Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease with no effective curative therapies. Necroptosis, a regulated necrotic cell death pathway controlled by receptor-interacting protein kinase 1 (RIPK1), has emerged as a potential driver of inflammation and fibrosis in chronic liver disorders. We investigated the role of necroptosis in PSC and whether RIPK1 inhibition could modify disease course and progression. Spatial profiling of human PSC biopsies revealed that necroptosis primarily affects cholangiocytes, while apoptosis was more frequent in hepatocytes and nonbiliary cells. In vitro, necroptosis inhibition protected cholangiocytes, and RIPK1 deletion conferred resistance to TNF-mediated cytotoxicity. In a murine model of PSC, pharmacological RIPK1 inhibition reduced cholestatic injury, hepatic inflammation, and biliary fibrosis. Multiomic analyses comprehensively demonstrated reprogramming toward wild-type-like profiles following treatment. These findings identify necroptosis as a critical effector in PSC and highlight RIPK1 inhibition as a promising disease-modifying approach, opening the door to targeted necroptosis-based therapies for this otherwise untreatable disease.

Current treatment strategies for hepatitis B virus (HBV) are lifelong and rarely curative. Finite therapeutic strategies aim to restore efficient immune responses in chronically infected patients for functional cures. Here, we investigate the impact of treatment with the capsid assembly modulator (CAM) GLP-26 in an immunocompetent humanized mouse model of chronic HBV. GLP-26 treatment clears viremia and HBV surface antigen (HBsAg), reduces hepatitis, and potentiates anti-HBs antibody (HBsAb) development. Remarkably, after discontinuing GLP-26, either viral control or viral rebound occurs in half the cohort. In particular, the viral controllers display seroconversion (HBsAg-/HBsAb+) and development of neutralizing antibodies. In the liver, cytotoxic natural killer cells and activated memory T cells are enriched, including HBV-specific polyfunctional T cell responses. Thus, finite treatment with a potent antiviral CAM results in viral clearance and antigen depletion, enabling effective adaptive immune responses and viral control off therapy, suggestive of a functional cure.

Résumé Les carcinomes hépatocellulaires (CHC) sont les tumeurs hépatiques primitives du foie les plus fréquentes. Ils représentent la seconde cause de mortalité liée au cancer dans le monde. Les principaux facteurs étiologiques incluent les hépatites virales B et C, la prise chronique d’alcool et les syndromes métaboliques. Nous focaliserons cette revue sur l’impact de l’infection par le virus de l’hépatite B (VHB) et la progression de l’atteinte hépatique vers le CHC. Des mécanismes à la fois directs et indirects à l’infection par le VHB contribuent à l’émergence d’un CHC. Nous aborderons ces deux types de mécanismes en nous concentrant sur les effets viraux directs, explorés dans de très nombreuses études in vivo et in vitro.Les résultats publiés peuvent parfois être contradictoires. Ils varient notamment en fonction du modèle d’étude, de la séquence virale, du niveau d’expression du VHB, du type de génome viral transféré (partiel ou complet), de la méthode de transfert, générant une expression stable ou transitoire, et de bien d’autres paramètres. Néanmoins, les connaissances acquises récemment sur le cycle viral, et en particulier sur la nature du récepteur hépatocytaire du VHB, permettent aujourd’hui de reproduire in vitro un cycle viral complet dans différents modèles expérimentaux et ainsi de mieux apprécier les activités pro-tumorales de l’infection par le VHB.Cette revue est bien évidemment non exhaustive et les connaissances, notamment sur la prise en charge clinique et thérapeutique du CHC qu’il soit associé à la présence du VHB ou non, sont actuellement en pleine évolution avec l’essor des immunothérapies combinées.

Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry. In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.

Diethylnitrosamine (DEN) is a chemical hepatocarcinogenic agent that triggers a large array of oncogenic mutations after a single injection. Initiated hepatocytes subsequently undergo clonal expansion within a proliferative environment, rendering the DEN model a comprehensive carcinogen. In rodent studies, DEN finds extensive utility in experimental liver cancer research, mimicking several aspects of human hepatocellular carcinoma (HCC), including angiogenesis, metabolic reprogramming, immune exhaustion, and the ability to metastasize. Beyond the wealth of scientific insights gleaned from this model, the objective of this chapter is to review morphological, genomic, and immunological characteristics associated to DEN-induced HCC. Furthermore, this chapter provides a detailed procedural guide to effectively induce hepatocarcinogenesis in mice through a single intraperitoneal injection of DEN.

Supplementary Table 5: Difference in gene expression between Immune High (IH) and Immune Low (IL) subgroups from the validation dataset (n=20, Coulouarn et al, Carcinogenesis, 2012) (33).

Background and Aims: Hepatitis B virus (HBV) infection causes oxidative stress (OS) and alters mitochondria in experimental models. Our goal was to investigate whether HBV might alter liver mitochondria also in humans, and the resulting mitochondrial stress might account for the progression of fibrosis in chronic hepatitis B (CHB). Approach and Results: The study included 146 treatment‐naïve CHB mono‐infected patients. Patients with CHB and advanced fibrosis (AF) or cirrhosis (F3‐F4) were compared to patients with no/mild‐moderate fibrosis (F0‐F2). Patients with CHB were further compared to patients with chronic hepatitis C (CHC; n = 33), nonalcoholic steatohepatatis (NASH; n = 12), and healthy controls ( n = 24). We detected oxidative damage to mitochondrial DNA (mtDNA), including mtDNA strand beaks, and identified multiple mtDNA deletions in patients with F3‐F4 as compared to patients with F0‐F2. Alterations in mitochondrial function, mitochondrial unfolded protein response, biogenesis, mitophagy, and liver inflammation were observed in patients with AF or cirrhosis associated with CHB, CHC, and NASH. In vitro , significant increases of the mitochondrial formation of superoxide and peroxynitrite as well as mtDNA damage, nitration of the mitochondrial respiratory chain complexes, and impairment of complex I occurred in HepG2 cells replicating HBV or transiently expressing hepatitits B virus X protein. mtDNA damage and complex I impairment were prevented with the superoxide‐scavenging Mito‐Tempo or with inducible nitric oxide synthase (iNOS)–specific inhibitor 1400 W. Conclusions: Our results emphasized the importance of mitochondrial OS, mtDNA damage, and associated alterations in mitochondrial function and dynamics in AF or cirrhosis in CHB and NASH. Mitochondria might be a target in drug development to stop fibrosis progression.

The authors regret that in the original publication, in Table 1 IHC (3+) positivity for GEA biopsy was defined incorrectly as "≥1 Cluster of positive tumor cells with weak to moderate staining, regardless of proportion of positive cells". This should read "≥1 Cluster of positive tumor cells with strong staining, regardless of proportion of positive cells". The authors would like to apologise for any inconvenience caused. Targeting the complexity of ERBB2 biology in gastroesophageal carcinomaAnnals of OncologyVol. 33Issue 11PreviewERBB2 is the most prominent therapeutic target in gastroesophageal adenocarcinoma (GEA). For two decades, trastuzumab was the only treatment available for GEA overexpressing ERBB2. Several drugs showing evidence of efficacy over or in complement to trastuzumab in breast cancer failed to show clinical benefit in GEA. This resistance to anti-ERBB2 therapy is peculiarly recurrent in GEA and is mostly due to tumor heterogeneity with the existence of low expressing ERBB2 tumor clones and loss of ERBB2 over time. Full-Text PDF Open Archive

IntroductionOral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and MethodsCells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. ResultsNormal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4(+) T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4(+) T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8(+) T lymphocytes among CD45(+) cells in NSND patients was associated with a better disease-free survival (p=0.004). ConclusionGranulocytes, monocytes-macrophages, and CD4(+) T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4(+) T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8(+) T cells in NSND patients could help defining the prognostic of OSCC patients.

AbstractPurpose:Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare malignancy associated with an overall poor prognosis. We aimed to investigate the immune profile of cHCC-CCA and determine its impact on disease outcome.Experimental Design:We performed a multicenter study of 96 patients with cHCC-CCA. Gene expression profile was analyzed using nCounter PanCancer IO 360 Panel. Densities of main immune cells subsets were quantified from digital slides of IHC stainings. Genetic alterations were investigated using targeted next-generation sequencing.Results:Two main immune subtypes of cHCC-CCA were identified by clustering analysis: an “immune-high” (IH) subtype (57% of the cases) and an “immune-low” (IL) subtype (43% of the cases). Tumors classified as IH showed overexpression of genes related to immune cells recruitment, adaptive and innate immunity, antigen presentation, cytotoxicity, immune suppression, and inflammation (P < 0.0001). IH cHCC-CCAs also displayed activation of gene signatures recently shown to be associated with response to immunotherapy in patients with HCC. Quantification of immunostainings confirmed that IH tumors were also characterized by higher densities of immune cells. Immune subtypes were not associated with any genetic alterations. Finally, multivariate analysis showed that the IH subtype was an independent predictor of improved overall survival.Conclusions:We have identified a subgroup of cHCC-CCA that displays features of an ongoing intratumor immune response, along with an activation of gene signatures predictive of response to immunotherapy in HCC. This tumor subclass is associated with an improved clinical outcome. These findings suggest that a subset of patients with cHCC-CCA may benefit from immunomodulating therapeutic approaches.

ERBB2 is the most prominent therapeutic target in gastroesophageal adenocarcinoma (GEA). For two decades, trastuzumab was the only treatment available for GEA overexpressing ERBB2. Several drugs showing evidence of efficacy over or in complement to trastuzumab in breast cancer failed to show clinical benefit in GEA. This resistance to anti-ERBB2 therapy is peculiarly recurrent in GEA and is mostly due to tumor heterogeneity with the existence of low expressing ERBB2 tumor clones and loss of ERBB2 over time. The development of new ERBB2 testing strategies and the use of antibody-drug conjugates having a bystander effect are providing new tools to fight heterogeneity in ERBB2-positive GEA. Co-amplifications of tyrosine kinase receptors, alterations in mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways and in proteins controlling cell cycle are well known to contribute resistance to anti-ERBB2 therapy, and they can be targeted by dual therapy. Recently described, NF1 mutations are responsible for Ras phosphorylation and activation and can also be targeted by MEK/ERK inhibition along with anti-ERBB2 therapy. Multiple lines of evidence suggest that immune mechanisms involving antibody-dependent cell-mediated cytotoxicity are preponderant over intracellular signaling in anti-ERBB2 therapy action. A better comprehension of these mechanisms could leverage immune action of anti-ERBB2 therapy and elucidate efficacy of combinations associating immunotherapy and anti-ERBB2 therapy, as suggested by the recent intermediate positive results of the KEYNOTE-811 trial.

The incidence of oropharyngeal cancers (OPC) is increasing in the world. Among OPC, those induced by human papillomaviruses have a better prognosis than non-HPV-associated OPC. The objective of this study was to highlight the relevance of HPV16 load, HPV16 DNA integration and HPV16-L1 serology on progression-free survival and overall survival of OPC patients. The PAPILLOPHAR cohort consists of 362 patients with oropharyngeal squamous cell carcinomas prospectively followed up for 5 years after treatment. Tumor biopsies and sera were collected at inclusion to investigate tumor HPV DNA/RNA characteristics and HPV16 L1 serology, respectively. Twenty-seven percent of tumor biopsies were HPV DNA- and RNA-positive and HPV16 represented 93% of HPV-positive cases. Among them, neither HPV16 viral load nor HPV16 DNA integration was associated with overall survival (OS) or progression-free survival (PFS). In contrast, high anti-HPV16 L1 antibody titers were significantly associated with a better OS and PFS. This study reveals that HPV16 load and integration are not relevant prognosis biomarkers in OPC patients. Clinical Relevance: High levels of HPV16 L1 antibodies may be useful to predict OPC patient outcome following treatment. ClinicalTrials.gov Identifier: NCT00918710, May 2017.

Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle. Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBVnaf). In vitro infection assays were performed to address the impact of HBVnaf. Hierarchical clustering individualized two clusters of HBVnaf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 ± 6.4 vs 6.6 ± 6.2 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1