Consensus Guidelines: Best Practices for the Prevention, Detection and Management of Hepatitis B Virus Reactivation in Clinical Trials with Immunosuppressive/Immunomodulatory Therapy.

Hepatitis B virus reactivation (HBVr) during and after immunosuppressive/immunomodulatory (IS/IM) therapy is associated with significant morbidity and mortality, including hepatic decompensation and acute liver failure. The risk of HBVr with IS/IM has been heterogeneous and often unpredictable. As a result, patients with active or previous HBV infection are often excluded from clinical drug trials of such agents. Thorough screening for HBV infection, antiviral prophylaxis, and careful monitoring for HBVr have proven to be effective in reducing the rate of HBVr and improving its outcome in the context of IS/IM. Therefore, safe enrollment and management of certain HBV-marker-positive patients in clinical trials is possible. There is a great, unmet need for consistent, evidence-based recommendations for best practices pertaining to enrollment, monitoring, and management of HBVr in clinical trial participants receiving IS/IM. The aim of these consensus guidelines is to provide a step-by-step blueprint to safely enroll, monitor and manage the patient with inactive chronic or resolved HBV in IS/IM clinical trials from the time of screening through to the end of post-treatment follow up.

Immunostaining for hepatitis B viral antigens in liver: Association with clinical, biochemical, and virologic features of disease.

BACKGROUND AND AIM: Staining for hepatitis B viral antigens is often done in liver biopsies from patients with chronic hepatitis B, but its correlates with clinical phenotypes are not well described. METHODS: Biopsies were collected from a large cohort of adults and children with chronic hepatitis B viral infection through the Hepatitis B Research Network. Immunohistochemical staining of sections was done for hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) and then centrally read by the pathology committee. The degree of liver injury and pattern of staining were then correlated with clinical characteristics, including the clinical phenotype of hepatitis B. RESULTS: Biopsies from 467 subjects were studied, including 46 from children. Immunostaining for HBsAg was positive in 417 (90%) with scattered hepatocyte staining being the most common pattern. HBsAg staining correlated best with serum levels of HBsAg and hepatitis B viral DNA; the absence of HBsAg staining was often a prelude to loss of HBsAg from serum. HBcAg staining was positive in 225 (49%), and, while cytoplasmic staining was more frequent than nuclear staining, both nuclear and cytoplasmic positivity were often seen in the same specimen. Staining for HBcAg correlated with both level of viremia and liver injury. No biopsies from inactive carriers had stainable HBcAg, while 91% of the biopsies from those with hepatitis B e antigen-positive chronic hepatitis B stained positively for HBcAg. CONCLUSION: Immunostaining for hepatitis B viral antigens may yield helpful insights into liver disease pathogenesis but appears to add little to commonly used serological and biochemical blood tests.

Withdrawal of Long-Term Nucleotide Analog Therapy in Chronic Hepatitis B: Outcomes From the Withdrawal Phase of the HBRN Immune Active Treatment Trial.

INTRODUCTION: Withdrawal of nucleos(t)ide analog therapy is increasingly being evaluated in chronic hepatitis B infection as a strategy to induce hepatitis B surface antigen (HBsAg) loss. The Hepatitis B Research Network Immune-Active Trial evaluated treatment with tenofovir (TDF) for 4 years ± an initial 6 months of peginterferon-α (PegIFN) (NCT01369212) after which treatment was withdrawn. METHODS: Eligible participants (hepatitis B e antigen [HBeAg]-/anti-HBe+, hepatitis B virus [HBV] DNA <10 3 IU/mL, no cirrhosis) who discontinued TDF were followed for at least 1 year with optional follow-up thereafter. Retreatment was based on predefined criteria. RESULTS: Among 201 participants who received 4 years of treatment, 97 participants (45 TDF and 52 TDF + PegIFN arm, 79 Asian) discontinued TDF. HBsAg loss occurred in 5 participants, 2 within 25 weeks and 3 within 89-119 weeks postwithdrawal (cumulative rate 4.3% by 2 years). Alanine aminotransferase (ALT) flares (>5× upper limit of normal) after TDF withdrawal occurred in 36 (37.1%) participants and occurred more frequently and earlier in those HBeAg- compared with HBeAg+ at treatment initiation. ALT flares were associated with older age and higher HBV DNA pretreatment and at the visit before the flare. ALT flares were not significantly associated with HBsAg decline or loss but were associated with immune active disease at 1 year (70.6% vs 11.9%, P < 0.0001) and 2 years (66.7% vs 25.9%, P = 0.03) postwithdrawal. Treatment reinitiation was required in 13 (13.4%) participants, and 13 others remained in a sustained inactive carrier state by the end of the study follow-up. No criteria reliably predicted safe treatment withdrawal. DISCUSSION: Results from this trial do not support TDF withdrawal as a therapeutic strategy. HBsAg loss was infrequent within 2 years of stopping long-term TDF. If withdrawal is considered, HBV DNA should be carefully monitored with reinitiation of therapy if levels rise above 4 log 10 IU/mL to reduce the risk of ALT flares, as they were not associated with subsequent HBsAg decline or loss.

Randomized Trial of Tenofovir With or Without Peginterferon Alfa Followed by Protocolized Treatment Withdrawal in Adults With Chronic Hepatitis B.

INTRODUCTION: Hepatitis B surface antigen (HBsAg) loss is associated with improved long-term outcomes of patients with chronic hepatitis B but is infrequently achieved with current monotherapies. We assessed whether combination strategies that included treatment withdrawal enhanced HBsAg loss. METHODS: A randomized (1:1) trial of tenofovir disoproxil fumarate (TDF) for 192 weeks with or without peginterferon (PegIFN) alfa-2a for the first 24 weeks, followed by withdrawal of TDF at week 192 with 48 weeks of off-treatment follow-up to week 240. The primary end point was HBsAg loss at week 240. RESULTS: Of 201 participants (52% HBeAg positive, 12%/6% genotype A/A2, 7% cirrhosis) randomized to TDF + PegIFN (n = 102) or TDF alone (n = 99), 6 participants had lost HBsAg at the end of the treatment phase (week 192), 5 (5.3%) in the combination group, and 1 (1.0%) in the TDF alone group ( P = 0.09). By week 240, 9 participants had cleared HBsAg, 5.3% in combination, and 4.1% in monotherapy arms ( P = 0.73). HBsAg decline and loss occurred earlier with TDF + PegIFN than TDF, with a ≥1-logIU/mL qHBsAg decline by week 24 in 28% in TDF + PegIFN compared with 6% in TDF ( P = 0.04). HBsAg loss occurred in 7 of 12 (58%) with hepatitis B virus subgenotype A2 (all HBeAg positive) compared with only 2 of 189 (1%) with other hepatitis B virus genotypes and in 8 of 93 (8.6%) HBeAg positive vs 1 of 87 (1.1%) HBeAg negative. DISCUSSION: PegIFN combined TDF followed by protocolized TDF withdrawal led to earlier but not higher percentages of HBsAg clearance. Pretreatment HBeAg positivity and subgenotype A2 were strongly associated with HBsAg clearance.

A Randomized, Dose-Finding, Proof-of-Concept Study of Berberine Ursodeoxycholate in Patients With Primary Sclerosing Cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a fibroinflammatory disease of the bile ducts leading to cirrhosis and hepatic decompensation. There are no approved pharmaceutical therapies for PSC. Berberine ursodeoxycholate (HTD1801) is an ionic salt of berberine and ursodeoxycholic acid with pleiotropic mechanisms of action. METHODS: An 18-week proof-of-concept study was conducted to assess the safety and efficacy of HTD1801 in PSC. This study had three 6-week periods: (i) a placebo-controlled period, (ii) a treatment extension period, and (iii) a randomized treatment withdrawal period. The primary end point was change from baseline in alkaline phosphatase (ALP) at week 6. RESULTS: Fifty-five patients were randomized and treated; 35 (64%) had inflammatory bowel disease and 22 (40%) had previously received ursodeoxycholic acid. Patients were initially randomized to placebo (n = 16), HTD1801 500 mg BID (n = 15), or HTD1801 1000 mg BID (n = 24). At baseline, mean (range) ALP values were 414 U/L (138-1,048), 397 U/L (237-773), and 335 U/L (122-882) for the placebo, HTD1801 500 mg BID, and HTD1801 1,000 mg BID groups, respectively. At week 6, a significant decrease in ALP was observed with HTD1801 (least square mean; HTD1801 500 mg BID = -53 U/L, P = 0.016; HTD1801 1000 mg BID = -37 U/L, P = 0.019) compared with placebo (98 U/L). ALP reductions were sustained through week 18 in those who remained on therapy, whereas ALP increased in those who crossed over to placebo during period 3. HTD1801 was generally well tolerated; 4 patients experienced serious adverse events, none attributed to HTD1801. DISCUSSION: HTD1801 is associated with significant improvement in ALP and warrants further study as a treatment for PSC.

Within-host quantitation of anellovirus genome complexity from clinical samples.

Anellovirus (AV) is a ubiquitous and diverse virus in the human population. An individual can be infected with multiple AV genera and species that form a heterogeneous repertoire, called the anellome. Due to its exceptional genetic diversity, efficient evaluation of anellome complexity remains a methodological challenge. In the current study, AV genome was first enriched from patient serum samples through two-phase rolling circle amplification. Following Illumina sequencing, anellome was analyzed with an advanced bioinformatics pipeline, including read extraction at three similarity levels, de novo assembly, species assignment, and determination of relative abundance among AV variants. The method was validated in the mock sample and then applied to 21 hepatitis C virus (HCV) patients with and without hepatocellular carcinoma (HCC). Overall, there was a large variance regarding AV richness, ranging from 2 to 51 AV species. In contrast to HCV patients without HCC, HCC incidence was associated with reduced richness (12.6 ± 14.4 vs. 35.4 ± 13.6, p = 0.001) and Shannon entropy (0.4 ± 0.34 vs. 0.61 ± 0.12, p = 0.095) at the AV species level. Interestingly, AV genus beta and gamma expanded in the anellome in 7 of 10 HCC patients. These observations shed light on the potential association between anellome and HCC incidence in patients with chronic HCV infection. The method presented here represents a valuable tool to investigate the role of anellome in human health and disease.

A phase 2, proof of concept, randomised controlled trial of berberine ursodeoxycholate in patients with presumed non-alcoholic steatohepatitis and type 2 diabetes.

Non-alcoholic steatohepatitis is frequently associated with diabetes and may cause progressive liver disease. Current treatment options are limited. Here we report on a prospective, randomised, double-blind, placebo-controlled trial of two doses of HTD1801 (berberine ursodeoxycholate, an ionic salt of berberine and ursodeoxycholic acid), versus placebo that was conducted in 100 subjects with fatty liver disease and diabetes (NCT03656744). Treatment was for 18 weeks with a primary endpoint of reduction in liver fat content measured by magnetic resonance imaging proton density fat fraction. Key secondary endpoints included improvement in glycemic control, liver-associated enzymes and safety. The pre-specified primary endpoint was met. Thus, subjects receiving 1000 mg twice a day of berberine ursodeoxycholate had significantly greater reduction in liver fat content than in placebo recipients (mean absolute decrease -4.8% vs. -2.0% (p = 0.011). Compared to placebo, subjects receiving this dose also experienced significant improvement in glycemic control as well as reductions in liver-associated enzymes and significant weight loss. Diarrhea and abdominal discomfort were the most frequently reported adverse events. We conclude that berberine ursodeoxycholate has a broad spectrum of metabolic activity in patients with presumed NASH and diabetes. It is relatively well tolerated and merits further development as a treatment for NASH with diabetes.

Evaluation of Serum-Derived Bovine Immunoglobulin Protein Isolate in Subjects With Decompensated Cirrhosis With Ascites.

Background Bacterial translocation plays a pivotal role in the natural course of cirrhosis and its complications. Serum-derived bovine immunoglobulin (SBI) is an oral medical food that has been shown to both reduce inflammation in the intestines and neutralize bacteria. It represents a unique intervention that has not been studied in this population. Methodology We conducted a prospective open-label trial with an eight-week treatment phase of SBI. Individuals were assessed using lactulose breath testing, serum markers for enterocyte damage and bacterial translocation, and the Chronic Liver Disease Questionnaire (CLDQ) prior to and after completion of the treatment phase. Results We evaluated nine patients with a diagnosis of decompensated cirrhosis with ascites. Subjects had a mean Model for End-Stage Liver Disease (MELD) score of 11.6 ± 3.0 and were not taking lactulose or antibiotics. All subjects tolerated SBI well with no significant adverse events or changes to any of the six domains of the CLDQ. Laboratory tests including liver tests and MELD score remained stable over the course of treatment. There were no significant changes in the rates of small intestinal bacterial overgrowth (55.6% vs 55.6%, p = 1.00) or serum levels of lipopolysaccharide-binding protein, intestinal fatty acid-binding protein, or soluble CD14 (p-values 0.883, 0.765, and 0.748, respectively) when comparing values prior to and immediately after treatment. Conclusions No adverse events or significant changes to the quality of life were detected while on treatment. There were no statistically significant differences in our outcomes when comparing individuals before and after treatment in this small prospective proof-of-concept pilot study. Further prospective randomized studies could be beneficial.

A Pragmatic, Randomized Controlled Trial of Oral Antivirals for the Treatment of Chronic Hepatitis C: The PRIORITIZE Study.

BACKGROUND AND AIMS: Multiple direct-acting antiviral (DAA) regimens are available to treat HCV genotype 1 infection. However, comparative effectiveness from randomized controlled trials of DAA regimens is unavailable. APPROACH AND RESULTS: We conducted a pragmatic randomized controlled trial (NCT02786537) to compare the effectiveness of DAAs for HCV genotype 1a or 1b on viral response, safety, tolerability, and medication nonadherence. Adults with compensated liver disease, HCV genotype 1, not pregnant or breastfeeding, and with health insurance likely to cover ledipasvir/sofosbuvir (LDV/SOF) were recruited from 34 US viral hepatitis clinics. Participants were randomized (± ribavirin) to LDV/SOF, elbasvir/grazoprevir (EBR/GZR), and paritaprevir/ritonavir/ombitasvir+dasabuvir (PrOD; treatment arm stopped early). Primary outcomes included sustained viral response at 12 weeks (SVR12), clinician-recorded adverse events, patient-reported symptoms, and medication nonadherence. Between June 2016 and March 2018, 1,609 participants were randomized. Among 1,128 participants who received ≥1 dose of EBR/GZR or LDV/SOF (± ribavirin), SVR12 was 95.2% (95% CI, 92.8%-97.6%) and 97.4% (95% CI, 95.5%-99.2%), respectively, with a difference estimate of 2.2% (-0.5% to 4.7%), falling within the "equivalence" interval (-5% to 5%). While most (56%) participants experienced adverse events, few were serious (4.2%) or severe (1.8%). In the absence of ribavirin, discontinuations due to adverse events were rare. Patient-reported symptoms and medication nonadherence were similar. Study limitations were dropout due to insurance denial and loss to follow-up after treatment, limiting the ability to measure SVR12. CONCLUSIONS: This pragmatic trial demonstrated high SVR12 for participants treated with EBR/GZR and LDV/SOF with few adverse effects. Overall, the two regimens were equivalent in effectiveness. The results support current HCV guidelines that do not distinguish between ribavirin-free EBR/GZR and LDV/SOF.

A Real-World Observational Cohort of Patients with Hepatocellular Carcinoma: Design and Rationale for TARGET-HCC.

This study describes the design of the TARGET-hepatocellular carcinoma (HCC) cohort and descriptive characteristics of the patient population at diagnosis among those who were enrolled in the cohort across academic and community clinical centers. TARGET-HCC is a 5-year, longitudinal, observational cohort of patients with HCC receiving care in usual clinical practice. Redacted clinical information, obtained from medical records, captures the natural history and management of the disease, including the safety and efficacy of treatment interventions used in usual clinical practice. Patients can complete patient-reported outcome measures and provide biological specimens for future translational studies. The TARGET-HCC study includes adults with histologic, cytologic, or radiologic diagnosis of HCC from academic and community centers in both the United States and Europe. A total of 1,841 participants were enrolled between January 9, 2017, and July 23, 2019, at 67 sites in the United States and Europe. To date, the most common liver disease etiology in the cohort continues to be hepatitis C, although nearly half had a nonviral etiology, including alcohol-related liver disease or nonalcoholic steatohepatitis. Most included patients were diagnosed at an early stage (Barcelona Clinic Liver Cancer Stage [BCLC] 0/A), but only approximately one third underwent curative treatment. Systemic therapy has been used in 7.3% of enrolled patients, including 45.7% of those with BCLC stage C tumors. Conclusion: Overall, the TARGET-HCC cohort allows for the assessment of patient characteristics and investigation of new treatment paradigms and sequencing with existing agents as well as novel regimens for HCC.

Patient-reported outcomes 12 months after hepatitis C treatment with direct-acting antivirals: Results from the PROP UP study.

BACKGROUND & AIMS: The long-term impact of hepatitis C virus (HCV) therapy with all-oral direct-acting antivirals (DAAs) on patient-reported outcomes (PROs) has not been well-described. We characterized changes in PROs from pre-treatment to 12 months post-treatment in a real-world cohort. METHODS: PROP UP was a multi-centre observational cohort study of 1601 patients treated with DAAs at 11 US gastroenterology/hepatology practices from 2015 to 2017. PROs were evaluated pre-treatment (T1) and 12 months post-treatment (T5). A minimally important change (MIC) threshold was prespecified as >5% change in PRO scores from T1 to T5. Multivariable analyses identified predictors of change. RESULTS: Three-quarters of patients were 55 or older; 45% were female, 60% were white, 33% were black, nearly half had cirrhosis. The most commonly-prescribed DAA regimens were sofosbuvir-based (83%) and grazoprevir/elbasvir (11%). Study retention was >95%. On average, small improvements were observed at 3 months post-treatment in all PROs and sustained at 12 months post-treatment among patients with sustained virologic response (SVR). Clinically meaningful improvements were achieved in fatigue (mean change score: -3.7 [-4.2, -3.1]), sleep (mean change score: -3.1 [-3.7, -2.5]), abdominal pain (mean change score: -2.6 [-3.3, -1.9]) and functional well-being (mean change score: -7.0 [-6.0, -8.0]). Symptom improvements were generally not sustained with no SVR (n = 52). Patients with cirrhosis and MELD ≥12 had the greatest improvements in functional well-being (-12.9 [-17.6, -8.1]). CONCLUSIONS: The improvements in patient-reported outcomes reported by patients who achieved SVR following HCV DAA therapy were durable at 12 months post-treatment.

Low Incidence of Adverse Outcomes in Adults With Chronic Hepatitis B Virus Infection in the Era of Antiviral Therapy.

BACKGROUND AND AIMS: Outcomes of persons with chronic hepatitis B virus (HBV) infection in the era of antiviral therapy (AVT) are not well characterized. We determined the incidence and factors associated with clinical outcomes in a multiethnic, North American cohort of adults with chronic HBV infection, who were not on AVT at enrollment. APPROACH AND RESULTS: Adults with chronic HBV infection, not receiving AVT, and without a history of decompensation, HCC, or liver transplantation (LT), were prospectively followed. Participants with known human immunodeficiency virus (HIV), hepatitis C virus, or hepatitis D virus (HDV) coinfection were excluded. During follow-up, treatment could be initiated per standard of care. Clinical outcomes included: incident cirrhosis, decompensation, HCC, OLT, and HBV-related death. Among 1,418 participants analyzed, 51.5% were women, median age was 41.1 years, 75% were Asian, 10% White, 13% Black, 24% HBeAg(+), and 1.5% cirrhosis at baseline. During the study, 274 started treatment, 83 had an alanine aminotransferase flare, 118 of 330 initially HBeAg(+) became HBeAg(-), and 90 of 1,329 became HBsAg(-). After 6,641 person-years follow-up, 8 participants (4 of 21 with baseline cirrhosis) had 12 clinical outcomes (2 decompensation, 5 HCC, 2 OLT, and 3 HBV-related deaths) and 19 of 1,397 had incident cirrhosis. Twenty-one of 26 participants had first outcome before treatment, none had become HBsAg(-), whereas 5/9 HBeAg(+) had become HBeAg(-) at time of first outcome. Cumulative percentage of clinical outcomes was 16% at year 4 in participants with baseline cirrhosis and 2% (including incident cirrhosis) at year 7 in those without. CONCLUSIONS: Incidence of adverse outcomes was low in this closely monitored, large cohort of North American adults with predominantly inactive, chronic HBV without cirrhosis. Our data highlight the benefits of HBsAg loss and the importance of early diagnosis and treatment to prevent cirrhosis and other complications of chronic HBV infection.

Consensus Guidelines: Best Practices for Detection, Assessment and Management of Suspected Acute Drug-Induced Liver Injury During Clinical Trials in Adults with Chronic Viral Hepatitis and Adults with Cirrhosis Secondary to Hepatitis B, C and Nonalcoholic Steatohepatitis.

With the widespread development of new drugs to treat chronic liver diseases (CLDs), including viral hepatitis and nonalcoholic steatohepatitis (NASH), more patients are entering trials with abnormal baseline liver tests and with advanced liver injury, including cirrhosis. The current regulatory guidelines addressing the monitoring, diagnosis, and management of suspected drug-induced liver injury (DILI) during clinical trials primarily address individuals entering with normal baseline liver tests. Using the same laboratory criteria cited as signals of potential DILI in studies involving patients with no underlying liver disease and normal baseline liver tests may result in premature and unnecessary cessation of a study drug in a clinical trial population whose abnormal and fluctuating liver tests are actually due to their underlying CLD. This position paper focuses on defining best practices for the detection, monitoring, diagnosis, and management of suspected acute DILI during clinical trials in patients with CLD, including hepatitis C virus (HCV) and hepatitis B virus (HBV), both with and without cirrhosis and NASH with cirrhosis. This is one of several position papers developed by the IQ DILI Initiative, comprising members from 16 pharmaceutical companies in collaboration with DILI experts from academia and regulatory agencies. It is based on an extensive literature review and discussions between industry members and experts from outside industry to achieve consensus regarding the recommendations. Key conclusions and recommendations include (1) the importance of establishing laboratory criteria that signal potential DILI events and that fit the disease indication being studied in the clinical trial based on knowledge of the natural history of test fluctuations in that disease; (2) establishing a pretreatment value that is based on more than one screening determination, and revising that baseline during the trial if a new nadir is achieved during treatment; (3) basing rules for increased monitoring and for stopping drug for potential DILI on multiples of baseline liver test values and/or a threshold value rather than multiples of the upper limit of normal (ULN) for that test; (4) making use of more sensitive tests of liver function, including direct bilirubin (DB) or combined parameters such as aspartate transaminase:alanine transaminase (AST:ALT) ratio or model for end-stage liver disease (MELD) to signal potential DILI, especially in studies of patients with cirrhosis; and (5) being aware of potential confounders related to complications of the disease being studied that may masquerade as DILI events.

Pharmacokinetics and pharmacodynamics of HTD1801 (berberine ursodeoxycholate, BUDCA) in patients with hyperlipidemia.

BACKGROUND: Reduction in elevated serum cholesterol concentrations is important in the management of individuals at risk of atherosclerotic cardiovascular disease (ASCVD), such as myocardial infarction and thrombotic stroke. Although HMGCoA reductase inhibitors ("statins") are frequently used for this purpose, a significant proportion of patients remain at increased residual risk of ASCVD as they do not adequately address some of the associated co-morbidities such as diabetes and fatty liver disease. METHODS: A double-blind, randomized, placebo-controlled, dose ranging study was carried out that compared three doses of berberine ursodeoxycholate (BUDCA) to placebo in a cohort of subjects with a history of hypercholesterolemia and serum LDL cholesterol levels above 2.59 mmol/L (> 99.9 mg/dL). BUDCA was administered in two divided doses each day for 28 days. The primary endpoints of the study were safety and tolerability of this new compound, as well as its effect in lowering serum lipid and lipoprotein concentrations. RESULTS: A total of 50 subjects were enrolled into three dose cohorts in this study. BUDCA was generally well tolerated, even at doses of 2000 mg per day (the highest dose group); there were no significant adverse effects reported and this highest dose was associated with significant reductions in LDL cholesterol. By day 28 and with the highest dose of BUDCA, there were significant reductions in the serum concentrations of total cholesterol by 8.2% (P = 0.0004) and LDL cholesterol by 10.4% (P = 0.0006), but no significant changes in triglyceride and HDL cholesterol concentrations. CONCLUSIONS: BUDCA is a new single molecular entity that has a significant but modest effect in safely lowering serum LDL-cholesterol concentrations in individuals with a history of hypercholesterolemia. It has a potential use for treating hypercholesterolemia in individuals who cannot take statins, and possibly as adjunctive to other agents, such as ezetimibe or bempedoic acid. TRIAL REGISTRATION: The study was registered on Clinicaltrials.gov ( NCT03381287 ).

A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion.

OBJECTIVE: Owing to the overwhelming dominance of human and commensal microbe sequences, low efficiency is a major concern in clinical viral sequencing using next-generation sequencing. DNA composed of 7-deaza-2'-deoxyguanosine 5'-triphosphate (c7dGTP), an analog of deoxyguanosine triphosphate (dGTP), is resistant to selective restriction enzymes. This characteristic has been utilized to develop a novel strategy for target enrichment in next-generation sequencing. RESULTS: The new enrichment strategy is named target enrichment via enzymatic digestion in next-generation sequencing (TEEDseq). It combined 7-deaza-2'-deoxyguanosine 5'-triphosphate (c7dGTP)-involved primer extension, splinter-assisted intracellular cyclization, c7dGTP)-resistant enzymatic digestion, and two-phase rolling cycle amplification. We first estimated c7dGTP for its efficiency in PCR amplification and its resistance to three restriction enzymes, AluI, HaeIII, and HpyCH4V. We then evaluated TEEDseq using a serum sample spiked with a 1311-bp hepatitis B virus (HBV) fragment. TEEDseq achieved an HBV on-target rate of 3.31 ± 0.39%, which was equivalent to 454× the enrichment of direct Illumina sequencing. Therefore, the current study has provided a concept proof for TEEDseq as an alternative option for clinical viral sequencing that requires an enrichment in next-generation sequencing.

Genome-wide capture sequencing to detect hepatitis C virus at the end of antiviral therapy.

BACKGROUND: Viral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Next-generation sequencing (NGS) provides an alternative option for viral detection in a genome-wide manner. However, owing to the overwhelming dominance of human genetic content in clinical specimens, direct detection of HCV by NGS has a low sensitivity and hence viral enrichment is required. METHODS: Based on template-dependent multiple displacement amplification (tdMDA), an improved method for whole genome amplification (Wang et al., 2017. Biotechniques 63, 21-27), we evaluated two strategies to enhance the sensitivity of NGS-based HCV detection: duplex-specific nuclease (DSN)-mediated depletion of human sequences and HCV probe-based capture sequencing. RESULTS: In DSN-mediated depletion, human sequences were significantly reduced in the two HCV serum samples tested, 65.3% → 55.6% → 33.7% (#4727) and 68.6% → 56% → 21% (#4970), respectively for no normalization, self- and driver-applied normalization. However, this approach was associated with a loss of HCV sequences perhaps due to its micro-homology with the human genome. In capture sequencing, HCV-mapped sequencing reads occupied 96.8% (#4727) and 22.14% (#4970) in NGS data, equivalent to 1936x and 7380x enrichment, respectively. Capture sequencing was then applied to ten serum samples collected at the end of HCV antiviral therapy. Interestingly, the number of HCV-mapped reads was significantly higher in relapsed patients (n = 5) than those from patients with sustained virological response (SVR) (n = 5), 102.4 ± 72.3 vs. 2.6 ± 0.55, p = 0.014. CONCLUSIONS: Our data provides concept evidence for a highly sensitive HCV detection by capture sequencing. The abundance difference of HCV sequencing reads at the end of HCV antiviral therapy could be applied to predict treatment outcomes.

Best practices for detection, assessment and management of suspected immune-mediated liver injury caused by immune checkpoint inhibitors during drug development.

Immune checkpoint inhibitors (ICIs) have shown significant efficacy in patients with various malignancies, however, they are associated with a wide range of immune-related toxicities affecting many organs, including the liver. Immune-mediated liver injury caused by checkpoint inhibitors (ILICI) is a distinctive form of drug induced liver injury (DILI), that differs from most DILI types in presumed underlying mechanism, incidence, and response to therapeutic interventions. Despite increased awareness of ILICI and other immune-related adverse effects of ICIs reflected by recent guidelines for their management in post marketing clinical practice, there is lack of uniform best practices to address ILICI risk during drug development. As efforts to develop safer and more effective ICIs for additional indications grow, and as combination therapies including ICIs are increasingly investigated, there is a growing need for consistent practices for ILICI in drug development. This publication summarizes current best practices to optimize the monitoring, diagnosis, assessment, and management of suspected ILICI in clinical trials using ICI as a single agent and in combination with other ICIs or other oncological agents. It is one of several publications developed by the IQ DILI Initiative in collaboration with DILI experts from academia and regulatory agencies. Recommended best practices are outlined pertaining to hepatic inclusion and exclusion criteria, monitoring of liver tests, ILICI detection, approach to a suspected ILICI signal, causality assessment, hepatic discontinuation rules and additional medical treatment.

DAA therapy and long-term hepatic function in advanced/decompensated cirrhosis: Real-world experience from HCV-TARGET cohort.

BACKGROUND & AIMS: Direct-acting antiviral (DAA) therapy is used in patients with HCV-related decompensated cirrhosis with the expectation of improving hepatic function. However, little is known about the long-term hepatic benefit of successful antiviral treatment. METHODS: Patients with advanced/decompensated cirrhosis (model for end-stage liver disease [MELD] ≥10), in whom NS5A-containing DAA therapy was initiated prior to September 2018, were included (from the HCV-TARGET cohort). Treatment outcomes and the impact of treatment on short-term and long-term hepatic function were examined. RESULTS: A total of 642 patients were analyzed. The mean age was 60 years, 68% were male. The median baseline MELD was 12 (range 10-39) and 64% had prior decompensation. Among patients with available virologic outcomes, 90.5% achieved a sustained virologic response at 12 weeks (SVR12). Eighty (24%) patients achieved a clinically significant decrease in MELD by ≥3 points during short-term follow-up (9-26 weeks after the end of treatment). However, in long-term follow-up (median of 4 years after treatment), mean changes in MELD (-0.30 points), total bilirubin (+0.23 mg/dl) and albumin (+0.36 g/dl) were marginal. Fifty-one patients died and 22 underwent liver transplant. In long-term follow-up, a clinically meaningful decrease in MELD of ≥3 occurred in 29% and a final MELD score of <10 was achieved in 25%. CONCLUSION: In a large real-world experience of patients with advanced/decompensated HCV-related cirrhosis treated with DAAs, there were only marginal improvements in MELD, total bilirubin, or albumin at long-term follow-up (after achieving SVR12). These patients may remain at high risk of decompensation and must continue to be closely monitored. CLINICALTRIALS.GOV: NCT01474811. LAY SUMMARY: Hepatitis C virus infection can now be cured with medications, even in patients who have advanced scarring of the liver (cirrhosis). In this study, we evaluated whether liver function improves or deteriorates in the long-term, following successful treatment of hepatitis C in patients with cirrhosis. We found that overall liver function was relatively stable with only 29% of patients achieving a clinically meaningful improvement in liver function, and we therefore believe that these patients require ongoing monitoring.