Burden of grade 3 or 4 liver injury associated with immune checkpoint inhibitors.

Background & Aims: There is concern about the burden of liver injury in patients with cancer exposed to immune checkpoints inhibitors (ICIs). Methods: In a retrospective cohort study, we evaluated the likelihood of grade 3/4 liver injury, of grade 3/4 cholestatic liver injury, and of liver failure, as per the Common Terminology Criteria for Adverse Events (CTCAE) version 5, following treatment with ICIs. We compared these occurrences with a group of cancer patients who were propensity-matched and treated with conventional chemotherapy. For all ICI patients experiencing grade 3/4 liver injury, we conducted a causality assessment using the RUCAM method and examined patient outcomes. Results: Among 952 patients (median [IQR] age 66 [57-73] years, 64% males) who were treated with ICI between January 1, 2015, and December 31, 2019, a total of 86 (9%) progressed to grade 3/4 liver injury, and liver failure was not observed. Anti-PD-(L)1/anti-CTLA-4 antibodies combinations (adjusted hazard ratio 3.36 [95% CI: 1.67-6.79]; p <0.001), and chronic hepatitis B (adjusted hazard ratio 5.48 [95% CI: 1.62-18.5]; p = 0.006], were independent risk factors. Liver injury was attributed to ICI treatment in 19 (2.0%) patients. Patients with ICI toxicity typically presented with granulomatous hepatitis or cholangiocyte inflammation. ICI withdrawal was associated with cancer progression and mortality. Re-introduction of ICI was not associated with recurrent grade 3/4 liver injury. Compared with matched patients treated with conventional, non-ICI-based chemotherapy, anti-PD-(L)1/anti-CTLA-4 combinations (p <0.001) and anti-PD-(L)1 monotherapies (p = 0.053) increased the risk of grade 3/4 liver injury and of grade 3/4 cholestatic liver injury, respectively. Conclusions: An increased risk of grade 3/4 liver injury under anti-PD-(L)1/anti-CTLA-4 antibodies was observed, whereas no substantial increase in the likelihood of liver failure occurred even after treatment reintroduction. Impact and implications: There is concern about liver injury in patients with cancer exposed to immune checkpoints inhibitors (ICIs). We investigated the burden of grade 3/4 liver injury after treatment with ICIs in a multicentric cohort of patients with cancer. Overall, a 9% incidence of grade 3/4 liver injury was detected after ICIs, and direct ICI hepatotoxicity was demonstrated in 2% of patients. Anti-PD-(L)1/Anti-CTLA-4 antibody combinations, and chronic HBV infection were independent risk factors. ICI withdrawal for grade 3/4 liver injury was associated with cancer progression. Re-introduction of ICI treatment was not associated with recurrent grade 3/4 liver injury.

Burden of liver disease progression in hospitalized patients with type 2 diabetes mellitus.

BACKGROUND & AIMS: There are uncertainties regarding the burden of liver disease in patients with type 2 diabetes (T2D). Thus, we aimed to quantify the burden of liver disease, identify risk factors, and estimate attributable risks in patients with T2D. METHODS: We measured adjusted hazard ratios of liver disease progression to hepatocellular carcinoma and/or decompensated cirrhosis in a 2010-2020 retrospective, bicentric, longitudinal, cohort of 52,066 hospitalized patients with T2D. RESULTS: Mean age was 64±14 years and 58% were men. Alcohol use disorders accounted for 57% of liver-related complications and were associated with all liver-related risk factors. Non-metabolic liver-related risk factors accounted for 37% of the liver burden. T2D control was not associated with liver disease progression. The incidence (95% CI) of liver-related complications and of competing mortality were 3.9 (3.5-4.3) and 27.8 (26.7-28.9) per 1,000 person-years at risk, respectively. The cumulative incidence of liver disease progression exceeded the cumulative incidence of competing mortality only in the presence of well-identified risk factors of liver disease progression, including alcohol use. The incidence of hepatocellular carcinoma was 0.3 (95% CI 0.1-0.5) per 1,000 person-years in patients with obesity and it increased with age. The adjusted hazard ratios of liver disease progression were 55.7 (40.5-76.6), 3.5 (2.3-5.2), 8.9 (6.9-11.5), and 1.5 (1.1-2.1), for alcohol-related liver disease, alcohol use disorders without alcohol-related liver disease, non-metabolic liver-related risk factors, and obesity, respectively. The attributable fractions of alcohol use disorders, non-metabolic liver-related risk factors, and obesity to the liver burden were 55%, 14%, and 7%, respectively. CONCLUSIONS: In this analysis of data from 2 hospital-based cohorts of patients with T2D, alcohol use disorders, rather than obesity, contributed to most of the liver burden. These results suggest that patients with T2D should be advised to drink minimal amounts of alcohol. LAY SUMMARY: There is uncertainty on the burden of liver-related complications in patients with type 2 diabetes. We studied the risks of liver cancer and complications of liver disease in over 50,000 patients with type 2 diabetes. We found that alcohol was the main factor associated with complications of liver disease. This finding has major implications on the alcohol advice given to patients with type 2 diabetes.

Histopathologic Features of Chilblainlike Lesions Developing in the Setting of the Coronavirus Disease 2019 (COVID-19) Pandemic.

CONTEXT.­: During the coronavirus disease 2019 pandemic, several studies have described a distinctive cutaneous manifestation with a clinical picture resembling chilblains or chilblain lupus in young patients. OBJECTIVE.­: To report the histopathologic description of a series of chilblainlike lesions appearing in the context of the severe acute respiratory syndrome coronavirus 2 epidemic. DESIGN.­: The study included 13 patients with cutaneous acral lesions resembling chilblains occurring in the setting of suspected severe acute respiratory syndrome coronavirus 2 infection with available skin biopsy. RESULTS.­: Two main histopathologic patterns were observed: a chilblainlike histopathologic pattern (10 of 13 cases; 77%) and a thrombotic vasculopathy pattern (3 of 13 cases; 23%). The chilblainlike histopathologic pattern featured a superficial and deep perivascular infiltrate of lymphocytes of varying intensity. This infiltrate was sometimes peri-eccrine and alterations of eccrine glands were present in most cases. Vacuolar alteration of the basal layer of the epidermis was found in a majority of patients. Lichenoid interface dermatitis was rarely present. The thrombotic vasculopathy pattern featured an absent or mild inflammatory infiltrate, multiple intraluminal fibrin thrombi, and ischemic epidermal necrosis. In both patterns, no true vasculitis was observed. No patient tested positive for severe acute respiratory syndrome coronavirus 2 by polymerase chain reaction, possibly because these lesions may represent late cutaneous manifestations of the disease or are associated with an early effective immune response. CONCLUSIONS.­: The relationship of chilblainlike lesions to severe acute respiratory syndrome coronavirus 2 requires further investigations. Histopathologic features mimic chilblains, chilblain lupus, and, less frequently, a thrombotic vasculopathy. Response to viral infection might trigger diverse mechanisms leading to the 2 histopathologic patterns described.

Gene Therapy in Patients with Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Gene Therapy in a Patient with Sickle Cell Disease.

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

Production of TNF-alpha ex vivo is predictive of an immune response to flu vaccination in a frail elderly population.

OBJECTIVE: To investigate the relationship between the response to influenza vaccination and the ability to produce proinflamatory cytokines in elderly subjects. METHODS: Whole blood samples from 25 elderly subjects collected before influenza vaccination were stimulated with the influenza vaccine in order to evaluate the secretion of five specific cytokines: TNFα, IFNα, IFNγ, IL2 and IL10. The results were correlated with the increased HAI antibody titres two weeks after vaccination. RESULTS: Only 30% of elderly individuals seroconverted after vaccination. Although 50 to 70% of the cohort did not produce TNFα, IFNα, IFNγ, IL2 or IL10, all of the individuals who seroconverted were able to produce TNFα. Furthermore production of IFNγ, with or without production of IFNα/ß, was not associated with a better response to the vaccine. CONCLUSION: Production of TNFα appears to be primordial for an efficient vaccine response, and may provide a predictive marker for the humoral response to vaccination. It may also provide the basis for evaluating agents designed to rescue TNFα-producing cells. This study emphasises a need to rescue TNF-producing cell function.

Involvement of NK Cells and NKp30 Pathway in Antisynthetase Syndrome.

Antisynthetase syndrome (aSS) is characterized by the association of interstitial lung disease and myositis with anti-tRNA synthetase autoantibodies. Immune mechanisms leading to aSS could be initiated in the lungs, but the role of NK cells has not yet been studied. Both extensive NK cell phenotype and functions were compared between 33 patients and 26 controls. Direct and redirected polyfunctionality assays (degranulation and intracellular production of TNF-α and IFN-γ) were performed spontaneously or after IL-12 plus IL-18 stimulation in the presence of K562 or P815 target cells, respectively. NK cells from inactive patients showed normal phenotype, whereas active aSS revealed a differentiated NK cell profile, as indicated by increased CD57 and Ig-like transcript 2 and an inability to produce IFN-γ (p = 0.002) compared with controls. Importantly, active aSS was more specifically associated with a significant NKp30 decrease (p = 0.009), although levels of mRNA and intracellular protein were similar in aSS and healthy controls. This NKp30 decrease was strongly correlated with reduced NK cell polyfunctionality in both direct and redirected killing assays with anti-NKp30 Abs (p = 0.009 and p = 0.03, respectively), confirming its important impact in aSS. Histological studies revealed massive infiltrations of NK cells inside the lungs of aSS patients (148 versus 11/mm(2)). Taken together, these data suggest that NK cells and NKp30 could play a role in aSS pathogenesis.

Recognition of core-derived epitopes from a novel HBV-targeted immunotherapeutic by T-cells from patients infected by different viral genotypes.

Hepatitis B virus (HBV) infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Current therapies based on nucleos(t)ide analogs or pegylated-interferon-α lead to control of viral replication in most patients but rarely achieve cure. A potential strategy to control chronic hepatitis B is to restore or induce functional anti-HBV T-cell immune responses using HBV-specific immunotherapeutics. However, viral diversity is a challenge to the development of this class of products as HBV genotypes display a sequence diversity of up to 8%. We have developed a novel HBV-targeted immunotherapeutic, TG1050, based on a non-replicative Adenovirus vector encoding a unique and large fusion protein composed of multiple antigenic regions derived from a HBV genotype D sequence. Using peripheral blood mononuclear cells from 23 patients chronically infected by five distinct genotypes (gt A, B, C, D and E) and various sets of peptides encompassing conserved versus divergent regions of HBV core we have measured ability of TG1050 genotype D core-derived peptides to be recognized by T-cells from patients infected by various genotypes. Overall, PBMCs from 78% of genotype B or C- and 100% genotype A or E-infected patients lead to detection of HBV core-specific T-cells recognizing genotype D antigenic domains located both in conserved and variable regions. This proof-of-concept study supports the clinical development of TG1050 in large patient populations independently of infecting genotypes.

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice.

OBJECTIVE: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). METHODS: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. RESULTS: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. CONCLUSIONS: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Benefits associated with antiviral treatment in kidney allograft recipients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is more frequent in kidney recipients than in the general population with a higher rate of liver-related morbidity and mortality. We evaluated the benefit associated with HBV viral suppression by nucleos(t)ide analogues treatment in HBV-infected kidney recipients. METHODS: This retrospective study included 42 HBsAg-positive kidney recipients, 33 males, 9 females, median age 54 years, followed up during a mean of 15.4±11.8 years after kidney transplantation. Mean treatment duration by single or combined nucleos(t)ide analogues was 6.8±4.3 years. Fibrosis, before treatment, according to Metavir score was: F4 for 6 patients, F3 for 10, F2 for 6, and F0-F1 for 20 patients. The primary end point, the patient survival, was defined as patient death or liver transplantation, the secondary end point was graft survival. RESULTS: HBV DNA at the last evaluation was undetectable (<12 IU/ml) in 92.8% of patients. During the follow-up, 8 patients died (17.7%), death being related to hepatocellular carcinoma in 4 (9.5%), including 1 patient with baseline mild fibrosis, and to extrahepatic causes in 4. This mortality rate is strikingly lower than that previously reported in HBV-infected kidney recipients before oral antiviral therapies. Graft survival seems to be improved when compared to the former series. CONCLUSIONS: Suppression of HBV replication associated with nucleo(s)tide analogues treatment improves the survival of HBV-infected kidney recipients. Viral suppression does not exclude regular follow-up given the risk of occurrence of hepatocellular carcinoma even in non-cirrhotic patients.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists.

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes.

BACKGROUND & AIMS: Although hepatitis C virus (HCV) can be grown in the hepatocarcinoma-derived cell line Huh-7, a cell-culture model is needed that supports its complete, productive infection cycle in normal, quiescent, highly differentiated human hepatocytes. We sought to develop such a system. METHODS: Primary cultures of human adult hepatocytes were inoculated with HCV derived from Huh-7 cell culture (HCVcc) and monitored for expression of hepatocyte differentiation markers and replication of HCV. Culture supernatants were assayed for HCV RNA, core antigen, and infectivity titer. The buoyant densities of input and progeny virus were compared in iodixanol gradients. RESULTS: While retaining expression of differentiation markers, primary hepatocytes supported the complete infectious cycle of HCV, including production of significant titers of new infectious progeny virus, which was called primary-culture-derived virus (HCVpc). Compared with HCVcc, HCVpc had lower average buoyant density and higher specific infectivity; this was similar to the characteristics of virus particles associated with the very-low-density lipoproteins that are produced during in vivo infection. These properties were lost after re-culture of HCVpc in poorly differentiated Huh-7 cells, suggesting that authentic virions can be produced only by normal hepatocytes that secrete authentic very-low-density lipoproteins. CONCLUSIONS: We have established a cell-culture-based system that allows production of infectious HCV in physiologically relevant human hepatocytes. This provides a useful tool for the study of HCV interactions with its natural host cell and for the development of antiviral therapies.

One-step assay for quantification of neutralizing antibodies to biopharmaceuticals.

Assessment of immunogenicity is an important part of biopharmaceutical drug safety evaluation and a prerequisite for the development of less immunogenic and safer biopharmaceuticals since anti-drug antibodies can impair the activity and compromise the safety of biopharmaceuticals. Although regulatory authorities recommend cell-based assays for detection of neutralizing antibodies (NAbs), such assays are difficult to standardize, and ill adapted to high-throughput analysis. These limitations have been overcome by the development of a unique one-step cell-based assay that allows both drug activity and drug NAbs to be quantified rapidly and with a high degree of precision simply be adding reporter cells to a sample. The reporter cells have been engineered to express firefly luciferase (FL) under the control of a drug-responsive promoter, and to express the drug of interest, the production of which is normalized relative to the expression of Renilla luciferase (RL) transcribed from a common doxycycline-inducible promoter. Residual drug levels present in a sample are first quantified by determination of FL expression, autocrine drug synthesis is then induced, and NAb activity is quantified from the difference in the ratio of FL/RL expression in the presence or absence of the sample. Since assay results are normalized relative to the expression of an internal standard, results are independent of cell number or differences in cell viability thus affording a high degree of assay precision and reducing serum matrix effects to a minimum. This unique assay platform is ideally suited for high-throughput analysis, is applicable to most biopharmaceuticals, and will facilitate standardization and comparison of immunogenicity data. The performance of the one-step assay is illustrated for interferon alpha2 (IFNalpha2) used widely to treated chronic hepatitis C (HCV) infection and neoplastic disease.

Human bocavirus infection in hospitalized children during winter.

Human bocavirus (HBoV) has recently been described as a common agent of acute upper and lower respiratory tract infections in children. We screened by polymerase chain reaction for HBoV nucleic acid nasopharyngeal aspirates from hospitalized children with negative culture and immunofluorescence assay for respiratory syncytial virus, influenza viruses, adenovirus, and parainfluenza viruses. HBoV was detected in 32 children (5.5%) and was the second virus identified in nasopharyngeal aspirates after respiratory syncytial virus. Most of the children had severe disease.

Culture-proven herpetic keratitis after penetrating keratoplasty in patients with no previous history of herpes disease.

OBJECTIVE: To report three cases of herpetic infection in recipients of organ-cultured donor corneas among 586 consecutive corneal transplantation procedures. METHODS: Three patients with no history of symptomatic herpes infection underwent corneal transplantation for keratoconus (2 patients) and Fuchs dystrophy (1 patient). Two patients developed keratouveitis and primary graft failure. The third patient developed dendritic keratitis in the graft. Culture of corneal scrapings and the patient's bandage contact lens were positive for herpes simplex virus type 1 (HSV-1). Donor and recipient sera were tested for HSV serology by EIA. Recipient corneal buttons were studied by means of transmission electron microscopy and immunohistochemistry. The three HSV-1 strains were genotyped by sequencing part of a variable antigenic domain of glycoprotein B (gB). RESULTS: None of the donor corneas showed endothelial cell necrosis after organ culture. All keratoplasties performed with the three mate donor corneas had an uncomplicated course. All three donor sera were positive for HSV. Preoperative recipient sera were positive for HSV. Analysis of the recipient corneal buttons showed no evidence of herpetic infection. Sequence analysis revealed three different gB genotypes. CONCLUSION: Ascertaining that a postoperative herpetic infection in a corneal transplant originates from the donor tissue is still difficult. Although some features of the reported cases suggest donor-to-host transmission of herpes simplex virus, the recipients could have been the source of the virus.