PREDICTIVE FACTORS OF THROMBOSIS AND VASCULAR RECANALISATION IN VASCULAR LIVER DISEASES IN THE ABSENCE OF CIRRHOSIS

In vascular liver diseases, a risk factor is found in more than 50% of cases, associated in more than 30% of cases. However, genetic and familial causes remain poorly characterized. Curently, in the absence of risk factors, the impact of long-term anticoagulant therapy on patency and thrombotic risk has not been demonstrated.

RESUME ETUDES DES FACTEURS PREDICTIFS DE THROMBOSE ET DE REPERMEABILISATION VASCULAIRE AU COURS DES MALADIES VASCULAIRES DU FOIE EN DEHORS DE LA CIRRHOSE

Au cours des maladies vasculaires du foie, un facteur systémique est retrouvé dans plus de 50% des cas et plusieurs facteurs systémiques dans plus d'un tiers des cas. Cependant, certaines causes génétiques et familiales restent mal ou non caractérisées. Enfin, en l'absence de cause, l'impact de la poursuite du traitement anticoagulant au long cours n'est pas démontré.

1. Première partie : préciser les facteurs héréditaires pouvant favoriser le développement des maladies vasculaires du foie.

-Etude 1. Nous avons comparé une cohorte de patients mutés sur les gènes de la maintenance des télomères (TRG) à 396 patients contrôles d'une population d'un centre de soin primaire. Les patients TRG ont été divisés en 2 groupes : i) « avec anomalie hépatique (AH) » (transaminases > à 30 UI/L et/ou anomalie à l'imagerie) ; et ii) « sans AH ». Parmi 132 patients porteurs de mutations TRG, 95 avaient une maladie hépatique (19 transaminases > 30 UI/L, 12 un foie dysmorphique et 64 les deux). La présence d'une mutation TRG était associée à un risque accru de maladie du foie : Hazard Ratio 12,9 IC95 % 7,8-21,3. (p<0,001). La biopsie hépatique chez 52/95 patients a identifié des lésions hétérogènes : 42 % de maladie vasculaire porto-sinusoïdale, 15 % de fibrose/cirrhose avancée et 13 % de NASH. La survie globale et la survie sans TH étaient respectivement de 79 % et 69 %. Un score FIB-4 ' 3,25 et au moins un cofacteur de cirrhose du foie sont associés à une faible survie sans TH.

2. Deuxième partie : Analyse de l'impact du traitement de de la cause sur les complications de la maladie vasculaire du foie.

-Etude 2. Le pronostic particulièrement sombre de l'hémoglobinurie paroxystique nocturne -sans traitement -est lié aux thromboses artérielles et veineuses observées chez 30% de ces patients non traités. L'Eculizumab, anticorps anti C5, a permis de réduire l'incidence cumulative des événements thrombotiques entre 0.8 et 3%. Il n'y a pas de données publiées sur l'impact de l'Eculizumab sur l'évolution de la maladie vasculaire du foie en particulier. Nous avons observé un impact significatif de l'Eculizumab sur la survie avec une incidence de mortalité significativement plus faible chez les patients exposés vs non exposés. (IR 2.62 % patients-year; vs 8.7 % patients-year, IRR 2.99 [1][2][3][4][5][6][7][8][9][10] (p value= 0.035)

3. Troisième partie : En l'absence de cause forte de thrombose portale, impact du traitement anticoagulant sur le risque de récidive et analyse de facteurs de risque récidive -Etude 3. L'objectif principal de l'étude était d'évaluer l'efficacité préventive du rivaroxaban sur le risque de récidive de thrombose veineuse profonde de toute localisation, et de décès, chez des patients ayant une thrombose portale chronique, sans thrombophilie à haut risque, randomisés au rivaroxaban 15 mg/jour ou sans anticoagulation. Le critère d'évaluation principal était la survie sans thrombose. La survenue d'événements hémorragiques majeurs constituait un critère d'évaluation secondaire. Une analyse intermédiaire demandée par le promoteur, montrait un taux d'incidence de thrombose de 0 pour 100 personnes-années (PA) dans le bras rivaroxaban, et 19,71 pour 100 AP (IC à 95 % [7, 49 31,92]) dans le bras sans anticoagulation (valeur p du log-rank = 0,0008) avec une médiane de 11,8 mois (IC à 95 % [8,[8][9][10][11][12][13]2]). Sur la base de ces données, le comité indépendant de surveillance a recommandé de switcher les patients du groupe sans anticoagulation à un traitement anticoagulant. Après un suivi médian de 30,3 mois (IC à 95 % [29,9]), une hémorragie sévère est survenue chez deux patients recevant du rivaroxaban et un sans anticoagulant. Dans le groupe sans anticoagulation, les D-dimères > 500 ng/mL à 1 mois étaient associés à une récidive de thrombose (HR 7,78 [1,[START_REF] Bounameaux | Duration of anticoagulation therapy for venous thromboembolism[END_REF]), avec une valeur prédictive négative de 93,5 %.

Mots clés : maladies vasculaires du foie, anticoagulants, cause, thrombophilie, récidive thrombose, téloméropathie, gènes de la maintenance des télomères, hémoglobinurie paroxystique nocturne, Eculizumab, rivaroxaban 1. Specify hereditary factors that can promote the development of vascular liver diseases -Study 1. We compared a cohort of telomere maintenance genes (TRG) mutated patients with 396 control patients from a population of a primary care center. TRG patients were divided into 2 groups: i) "with hepatic abnormality (HA)" (transaminases> 30 IU / L and / or imaging abnormality); and ii) "without HA". Of 132 patients with TRG mutations, 95 had liver disease (19 transaminases> 30 IU / L, 12 with dysmorphic liver and 64 both). The presence of a TRG mutation was associated with an increased risk of liver disease: Hazard Ratio 12.9 95% CI 7.8-21.3. (p <0.001). Liver biopsy in 52/95 patients identified heterogeneous lesions: 42% portosinusoidal vascular disease, 15% advanced fibrosis / cirrhosis and 13% NASH. Overall survival and LT-free survival were 79% and 69%, respectively. A FIB-4 score > 3.25 and at least one cofactor of liver cirrhosis are associated with poor LT-free survival.

2. Analysis of the impact of treating the causes of vascular liver disease, on VLD survival and complications.

-Study 2. The poor prognosis of paroxysmal nocturnal hemoglobinuria -without treatment -is linked to arterial and venous thrombosis observed in 30% of these untreated patients. Eculizumab, an anti-C5 antibody, reduced the cumulative incidence of thrombotic events to between 0.8% and 3%. There are no published data on the impact of Eculizumab on the course of vascular liver disease. We observed a significant impact of Eculizumab on survival with a significantly lower incidence of mortality in exposed vs. unexposed patients. (IR 2.62% patients-year; vs 8.7% patients-year, IRR 2.99 [1][2][3][4][5][6][7][8][9][10] (p value = 0.035) 3. In the absence of a major cause of portal thrombosis, impact of anticoagulant treatment on the risk of recurrence and analysis of risk factors for recurrence -Study 3. The main objective of the study was to assess the efficacy of rivaroxaban on the risk of deep vein thrombosis recurrence, and of death, in patients with chronic portal thrombosis, in the absence of major risk factors of recurrent thrombosis, in petients randomized to rivaroxaban 15 mg / day or without anticoagulation. The primary endpoint was thrombosis-free survival. The occurrence of major bleeding events was a secondary endpoint. An interim analysis requested by the promoter, showed an incidence rate for thrombosis of 0 per 100 person-years (PY) in the rivaroxaban arm, and 19.71 per 100 PY (95% CI [7.49 -31.92]) in the no-anticoagulation arm (logrank p-value=0.0008) with a median follow-up of 11.8 months (95% CI [8.8-13.2]). On the basis of these data, the independent monitoring committee recommended switching patients from the group without anticoagulation to anticoagulation treatment. After a median follow-up of 30.3 months (95% CI [29.8-35.9]), severe bleeding occurred in two patients receiving rivaroxaban and one without anticoagulant. In the group without anticoagulation, D-dimers> 500 ng / mL at 1 month were associated with recurrent thrombosis (HR 7.78 [1.49-40.67]), with a negative predictive value of 93.5 %.

Key words: mutations of telomere-related genes (TRG), paroxysmal nocturnal hemoglobinuria, Eculizumab, rivaroxaban

REMERCIEMENTS

« Si tu veux tracer ton sillon droit, accroche ta charrue à une étoile. » Merci Dominique de m'avoir guidée toutes ces années pour tracer ce sillon afin que tous ensemble nous ajoutions une petite pierre à la recherche des maladies vasculaires du foie. Votre exigence à tout égard doublée de bienveillance est un exemple pour nous tous.

Merci aux

Vascular liver diseases: Definitions and classifications

I-Definitions

Vascular liver diseases (VLD) are rare diseases mostly occuring in the context of liver venous inflow or outflow obstruction. Non cirrhotic portal vein thrombosis (PVT), Budd Chiari syndrome (BCS), and porto sinusoidal vascular diseases (PSVD) are the three most frequent diseases affecting the venous system, and although these disorders affect distinct venous sites, simultaneous involvment is frequently encountered. These diseases are related to alterations in hepatic macrocirculation (hepatic veins, portal vein) or microcirculation (portal veinules). Even though various types of VLD differ in presentation and etiology, they still share in part underlying causes and mechanisms.

Venous circulation of the liver involve the portal vein and its branches that supply blood flow from intestinal tract to the liver. The portal vein is formed by the union of the superior mesenteric vein and the splenic vein. It subdivides in left and right branches to segmental veins, and to terminal portal veinules in the portal tract. Theses venules drain into the sinusoids, then to the hepatic vein circulation from small hepatic veins to inferior vena cava.

1-Non cirrhotic portal vein thrombosis (Non cirrhotic PVT)

Non cirrhotic PVT is defined as the obstruction of the extrahepatic portal vein, and/or right or left branches, associated or not to obstruction of other segments of the splanchnic venous axis. [1] It does not include isolated thrombosis of the splenic or superior mesenteric veins. Portal vein obstruction secondary to malignant tumor (frequently but improperly referred to as malignant thrombosis) is considered as a different entity, related to encasement or invasion of the veins by malignant tumors, including primary hepatobiliary malignancy most often in the presence of cirrhosis. We will discuss only non-malignant non cirrhotic portal vein thrombosis. Non cirrhotic portal vein thrombosis is either diagnosed at a recent stage (so called recent or acute) or at a chronic stage, porto-portal collaterals as a sequel of portal vein obstruction constitute a so called portal cavernoma. These 2 stages of a same disease share similar causes (chapter 2). Complications of portal hypertension and mesenteric ischemia due to thrombosis extension to mesenteric veins are the most dreaded complications of PVT [2,3]. The consequences of portal vein thrombosis depend on the extension of the thrombus. Mesenteric ischemia results from the extension of the thrombus towards the mesenteric venous arches. When ischemia is prolonged, an intestinal infarction may occur. Mortality from intestinal infarction is 20-50%, death is due to peritonitis and multivisceral failure. Intestinal resection following mesenteric-portal venous thrombosis is one of the main causes of short small bowel syndrome. Stenosis of the small intestine may be a late sequelae of mesenteric venous ischemia. [4][5][6] Downstream of portal vein thrombosis, the hepatic consequences appear minor. Clinical and biological signs of hepatic suffering are absent or transient. Collateral veins appear at the periphery or within the structures adjacent to the obstructed portion of the portal vein: main bile duct, gallbladder, pancreas, gastric antrum, duodenum. When the obstruction of the trunk of the portal vein persists, the collateral veins tend to progressively enlarge to form a portal cavernoma. Despite this, portal pressure increases which maintains hepatic blood perfusion. Episodes of gastrointestinal hemorrhage due to rupture of oesogastric varices or portal hypertension gastropathy may occur within an unpredictable period of time. The incidence of GI hemorrhage is 12-20% per year. [1,7]The varicose veins responsible for the hemorrhage may belong to a porto-systemic collateral circulation (esophageal, gastric or fundic varices) or to the veins of the cavernoma (gastric antrum and duodenum). Diagnosis of PVT is based on findings at liver doppler ultrasound associated to 3-phase contrast enhance imaging. Multiple classifications of PVT have been suggested, based broadly on anatomical location, degree of obstruction, presence or absence of collaterals, functional implications or combinations of these. The latest classification of PVT includes 3 parameters: time course, transversal and longitudinal occlusion of main PV, response to treatment or interval change. [1]

2-Budd Chiari syndrome

BCS is defined as the obstruction of hepatic venous outflow that can be located from the small hepatic veins up to the entrance of the IVC into the right atrium. [8]Hepatic outflow obstruction related to cardiac disease, pericardial disease or sinusoidal obstruction syndrome (SOS) are excluded from this definition. BCS is classified into: a) primary, most often caused by thrombosis; and b) secondary in the presence of compression by space occupying lesions, or invasion by malignancy or parasites otherwise. Only primary BCS will be discussed in this manuscript.

BCS is a rare disease, and epidemiological data is scarce. Incidence and prevalence were respectively 0.68/4.1 in France in 2010 in a recent French study reporting prospective patients from hepatology units. A recently published meta-analysis reported an estimated pooled incidence of 1 case per million inhabitants per year. [9,10] Manifestations are diverse and range from an absence of symptoms to fulminant hepatic failure depending on the site of obstruction, the number of hepatic veins obstructed, and the presence of hepatic venous collaterals. In a multicenter European study, ascites, present in 83% of patients, hepatomegaly in 67% and abdominal pain in 61%, were the main clinical features. Diagnosis is performed with liver doppler ultrasound by an experienced and avised operator. Three contrast enhance phase imaging is helpful to complete assesment.

3-Porto sinusoidal vascular disease (PSVD)

Porto-sinusoidal vascular liver disorder (PSVD), is a rare liver disorder, accounting for up to 10% of the causes of portal hypertension in Europe and North America. [11]It is characterized by i) microvascular liver abnormalities that may cause portal hypertension, and ii) the absence of cirrhosis. PSVD pathogenesis is unknown. PSVD has been associated with diverse systemic diseases (including among others, various immune system or inherited disorders) which, however, are absent in around 30% of the patients. The course of PVSD is highly variable ranging from the absence of clinical manifestations to lifethreatening complications. PSVD mostly affects young adults. A large proportion of patients develop severe complications related to portal hypertension, including variceal bleeding (in 40% of patients), portal vein thrombosis (in up to 40% at 5 years), or ascites (in 20-50% at 10 years). Liver tumors and lung disorders can also occur. In patients with such severe complications, transjugular intrahepatic portosystemic shunt or liver transplantation have to be considered. By contrast, in other patients who do not have clinically significant portal hypertension, manifestations are limited to abnormal liver blood tests. Due to its rarity, PVSD is frequently overlooked or misdiagnosed as cirrhosis; and knowledge is scarce, coming from small-sized studies of heterogeneous patient cohorts, without long-term data. [11] There is no simple blood test or imaging technique that can lead to a diagnosis of non-cirrhotic portal hypertension. Histology is essential for diagnosis. In terms of the causes of non-cirrhotic portal hypertension there are multiple terms in the literature for the same histologic findings in the liver, there are multiple diseases associated with the same hepatic histology, and there may be multiple histologic findings in the liver over time in the same patient. [12] As an illustration the terms idiopathic non-cirrhotic portal hypertension, incomplete septal cirrhosis, non-cirrhotic portal fibrosis, non-cirrhotic intrahepatic portal hypertension, benign intrahepatic portal hypertension, and noncirrhotic perisinusoidal hepatic fibrosis have all been used to refer to similar histology. Recently, the Vascular Liver Diseases Interest Group (VALDIG) proposed diagnostic criteria combining the exclusion of cirrhosis, particular histopathologic alterations, and specific portal hypertension manifestations (figure 1). [11] Prognosis of patients with VLD is also determined by the underlying cause. Recent data suggest that it may be a determinant factor for survival in particular in PSVD and PVT.

Figure 1 Definition of PSVD

II-Causes and mecanisms of thrombosis in VLD

Prothrombotic disorders are found in 65 to 85% of patients with BCS or PVT, but only 20-40% of PSVD patients. By contrast, chronic inflammatory conditions are more frequently encountered in patients with PSVD and PVT than in those with BCS. There is evidence for a significant genetic influence in patients with PSVD. Still, in over 40% of PSVD patients the cause remains unknown. The reason for these differences in causes among the various VLD, as well as the mechanisms for specific sites of thrombosis according to the underlying conditions remain obscure. Identified causes and prevalence according to VLD are presented table 1. Evidence of prothrombotic disease is an important consideration in the decision to continue long-term anticoagulant therapy.

1-Acquired conditions

A hereditary or acquired prothrombotic condition (Table 1) is found in approximately 10-50% of cases and an exogenous risk factor for venous thrombosis (hormonal, etc.) is present in approximately 6-30% of cases. Several factors are associated in the same patient in 10% of cases, which is more frequent than expected by chance alone . Therefore, the identification of a risk factor should not suspend the search for another factor. However, some conditions are difficult to diagnose, such as coagulation inhibitor deficiencies and antiphospholipid syndrome. Indeed, a non-specific decrease in coagulation inhibitors and the presence of non-specific antiphospholipid antibodies with low positive levels may be the consequence (and not the cause) of portal thrombosis. Strict diagnostic criteria for anti phospholipid syndrome and inhibitor deficiencies are mandatory to caracterise high or low risk thrombotic factors in VLD.

An antiphospholipid syndrome is confirmed if patients with portal thrombosis have the following findings:

-anti-cardiolipid antibodies > 40, or >99th percentile -or antiB2GP1 antibodies >99th percentile -or positive circulating anticoagulant lupus with 2 biological tests at least 12 weeks apart -or a protein C deficiency defined by a functional protein C level of less than 70% -or a protein S deficiency is defined by a functional protein S level of less than 60% in men, less than 50% in young women, less than 55% in postmenopausal women, in the absence of vitamin K antagonist treatment, or estrogenic treatment in women, associated decreases in coagulation factors II, VII and X. If in doubt, family screening should be performed. Genetic testing for known mutations may also be carried out -or antithrombin deficiency is defined as a functional antithrombin level of less than 80% amidolytic activity, in the absence of heparin or derivatives and in the absence of an associated decrease in coagulation factors II, V, VII and X.

a) Myeloproliferative neoplasm

Myeloproliferative neoplasm (MPN) is the most common cause of vascular liver disorder. Most patients do not meet the classical haematological diagnostic criteria due to haemodilution and hypersplenism as a consequence of portal hypertension. Therefore, screening for MPN in patients with VLD, especially with portal hypertension, should be performed irrespective of blood cell count. Diagnosis of MPN is now easier by testing a peripheral blood sample for the V617F JAK2 mutation. Indeed, it has been detected in 90% of BCS patients with MPN and in 30-45% of BCS patients. [13]This mutation is present in 21-37% of patients with portal thrombosis outside the context of cancer and cirrhosis. Finally, in approximately 20% of patients with portal thrombosis, no cause is found despite exhaustive explorations. Other somatic mutations, such as JAK2 exon 12 mutations mutations, MPL mutations and, in the gene encoding calreticulin (CALR ), are less frequently encounterered in VLD patients. [14] Recent data identified high-risk patients for adverse hematologic outcomes (hematological transformation, and poorer survival) in MPN/SVT patients. Indeed in a cohort of 80 MPN /SVT patients, 29% of the cohort harboured at least one molecular risk factor: JAK2 mutant allele burden ≥50%, or presence of chromatin/spliceosome/TP53 mutation. These high-risk patients had wors event free survival (81% versus 100%, p=0.001) and overall 10 years survival(89% versus 100%, p=0.01) than low-risk patients. [15] b) Paroxysmal nocturnal hemoglobinuria (PNH) Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder of hematopoietic stem cells, related to a somatic mutation in the Phosphatidilinositol Glycan class A (PIG-A), X-linked gene, responsible for a deficiency in glycosyl phosphatidilinositol-anchored proteins (GPI-AP). The lack of one of the GPI-AP complement regulatory proteins (CD59) leads to hemolysis. The disease is often diagnosed with hemolytic anemia, bone marrow failure and episodes of venous thrombosis. Venous thrombosis occurs in 15 to 30% of the patients and mainly affects cerebral, deep limb or splanchnic veins. In a recent study, thrombosis was the major prognostic factor affecting outcome. Risk factors for thrombosis included older age, thrombosis at diagnosis, transfusions, and decreased risk after immunosuppressive therapy. [16] Splanchnic vein thrombosis in PNH has been described in literature, in larger studies for Budd-chiari syndrome and in small studies or case reports for portal and mesenteric veins. Clinical presentations of patients with PNH and haemolytic anaemia are often associated to severe abdominal pain and inflammatory syndrome, with no clear explanation. Improvement in imaging acquisitions enhances thrombosis recognition in splanchnic vein thrombosis. Furthermore enthusiastic results have been published using new therapeutic agents, such as Eculizumab, a C5 inhibiting recombinant humanized monoclonal antibody. [17] The role of eculizumab on thrombosis outcome in patients with splanchnic thrombosis is not well known. Behcet's disease is highly prevalent in BCS reported around the Mediterranean basin, in Turkey (20-30%), and in Egypt (14.4%), whereas it is found in approximately 5% of patients with BCS in western countries. In a French cohort of BD patients, BCS had a high prognosis impact, increasing the risk of death by 9-fold. Early treatment of BD with immunosuppresive regimen may improve BCS outcome, and avoid the use of recanalisation procedures in BCS patients with initially severe liver disease. [22,23] 2-Genetic conditions a) AT, PC, PS deficiency AT, PC, PS deficiency were reported in respectively i highly variable prevalences in VLD (table1). Diagnosis of inherited AT, PC, and PS deficiency based on plasma measurements is often difficult in these patients due to liver disease which has a significant influence on the synthesis and clearance of these coagulation inhibitors. Indeed acquired abnormalities are frequently due to liver failure or acute thrombosis.Moreover, assessing the inherited character of these deficiencies by usual means requires family investigations which may be difficult to perform or non informative. Therefore, the extrapolation of these results as permanent prothrombotic condition deserves caution.

b) Other syndromic diseases associated to genetic conditions

Other genetic conditions unrelated to coagulation pathway have been described as possibly associated to vascular liver disease, and especially to PSVD. A genetic component is suggested by findings from exome sequencing in familial PSVD and by the association with Adams-Oliver syndrome,Turner's syndrome or telomeropathies in very limited studies. Telomeropathy susceptibility to PSVD merits further evaluation in a large cohort of well-characterized patients.

A French pediatric survey of porto-sinusoidal disease found 40% syndromic pathology (Noonan syndrome, Turner,..) and 16% familial involvement, some of which have missense mutations of Notch1, a gene involved in vascular remodeling. In adults, portosinusoidal disease is also reported during syndromic pathologies such as Turner syndrome, genetic diseases such as cystic fibrosis and telomerase dysfunction due to telomeres regulatory gene (TRG) mutations. [24][25][26]

c) Telomeres regulatory gene (TRG) mutations

Other genetic conditions unrelated to coagulation pathway have been described as possibly associated to vascular liver disease, and especially to PSVD. A genetic component is suggested by findings from exome sequencing in familial PSVD and by the association with Adams-Oliver syndrome,Turner's syndrome or telomeropathies in very limited studies. Telomeropathy susceptibility to PSVD merits further evaluation in a large cohort of well-characterized patients.

Telomeres are essential to maintain chromosome integrity at each cell division. Telomerase repair defective telomeres and regulate telomere length. Telomere shortening is associated to advanced age. Telomere maintenance is essential to avoid premature cell senescence due to telomere shortening. Premature cell senescence and genetic instability are the consequences of telomerase dysfunction. [27]Germline lossof-function rare variations in genes that encode components of the telomere repair complex, called telomere-related genes (TRG), accelerate telomere shortening and premature cellular senescence. Defective telomere repair has been causally associated with several human diseases with great diversity in severity, leading to several appellations in the literature: Hoyeraal-Hreidarsson syndrome and dyskeratosis congenita for the most severe of this variants. Telomere diseases comprise dysfunction of various organs including bone marrow failure, immune deficiency, hepatic disorders, pulmonary fibrosis and an increased risk of cancer. Bone marrow failure is a major complication of TRG mutations. [28,29] Macrocytosis, cytopenias, medullar aplasia ar the main characteristics. [30] Myelodysplasic syndrome and acute myeloid leukemia are the most frequently described cancers in TRG mutations, present in 75% of patients with cancer and TRG mutation. [30] Haematologic disorders are the consequence of quantitative and functionnal alteration of the hematopoietic stem cells. The consequences are a quantitative and qualitative impairment in mature cells (leukocytes, platelets and red cells) associated to lymphocyte B and T alterations (figure ). Similarly, it has been shown that pulmonary stem cells were affected in TRG mutation disorders. Consequently lung mesenchymal and epithelial can be equally affected (figure ). [31] Thus, pulmonary phenotype in TRG mutations varies from pulmonary fibrosis to isolated or associated pulmonary emphysema (figure ). Unexplained intersticial pneumonia, obliterative bronchiolitis are other described pulmonary lesions. Environment has a major impact on pulmonary disease prognosis. Smoking and toxics impair idiopathic pulmonary fibrosis outcome. Liver impairment in telomere diseases has been described in 9 studies which included 4-40 patients, with a total of 86 patients. [32][33][34][35][36][37] The prevalence of liver impairment varies from 5 to 40%, according to patient's selection. In these series, patients could either have an identified TRG mutation or a phenotype of telmeropathy in the absnce of identified mutation. In this setting, liver impairment's prognosis is unknown, possibly underestimated due to the severity of other organs (pulmonary, bone marrow, etc) impairment. Several cases of non -cirrhotic portal hypertension have been described, associated to this condition. Hepatopulmonary syndrome seems to be frequently associated to TRG mutations identified in 25% of patients in a serie of patients with pulmonary disease. [35] Data on prevalence of TRG mutation in chronic liver disease is scarce. One multicentric European studie identified 2,6% TRG mutations in a group of patients with cirrhosis all causes, compared to 0,5% in a control group with no cirrhosis, [RR] 1.859; 95% CI 1.552-2.227. [32] An overview of these patient's clinical characteristics seemed to identify shorter telomeres, younger patients and more aggressive liver diseases. Similarly the presence of new TERT mutations indentified in 3 patients with NASH, cirrhosis and HCC led to the hypothesis that TRG mutations may play a role in accelerating fibrosis and eventually HCC occurrence . [38] Clarification of liver disease characteristics and outcome in TRG mutations is crucially needed. More over the impact of TRG mutations, regarding other risk factors for liver disease has never been assessed. The prevalence of local causes varies according to vascular liver disease, 4 times more frequent in non-cirrhotic portal vein thrombosis than in Budd-Chiari syndrome. [3,20] Intra-abdominal septic foci are the most common. Local inflammation or infection, with or without systemic inflammatory response, are responsible for a prothrombotic state. Indeed, activation of coagulation is a major response in the host's defense against infection. Leukocytes, platelets and endothelial cells play an important role in thrombus development in combination with activation of the coagulation system [39]. Septic pylephlebitis is a septic portal vein thrombosis, frequently associated with the presence of intra-abdominal infection/inflammation, occurring in a territory drained by the splanchnic veins. It is associated with bacteremia in 44% of cases and most often discribed after appendicitis or diverticulitis.CMV infection has both a local and systemic role in coagulation activation system. Nevertheless only few cases are reported [40,41]. Characteristics and outcome of patients with PVT associated with CMV disease are not described. PVT can also occur after intra-abdominal surgery following intraoperative vascular manipulation or postoperative local inflammation. The natural history of postoperative DVT is influenced by the type of surgery, the degree of occlusion and the type of anticoagulation used. The presence of abdominal neoplasia is associated with a complex and multifactorial coagulopathy state. A pro-thrombotic state, compression or vascular invasion may favor the occurrence of thrombotic events. [42] b) Central obesity and metabolic syndrome Obesity is a major risk factor for cardiovascular disease. High body mass index (BMI) is associated with venous and arterial thromboembolic risks. Suggested mechanisms for the development of obesity-related thrombosis are coagulation activation (hypofibrinolysis, increased factor VII and plasminogen activator inhibitor (PAI)-1), platelet dysfunction, and endothelial dysfunction. Recent studies suggest that central obesity and metabolic syndrome are risk factors for idiopathic portal vein thrombosis. Similarly, in a recent epidemiological study, 50% of patients with idiopathic SBC had a BMI>25 kg/m2 compared to only 27% when a cause was identified. [9,21]. Data on glucose metabolism in this population is needed.

c) Hormones and VLD

The association of BCS with oral oestrogene containing contraceptive use has been evaluated in two case-control studies from 1970 to 1983 (OR 2.37 (95% CI 1.05-5.34, p < 0.02) and 1985 to 2000 (OR 2.4, 95% CI 0.9-6.2). Pregnancy also seems to be a trigger for BCS, based on the temporal association between both conditions, although it is not so clear for PVT. [43,44] Data on cycle disorders and endocrine parameters lack in women with VLD. Appropriate contraception remains challenging after VLD diagnosis and there is currently no data to support the use of progestin-only contraception.

d) Other causes

Hypereosinophilic syndromes, granulomatous venulitis, ulcerative colitis, celiac disease, have been described in VLD. Human immunodeficiency virus (HIV) infection frequently associated with PSVD and PVT. Exposure to didanosine and acquired decrease in protein S activity may contribute to the PSVD venopathy.

III-Treatment 1-Medical therapy: Anticoagulation and treatment of the cause

A-Anticoagulation a) Indications

The outcome of VLD has been recently evaluated in large prospective European studies. In BCS, a recent update in a large European cohort showed a good outcome with a consecutive strategy from less to more invasive treatments. [20,45]This strategy used anticoagulation therapy and, if inefficient, angioplasty, stenting, TIPS, and ultimately liver transplantation. Similarly, anticoagulation at the early stage of PVT has been shown to prevent thrombus extension and, in 40% of the cases, to be accompanied by recanalization. [3] By contrast, for patients with chronic PVT prospective follow-up data are not available. Retrospective data suggest that anticoagulation is not associated with excessive bleeding. However, in patients at risk for gastrointestinal bleeding due to portal hypertension and a mild or moderate risk of recurrent thrombosis, the benefit-risk ratio of anticoagulation therapy is particularly unclear. Furthermore, the time point, if any, when anticoagulation shifts from a beneficial to a detrimental effect, has not been explored.

Patients with BCS receive anticoagulant therapy as soon as possible for an indefinite period of time in an attempt to reduce the risk of clot extension and new thrombotic episodes. Anticoagulation is the first step of a step by step therapeutic strategy, where therapeutic procedures are performed with increased invasiveness, according to the response to previous therapy.

Figure 5: Therapeutic strategy in Budd Chiari syndrome

There are no randomised studies for anticoagulation in BCS. Nevertheless, the rationales for long term anticoagulation include an improved outcome in nontransplant as well as transplant patients since the implementation of systematic anticoagulation, a high risk of thrombosis recurrence due to a high association rate to prothrombotic diseases; case studies describing improvement following oral anticoagulant treatment, worsening when treatment is stopped and a noticeable improvement when it is reintroduced, as well as recanalization in rare cases of thrombosed hepatic veins. [46] In most centers, low molecular heparin has been used followed by long-term administration of coumarine derivatives, whatever the underlying thrombotic risk factor. Portal hypertension primary and secondary prophylaxis is similar to that for cirrhosis.

Patients with recent or acute PVT receive anticoagulant therapy for 6 months in the absence of severe bleeding contraindication. Usually permanent anticoagulation is recommended in patients harboring high risk associated causes and past history of gut resection for mesenteric infarction. [1,8,47]In expert recommendations for deep vein thrombosis, high risk associated causes are myeloproliferative neoplasms, antiphospholipid syndrome or homozygous or composite heterozygous G20210A factor II and G1691A factor V mutations, personal or 1st degree unprovoked family history of venous thrombosis. The benefit of permanent anticoagulation in patients with low risk factors for thrombosis is unknown.

Anticoagulation in patients with PSVD has been proposed to treat associated portal, splenic and mesenteric vein thrombosis and avoid development of chronic portal vein thrombosis and associated complications, but also to prevent intrahepatic thromboses and consequently liver disease progression. This view is supported by a retrospective study from our group suggesting that patients receiving anticoagulation have a better outcome than those without. [12] Moreover, liver histological and imaging findings suggest that intrahepatic thrombosis could play a major role. The most common histological findings are thickening or obliteration of intrahepatic portal venules, that could result from previous thrombotic events, or nodular regenerative hyperplasia where atrophy of hepatocytes might be due to ischemia. At imaging, abnormal intrahepatic portal branches are observed in half the patients. New portal vein thrombosis develops in 20% of the patients at 2 years. This incidence if twice higher in patients infected with HIV than in HIV negative patients. [19,48]

b) Type of anticoagulation

According to recommendations for deep vein thrombosis, patients are first treated with low molecular weight heparin (LMWH) in the absence of invasive procedure, followed with oral anticoagulant treatment with coumarine derivatives. LMWH is stopped when INR is within the target range, aiming at an international normalised ratio (INR) between 2 and 3.

In the absence of severe liver failure or portal hypertension, or portosystemic shunting, direct-acting oral anticoagulants (DOACs) may change the therapeutic scenario of patients requiring short-and long-term anticoagulation. The evidence gathered so far (from pre-approval pivotal trials to real-world postmarketing observational data) consistently confirms that DOACs are overall comparable to vitamin-K antagonists (VKAs) in terms of safety, efficacy, effectiveness and unequivocally documents a clinically relevant reduced risk of intracranial bleeding in the settings of non-valvular atrial fibrillation (AF) and venous thromboembolism. [START_REF] Kearon | Antithrombotic therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines[END_REF][START_REF] Kirchhof | ESC Guidelines for the management of atrial fibrillation developed in collaboration with EACTS[END_REF] The metabolism of DOACs is modified in renal and hepatic failure and differently according to the molecule. [START_REF] Graff | Anticoagulant therapy with the oral direct factor Xa inhibitors rivaroxaban, apixaban and edoxaban and the thrombin inhibitor dabigatran etexilate in patients with hepatic impairment[END_REF][START_REF] Potze | Decreased in vitro anticoagulant potency of Rivaroxaban and Apixaban in plasma from patients with cirrhosis[END_REF] Thus patients with coagulopathy or severe liver failure were excluded from major studies. Data on direct-acting oral anticoagulants (DOACs) in patients with VLD is limited, although provide some preliminary data on the efficacy and safety of these drugs in VLD patients. [START_REF] Intagliata | Direct Oral Anticoagulants in Cirrhosis Patients Pose Similar Risks of Bleeding When Compared to Traditional Anticoagulation[END_REF][START_REF] De Gottardi | Antithrombotic treatment with direct-acting oral anticoagulants in patients with splanchnic vein thrombosis and cirrhosis[END_REF]One series reported 94 patients from 17 centers including 36 patients with cirrhosis, 58 splanchnic vein thrombosis and only 4 BCS patients. Child-Pugh score was 6 (range 5-8), and MELD score 10.2 (range 6-19). Indications for anticoagulation were splanchnic vein thrombosis (75%), deep vein thrombosis (5%), atrial fibrillation (14%) and others (6%). DOACs used were rivaroxaban (83%), dabigatran (11%) and apixaban (6%). Patients were followed up for a median duration of 15 months (cirrhotic) and 26.5 months (non-cirrhotic). One had recurrent portal vein thrombosis and five had bleeding. Treatment with DOACs was stopped in three cases. Renal and liver function did not change during treatment. [START_REF] De Gottardi | Antithrombotic treatment with direct-acting oral anticoagulants in patients with splanchnic vein thrombosis and cirrhosis[END_REF] The second series compared rates of bleeding in cirrhosis patients treated with DOACS to traditional anticoagulation (warfarin and low molecular weight heparin), over 3 years from a research database. Clinical characteristics between the two groups were similar. There were no differences in terms of bleeding among both groups (p = 0.9) with two major bleeding events in the traditional anticoagulation group and one in the DOAC group. [START_REF] Intagliata | Direct Oral Anticoagulants in Cirrhosis Patients Pose Similar Risks of Bleeding When Compared to Traditional Anticoagulation[END_REF]A third series analyzed patients with cirrhosis and atrial fibrillation treated with anticoagulation over a 3-year period for thrombosis or prevention of stroke. The primary outcomes were bleeding events and recurrent thrombosis or stroke: Both groups had similar total bleeding events (8 DOAC vs 10 other, P=.12).and recurrent thrombosis. [START_REF] Chokesuwattanaskul | Efficacy and safety of anticoagulation for atrial fibrillation in patients with cirrhosis: A systematic review and meta-analysis[END_REF]The results of these papers strongly suggest that this new class of anticoagulants could be safe in cirrhotic patients in the absence of severe liver failure, with an incidence of major bleedings and other drug-induced side effects seemed close to the incidence reported in patients treated with conventional anticoagulation. [START_REF] Intagliata | Direct Oral Anticoagulants in Cirrhosis Patients Pose Similar Risks of Bleeding When Compared to Traditional Anticoagulation[END_REF][START_REF] De Gottardi | Antithrombotic treatment with direct-acting oral anticoagulants in patients with splanchnic vein thrombosis and cirrhosis[END_REF][START_REF] Chokesuwattanaskul | Efficacy and safety of anticoagulation for atrial fibrillation in patients with cirrhosis: A systematic review and meta-analysis[END_REF][START_REF] Hum | The efficacy and safety of direct oral anticoagulants vs traditional anticoagulants in cirrhosis[END_REF] Data on direct-acting oral anticoagulants (DOACs) in patients with SVT are very limited, and until more data is available, DOAC s are currently not routinely recommended as first line therapy in SVT, in the absence of cirrhosis. Since DOACs are increasingly used off label in this indication, more data about safety and efficacy are urgently needed.

c) Bleeding complications in patients treated by anticoagulant treatment for deep vein thromboses or splanchnic vein thrombosis

The bleeding risk of patients treated with VKA during DVT was assessed in a meta-analysis of 33 studies and 4374 patients. It is significant since the rate of major hemorrhage was respectively 2.06/100 BP before 3 months (95% CI 2.04-2.08) and 2.74/100 BP after 3 months (95% CI 2.71 to 2.77). Fatal hemorrhage rates over these 2 periods were 0.37 (95% CI 0.36-0.38) and 0.63/100 BP (95% CI 0.61-0.65). The case-to-death ratio was 13.4% (95% CI 9.4-17.4) with an intracerebral hemorrhage rate of 1.15/100 BP (95% CI 1.14-1.16). When treatment was continued beyond 3 months the risk was 9.1% (95% CI 2.5-21.7), with an intracerebral hemorrhage rate of 0.65/100 BP (95% CI 0.63-0.68).

The annual risk of major bleeding on anticoagulant treatment is highly variable in observational studies from 2% to 29%. Many scores have evaluated the risk of bleeding [START_REF] Torn | Risks of oral anticoagulant therapy with increasing age[END_REF][START_REF] Gage | Clinical classification schemes for predicting hemorrhage: results from the National Registry of Atrial Fibrillation (NRAF)[END_REF] Severe bleeding seems to be much lower now with DOACS. Indeed, intracranial hemorrhage is about half with DOACs in comparison to standard antithrombotic treatment [START_REF] Caldeira | Intracranial hemorrhage risk with the new oral anticoagulants: a systematic review and meta-analysis[END_REF] Table 2: HEMORR2HAGES bleeding score and RIETE registry bleeding score [START_REF] Torn | Risks of oral anticoagulant therapy with increasing age[END_REF][START_REF] Gage | Clinical classification schemes for predicting hemorrhage: results from the National Registry of Atrial Fibrillation (NRAF)[END_REF] Current data estimates case-fatality rate is 8% for DVT, 12% for pulmonary embolism for recurrent VTE, and about 10% for major bleed, measured during and after anticoagulation. [START_REF] Carrier | Systematic review: case-fatality rates of recurrent venous thromboembolism and major bleeding events among patients treated for venous thromboembolism[END_REF] In BCS, the bleeding rates and heparin-induced thrombopenia are more frequent and more severe in BCS anticoagulated patients than in those receiving anticoagulation for deep venous thromboembolism (VTE). Invasive procedures and portal hypertension are major causes of bleeding in BCS anticoagulated patients, while excess anticoagulation probably plays a secondary role. In a study with 94 consecutive patients, major bleeding episodes occur in 22.8 per 100 patient-years [START_REF] Rautou | Bleeding in patients with Budd-Chiari syndrome[END_REF][START_REF] Zaman | Increased prevalence of heparin-induced thrombocytopenia in patients with Budd-Chiari syndrome: a retrospective analysis[END_REF].

In the study by Condat et al, the incidence of gastro intestinal hemorrhage was 12.5/100 BP (95% CI [10][11][12][13][14][15]. The presence of large varicose veins was an independent risk factor for gastrointestinal hemorrhage. Anticoagulant treatment, age, sex, age of thrombosis, and history of GI hemorrhage did not increase the risk of hemorrhage [2]. In the American study, the incidence of major hemorrhage was 6.9/100 BP. The risk of hemorrhage was significantly higher in cases of large varicose veins (HR 2.63, 1.72-4.03; p<0.001) or treatment with warfarin (HR 1.91, 1.25-2.92; p=0.003).

The risk of hemorrhage with DOACS in DVT is comparable or lower with DOACS compared to the risk with warfarin. In the literature, this risk is estimated at 0.7% of major bleeding vs 7% minor bleedings. With DOACS the risk of digestive hemorrhage is slightly higher, while the risk of severe bleeding in particular of intracranial hemorrhage is lower. Retrospective limited data shows comparable bleeding risk with DOACS compared to other anticoagulation in splanchnic vein thrombosis.

Although the last decade have provided a marked impetus to the field of VLD, crucial information is still lacking, particularly in the areas of etiology, inherited factors, and treatment. Etiology is still unknown in 40% of the patients; genetic data are incomplete, therapeutic trials, although needed have never been performed, so that recommendations for treatments (with potentially fatal side effects in these high bleeding risk population) rely on limited data.

d) Treatment of the cause

In BCS the prevalence of underlying thrombophilia patients is high, since 87% of patients have an underlying risk factor for thrombosis and about 25% have several causal factors. Similarly, in PSVD the prevalence of inflammatory systemic diseases is high. Even though treatments are available for certain of these causes (i.e., myeloproliferative neoplasms, antiphospholipid syndrome, paroxysmal nocturnal hemoglobinuria, and Behcet's disease), data on the impact of these treatments on VLD outcome or survival is very limited. Promising results have been presented in MPN: One published showing that ruxolitinib is effective in reducing spleen size and disease-related symptoms and one published as an abstract showed improved survival with hydroxyurea and pegylated interferon. [START_REF] Derrode | Impact of cytoreductive therapy on the outcome of patients with myeloproliferative neoplasms and hepato-splanchnic vein thrombosis[END_REF][START_REF] Pieri | Safety and efficacy of ruxolitinib in splanchnic vein thrombosis associated with myeloproliferative neoplasms -PubMed n[END_REF]. More recently, 2 studies in BCS and Behcet's disease have shown that in patients treated with anticoagulation therapy, corticosteroids and immunosuppressive therapy about two thirds of patients did not require futher more invasive treatment. Indeed, invasive procedures are usually performed in 60 to 90% of the patients. In both cohorts medical treatment was solely performed in 67% and 62% in Behcet's disease compared to 30% BCS patients without BD, with similar survival rates. [22,[START_REF] Sakr | Characteristics and outcome of primary Budd-Chiari syndrome due to Behçet's syndrome[END_REF]. More data is needed in other acquired conditions.

IV-Risk of thrombosis recurrence in deep vein thrombosis and identified risk factors

Studies assessing the impact of permanent anticoagulation vs 3 months anticoagulation in deep vein thrombosis are based on estimating a balance between reduction in recurrent DVT and increase in major bleeding. An estimation of both indicators will then determine recommendations for permanent anticoagulation The risk of DVT recurrence is higher in:

-Idiopathic events: 10% per year the first two years with an odds ratio of 2.4, -In male subjects: before the age of 60, with an odds ratio of 2-4, -In patients with persistently elevated D-dimer level (odds ratio of 2.3, compared with normal level); especially in low risk factor patients [START_REF] Avnery | Ddimer levels and risk of recurrence following provoked venous thromboembolism: findings from the RIETE registry[END_REF],

-During the first two years after discontinuation of treatment.

Therefore provoking factors for DVT, localization of DVT, past personal or familial history of DVT, male sex and d-dimer, thrombophilia, and residual thrombus balanced to the bleeding risk are indicators that will influence the decision to continue or stop anticoagulation after 3 months. [START_REF] Kearon | Antithrombotic therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines[END_REF][START_REF] Douketis | Patient-level meta-analysis: effect of measurement timing, threshold, and patient age on ability of D-dimer testing to assess recurrence risk after unprovoked venous thromboembolism[END_REF][START_REF] Adeboyeje | Major Bleeding Risk During Anticoagulation with Warfarin, Dabigatran, Apixaban, or Rivaroxaban in Patients with Nonvalvular Atrial Fibrillation[END_REF][START_REF] Marchiori | The risk of recurrent venous thromboembolism among heterozygous carriers of factor V Leiden or prothrombin G20210A mutation. A systematic review of prospective studies[END_REF][START_REF] Bounameaux | Duration of anticoagulation therapy for venous thromboembolism[END_REF] V-Risk of recurrence in VLD and identified risk factors 1-Risk of recurrence in Budd Chiari syndrome with or without anticoagulant treatment.

Even though there are no randomized studies comparing anticoagulation versus anticoagulation interruption, long term anticoagulation is recommended in patients with BCS. The rational for long term anticoagulation is the improved survival rate after anticoagulation systematic introduction in 1984, the high rate of high risk thrombophilia, the severity of liver failure in case of recurrent thrombosis. Currently, the risk of recurrent thrombosis in patients treated with anticoagulation seems low, although it may be higher in patients with myeloproliferative neoplasms [START_REF] Stefano | Splanchnic vein thrombosis in myeloproliferative neoplasms: risk factors for recurrences in a cohort of 181 patients[END_REF][START_REF] Hayek | Longterm Outcome and Analysis of Dysfunction of Transjugular Intrahepatic Portosystemic Shunt Placement in Chronic Primary Budd-Chiari Syndrome[END_REF].

• In a French radiologic study on TIPS in BCS patients, myeloproliferative neoplasm was a risk factor for TIPS dysfunction during follow-up. [25]. After liver transplantation (LT), long term anticoagulation regimen is not clearly recommended. In a study, including 23 patients with LT, 5 patients, not or inadequately treated with antithrombotic therapy, had recurrent thrombosis. Moreover recurrent BCS after liver transplantation seem to occur in 21 to 27 % of not anticoagulated patients. [START_REF] Mentha | Liver transplantation for Budd-Chiari syndrome: A European study on 248 patients from 51 centres[END_REF][START_REF] Cruz | High incidence of recurrence and hematologic events following liver transplantation for Budd-Chiari syndrome[END_REF][START_REF] Halff | Liver transplantation for the Budd-Chiari syndrome[END_REF]

2-Risk of recurrence or extension of portal thrombosis with or without anticoagulant treatment.

Currently, duration of anticoagulation in patients with SVT is unclear. Expert recommendations suggest permanent anticoagulation in high risk associated causes The rational for permanent anticoagulation in population relies on multiple myeloproliferative neoplasm studies, although all uncontrolled retrospective studies:  In a European cohort of 604 patients with splanchnic vein thrombosis, including 28% of cirrhotic patients, a thrombotic event occurred in 7.3 per 100 patientyears. In 465 patients who had anticoagulation the rate was 5.6 per 100 patients-years. They observed a high recurrent thrombotic rate of 10.5 per 100 patient-years (6.8-16.3) after anticoagulation discontinuation, whereas recurrent rate while on VKA was still 3.9 per 100 pt-years. Common risk factors for SVT in this population were MPN in 20%, cancer in 22% and cirrhosis in 18%.The case fatality rate of thrombotic events was 13.2%(95%CI, 6.60%-24.15%). Male sex, solid cancer, myeloproliferative neoplasms, and unprovoked SVT were associated with an increased risk of vascular events [START_REF] Ageno | Long-term Clinical Outcomes of Splanchnic Vein Thrombosis: Results of an International Registry[END_REF]  In a European cohort of 181 patients with myeloproliferative neoplasms and splanchnic vein thrombosis, the recurrent thrombosis incidence rate was 4.2 per 100 pt-years [START_REF] Stefano | Splanchnic vein thrombosis in myeloproliferative neoplasms: risk factors for recurrences in a cohort of 181 patients[END_REF] It was reduced to 3.9 per 100 pt-years in patients treated with VKA:, whereas in the small fraction (15%) not receiving VKA it was as high as 7.2 per 100 pt-years.  In a small Irish cohort of 14 SVT patients, recurrence of SVT occurred mostly in the setting of interventional liver procedure. Recurrent thrombosis outside of the splanchnic venous system occurred in 28.5% of patients, predominantly off therapeutic anticoagulation. [START_REF] Greenfield | Splanchnic venous thrombosis in JAK2 V617F mutation positive myeloproliferative neoplasms -long term follow-up of a regional case series[END_REF]  In a series of 60 patients with chronic mesenteric venous thrombosis, the two factors that significantly improved survival were anticoagulant treatment and beta-blocker treatment.


Another recent retrospective study compared 512 patients with splanchnic thrombosis to 320 patients with DVT. This American study found different results from previously reported series. Recurrence-free survival at 10 years was comparable in both groups (76% for splanchnic thrombosis and 68% for DVT) and anticoagulant treatment was not associated with prolonged recurrence-free survival. Only hormonal treatment was associated with thrombotic recurrence.

There is no assessment in terms of anticoagulant used, as patients in these retrospective studies receive either heparins or VKAs without any real distinction in the studies.

Thus data is still controversial for the use of permanent anticoagulation in portal vein thrombosis. There is no convincing data in patients harboring low risk factor for thrombosis in this setting.

THESIS WORK I-Background and aims

This work focuses on the usefulness of extensive knowledge and workup of causes in VLD patients to improve the care of patients and stratify management accordingly. We wanted to explore new genetic causes and their impact on VLD prognosis, the impact of treating causes on prognosis and the risk of recurrence when these causes were absent or considered as low risk factors for thrombosis.

The first work focused on the description of liver disease in patients with TRG mutations. It has been identified as a possible risk factor for cirrhosis, but further data seems to describe non cirrhotic portal hypertension in patients harboring these mutations. Therefore, the aim of this study was to characterize patients with liver disease, and to assess the impact of these mutations as a new causal factor for liver disease.

The second work focused on the usefulness of treating with anti-complement monoclonal antibodies, paroxysmal nocturnal hemoglobinuria patients with splanchnic vein thrombosis.

The third work focuses on the usefulness of treating with anticoagulants patients with no or low risk factors for thrombosis, and identifying biomarkers of recurrence.

II-Results

1-Article 1

Introduction

Telomeres are repeated hexanucleotides/protein complexes, located at the end of linear chromosomes (1,2). Telomere length is incrementally shortened with each mitosis (3). When telomeres become critically short, a DNA-damage program is activated by the telomerase complex (1-3). Rare germline loss-of-function variations in genes that encode components of the telomere repair complex, called telomere-related genes (TRG), accelerate telomere shortening and premature cellular senescence. Defective telomere repair has been causally associated with several human diseases of variable severity named telomere biology disorders (TBDs) or telomeropathies (4)(5)(6). Telomeropathies diversely combine dysfunction of various organs including bone marrow failure, immune deficiency, liver disease, pulmonary fibrosis and a narrow cancer spectrum (acute myeloide leukemia, myelodisplastic syndrome, and squamous cell carcinoma) (7)(8)(9)(10)(11)(12)(13)(14). Telomeropathies have a highly variable penetrance (7,8).

Liver disease has been rarely described in telomeropathies (15,16). Based on a limited number of patients with biopsy, histopathological findings are varied (6,15,16). The outcome of patients with TRG mutations and liver disease is unknown (6,15). Therefore, the aims of this study were: (a) to determine whether TRG mutations are associated with an increased risk for liver disease; (b) to thoroughly characterize TRG mutations associated liver disease; and (c) to identify risk factors for a poor outcome in patients with TRG mutations.

Methods

Patients

This was a retrospective, multicenter observational study of patients with TRG mutations, identified i) through a genetic registry for TRG mutations (1990-2019); and ii) clinical referral networks for rare diseases registries (CRMVF, national network for vascular liver disease; OrphaLung, national referral center for rare lung diseases; and referral center for aplastic anemia). A previously described community-based cohort of 1190 unselected subjects screened for liver disease was used for community controls (17).

TRG mutations cohort

Patients were included if they had a confirmed pathogenic TRG mutation, at least one organ disease (blood, liver or lung) associated with TRG mutations for the probands, regardless of the telomere length (18,19). Mutations were interpreted as pathogenic variations according to the American College of Genetics and Genomics guidelines, and the European Society for Human Genetics recommendations (18,19). Date of genetic diagnosis was defined as the date the blood sample was sent to the genetics laboratory. Date of first organ disease was defined as the date of diagnosis of one organ disease (blood, liver, or lung) associated with TRG mutations. Date of liver disease was defined as the date of first visit identifying liver transaminases and/or imaging abnormalities. Date of lung disease was defined as the date of first available abnormal chest CT scan or, when unavailable, as the date of lung biopsy. Date of blood disease was defined as the date of bone marrow biopsy or aspiration or, when unavailable the date of first visit identifying blood count abnormalities. Duration of liver disease follow-up started from the date of liver disease to the last follow-up or death. Follow-up durations for lung and blood diseases were defined similarly.

Exclusion criteria were any of the following: the absence of pathogenic genetic variant despite a clinical phenotype suggestive of TRG mutation; "coats-like" disease (CTC1 mutation,); missing data; and absence of patient consent. This TRG mutations cohort was divided into 2 groups (Figure 1): a first group of patients with liver disease characterized by AST and/or ALT >30 IU/L (20-24) and/or abnormal liver imaging (dysmorphic and/or hyperechogenic liver and/or features of portal hypertension). A second group of patients with TRG mutations without liver disease i.e., with AST and ALT ≤ 30 IU/L and no liver imaging abnormality.

Control group

Controls were selected from a cohort of unselected subjects, aged 45 years or more, scheduled for free medical check-up in a social medical centre (Hôpital Avicenne, AP-HP, Bobigny, France) between September 2005 and February 2008, who underwent liver stiffness measurement as a screening procedure for liver disease, as previously described by Roulot et al. (17). Subjects were stratified by age and matched 3:1 using a propensity score according to sex, overweight status, excessive alcohol consumption, arterial hypertension, and diabetes. In this cohort, liver disease was defined on the same criteria as in the TRG mutations cohort.

Pathogenic variations sequencing and telomere measurement

Sequencing was performed in the genetics laboratory of Bichat Hospital in Paris (national referral laboratory for genetic diagnosis of TBD) except for 7 patients for whom molecular exploration was performed in another laboratory. Methods for sequencing are presented in supplementary material. Rare variants were retained and classified according to the American College of Genetics and Genomics guidelines (18,19). Variants identified in previous reports of telomeropathy were deemed disease-causing in this study. In summary, disease-causing variants are subsequently referred to as confirmed TRG mutations.

Assessment of telomere length was performed by flow-FISH method in Hematology

Laboratory of Robert Debré Hospital in Paris as previously described (25). Telomere length was defined based on leukocyte telomere lengths measured relative to age-matched apparently healthy individuals.

Patients' characteristics

Patients' characteristics including risk factors for chronic liver disease [history of arterial hypertension, diabetes, hyperlipidemia, body mass index (BMI) (overweight was defined as BMI >25kg/m²), alcohol consumption (excessive alcohol consumption was defined as >100g per week), B and C hepatitis status] were collected.

For the group of patients with TRG mutations and liver disease, data were collected at the time of liver disease diagnosis, and follow-up outcomes were reported since liver disease diagnosis. For the group of patients with TRG mutations but no liver disease, data were collected at the last follow-up visit which did not show liver disease and follow-up outcomes were reported since the diagnosis of the first organ disease. Family history, defined as a history of anyfirst-or second-degree TBD, was also collected for all patients with TRG mutations.

For the control cohort, data on history of arterial hypertension, diabetes, hyperlipidemia, Body Mass Index (BMI), alcohol consumption, hepatitis B or C virus infection, laboratory data, and liver stiffness were prospectively collected as part of the initial study. (17).

Organs Assessment

Liver, lung, and blood assessments are described in supplementary material. Data from dermatological examination, bone densitometry, immune disease, ophalmological examination, and occurrence of neoplasia were also collected, when available.

Liver biopsies

Liver biopsies were reviewed in the pathology department of Beaujon Hospital except for one patient for whom the review was performed, by the local pathologist according to the predefined grid of pathological features described in supplementary material.

Statistical Analysis

Data are presented as median (interquartile range) or number (proportion) as appropriate. Comparisons of quantitative and qualitative variables were made using Mann-Whitney test and Chi2 or Fisher exact test, respectively.

The comparative control group was derived from the prospective cohort of 1322 patients subjects from community-based population, aged 45 years or more, scheduled for free medical check-up in a social medical center (Bobigny, France) between September 2005 and February 2008 who had liver stiffness measurement as a screening procedure for liver disease (17). We stratified patients with TRG mutations and controls according to their age (under 50, 50 to 60, 60 to 70 and over 70 years old). To control for confounding variables, we used propensity score matching in each age group. We estimated the propensity score for every patient using a logistic regression model. Covariates in the model included sex, excessive alcohol consumption (consumption > 10 g daily), overweight status (BMI>25kg/m²), arterial hypertension and diabetes. The model discriminated well between the TRG and control cohorts in each age group (area under the receiver operating characteristic curve: 0.83, 0.77, 0.93, 0.75, respectively), allowing for a 3-to-1 matching of patients.

To identify variables associated with liver disease in the whole study population (TRG mutations cohort and controls), a binary logistic regression was performed. Hazard ratios (HRs) for binary logistic regression were provided with their 95% confidence intervals (CI).

To avoid survivorship bias, survival analysis was performed only in patients who had diagnosis of both liver disease and TRG mutation after 2004. Univariate and multivariate Cox model regression analysis were performed to determine prespecified baseline factors associated with overall survival and liver transplantation-free (LT-free) survival in patients with TRG mutations and liver disease. Multivariate analysis was performed on variables with <0.01 significance in univariate analysis and for which more than 95% of the data were available.

Hazard ratios (HRs) for Cox model regression were provided with their 95% confidence interval (CI). Kaplan-Meier analysis was applied to overall and LT-free survival in patients with TRG mutations and liver disease. LT-free survival in patients with TRG mutations and liver disease according to prognostic factors were compared using a stratified Log-Rank test.

All tests were two-sided, and p≤0.05 was considered significant. Data handling and analysis was performed with SPSS 25.0 (SPSS Inc., Chicago, IL).

Results

Between October 2018 and August 2019, we screened 189 patients from 25 centers: 27 were excluded; 132 were included, comprising 95 (72%) with liver disease and 37 (28%) without liver disease (Figure 1).

TRG mutations cohort

TERT mutations were the most frequently identified mutations, observed in 78 cases (59%). TERT, TERC, RTEL1, DKC1, TINF2, PARN, and NHP2 mutations were similarly represented in patients with or without liver disease. Gene variants are reported in Supplementary data 1. The pedigrees of 3 families with TRG mutations are reported in Supplementary data 2 and showed more severe phenotypes or/and an earlier onset along generations. Among 68 patients in whom telomere length was assessed, shortened telomere length<1 st percentile was identified in 89% of patients with liver disease, similar to patients without liver disease (Table 1 and Supplementary data 1).

In the TRG mutations cohort, ninety-four patients were male (71%), aged 57 years. Ninety-five patients (72%) had liver disease, 92 (70%) had blood disease and 104 (79%) had lung disease (Table 1).

In total, n=95 (72%) patients had liver disease: n=19 (20%) with isolated serum transaminase elevation, n=12 (13%) with at least one anomaly on imaging and normal serum transaminase value, while n=64 (67%) had both.

Features of liver disease at diagnosis are described in Table 1 and2. All patients underwent at least one imaging test at diagnosis: doppler-ultrasound (n=88), abdominal CT scan (n=67) and/or hepatic MRI (n=48) (Table 2). Three patients had ascites and one had hepatic encephalopathy at diagnosis (Table 2).

Patients with liver disease presented telomeropathy-related related lung, blood, cutaneous, rheumatologic, ophthalmic disease in 82%, 77%, 55%, 39% and 30% of cases, respectively (Table 1). Only 3 patients had isolated liver disease. Rheumatologic and ophthalmic disease are reported in 39% and 30% of cases, respectively (Supplementary data 3). Mean platelet and leukocyte counts were lower and mean corpuscular volume was increased in the patients with liver disease (p<0.005) (Table 1). Median age of the patients with liver disease was similar to the patients without liver disease.

Hepatic venous pressure gradient (HVPG) (measured in 25 patients) was 6 (4 -11) mmHg and liver stiffness (measured in 54 patients) 8.2 (5.6 -14.9) kPa (Table 2). Liver stiffness was significantly higher in patients with large esophageal varices (grade II or III) (p=0.027) (Supplementary data 4).

Liver biopsy (n=52) identified porto-sinusoidal vascular disease specific pathology lesions (PSVD) in 22 patients(42%) including 6 with advanced fibrosis, 7 had non fibrotic NASH and 8 (15%) had advanced fibrosis in the absence of vascular lesions. (Table 2, Supplementary data 5A and 5B). Among 14 patients with minimal lesions, mainly sinusoidal distension and minimal regenerative anomalies, 3 had severe portal hypertension, suggestive of additional PSVD in the context. TERT mutation, low platelet count, high ALP, and myelodysplasia, were significantly associated to PSVD compared to cirrhosis (Supplementary data 6). Among twelve patients with isolated transaminase elevation or with transaminases ≤30 IU/L and with hepatic dysmorphia, biopsy identified 3 with PSVD, 1 with cirrhosis and 3 with NASH.

Outcome in all patients with TRG mutations

Follow-up outcomes in patients with and without liver disease in the TRG mutations cohort (N=132) are presented in Supplementary data 7. After a median follow-up of 21 (12 -54) months, ascites occurred in 14% of patients with liver disease, variceal bleeding in 13%, hepatopulmonary syndrome (HPS) in 13%, portal vein thrombosis in 5%, hepatic encephalopathy in 4%, and hepatocellular carcinoma (HCC) in 2%, respectively. In Supplementary data 8, features of mutated patients who died of hepatic causes and/or underwent a liver transplantation (N=15) are described precisely.

Thirteen patients underwent lung transplantation; 12, liver transplantation (LT); and 5, hematopoietic stem cell transplantation. Among the 12 patients who underwent a liver transplantation, 4 had hematopoietic stem cell transplantation (2 before and 2 after liver disease diagnosis), 4 had lung transplantation after liver disease diagnosis (2 combined and 2 sequential). Eventually, 9 of the 12 patients with HPS received a liver transplantation, among which only 1 patient had a double lung-liver transplantation. In one patient, HCC was diagnosed on liver explant.

Outcome analysis in patients with TRG mutations and liver disease

In total, 89/95 (94%) were included in survival analysis. Among, 89 patients (94%) 27 died, 15 before any transplantation and 5 after liver transplantation. Causes of deaths were hepatic, pulmonary, or hematological in 7, 18 and 2 respectively (Supplementary data 7). Five years overall survival and LT-free survival rates were 79% and 69% respectively (Figure 2A).

In univariate analysis, DKC1 mutation, cofactor for liver disease, ascites, MELD score, FIB-4 score, liver stiffness, HPVG and MCV remained significantly associated with LT or death (Table 3). Telomere length <1 st percentile was not significantly associated with LT or death (Table 3). In multivariate analysis, a FIB-4 score ≥3.25 and cofactor for liver disease (alcohol consumption >100g/week, HBsAg +, BMI>25 kg/m², ferritin>1000 µg/L) remained significantly associated with LT or death (Table 3). LT-free survival rate was significantly better when FIB-4 score was inferior to 3.25 and in the absence of a cofactor for liver disease (p<0.001) (Figure 2B). Prognostic variables associated with overall survival in univariate and multivariate analysis are shown in Supplementary data 9.

Comparison of TRG mutations cohort and controls

Characteristics of patients in the TRG mutations cohort (n=132) versus patients in the control cohort stratified by age and matched 3:1 with propensity score matching using sex, overweight status, excessive alcohol consumption, arterial hypertension, and diabetes (n=396) are reported in Supplementary data 10. Compared to controls, liver disease defined as AST or ALT > 30 UI/L, and/or liver imaging abnormality was significantly more frequent in patients with TRG mutations than in controls: respectively 95/132 (72%) vs 80/396 (20%) (p<0.001).

Patients with TRG mutations had significantly higher liver stiffness than controls (7.4 (5.3-14.1) kPa versus 5.3 (4.3 -6.4) kPa respectively, p<0.001) and frequent blood tests abnormalities (thrombopenia 127 (61-218) 10 9 /L vs 245 (213-288) 10 9 /L and higher mean corpuscular volume (MCV) (92-103) fL vs 89 (86-92) fL; p<0.001)

In multivariate analysis, male sex, presence of a TRG, excessive alcohol consumption and BMI>25 kg/m² were independently associated with liver disease (p<0.001, p<0.001, p=0.007, and p<0.001, respectively) (Table 4).

Discussion

We found a high prevalence of liver disease (72%) in patients with TRG mutations. Indeed, the prevalence of liver impairment varied from 5 to 40%, according to patient's selection in 9 studies which included 4-40 patients, with a total of 86 patients. (15,16) (Supplementary data 11). In these series, patients could either have an identified TRG mutation or a phenotype of telomeropathy in the absence of identified mutation. (6,15,16,26).

In our study, a pathogenic TRG mutation was a mandatory inclusion criterion and consequently present in 100% of the patients. Differences can also be explained: by the heterogeneity of criteria to define liver disease (Supplementary data 11). We chose to use current criteria used for patients with metabolic disease or chronic viral hepatitis (20)(21)(22)(23) and identified clinically relevant pathologic lesions in 60% of liver biopsies.

This study fills a knowledge gap by demonstrating that TRG mutations have a major independent impact on the occurrence of liver disease. Compared to unselected communitybased controls, liver disease was far more prevalent (HR of 12.89 (7.87-21.35)) in TRG mutations carriers. Furthermore, liver disease when present was more severe, more frequently associated to cytopenias and macrocytosis among patients with TRG mutations than in controls.

Data on prevalence of TRG mutation in chronic liver disease is scarce. One multicentric European studie identified 2,6% TRG mutations in a group of patients with cirrhosis all causes, compared to 0,5% in a control group with no cirrhosis, [RR] 1.859; 95% CI 1.552-2.227. An overview of these patient's clinical characteristics seemed to identify shorter telomeres, younger patients and more aggressive liver diseases. Similarly the presence of new TERT mutations indentified in 3 patients with NASH, cirrhosis and HCC led to the hypothesis that TRG mutations may play a role in accelerating fibrosis and eventually HCC occurrence (27)(28)(29)(30) (Supplementary data 11). Thus TRG mutation has been identified as a possible risk factor for cirrhosis, but further data seems to describe non cirrhotic portal hypertension in patients harboring these mutations. Our data show that liver disease in patients with TRG mutation occurred at a younger age, with past personal or familial history of lung, hematological, cutaneous disease in the majority. Early and severe osteoporosis and ophthalmologic involvement occurred in 10% and 20% of cases respectively. Apart from liver tests and imaging abnormalities, lower mean platelet and leukocyte counts, and higher mean corpuscular volume were significant distinctive features among TRG mutations patients with liver disease.

Centralized analysis of liver biopsies in more than half of patients, helped characterizing liver disease (16) and raised physiopathology issues in 3 situations: first, among patients with features of portal hypertension, portosinusoidal vasculaire disease (31) was diagnosed in 32 (42%) cases (all had portal hypertension signs on imaging or on endoscopy), and was more frequently encountered than advanced fibrosis or cirrhosis (15%). Low hepatic vein pressure gradient (HVPG) suggested PSVD in these patients with portal hypertension (31).

Interestingly, patients with PSVD had frequent associated severe hematologic disease.

Secondly, NASH prevalence in our study seemed close to the general French population estimate, contrasting with a low prevalence of diabetes (only 8%) in TRG mutated patients with liver disease. Furthermore, we currently identified 27% with "minor" unspecific lesions whose natural history progression to specific lesions of PSVD or cirrhosis deserves clarification in the future.

Although TRG mutation is independantly associated to liver disease, concomitant environmental cofactors for cirrhosis (BMI>25 kg/m², excessive alcohol consumption,..) were independently predictive of death or liver transplantation in this population. The diversity of associated liver lesions and risk factors for cirrhosis suggests a multifactorial pathophysiology.

Similary, gene environment and shortened telomeres have been shown to severely impact prognosis in idiopathic pulmonary fibrosis (33): in an American cohort of 134 patients from 21 families with TERT mutation, almost all patients with interstitial lung disease (96%) were smokers or declared a pneumotoxic exposure (12).

In our serie telomere length was below 1st percentile in 90% of tested patients, with non direct impact on prognosis. Conversely, liver stiffness ≥20 kPa, FIB-4 score ≥3.25 and HVPG ≥10 mmHg at baseline correlated with poor survival. Furthermore, liver stiffness (FibroScan®) correlated to hepatic venous pressure gradient and presence of grade II or III esophageal varices.

In cirrhosis, the association between liver stiffness and the degree of portal hypertension as well as with patients' outcomes is clearly established (34). Conversely, liver stiffness < 10 kPa strongly suggests PSVD in patients with signs of portal hypertension, whereas 10k Pa> liver stiffness <20kPa suggests the presence of an extrahepatic condition (35). An increasing amount of data supports the use of FIB-4 as a screening tool in general diabetic populations (36,37), and in predicting the clinical prognosis in NASH and viral hepatitis (38)(39)(40)(41)(42)(43). Thus, our findings suggesting that liver stiffness, FIB-4 and HPVG measurement are predictors of major outcomes in TRG mutation context, support literature in other liver diseases but warrant confirmation.

In conclusion, TRG mutations dramatically increase the risk for liver disease, independently from other risk factors. PSVD and dysmetabolic cirrhosis are the 2 most frequently observed liver related phenotypes in TRG mutation suggesting a multifactorial mechanism. These results support routine screening for TRG mutations in patients with unexplained liver disease (early onset age, personal or familial extra liver disease…). When TRG mutation is identified, other causes for liver disease and/or a FIB-4 score ≥ 3.25 are at particularly high risk for severe liver failure, portal hypertension complications or hepatocellular carcinoma. (25,26).

* Including 75 previously reported patients

Liver disease was defined by raised serum transaminases (AST and/or ALT) >30 IU/L and/or abnormal liver imaging (dysmorphic and/or hyperechogenic liver and/or features of portal hypertension). HSCT before liver disease diagnosis 4 (4) --

Immunologic disease

Gammaglobulins (g/L)

Table 2. Features of liver disease in patients with TRG mutations at the time of liver disease diagnosis (N=95).

Patients with liver diseases (N=95)

Clinical characteristics

Ascites N -(%)

Patients with liver (N=95). B. Patients without liver disease (N=37).

A. Patients with liver disease (N=95). In medical charts, the information that the patient was carrier of a mutation was indicated without the nomenclature.

Patients

Supplementary data 9. Univariate and multivariate analysis of factors associated with overall survival in patients with liver disease and TRG mutations (N=95).

Overall survival

Univariate analysis

Age at liver disease diagnosis We want to thank Kamal Zekrini and Dahia Secour for their collaboration kamal.zekrini@aphp.fr

Background and aims:

Primary Budd-Chiari syndrome (BCS) is a rare disorder defined as a blocked hepatic venous outflow tract by thrombosis at various levels from small hepatic veins to the terminal portion of the inferior vena cava [1]. Nonmalignant non-cirrhotic extrahepatic portal vein thrombosis (PVT) is characterized by a thrombus developed in the main portal vein and/or its right or left branches and/or splenic or mesenteric veins, or by the permanent obliteration that results from a prior thrombus [2] . The pathogenesis of these vascular liver diseases (VLD) is largely dependent on the presence of systemic prothrombotic conditions that promote thrombus formation in the respective splanchnic veins [3,4].

Four to nineteen percent of patients with BCS have paroxysmal nocturnal hemoglobinuria (PNH) and small studies or case reports also described PNH in patients with portal and mesenteric vein thrombosis [1,5,6]. PNH is a rare acquired disorder of hematopoietic stem cells, related to a somatic mutation in the phosphatidyl-inositol Glycan class A (PIG-A), X-linked gene, responsible for a deficiency in glycosyl phosphatidilinositol-anchored proteins (GPI-AP).

The lack of GPI-AP complement regulatory proteins leads to hemolysis. PNH often presents with hemolytic anemia, bone marrow failure and episodes of venous thrombosis. Venous thrombosis occurs in 15 to 30% of the patients with PNH and mainly affects cerebral, deep limb or splanchnic veins. Thrombosis is the major factor affecting outcome [7]. Risk factors for thrombosis include older age, thrombosis at diagnosis, and the need of transfusions. Recently reported approaches have used new therapeutic agents, such as eculizumab, a C5 inhibiting recombinant humanized monoclonal antibody [8][9][10]. In PNH patients, uncontrolled terminal complement activation and the resulting complement-mediated intravascular haemolysis are

Patients and Methods

Methods and results are reported according to the STROBE statement [12].

Design and setting

We performed a retrospective study including patients with PNH and a diagnosis of BCS or PVT made between 1997 and 2019 in centers of the Vascular Liver Disease interest Group ("VALDIG"). Diagnosis of PNH preceded or followed diagnosis of vascular liver disease. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board (IRB n 2003/21, Paris; France).

Demographic and patient's data were collected at the diagnosis of vascular liver disease and during follow-up. Complications related to thrombosis, bleeding, portal hypertension or blood disease were recorded.

Definitions

BCS diagnosis was based on ultrasonography and/or multidetector computed tomography and/or magnetic resonance imaging, and/or venography. Diagnostic criteria for PVT included recent portal, and/or splenic and/or mesenteric venous thrombosis or portal cavernoma. PVT patients with cirrhosis or abdominal malignancies were not included. In the VALDIG group, patients with BCS were treated according a stepwise therapeutic strategy, since 1997 [1,5].

The first step consists of anticoagulation therapy, specific therapy for underlying conditions, and medical or endoscopic management of liver-related complications. Recanalization of stenosis using angioplasty or stenting is routinely considered. In patients who do not respond to this first step therapy, transjugular intrahepatic portosystemic shunt (TIPS) is proposed and, as a fourth step, orthotopic liver transplantation (OLT). Patients with acute portal or mesenteric vein thrombosis are treated with anticoagulants since the 1990's [3,6]. Patients with chronic portal vein thrombosis are treated with anticoagulation when a severe thrombophilic factor is identified, and/or past history of mesenteric infarction or personal or family history of deep vein thrombosis, or on a case to case basis for others [1,13,14]. Anticoagulants therapy is based on low molecular weight heparin during the acute phase, or in patients with high bleeding risk situations, and switch to warfarine derivatives in stabilized conditions [1,4,5,14].

PNH diagnosis was based on the detection of GPI-deficient WBCs by flow cytometry assay.

To avoid biases related to the evolution of flow cytometry techniques a threshold of 5% was used to define a PNH clone. As previously proposed, PNH were categorized as follows [7]: the Classic PNH subcategory includes patients with clinical evidence of intravascular hemolysis but no evidence of other defined bone marrow failure criteria (neutrophils and platelets were per criteria higher than 1,500/L and 120,000/L, respectively). Aplastic anemia-PNH syndrome (AA-PNH syndrome) subcategory includes patients who were diagnosed with PNH (with a PNH clone at diagnosis) and with, at least, 2 or 3 peripheral blood cytopenias (hemoglobin < 100g/L, platelets < 80 × 10 9 /L, neutrophils <1, 000/L). Patients who did not fulfill the last 2 groups' criteria were assigned to a third group of patients called intermediate PNH: this category includes patients with myelodysplastic (MDS) and myeloproliferative neoplasm (MPN).

Temporality of PNH and VLD was categorized as PNH before VLD, PNH after VLD, and contemporary diagnoses when PNH was diagnosed within a month of VLD diagnosis (before or after).

Eculizumab therapy was first available in 2007. In a sensitivity analysis, we considered only patients with a VLD diagnosis made after 2007 (after first availability of eculizumab).

The PNH dosing regimen of eculizumab was the following: of a 4-week initial phase followed by a maintenance phase: Initial phase: 600 mg of Soliris administered via a 25 -45 minute intravenous infusion every week for the first 4 weeks.

Maintenance phase: 900 mg of Soliris administered via a 25 -45 minute intravenous infusion for the fifth week, followed by 900 mg of Soliris administered via a 25 -45 minute intravenous infusion every 14 ± 2 days [9].

Endpoints

Primary endpoint was mortality. Secondary outcomes were as follows: new venous thrombosis (including deep vein thrombosis, new splanchnic vein thrombosis, cerebral thrombosis and TIPS thrombosis) occurring after VLD diagnosis; arterial ischemic event (arterial stroke, myocardial infarction, obliterating arteriopathy), bleeding event including upper gastrointestinal bleeding (UGIB) or other severe bleeding, bacterial infection, and liver complications (including spontaneous bacterial peritonitis (SBP), UGIB, hepato-renal syndrome, hepatic encephalopathy, or ascites (resolved or recurrent)). Ascites resolution was assessed among patients with ascites at VLD diagnosis; and ascites recurrence, among patients with ascites resolution Follow-up Patients were followed according to local practice from the date of VLD diagnosis until death or last visit of follow-up or 10 years follow-up (predefined as the most clinical significant period) after VLD diagnosis, whichever occurred first. For five patients with BMT, follow-up was censored at the date of BMT (the patient being therefore considered as cured).

Investigations for thrombotic risk factors.

Patients were tested according to previously reported methods for the following thrombotic risk factors [4,5]: factor V R 506 Q mutation (factor V Leiden); G 20210 A factor II gene mutation; JAK2 V617F , mutations, deficiencies in protein C, protein S, or antithrombin (regarded as primary deficiencies only in conjunction with a prothrombin index ≥ 80%); PNH; and anti-phospholipid antibodies. Oral contraceptive use was considered as a thrombotic risk factor when taken within the 3 months preceding diagnosis of VLD [15].

Statistical analysis

Data were described using median (interquartile range) or number (proportion) as appropriate.

In order to account and control for immortal time bias [16,17], we defined exposed and unexposed person-time by using start and end date of eculizumab. Incidence rates for individual outcomes were calculated by dividing the incident number of events by the number of person-years (PY) over the corresponding period (either exposed or unexposed period).

Incidence rate ratio (IRR) was estimated by Poisson's regression with their 95% confidence interval (95%CI). In a sensitivity analysis, patients diagnosed and managed after 2007 (i.e. the opportunity of having eculizumab therapy) were analysed separately.

Analyses were performed using the R software (The R Foundation for Statistical Computing Plateform), version 3.6.

Results

Patient characteristics

Patients' characteristics are shown in table 1. Sixty-two patients (33 women, 53%) were included, including 50 with BCS, and 12 with PVT, with a median age of 35 (28 -48) JAK2 V617F myeloproliferative neoplasm was detected in 3 (7%) patients with BCS.

Sixty patients (97%) were treated with anticoagulants: The two patients who were not treated with anticoagulants had severe thrombocytopenia (<40 000 platelets) or/and active bleeding at diagnosis.

PNH characteristics

Median clone size among neutrophils, was 80% (70-90), hemoglobin 10 (8-11)

Patients with BCS

In patients with BCS, hepatic venous outflow obstruction was due to occlusion of one, two and 3 hepatic veins in 7, 7, and 33 patients, respectively, associated or not to obstruction of the suprahepatic segment of the inferior vena cava in 5 patients. Fifteen (31%) out of the 50 patients with BCS also had a PVT, four had an obstruction of the mesenteric vein (one had a mesenteric infarction with small bowel resection), and eight of the splenic vein. Duration between symptoms and BCS diagnosis was longer than 6 months in 17 (37%) patients. One, two and three inconclusive imaging of the liver were performed respectively in 20, 7, and 1 patients before diagnosis of BCS was eventually confirmed. Ten patients underwent a liver biopsy. In 1 patient, imaging was inconclusive whereas liver biopsy found a "small vessel BCS".

Thirty-one (65%) had ascites, 16 (38%) oesophageal varices (4 variceal bleeding). Median Child-Pugh score, Clichy score and Rotterdam score at diagnosis were respectively of 10 (8-11), 6 (5-7) and 1.2 (0.1-1.31).

Patients with PVT

Mesenteric vein obstruction was present in 6 (60%), splenic infarction in 1, and mesenteric infarction with bowel resection, in 1 patient. At diagnosis, 2 patients had oesophageal varices and none had bled.

Therapeutic management

All BCS patients received a stepwise therapeutic strategy, including anticoagulation (n=49, 98%), hepatic venous angioplasty/stenting (n=3; 6%), transjugular intrahepatic portosystemic shunt (n=16, 32%). No patient had liver transplantation.

Patients diagnosed and managed with VLD before and after 2007 were comparable in terms of initial severity, and management (table 3).

Outcomes

Hepatic or portal vein recanalization was obtained in 12 patients, all on anticoagulation therapy.

Overall, 17 (27%) patients died (8 died from severe liver disease complications, 4 from infection and multiple organ failure, 3 from cancer or leukemia, and 2 cerebral bleeding) and fifteen of them had persistent ascites and portal hypertension at the time of death. The mortality was significantly lower with eculizumab therapy (table 4): 2.6 vs 8.7 per 100 PY, IRR 0.30 95%CI (0.10 -0.92) (p value= 0.035).

During follow-up, there were 25 venous thrombosis, including 8 TIPS thrombosis and cerebral thrombosis. New venous thrombosis, in the splanchnic or extrasplanchnic veins, occurred less frequently in patients during exposure time to eculizumab: 2.62 vs 14.2 per PY, IRR 0.22 (0.07-0.64).

Other secondary end-points (bleeding, arterial ischemic lesions, ascites resolution and recurrence, infection and liver complications) were less common in patients exposed versus non-exposed period, although not reaching statistical significancy (table 4). All patients but one have continued long term eculizumab therapy until bone marrow stem cell transplantation, or death. In 8 patients, the dose of eculizumab was temporally increased to 1200mg/every weeks, due to inefficacy on hematological manifestations and on complement blockade: among them, 6 patients had ascites, and 4 had a TIPS. At the end of follow-up, one of these patients was deemed to require BMT, because of severe bone marrow failure. This patient had persisting moderate ascites despite functional TIPS (porto caval gradient below 8 mm Hg).

One patient stopped eculizumab during follow-up, and had recurrent thrombosis despite anticoagulation. Eight (13%) patients underwent haematopoietic stem cell transplantation for aplastic anemia, including 4 who were previously treated with eculizumab.

Twenty-six (42%) still needed regular blood transfusion at the end of follow-up vs. 50 (81%) at diagnosis. Eighteen patients have had severe infection during follow up, which was synchronous to venous thrombosis episode in 3.

When restraining analyses of primary and secondary outcomes to the "after 2007 period", era of eculizumab theapy, we observe significant reduced venous thrombosis and arterial events, and a trend in favour of eculizumab efficacy on survival. (table 5)

Discussion

The most important result from this multicentric cohort is the significant impact on mortality of eculizumab in patients with severe vascular liver diseases complications of PNH, namely BCS or PVT, with an incident rate ratio of death of 8.74 per 100 PY in patients not treated with eculizumab compared to 2.62 per 100 PY in patients treated with eculizumab. To avoid immortal bias, which may arise in cohort studies of drug effects, we have used incidence rates of exposition to assess the effect of eculizumab on endpoints [16,17]. This bias leads, in general, to an estimation in favor of the treatment under study by conferring a spurious advantage to the treated group. Assessing the effect of eculizumab by means of incidence rate ratios permits to limit the bias induced by "time fixed analysis" [16,17]. The drawback of this methodology is that the study spans over a long period of time (1997-2019), with Eculizumab available only after 2007. Nevertheless, this period has the advantage that VLD management otherwise than Eculizumab administration was stable over time among the Valdig group. Indeed, management of BCS or PVT was homogeneous, in terms of anticoagulation therapy for all BCS or PVT patients, and "BCS therapeutic strategy" in BCS within the Valdig group [4,5]. Indeed, most of these centers routinely record VLD patients in a registry for BCS or PVT so called the VALDIG registry, and manage the patients according to a protocol implemented for ENVie studies [3,4]. It is also important to note BCS or PVT patients were comparable before and after 2007, in terms of extension of thrombosis, severity of PNH, severity of VLD (number of BCS and/or PVT) and in BCS patients, prognosis indexes (table 3). Finally, when restricting the analysis from 2007, Eculizumab has similarly a significant impact on thrombosis and arterial complications, and trends to be significant on survival. It is still unclear in what way eculizumab decreases mortality, as immediate causes for deaths varied from severe liver disease complications to septic causes or cerebral bleeding. We have not found a significant impact of eculizumab therapy on liver or portal hypertension complications and conversely we have found that mortality and recurrent thrombotic events are reduced with eculizumab therapy. Indeed, the second main result of our study shows that recurrent thrombosis is less common in PNH patients with VLD treated with eculizumab, with an incident rate ratio of recurrent thrombosis of 14.12 per 100 PY in patients not treated with eculizumab, significantly higher compared to 2.62 per 100 PY in patients treated with eculizumab. (table 4). Although it could have been a hypothesis that patients described with other causes for VLD, would be at higher risk for thrombosis, this risk was not increased in the 11 patients with another cause.

One hypothesis to explain the reduction of mortality in VLD-PNH patients treated with eculizumab could be the persistence of prothrombotic mechanisms in non ecuilzumab-treated patients, despite anticoagulation. Preliminary results presented in 2012, seem to support this hypothesis: survival after eculizumab therapy was a 100% in 19 BCS patients, after a median follow-up of 7 years with eculizumab therapy, recurrent thrombosis did not occur in 6/19 patients after eculizumab late therapy, while 3 had recurrent thrombosis prior eculizumab administration [18]. Development of thrombosis despite anticoagulation therapy was a severe complication prior eculizumab era [7]. Initial studies have shown a significant impact of eculizumab on first incident episode of thrombosis in PNH patients: thrombo-embolic event rate was reduced from 7.3 events per 100 patient-years before eculizumab to 1.07 events per 100 patient-years during eculizumab treatment (P< 0 .001) [9,11] . In these studies, the mean pas history thrombotic event rate at inclusion, was 19 to 43 % [8,9,11]. In our study, all patients (100%) had recent vascular liver disease, and 97% were treated with antithrombotic therapy.

To conclude, we show that recurrent thrombotic risk is much lower in patients with eculizumab therapy, although it still exists in very few patients with other possible local or general co-factors for thrombosis.

Combining PVT and BCS in this study was noteworthy, as it has previously been shown that PNH mainly affects thrombosis in small vessels, sometimes difficult to characterize [19], and associates multiple splanchnic veins sharing similar severe complications such as mesenteric vein infarction [20]. One European study had compared PNH patients to other causes in BCS and identified a significant increase of associated PVT [20]. This is clearly confirmed in this study. Venous thrombosis in other territories occurred in 40% of the patients, mesenteric or splenic infarction occurred in 12%, and BCS was associated with PVT in more than 30% of BCS patients.

This study demonstrates that treating the underlying cause favorably impacts on survival in VLD patients. Actually, a treatable cause for thrombosis is common in VLD patients, including for example myeloproliferative neoplasms, paroxysmal nocturnal haemoglobinuria, or Behcet's disease, collectively found in 50-80% of patients with BCS, and 40-60% of patients with PVT [1].

The management of causes and their treatment could have a major effect on the course of liver disease. Yet, studies on this point are scarce [21]. This important result supports a recommendation for an early treatment for underlying conditions in PNH/VLD. This study also confirms that other causes, such as myeloproliferative neoplasms or antiphospholipid syndrome, can be associated in VLD patients, and that diagnosing one cause should not jeopardise a complete screening of all causes, especially now that we know that treating PNH is efficient on VLD course. It is also interesting to note that eight patients were deemed to require increased doses of eculizumab to reduce hemolysis and obtain complement inhibition as previously shown [22]. Still, few patients died despite eculizumab therapy, or had BMT because of persisting severe hematological prognosis. The impact of persisting ascites in the ability to efficiently block complement with eculizumab needs to be investigated.

In conclusion, eculizumab is significantly associated with a lower mortality and less thrombotic recurrences in the overall VLD/PNH population. Nevertheless, close follow-up is

Introduction

Non-malignant extrahepatic portal vein obstruction in adults is predominantly related to thrombosis and referred to as portal vein thrombosis (PVT). In the absence of cirrhosis, long-standing obstruction due to PVT causes portal hypertension despite the development of hepatopetal veins supplying the liver called portal cavernoma (1)(2)(3)(4). PVT is associated with one or several underlying risk factors of thrombosis in 60-70% of cases (2,(5)(6)(7). The spectrum of the latter covers conditions associated with a major prothrombotic risk factor (e.g. myeloproliferative neoplasms, antiphospholipid syndrome, or homozygous factor V Leiden), to conditions associated with a mild to moderate risk (e.g. heterozygous G20210A

factor II or G1691A factor V mutations, low titer antiphospholipid antibodies, C677T polymorphism in MTHFR gene) (2,4,6). It should be noted that the underlying risk factors cannot be identified in up to 25% of patients using current techniques thus, their risk of recurrent thrombosis is unknown.

The two major complications of long term PVT are gastrointestinal bleeding related to portal hypertension, and recurrent thrombosis. Intestinal infarction is the most severe outcome of recurrent thrombosis. Indeed, this event is associated with a mortality rate of 20 to 60%, while survivors may have severe disability due to extended resection or postischemic intestinal stenosis (1)(2)(3)(4).

Retrospective studies in patients with a previous history of PVT without cirrhosis have suggested that permanent anticoagulation therapy significantly decreases the incidence of recurrent thrombosis without increasing the occurrence or the severity of gastrointestinal bleeding (8)(9)(10)(11). Furthermore, a retrospective study in patients with portomesenteric vein thrombosis provided some evidence that anticoagulation therapy independently improved survival (10). In another multicenter registry study of incidentally diagnosed splanchnic vein thrombosis, anticoagulant therapy reduced the incidence of thrombotic events without increasing the risk of major bleeding (p<0.05) (12). However, this study was limited by a high proportion (28%) of patients with cirrhosis. The scope and strength of existing recommendations from scientific societies are limited by an absence of randomized controlled therapeutic trials (2)(3)(4)13,14). Thus, clarifying the benefit-risk ratio of anticoagulation therapy in patients with a previous history of PVT and without highrisk factors for recurrent thrombosis is an important issue. Moreover, the optimal anticoagulation management in PVT patients has not yet been determined. For the moment the safety and efficacy of direct oral anticoagulants (DOACs) in patients with portal hypertension can only be evaluated from retrospective analyses of small groups of hepatocellular carcinoma or childhood cavernoma were not eligible. Patient eligibility was reviewed by a central eligibility committee including 3 hepatologists and 3 hematologists.

Estroprogestatives were stopped before inclusion. The complete study protocol including a list of eligibility criteria is provided in the Appendices.

Outcomes

The primary endpoint was the occurrence of a venous thromboembolic event at any site and/or death within 2 years of randomization. According to the Prospective Randomized Open Blinded Endpoint (PROBE) methodology, to limit the risk of misclassification due to the open-blinded design, an independent committee including a hepatologist and a radiologist blinded to the treatment group assessed all abdominal CT scans and clinical data (18).

Secondary endpoints included death, pulmonary embolism, deep vein thrombosis, major bleeding according to ISTH (International Society on Thrombosis and Haemostasis) guidelines definition (16), portal hypertension related bleeding, non-bleeding complications of portal hypertension (i.e. ascites, portal cholangiopathy), minor bleeding, liver toxicity (as defined by an otherwise unexplained increase in serum aminotransferase levels) and any other adverse events.

Randomization and treatment

A randomization sequence was used for treatment assignments, with stratification according to anticoagulant therapy at inclusion and center. Patients were randomly allocated in a 1:1 ratio to receive either oral rivaroxaban 15 mg once daily for the duration of follow-up, or no anticoagulant medication. A dose of 15 mg daily was chosen for the current study because PVT patients are at risk of portal-hypertension related bleeding.

Thus the 15 mg dose was based on studies for atrial fibrillation which used a dose of 15 mg of rivaroxaban daily in patients with high risk of bleeding due to altered renal function (17)

Follow-up visits

Patients were seen at randomization (baseline) and, at 1, 3, and 6 months, then every 6 months thereafter for 24 months to 48 months. Demographic data were collected at baseline. Clinical, imaging and laboratory data were obtained at baseline and at each follow-up visit. At each follow-up visit, the investigator collected information on prespecified complications.

Sample size and statistical analyses

We calculated that a sample size of 296 patients was needed to detect 3.5% recurrent thrombosis in the non-treated group versus 0.8% in the rivaroxaban group, with a power of 80%, a two-sided type I error rate of 0.05 and 6% of lost to follow-up patients. We assumed that 24 months were needed to recruit patients, with a minimum follow-up of 24 months. However, after temporary suspension of inclusions due to 5 thrombotic events in the non-treated group within 21 weeks after the study began (first interim analysis), the data monitoring and safety committee recommended that the study continue and that a second interim analysis be performed. The study protocol was therefore amended with an additional interim analysis planned after 10 thrombotic events.

Analyses of the primary and secondary outcomes included all patients who underwent randomization (intent-to-treat population). Analysis of the primary outcome was based on a group-sequential design with two interim analyses with O'Brien & Fleming stopping boundaries (i.e. respectively 0.0005 and 0.014), and stratified two-sided log-rank tests comparing patients randomized in the rivaroxaban group with those in the nonanticoagulation group.

Inclusions were discontinued on January 19, 2018 after the second interim analysis (after 28 months of patient enrolment). All patients were thereafter offered anticoagulant therapy and followed-up for at least 24 months after randomization and up to 48 months. Rivaroxaban 15 mgonce daily was suggested but the attending physicians and patients were allowed to make their own decision. Thus, there were two distinct periods to the study: Period 1 (randomized period) from randomization to January 19, 2018 and the last inclusions with a proposal to switch patients in the no anticoagulation group to anticoagulants, and Period 2 (follow-up period): from January 19, 2018 to end of study on January 30, 2020 (Supplementary figure 1).

Major and minor bleeding events as well as non-bleeding outcomes were analyzed in randomized patients and followed according to their effective treatment received throughout the entire study (both period 1 and period 2). We estimated person-times on treatment with rivaroxaban, on other anticoagulant therapies and with no treatment using the start and stop dates of each type of treatment. The incidence rates of bleeding events and non-bleeding outcomes were calculated by dividing the number of incident events by the number of person-years during the corresponding period. Incidence rate ratios (IRR) were estimated by Poisson's regression with a 95% confidence intervals (95%CI).

Univariate Cox proportional hazard models were used to assess whether thrombophilia, other known risk factors for thrombosis, imaging data including extension of thrombosis (intra, extra hepatic, and recanalized PVT), laboratory data (PT, coagulation factors and factor VIII, fibrinogen, transaminases) collected at randomization, and D-dimer and factor VIII values at one month after randomization, were associated with recurrent thrombosis among patients randomized in the no-anticoagulation group. In this analysis, patients were followed from randomization until January 19, 2018 (second interim analysis and inclusions' discontinuation).

A P-value of 0.05 or less was considered to be statistically significant. Data handling and analysis were performed with SAS statistical software, version 9.4 (SAS Institute, Cary, NC, USA).

Results

Trial Population

Overall, 193 patients with PVT were screened by participating hospitals. The number of included and randomized patients is described in figure 1A. We analyzed 55 patients in the rivaroxaban group and 56 in the "no anticoagulation" group (figure 1A). Median followup for the whole study was 30.3 months [29.8-35.9]. Three patients were lost to follow-up.

The median time from PVT diagnosis to inclusion was 1.7 [0.9-5.3] years.

Ninety-two patients were included in Hôpital Beaujon, and 19 patients in the seven other French centers (Supplementary Table 1).

Median follow-up for period 1 (i.e. randomized period) was 11.8 months [8.8-13.2]. Baseline characteristics were similar in the 2 study groups (Table 1). As shown in table 1, low-risk thrombophilia were identified in 48 patients (50%). When combining low risk thrombophilia and other minor identified risk factors for thrombosis (male gender, BMI>30, over 60 years old, non-O blood group, Systemic disease, PSVD, induced 1st degree family history ≥2, unprovoked 2nd degree family history ≥1, Factor VIII > 150) , at least one persisting low risk factor was identified in 94% of patients. One patient with protein S deficiency had a previously identified detrimental PROS1 heterozygous mutation. When PVT was diagnosed, a local cause was found in 17 (15%) patients, including 2 liver abscesses, 6 infectious colitis, 3 acute pancreatitis and 6 other splanchnic or systemic infections, which had all been cured at inclusion. At inclusion, a portal cavernoma was present in 70% of patients. Obstruction of the portal vein trunk or concomitant obstruction of both right and left portal branches was present in 73% of patients. The location of intra or extra hepatic obstruction according to the study group is shown in Table 1. A detailed description of the splanchnic vein obstruction at diagnosis is provided in Supplementary Figure 2. Porto-systemic collaterals were present in 53% of patients, esophageal varices in 29% and gastric varices in 9%.

At randomization, 82 patients (74%) were on anticoagulation therapy. Nineteen of the 29 remaining patients not treated with anticoagulants at inclusion, had had prior anticoagulation therapy and 10 had not.

Sixteen of the 29 patients without anticoagulants at inclusion were randomized to the rivaroxaban group and 13 to the no-anticoagulation group.

Primary Outcome

During period 1 and up to the second interim analysis, venous thrombosis recurred in 10 patients, involving the splanchnic territory in 4 patients, the lower limb veins in 3, and the pulmonary veins in 3 (Supplementary Table 2). There were no deaths. The incidence rate of thrombotic events was 19.71 per 100 PY (95% CI [7.49 -31.92]) in the control group, while no thrombotic events occurred in the rivaroxaban group. Event-free survival significantly differed between treatment groups (p=0.0008, Figure 2).

Risk factors for recurrent thrombosis

During period 1, univariate analysis was performed in the no-anticoagulation group to identify risk factors for thrombosis recurrence, including thrombophilia factors, imaging data including extension of thrombosis (intra, extra hepatic, and recanalised PVT), laboratory data, and D-dimer and factor VIII values at one month. None of the thrombophilia factors were associated with recurrent thrombosis. One patient with recurrent thrombosis (pulmonary embolism) had been shown to have detrimental PROS1 heterozygous mutation (Supplementary Table 2). D-dimer ≥ 500 ng/mL at 1 month was significantly associated to recurrent thrombosis (HR=7.78 [1.49-40.67]) (Figure 3) with a predictive positive value (PPV) of 37.5 % and a negative predictive value (NPV) of 93.8%. Isolated and resolved causes for PVT (local causes or estrogen containing oral contraceptives) were associated with an absence of recurrent thrombosis (Table 2).

Follow-up period (Period 2)

Period 2 started on January 19, 2018, and lasted until the last patient's visit on January 30, 2020. During Period 2, all randomized patients were followed for a mean 20.6 months (±3.7 months). Anticoagulation therapy was proposed to all patients Forty six of the 55 patients from the rivaroxaban arm continued rivaroxaban, 4 switched to another anticoagulant and 5 received no anticoagulants Thirty two of the 56 patients from the no anticoagulation arm received rivaroxaban, 17 another anticoagulant, and 7 no anticoagulants (figure 1B).

During Period 2, 3 patients not exposed to recent anticoagulation, developed 3 new thrombotic events. The incidence rate of thrombotic events was 2. 2) Additionally, patient # 2, who had already had a recurrent deep vein thrombosis during Period 1 and was treated with warfarin, had recurrent portal vein thrombosis associated with a liver abscess.

A detailed description of patients with recurrent venous obstruction is provided in Supplementary Table 2.

Secondary Outcomes

Major bleeding occurred in 3 patients throughout the study period, 2 in patients treated with rivaroxaban, and 1 in a patient not treated by an anticoagulant (Table 3). Rivaroxaban was not definitively interrupted in any of them. Complications were comparable throughout the periods of exposure (either DOACs, other anticoagulants or untreated period) for major bleeding, portal hypertension related bleeding and other complications.

Fifty-seven minor bleeding episodes occurred in 36 patients: 41 episodes in 24 patients in the rivaroxaban group vs 16 in 12 patients in the no-anticoagulation group. Bleeding episodes mainly involved epistaxis (23%), uterine bleeding (11%), and rectal bleeding (16%). Minor bleeding occurred more frequently during exposure to DOACs than during the period without treatment (IRR 4.86 [1.75 -13.50]). In one patient, recurrent minor bleeding episodes (rectal bleeding) led to permanent discontinuation of anticoagulation therapy.

Twenty-two other adverse events were reported in 17 patients, including 4 episodes of ascites, and 3 episodes of portal cholangiopathy related symptoms (Supplementary table 3). Four episodes of acute pancreatitis occurred in three patients treated with rivaroxaban, 2 of whom had taken codeine known as as a possible drug cofactor. None of these adverse events were directly related to rivaroxaban. There was no evidence for liver toxicity from rivaroxaban (supplementary figure 2).

Discussion

This first randomized controlled trial fills the knowledge gap on the risk of recurrent thrombosis in patients with a previous history of non-cirrhotic portal vein thrombosis.

Despite a careful exclusion of high-risk factors for recurrent venous thrombosis, the incidence of thrombotic episode was close to 20 p.100 PY in the 56 patients randomized to no anticoagulation therapy. The severity of recurrent thrombosis should be highlighted, as the splanchnic veins were involved in 4 cases and pulmonary embolisms in 3. It is important to note that the results in Period 2 confirm the high risk of recurrence found in Period 1, the randomized phase of the study. Indeed, during Period 2, 3/12 patients who stopped anticoagulation developed recurrent episodes of thrombosis. Thus, this study shows that a past history of portal vein thrombosis per se should be considered as a major risk factor for recurrent thrombosis even in the absence of recognizable prothrombotic conditions. It should be noted that although 50% of our patients had identified low-risk thrombophilia the presence and nature of the underlying conditions had no relationship to recurrent thrombosis. Thus far, this comprises the first randomized controlled trial and hence, the previously published data cannot be fully compared to the present findings because of the retrospective design of those previous studies, and the heterogenous patient characteristics and management (8)(9)(10)(11). Nevertheless, the incidence of recurrent thrombosis in the no-anticoagulation group from the present study is clearly higher than reported in retrospective studies which merged patients with and without anticoagulation therapy (8)(9)(10)(11).

On the other hand, the 55 patients allocated to the anticoagulant group that received rivaroxaban 15 mg daily were fully protected from a new episode of thrombosis. The protective effect of rivaroxaban appeared so early in the study that an early interruption was requested by the independent data monitoring and safety committee. These prospective findings clearly validate conclusions from retrospective data, showing that anticoagulation plays an independent in preventing recurrent thrombosis in patients with non-cirrhotic PVT (8)(9)(10)(11)(12)18).

The benefit of preventing recurrent thrombosis with rivaroxaban must be balanced to the risk of bleeding during therapy. The modification in patients' management as requested by the independent data safety committee (all patients were proposed anticoagulation therapy during Period 2) limits full comparison to the control group. Nevertheless, Period 2 has allowed us to follow 99 patients receiving anticoagulation out of the 111 enrolled.

Most of the patients on anticoagulation therapy (78/99) received rivaroxaban 15 mg per day. As expected, the absolute incidence of a minor bleeding episode was high (16)(17)(18)(19)(20)(21)(22)(23)(24) p.100 PY), and significantly higher than in the absence of anticoagulation therapy. On the other hand, the incidence of major bleeding (incidence 1.02 per100 PY) and portalhypertension related bleeding (incidence 1.5-2.7 per100 PY) were rare. In the absence of portal hypertension, the overall risk of major bleeding during anticoagulation therapy with warfarin and DOACs assessed in pivotal meta analyses and real life studies in atrial fibrillation was found to be close to 5 to 6 per 100 PY (19)(20)(21). DOACs have been shown to be associated with an increased frequency of gastrointestinal bleeding compared to warfarin, athough the severity of these episodes does not seem to be increased (22,23).

Furthermore, although it is based on limited data, the risk of bleeding in patients with cirrhosis and portal hypertension seems to be lower with DOACS than with vitamin K antagonists (24)(25)(26)(27). Up to now, evidence for the safety and efficacy of DOACS in PVT in the absence of cirrhosis has been limited (15).

Our results do not show any increased risk of major bleeding or portal-hypertensionrelated bleeding, and strongly support the use of rivaroxaban 15 mg daily in this indication.

It is important to note that there were no signs of liver toxicity from rivaroxaban in the present study.

The findings of this study may pave the way for personalized treatment by clarifying the risk factors of recurrent thrombosis in the absence of anticoagulation therapy. Indeed, like in deep vein thrombosis (28)(29)(30), a D-dimer value <500 ng/ml at 1 month after discontinuation of anticoagulants is strongly associated with a low risk of recurrence, given a negative predictive value of 93.5 %. None of the common causes of thrombosis were significantly related to the risk for recurrence. Obesity and advanced age were not identified as additional risk factors for recurrence. Furthermore, none of the patients who had mainly local infectious or inflammatory causes of PVT at diagnosis that were resolved at inclusion or who took estroprogestative oral contraceptives had recurrent thrombosis. Thus, in patients with isolated and resolved causes of PVT, discontinuation of anticoagulants could be considered based on D-dimer values at 1 month. These conclusions should be validated in a large independent group of patients.

Conclusion

In non-cirrhotic patients with a past history of PVT without major prothrombotic risk factors, long term rivaroxaban p.o. at 15 mg once daily reduced the incidence of recurrent venous thrombosis but did not increase the occurrence of major bleeding. D-dimer<500ng/ml one month after anticoagulation is discontinued predicted a low risk of recurrence. As this is the first randomized clinical trial in the setting of PVT and thrombosis recurrence to date, the predictive value of D-dimers for the risk of recurrence should be validated in further studies.

CONCLUSION I-Conclusion and perspectives

Ours first study has shown the dramatic impact of TRG mutations on the risk for liver disease, independently from other risk factors, especially for PSVD. These results support routine screening for TRG mutations in patients with unexplained liver disease (early onset age, personal or familial extra liver disease…). When TRG mutation is identified, other causes for liver disease and/or a FIB-4 score ≥ 3.25 are at particularly high risk for severe liver failure, portal hypertension complications or hepatocellular carcinoma. Our second study assesses for the first time the impact of eculizumab for PNH on survival, and complications in patients with vascular liver diseases. The impact on clinical practice is major in the field of hematology, hepatology and gastroenterology as for the first time it is clear that in this specific setting of splanchnic vein thrombosis, early administration of eculizumab is mandatory, in addition to standard of care therapy for BCS or PVT, to avoid death from severe liver failure in BCS and mesenteric infarction in PVT due to recurrent thrombosis Early initiation of eculizumab and close monitoring of clinical and biological markers, concomitant to VLD treatment prevents severe VLD and may avoid the use of invasive procedures. Our third study shows that in non-cirrhotic patients with a past history of PVT without major prothrombotic risk factors, long term rivaroxaban at 15 mg once daily reduced the incidence of recurrent venous thrombosis but did not increase the occurrence of major bleeding. We identified D-dimers as specific biomarkers to predict the risk of recurrence In conclusion this thesis work provides new insights into the role of causes for vascular liver diseases, and their impact on prognosis. It also discloses new strategies for therapeutic management, emphasizing the need to treat the cause of VLD concomitantly to the VLD. It also modifies the dogmas, where patients with no cause identified are probably at higher risk of thrombosis, and necessitate permanent anticoagulation.

Additional works would be useful to clarify the role of telomerase mutations in the mechanisms of PSVD, in particular the role of these mutations on telomeres length before and after liver transplantation in leucocytes and in liver tissue.

Analysis of NGS added to D dimers of patients with currently no cause for PVT may also be helpful to adapt personalized therapeutic strategies in these patients.

  ................................................................................................. a) AT, PC, PS deficiency...................................................................................... b) Other syndromic diseases associated to genetic conditions......................... c) Telomeres regulatory gene (TRG) mutations................................................. 3-Other risk factors..................................................................................................... a) Local cause..................................................................................................... b) Central obesity and metabolic syndrome...................................................... c) Hormones and VLD........................................................................................ d) Other causes.................................................................................................. III-Treatment……………………………………………………………………………………………………….………. 1-Medical therapy: Anticoagulation and treatment of the cause............................... A-Anticoagulation................................................................................................... a) Indications...................................................................................................... b) Type of anticoagulation................................................................................. c) Bleeding complications in patients treated by anticoagulant treatment for deep vein thromboses or splanchnic vein thrombosis.................................. d) Treatment of the cause................................................................................. IV-Risk of thrombosis recurrence in deep vein thrombosis and identified risk factors... V-Risk of recurrence in VLD and identified risk factors.................................................. 1-Risk of recurrence in Budd Chiari syndrome with or without anticoagulant treatment............................................................................................................... 2-Risk of recurrence or extension of portal thrombosis with or without anticoagulant treatment...............................................................................................................

Figure 2 :Figure 3 :

 23 Figure 2: Hematologic stem cell is affected quantitatively and functionnaly in TRG mutations

Figure 4 : 3 -

 43 Figure 4: Hypothesis regarding TRG mutation impact in NASH, cirrhosis and HCC, in addition to acquired risk factors[38]

  Article type: Original Research Title: Mutations of telomere-related genes, a risk factor for liver disease: a multicentric study. Keys words: Liver disease, telomere biology disorder, mutations of telomere-related genes, porto sinusoidal vascular disease, Nonalcoholic steatohepatitis, fibrosis, FIB-4 score. (14) Centre Hospitalier Universitaire de Montpellier, Hépatologie, Montpellier, France (15) Centre Hospitalier Régional Universitaire de Lille, Hépatologie, Lille, France (16) Hôpital Foch, Hépatologie, Suresnes, France (17) Hôpital Cochin, Hépatologie, Paris, France (18) Hôpital Nord, Pneumologie, Marseille, France (19) Centre Hospitalier Régional Universitaire de Lille, Pédiatrie, Lille, France (20) Hôpital Foch, Médecine Interne, Suresnes, France (21) Hôpital Universitaire Dupuytren, Hépatologie, Limoges, France (22) Hôpital Paul-Brousse, AP-HP, Hépatologie, Villejuif, France (23) Hôpitaux Universitaires de Genève (HUG), Pathologie, Genève, Suisse (24) Centre Hospitalier Universitaire Vaudois, Pneumologie, Lausanne, Suisse (25) Hôpitaux Universitaires de Genève (HUG), Hépatologie, Genève, Suisse (26) Centre Hospitalier Universitaire -Haut-Lévêque, Hépatologie, Pessac, France (27) Centre Hospitalier Universitaire, Hépatologie, Clermont-Ferrand, France (28) Centre Hospitalier Universitaire, Hématologie, Clermont-Ferrand, France (29) Centre Hospitalier Universitaire, Hépatologie, Rennes, France (30) Centre Hospitalier Universitaire, Pneumologie, Rennes, France (31) Centre Hospitalier Régional Universitaire de Lille, Pneumologie, Lille, France (32) Hôpital Saint Louis AP-HP, Pneumologie, France (33) Centre Hospitalier Universitaire de Caen Normandie, Hépatologie, Caen, France (34) Centre Hospitalier Universitaire Amiens-Picardie Site Sud, Hépatologie, Amiens, France (35) Hôpital Bichat-Claude Bernard AP-HP, Génétique, Paris, France (36) Hôpital Avicenne AP-HP, Hépatologie, Bobigny, France (37) Hôpital Beaujon AP-HP, Radiologie, Clichy, France (38) Hôpital Purpan, Hépatologie, Toulouse, France (39) Hôpital Avicenne AP-HP, Pneumologie, Bobigny, France (40) Hôpital Beaujon AP-HP, Anatomopathologie, Clichy, France * Both authors contributed equally to this work. Centres de Références des Maladies Rares (CRMR) (Referral centre for rare diseases) Centre de Référence des Maladies Pulmonaires Rares (Orphalung) (Rare Referral centre for Lung Disease) Centre de Référence des Maladies Vasculaires du Foie (CRMVF) (National network for Vascular Liver Disease) Centre de Référence Aplasies (Rare Referral centre for Aplastic Anemia)

  This first cohort consisted in patients managed for TRG mutations in twenty-five centers. Screening for liver disease was performed between October 2018 and August 2019 in all known TRG mutations patients, diagnosed between 1990 and 2019. Molecular explorations results were reviewed to confirm mutation pathogenicity. Patients or relatives gave a signed consent for germline genetic analysis. Those patients had been included in registers and consented to the use of their data for research purposes. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board (IRB n 2003/21, Paris; France).

*

  iron deposition on MRI -N/investigated (%)14/48 (29) Cofactor for liver disease: excessive alcohol consumption >100 g/week, positive HBs Ag, metabolic syndrome (Defined by the presence of at least 3 of the following criteria: hypertension, diabetes, overweight BMI >25 kg/m², HDLc <0.4 g/L in men and <0.5 g/L in women), BMI >25 kg/m², iron overload (ferritin >1000 µg/L). BMI: body mass index (kg/m²).

Figure 2A .

 2A Figure 2A. Overall survival and liver transplantation-free survival in patients with liver disease and TRG mutations (N=89) (Kaplan Meir) (89/95 patients had a diagnosis of liver disease and genetic mutation between 2004 and 2019 were included in survival analysis). 2B. Liver transplantation-free survival according to prognostic factor of patients with liver disease who received diagnoses of liver disease and genetic mutation between 2004 and 2019 (N=85).

  TINF2 c.849delC, p.(Ser284Leufs*67) <1st TERC r.119delC <1st TINF2 c.873G>C,p.(Arg291Ser) <1st TERT c.2224C>T,p.(Arg742Cys) <1st TERC r.448 A>U <1st TERC r.58C>U <1st TERT c.2446C>G, p.(His816Asp) ; TERC r.434G>U <1st DKC1 § NA TERC r.110_113delGACT NA TERT c.2537A>G,p.(Tyr846Cys) <1st TERT c.2011C>T,p.(Arg671Trp) <1st RTEL1 c.2248C>T,p.(Arg750Cys) NA RTEL1 c.2869C>T, p.(Arg957Trp) NA TERT c.2843+1G>A NA TERC r.130G>A NA TERC r.37A>G NA TERT c.2935 C>T,p.(Arg979Trp) <1st RTEL1 c.2266-1G>C <1st RTEL1 c.3493dupC, p.(Gln1165Profs*22) <1st TERT c.2224C>T,p.(Arg742Cys) <1st TERT c.2383-2A>G NA TERT c.2159T>C, p.(Ile720Thr) NA TERT c.446T>A, p.(Leu149Gln) NA TERC r.313C>U <1st TERT r.448A>T <1st TERT c.3286C>T, p.(Leu1096Phe) <1st # TERC r.36C>U <1st TERT c.1657G>A, p.(Val553Ile) 25th TERT c.2225G>A, p.(Arg742His) <1st TERT c.2080G>A, p.(Val694Met) NA TERT c.2989G>A, p.(Val997Met) <1st TERC r.448A>U NA TERT c.2935C>T, p.(Arg979Trp) NA TERT c.2935C>T, p.(Arg979Trp) NA TERT c.2224C>T, p.(Arg742Cys) <1st TERC r.67G>A NA TERT c.2147C>T, p.(Ala716Val) <1st DKC1 c.851G>A, p.(Arg284Gln) <1st TERC r.35C>U <1st TERC r.235C>G 25th TERT c.2516C>T, p.(Thr839Met) <1st RTEL1 c.2946C>G, p.(His982Gln) 10th TERT c.2968C>T, p.(Gln990*) <1st & ? RTEL1 c.2892T>G, p.(Phe964Leu) ; c.3802T>C, p.(Cys1268Arg) <1st NHP2 c.122G>A, p.(Arg41His) <1st DKC1 c.1318G>A, p.(Glu440Lys) <1st TERT c.2968C>T p.(Gln990*) NA TERC r.441C>U NA TERT c.2287-2A>G <1st TERT c.2665C>T, p.(Arg889*) NA TERT r.182delG <1st TERT c.2227C>T, p.(Arg743Trp) 50-75th PARN § NA TERT c.1072C>T, p.(Arg358Trp) 10th TERT § NA TERC r.323C>G <1st TERC r.270G>C NA NHP2 c.289_290delAT, p.(Met97Valfs*2) <1st RTEL1 c.2767A>C, p.(Lys 923Gln) NA & ? RTEL1 c.394C>T, c.394C>T, p.(Arg132Trp) ; c.1537 dup, p.(Val513Gly fs*18) NA TERT c.2377G>A, p.(Glu793Lys) <1st TERC r.179U>C <1st TERT c.2147C>T, p.(Ala716Val) <1st TERT c.1630T>C, p.(Phe544Leu) NA TERC r.236C>T NA RTEL1 c.1451C>T, p.(Pro484Leu) NA RTEL1 c.146C>T, p.(Thr49Met) NA TERT c.1511C>T, p.(Ser504Leu) NA TERT c.3216G>A, p.(Trp1072*) <1st TERT c.2911C>T, p.(Arg971Cys) <1st TERT c.2093G>A, p.(Arg698Gln) NA TERT c.2911C>T, p.(Arg971Cys) <1st TERT c.2836T>C, p.(Tyr946His) NA TERT c.1864C>T, p.(Arg622Cys) NA TERT c.2287-2A>C NA TERC r.448A>U <1st TERT c.2945G>A, p.(Cys982Tyr) NA TERC r.65G>C NA TERT c.2225G>T, p.(Arg742Leu) <1st TERT c.1990 G>A, p.(Val664Met) <1st TERT c.2552A>T, p.(Asn851Ile) <1st TERT c.2678A>T, p.(Glu893Val) NA TERT c.2960T>C, p.(Leu987Pro) NA TERT c.1710G>C, p.(Lys570Asn) NA TERT c.2225G>A, p.(Arg742His) <1st TERC r.180C>U <1st TERT c.2911C>T, p.(Arg971Cys) NA TERT c.2911C>T, p.(Arg971Cys) NA TERT c.191C>T, p.(Pro64Leu) NA TERT c.2320C>T, p.(Arg774*) NA TERT c.3040G>C, p.(Ala1014Pro) NA TERT c.3040G>C, p.(Ala1014Pro) NA TERT c.265T>C, p.(Cys89Arg) NA B. Patients without liver disease (N=37). Patients Mutations (most mutations were identified at heterozygous state, excepted those indicated by a #) Telomere lengths* TERT c.2542G>A, p.(Asp848Asn) <1st TERT c.2542G>A, p.(Asp848Asn) <1st TERC r.67G>A NA RTEL1 c.2879 A>G, p.(His960Arg) 15th TERT c.1891C>T, p.(Arg631Trp) NA RTEL1 c.146C>T, p.(Thr49Met) NA RTEL1 c.146C>T, p.(Thr49Met) NA RTEL1 c.146C>T, p.(Thr49Met) NA RTEL1 c.146C>T, p.(Thr49Met) NA TERT c.22_43dup22, p.(Arg15ProfsX) NA TERT c.2011C>T, p.Arg671Trp NA TERT c.1990 G>A, p.(Val664Met) <1st TERT § NA PARN c.948_949del AT, p.(Val318Phe fs*9) NA PARN c.948_949del AT, p.(Val318Phe fs

  for the French national network for vascular liver diseases 1 Hôpital Beaujon, AP-HP Nord-Université de Paris, DHU Unit, Service d'Hépatologie, Centre de Référence des Maladies Vasculaires du Foie, Inserm U1149, Centre de Recherche sur l'Inflammation (CRI), Paris, Université de Paris, ERN Rare liver Clichy, France BACKGROUND Paroxysmal nocturnal hemoglobinuria (PNH) is identified in 2-10% of vascular liver disease (VLD) patients. Eculizumab is indicated in PNH patients with hemolysis and clinical symptoms indicative of high disease activity. FINDINGS Eculizumab reduces mortality and thrombotic recurrence in patients with VLD and PNH, in addition to standard of care therapy for VLD (anticoagulation and recanalization procedures according to response to medical therapy). IMPLICATIONS FOR PATIENT CARE. Early initiation of eculizumab and close monitoring of clinical and biological markers, concomitant to VLD treatment prevents severe VLD and may avoid the use of invasive procedures. List of Abbreviations PNH Paroxysmal nocturnal hemoglobinuria; BCS, Budd-Chiari syndrome; PVT portal vein thrombosis; VLD vascular liver disease; JAK2, Janus kinase 2 gene; MPN, Myeloproliferative neoplasm; VALDIG European vascular liver disease network; SBP, spontaneous bacterial peritonitis; UGIB, upper gastrointestinal bleeding; TIPS transjugular intrahepatic portosystemic shunt; OLT, orthotopic liver transplantation; BMT, Bone marrow transplantation Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, Budd-Chiari syndrome, Portal vein thrombosis, splenomegaly, arterial ischemic event, cohort study, mortality, eculizumab

  years at the time of VLD diagnosis. Median follow-up after VLD diagnosis was 4.7 years (1.2-9.5). In 13 patients, PNH was diagnosed after VLD, with a median interval of 0.2 (0-1.7) years. In 4 patients, PNH was diagnosed at the time of VLD diagnosis, and in 45 patients PNH was diagnosed before VLD diagnosis with a median interval of 3.6 (1.3-7.1) years (table 2). Venous and arterial thrombosis elsewhere at diagnosis had occurred in 34 including 8 (13%) cerebral thrombosis.

Out of the 62

 62 patients, 42 (68%) patients received eculizumab, including 29 within the first 4 months after VLD diagnosis; 34/42 (80%) had BCS, and 8/42 (20%) had PVT. Eculizumab administration was delayed in 8 patients with a median delay of 4.2 (3.4 -5.9) years. In 4 of these 8 patients diagnosis of PNH was reached after the diagnosis of VLD. Eculizumab median exposure time was 40.05 [9.33:72.55] months. All had Eculizumab until death or bone marrow stem cell transplantation, or patient's interruption (n=1). Eculizumab therapy according to BCS or PVT diagnosis is described figure 1.

Figure Legends

  Figure Legends

Figures and tables: 3

 3 Figures and tables:3 tables and 3 figures / 3 supplementary tables and 3

  14 per 100 PY (95% CI [0.04-4.24]) during period 2: Patient #11 had stopped anticoagulation therapy for personal reasons 15 months before the recurrent episode of thrombosis. He had a protein S deficiency and portosinusoidal vascular disease. Patients # 12 and # 13 had superficial venous thrombosis and splenic vein thrombosis, 2 days and 23 months after anticoagulation interruption. (Supplementary Table

Figure 1A: flow chart period 1 Figure 1B: flow chart period 2 Figure 3

 123 Figure 1A: flow chart period 1

  Professeurs Ariane Mallat et Nathalie Ganne, d'avoir accepté d'être rapporteurs de ce travail. Vous portez chacune notre voix et représentez nos efforts. Merci au Professeur Christophe Bureau d'avoir accepté d'être examinateur. Vous avez ensemble travaillé pour tracer le sillon de notre spécialité afin qu'elle progresse avec l'AFEF, lieu d'échange et de collaboration. Merci au Docteur Kannengiesser, de m'avoir éduquée avec tellement de bienveillance aux approches génétiques des téloméropathies. Beaucoup d'amitiés et de beaux souvenirs de travail avec les Docteur Didier Lebrec et le Docteur Richard Moreau. Richard, ce sont ces premiers travaux de recherche avec toi qui m'ont donné envie de poursuivre dans cette voix.

	Merci Danielle, Stéphane, aux patients et tous les amis de l'AMVF. Quelle découverte de travailler
	avec vous.
	Aux chers absents mon père Jacques Plessier, Olga, Grane et Clothilde, Caroline, Manuel,
	A mes proches qui m'entourent : A ma mère Marie-Amélie Anquetil, à Suzanne
	A Philippe tellement « supportive », A nos chers enfants Arthur, Léo, Marin, Sarah, Victor, William,

Merci Pierre Emmanuel, d'être là et partout à la fois.

Merci à toute l'équipe motivée du centre de référence. A Kamal Zekrini et Djalila Seghier pour votre travail de recherche auprès des malades et des médecins, à Marie Santin pour son rôle de formation à l'empowerment et à Valérie de Bremand sans qui ce travail n'aurait pu aboutir. Merci à l'ensemble du réseau français des maladies vasculaires du foie. Merci à tous mes collègues et amis de Beaujon, personnel médical et para médical, des maladies vasculaires du foie, des maladies rares, et de thèse, Audrey, Sabrina, Olivier, Mohamed, Laure, Sophie, Odile, Julie, Lucie, Lucile, Valérie « s », Onorina, Emmanuelle, Larbi, Claire, Dominique, Aurélie, Laurent, Tarik, François, Jacques et Françoise, Régis et Flore, Raphael, Olivier, Francisca, Vinciane, Violaine, Isabelle, Odile…et tous les autres…avec qui j'ai partagé et partage encore le quotidien.

A Nils et Djoher, Marine, Clément et Jean-Jacques. A Françoise, Valentine, Marie-Pierre, Benoit, Hamid et Jo, Marie-Odile, Caline, Isabelle, Alexandra, Pilar, aux compagnons de voile Laurent « s », Florian, Fred et Maurizio

TABLE OF CONTENTS

 OF RESUME…………………….………………………………………………….…........................................…..…. ABSTRACT………………...……………………………………………….….......................................….…...…. ABREVIATIONS USED…………………………………………………………….......................................…... Vascular liver diseases: Definitions and classifications I-Definitions……………………………………………………………......…..…........................................ 1-Non cirrhotic portal vein thrombosis (Non cirrhotic PVT)………......…...................... 2-Budd Chiari syndrome………………………………………………….....................................….. 3-Porto sinusoidal vascular disease (PSVD)……………………...………............................... II-Causes and mecanisms of thrombosis in VL……………………………………....................

…… 1-Acquired conditions................................................................................................ a) Myeloproliferative neoplasm……………………………………………...................….…. b) Paroxysmal nocturnal hemoglobinuria (PNH)................................................ c) Behcet's disease (BD)..................................................................................... 2-Genetic conditions.

3 -

 3 Risk of extension to portal vein thrombosis in PSVD with or without anticoagulant treatment............................................................................................................... THESIS WORK..................................................................................................................... I-Background and aims..................................................................................................

	..
	2-Article 2………………………………………………………………………………………………….….…….
	3-Article 3……………………….………………………………………………………………………….……….
	Annex 1………………………………………………………………………………………….…………….………..
	Annex 2……………………………………………………………………………………………….………….……..
	CONCLUSION……………………………………………………………………………………………………….……….

II-Results......................................................................................................................... 1-Article 1...............................................................................................................I-Conclusion and perspectives...................................................................................... REFERENCES………………………………………………………………………………………………………………….

Table 1 : Prevalence of causal factors in vascular liver diseases [1,3,8,11,18-21] Causal factor prevalence PVT BCS PSVD Acquired conditions

 1

	Myeloproliferative neoplasm (MPN) %*	20	40-50	10-15
	Antiphospholipid Syndrome (APLS) %*	8-12	4-25	4-8
	Paroxysmal nocturnal hemoglobinuria (PNH) %*	0-2	4-19	NA
	Behcet's disease %*	0-30.	0-33	NA
	Hyperhomocysteinemia %	12-22.	11-37	NA
	Hereditary or Genetic conditions
	Factor V Leiden %	5	6-32	0
	Factor II gene mutation%	10-15	5-7	3
	Protein C deficiency%	0-26	4-30	3
	Protein S deficiency%	2-30	3-20	3
	Antithrombin deficiency %	1	10	0
	Risk factors for thrombosis
	Oral contraceptives %	40	6-60	NA
	Pregnancy%	6-30	6-12.	NA
	Metabolic syndrome%	25-47	NA	NA
	Local cause %	20-25	5	NA
	Systemic Disease %	23	4	17
	>1 Risk factor %	50	45	5-15
	*High risk thrombotic associated causes+ Homozygous or composite heterozygous fV leiden
	or Factor II

gene mutation c) Behcet's disease (BD)

  thromboses, 5 pulmonary embolisms, 8 mesenteric venous infarctions and 1 splenic infarction. In multivariate analysis, only the absence of thrombophilia and the presence of anticoagulant treatment were significantly associated with survival without new thrombotic events. A history of thrombosis, age, gender, and age of thrombosis were not predictive of thrombosis recurrence. With documented thrombophilia, the risk of recurrence in the portal system and of mesenteric or splenic infarction was 0.

	82
	and 5.19/100 BP with and without anticoagulants, respectively (RR 6.3, 95% CI
	1.3-30.4; p = 0.01). Only 2 patients had a mesenteric infarction on
	anticoagulants. Overall, this study suggests a benefit of anticoagulant
	treatment on the risk of thrombosis in patients with thrombophilia without
	increasing the risk of bleeding. [2]
	In a cohort of 44 PVT /MPN patients with a long term follow-up (median 5.8
	years (0.4-21 years), recurrent thrombosis occurred in 12 patients (24%). It was
	the cause of death in 18% of the patients. [74]
	 In the retrospective study by Condat et al. which included 136 patients with
	acute or chronic portal venous thrombosis followed for 4 years, 84 patients

received anticoagulant treatment, 30 of whom received treatment for a limited time only. Fifty-two patients did not receive anticoagulant treatment

(5)

. Thirty-six thrombotic events were reported in 26 patients, including 18 lower limb venous

Table 3 : Incidence per 100 patient years of recurrent thrombosis in SVT cohorts Recurrent thrombosis incidence per 100 patient-years

 3

	Ageno N =604	Condat N=136*

Risk of extension to portal vein thrombosis in PSVD with or without anticoagulant treatment

  Portal vein thrombosis is common in PSVD with portal hypertension and affects one third of the patients at five years. HIV infection or the presence of variceal bleeding at diagnosis are independent risk factors for portal vein thrombosis. Anticoagulation is proposed in PSVD patients with or without portal vein thrombosis, although there is no evidence yet, to support anticoagulation in this context.

* With documented thrombophilia

Table 1 . Characteristics of patients with and without liver diseases in TRG mutations cohort (N=132).

 1

	Patients with	Patients without	p
	liver disease	liver disease
	(N=95)	(N=37)

One patient had TERT and TERC mutations, both classified as pathogenic.

  *Family history was defined as a history of any telomere disease (first or second degree)

	¤ ALP: alkaline phosphatase, ALT: alanine aminotransferase, AST: aspartate aminotransferase, BMI:
	body mass index (kg/m²), HSCT: hematopoietic stem cell transplantation, MDS: myelodysplastic
	syndrome, GGT: gamma glutamyl transferase, ILD: interstitial lung disease, TLCO: transfer factor of
	the lung for carbone Monoxide.
	*** Cancers in patients with liver disease: Chondrosarcoma, Cutaneous cancer without precisions,
	Breast Cancer. Cancers in patients without liver disease: Kidney Cancer, two Cervical Cancers, Breast
	Cancer.
		13.6 (10.2-19.2)	13.6 (11.8-16.3)	0.665
	Immunologic disease N/investigated	11/58 (19)	4/24 (17)	1.000
	(%)
	Cutaneous disease	51 (54)	15 (46)	0.163
	Premature hair greying before the	34 (36)	7 (19)	0.060
	age of 30
	Skin hyper / hypopigmentation	18 (19)	5 (14)	0.460
	Ungual dysplasia / dystrophia	15 (16)	2 (5)	0.150
	Mucosal leucoplasia	12 (13)	4 (11)	1.000
	Rheumatologic disease
	Osteoporosis -N/investigated (%)	18/46 (39)	4/14 (29)	0.473
	Ophthalmologic disease -N/investigated	9/29 (30)	0/9 (0)	0.085
	(%)
	Cancer history (other than blood
	cancer)***

*

Table 3 . Univariate and multivariate analysis of factors associated with liver transplantation-free survival in patients with liver disease in TRG mutations cohort (N=95).

 3

	Transplantation-free Survival
		HR	95% IC	p
	Univariate analysis
	Age at liver disease diagnosis	1.018	0.997	1.039	0.090
	Age at lung diagnosis	1.006	0.982	1.029	0.637
	Age at blood disease diagnosis	1.020	0.997	1.044	0.096
	Age at mutation diagnosis	1.016	0.995	1.037	0.145
	Male sex	1.327	0.457	3.856	0.604
	TERT mutation	1.467	0.641	3.354	0.364
	TERC mutation	0.464	0.139	1.541	0.210
	DKC1 mutation	12.466	2.623	59.255	0.002
	Telomere lengh <1st percentil	1.494	0.192	11.636	0.701
	Cofactor for liver disease*	5.281	1.798	15.511	0.002
	Excessive alcohol consumption**	2.813	1.040	7.607	0.042
	BMI >25 kg/m ²	1.780	0.833	3.801	0.137
	Ascites	5.978	1.335	26.775	0.019
	Portal venous thrombosis	7.478	0.946	59.094	0.056
	AST (IU/L)	1.012	0.999	1.025	0.063
	ALT (IU/L)	0.976	0.956	0.997	0.026
	ALP (IU/L)	1.005	1.000	1.010	0.021
	GGT (IU/L)	1.004	1.001	1.008	1.008
	Serum albumin (g/L)	0.860	0.801	0.925	< 0.001
	Ferritin (µg/L)	1.001	1.000	1.001	0.046
	Transferrin saturation (%)	1.040	1.012	1.069	0.004

*Cofactor for liver disease: excessive alcohol consumption >100 g/week, positive HBs Ag, metabolic syndrome (Defined by the presence of at least 3 of the following criteria: hypertension, diabetes,

Table 4 . Variables associated with liver disease in the combined population (N=628) of TRG mutations cohort (N=132) and propensity score-matched controls (N = 396) . Binary logistic regression. Matching based on sex, overweight status, excessive alcohol consumption, arterial hypertension, and diabetes

 4

		HR	CI95%	p
	Univariate analysis
	Male sex	2.96	1.93 4.55	< 0.001
	At least one TRG mutation	10.14	6.45 15.94	< 0.001
	Excessive alcohol consumption*	3.58	1.83 7.02	< 0.001
	BMI > 25 kg/m²	1.57	1.09 2.26	0.015
	Arterial hypertension	1.56	0.78 3.13	0.210
	Diabetes	1.31	0.65 2.62	0.449
	Multivariate analysis
	Male sex	3.34	1.99 5.60	< 0.001
	At least one TRG mutation	12.89	7.78 21.35	< 0.001
	Excessive alcohol consumption*	2.96	1.34 6.51	0.007
	BMI > 25 kg/m²	2.33	1.47 3.68	< 0.001
	*According to WHO definition (>100g per week)
	BMI: body mass index (kg/m²)

. TRG mutations cohort: genes variants and telomere length. A.

Article 2 Paroxysmal nocturnal hemoglobinuria (PNH) and vascular liver disease (VLD): eculizumab therapy decreases mortality, and thrombotic

  During the study period, samples from biopsies or explants of liver transplant recipients were systematically collected, formalin fixed, paraffin embedded, stained for histological examination, and stored.Hematein and eosin, picrosirius and Perls stained tissue sections were retrospectively reviewed by an expert pathologist (Valerie Paradis) unaware of clinical, laboratory or imaging result.

	FIB-4 score ≥3.25		2.278	0.887	5.851	0.087
	Liver stiffness (FibroScan®)		0.991	0.917	1.071	0.815
	Liver stiffness (FibroScan®) ≥20 kPa	1.654	0.269	10.160	0.587
			complications
	HVPG (mmHg)	1.127 (15 characters excluding space) 0.123	10.283	0.916
	HVPG ≥10 mmHg Authors:		1.040 0.776	1.011 0.358	1.069 1.686	0.006 0.522
	Age at lung disease diagnosis Leucokytes (10 9 /L) Histological features Aurélie Plessier 1 Lengh, millimeter Marina Esposito-Farèse 2		1.034 1.000	1.002 1.000	1.068 1.000	0.036 0.262
	Age at blood disease diagnosis Hemoglobin (g/dL) Number of portal tract Anna Baiges 3 Porto-sinusoïdal vascular disease Akash Shukla 4	1.045 1.050	1.012 0.881	1.079 1.251	0.007 0.583
	Age at mutation diagnosis Platelets (10 9 /L) Juan Carlos Garcia Pagan 3 Specific lesions Emmanuelle De Raucourt 5		1.037 1.000	1.009 1.000	1.067 1.000	0.010 0.607
	Male sex MCV (fL) Isabelle Ollivier-Hourmand 6 RNH OPV Jean-Paul Cervoni 7		0.860 1.062	0.284 1.008	2.609 1.119	0.791 0.023
	TERT mutation TERC mutation Tabacco exposure Multivariate analysis*** Victor De Ledinghen 8 Incomplete septal cirrhosis Zoubida Tazi 9 Unspecific lesions Jean-Baptiste Nousbaum 10 Sinusoidal distension René Bun 2	1.697 0.695 1.776	0.611 0.202 0.711	4.717 2.388 4.434	0.310 0.564 0.219
	DKC1 mutation Cofactor for liver disease* Christophe Bureau 11		11.397 13.382	1.323 1.747	98.164 102.506	0.027 0.013
	Telomere lenght <1st percentil FIB-4 score ≥3.25 Christine Sylvain 12 Cirrhosis Olivier Tournilhac 13		0.882 1.074	0.110 1.024	7.100 1.127	0.906 0.003
	Cofactor for liver disease* Mathieu Gerfaud Valentin 14 Excessive alcohol consumption** François Durand 1 Advenced Fibrosis F3 Odile Goria 15 Cirrhosis F4 Luis Tellez 16	16.857 3.345	2.222 1.082	127.857 10.348	0.006 0.036
	BMI >25 kg/m ² Agustin Albillos 16 Ascites Stefania Gioia 17 NASH Oliviero Riggio 17 Andrea De Gottardi 18		2.878 4.450	1.128 0.554	7.346 35.720	0.027 0.160
	Portal venous thrombosis AST (IU/L) ALT (IU/L) ALP (IU/L) Audrey Payance 1 Hepatocellular ballooning Pierre-Emmanuel Rautou 1 Hepatocellular inflammation Louis Terriou 19 Steatosis Aude Charbonnier 20 SAF score 21 Laure Elkrief NAS score Regis Peffault de la Tour 22 Dominique-Charles Valla 1	12.920 0.994 0.962 0.996	1.506 0.971 0.933 0.987	110.810 1.018 0.992 1.005	0.020 0.608 0.014 0.393
	GGT (IU/L) Nathalie Gault 2,23 Associated lesions Flore Sicre de Fontbrune 22		1.002	0.997	1.007	0.501
	Serum albumin (g/L)		0.893	0.817	0.976	0.012
	Ferritin (µg/L) Lobular cholestasis Iron overload		1.000	1.000	1.001	0.203
	Transferrin saturation (%)		1.052	1.020	1.085	0.001
	MELD score Normal liver biopsy		1.436	1.022	2.018	0.037
	FIB-4 score		1.084	1.034	1.138	0.001

*Cofactor for liver disease: excessive alcohol consumption >100 g/week, positive HBs Ag, metabolic syndrome, BMI >25 kg/m², iron overload (ferritin >1000 µg/L). **According to WHO definition (>100g per week) ***Multivariate analysis was performed on variables with <0.01 significance in univariate analysis and for which more than 95% of the data were available. ALP: alkaline phosphatase, ALT: alanine aminotransferase, AST: aspartate aminotransferase, GGT: gamma glutamyl transferase, HVPG: hepatic venous pressure gradient, MCV: Mean corpuscular volume.

OPV: obliterative portal venopathy; NRH: nodular regenerative hyperplasia

2-

  g/dL, LDH 736 (482-1744) IU/L; the size of clone was not different over time, before and after 2007 (table 3); 21 patients (37%) had classic PNH, 12 (21%) had aplastic anemia-PNH syndrome (AA-PNH syndrome), and 23 (41%) had intermediate PNH. Among intermediate PNH, 7 had a diagnosis of MDS and 3 a diagnosis of MPN, others not fulfilled the diagnosis of AA-PNH.

Table 1 .

 1 Characteristics at Budd-Chiari Syndrome (BCS) or Portal Vein Thrombosis (PVT) diagnosis, risk Factors for Thrombosis and BCS or PVT treatments.

	Total	PVT	BCS
	(n=62)	(n = 12)	(n = 50)

Table 2 -

 2 Sequence of diagnosis and delay of diagnosis

	Total	PVT	BCS

Table 3 .

 3 Characteristics of BCS or PVT patients at diagnosis before and after 2007

		SVT before 2007	SVT after 2007	p
	Number	24	38
	Age, years	36 (30-49)	35 (27-48)	0.64
	Females	15 (62)	18 (47)	0.30
	SVT			0.75
	BCS	20 (83)	30 (79)
	PVT	4 (17)	8 (21)
	Acquired thrombophilia
	MPNs	1 (6)	2 (8)	0.99
	Antiphospholipid antibody syndrome	3 (16)	0 (0)	0.07
	Hemoglobin, g/dL	9.9 (8.6-10.8)	10.3 (8.5-11.2)	0.47
	Platelet count, x10 9 /L	78 (62-113)	115 (61-205)	0.10
	Clone size%, IQR	83 (73-90)	83 (70.8 -95.5)	0.52
	AST, U/L	58.5 (29.0-76.8)	54.0 [31.5-86.0)	0.90
	ALT, U/L	33.5 (23.5-73.5)	40.0 (30.5-74.5)	0.52
	Serum bilirubin, µmol/L	34 (16.8-55.2)	24.5 (15.6-50.8)	0.49
	Serum creatinine, µmol/L	66 (55.3-86.3)	62.5 (53.2-85)	0.64
	Serum albumin, g/L	35 (29-42.3)	32 (28-37)	0.20
	Factor V, %	74 (67-84)	83 (60-93)	0.83
	Child Pugh score (among BCS patients, N = 50)	10 (8.5-11.3)	10 (8-11)	0.68
	Clichy score (among BCS patients, N = 50)	6 (5.3-6.7)	5.9 (5.5-6.7)	0.99
	Anticoagulation	22 (92)	38 (100)	0.15
	Stent	2 (8)	1 (3)	0.55
	TIPS	7 (29)	9 (24)	0.77
	Liver transplantation	0 (0)	0 (0)	0.99
	Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; BCS, Budd-Chiari syndrome, MPN,
	myeloproliferative neoplasm; PVT, portal venous system thrombosis;
	Values are n (%) or median (interquartile range).

Table 4 :

 4 Complications during follow-up Venous thrombosis included deep vein thrombosis, new splanchnic vein thrombosis, cerebral thrombosis and TIPS thrombosis occurring after BCS or PVT diagnosis *** For ascites resolution, analyses were performed among patients with ascites at BCS or PVT diagnosis. **** For recurrent ascites, analyses were performed among patients with ascites resolution

		Complication	Exposure	Number of event	Person-years (PY)	Incidence rate per 100 PY	IRR (95% CI)
		Death	yes	4	152.6	2.62	0.30 (0.10 -0.92)
			no	13	148.7	8.74
			yes	5	152.6	3.28	0.41 (0.14-1.15)
		Bleeding
			no	12	148.7	8.07
			yes	4	152.6	2.62	0.43 [0.13-1.41]
		Liver complications*
			no	9	148.7	6.05
			yes	6	152.6	3.93	0.53 [0.2-1.44]
		Infection
			no	12	148.7	8.07
			yes	1	152.6	0.66	0.16 (0.02-1.35)
	Arterial ischemic event
			no	8	148.7	5.38
			yes	4	152.6	2.62	0.22 (0.07-0.64)
	Venous thrombosis**
			no	21	148.7	14.12
	Among 33 patients with ascites
			yes	13	80.9	16.1	0.29 [0.08 -1.05]
		Ascites resolution***
			no	11	86.8	12.7
	Among 24 patients with resolved ascites
			yes	1	71.5	1.40	0.97 [0.06 -15.5]
		Recurrent ascites****
			no	5	69.3	7.21
	*	Hepatic encephalopathy/ hepatorenal syndrome/ péritonitis/ ascites
	**

Table 1 . Characteristics of patients at inclusion

 1

		TOTAL (N=111)	Rivaroxaban (N=55)	No (N=56) anticoagulation	p-value
	Age	49.3 [41.9-60.8]	50.5 [41.6-60.8]	48.1 [42.3-61.5]	0.92
	Male gender N %	64 (58)	31 (56)	33 (59)	0.78
	Ongoing anticoagulation at inclusion N %	82 (74)	39 (71)	43 (77)	0.48
	Past anticoagulation therapy in
	patients without anticoagulation	19 (66)	9 (56)	10 (77)	0.43
	at inclusion N %
	Low-risk Thrombophilia N %
	Heterozygous G1691A factor V mutation	1 (1)	0 (0)	1 (2)	1.00
	Heterozygous G20210A factor II mutation	8 (7)	6 (11)	2 (4)	0.16
	Protein S deficiency*	16 (15)	10 (18)	6 (11)	0.28
	Protein C deficiency*	22 (20)	14 (26)	8 (14)	0.14
	Antithrombin deficiency*	2 (2)	1 (2)	1 (2)	1.00
	Hyperhomocysteinemia	24 (26)	12 (27)	12 (25)	0.80
	Total thrombophilia	48 (50)	28 ( 60)	20 (42)	0.08
	Other risk factors for
	thrombosis at the time of PVT
	diagnosis N %
	Oestroprogestative or
	pregnancy 3 months prior PVT	14 (30)	7 (29)	7 (30)	0.92
	among 47 women
	Systemic disease	1 (1)	1 (2)	0 (0)	0.50
	Local risk factors at diagnosis
	-Abdominal or pelvic surgery within 3 months prior PVT	13 (12)	8 (15)	5 (9)	0.36
	-Local inflammation or
	infection at the time of	17 (15)	8 (15)	9 (16)	0.82
	thrombosis diagnosis
	PSVD (out of 15 liver biopsies)	6 (5)	5 (9)	1 (2)	0.11
	Other cardiovascular risk
	factors
	Past cerebral stroke N %	2 (2)	1 (1)	1 (1)	1.00
	Type-2 diabetes N %	6 (5)	4 (7)	2 (4)	0.44
	Arterial hypertension N %	19 (17)	14 (26)	5 (9)	0.021
	BMI (kg/m2)	26.8 [24.0-30.2]	26.9 [24.0-29.9]	26.8 [24.1-30.3]	0.97
	BMI >30 N %	29 (26)	13 (24)	16 (29)	0.59
	Ongoing smoking N %	12 (11)	6 (11)	6 (11)	1.00
	At least one persisting low-risk factors ** N %	102 (94)	47 (90)	55 (98)	0.06
	Number of persisting low-risk
	factors : N % 1	13 (12)	4 ( 7.5)	9 (16)	0.012
	2	22 ( 20)	5 ( 9)	17 (30)
	3	25 ( 23)	11 ( 21)	14 (25)
	4	26 ( 24)	16 ( 30)	10 (18)
	5	10 ( 9)	7 ( 13)	3 (5)

(3)

(11) 0.096 Data are presented as counts N (%) or medians (interquartile range) *According to WHO definition (>100g per week)

(33) -Retinal detachment 3 (33) -Keratoconus 1 (11) -Macular degeneration at early age 1 (11) -Congenital cataract 1 (11) -

13/32(41) -Variceal bleeding 12 (13) -

(13) -

(3) -

(45) 396 (100) < 0.001

Acknowledgements: Dahia Sekour (Clinical research assistant, Hepatology department, Beaujon Hospital, APHP, France) for data collection, Kamal Zekrini (Clinical research assistant, Hepatology department, Beaujon Hospital, APHP, France) for patients screening, Christelle Ménard and Claire Oudin (Genetic laboratory technicians, Genetics department, Bichat Hospital, APHP, France). Corresponding author : Dr Aurélie Plessier, MD Service d'Hépatologie, Hôpital Beaujon, Assistance Publique des Hôpitaux de Paris, Clichy, France Telephone: +33 1 40 87 55 01 Fax: +33 1 40 87 55 30 E-mail: aurelie.plessier@aphp.fr Author's contributions: * The authors contributed equally. Concept and design: SS, AP, FS, RB, CK, DV, PER, VP Acquisition of data: SS Statistical analysis: SS, AP, PER Interpretation of data: SS, AP, PER, DV Drafting and critical revision of manuscript: SS, AP, FS, RB, CK, DV, PER, VP, EL, OG, JC, JMN, VC, BC, VB, JD, EJ, JBN, SH, AB ,MM, SD, SH, VM, MRG, LT, FG, WAC, JEK, PC, Acknowledgements We would like to express our gratitude to Djalila Seghier, Kamal Zekrini and the French national network for vascular liver diseases for their collaboration in data acquisition, the Association des Malades des Vaisseaux du Foie (AMVF) and the data safety monitoring board: Pr Ariane Mallat, Pr Philippe Sogni , and Pr Michaela Fontenay for their scientific support, URC Paris Nord and Estelle Marcault for regulatory and logistic support, and Dr Didier Lebrec, Pr Sarwa Darwish Murad and Dale Lebrec for their critical and English revision of the manuscript.

Financial Support

This work was supported by the Centre de reference des maladies vasculaires du foie and a fund from the Assistance Publique Hôpitaux de Paris CRC 2017 Financial Support This work was supported by a grant Programme Hospitalier de Recherche Clinique (PHRC N° P110150), from the French Ministry of Health; and by a grant from the Association de Malades des Vaisseaux du foie (AMVF) ClinicalTrials.gov identifier NCT02555111; Word count: words (including references) (≤ 3500 words) References: #30

Data are presented as counts N (%) or medians (interquartile range). GFR: glomerular filtration rate.

Data availability statement:

The datasets generated and analysed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

ABBREVIATIONS USED:

APLS

Authors:

Sabrina Sidali (1,2), Raphaël Borie* (3), Flore Sicre de Fontbrune* (4), Elodie Lainey (5), Pierre-Emmanuel Rautou (1), Kinan El Husseini (6), Odile Goria (1,2), Jacques Cadranel (7), Jean-Marc Naccache (7), Vincent Cottin (8), Bruno Crestani (3), Vincent Bunel (3), Jérôme Dumortier (9), Emmanuel Jacquemin (10), Nousbaum Jean-Baptiste (11), Sandrine Hirschi (12), Arnaud Bourdin (13), Magdalena Meszaros (14), Sebastien Dharancy (15), Sophie Hilaire (16), Vincent Mallet (17), Martine Reynaud-Gaubert (18), Louis Terriou (19), Frédéric Gottrand (19), Wadih Abou Chahla (19), Jean-Emmanuel Khan (20), Paul Carrier (21), Faouzi Saliba (22), Laura Rubbia-Brandt (23), John-David Aubert (24), Laure Elkrief (25), Victor de Lédinghen (26), Armand Abergel (27), Tournilhac Olivier (28), Pauline Houssel (29), Stephane Jouneau (30), Ludivine Wemeau (31), Anne Bergeron (32), Yasmina Chouik (9), Antoine Coupier (9), Thierry Leblanc (5), Isabelle Ollivier-Hourmand (33), Eric Nguyen Khac (34), Hélène Morisse-Pradier (6), Ibrahima Ba (35), Françoise Roudot-Thoraval (36), Dominique Roulot (36), Valérie Vilgrain (37), Christophe Bureau (38), Hilario Nunes (39), François Durand (1), Catherine Boileau (35), Claire Francoz (1), Dominique Valla (1), Valérie Paradis (40), Caroline Kannengiesser (35), Aurélie Plessier (1).

Affiliations :

(1) Université de Paris, AP-HP, Hôpital Beaujon, Service d'Hépatologie, DMU DIGEST, Centre de Référence des Maladies Vasculaires du Foie, FILFOIE, ERN RARE-LIVER, Centre de recherche sur l'inflammation, Inserm, UMR 1149, Paris, France (2) Centre Hospitalier Universitaire Charles Nicolle, Hépatogastro-entérologie, Rouen, France (3) Hôpital Bichat AP-HP, Pneumologie A, Paris, France, Orphalung network (4) Hôpital Saint Louis, Hematology and Transplant Unit, APHP, CRMR rare referral center for aplastic anemia, 75475 Paris Cedex 10, France (5) Hôpital Robert Debré, Hématologie, Paris, France (6) Centre Hospitalier Universitaire Charles Nicolle, Pneumologie, Rouen, France (7) Hôpital Tenon AP-HP, Pneumologie, Paris, France (8) Centre Hospitalier Universitaire Lyon Sud, Pneumologie, Pierre-Bénite, France (9) Hôpital Édouard Herriot, Hépatologie, Lyon, France (10) Hôpital Kremlin-Bicêtre AP-HP, Hépatologie Pédiatrique, Le Kremlin-Bicêtre, France (11) Centre Hospitalier Régional Universitaire Morvan, Hépatologie, Brest, France Results: Among 132 patients with TRG mutations, 95 (72%) had liver disease (19 AST and/or ALT > 30 IU/L, 12 a dysmorphic liver; and 64 both) versus 80/396 (20%) of controls (Hazard Ratio 12.9 CI95% 7.8-21.3, p<0.001). Liver biopsy performed in 52 TRG patients with liver disease identified porto-sinusoidal vascular disease in 42%, and advanced fibrosis/cirrhosis in 15%. Patients with liver disease presented TRG lung, blood, cutaneous, rheumatologic, ophthalmic disease in 82%, 77%, 55%, 39% and 30% of cases, respectively.After a median follow-up of 21 months (CI95% 12 months-54 months), ascites, variceal bleeding, and hepatocellular carcinoma occurred in 14%, 13%, and 2% of TRG patients with liver disease, respectively. Overall survival and LT-free survival were 79% and 69% respectively. FIB-4 score ≥ 3.25 and at least one risk factor for cirrhosis were associated with a poor LTfree survival.

Conclusion:

PSVD and dysmetabolic cirrhosis are the 2 most frequently observed liver related phenotypes in TRG mutation suggesting a multifactorial mechanism. Environment has a major impact on TRG liver disease's prognosis. TRG mutations constitute a strong genetic risk factor of liver diseases. PSVD -N/investigated (%) 22/52 (42) NASH -N/investigated (%) 7/52 (13) Advanced fibrosis -Cirrhosis -N/investigated (%) 8/52 (15) Aspecific isolated lesions -N/investigated (%) A.

B.

HVPG: hepatic venous pressure gradient. * Among the patients with aspecific isolated lesions, three had a liver stiffness > 7 kPa and three had severe portal hypertension.

Supplementary data 6. Genetic and general characteristics in patients with PSVD (N=22)

and

Lung Assessment

Have been collected: toxic exposure (tobacco, fibrogenic exposure, avian or to domestic precipitins), pulmonary functional signs, results from chest imaging, blood gases and pulmonary function tests (PFT) (vital capacity, carbon monoxide diffusion capacity), cardiac transthoracic contrast echography, and the occurrence of complication during the follow-up.

Blood Assessment

For blood disease, results were obtained from blood analysis, electrophoresis of plasma proteins, bone marrow biopsy and aspiration, cytogenetic exam, and the occurrence of complication during follow-up.

Supplementary materiel: Pathological features predefined to liver biopsy reviewing.

Contact Information

Keywords

blocked with eculizumab treatment. Chronic administration of eculizumab resulted in a rapid and sustained reduction in complement-mediated haemolytic activity [9][10][11]. EMA information summary of product characteristics assesses that eculizumab is currently indicated in adult and children PNH patients with hemolysis with clinical symptoms indicative of high disease activity, regardless of transfusion history [12]. Therefore, availability has varied over the last 15 years according to time in each country. The effect of eculizumab on outcome of patients with vascular liver disease is unknown.

We aimed to assess the impact of eculizumab therapy on mortality, liver disease complications and thrombotic or bleeding complications, in patients with BCS or PVT.

still needed in these patients who may still have severe, lethal complications, often triggered by bacterial infection. Prospective studies are warranted to evaluate if an early initiation of eculizumab (before 2 weeks) and complement blockade monitoring will help to avoid the use of stent and TIPS and prevent severe VLD in these patients. This is of particular importance in those patients with hematologic disease (aplastic anemia, MDS and MPN) that could be candidates for bone marrow transplantation. Background. In non-cirrhotic patients with a previous history of portal vein thrombosis (PVT), the benefit of long-term anticoagulation in the absence of major risk factors for thrombosis is unknown. We assessed the effects of rivaroxaban on the risk of recurrent venous thrombosis.

Patients and Methods

In this multicenter, open label randomized controlled trial, we assigned patients with a history of previous PVT without major prothrombotic risk factors to rivaroxaban 15 mg/day versus no anticoagulation. Primary endpoint was 2-year thrombosis-free survival. The occurrence of major bleeding events was a secondary endpoint.

Results

The second interim analysis, as requested by the sponsor after the occurrence of 10 thrombotic events in the 111 included patients showed an incidence of thrombosis of 0 per 100 person-years (PY) in the rivaroxaban group, and 19.71 per 100 PY (95% CI [7.49 -31.92]) in the no-anticoagulation group (log-rank p-value=0.0008). Based on these data, the independent safety monitoring board recommended switching patients from the noanticoagulation group to anticoagulation.

After a median follow-up of 30.3 months (95%CI [29.8-35.9]), major bleeding occurred in two patients receiving rivaroxaban, and in one without anticoagulant. In the noanticoagulation group, plasma D-dimer concentrations ≥ 500 ng/mL 1 month after inclusion were significantly associated with recurrent thrombosis (HR 7.78 [1.49-40.67] with a negative predictive value of 94%. There were no deaths.

Conclusion

In patients with previous PVT without cirrhosis and without major prothrombotic risk factors, rivaroxaban reduced the incidence of recurrent venous thrombosis without excess bleeding risk. D-dimer < 500ng/ml one month after anticoagulation discontinuation predicted a low risk of recurrence.

Registration number /registry name: 2015-001190-10 /NCT02555111

selected patients with markedly different doses, agents, and underlying risk factors of thrombosis (15).

Thus, the goal of this trial was to assess the efficacy of rivaroxaban 15 mg/day to prevent the recurrence of thromboembolic events or death in patients with a prior history of noncirrhotic PVT and without major risk factors for thrombosis.

Methodology Design & settings

This randomized, open-label controlled trial was performed between September 16, 2015 and January 30, 2020 (NCT02555111), in 8 centers in France with experience in the management of vascular liver disease.

Ethics

The institutional review board (IRB Ile-de-France IV n°53-15 on September 2, 2015, and the French Medicine Agency on July 28 th , 2015) approved the protocol. All participants were fully informed about the study protocol and provided written informed consent.

Population

Eligible subjects were adult patients, with a previous history of PVT defined as chronic portal vein thrombosis (including those with portal cavernoma) or signs of recent PVT, without major prothrombotic risk factors, diagnosed more than 6 months prior to inclusion.

The diagnosis of portal vein thrombosis was based on previously defined EASL and ASSLD guidelines (2,4), and patients may or may not have been treated with anticoagulants; For patients initially diagnosed with recent PVT, i) the event occurred more than 6 months prior to inclusion; ii) PVT was documented by an contrast-enhanced multiphasic CT or MR, iii) PVT was followed by recanalization or persistence of obstruction; and iv) thrombosis affected the trunk or the right or left branches of the portal vein, with or without splenic or mesenteric vein involvement, with or without ischemic intestinal damage. PVT was classified according to the classification suggested in the recent AASLD practice guidance document (4).

Major prothrombotic risk factors included myeloproliferative neoplasms, antiphospholipid syndrome, homozygous or composite heterozygous G20210A factor II or G1691A factor V mutations, and a personal or 1 st degree family history of unprovoked venous thrombosis.

Patients with intestinal resection (due to past mesenteric infarction), cirrhosis,