Cholangiocarcinoma 2020: the next horizon in mechanisms and management

[EN] Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non- invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlightedJ.M.B. received EASL Registry Awards 2016 and 2019 (European CCA Registry, ENS-CCA). J.M.B. and M.J.P. were supported by: the Spanish Ministry of Economy and Competitiveness (J.M.B.: FIS PI12/00380, FIS PI15/01132, FIS PI18/01075 and Miguel Servet Programme CON14/00129; M.J.P.: FIS PI14/00399, FIS PI17/00022 and Ramon y Cajal Programme RYC-2015-17755, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER)); ISCIII CIBERehd; “Diputación Foral de Gipuzkoa” (J.M.B: DFG15/010, DFG16/004), and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD); the Department of Health of the Basque Country (M.J.P.: 2015111100; J.M.B.: 2017111010), and “Fundación Científica de la Asociación Española Contra el Cancer” (AECC Scientific Foundation) (J.M.B.). J.M.B. and J.W.V. were supported by the European Commission Horizon 2020 programme (ESCALON project 825510). The laboratory of J.B.A. is supported by competitive grants from the Danish Medical Research Council, the Danish Cancer Society, and the Novo Nordisk and A.P. Møller Foundations. J.J.G.M. and R.I.R.M. were supported by the Carlos III Institute of Health, Spain (PI16/00598 and PI18/00428) and were co-financed by the European Regional Development Fund. J.M.B. and J.J.G.M. were supported by the Ministry of Science and Innovation, Spain (SAF2016-75197-R), and the “Asociación Española Contra el Cancer”, Spain (AECC-2017). R.I.R.M. was supported by the “Centro Internacional sobre el Envejecimiento”, Spain (OLD-HEPAMARKER, 0348-CIE-6-E). A.L. received funding from the Christie Charity. M.M. was supported by the Università Politecnica delle Marche, Ancona, Italy (040020_R.SCIENT.A_2018_MARZIONI_M_STRATEGICO_2017). M.S. was supported by the Yale Liver Center Clinical and Translational Core and the Cellular and Molecular Core (DK034989 Silvio O. Conte Digestive Diseases Research Center). C.C. is supported by grants from INSERM, Université de Rennes, INCa, and ITMO Cancer AVIESAN dans le cadre du Plan Cancer (Non-coding RNA in Cancerology: Fundamental to Translational), Ligue Contre le Cancer and Région Bretagne. J.Bruix was supported by grants from Instituto de Salud Carlos III (PI18/00763), AECC (PI044031) and WCR (AICR) 16-0026. A.F. was supported by grants from ISCIII (PI13/01229 and PI18/00542). CIBERehd is funded by the Instituto de Salud Carlos III. V.C., D.M., J. Bridgewater and P.I. are members of the European Reference Network - Hepatological Diseases (ERN RARE-LIVER). J.M.B. is a collaborator of the ERN RARE-LIVER

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein Barr virus induced G protein coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in livers of NASH patients by liquid chromatography-mass spectrometry and tested the role of the EBI2-7α,25-diHC-system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared to controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype related differences were observed in Ebi2-/- animals and animals with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared to wildtype littermate controls,arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by enhanced level of 7α- hydroxycholest-4-en-3-one, and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment

Studies on the anti-apoptotic effect of the proteasome activator PA28gamma

Die bisher identifizierten Zielproteine des nukleären Proteasomaktivators PA28gamma weisen darauf hin, dass dieser an der Regulation wichtiger zellulärer Prozesse wie der Zellproliferation, der DNA-Reparatur und der Tumorentstehung beteiligt ist. Es konnte auch gezeigt werden, dass PA28gamma ein negativer Regulator der Apoptose sein kann, wobei die zugrundeliegenden Mechanismen noch wenig untersucht sind. Ziel der vorliegenden Arbeit war es daher, den Einfluss von PA28gamma auf wichtige apoptotische Ereignisse zu untersuchen. Dafür wurden in vitro-Experimente mit Zelllinien durchgeführt, bei denen die PA28gamma-Expression durch exogene DNA verstärkt bzw. durch miRNA-vermittelten Knockdown verringert war. Die Ergebnisse bestätigen PA28gamma als einen anti-apoptotischen Regulator und zeigen, dass PA28gamma verschiedene apoptotische Ereignisse beeinflusst. Im Besonderen führt PA28gamma unter apoptotischen Bedingungen zur Akkumulation von Cytochrom c im Mitochondrium, zur Inhibierung der Caspaseaktivität, zur Phosphorylierung von nukleärem p53 und letztendlich zu einer verringerten Apoptose. Die in dieser Arbeit gewonnen Daten und die daraus resultierenden Hypothesen legen die Grundlage für weitere detaillierte Untersuchungen der anti-apoptotischen Funktion von PA28gamma. Verschiedene Tumorarten können mit erhöhten PA28gamma- Level assoziiert sein. Aber auch bei systemischen Autoimmunerkrankungen scheint PA28gamma involviert zu sein. Daher sollte ein Sandwich-ELISA zur Quantifizierung von PA28gamma in humanen Seren entwickelt und anschließend die Relevanz von PA28gamma als diagnostischer und prognostischer Marker bei diesen Erkrankungen untersucht werden. Es konnte in allen getesteten Tumor- bzw. Autoimmunerkrankungen erhöhte PA28gamma-Serumlevel detektiert werden. Der Vergleich von Patienten mit rheumatoider Arthritis, die mit Abatacept behandelt wurden, ergab, dass das PA28gamma-Serumlevel signifikant mit dem Krankheitsaktivitätsindex (DAS28, disease activity score 28) und dem Entzündungsparameter ESR (Erythrozytensedimentationsrate) korreliert. Die Ergebnisse dieser Studie zeigen, dass das PA28gamma-Serumlevel in den untersuchten Erkrankungen erhöht, aber nicht krankheitsspezifisch ist. Bei Patienten mit rheumatoider Arthritis kann PA28gamma als neuer zusätzlicher Marker für den Verlauf der Erkrankung bzw. für die Untersuchung des Therapieverlaufs eingesetzt werden, da das PA28gamma-Serumlevel mit den spezifischen Markern für diese Erkrankung korreliert. Die Kenntnis über die genaue Bindungsstelle eines Autoantikörpers an ein Autoantigen ist für das Verständnis von Autoimmunerkrankungen und zur Entwicklung von Antikörper- basierten Assays bzw. Therapien von großem Nutzen. Daher sollte u.a. mit Hilfe des für den Sandwich-ELISA hergestellten PA28gamma-Hyperimmunserums validiert werden, ob ein Microbead-basierter Multiplex-Immunoassay zum Epitopmapping von Antikörpern eingesetzt werden kann. Dabei wurden Streptavidin-beschichtete, Fluoreszenz- und Größen-codierte Microbeads mit überlappenden, biotinylierten Peptiden des Antigens beladen und mit dem zu testenden Serum inkubiert. Der Multiplex-Immunoassay identifizierte die gleichen Peptide wie ein entsprechender ELISA. Mit dem erfolgreich validierten Microbead-basierten Multiplex-Immunoassay ist es gelungen ein kostengünstiges und zeitsparendes Verfahren zum Epitopmapping von Antikörpern zu etablieren.Proteasome activator PA28gamma is an important regulator of many cellular processes among them proliferation, DNA damage response and tumor development. Different studies demonstrated that PA28gamma is also involved in apoptosis but the underlying mechanism has not been investigated yet in detail. The aim of the study was to study the impact of PA28gamma on characteristic apoptotic events. Therefore, we employed cell lines with gain of function and miRNA- mediated gene silencing. It has been demonstrated that PA28gamma affects characteristic apoptotic events in vitro. In detail, under apoptotic conditions PA28gamma leads to an accumulation of mitochondrial cytochrome c, to an inhibition of caspase activity, to an enhanced phosphorylation of nuclear p53, and consequently to a reduction of apoptosis. The results of this study and the resulting hypothesis provide the basis for further investigations of the anti-apoptotic role of PA28gamma. Different tumors are associated with enhanced PA28gamma expression. Furthermore, PA28gamma is also involved in systemic autoimmune diseases. Therefore, the aim of this study was to develop a PA28gamma-specific sandwich ELISA for the quantification of PA28gamma in human sera. In addition, it was intended to use this ELISA to investigate the relevance of PA28gamma as a diagnostic and prognostic marker in different tumor and autoimmune diseases. The developed sandwich ELISA was able to detect enhanced levels of PA28gamma in all investigated sera of tumor and autoimmune diseases. Comparison of patients with rheumatoid arthritis, which were treated with abatacept, showed that PA28gamma sera level significantly correlates with the disease activity score 28 (DAS28) and the inflammation parameter ESR (erythrocyte sedimentation rate). The results obtained in this study reveal that PA28gamma is elevated in the sera of patients with tumor or autoimmune diseases but, unfortunately, this is not diseases specific. However, PA28gamma correlates with different markers of rheumatoid arthritis and could be a new additional marker for the investigation of progression and treatment of this disease. To understand autoimmune diseases and to develop appropriate assays and immune therapies, the knowledge of the antibody binding site is important. The aim of this study was to validate if a microbead-based multiplex immunoassay can be used for epitope mapping of antibodies. Therefore, recombinant synthesized overlapping peptides were coupled via biotin with streptavidin-coated, fluorescence- and size-encoded microbeads and incubated with the sera. The multiplex immunoassay was able to detect the same peptides as a corresponding sandwich ELISA. The results of this study reveal a fast and inexpensive method for epitope mapping of antibodies

Increased proteasome activator 28 gamma (PA28γ) levels are unspecific but correlate with disease activity in rheumatoid arthritis

Background PA28γ (also known as Ki, REG gamma, PMSE3), a member of the ubiquitin-and ATP-independent proteasome activator family 11S, has been proved to show proteasome-dependent and -independent effects on several proteins including tumor suppressor p53, cyclin-dependent kinase inhibitor p21 and steroid receptor co-activator 3 (SCR-3). Interestingly, PA28γ is overexpressed in pathological tissue of various cancers affecting e. g. breast, bowl and thyroids. Furthermore, anti-PA28γ autoantibodies have been linked to several autoimmune disorders. The aim of this study was to develop and evaluate a novel and sensitive PA28γ sandwich ELISA for the quantification of PA28γ serum levels in patients with cancer and autoimmune diseases for diagnostic and prognostic purposes. Methods PA28γ-specific polyclonal antibodies and recombinant His-tagged PA28γ were purified and used to develop a sandwich ELISA for the detection of circulating PA28γ. With this new assay, PA28γ serum levels of patients with various cancers, rheumatoid arthritis (RA), Sjögren’s syndrome (SS), adult-onset Still’s disease (AOSD) and different connective- tissue diseases (CTD) were compared with healthy control subjects. Anti-PA28γ autoantibodies were additionally confirmed using a newly developed microbead assay. Results The developed PA28γ sandwich ELISA showed a high specificity with a detection limit of 3 ng/ml. A significant up-regulation of circulating PA28γ was detected in the sera of patients with cancer, RA, SS and CTD. A significant correlation was observed dependent on age as well as anti-PA28γ autoantibody levels with circulating PA28γ protein levels. Furthermore, PA28γ serum levels showed a correlation with disease activity in patients with RA under treatment with the T-cell directed biological compound abatacept according to disease activity score 28 (DAS28) and erythrocyte sedimentation rate (ESR). Conclusion The application of PA28γ as a novel biomarker for diagnostic purposes of a specific disease is limited, since elevated levels were observed in different disorders. However, the correlation with disease activity in patients with RA suggests a prognostic value, which needs to be addressed by further studies. Therefore our results show that PA28γ is a useful marker which should be included in studies related to novel treatments, e.g. abatacep

The tumour microenvironment and immune milieu of cholangiocarcinoma

Tumour microenvironment is a complex, multicellular functional compartment that, particularly when assembled as an abundant desmoplastic reaction, may profoundly affect the proliferative and invasive abilities of epithelial cancer cells. Tumour microenvironment comprises not only stromal cells, mainly cancer-associated fibroblasts, but also immune cells of both the innate and adaptive system (tumour-associated macrophages, neutrophils, natural killer cells, and T and B lymphocytes), and endothelial cells. This results in an intricate web of mutual communications regulated by an extensively remodelled extracellular matrix, where the tumour cells are centrally engaged. In this regard, cholangiocarcinoma, in particular the intrahepatic variant, has become the focus of mounting interest in the last years, largely because of the lack of effective therapies despite its rising incidence and high mortality rates worldwide. On the other hand, recent studies in pancreatic cancer, which similarly to cholangiocarcinoma, is highly desmoplastic, have argued against a tumour-promoting function of the tumour microenvironment. In this review, we will discuss recent developments concerning the role of each cellular population and their multifaceted interplay with the malignant biliary epithelial counterpart. We ultimately hope to provide the working knowledge on how their manipulation may lead to a therapeutic gain in cholangiocarcinoma

NASH limits anti-tumour surveillance in immunotherapy-treated HCC

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment

Cholangiocarcinoma 2020: the next horizon in mechanisms and management

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted

Excellent outcome of direct antiviral treatment for chronic hepatitis C in Switzerland

BACKGROUND The introduction of direct acting antivirals (DAAs) for the therapy of chronic hepatitis C (CHC) has revolutionised treatment and marks a paradigm shift in the approach to this disease, rendering interferon-based therapies obsolete. AIMS OF THE STUDY We retrospectively and prospectively evaluated treatment results after the introduction of DAA in Switzerland in a cohort of patients with CHC. METHODS We examined 565 patients who received DAA treatment for CHC between November 2013 and June 2016 with regard to HCV genotype, fibrosis stadium, treatment and outcome. In addition, outcome of re-treatment and resistance-associated substitutions (RAS) in patients that did not achieve sustained virological response (SVR) were evaluated. The majority of patients participate in the Swiss Hepatitis C Cohort Study. Data were evaluated in an intention-to-treat and a modified intention-to-treat analysis. RESULTS Overall SVR rate for all patients was 94% (530 of 565, 95% CI 92-96%). Of 350 patients with HCV genotype 1 CHC, 335 achieved SVR, resulting in an SVR rate of 96% (335 of 350, 95% CI 94-98%). Patients with HCV genotype 2 achieved SVR in 94% (48 of 51, 95% CI 87-100%). Patients with HCV genotype 3 showed SVR of 92% (98 of 107, 95% CI 87-97%). In patients with HCV genotype 4, the SVR rate was substantially lower at 85% (49 of 57, 95% CI 76-94%). The rate of advanced liver fibrosis (Metavir F3/F4) assessed by means of liver biopsy or Fibroscan® in the entire patient population was 71% (404 of 565). Out of 35 patients that did not achieve SVR after DAA treatment, 32 had a relapse and 3 patients showed viral breakthrough. In 17 of 35 cases (49%) patients were treatment naïve and 21 of 35 patients (60%) were cirrhotic. RAS genotyping of HCV was performed in 14 patients. Nine of these 14 patients (60%) carried mutations in the NS5A region of the virus genome. Twenty-seven percent of patients who experienced treatment failure were not treated with recommended regimens as a result of drug availability and reimbursement limitations. CONCLUSION In Switzerland, novel DAA treatments for CHC reflect the positive results from registration trials. Genotypes 2 and 4 remained more difficult to treat between 2014 and 2016. Patients who experienced a relapse after DAA treatment in Switzerland predominantly showed mutations in the NS5A region of the virus genome. DAA treatment limitations in Switzerland did prevent optimal treatment regimens in some patients