Telomere maintenance in liver carcinogenesis : functional and translational study

Contexte : L'activation de la télomérase est l'événement le plus précoce dans le développement du carcinome hépatocellulaire (CHC) et est fréquente dans les tumeurs du foie. Nous avons cherché à élucider le rôle de la maintenance des télomères dans le foie non-tumoral, dans la carcinogenèse et la progression tumorale. Nous avons évalué le potentiel thérapeutique de l'inhibition de la télomérase. Méthodes : Nous avons développé une méthode interne pour estimer la longueur absolue des télomères (LT) par PCR quantitative, dans le foie tumoral et non tumoral d'une grande collection de tissus provenant de 1 502 patients (978 atteints de CHC). Nous avons déterminé les facteurs cliniques (démographie, exposition aux facteurs de risque environnementaux) et moléculaires (polymorphismes mononucléotidiques par GWAS) de la longueur dans le foie non tumoral. Nous avons intégré la LT aux altérations et à l'expression de TERT, ainsi qu'aux caractéristiques cliniques et moléculaires (analysées par séquençage du génome, de l'exome, ciblé et/ou de l'ARN) du CHC. Nous avons évalué l'efficacité préclinique d'oligonucléotides antisens (ASO) anti-TERT in vitro dans 26 lignées cellulaires et in vivo dans un modèle de souris xénogreffée et avons exploré l'hétérogénéité de la réponse à l'inhibition de la télomérase. Résultats : Le vieillissement, la fibrose hépatique, le sexe masculin et la consommation excessive d'alcool étaient des déterminants indépendants de l'attrition des télomères dans le foie. Une étude préliminaire d'association pangénomique a identifié le rs2736100 de TERT, ainsi que plusieurs SNP intergéniques spécifiques à l'étiologie qui pourraient moduler la LT dans le foie. Dans le CHC, le gène TERT est altéré dans environ 80 % des cas. La longueur des télomères dans le foie non tumoral était associée aux caractéristiques cliniques et moléculaires des tumeurs qui s'y développent. Les CHC qui se sont développés dans des foies avec de longs télomères présentaient souvent un profil TERT sauvage, avec des caractéristiques progéniteur et des mutations de BAP1. En revanche, les CHC qui se sont développés sur des foies avec des télomères courts étaient plus fréquemment de la classe des CHC non proliférants et présentaient plus souvent des mutations somatiques du promoteur de TERT. Dans les CHC et dans le foie non tumoral, la LT est stabilisée dans une fourchette biologique étroite similaire autour de 5,7 kb, par divers mécanismes qui activent l'expression de TERT. Les télomères longs sont caractéristiques de CHC très agressifs, associés à la sous-classe transcriptomique G3, aux altérations de TP53 et à un mauvais pronostic. La maintenance des télomères est associé à la transformation maligne des tumeurs hépatiques bénignes et des nodules dysplasiques chez les patients cirrhotiques. L'inhibition de TERT par ASO était efficace dans les lignées cellulaires de CHC hautement prolifératives et peu différenciées. Le traitement pendant 3 à 16 semaines a induit un arrêt de la prolifération dans 12 lignées cellulaires par raccourcissement des télomères, dommages à l'ADN et activation de l'apoptose. L'effet thérapeutique a également été confirmé dans un modèle de xénogreffe de souris. Principales conclusions : La longueur des télomères joue un rôle majeur dans la carcinogenèse hépatique. La LT du foie est déterminée par le vieillissement, le sexe, la progression de la fibrose, l'exposition à la consommation d'alcool et des traits génétiques constitutionnels. Les altérations de la télomérase sont les altérations les plus fréquentes dans le CHC. La maintenance des télomères est associé aux caractéristiques moléculaires de la tumeur, à sa progression clinique et à son agressivité. Le traitement par ASO anti-TERT a révélé l'addiction oncogénique à TERT dans le CHC, fournissant un rationnel préclinique pour tester l'inhibition directe de la télomérase dans des essais cliniques.Background: Telomerase activation is the earliest event in hepatocellular carcinoma (HCC) development and frequent in liver tumors. We aimed to elucidate the role of telomere length maintenance in the non-tumoral liver, in liver carcinogenesis and progression. We aimed to assess therapeutic actionability of telomerase alterations. Methods: We developed an in-house method to estimate absolute telomere length (TL) by quantitative PCR. We measured TL in the tumor and non-tumor liver of a large collection of tissues from 1,502 patients (978 with HCC). We determined clinical (demographics, exposure to environmental risk factors) and molecular (single nucleotide polymorphisms using GWAS) determinants of non-tumor liver TL. We integrated TL with TERT alterations and expression, as well as clinical and molecular (analyzed by genome, exome, targeted and/or RNA-sequencing) features of HCC. We assessed the preclinical efficacy of anti-TERT antisense oligonucleotides (ASO) in vitro in 26 cell lines and in vivo in a xenograft mouse model and explored the heterogeneity of response to telomerase inhibition. Results: Aging, liver fibrosis, male sex and excessive alcohol consumption were independent determinants of liver telomere attrition. A preliminary GWAS identified TERT rs2736100, as well as several etiology-specific intergenic SNPs that may contribute to liver telomere maintenance. In HCC, TERT gene is altered in approximately 80% of tumors. Non-tumor liver telomere length was associated with liver tumor clinical and molecular features. HCC that developed in livers with long telomeres frequently had wildtype TERT with progenitor features and BAP1 mutations. In contrast, HCC that developed on livers with short telomeres were enriched in the non-proliferative HCC class and frequently had somatic TERT promoter mutations. In HCCs, telomere length is stabilized in a narrow biological range around 5.7 kb, similar to non-tumor livers, by various mechanisms that activate TERT expression. Long telomeres are characteristic of very aggressive HCCs, associated with the G3 transcriptomic subclass, TP53 alterations and poor prognosis. Telomere maintenance was associated with malignant transformation of benign liver tumors and dysplastic nodules in cirrhotic patients. In HCC cell lines, TERT silencing with ASO was efficient in highly proliferative and poorly differentiated cells. Treatment for 3 to 16 weeks induced cell proliferation arrest in 12 cell lines through telomere shortening, DNA damage and activation of apoptosis. The therapeutic effect was also confirmed in a xenograft mouse model. Main Conclusions: Telomere length is key to liver carcinogenesis. Liver TL is determined by aging, sex, fibrosis progression, exposure to alcohol consumption and constitutional genetic traits. Telomerase alterations are the most frequent alterations in HCC. Telomere maintenance is associated with tumor molecular features, clinical progression and aggressiveness. Anti-TERT ASO treatment revealed the oncogenic addiction to TERT in HCC, providing a preclinical rationale for direct telomerase inhibition in HCC clinical trials

Benefits of tailored hepatocellular carcinoma screening in patients with cirrhosis on cancer‐specific and overall mortality: A modeling approach

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Stereotactic Body Radiation Therapy for the Management of Hepatocellular Carcinoma: Efficacy and Safety

This study aimed to describe patient characteristics, treatment efficacy, and safety in patients with hepatocellular carcinoma (HCC) undergoing stereotactic body radiation therapy (SBRT). We retrospectively analyzed data of 318 patients with 375 HCC treated between June 2007 and December 2018. Efficacy (overall survival [OS], relapse-free survival, and local control) and acute and late toxicities were described. The median follow-up period was 70.2 months. Most patients were treated with 45 Gy in three fractions. The median (range) PTV volume was 90.7 (2.6–1067.6) cc. The local control rate at 24 and 60 months was 94% (91–97%) and 94% (91–97%), respectively. Relapse-free survival at 12, 24, and 60 months was 62% (55–67%), 29% (23–36%), and 13% (8–19%), respectively. OS at 12, 24, and 60 months was 72% (95%CI 67–77%), 44% (38–50%), and 11% (7–15%), respectively. Approximately 51% and 38% experienced acute and late toxicity, respectively. Child-Pugh score B-C, high BCLC score, portal thrombosis, high GTV volume, and higher PTV volume reported on total hepatic volume ratio were significantly associated with OS. SBRT is efficient for the management of HCC with a favorable toxicity profile. The outcome is highly related to the natural evolution of the underlying cirrhosis

Neo-Adjuvant Use of Sorafenib for Hepatocellular Carcinoma Awaiting Liver Transplantation

International audienceData on efficacy and safety of sorafenib in a neoadjuvant setting for HCC awaiting liver transplantation (LT) are heterogeneous and scarce. We aimed to investigate the trajectory of patients treated with sorafenib while awaiting LT. All patients listed for HCC and treated with sorafenib were included in a monocentric observational study. A clinical and biological evaluation was performed every month. Radiological tumor response evaluation was realized every 3 months on the waiting list and every 6 months after LT. Among 327 patients listed for HCC, 62 (19%) were treated with Sorafenib. Sorafenib was initiated for HCC progression after loco-regional therapy (LRT) in 50% of cases and for impossibility of LRT in 50% of cases. The mean duration of treatment was 6 months. Thirty six patients (58%) dropped-out for tumor progression and 26 (42%) patients were transplanted. The 5-year overall and recurrent-free survival after LT was 77% and 48% respectively. Patients treated for impossibility of LRT had acceptable 5-year intention-to-treat overall and post-LT survivals. Conversely, patients treated for HCC progression presented high dropout rate and low intention-to-treat survival. Our results suggest that it is very questionable in terms of utility that patients treated for HCC progression should even be kept listed once the tumor progression has been observed

Bariatric Surgery Provides Long-term Resolution of Nonalcoholic Steatohepatitis and Regression of Fibrosis

International audienceBackground and aims: Studies are needed to determine the long-term effects of bariatric surgery for patients with nonalcoholic steatohepatitis (NASH). We evaluated sequential liver samples, collected the time of bariatric surgery and 1 and 5 years later, to assess the long-term effects of bariatric surgery in patients with NASH.Methods: We performed a prospective study of 180 severely obese patients with biopsy-proven NASH, defined by the NASH clinical research network histologic scores. The patients underwent bariatric surgery at a single center in France and were followed for 5 years. We obtained liver samples from 125 of 169 patients (76%) having reached 1 year and 64 of 94 patients (68%) having reached 5 years after surgery. The primary endpoint was the resolution of NASH without worsening of fibrosis at 5 years. Secondary end points were improvement in fibrosis (reduction of ≥1 stage) at 5 years and regression of fibrosis and NASH at 1 and 5 years.Results: At 5 years after bariatric surgery, NASH was resolved, without worsening fibrosis, in samples from 84% of patients (n = 64; 95% confidence interval, 73.1%-92.2%). Fibrosis decreased, compared with baseline, in samples from 70.2% of patients (95% CI, 56.6%-81.6%). Fibrosis disappeared from samples from 56% of all patients (95% CI, 42.4%-69.3%) and from samples from 45.5% of patients with baseline bridging fibrosis. Persistence of NASH was associated with no decrease in fibrosis and less weight loss (reduction in body mass index of 6.3 ± 4.1 kg/m2 in patients with persistent NASH vs reduction of 13.4 ± 7.4 kg/m2; P = .017 with resolution of NASH). Resolution of NASH was observed at 1 year after bariatric surgery in biopsies from 84% of patients, with no significant recurrence between 1 and 5 years (P = .17). Fibrosis began to decrease by 1 year after surgery and continued to decrease until 5 years (P < .001).Conclusions: In a long-term follow-up of patients with NASH who underwent bariatric surgery, we observed resolution of NASH in liver samples from 84% of patients 5 years later. The reduction of fibrosis is progressive, beginning during the first year and continuing through 5 years

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules.

International audienceIn liver transplantation, organ shortage leads to the use of marginal grafts that are more susceptible to ischemia-reperfusion (IR) injury. We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in IR. Herein, we aimed to elucidate the role of NOD1 in IR injury, particularly focusing on its effects on the endothelium and hepatocytes.Nod1 WT and KO mice were treated with NOD1 agonists and subjected to liver IR. Expression of adhesion molecules was analyzed in total liver, isolated hepatocytes and endothelial cells. Interactions between PMNs and hepatocytes were studied in an ex vivo co-culture model using electron microscopy and lactate dehydrogenase levels. We generated NOD1 antagonist-loaded nanoparticles (np ALINO).NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in Nod1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. Compared to untreated hepatocytes, a NOD1 agonist markedly increased hepatocyte lysis in co-culture with PMNs as shown by the increase of lactate dehydrogenase in supernatants. Interaction between hepatocytes and PMNs was confirmed by electron microscopy. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, aminotransferase levels and ICAM-1 expression.NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocyte-PMN interactions. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and provide a potential approach to prevent IR, especially in the context of liver transplantation.Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in ischemia-reperfusion. Here, we show that the NOD1 pathway targets liver adhesion molecule expression on the endothelium and on hepatocytes through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion emphasizes the importance of adhesion molecules in liver injury. In this study we generated nanoparticles loaded with NOD1 antagonist. These nanoparticles reduced liver necrosis by reducing PMN liver infiltration and adhesion molecule expression

NEW INSIGTHS IN CELLULAR MECHANISMS INVOLVED IN THE LIVER REGENERATION DEFECT DESCRIBED IN PATIENTS WITH SEVERE ALCOHOLIC HEPATITIS.

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Morbid obesity increases death and dropout from the liver transplantation waiting list: A prospective cohort study

International audienceAbstract Liver transplant (LT) candidates with a body mass index (BMI) over 40 kg/m 2 have lower access to a liver graft without clear explanation. Thus, we studied the impact of obesity on the waiting list (WL) and aimed to explore graft proposals and refusal. Method Data between January 2007 and December 2017 were extracted from the French prospective national database: CRISTAL. Competing risk analyses were performed to evaluate predictors of receiving LT. Competitive events were (1) death/WL removal for disease aggravation or (2) improvement. The link between grade obesity, grafts propositions, and reason for refusal was studied. Results 15,184 patients were analysed: 10,813 transplant, 2847 death/dropout for aggravation, 748 redirected for improvement, and 776 censored. Mortality/dropout were higher in BMI over 35 (18% vs. 14% 1 year after listing) than in other candidates. In multivariate analysis, BMI>35, age, hepatic encephalopathy, and ascites were independent predictors of death/dropout. Candidates with a BMI ≥ 35 kg/m 2 had reduced access to LT, without differences in graft proposals. However, grafts refusal was more frequent especially for ‘morphological incompatibility’ (14.9% vs. 12.7% p < 0.01). Conclusion BMI over 35 kg/m 2 reduces access to LT with increased risk of dropout and mortality. Increased mortality and dropout could be due to a lower access to liver graft secondary to increased graft refusal for morphological incompatibility

Real life experience of mycophenolate mofetil monotherapy in liver transplant patients

International audienceBACKGROUND: Mycophenolate mofetil (MMF) monotherapy following liver transplantation (LT) remains controversial due to a risk of acute rejection. The aim of this study was to report the largest multicenter experience of the use a MMF monotherapy guided by therapeutic drug monitoring using pharmacoslope modeling and Bayesian estimations of the MPA inter-dose AUC (BEMPAMETHODS: MMF daily doses were adjusted to reach the BEMPARESULTS: From 2000-2014, in 2 transplantation centers, 94 liver transplant recipients received MMF monotherapy 6.5±4 years after LT. The mean BEMPACONCLUSIONS: MMF monotherapy regimen appears usually safe and beneficial, with low risk of acute rejection and eGFR improvement. Therapeutic drug monitoring strategy seemed useful by identifying 14% of patients with low MMF exposure