HCC after achieving an SVR in individual patients. METHODS: In this multicenter cohort study, 1742 patients with chronic hepatitis C who achieved an SVR were enrolled. Five ML models were developed including DeepSurv, gradient boosting survival analysis, random survival forest (RSF), survival support vector machine, and a conventional Cox proportional hazard model. Model performance was evaluated using Harrel' c-index and was externally validated in an independent cohort (977 patients). RESULTS: During the mean observation period of 5.4 years, 122 patients developed HCC (83 in the derivation cohort and 39 in the external validation cohort). The RSF model showed the best discrimination ability using seven parameters at the achievement of an SVR with a c-index of 0.839 in the external validation cohort and a high discriminative ability when the patients were categorized into three risk groups (P <0.001). Furthermore, this RSF model enabled the generation of an individualized predictive curve for HCC occurrence for each patient with an app available online. CONCLUSIONS: We developed and externally validated an RSF model with good predictive performance for the risk of HCC after an SVR. The application of this novel model is available on the website. This model could provide the data to consider an effective surveillance method. Further studies are needed to make recommendations for surveillance policies tailored to the medical situation in each country. IMPACT AND IMPLICATIONS: A novel prediction model for HCC occurrence in patients after hepatitis C virus eradication was developed using machine learning algorithms. This model, using seven commonly measured parameters, has been shown to have a good predictive ability for HCC development and could provide a personalized surveillance system.

Change in hepatitis C virus positivity among needle-stick injury source patients: a 10-year experience in a Japanese tertiary hospital.

BACKGROUND: As a blood-borne pathogen, hepatitis C virus (HCV) has long been a major threat associated with needle-stick injuries (NSIs) mainly because no vaccine is available for HCV. Following an NSI, we usually test the source patient for HCV antibody (HCV-Ab). Since HCV-Ab positivity does not necessarily indicate current infection, HCV RNA is further examined in patients positive for HCV-Ab. Direct-acting antivirals (DAAs) have enabled us to treat most HCV-infected patients; therefore, we speculate that the rate of HCV RNA positivity among HCV-Ab-positive patients decreased after the emergence of DAAs. This cross-sectional study was performed to investigate the change in the actual HCV RNA positivity rate in source patients before and after the interferon (IFN)-free DAA era. METHODS: This was a cross-sectional study of NSI source patients at a tertiary academic hospital in Japan from 2009 to 2019. IFN-free DAA regimens were first introduced in Japan in 2014. Accordingly, we compared HCV status of NSI source patients that occurred between 2009 and 2014 (the era before IFN-free DAAs) with those that occurred between 2015 and 2019 (the era of IFN-free DAAs) in a tertiary care hospital in Japan. RESULTS: In total, 1435 NSIs occurred, and 150 HCV-Ab-positive patients were analyzed. The proportion of HCV RNA-positive patients significantly changed from 2009 through 2019 (p = 0.005, Cochran-Armitage test). Between 2009 and 2014, 102 source patients were HCV-Ab-positive, 78 of whom were also positive for HCV RNA (76.5%; 95%CI, 67.4-83.6%). Between 2015 and 2019, 48 patients were HCV-Ab-positive, 23 of whom were also positive for HCV RNA (47.9%; 95%CI, 34.5-61.7%; p = 0.0007 compared with 2009-2014). In the era of IFN-free DAAs, 9 of 23 HCV RNA-negative patients (39.1%) and 2 of 22 HCV RNA-positive patients (9.1%) were treated with an IFN-free combination of DAAs (p = 0.0351). Regarding the departments where NSIs occurred, HCV RNA-negative patients were predominant in departments not related to liver diseases in the era of IFN-free DAAs (p = 0.0078, compared with 2009-2014). CONCLUSIONS: Actual HCV RNA positivity in source patients of NSIs decreased after the emergence of IFN-free DAAs. IFN-free DAAs might have contributed to this reduction, and HCV RNA-negative patients were predominant in departments not related to liver diseases in the era of IFN-free DAAs.

Helicobacter pylori infection is not associated with fatty liver disease including non-alcoholic fatty liver disease: a large-scale cross-sectional study in Japan.

BACKGROUND: Fatty liver disease (FLD) including non-alcoholic fatty liver disease (NAFLD), a rapidly emerging and widely recognized liver disease today, is regarded as a hepatic manifestation of metabolic syndrome. Helicobacter pylori, one of the most common pathogens worldwide, has been reported to be associated with metabolic syndrome, but whether there is a direct association with FLD is as of yet unclear. The aim of this study was to clarify the association of FLD and NAFLD with causative background factors including Helicobacter pylori infection. METHODS: This was a cross-sectional study of Japanese adults who received medical checkups at a single medical center in 2010.Univariate and multivariate statistical analysis was performed to evaluate background factors for ultrasonography diagnosed FLD. Subjects free from alcohol influence were similarly analyzed for NAFLD. RESULTS: Of a total of 13,737 subjects, FLD was detected in 1,456 of 6,318 females (23.0 %) and 3,498 of 7,419 males (47.1%). Multivariable analyses revealed that body mass index (standardized coefficients of females and males (ß-F/M) =143.5/102.5), serum ALT (ß-F/M = 25.8/75.7), age (ß-F/M = 34.3/17.2), and platelet count (ß-F/M = 17.8/15.2) were positively associated with FLD in both genders. Of the 5,289 subjects free from alcohol influence, NAFLD was detected in 881 of 3,473 females (25.4%) and 921 of 1,816 males (50.7%). Body mass index (ß-F/M = 113.3/55.3), serum ALT (ß-F/M = 21.6/53.8), and platelet count (ß-F/M = 13.8/11.8) were positively associated with NAFLD in both genders. Metabolic syndrome was positively associated with FLD and NAFLD only in males. In contrast, Helicobacter pylori infection status was neither associated with FLD nor NAFLD regardless of gender. CONCLUSIONS: Body mass index, serum ALT and platelet count were significantly associated with FLD and NAFLD, whereas infection of Helicobacter pylori was not.

Impact of Obesity and Heavy Alcohol Consumption on Hepatocellular Carcinoma Development after HCV Eradication with Antivirals.

BACKGROUND AND AIMS: It remains unclear whether obesity increases the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C who achieved a sustained virological response (SVR) with antiviral therapy. METHODS: In this multicenter cohort study, we enrolled patients with chronic hepatitis C who achieved SVR with interferon (IFN)-based therapy (IFN group) or direct-acting antiviral (DAA) therapy (DAA group) between January 1, 1990, and December 31, 2018. The patients underwent regular surveillance for HCC. Cumulative incidence of and the risk factors for HCC development after SVR were assessed using the Kaplan-Meier method and Cox proportional hazard regression analysis, respectively. RESULTS: Among 2,055 patients (840 in the IFN group and 1,215 in the DAA group), 75 developed HCC (41 in the IFN group and 34 in the DAA group) during the mean observation period of 4.1 years. The incidence rates of HCC at 1, 2, and 3 years were 1.2, 1.9, and 3.0%, respectively. Multivariate analysis revealed that in addition to older age, lower albumin level, lower platelet count, higher alpha-fetoprotein level, and absence of dyslipidemia, obesity (body mass index ≥25 kg/m2) and heavy alcohol consumption (≥60 g/day) were independent risk factors for HCC development, with adjusted hazard ratio (HR) of 2.53 (95% confidence interval [CI]: 1.51-4.25) and 2.56 (95% CI: 1.14-5.75), respectively. The adjusted HR was not significant between the 2 groups (DAA vs. IFN; HR 1.19, 95% CI: 0.61-2.33). CONCLUSIONS: Obesity and heavy alcohol consumption increased the risk of HCC development after SVR.

Heterozygous knockout of Bile salt export pump ameliorates liver steatosis in mice fed a high-fat diet.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing worldwide, including in Asian countries. We reported that the hepatic expression of bile salt export pump (BSEP) was downregulated in patients with NASH, suggesting that BSEP is involved in the pathogenesis of NASH. To identify the underlying mechanism, we analyzed Bsep heterozygous knock-out (Bsep+/- mice) and wild-type (WT) C57BL/6J mice fed a high-fat diet (HFD) (32.0% animal fat) or normal diet. We examined histological changes, levels of hepatic lipids and hepatic bile acids, and expression of genes related to bile acid and cholesterol metabolism. HFD-fed Bsep+/- mice exhibited milder hepatic steatosis and less weight gain, compared to HFD-fed WT mice. The concentrations of total bile acid, triglycerides, and cholesterols were reduced in the liver of HFD-fed Bsep+/- mice. Regarding hepatic bile acid metabolism, the expression levels of Farnesoid X receptor (Fxr) and Multidrug resistance-associated protein 2 were significantly upregulated in HFD-fed Bsep+/- mice, compared to HFD-fed WT mice. Furthermore, several alterations were observed in upstream cholesterol metabolism in the liver. The expression levels of bile acid metabolism-related genes were also altered in the intestine of HFD-fed Bsep+/- mice. In conclusion, HFD-fed Bsep+/- mice exhibited significant alterations of the expression levels of genes related to bile acid and lipid metabolism in both the liver and ileum, resulting in alleviated steatosis and less weight gain. These results suggest the importance of BSEP for maintenance of bile acid and cholesterol metabolism. Further investigations of the involvement of BSEP in the pathogenesis of NASH will provide greater insight and facilitate the development of novel therapeutic modalities.

Improved liver function in patients with cirrhosis due to chronic hepatitis C virus who achieve sustained virologic response is not accompanied by increased liver volume.

BACKGROUND: Serum albumin level improves in patients with chronic hepatitis C virus (HCV) infection who achieve sustained virologic response (SVR) with antiviral therapy. However, it remains controversial whether liver volume increases along with SVR. METHODS: Patients with chronic HCV infection with a history of hepatocellular carcinoma (HCC) who achieved SVR with anti-HCV treatment from March 2003 to November 2017 were enrolled. Patients were followed up with periodic computed tomography (CT) scans to detect HCC recurrence. Patients who underwent treatment for HCC recurrence within 1 year after initiation of anti-HCV treatment were excluded. Laboratory data, including alanine aminotransferase (ALT) level, serum albumin level, and platelet count, were collected at baseline and timepoints after treatment initiation. Liver volume was evaluated at baseline and 24 and 48 weeks after treatment initiation using a CT volume analyzer. A linear mixed-effects model was applied to analyze the chronologic change in liver volume. The correlations between changes in ALT level, albumin level, and liver volume were also evaluated. RESULTS: Of 108 enrolled patients, 78 had cirrhosis. Serum albumin level continued to increase through 48 weeks after treatment initiation. A significant increase in liver volume was observed only in patients without cirrhosis (P = 0.005). There was a significant correlation between ALT level decrease and albumin level increase (P = 0.018). CONCLUSIONS: Improved liver albumin production with SVR was contributed by improved liver cell function rather than increased liver volume in patients with cirrhosis.

Hepatic FATP5 expression is associated with histological progression and loss of hepatic fat in NAFLD patients.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are characterized by the accumulation of excess hepatic fat. However, in the progression from NASH to cirrhosis, hepatic fat is often lost. Our aim was to elucidate the mechanism underlying hepatic fat loss during NASH progression. METHODS: Liver biopsies were performed at The University of Tokyo Hospital between November 2011 and March 2016 on 146 patients with NAFLD and 14 patients with cryptogenic cirrhosis who were not being treated with any diabetes or dyslipidemia drugs. Among them, 70 patients underwent liver biopsy after an overnight fast, and 90 patients were biopsied 5 h after an oral glucose tolerance test. Expression differences in genes encoding several fatty acid metabolism-related factors were examined and correlated with hepatic histological changes based on NAFLD activity scores. Prospective patient follow-up continued until June 2018. RESULTS: The level of fatty acid transport protein 5 (FATP5), which is associated with free fatty acid intake, was significantly and inversely correlated with features of histological progression, including ballooning and fibrosis. This was confirmed by immunohistochemical analysis. Transcript levels of genes encoding fatty acid metabolism-related proteins were comparable between NASH with severe fibrosis and cryptogenic cirrhosis. Furthermore, a prospective cohort study demonstrated that low FATP5 expression was the most significant risk factor for hepatic fat loss. CONCLUSIONS: Decreased hepatic FATP5 expression in NAFLD is linked to histological progression, and may be associated with hepatic fat loss during NASH progression to cirrhosis.

Noninvasive diagnostic criteria for nonalcoholic steatohepatitis based on gene expression levels in peripheral blood mononuclear cells.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) consists of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH); the latter progresses to liver cirrhosis and hepatocellular carcinoma. Discriminating NASH from NAFL typically involves liver biopsy. The mechanism of NASH progression is unclear but may involve immunological pathways. In this study, we examined expression levels of cytokine- and chemokine-encoding genes in peripheral blood mononuclear cells (PBMCs) from NAFLD patients and established immunological criteria for discriminating NASH from NAFL. METHODS: PBMCs were obtained from 54 patients diagnosed histologically with NAFLD (NAFL, 18; NASH, 36). mRNA was extracted from PBMCs, and expression levels of cytokine- and chemokine-encoding genes were determined by quantitative real-time PCR. Statistical analysis was performed by nonparametric test. RESULTS: Expression levels of interferon (IFN)γ, interleukin (IL)2, IL15, C-C-motif chemokine ligand (CCL)2, IL10, and C-X-C-motif chemokine ligand (CXCL)11 were significantly upregulated in NASH patients compared with NAFL patients. Moreover, their expression levels were positively correlated with the degree of ballooning of hepatocytes but not of steatosis or lobular inflammation. We focused on those encoding IL10, IFNγ, and CCL2, and developed a scoring system to discriminate NASH from NAFL. The discriminatory power of the criteria was validated in an independent cohort. CONCLUSIONS: Expression levels of the cytokine- and chemokine-encoding genes in PBMCs were positively correlated with ballooning, suggesting their utility for the diagnosis of NASH. The data indicate that peripheral as well as intrahepatic immunity is involved in the progression of NASH. Our findings afford new insight into immunological mechanisms of NASH and will facilitate its noninvasive diagnosis.

Helicobacter pylori infection and liver diseases: Epidemiology and insights into pathogenesis.

Both Helicobacter pylori (H. pylori) infection and liver diseases, including nonalcoholic fatty liver disease (NAFLD), viral hepatitis, and hepatocellular carcinoma (HCC), have high prevalences worldwide, and the relationship between H. pylori infection and liver disease has been discussed for many years. Although positive correlations between H. pylori and NAFLD have been identified in some clinical and experimental studies, negative correlations have also been obtained in high-quality clinical studies. Associations between H. pylori and the pathogenesis of chronic viral hepatitis, mainly disease progression with fibrosis, have also been suggested in some clinical studies. Concerning HCC, a possible role for H. pylori in hepatocarcinogenesis has been identified since H. pylori genes have frequently been detected in resected HCC specimens. However, no study has revealed the direct involvement of H. pylori in promoting the development of HCC. Although findings regarding the correlations between H. pylori and liver disease pathogenesis have been accumulating, the existing data do not completely lead to an unequivocal conclusion. Further high-quality clinical and experimental analyses are necessary to evaluate the efficacy of H. pylori eradication in ameliorating the histopathological changes observed in each liver disease.

Hepatic IRS1 and ß-catenin expression is associated with histological progression and overt diabetes emergence in NAFLD patients.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes. Our aim was to investigate the relationship between NAFLD and impaired glucose metabolism in terms of insulin receptor substrate 1 and 2 (IRS1 and IRS2) expression in the liver. METHODS: Liver biopsy was performed at the University of Tokyo Hospital between November 2011 and March 2016 on 146 patients with NAFLD who were not being treated with any diabetes or dyslipidemia drugs. Among them, 63 underwent liver biopsy after an overnight fast, and 83 at 5 h after an oral glucose tolerance test (OGTT). Differences in messenger RNA (mRNA) levels of several glucose metabolism-related factors were determined and correlated with hepatic histological changes assessed by NAFLD activity score. We prospectively followed up with the patients until May 2017. RESULTS: Hepatic necroinflammation was significantly correlated with serum insulin levels and inversely correlated with IRS1 mRNA levels. In specimens obtained after an OGTT, hepatic necroinflammation and IRS1 expression correlated significantly with both peripheral and hepatic insulin resistance. We also found that hepatic ß-catenin and glucokinase mRNA levels were elevated in patients undergoing liver biopsy after an OGTT, especially in those with less hepatic necroinflammation and a lower degree of fibrosis. A prospective cohort study showed that ballooning is the most significant risk factor for developing diabetes. CONCLUSIONS: The decreased hepatic expression of IRS1 and ß-catenin in NAFLD is linked to histological progression such as ballooning, and might lead to diabetes as a result of impaired glucose metabolism.

Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver.

The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2'-5' oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity.

The intrahepatic expression levels of bile acid transporters are inversely correlated with the histological progression of nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) presents as a spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter is progressive, and its pathogenesis remains poorly understood. Recently, bile acid (BA) metabolism has become a therapeutic focus in NASH patients. The aim of the present study was to explore changes in bile acid metabolism in NAFLD patients in the context of disease progression. METHODS: We prospectively enrolled patients with clinically suspected NAFLD. Patients taking ursodeoxycholic acid were excluded. The intrahepatic expression levels of genes associated with BA metabolism were determined by quantitative PCR and immunohistochemistry. RESULTS: Seventy-eight patients (male:female = 49:29) histologically diagnosed with NAFLD were analyzed. The expression levels of farnesoid X receptor, liver receptor homolog 1, and small heterodimer partner, key proteins in BA synthesis, significantly decreased as the NAFLD activity score (NAS) increased in either males or females. The levels of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of BA synthesis, were not changed. Notably, the expression levels of a main export transporter, bile salt export pump (BSEP), significantly decreased as the NAS and the each NAS component increased in both genders. The decreases of BSEP levels were also observed by immunohistochemistry, particularly in areas with pronounced fatty changes in cases with high NAS. CONCLUSIONS: The expression levels of the BA export transporter BSEP were inversely correlated with NAS in NAFLD patients. Such down-regulation may cause excessive BA levels in hepatocytes, leading to cell injury. Our findings may afford new insights into the pathogenesis of NASH.

Recurrent Acute Liver Failure Because of Acute Hepatitis Induced by Organic Solvents: A Case Report.

The authors present a case of recurrent acute liver failure because of occupational exposure to organic solvents. A 35-year-old man with a 3-week history of worsening jaundice and flu-like symptoms was admitted to our hospital. Viral hepatitis serology and autoimmune factors were negative. The authors considered liver transplantation, but the patient's liver function spontaneously recovered. Liver biopsy revealed massive infiltration of neutrophils, but the cause of the acute hepatitis was not identified. Four months after discharge, the patient's liver function worsened again. The authors considered the possibility of antinuclear antibody-negative autoimmune hepatitis and initiated steroid treatment, which was effective. Four months after discharge, the patient was admitted for repeated liver injury. The authors started him on steroid pulse therapy, but this time it was not effective. Just before the first admission, he had started his own construction company where he was highly exposed to organic solvents, and thus the authors considered organic solvent-induced hepatitis. Although urine test results for organic solvents were negative, a second liver biopsy revealed severe infiltration of neutrophils, compatible with toxic hepatitis. Again, his liver function spontaneously improved. Based on the pathology and detailed clinical course, including the patient's high exposure to organic solvents since just before the first admission, and the spontaneous recovery of his liver damage in the absence of the exposure, he was diagnosed with toxic hepatitis. The authors strongly advised him to avoid organic solvents. Since then, he has been in good health without recurrence. This is the first report of recurrent acute liver failure because of exposure to organic solvents, which was eventually diagnosed through a meticulous medical history and successfully recovered by avoiding the causative agents. In acute liver failure with an undetermined etiology, clinicians should rule out organic solvent-induced hepatitis.