Liver steatosis is a major predictor of poor outcomes in chronic hepatitis C patients with sustained virological response.

Sustained virological response (SVR) results in reduced incidence of hepatocellular carcinoma (HCC) and mortality among chronic hepatitis C (CHC) patients with advanced fibrosis. Since both advanced fibrosis and liver steatosis (LS) may coexist in CHC patients, we evaluated their individual effects on a composite outcome of all-cause mortality and HCC in CHC patients with SVR following direct-acting antivirals (DAA) treatment. We retrospectively evaluated inception cohort of 515 CHC patients who achieved SVR following treatment with DAA, with a mean follow-up of 24 months. Baseline liver fibrosis was assessed by transient elastography, and LS was validated by at least three independent ultrasonographic examinations. 211 of 515 patients (41%) had baseline LS. Patients with LS had a higher cumulative rate of all-cause mortality and HCC at 2 years of follow-up compared to patients without LS (15.75% and 2.79%, respectively, P < 0.001), although they did not have increased incidence of advanced fibrosis or cirrhosis. Consistently, multivariate analysis showed that LS was associated with a significant 7.5-fold increased risk of all-cause mortality and HCC (HR 7.51, 95% C.I 3.61-13.36, P < 0.001) even upon adjustment to components of the metabolic syndrome, whereas advanced fibrosis showed only a trend towards statistical significance (HR 2.32, 95% C.I 0.97-6.59, P = 0.06). In conclusion, LS is a major predictor of all-cause mortality and HCC in patients who achieved SVR following DAA treatment regardless of fibrosis stage. These patients should be rigorously screened for HCC.

The Pattern of Elevated Liver Function Tests in Nonalcoholic Fatty Liver Disease Predicts Fibrosis Stage and Metabolic-Associated Comorbidities.

BACKGROUND: Patients with nonalcoholic fatty liver disease (NAFLD) and with abnormal liver function tests (LFTs) most commonly present with elevated hepatocellular enzymes (H pattern), but a subset of patients is found to have elevated cholestatic enzymes (C pattern) or a mixed (M) pattern. AIMS AND METHODS: To determine whether the epidemiologic background and comorbidities, as well as the degree of liver fibrosis, differ between NAFLD patients with different patterns of elevated LFTs by retrospectively analyzing data of 106 patients with a biopsy-proven diagnosis of NAFLD. The pattern of elevated LFTs was determined by adopting the "R-Ratio" formula commonly used for drug-induced liver injury. RESULTS: Advanced fibrosis (F > 2) was found in 15 out of 48 (31.3%) patients with a C pattern of elevated LFTs as compared to 2 out of 44 (4.5%) in M patients and 2 out of 11 (18.2%) in H patients (p = 0.004). Group C patients are older and also had a higher prevalence of diabetes, a higher mean hemoglobin A1c, and a higher prevalence of hypertension, as well as a trend for a higher prevalence of hypertriglyceridemia. CONCLUSIONS: Using a simple formula incorporating routine LFTs can help to categorize NAFLD patients as low or high risk for advanced fibrosis stage and metabolic-associated comorbidities.

Enhanced hepatocarcinogenesis in mouse models and human hepatocellular carcinoma by coordinate KLF6 depletion and increased messenger RNA splicing.

UNLABELLED: KLF6-SV1 (SV1), the major splice variant of KLF6, antagonizes the KLF6 tumor suppressor by an unknown mechanism. Decreased KLF6 expression in human hepatocellular carcinoma (HCC) correlates with increased mortality, but the contribution of increased SV1 is unknown. We sought to define the impact of SV1 on human outcomes and experimental murine hepatocarcinogenesis and to elucidate its mechanism of action. In hepatitis C virus (HCV)-related HCC, an increased ratio of SV1/KLF6 within the tumor was associated with features of more advanced disease. Six months after a single injection of diethylnitrosamine (DEN), SV1 hepatocyte transgenic mice developed more histologically advanced tumors, whereas Klf6-depleted mice developed bigger tumors compared to the Klf6fl(+/+) control mice. Nine months after DEN, SV1 transgenic mice with Klf6 depletion had the greatest tumor burden. Primary mouse hepatocytes from both the SV1 transgenic animals and those with hepatocyte-specific Klf6 depletion displayed increased DNA synthesis, with an additive effect in hepatocytes harboring both SV1 overexpression and Klf6 depletion. Parallel results were obtained by viral SV1 transduction and depletion of Klf6 through adenovirus-Cre infection of primary Klf6fl(+/+) hepatocytes. Increased DNA synthesis was due to both enhanced cell proliferation and increased ploidy. Coimmunoprecipitation studies in 293T cells uncovered a direct interaction of transfected SV1 with KLF6. Accelerated KLF6 degradation in the presence of SV1 was abrogated by the proteasome inhibitor MG132. CONCLUSION: An increased SV1/KLF6 ratio correlates with more aggressive HCC. In mice, an increased SV1/KLF6 ratio, generated either by increasing SV1, decreasing KLF6, or both, accelerates hepatic carcinogenesis. Moreover, SV1 binds directly to KLF6 and accelerates its degradation. These findings represent a novel mechanism underlying the antagonism of tumor suppressor gene function by a splice variant of the same gene.

Gender specific survival rates after deceased donor liver transplantation: A retrospective cohort.

Background: According to the literature, there are sex allocation inequalities in liver transplantation (LT). Sex disparities in outcomes after LT have been debated. This study aimed to evaluate sex-specific outcomes after LT, specifically short-term mortality and long-term survival rates. Methods: A retrospective cohort of the entire LT series from to 2010-2019 in a single center in which the inclusion criteria were adults ≥18 YO age who underwent primary deceased donor LT. Mortality rate was evaluated within 30 days and 6 months. Survival rate was evaluated at 1,3 and 5 years of age. Results: A total of 240 primary and deceased donor LTs (153 men and 87 women) were included. Mean age 55.2Y men and 51.6Y women (p = 0.02). Hepatocellular carcinoma (HCC) was the direct indication in 32.7% of the men and only 17.4% of the women. The leading primary liver morbidities were viral hepatitis (B, C, and D) in 38.3% (N = 92) and nonalcoholic steatohepatitis (NASH) in 20.8% (N = 50) of patients. Thirty-day mortality was 14%, which was significantly higher in men (18%) than in women (8%). Survival rates after 5 years were 64.9% and 78.3%, respectively. Multivariate analysis through logistic regression that included age, direct indication, MELD, and primary liver morbidity revealed statistically significant female to male Odds-Ratio of 0.4 in 30 days, 6 m mortality and a statistically significant higher long-term survival. Conclusions: Our observations revealed better female outcomes, namely, lower short-term mortality and higher long-term survival. Given the consistency after stratification and given the multivariate analysis, this is unlikely to be attributable to confounders. Such findings suggesting consistently better female outcomes have not been previously reported; hence, multi center study is encouraged.

[¹¹C] choline as a potential PET/CT biomarker of liver cirrhosis: A prospective pilot study.

AIM OF THE STUDY: To compare [¹¹C] choline PET/CT findings between patients with cirrhosis and normal liver controls. METHODS: Included 11 patients with cirrhosis and 14 controls. All underwent a dynamic [11C] choline PET/CT. The maximal standard uptake values (SUVmax), the area under the curve (AUC) and kinetic parameters (K1 and K2), clinical and laboratory data, were compared between groups. RESULTS: Patients mean age was 68.4 ±â€¯10.7 and controls, 69.7 ±â€¯7.3 years. Mean SUVmax was higher in patients than controls (right lobe, 10.06 ±â€¯12 vs. 6.3 ±â€¯1.6, P = 0.011; left lobe, 8.6 ±â€¯11.6 vs. 5.4 ±â€¯0.9, P = 0.024; spleen 17.99 ±â€¯27.8 vs. 13.4 ±â€¯2.6, P = 0.027; kidney, 35.9 ±â€¯59.5 vs. 19.3 ±â€¯4.8, P = 0.025) and also AUC values (right lobe, 13,538 ±â€¯20,020 vs. 8427.3 ±â€¯1557.9, P = 0.026; left lobe 12,304 ±â€¯18,871 vs. 6878.9 ±â€¯1294.3, P = 0.024; spleen, 12,875 ±â€¯17,930 vs. 8263.9 ±â€¯1279.2, P = 0.023; kidney, 24,623 ±â€¯36,025 vs. 13,667 ±â€¯3873.9, P = 0.032). No difference in kinetic parameters was found. No correlations between severity of clinical signs and imaging-derived parametric data were found among patients with cirrhosis. CONCLUSIONS: [11C] choline PET/CT may serve as a noninvasive biomarker for patients with cirrhosis.

Response guided therapy for reducing duration of direct acting antivirals in chronic hepatitis C infected patients: a Pilot study.

The advent of direct-acting antivirals (DAAs) has transformed the landscape of hepatitis C virus (HCV) management. We aimed to prospectively (real-time) evaluate the feasibility of using a response-guided therapy approach, based on mathematical modeling of early viral kinetics, to reduce the duration of DAAs therapy. Patients were treated with DAAs according to the physicians' preference. HCV was measured at baseline and at day 2 and weeks 1, 2 and 4 after treatment initiation. The primary endpoint was the proportion of patients with sustained-virological response (SVR) at 12 and/or 24 weeks post-treatment. Twenty-nine patients (mean age 54 ± 16, 44% females, 73% with HCV genotype 1), were enrolled and all completed therapy. Treatment duration was shortened in 11 of the 29 patients (38%). SVR was achieved in 28 of the 29 patients (97%). Relapse occurred post treatment in a single case of a non-cirrhotic male with genotype 3, who was treated with sofosbuvir/velpatasvir for 6 weeks. Virus sequencing did not identify baseline or treatment emergent resistance associated substitutions. Real-time mathematical modeling of early HCV kinetics can be utilized for shortening DAAs duration in approximately 40% of patients without compromising treatment efficacy.Clinical trial registration: ClinicalTrials.gov Identifier: NCT03603327.

Liver steatosis is a strong predictor of mortality and cancer in chronic hepatitis B regardless of viral load.

Liver steatosis may occur concomitantly in patients with chronic hepatitis B infection (CHB) and is implicated in increased morbidity and mortality. Hepatitis B virus (HBV) viral load is a marker for disease progression and long-term outcomes in CHB. We investigated the association between liver steatosis and HBV viral load and their individual effects on all-cause mortality and the development of cancer in patients with CHB and liver steatosis. METHODS: This retrospective study included 524 treatment-naïve patients with CHB, with a mean follow-up of 6 years. Liver biopsy was available for 170 patients and liver steatosis was validated by at least 3 ultrasonographic examinations. RESULTS: A total of 241/524 (46%) patients with CHB had liver steatosis, with a strong correlation between the degree of liver steatosis as assessed by ultrasonography or by liver biopsy (r = 0.9, p < 0.001). Although liver steatosis was not significantly associated with advanced fibrosis, a multivariate analysis showed that liver steatosis was associated with a 4-fold increased risk of all-cause mortality and cancer (hazard ratio 4.35; 95% CI 1.69-8.99; p < 0.001), irrespective of other major metabolic factors. However, baseline HBV viral load was not significantly associated with this composite outcome (hazard ratio 1.65; p = 0.29). In addition, liver steatosis was inversely associated with HBV viral load. CONCLUSION: Patients with CHB and liver steatosis have an increased risk of all-cause mortality and cancer development compared to patients with CHB without liver steatosis, regardless of their baseline HBV viral load. Although tending to have a lower baseline viral load, patients with CHB and liver steatosis should be closely monitored irrespective of viral load. LAY SUMMARY: Patients with chronic hepatitis B infection (CHB) may have liver steatosis at the same time. Here we show that in patients with CHB, liver steatosis is significantly associated with all-cause mortality and cancer, irrespective of other major metabolic factors, and the effect of liver steatosis on mortality and cancer is stronger than the effect of hepatitis B viral load on these outcomes. Thus, patients with CHB and liver steatosis should be closely monitored, irrespective of their viral load.

High frequency of multiclass HCV resistance-associated mutations in patients failing direct-acting antivirals: real-life data.

BACKGROUND: Direct-acting antiviral (DAA) therapy has dramatically increased sustained virological response rates in HCV-infected patients. However, resistance-associated substitutions (RAS) interfering with NS3- and NS5A-targeted therapy, still emerge. This real-life study analysed the type and frequency of RAS in rare cases of patients failing DAA regimens in 12 clinical centres in Israel. METHODS: Blood samples and clinical data from 49 patients who failed various DAAs were collected. RAS identified in the NS3 and NS5A regions by population (Sanger) and next-generation sequencing (NGS) were compared by treatment regimen and HCV subtypes. RESULTS: The majority (71.4%, 35/49) of patients were infected with the genotype (GT)1b strain, while 12.2% (6/49) carried GT1a and 14.3% GT3a/b (7), GT4a (1) and GT1b/GT3a. RAS were identified in 85.7% (42/49) of failures, of which 90.5% (38/42) were clinically relevant RAS (known to be associated with a specific GT and DAA in patients failing therapy or those with more than twofold change in in vitro replicon assays). The most abundant RAS were 168A/E/Q/G/N/V (32.6%, 16/49) identified in NS3, and 93H/N (61.2%, 30/49), 31I/M/V (34.7%, 17/49) and 30R/H/K (12.2%, 6/49), identified in NS5A. Significantly more clinically relevant RAS were identified in NS5A (82.2%, 37/45) than in NS3 (35.7%, 10/28; P<0.01). While RAS were identified in all GT1a, GT3b and GT4a failures (100%, 10/10), only 71.8% (28/39) of GT1b or GT3a failures had RAS (P=0.09). In four cases, NGS identified additional clinically relevant RAS and in one patient, NGS deciphered coexistence of GT3a and GT1b infections. CONCLUSIONS: Our findings, together with additional real-life data, will contribute to the optimization of retreatment in DAA failure, when cost-related and suboptimal regimens must be employed.

Noninvasive scoring systems predict hepatic and extra-hepatic cancers in patients with nonalcoholic fatty liver disease.

BACKGROUND: Liver fibrosis predicts liver-related morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD). Non-invasive scores correlate with the degree of liver fibrosis in these patients. AIMS AND METHODS: To investigate the accuracy of noninvasive scoring systems in predicting long-term outcomes and cancer incidence of patients with NAFLD, we performed a single-center retrospective study of patients with biopsy proven NAFLD. Mean follow up period was 100 months. Outcomes included liver-related complications, hospitalizations, overall mortality and the development of any malignancies. RESULTS: 32 patients had advanced fibrosis (F3-F4) per biopsy at baseline and 121 patients had mild to moderate fibrosis (F0-F2). Both advanced histologic fibrosis stage as well as higher non-invasive scores predicted repeated hospitalizations and longer hospitalization stays. In a multivariate analysis, liver fibrosis (p = 0.002), FIB-4 score (p<0.001), NFS (p<0.001) but not APRI score (p = 0.07) were predictors of overall mortality, and the occurrence of malignancies was associated with higher APRI (p<0.001), FIB-4 (p<0.001) and NFS (p = 0.008) scores, but not with advanced fibrosis, as determined by liver biopsy (p = 0.105). CONCLUSIONS: In NAFLD patients, noninvasive scoring systems are good predictors of morbidity and mortality and may have an additive value in predicting the development of hepatic and extra-hepatic cancers.

Stereotactic body radiation therapy (SBRT) for definitive treatment and as a bridge to liver transplantation in early stage inoperable Hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Stereotactic body radiotherapy (SBRT) is an emerging modality for definitive treatment of Hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This retrospective study included all early stage HCC patients who were not candidates for primary resection and/or local therapy, treated with SBRT between 11/2011 and 1/2016. RESULTS: Twenty-three patients were included. The median age was 62 years; 70% males; 30% females; 70% viral hepatitis carriers; 100% cirrhotic; 13 Child Pugh [CP]-A and 10 [CP]-B. The median tumor volume was 12.7cm3 (range, 2.2-53.6 cm3). Treatment was well tolerated. With the exception of one patient who developed RILD, no other patient had significant changes in 12 weeks of laboratory follow-up. SBRT was a bridge to transplantation in 16 patients and 11 were transplanted.. No surgical difficulties or complications were reported following SBRT, and none of the transplanted patients had local progression before transplantation. The median prescribed dose to the tumor was 54Gy (range, 30-54Gy), the median dose to the uninvolved liver was 6.0Gy(range, 1.6-12.6Gy). With a median follow-up time of 12 months, the median overall-survival for the 11 transplanted patients was not reached (range, 2.0-53.7+ months) and was 23 months for the 12 non-transplanted patients. The median progression-free survival for the transplanted patients was not reached (54+ months) and was 14.0 months for the non-transplanted patients. There was no SBRT-related mortality. Liver explant post SBRT revealed pathological complete response in 3(27.3%), pathological partial response in 6(54.5%), and pathological stable disease in 2(18.2%) tumors. CONCLUSIONS: SBRT is safe and effective and can be used as a bridge to transplantation without comprising the surgical procedure.

HCV genotype-1 subtypes and resistance-associated substitutions in drug-naive and in direct-acting antiviral treatment failure patients.

BACKGROUND: Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. METHODS: In a multicentre cohort study (n=308), NS3 or NS5B sequencing (n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. RESULTS: GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. CONCLUSIONS: NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.

Radiotherapy and Sorafenib in the Management of Patients with Hepatocellular Carcinoma Have Led to Improved Survival: A Single Center Experience.

BACKGROUND & AIMS: Hepatocellular Carcinoma (HCC) is the sixth most common malignancy and the third most common cause of cancer mortality worldwide. We aimed to assess the effect of novel treatment options on the survival of HCC patients. METHODS: This retrospective study included all HCC patients diagnosed between 2000 and 2013 referred to the Davidoff center and treated by a multidisciplinary team. RESULTS: The analysis included 321 patients (median age, 64 years; 74.8% males; 74.1% viral carriers; 76.0% cirrhosis; 56.7% diagnosis at an early stage). The estimated hazard ratio by multivariate analysis for the effect of the period of diagnosis (2007-2013 vs. 2000-2006) on survival was 0.72 (95% CI: 0.54-0.96; p=0.027). There was no difference in the distribution by CP score, by BCLC stage at diagnosis or in the proportion of patients undergoing surgical procedures (liver transplantation or resection). In the later time frame, there was a significant decrease in the proportion of patients undergoing percutaneous treatments (14.6% vs.4.2%, p=0.004) and embolization (46.9% vs.24.6%, p=0.001), and a significant increase in radiotherapy (1.5% vs. 8.4%, p=0.009) and treatment with sorafenib (6% vs. 18.3%, p=0.002). CONCLUSION: Technological/pharmaceutical innovations have led to advancement in HCC treatment. Since there was no significant difference in the proportion of patients undergoing surgical procedures during the evaluated timeframe, the improved survival may stem from better management of advanced stage patients by a multidisciplinary team.

Hepatocyte growth factor enhances alternative splicing of the Kruppel-like factor 6 (KLF6) tumor suppressor to promote growth through SRSF1.

Alternative splicing of the Krüppel-like factor 6 (KLF6) tumor suppressor into an antagonistic splice variant 1 (SV1) is a pathogenic event in several cancers including hepatocellular carcinoma (HCC) because elevated SV1 is associated with increased tumor metastasis and mortality. Ras activation is one factor that can enhance KLF6 splicing in cancer cells, however pathways driving KLF6 splicing are unknown. Splice site selection is regulated by splice factors that include serine/arginine-rich (SR) proteins such as SRSF1 (ASF-SF2), which in turn is controlled by phosphoinositide 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) signaling pathway. Because signaling pathways downstream of the liver mitogen hepatocyte growth factor (HGF) include Akt, we explored whether HGF induces KLF6 alternative splicing. In HepG2 cells, HGF (25 ng/mL) significantly increases the ratio of SV1/KLF6 full by 40% through phosphorylation of Akt and subsequent downregulation of two splicing regulators, SRSF3 (SRp20) and SRSF1. Decreased SRSF3 levels regulate SRSF1 levels by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD), which stimulates cell growth by decreasing p21 levels. Enhanced cell replication through increased KLF6 alternative splicing is a novel growth-promoting pathway of HGF that could contribute to the molecule's mitogenic activity in physiologic liver growth and hepatocellular carcinoma.

Current status of novel antifibrotic therapies in patients with chronic liver disease.

Fibrosis accumulation is a dynamic process resulting from a wound-healing response to acute or chronic liver injury of all causes. The cascade starts with hepatocyte necrosis and apoptosis, which instigate inflammatory signaling by chemokines and cytokines, recruitment of immune cell populations, and activation of fibrogenic cells, culminating in the deposition of extracellular matrix. These key elements, along with pathways of transcriptional and epigenetic regulation, represent fertile therapeutic targets. New therapies include drugs specifically designed as antifibrotics, as well as drugs already available with well-established safety profiles, whose mechanism of action may also be antifibrotic. At the same time, the development of noninvasive fibrogenic markers, and techniques (e.g. fibroscan), as well as combined scoring systems incorporating serum and clinical features will allow improved assessment of therapy response. In aggregate, the advances in the elucidation of the biology of fibrosis, combined with improved technologies for assessment will provide a comprehensive framework for design of antifibrotics and their analysis in well-designed clinical trials. These efforts may ultimately yield success in halting the progression of, or reversing, liver fibrosis.