M Savale

M.-R Jevnikar

J Ghigna

S Weatherald

MD Andrei Seferian

MD Xavier Jaïs

MD, PhD Laurent Savale

MD Mitja Jevnikar

MD Maria-Rosa Ghigna

MD Jason Weatherald

MD Sandra Assoun

MD, PhD Elie Fadel

MD Gérald Simonneau

MD, PhD Olivier Sitbon

MD, PhD Marc Humbert

MD, PhD David Montani
email: david.montani@aphp.fr

MD Montani David

Klippel-Trenaunay syndrome as a rare cause of chronic thromboemboembolic pulmonary hypertension

Keywords: Klippel-Trenaunay syndrome, Pulmonary hypertension, Chronic thromboembolic pulmonary hypertension, endarterectomy

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The Klippel-Trenaunay syndrome (KTS) is a rare congenital disorder characterized by the clinical triad of: 1) capillary malformations (port-wine stain); 2) soft tissue and bone hypertrophy or, occasionally hypotrophy; and 3) atypical, mostly lateral varicosity, without significant arteriovenous shunting. KTS is, in most of the cases, a sporadic event with an incompletely elucidated etiology, but most likely due to a mesodermal abnormality that occurs during early fetal development [START_REF] Gloviczki | Klippel-Trenaunay syndrome: current management[END_REF] . It is associated with a higher risk for thromboembolic events of up to 22% [START_REF] Gianlupi | Recurrent pulmonary embolism associated with Klippel-Trenaunay-Weber syndrome[END_REF][START_REF] Baskerville | Thromboembolic disease and congenital venous abnormalities[END_REF] . Incomplete resolution of the vascular obstruction after a pulmonary embolism may lead to chronic thromboembolic pulmonary hypertension (CTEPH). The most effective therapy for CTEPH is pulmonary thromboendarterectomy, which is reserved for the proximal forms of the disease [START_REF] Dartevelle | Chronic thromboembolic pulmonary hypertension[END_REF] . Recently, for technically inoperable cases balloon pulmonary angioplasty (BPA) and pulmonary hypertension (PH) -targeted medical therapy became available as part of a multimodal, individualized patient approach in specialized PH centers 5- 7 .

We present a series of 5 cases of patients with KTS and CTEPH referred to the French Referral Centre for Pulmonary Hypertension. Clinical, functional and hemodynamic parameters, management and survival of these patients are presented in Table 1.

Case 1

A 50-year-old male with KTS diagnosed in childhood was directly admitted to intensive care unit with acute pulmonary embolism. He had a previous history of pulmonary embolism seven years prior and, at that time, life-long oral anticoagulation therapy (OAT) therapy had been initiated. Elevated pulmonary arterial pressures on echocardiography suggested a severe pulmonary hypertension (PH), confirmed on right heart catheterization (RHC): mean pulmonary arterial pressure (mPAP) of 61 mmHg, cardiac index (CI) of 1.46l/min/m 2 and pulmonary vascular resistance (PVR) of 18.3 Woods unit (WU). Ventilation perfusion lung scan, angiography and chest computer tomography were more in keeping with CTEPH with proximal lesions (Figure 1 A-D). Thus, pulmonary thromboendarcterectomy was performed after a 3-month period of bridge-therapy by oral phosphodiesterase type 5 inhibitors (PDE5i). Surgery resulted in good reperfusion of the pulmonary arteries (Figure 1E). Typical intimal fibrosis, associated with chronic and more recent thromboembolic material were found on histological analysis. Six months later, the patient was in NYHA FC I and had a normal CI that allowed the discontinuation of PDE5-i. The patient was lost on follow-up 48 months after diagnosis of CTEPH.

Case 2

A 42-year-old female with KTS diagnosed at birth was admitted for acute sub-segmental pulmonary embolism, with normal right ventriclar function on echocardiography. In the context of KTS, she had a history of splenectomy for a giant splenic angioma and multiple amputations of her right lower limb due to frequently infected superficial low-flow arteriovenous malformations extending into the pelvis (Figure 2A). The patient was treated with low molecular weight heparin (LMWH) and placement of an inferior vena cava filter was not feasible. Two months later, despite well-conducted anticoagulation therapy the patient had a second episode of pulmonary embolism, complicated by a stroke secondary due to systemic embolization through a patent foramen ovale. After two months, the patient had a complete neurological recovery and was in NYHA FC III. RHC showed moderate PH with mPAP of 46mmHg, CI of 3.1 l/min/m 2 and PVR 7.41 WU. Radiological features confirmed an absence of surgically accessible thromboembolic material (Figure 2B-D), and an oral dual endothelin receptor antagonist was added with significant improvement (mPAP= 33 mmHg, CI= 4.2 l/min/m 2 , PVR= 3.6 WU). The patient died from pelvic hemorrhagic complications 44 months after diagnosis of CTEPH.

Case 3

A 42-year-old male with KTS known since adolescence was referred for evaluation of progressive exertional dyspnea, which had progressed over the last 6 months. He was previously treated with oral anticoagulants for one year after an episode of deep vein thrombosis with concurrent asymptomatic sub-segmental pulmonary embolism. The patient was in NYHA FC III and RHC demonstrated severe PH (mPAP= 54 mmHg, CI of 2.54 l/min/m 2 , PVR= 11 WU). No proximal thromboembolic material was found on radiological examinations and inhaled prostacyclin was initiated but later switched to an oral dual endothelin receptor antagonist after 1 year because of prostacyclin-related side-effects. After 6 years, a decrease in the CI prompted the addition of a PDE5-i with satisfactory response.

The patient is still alive 16 years after initial diagnosis on dual oral therapy.

Case 4

A 26-year-old female, with a history of typical KTS diagnosed when she was 2-years-old and splenectomy for splenic angioma was referred to our department three months after an episode of left femoral deep vein thrombosis complicated by an acute pulmonary embolism. She was in NYHA FC III with a 6-minute walking distance of 383 m. RHC showed severe pre-capillary PH (mPAP of 70 mmHg, CI of 2.68 l/min/m 2 , PVR= 11.9 WU). The ventilation perfusion lung scan showed sub-segmental non-matched perfusion defects confirmed by angiography and chest computer tomography. Oral dual endothelin receptor antagonist was initiated and an inferior vena cava filter was inserted. The patient deteriorated after 13 months requiring addition of a PDE5-i and continuous intravenous prostacyclin. No improvement was observed, and the patient was listed for lung transplantation, but died due to irreversible end-stage right heart failure 11 months later.

Case 5

A 46-year-old male with KTS since birth was referred for evaluation of progressive exertional dyspnea, which progressed over the last 12 months. At

DISCUSSION

KTS is a rare complex vascular malformation that generally involves capillaries, veins and lymphatics. Recently, genetic studies in patients with vascular malformations showed that mutations in PI3K-AKT pathway are found in patients with KTS [START_REF] Kurek | Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome[END_REF][START_REF] Luks | Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA[END_REF] opening the door for clinical studies for the use of targeted molecules like rapamycin an inhibitor of mTOR1 that interacts with the altered pathway [START_REF] Youssefian | Fibroadipose hyperplasia versus Proteus syndrome: segmental overgrowth with a mosaic mutation in the PIK3CA gene[END_REF] . A multidisciplinary approach is needed from the early stages of the disease usually during childhood with the aim of preventing the most severe complications: hemorrhage, recurrent infections and thromboembolic events [START_REF] Gloviczki | Klippel-Trenaunay syndrome: current management[END_REF][START_REF] Gianlupi | Recurrent pulmonary embolism associated with Klippel-Trenaunay-Weber syndrome[END_REF][START_REF] Muluk | Klippel-Trenaunay syndrome with multiple pulmonary emboli--an unusual cause of progressive pulmonary dysfunction[END_REF] . The risk for thromboembolic events is up to 22% in patients with KTS [START_REF] Gloviczki | Klippel-Trenaunay syndrome: current management[END_REF][START_REF] Gianlupi | Recurrent pulmonary embolism associated with Klippel-Trenaunay-Weber syndrome[END_REF][START_REF] Baskerville | The Klippel-Trenaunay syndrome: clinical, radiological and haemodynamic features and management[END_REF] and is probably due to a hypercoagulability mechanism at sites of vascular malformations, with blood stagnation and activation of coagulation. This phenomenon suggested by high levels of specific coagulation markers like fibrinogen, prothrombin, D-dimer or von Willebrand factor, occurs within the distorted, enlarged veins and is the trigger of thrombotic events [START_REF] Gianlupi | Recurrent pulmonary embolism associated with Klippel-Trenaunay-Weber syndrome[END_REF][START_REF] Mazoyer | Coagulation abnormalities associated with extensive venous malformations of the limbs: differentiation from Kasabach-Merritt syndrome[END_REF][START_REF] Huiras | Pulmonary thromboembolism associated with Klippel-Trenaunay syndrome[END_REF] .

Like previously reported KTS-associated CTEPH patients, deep vein thrombosis has been objectively described in 3 of our patients. In addition, four patients had a history of acute symptomatic pulmonary embolism. Anticoagulation therapy with vitamin K antagonists was preferred for 4 patients and an inferior vena cava filter was placed in 2 patients. Pulmonary embolism recurrence was recorded in one patient. Despite previous reports that suggested that LMWH are more effective for patients with extensive vascular malformations and thromboembolic events [START_REF] Mazoyer | Coagulation abnormalities associated with extensive venous malformations of the limbs: differentiation from Kasabach-Merritt syndrome[END_REF] , the patient treated with LMWH experienced also recurrence of pulmonary embolism. Magnetic resonance venography by showing extensive pelvis and abdominal malformations ruled out placement of IVCF for the rest of the patients. There is sometimes a difficult balance between the risk for bleeding associated with the malformations and the need for a anticoagulant therapy. Our data suggest that efficacy of the anticoagulant therapy depends on the extension of vascular malformations and that perhaps local surgery could play a more important role. None of our patients received direct oral anticoagulants, mainly as we present a historic cohort as there are some drug interactions between direct oral anticoagulants and PAH targeted therapies [START_REF] Margelidon-Cozzolino | Indications and potential pitfalls of anticoagulants in pulmonary hypertension: Would DOACs become a better option than VKAs?[END_REF] . As part of their follow-up for KTS, all our patients were also periodically evaluated by phlebologists, hematologists and plastic surgeons.

The incidence of CTEPH after symptomatic pulmonary embolism ranges from 0.4% to 6.2%, giving a pooled incidence of 3.4% [16][START_REF] Pengo | Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism[END_REF][START_REF] Becattini | Incidence of chronic thromboembolic pulmonary hypertension after a first episode of pulmonary embolism[END_REF] . In the literature, there are previously reported cases of CTEPH complicating KTS [START_REF] Johnson | Pulmonary thromboendarterectomy in Klippel-Trenaunay syndrome[END_REF][START_REF] Walder | Successful pulmonary thromboendarterectomy in a patient with Klippel-Trenaunay syndrome[END_REF][START_REF] Oduber | Vascular malformations as underlying cause of chronic thromboembolism and pulmonary hypertension[END_REF] or other extensive vascular malformations but to our knowledge the present study represents the most extensive series of CTEPH cases complicating KTS. In addition to the vascular malformations, splenectomy, a known risk factor for CTEPH [START_REF] Jais | Splenectomy and chronic thromboembolic pulmonary hypertension[END_REF] was noted in 2 patients. Clinical, functional and hemodynamic data at time of diagnosis showed severe disease for all 5 patients, in accordance with previous reports [START_REF] Gianlupi | Recurrent pulmonary embolism associated with Klippel-Trenaunay-Weber syndrome[END_REF][START_REF] Oduber | Vascular malformations as underlying cause of chronic thromboembolism and pulmonary hypertension[END_REF][START_REF] Bonderman | Bosentan therapy for inoperable chronic thromboembolic pulmonary hypertension[END_REF] . The outcome was favorable in the patient with proximal disease that returned to normal hemodynamics after pulmonary thromboendarterectomy. The others had distal forms and specific PAH therapies available at time of diagnosis (prostacyclin agonists, endothelinreceptor antagonists, phosphodiesterase type-5 inhibitors) were initiated with variable responses. One patient is still in follow-up in our clinic 16 years later with the same evolution pattern as commonly observed in CTEPH.

Besides lifelong anticoagulation, surgical endarterectomy is the treatment of choice in patients with proximal CTEPH affecting large pulmonary arteries. However, around half of CTEPH patients are not operated, mainly because of distal lesions inaccessible to surgery.

For technically inoperable cases, PH medical therapy is recommended with or without balloon pulmonary angioplasty performed in experienced centers [START_REF] Kim | Chronic thromboembolic pulmonary hypertension[END_REF] .

For severe PH patients refractory to treatment, lung transplantation is the last therapeutic option [START_REF] Walder | Successful pulmonary thromboendarterectomy in a patient with Klippel-Trenaunay syndrome[END_REF] . In recent years, careful timing of the decision helped improve overall survival that however remains rather low at about 50% at 5 years [START_REF] Fadel | Long-term outcome of double-lung and heart-lung transplantation for pulmonary hypertension: a comparative retrospective study of 219 patients[END_REF] . KTS was not considered a contraindication for transplantation. Two patients were listed for lung transplantation, but unfortunately both died: one before the procedure due to irreversible, end-stage heart failure, while the other was transplanted and died of post-transplant lymphoproliferative disorder.

From the histological analysis of the explanted lungs we can assume that there is no specific pattern in CTEPH associated with KTS compared to CTEPH patients both exhibiting chronic and more recent thromboembolic material.

In conclusion, KTS requires specific multidisciplinary follow-up and prompt evaluation of unexplained dyspnea. CTEPH is a rare but severe complication of pulmonary embolism occurring in the context of KTS. Distal forms of CTEPH were associated with a poor prognosis. No standard therapy could be determined and therefore each case must be examined individually. In the modern management era of CTEPH, balloon pulmonary angioplasty will certainly be an interesting option in patients with inoperable disease.

Proximal CTEPH associated with KTS had a good prognosis in one patient and therefore high-resolution computer tomography of the chest and pulmonary angiography are needed to discriminate these patients.

  18 years of age, varicose veins and a deep vein thrombosis of the left lower limb required thrombolysis and placement of an IVCF that was still patent. The patient was in NYHA FC II and RHC showed a mPAP of 51 mmHg, a CI of 3.9 l/min/m 2 and PVR of 5.6 WU. Radiological features suggested a distal form of CTEPH. At the time of diagnosis, epoprostenol was the only available specific PAH therapy and this option was refused by the patient. Six years later, inhaled prostacyclin was initiated in the context of increasing dyspnea (NYHA FC III) and hemodynamic deterioration. Sideeffects associated with inhaled iloprost and further clinical and hemodynamical deterioration prompted a switch to i.v. epoprostenol after 4 years. Because of clinical worsening, an oral dual endothelin receptor antagonist was added but right heart failure prompted for urgent lung transplantation 15 years after diagnosis of CTEPH. Histological analysis of the explanted lungs showed typical chronic thromboembolic material with recent thrombosis (Figure 3). The patient died of post-transplant lymphoproliferative disorder 12 months after transplantation.

Figure 1 Figure 2

 12 Figure 1 Review of a patient with KTS/CTEPH and proximal perfusion defects A. Contrast-enhanced high-resolution computed tomography of the chest -pulmonary arteries reformatted by using maximal intensity projection (MIP) showing bilateral proximal thromboembolic material; B. V/Q lung scan with multiple non-matched perfusion defects: right basal pyramid, right superior lobe, left inferior lobe; C. Angiography (right anterior view) showing proximal thromboembolic lesions; D. Fully dissected thromboembolic material extracted after thromboendarterectomy

Figure 3

 3 Figure 3 Histological analysis from explanted lungs of a KTS/CTEPH patient (Haematoxilin & Eosin Stain) A. Recent thromboembolic lesion; B. Organized thrombus in an elastic pulmonary artery; C., D. Organized thrombi within pre-acinar pulmonary arteries, forming "colander-like" lesions

Table 1 . Clinical, functional and hemodynamic parameters and outcomes of CTEPH associated with KTS patients

 1 New York Heart Association functional class; mPAP: mean pulmonary artery pressure, N/A: not available; NYHA: New York Heart Association; PE: pulmonary embolism; TPR: total pulmonary resistance; ERA: endothelin receptor antagonist; PDE5-i: phosphodiesterase type 5 inhibitor; PVR: pulmonary vascular resistance

	6MWD: 6-minute walk distance; CI: cardiac index; DVT: deep vein thrombosis; IVCF: inferior vena cava filter; LMWH: low-molecular weight heparin; NYHA
	FC:


Irina Filippi

Nicolas Hoertel

Eric Artiges

Guillaume Airagnes

Christophe Guérin-Langlois

Anne-Sophie Seigneurie

Pauline Frère

Manon Dubol

François Guillon

Hervé Lemaître

Christophe Guerin- Langlois

Pauline Frere

Mehdi Rahim

Dr Jean-Luc Martinot
email: jean-luc.martinot@inserm.fr

Frédéric Limosin

Family history of alcohol use disorder is associated with brain structural and functional changes in healthy first-degree relatives

Keywords: Alcohol Use Disorder, Childhood Trauma Questionnaire, Monetary Incentive Delay task, Relatives, Voxel-Based Morphometry, Vulnerability

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Alcohol use disorder (AUD) is a leading cause of mortality, morbidity and disability worldwide [START_REF] Gowing | Global statistics on addictive behaviours: 2014 status report[END_REF]. AUD has been attributed to genetic predisposition for up to 40 to 60% [START_REF] Bienvenu | Psychiatric 'diseases' versus behavioral disorders and degree of genetic influence[END_REF][START_REF] Osby | Genes and alcohol[END_REF], hence this risk in first-degree relatives of AUD individuals is much greater than in the general population [START_REF] Nurnberger | A family study of alcohol dependence: coaggregation of multiple disorders in relatives of alcohol-dependent probands[END_REF]. Neuroimaging studies conducted among AUD individuals discussed inherent pre-existing variations entangled with the neurotoxic effects of alcohol use per se [START_REF] Dupuy | Imaging the Addicted Brain: Alcohol[END_REF]. Hence, investigations in adolescents from AUD families provided evidence of structural [START_REF] Hill | Right amygdala volume in adolescent and young adult offspring from families at high risk for developing alcoholism[END_REF][START_REF] Benegal | Gray matter volume abnormalities and externalizing symptoms in subjects at high risk for alcohol dependence[END_REF][START_REF] Hill | Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families[END_REF][START_REF] Hill | Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: association with allelic variation in GABRA2 and BDNF[END_REF][START_REF] Cservenka | Neurobiological Phenotypes Associated with a Family History of Alcoholism[END_REF] and functional [START_REF] Schweinsburg | An FMRI study of response inhibition in youths with a family history of alcoholism[END_REF][START_REF] Heitzeg | Striatal dysfunction marks preexisting risk and medial prefrontal dysfunction is related to problem drinking in children of alcoholics[END_REF][START_REF] Cservenka | Risky decision-making: an FMRI study of youth at high risk for alcoholism[END_REF][START_REF] Hardee | Development of impulse control circuitry in children of alcoholics[END_REF] variations possibly reflecting vulnerability to AUD. In addition, there are prevalent confounders which may affect brain maturation such as childhood maltreatment, which is associated with psychopathology and risk of substance abuse [START_REF] Herringa | Childhood maltreatment is associated with altered fear circuitry and increased internalizing symptoms by late adolescence[END_REF][START_REF] Keyes | Childhood maltreatment and the structure of common psychiatric disorders[END_REF]. Therefore, the previous findings need to be re-evaluated to consider the prepotent influence of the maltreatment on structural deficits (i.e. smaller grey matter volumes) and functional overactivations in limbic regions that consistently include the hippocampus or the anterior cingulate cortex (17). Maltreatment, even below the threshold of reportable childhood maltreatment, can lead to significant changes in the brain's emotion-regulating circuitry in adolescents [START_REF] Herringa | Childhood maltreatment is associated with altered fear circuitry and increased internalizing symptoms by late adolescence[END_REF]. In addition, chronic maltreatment is associated with cognitive deficits such as poorer inhibitory control [START_REF] Teicher | Childhood maltreatment and its effect on neurocognitive functioning: Timing and chronicity matter[END_REF] as described in FH+ adults [START_REF] Gierski | Executive functions in adult offspring of alcohol-dependent probands: toward a cognitive endophenotype?[END_REF].

AUD adults indeed displayed grey matter volume (GMV) reductions in the hippocampus [START_REF] Beresford | Hippocampus volume loss due to chronic heavy drinking[END_REF], amygdala [START_REF] Wrase | Amygdala volume associated with alcohol abuse relapse and craving[END_REF], insula [START_REF] Chanraud | Brain morphometry and cognitive performance in detoxified alcohol-dependents with preserved psychosocial functioning[END_REF], caudate and putamen [START_REF] Sullivan | Striatal and forebrain nuclei volumes: contribution to motor function and working memory deficits in alcoholism[END_REF], cerebellum [START_REF] Sullivan | Contribution of alcohol abuse to cerebellar volume deficits in men with schizophrenia[END_REF] as well as in the brainstem [START_REF] Pfefferbaum | Corpus callosum, pons, and cortical white matter in alcoholic women[END_REF] compared to healthy controls using anatomical magnetic resonance imaging (MRI). GMV reductions were also observed in adolescents having a family history of AUD (called FH+) compared to those having no family history of AUD (called FH-) in the right amygdala [START_REF] Hill | Right amygdala volume in adolescent and young adult offspring from families at high risk for developing alcoholism[END_REF], parahippocampal gyrus, thalamus, cingulum, superior frontal gyrus [START_REF] Benegal | Gray matter volume abnormalities and externalizing symptoms in subjects at high risk for alcohol dependence[END_REF], orbitofrontal cortex [START_REF] Hill | Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families[END_REF] and cerebellum [START_REF] Hill | Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: association with allelic variation in GABRA2 and BDNF[END_REF]. GMV reductions have also been reported in the parahippocampal gyrus [START_REF] Sjoerds | Family history of alcohol dependence and gray matter abnormalities in nonalcoholic adults[END_REF] and in the amygdala [START_REF] Dager | Shared genetic factors influence amygdala volumes and risk for alcoholism[END_REF] in FH+ healthy adults. Interestingly, GMV reductions observed FH+ individuals may indicate brain structural changes associated with a family history of AUD.

The most acknowledged mechanisms of vulnerability to addiction include functional deregulations of the reward system [START_REF] Volkow | The Brain on Drugs: From Reward to Addiction[END_REF], intricately linked to deficits in inhibition [START_REF] Churchwell | Neuroimaging, adolescence, and risky behavior[END_REF] and sensation-seeking (30). Functional magnetic resonance imaging (fMRI) showed that FH+ adolescents did not show variations of reward-related neural response [START_REF] Cservenka | High and low sensation seeking adolescents show distinct patterns of brain activity during reward processing[END_REF][START_REF] Müller | No differences in ventral striatum responsivity between adolescents with a positive family history of alcoholism and controls[END_REF] but had a blunted brain response to inhibition during Go/No-Go fMRI tasks [START_REF] Schweinsburg | An FMRI study of response inhibition in youths with a family history of alcoholism[END_REF][START_REF] Heitzeg | Striatal dysfunction marks preexisting risk and medial prefrontal dysfunction is related to problem drinking in children of alcoholics[END_REF][START_REF] Hardee | Development of impulse control circuitry in children of alcoholics[END_REF]. However, shortterm detoxified AUD individuals [START_REF] Beck | Ventral striatal activation during reward anticipation correlates with impulsivity in alcoholics[END_REF] and FH+ adults [START_REF] Andrews | Individuals family history positive for alcoholism show functional magnetic resonance imaging differences in reward sensitivity that are related to impulsivity factors[END_REF][START_REF] Yau | Nucleus accumbens response to incentive stimuli anticipation in children of alcoholics: relationships with precursive behavioral risk and lifetime alcohol use[END_REF] had blunted activations in the ventral striatum during reward anticipation while performing a Monetary Incentive Delay (MID) task [START_REF] Balodis | Anticipatory reward processing in addicted populations: a focus on the monetary incentive delay task[END_REF]. Elucidating whether reward processing in FH+ individuals is affected at the earliest stages has been deemed essential to better characterize the addictive behaviour' predisposition.

Although some previous studies controlled (as exclusion criteria or confounding variable in the analyses) for the confounding effects of any psychiatric disorder and AUD [START_REF] Schweinsburg | An FMRI study of response inhibition in youths with a family history of alcoholism[END_REF][START_REF] Cservenka | Risky decision-making: an FMRI study of youth at high risk for alcoholism[END_REF], maternal AUD [START_REF] Hill | Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families[END_REF][START_REF] Cservenka | Risky decision-making: an FMRI study of youth at high risk for alcoholism[END_REF][START_REF] Müller | No differences in ventral striatum responsivity between adolescents with a positive family history of alcoholism and controls[END_REF], foetal alcohol syndrome [START_REF] Heitzeg | Striatal dysfunction marks preexisting risk and medial prefrontal dysfunction is related to problem drinking in children of alcoholics[END_REF][START_REF] Hardee | Development of impulse control circuitry in children of alcoholics[END_REF][START_REF] Cservenka | High and low sensation seeking adolescents show distinct patterns of brain activity during reward processing[END_REF] or even less frequently for maltreatment exposure [START_REF] Sjoerds | Family history of alcohol dependence and gray matter abnormalities in nonalcoholic adults[END_REF], it is still unclear whether brain structural and functional features observed in FH+ individuals persist when controlling simultaneously for these confounding factors. Moreover, there have been calls for neuroimaging research on brain structure in healthy FH+ adults [START_REF] Gierski | Executive functions in adult offspring of alcohol-dependent probands: toward a cognitive endophenotype?[END_REF], since developmental maturation is still ongoing in adolescents [START_REF] Mills | Methods and considerations for longitudinal structural brain imaging analysis across development[END_REF].

In addition, previous studies on brain structure mostly predefined regions of interest, having possibly neglected some brain variations in FH+ individuals. Finally, although deficits in differentiating a big versus small reward were reported in other addictive disorders [START_REF] Lole | Does size matter? An examination of problem gamblers' skin conductance responses to large and small magnitude rewards[END_REF] and impairments in the computation of expected reward value have been identified in AUD individuals [START_REF] Hariri | Preference for Immediate over Delayed Rewards Is Associated with Magnitude of Ventral Striatal Activity[END_REF][START_REF] Grodin | Altered Striatal Response During Effort-Based Valuation and Motivation in Alcohol-Dependent Individuals[END_REF], it is unknown whether they might contribute to vulnerability to AUD.

To address these limitations, the primary aim of the study was to explore the whole brain structure and processing of reward magnitude in adult first-degree relatives of AUD individuals having no prevalent confounding factors (i.e., severe maltreatment exposure, any clinically significant disorder, maternal AUD). The secondary aim was to assess inhibitory control to confirm inhibition deficits in a highly homogeneous sample. The Childhood Trauma Questionnaire (CTQ) score (41) was used as confounding variable in the statistical analyses.

The tertiary aim was to explore on the whole brain the effects of low-to-moderate CTQ scores on brain structure and processing of reward in the FH+ group.

Firstly, based on prior research, we hypothesized smaller GMV in FH+ individuals in regions detailed above, i.e., amygdala, parahippocampal gyrus, thalamus, cingulum, superior frontal gyrus, orbitofrontal cortex and cerebellum, and deficits in neural response to a big versus small win during a MID task. Secondly, we hypothesized poorer inhibition in cognitive tests in FH+ as compared to FH-participants. And thirdly, we hypothesized that exposure to low-tomoderate maltreatment can have a measurable impact on the brain i.e. structural deficits and reward-related overactivation in a group of FH+ individuals.

Material and methods

Participants' characteristics

Participants were recruited in the Department of Psychiatry and Addictology of the Corentin-Celton Hospital (Issy-les-Moulineaux, France). Participants from the FH+ group were mainly siblings of AUD patients followed in our department or participating in Alcoholics Anonymous, while participants from the FH-group were mainly psychology and medical students. The inclusion criteria were age ranging between 18 (age of civil majority) and 35, French native language, right-handed according to the Edinburgh Handedness Inventory scale. Healthy relatives of AUD individuals (FH+) were male and female defined as having two or more first or second-degree relatives with AUD including necessarily the father since inheritability to AUD has been found to be especially paternal [START_REF] Bernstein | Development and validation of a brief screening version of the Childhood Trauma Questionnaire[END_REF] and excluding the mother to prevent foetal exposure to maternal AUD. Controls (FH-) were male and female defined as having no family history of AUD in neither first nor second-degree relatives.

Since we aimed to identify brain correlates of vulnerability to AUD rather than those of alcohol use per se, we considered the following exclusion criteria for the whole sample assessed using the Diagnostic Interview for Genetic Studies (DIGS [START_REF] Mcgue | Origins and consequences of age at first drink. II. Familial risk and heritability[END_REF][START_REF] Preisig | Diagnostic interview for genetic studies (DIGS): inter-rater and test-retest reliability of the French version[END_REF]): past and current psychiatric diagnoses including abuse of or dependence to alcohol or other substances except tobacco according to DSM IV-TR criteria [START_REF]Diagnostic and Statistical Manual of Mental Disorders: DSM-IV, 4 th Ed Revised[END_REF]. Current psychiatric diagnoses, including posttraumatic stress disorder, were also examined during a face-to-face interview with a psychiatrist. Using the DIGS, participants were also excluded if they fulfilled at least one of the following conditions: more than three alcohol intakes per week or more than six drinks per week for women or more than nine drinks per week for men, more than one episode of binge drinking (defined as drinking on the same occasion, i.e. at the same time or within a couple of hours of each other, five or more drinks for men and four or more drinks for women [START_REF] Abuse | NIAAA council approves definition of binge drinking[END_REF]) over the past year and/or more than ten episodes in their lifetime; cannabis use in the last three months; use of prescribed and non-prescribed psychoactive drugs in the last six months; severe exposure to at least one form of childhood maltreatment, defined as having CTQ subscale scores ≥16, ≥13, ≥13, ≥18 and ≥13 for emotional abuse, physical abuse, sexual abuse, emotional neglect, and physical neglect, respectively [START_REF] Bernstein | Manual for the childhood trauma questionnaire[END_REF]. In addition to the excluded participants with maternal AUD, volunteers were not admitted to the study if they had a suspected foetal alcohol syndrome with generalized deficits in the processing and integration of information revealed by neurocognitive examination [START_REF] Kodituwakku | Neurocognitive profile in children with fetal alcohol spectrum disorders[END_REF].

Exclusion criteria also involved other significant medical conditions such as medical events in perinatal history (perinatal foetal distress or prematurity of more than three weeks); any clinically significant or unstable disease; organic diseases affecting the central nervous system such as neurocognitive disorders, intellectual disabilities, history of head trauma with loss of consciousness and/or requiring hospitalization or meningoencephalitis; and MRI contra-indications.

The protocol of the study was approved by the Ile-De-France VIII ethics committee and by the National Agency of Security for French biomedical researches. All participants signed an informed consent after receiving full information on the study. All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.

Data collection 2.2.1. Family history of AUD, clinical, sociodemographic and psychometric characteristics

Assessments have been performed face-to-face by the investigators. Family history of AUD was examined using the Family Informant Schedule and Criteria (FISC). Axis I psychiatric and substance use disorders were assessed with the DIGS during a face-to-face interview with a psychiatrist. Age, sex and education (in number of years of schooling) were also collected. Maltreatment exposure was assessed with the CTQ. Sensation seeking was assessed with the Sensation-Seeking Scale-Form V of Zuckerman [START_REF] Loas | Factorial structure of the Sensation-Seeking Scale-Form V: confirmatory factorial analyses in nonclinical and clinical samples[END_REF]. Trait impulsivity was assessed with the Barratt Impulsivity Scale (11 th version) (50).

Magnetic Resonance Imaging (MRI)

Acquisitions of high-resolution images were conducted with a 3.0 Tesla Siemens Trio scanner and a 12-channel coil at CENIR-ICM platform, Pitié-Salpêtrière Hospital (Paris, France). Pre-processing steps and statistical analyses were run with the Statistical Parametric Mapping software (SPM12, Welcome Trust Centre for Neuroimaging, University College London, UK).

Structural brain imaging

The anatomical 3D-T1-weighted sequence was carried out with the following parameters:

sagittal slice plane, Repetition Time (TR)=2. were excluded to control for errors of segmentation. Then, images were modulated and 10mm full-width at half-maximum (FWHM) Gaussian kernel smoothed. Overall, following visual quality control and outliers' detection, 18 (19.8%) out of 91 acquisitions were excluded. The remaining sample for analysis was composed of N=73 participants.

Functional brain imaging

An Echo-Planar Imaging sequence acquired 40 descending axial slices parallel to the anterior-posterior commissure for each n=191 time-series, with the following parameters:

thickness=2.4millimeters (mm), TR=2.2s, TE=30ms, matrix size 64 x 64, voxel size=3.4 x 3.4

x 2.4 mm and duration=427s. Excessive motion was prevented in the MRI with small wedges to fix head in place. Participants' blood oxygen level-dependent (BOLD) signal time course was recorded while performing an event-related revised MID task in which the main difference to the original version ( 52) is the omission of loss trials [START_REF] Nees | Determinants of Early Alcohol Use In Healthy Adolescents: The Differential Contribution of Neuroimaging and Psychological Factors[END_REF]. This task displayed sequences of clue, target and feedback phases. The clue indicated the amount of the gain and the participants were instructed to respond upon appearance of the target which occurred 4s after the clue. Response was followed by a 1.5s visual feedback informing the participants of their trial result (Supplementary Figure A.1). There were 42 trials (14 no wins; 14 small wins; 14 big wins).

A visual quality control has also been conducted on functional data. Images were excluded in cases of truncation (n=1), signal abnormality (n=2), major artefact (n=1) and sequence error Indeed, A quality control has been performed on fMRI "motor" contrasts (press left minus right, press right minus left) that usually displayed localized activations in the sensory / motor cortex and cerebellum. Therefore, any intense BOLD signal in the frontal, temporal or occipital lobe is atypical for motor contrasts (e.g. when the participant was not focusing on the task during the MID-task acquisition). Thus, out of the sample of 73 acquisitions, 13 (17.8%) were removed. Overall, the fMRI sample available for the second-level analyses was composed of N=60 participants.

Cognitive assessments of inhibition

Participants performed the following cognitive tests: Go/No-Go test [START_REF] Kamarajan | Alcoholism is a disinhibitory disorder: neurophysiological evidence from a Go/No-Go task[END_REF] and Hayling test [START_REF] Burgess | Response suppression, initiation and strategy use following frontal lobe lesions[END_REF]. Further details are given in the Supplementary Appendix A.1.

Statistical analyses

For all statistical analyses, age, sex and CTQ total score were included as confounding variables to account for residual variance between and within groups.

Voxel-Based Morphometry analyses

Voxel-Based Morphometry (VBM) analysis of grey matter images consisted of betweengroup voxel-to-voxel t-tests across the whole brain. The global GMV was added in the statistical model [START_REF] Peelle | Adjusting for global effects in voxel-based morphometry: Gray matter decline in normal aging[END_REF]. We used the xjView toolbox (http://www.alivelearn.net/xjview) to localise the regions of difference. Statistical maps had a height threshold at p<0.001

uncorrected and an extent threshold at p<0.05 Family-Wise Error (FWE) corrected (cluster size > 520 voxels). Because tobacco dependence [START_REF] Mcclernon | Neuroimaging of Nicotine Dependence: Key Findings and Application to the Study of Smoking-Mental Illness Comorbidity[END_REF] and cannabis use (58) may have confounding effects, we performed supplementary analyses with the number of pack-years of smoking or cannabis use as confounding variable.

We performed additional analyses to examine the confounding effects of the CTQ score on the GMV on the whole-brain within the FH+ group. Significance was set at a height threshold at p<0.05 uncorrected and an extent threshold at p<0.05 FWE corrected (cluster size > 8000 voxels).

Functional brain imaging analyses

Between-group comparisons of BOLD signal maps were also performed as voxel-to-voxel ttests across the whole brain for the contrast of anticipation of a "big versus small win" [START_REF] Nees | Determinants of Early Alcohol Use In Healthy Adolescents: The Differential Contribution of Neuroimaging and Psychological Factors[END_REF][START_REF] Büchel | Blunted ventral striatal responses to anticipated rewards foreshadow problematic drug use in novelty-seeking adolescents[END_REF] to assess neural response to the processing of reward magnitude; as well as for the two conditions independently, i.e. anticipation of a big win and anticipation of a small win.

Statistical maps had a height threshold at p<0.001 uncorrected and an extent threshold at p<0.05 FWE corrected (cluster size > 80 voxels) [START_REF] Eklund | Cluster failure: Why fMRI inferences for spatial extent have inflated false-positive rates[END_REF]. The BrainVISA/Anatomist software (http://brainvisa.info) was used to build the figure which displays both structural and functional results. Because tobacco dependence [START_REF] Mcclernon | Neuroimaging of Nicotine Dependence: Key Findings and Application to the Study of Smoking-Mental Illness Comorbidity[END_REF] and cannabis use (58) might have confounding effects, we performed supplementary analyses with the number of pack-years of smoking or cannabis use as confounding variable.

The additional analyses to examine the confounding effects of the CTQ score on the BOLD signal within the FH+ group were significant at a height threshold at p<0.05 uncorrected and an extent threshold at p<0.05 FWE corrected (cluster size > 1250 voxels).

Sociodemographic, psychometric and cognitive analyses

Between-group differences regarding individual factors were tested using unequal variance ttests or chi-square tests when the dependent variable was continuous or categorical, respectively. Between-group multivariable analyses were performed using linear or logistic regression when the dependent variable was continuous or binary, respectively. Statistical significance for cognitive data was determined using a two-sided alpha a priori set at 0.0036 (0.05 Bonferroni corrected i.e. divided by the number of tests, i.e. 14).

Results

Participants' characteristics

There were 73 participants included in the psychometric, structural and cognitive analyses;

37 participants with a family history of alcohol use disorder (FH+) and 36 controls having no such history (FH-). Participants' characteristics are given in Table 1. The total duration of education showed a non-clinically significant mean difference of 1.0 year. We found no significant between-group differences in age, sex, and substance use (all p>0.05). However, we found a significantly greater severity of exposure to all childhood trauma subtypes in the FH+ participants compared to the FH-controls, except for sexual abuse which was not significantly different between groups.

Voxel-Based Morphometry analyses

Between-group comparison of grey matter images showed that the FH+ participants had significantly smaller GMV than the FH-controls in four clusters (Table 2; Figure 1) A.1. There were significant negative associations between the CTQ score and GMV volumes in the bilateral hippocampus, left parahippocampal gyrus and bilateral cerebellum.

comprising

Functional brain imaging analyses

There were 60 participants included in the fMRI analyses (31 FH+ and 29 FH-). There was no significant between-group difference in their delayed final MID scores (means of 120.7s (SD=35.1) and 114.3s (SD=28.1) for FH+ and FH-participants, respectively; t=-0.12; p=0.90). Between-group comparisons of neural response to a big versus small win indicated that the FH-controls had a significantly greater activation in the bilateral middle cingulum and right supplementary motor area (SMA) compared to the FH+ participants (Table 3; Figure 1).

As to the brain activity in response to anticipation of a small win, the FH+ participants exhibited a significant greater activity than the FH-controls in the anterior cingulum (Table 3;

Figure 1). As to the brain activity in response to anticipation of a big win, no significant difference was observed between the groups. The same results were found when adding the number of pack-years of smoking or cannabis use as confounding variable (Supplementary

Figure B.2; Supplementary Figure D.2).

The confounding effects of the CTQ total score in the FH+ group are shown in

Supplementary Figure C.2; Supplementary Table A.2.

There were significant positive associations between the CTQ score and brain activation during the processing of reward magnitude in the bilateral parahippocampal gyrus, bilateral fusiform and lingual gyri, left cerebellum and right inferior frontal gyrus.

Cognitive measures

No significant between-group differences in the sensation-seeking and trait impulsivity scores were observed (p<0.001) (Table 4). In the Hayling test, compared to the FH-controls, the FH+ participants showed significant increases in time response per sentence and corrected time response, and a significant lower number of accurate responses. In the Go/No-Go test, the FH+ participants made significantly more errors of commission and omission than the FH-controls (p<0.001) (Table 4).

Discussion

This study sought to determine whether healthy adult first-degree relatives of individuals with AUD (FH+) differ from controls having no family history of AUD (FH-) in grey matter volumes (GMV), in brain response to big versus small win during a Monetary Incentive Delay (MID) task, and in inhibition performance (Hayling and Go/No-Go tests). We ruled out the confounding effects of childhood maltreatment, and of any psychiatric disorder or AUD, and of prenatal exposure to maternal AUD. We found that family history of AUD was associated with i) smaller GMV in the frontal and cingulate regions as well as in the bilateral nucleus accumbens and right insula, ii) a blunted activation in the middle cingulum during the processing of reward magnitude and a greater activation in the anterior cingulum in response to anticipation of a small win, iii) and deficits in the accuracy of the inhibition processes and in the latency of the act of control. Overall, the present findings confirm and extend research on vulnerability to AUD.

On one hand, our findings are in line with previous studies on FH+ adolescents that showed localised GMV deficits compared to controls [START_REF] Hill | Right amygdala volume in adolescent and young adult offspring from families at high risk for developing alcoholism[END_REF][START_REF] Benegal | Gray matter volume abnormalities and externalizing symptoms in subjects at high risk for alcohol dependence[END_REF][START_REF] Hill | Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families[END_REF][START_REF] Hill | Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: association with allelic variation in GABRA2 and BDNF[END_REF]. As previously identified in two distinct studies in FH+ adolescents [START_REF] Benegal | Gray matter volume abnormalities and externalizing symptoms in subjects at high risk for alcohol dependence[END_REF][START_REF] Hardee | Development of impulse control circuitry in children of alcoholics[END_REF], in the present study the middle cingulum was explicitly at the intersect of the brain structural (i.e. smaller GMV) and functional (i.e. blunted activation) differences between the FH+ and FH-participants. In healthy individuals, the volume of the middle cingulum underlies conflict monitoring (61) while its activity is typically involved during cognitive processing tasks [START_REF] Bush | Cognitive and emotional influences in anterior cingulate cortex[END_REF].

On the other hand, we did not find smaller hippocampus in the FH+ participants when controlling for low-to-moderate exposure to childhood maltreatment as confounding variable (i.e variable of no interest in the statistical analyses), unlike previous studies conducted in FH+ adolescents [START_REF] Benegal | Gray matter volume abnormalities and externalizing symptoms in subjects at high risk for alcohol dependence[END_REF], FH+ adults [START_REF] Sjoerds | Family history of alcohol dependence and gray matter abnormalities in nonalcoholic adults[END_REF] and AUD individuals [START_REF] Beresford | Hippocampus volume loss due to chronic heavy drinking[END_REF]. It remains unknown whether previous findings of hippocampal grey matter volume reductions were due to childhood trauma. However, childhood maltreatment exposure is known to impact the developing brain in numerous brain regions, the most consistently reported being the hippocampus as well as the anterior cingulate cortex (17) and our additional findings suggest that low-to-moderate maltreatment exposure may have an impact in FH+ participants on both brain structure and activity in the hippocampal and cerebellar regions.

Using the whole-brain method instead of the region-of-interest approach, additional brain regions were detected compared to earlier studies with FH+ individuals. We found that the FH+ participants had smaller GMV in the bilateral nucleus accumbens, a core region of the reward system involved in the addictive processes. Prior research conducted among AUD individuals showed GMV reductions in the caudate nucleus and putamen compared to healthy controls [START_REF] Sullivan | Striatal and forebrain nuclei volumes: contribution to motor function and working memory deficits in alcoholism[END_REF], supporting the hypothesis that this variation might pre-exist in the vulnerable individuals. Similarly, the insula, part of the paralimbic region, was altered in the present sample of FH+ participants on the right side, as previously described in AUD individuals [START_REF] Chanraud | Brain morphometry and cognitive performance in detoxified alcohol-dependents with preserved psychosocial functioning[END_REF] and tobacco smokers [START_REF] Naqvi | Damage to the Insula Disrupts Addiction to Cigarette Smoking[END_REF][START_REF] Artiges | IMAGING STUDY: Exposure to smoking cues during an emotion recognition task can modulate limbic fMRI activation in cigarette smokers[END_REF].

Despite similar MID-task performance between groups regarding brain function, the FH+ participants had a blunted activation in the middle cingulum in response to the processing of reward magnitude. Deficits in the computation of expected values have been linked to impaired reward-based decision making [START_REF] Galandra | The alcoholic brain: neural bases of impaired reward-based decision-making in alcohol use disorders[END_REF] thus may contribute to vulnerability to AUD.

However, the FH+ individuals showed an overactivity of the anterior cingulum compared to the FH-controls; a region that is thought to translate neural signals related to degree of reward expectancy [START_REF] Shidara | Anterior cingulate: single neuronal signals related to degree of reward expectancy[END_REF] and is involved in the reward system [START_REF] Volkow | The Brain on Drugs: From Reward to Addiction[END_REF]. Among detoxified AUD individuals, self-efficacy to abstain was positively associated with the neural response in the anterior cingulum during the MID anticipation phase (67), thus this overaction might denote a protective factor to AUD.

Deregulations of the inhibitory control have been largely reported in functional studies in FH+ adolescents [START_REF] Schweinsburg | An FMRI study of response inhibition in youths with a family history of alcoholism[END_REF][START_REF] Heitzeg | Striatal dysfunction marks preexisting risk and medial prefrontal dysfunction is related to problem drinking in children of alcoholics[END_REF][START_REF] Hardee | Development of impulse control circuitry in children of alcoholics[END_REF]. We confirmed that the FH+ group had deficits compared to the FH-group in both cognitive tasks of inhibition, supporting the validity of the present sample.

Deficient inhibitory control indicates poorer resistance to distractor or proactive interference (68), leading to a greater likelihood that a response will be executed rather than withheld (69). Although inhibitory control has been identified as a main component of impulsivity (70), we did not find significant between-group differences neither for the Barratt Impulsivity Scale score nor for the Sensation-Seeking Scale score that are associated with substance misuse in young individuals (53). Hence, the absence of such personality variations might denote a protective factor to AUD in the healthy FH+ individuals.

Despite the strengths of the study design involving participants with fewer confounding factors than previous studies, this design aiming to examine more specifically brain correlates of vulnerability to AUD, had some limitations. Firstly, there was no group of AUD individuals to compare with the FH+ participants. This would allow to disentangle the neural correlates of vulnerability to AUD from the mixed effects of alcohol intoxication observed in AUD individuals (5). Secondly, although we excluded participants with severe exposure to any childhood maltreatment type, FH+ participants still had substantially higher CTQ scores than FH-participants. Thirdly, we excluded participants with AUD and maternal AUD exposure, however we cannot exclude potential brain and/or cognitive alterations due to past minimal alcohol use or minimal foetal alcohol exposure. Fourthly, psychiatric disorders were assessed following DSM-IV-TR criteria and not DSM-5 criteria. Fifthly, future studies with greater statistical power would benefit in examining whether our results hold or differ in women and men. Finally, we cannot exclude that observed differences may reflect protective factors in the FH-group instead of vulnerability factors in the FH+ group.

Conclusions

We showed that adult first-degree relatives of AUD individuals display GMV reductions in the frontal and cingulate regions, as well as a blunted activation in the middle cingulum in response to the processing of reward magnitude during a MID task. Moreover, our findings highlight pre-existing structural deficits in the nucleus accumbens and right insula, and an overactivation in the anterior cingulum in response to anticipation of small non-drug rewards.

This study confirms and extents prior research on FH+ individuals, and encourages to systematically control for the confounding effect of childhood maltreatment among other confounders, i.e. any clinically significant disorder and exposure to maternal AUD, in the study of vulnerability to AUD.

Acknowledgements

This a Participants with a family history of alcohol use disorder had two or more first or second-degree relatives with alcohol use disorder (FH+)including necessarily the father and excluding the mother. Controls had no family history of alcohol use disorder in neither first nor second degree relatives (FH-). SD: standard deviation. µ Unstandardized β coefficients and their standard errors (SE) were estimated through linear regression and adjusted for age, sex and Childhood Trauma Questionnaire total score (df=4). p values in bold are statistically significant (alpha set a priori at 0.05/14=0.0036 Bonferroni corrected).

Figure 1. Brain regions of differences in grey matter volumes and bold signal changes during anticipation of reward in a MID task between individuals with and without a family history of alcohol use disorder.

Participants with a positive family history of alcohol use disorder (FH+) presented smaller grey matter volumes in four clust ers (blue colour) compared to participants having no family history of alcohol use disorder (FH-). FH+ individuals also had a blunted activation in response to a big versus small win (green colour) and a greater activation during anticipation of a small win (red colour) compared to FH-controls during the MID anticipation phase. Extent threshold was set at p<0.05 Family-Wise Error (FWE) corrected; Height threshold was set at p<0.001 uncorrected. Each point on the boxplots represents the adjusted (for age, sex and Childhood Trauma Questionnaire total score) mean value for each participant. The line joining the boxplots spots the group mean. Orientation R: Right; L: Left; A: Anterior; P: Posterior; voxel resolution 1.5x1.5x1.5 mm; Montreal Neurological Institute space. Sample size: n=37 FH+ and n=36 FH-in the structural analyses; n=31 FH+ and n=29 FH-individuals in the functional analyses.

(

  n=1). No excessive motion was visually detected. Per participant, pre-processing steps included timing correction of the 40 slices per volume; rigid body realignment of the 191 volumes to the mean image; nonlinear normalization to MNI standardized space, intensity bias correction, writing of 3mm 3 voxel-wrapped images which were finally 10-mm FWHM Gaussian filtered. Moreover, first-level analyses of BOLD signal changes were performed by adding anticipations and feedbacks as explanatory variables in the intra-subject model along with six motion realignment-computed parameters and derivatives. The canonical hemodynamic response function (HRF) accounted for lag between event onsets and effective BOLD signal changes. Participants not responding to all 'no win' conditions (n=2) and with no or atypical BOLD signals in response to handgrip press were excluded (n=6).

Figure C. 1 ;

 1 Figure C.1; Supplementary Table A.1. There were significant negative associations

  3 seconds (s), Echo Time (TE)=2.93 milliseconds (ms), 256 x 256 view matrix, 160 slices, voxel size=1.1 x 1.1 x 1.1 mm and duration=554s. A quality control has been conducted on T1 dataset from which images with a

benign abnormality (n=1), artifacts from excessive movements of the eyes's lens (n=4) and head (n=1) were excluded. The images were spatially normalized and segmented onto the MNI (Montreal Neurological Institute) tissue probability maps (TPM) into grey matter, white matter, cerebrospinal fluid, bone, soft tissue, background imagesby fitting iteratively to TPM voxel's intensities

[START_REF] Ashburner | Voxel-based morphometry--the methods[END_REF]

. Using the software R, the package "mvoutlier" detected extreme values from a normal distribution (https://cran.rproject.org/web/packages/mvoutlier/index.html) leading to the exclusion of outlier volumes (n=4). Then, covariances were calculated with the tool "check sample homogeneity" (http://www.neuro.uni-jena.de/vbm/check-sample-homogeneity/) and volumes with covariances greater than two standard deviations from the mean were detected with the R package "extreme values" (https://cran.rproject.org/web/packages/extremevalues/index.html) thus outlier covariance volumes (n=8)

Table 1 . Comparisons of sociodemographic characteristics, childhood trauma exposure and substance use between participants with (FH+) and without (FH-) a family history of alcohol use disorder. FH+ participants a (n = 37) FH-participants a (n = 36) FH+ participants vs FH- participants Sociodemographic characteristics Mean (SD) Mean (SD) t µ p value

 1 research was supported by the French Ministery of Health (Programme Hospitalier de

	Recherche Clinique P110132, 2011), the Fondation pour la Recherche Médicale
	(DPA20140629802, 2014) and the Fédération pour la Recherche sur le Cerveau
	(C16/0932A01, 2016). The study was sponsored by Assistance Publique Hopitaux de Paris,
	represented by the HEGP Clinical Research Unit (Pr Gilles Chatellier). We would like to
	thank the team of CENIR -Centre for NeuroImaging Research, ICM -Institut du Cerveau et
	de la Moelle épinière, Hôpital Pitié-Salpêtrière, Paris, France. We would also like to thank the
	IMAGEN consortium that provided the revised version of the Monetary Incentive Delay task
	and Joelle Francis for her advices.
	7. Competing interests
	Frédéric Limosin has received speaker and consulting fees from Astra Zeneca, Euthérapie-
	Servier, Janssen, Lundbeck, Otsuka Pharmaceuticals and Roche. The present work is
	unrelated to these relationships. Other authors have no conflicts of interest or financial
	disclosures to make.

Table 2 . Regions of reduction in grey matter volume in the participants having a family history of alcohol use disorder (FH+) compared to controls (FH-).

 2

	Region		Cluster level			Peak level	MNI Coordinates
		BA	Cluster size	p value	T	p value uncorrected	x	y	z
			(in voxels)	FWE corrected
	Cluster 1
	R middle frontal gyrus	9	1368	4.4072e-04	5.97	5.2166e-08	28	40	34
	R inferior frontal gyrus
	Orbital part	11			4.80	4.7218e-06	44	48	-9
	Triangular part	45			3.89	1.1968e-04	46	44	0
	Cluster 2
	R inferior frontal gyrus
	Opercular part	47	4599	1.5614e-09	5.79	1.0702e-07	48	10	2
	Triangular part				4.36	2.2891e-05	56	22	14
	L inferior frontal gyrus, Orbital part	11			4.23	3.6739e-05	-16	10	-20
	R olfactory cortex	47			5.04	1.9367e-06	18	10	-18
	L olfactory cortex	32			4.59	1.0342e-05	-6	20	-10
	R insula				4.82	4.3244e-06	40	-4	8
	L gyrus rectus	11			4.18	4.4344e-05	-6	33	-16
	R gyrus rectus	11			4.15	4.8556e-05	4	44	-21
	R nucleus accumbens				4.51	1.3629e-05	8	10	-10
	L nucleus accumbens				4.17	4.4885e-05	-6	12	-8
	Cluster 3
	Cingulum middle/posterior	31	1582	1.5793e-04	4.92	2.9949e-06	-2	-44	33
	R precuneus	7			4.62	9.2786e-06	4	-54	21
	L precuneus	31			4.21	3.8984e-05	-4	-57	32
	R Calcarine fissure/lingual				3.69	2.2512e-04	10	-60	10
	Cluster 4
	R precentral gyus	4	676	0.0192	4.47	1.5681e-05	44	-15	44
	R middle frontal gyrus	6			4.17	4.5851e-05	39	-3	54
	R postcentral gyrus				3.36	6.4635e-04	54	-8	36
	BA: Brodmann Area (if region described by Brodmann); MNI: Montreal Neurological Institute (coordinates in mm); R: Right; L: L eft; Extent threshold at p<0.05 Family-Wise Error (FWE) corrected

(cluster size > 520 voxels); Height threshold at p<0.001 uncorrected. p values in bold are statistically significant (p<0.05 FWE corrected). Sample size: n=37 FH+ and n=36 FH-.

Table 3 . Regions of BOLD signal changes in participants having a family history of alcohol use disorder (FH+) compared to controls (FH-) during a Monetary Incentive Delay (MID) task.

 3

	Region

Activation in FH+ greater than in FH- Anticipation of a small win

  Height threshold at p<0.001 uncorrected. p values in bold are statistically significant (p<0.05 FWE corrected). Sample size: n=31 FH+ and n=29 FH-.

	Anterior cingulum	166	0.008	4.94	3.7777e-06	0	38	8
	Anticipation of a big win
	Anterior cingulum	63	0.157	4.80	6.3054e-06	0	29	2
	BA: Brodmann Area (if region described by Brodmann); MNI: Montreal Neurological Institute (coordinates in mm); R: Right; L: L eft; Extent threshold at p<0.05 Family-Wise Error (FWE)
	corrected (cluster size > 80 voxels); 29

Table 4 . Comparisons of personality and cognitive measures between participants with (FH+) and without (FH-) a family history of alcohol use disorder.

 4

	FH+ participants (n = 37)	a	FH-participants (n = 36)	a	FH+ participants vs FH-participants
	Mean (SD)		Mean (SD)		β (SE) µ	p value	µ


F Fenoglietto

M.-P Bons

M-P Farcy-Jacquet

S Brengues

M Gourgou

Ozsahin

C Bourgier

C Lemanski

R Draghici

F Castan

P Fenoglietto

F Bons

M Brengues

S Gourgou

M Ozsahin

D Azria

Personnalisation de la dose, du volume et du fractionnement de la radiothérapie du sein

Keywords: Individual radiosensitivity, late effects, radiotherapy, breast

petits volumes mais également dans les atteintes plus importantes grâce à la réduction volumique du traitement systémique néoadjuvant. Cependant, certaine toxicité cardiaque, pulmonaire, mais aussi cutanée a altéré l'image et la qualité de vie à long terme d'un nombre limité de femmes traitées se considérant alors comme « surtraitées » ou traitées de manière inappropriée. Pour les tumeurs à bas risque de récidive, plusieurs essais ont exploré l'absence de radiothérapie après traitement conservateur. Le taux de récidive locale était supérieur en cas de chirurgie seule mais sans diminution de la survie globale. Des débats se sont alors multipliés afin de sélectionner le critère de jugement principal le plus pertinent et, à l'exception de rares écoles considérées comme rigoristes, un consensus a été admis sur l'intérêt de diminuer le taux de récidive locale en s'efforçant de développer des techniques peu invasives et en identifiant les patientes à risque de toxicité individuelle. Ainsi, dans le cas d'un risque carcinologique absolu évalué comme faible, les techniques de radiothérapie se sont développées pour permettre un traitement focal, notamment chez les patientes à haut risque de séquelles, permettant un compromis de réduction du taux de récidive locale, mais également d'apparition de séquelles ; notamment cutanées. De la même manière, les patientes atteintes de cancer à haut risque de récidive, les volumes à irradier nécessitent des techniques particulières pour mieux couvrir les cibles, en protégeant les organes critiques environnants, notamment cardiaque et pulmonaire. La modulation d'intensité et le complément d'irradiation de la zone locale peuvent nous permettre de mieux contrôler ces maladies de pronostic plus réservé mais à condition également d'intégrer la dimension biologique de risque de toxicité individuelle. Dans cet article, nous développons cette approche de radiothérapie personnalisée en fonction de l'évaluation du risque de récidive locale et d'apparition de toxicité radio-induite.

Mots clés

Radiosensibilité ; effets indésirables ; radiothérapie ; sein

Abstract

Randomized trials demonstrated similar overall survival between mastectomy and breast-conservative surgery followed by adjuvant radiation therapy. Breast-conservative surgery, with adjuvant radiation therapy, with or without neoadjuvant systemic therapy has become the standard of care for women with early or locally advanced breast cancer. Nevertheless, certain cardiac, lung or cutaneous toxicities may alter the long-term body image and the quality of life of a limited number of patients who consider having had "overtreatment" or treatment outside the best knowledge of science. In case of low-risk breast cancer, several trials have evaluated the carcinologic outcome in absence of radiation therapy after breast-conservative surgery. Local recurrences increased in case of breast-conservative surgery alone but without impact on overall survival. Multiple debates have emerged in order to select the most appropriate evaluation criteria. Finally, a large consensus has considered that reducing local recurrences is important but with modern technologies and after identifying patients of individual

Introduction

Le traitement conservateur du cancer du sein s'est généralisé grâce aux progrès continus des traitements locorégionaux. Après chirurgie conservatrice, la radiothérapie adjuvante de la totalité de la glande mammaire (45 à 50 Gy en 5 semaines puis un complément de 10 à 16 Gy dans le lit opératoire chez les patientes jeunes) est devenue un standard thérapeutique et a permis de réduire le taux de récidive locale de plus des deux tiers [START_REF] Darby | Effect of radiotherapy after breast-conserving surgery on 10-year recurrence and 15-year breast cancer death: meta-analysis of individual patient data for 10,801 women in 17 randomised trials[END_REF].

La radiothérapie a connu un essor considérable grâce aux progrès technologiques et informatiques permettant un ciblage plus précis des volumes tumoraux et une épargne des volumes critiques [START_REF] Belkacémi | Innovations en radiothérapie adjuvante du cancer du sein[END_REF]. Ces techniques permettent d'adapter le traitement adjuvant de manière plus personnalisée selon le risque absolu de récidive et la probabilité de séquelles à long terme et semble un compromis accepté par rapport à l'absence de radiothérapie, qui est un facteur de récidive locale indiscuté [START_REF] Hennequin | Radiothérapie du cancer du sein[END_REF].

Cette personnalisation utilise tous les paramètres radiobiologiques cliniques connus notamment le fractionnement (traitement hypofractionné), l'étalement (traitement accéléré), mais aussi le volume (irradiation partielle) et la dose totale. En outre, la délivrance de la dose selon la technique de modulation d'intensité semble également être un atout pour certaines indications [START_REF] Bourgier | Techniques d'irradiation du cancer du sein en 2016 : intérêt et indications de la radiothérapie conformationnelle avec modulation d'intensité[END_REF][START_REF] Lauche | Helical tomotherapy and volumetric modulated arc therapy: New therapeutic arms in the breast cancer radiotherapy[END_REF].

La possibilité d'éviter le risque de récidives locales en adaptant la technique de radiothérapie permet également de maitriser la dose aux tissus sains. Ceci est particulièrement pertinent chez les patientes identifiées comme ayant une radiosensibilité individuelle majorée. En pratique, cette notion majeure de la radiobiologie a cependant été marginalisée devant l'absence de test prédictif validé.

Récemment, l'étude multicentrique française a confirmé prospectivement la capacité du test d'apoptose radio-induit lymphocytaire de prédire une toxicité radio-induite, notamment cutanée, après radiothérapie [START_REF] Azria | Radiation-induced CD8 T-lymphocyte apoptosis as a predictor of breast fibrosis after radiotherapy: results of the prospective multicenter french trial[END_REF]. Avec l'avènement de la médecine personnalisée, un des enjeux actuels de la radiobiologie clinique est donc d'arriver à prédire la radiosensibilité individuelle de chaque patient afin de pouvoir délivrer un traitement ciblé grâce à des protocoles d'irradiation « sur-mesure » qui amélioreraient ainsi l'efficacité de la radiothérapie et les chances de guérison [START_REF] Azria | Personalizing breast cancer irradiation using biology: from bench to the accelerator[END_REF].

Dans cet article, nous ne détaillons que dans deux situations cliniques extrêmes (à bas risque et à haut risque de récidive), les techniques de radiothérapie peuvent être adaptées afin de contrôler le risque de récidive locorégionales tout en considérant la probabilité biologique individuelle de toxicité radio-induite.

Situations cliniques pour un traitement personnalisé : évaluation du risque tumoral

Les deux grandes situations cliniques qui sont actuellement étudiées pour une modification des paramètres radiobiologiques sont les tumeurs à risque faible ou élevé de récidive locorégionale.

Avant de développer les outils disponibles, il est particulièrement nécessaire de bien définir ces situations.

Tumeurs à faible risque de récidive

Les tumeurs à faible risque de récidive sont définies depuis les recommandations de Saint-Gallen comme des tumeurs de petite taille (moins de 2 cm) sans atteinte des ganglions axillaires, exprimant fortement les récepteurs hormonaux mais sans surexpression du human epidermal growth factor receptor 2 (HER2) et avec un indice de prolifération faible [START_REF] Goldhirsch | Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer[END_REF]. Récemment, l'indice de prolifération génomique a été intégré afin d'éliminer certaines tumeurs faussement rassurantes [START_REF] Jayasekera | Effects of radiotherapy in early-stage, low-recurrence risk, hormone-sensitive breast cancer[END_REF]. En effet, 1778 tumeurs issues de sept essais randomisés (9377 patientes) ont été analysées selon le score Oncotype.

La sélection a été rigoureusement effectuée selon plusieurs étapes successives :

• récepteurs hormonaux exprimés et HER non surexprimé (n=8816) ;

• taille tumorale inférieure ou égale à 2 cm et ganglions axillaires sains (n=6325) ;

• traitement conservateur du sein (n=4249) ;

• absence de chimiothérapie (n=3347) ;

• hormonothérapie (n=2974) ; À part le score plus récent d'Oncotype, les critères de sélection des patientes atteints de cancer à faible risque de récidive locorégionale sont donc généralement acceptées dans les essais de désescalade thérapeutique, avec quelques variantes notamment sur l'âge. Quoi qu'il en soit, il semble raisonnable d'autoriser une personnalisation de la dose, du fractionnement et du volume lorsque la patiente est âgée de plus de 60 ans, opérée de manière conservatrice pour une tumeur de moins de 2 cm, sans atteinte ganglionnaire, de faible indice pathologique de prolifération (si possible associé un score génomique faible), avec une forte expression des récepteurs hormonaux sans surexpression de HER2.

Tumeurs à haut risque de récidive

Les tumeurs à haut risque de récidive posent beaucoup de problèmes de définitions car le risque intrinsèque est bien connu à long terme depuis l'actualisation de la méta-analyse d'Oxford [START_REF] Darby | Effect of radiotherapy after breast-conserving surgery on 10-year recurrence and 15-year breast cancer death: meta-analysis of individual patient data for 10,801 women in 17 randomised trials[END_REF].

La population atteinte de tumeur localement évoluées colonisant les ganglions et traitées par irradiation après chirurgie conservatrice ont un gain de 21% à 10 ans de probabilité de récidive locale et de 8,5% à 15 ans de probabilité de survie spécifique par rapport aux patientes opérées sans irradiation adjuvante.

La population de patientes traitées par mastectomie bénéficie également de la radiothérapie en cas d'atteinte axillaire (un à trois ou plus de quatre ganglions atteints) avec un gain d'environ 10% en termes de probabilité de récidive et de 8,5% de probabilité de survie spécifique [START_REF] Mcgale | Effect of radiotherapy after mastectomy and axillary surgery on 10-year recurrence and 20-year breast cancer mortality: metaanalysis of individual patient data for 8135 women in 22 randomised trials[END_REF]. Les volumes d'irradiation en dehors de maladies associées multiples importantes, notamment cardiaques [START_REF] Darby | Risk of ischemic heart disease in women after radiotherapy for breast cancer[END_REF], A long terme, cet impact a également été confirmé par l'étude allemande avec plus de 10 ans de suivi [START_REF] Herskind | Predictive modelling of risk of breast fibrosis at 10 years after radiotherapy using the RILA assay[END_REF].

incluent
Grâce à sa validation internationale récente par des équipes indépendantes et à sa rapidité et sa simplicité, un tel examen (en combinaison avec d'autres facteurs cliniques, notamment l'âge, la prise d'hormonothérapie ou de chimiothérapie et l'intoxication tabagique) peut d'ores et déjà aider au choix décisionnel thérapeutique pour les patients.

Méthodes de personnalisation des paramètres radiobiologiques en fonction du risque de récidive et de toxicité

Les deux paragraphes précédents ont permis de détailler les outils utilisés afin d'identifier les populations qui seront les plus à même de bénéficier d'une personnalisation. Cette approche a été développée de manière matricielle et a été récemment présentée par notre groupe européen Requite [START_REF] Azria | Data-based radiation oncology: design of clinical trials in the toxicity biomarkers era[END_REF]. La méthodologie utilisée a identifié les essais de phase 3 disponibles dans une question posée et de l'appliquer ainsi à notre problématique [START_REF] De Ruysscher | Optimal design and patient selection for interventional trials using radiogenomic biomarkers: A REQUITE and Radiogenomics consortium statement[END_REF].

Patients atteints de tumeurs à faible risque de récidive

Les tumeurs à faible risque de récidive définies dans le premier paragraphe nous ont fait développer deux approches : doit-on éviter la radiothérapie ? Si non, existe-il des essais randomisés qui nous permettent d'adapter le traitement selon le risque de toxicité ?

À la première question, il est évident que le taux multiplié par cinq des récidives locales mais sans impact sur la survie globale dans les trois essais randomisés comparant une radiothérapie de l'ensemble du sein à une surveillance postopératoire (associée à une hormonothérapie adjuvante) nous incitent à proposer une radiothérapie la moins morbide possible, surtout en l'absence de maladies multiples associées et donc d'une espérance de vie longue [START_REF] Kunkler | Breast-conserving surgery with or without irradiation in women aged 65 years or older with early breast cancer (PRIME II): a randomised controlled trial[END_REF][START_REF] Fyles | Tamoxifen with or without breast irradiation in women 50 years of age or older with early breast cancer[END_REF][START_REF] Hughes | Lumpectomy plus tamoxifen with or without irradiation in women age 70 years or older with early breast cancer: long-term follow-up of CALGB 9343[END_REF][START_REF] Hughes | Lumpectomy plus tamoxifen with or without irradiation in women 70 years of age or older with early breast cancer[END_REF]. Cette notion est primordiale dans le choix de réaliser cette radiothérapie étant donné le risque accru (risque relatif, RR) de toxicité cutanée [START_REF] Azria | Facteurs de radiosensibilité tardive des tissus sains[END_REF], mais aussi le risque (faible mais réel) de cancer radio-induit (poumon : RR=1,61 ; oesophage : RR=2,06 ; leucémie :RR=1,71 ; sarcome : RR=2,34), de cancer du sein controlatéral (RR=1,18) et de décès d'origine cardiovasculaire (RR=1,25) [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF]. Comme présenté dans le paragraphe précédent, le test d'apoptose radio-induit lymphocytaire à notre disposition a été validé dans la prédiction des fibroses et des télangiectasies à long terme [START_REF] Azria | Radiation-induced CD8 T-lymphocyte apoptosis as a predictor of breast fibrosis after radiotherapy: results of the prospective multicenter french trial[END_REF][START_REF] Talbot | Analysis of biomarkers for late radiotherapy toxicity in the REQUITE project[END_REF][START_REF] Herskind | Predictive modelling of risk of breast fibrosis at 10 years after radiotherapy using the RILA assay[END_REF], mais également des sarcomes radio-induits [START_REF] Mirjolet | RILA blood biomarker as a predictor of radiation-induced sarcoma in a matched cohort study[END_REF]. Ainsi, les modifications thérapeutiques pourront être proposées en prenant en compte l'ensemble de ces paramètres selon les différents groupes à risque de récidive et de séquelles. Deux techniques d'irradiation partielle du sein très particulières sont également disponibles: la curiethérapie et la radiothérapie peropératoire qui dans des cas très sélectionnés permettent à des équipes entrainées de réaliser une irradiation très localisée de la zone opératoire sans sur-risque de récidives locales [START_REF] Strnad | 5-year results of accelerated partial breast irradiation using sole interstitial multicatheter brachytherapy versus wholebreast irradiation with boost after breast-conserving surgery for low-risk invasive and in-situ carcinoma of the female breast: a randomised, phase 3, non-inferiority trial[END_REF][START_REF] Vaidya | Risk-adapted targeted intraoperative radiotherapy versus whole-breast radiotherapy for breast cancer: 5-year results for local control and overall survival from the TARGIT-A randomised trial[END_REF].

Patientes atteintes de cancer à haut risque de récidive

Pour les patientes atteintes de cancer à haut risque de récidive, la discussion se fait essentiellement sur la bonne couverture du volume cible prévisionnel. Ainsi, la radiothérapie avec modulation d'intensité (RCMI) peut être une bonne indication lorsque ces volumes ne sont pas bien couverts en technique tridimensionnelle [START_REF] Bourgier | Techniques d'irradiation du cancer du sein en 2016 : intérêt et indications de la radiothérapie conformationnelle avec modulation d'intensité[END_REF]. Les résultats de la principale étude de phase 3 montrent une réduction de la toxicité, notamment aiguë ,grâce à la RCMI, sachant que la toxicité aiguë a été corrélée avec la fibrose sous-cutanée et les télangiectasies [START_REF] Pignol | Ten years results of the Canadian breast intensity modulated radiation therapy (IMRT) randomized controlled trial[END_REF]. Les auteurs mentionnent que toutes les patientes ne bénéficient pas de cette technique mais dans notre démarche, un haut risque de toxicité identifiées par le test

Conclusion

Les paramètres définissant une tumeur à bas risque sont communément reconnus comme étant un âge La radiothérapie mammaire adjuvante connaît un vrai changement de paradigme avec une personnalisation associant la technologie et la biologie.

•

  âge entre 40 et 74 ans (n=2390) • disponibilité des pièces anatomopathologiques pour l'évaluation du score Oncotype (n=1778). L'ensemble de ces étapes permet de confirmer que même dans une population parfaitement sélectionnée à très faible risque de récidive, l'omission de la radiothérapie (373 cas sur 1778) était associée à une augmentation du risque de récidive locorégionale d'un facteur 4, surtout lorsque le score Oncotype était supérieur à 11 et que les patientes étaient plus jeunes (moins de 60 ans contre audelà).

4. 1 . 1 .

 11 Patientes à faible risque de récidive et à faible risque de toxicitéPour les patientes à faible risque de récidive et à faible risque de toxicité, une radiothérapie hypofractionnée selon le schéma canadien ou anglais (START [Standardisation of breast cancer radiotherapy] B) semble tout à fait acceptable, mais sans boost[START_REF] Whelan | Long-term results of hypofractionated radiation therapy for breast cancer[END_REF][START_REF] Haviland | The UK Standardisation of Breast Radiotherapy (START) trials of radiotherapy hypofractionation for treatment of early breast cancer: 10-year follow-up results of two randomised controlled trials[END_REF]. Cela permet de traiter l'ensemble du sein sans risque de majoration de la toxicité avec un étalement raccourci (15 jours) et une dose par fraction autour de 2,6 Gy.4.1.2. Patients à faible risque de récidive et à haut risque de toxicitéPour les patientes à faible risque de récidive et à haut risque de toxicité, la radiothérapie doit être modifiée en réduisant le volume irradié afin de ne pas compromettre le bénéfice clinique. Le schéma disponible et publié en phase 3 correspond à celui de l'étude anglaise Import Low avec un suivi médian de 6 ans[START_REF] Coles | Partial-breast radiotherapy after breast conservation surgery for patients with early breast cancer (UK IMPORT LOW trial): 5-year results from a multicentre, randomised, controlled, phase 3, non-inferiority trial[END_REF]. L'hypofractionnement de l'ensemble du sein (40 Gy en 15 fractions) a été comparé à deux autres modalités : soit une irradiation de l'ensemble du sein de dose totale réduite (36 Gy) mais avec un boost de 4 Gy dans la zone chirurgicale, soit d'emblée une irradiation de 40 Gy sur dans volume réduit autour de la zone chirurgicale. Les résultats en termes de contrôle local sont rassurants et même en faveur de l'irradiation partielle du sein (0,5%: intervalle de confiance à 95 % [IC95%] : 0,2-1,4) ou du schéma à dose réduite (0,2%: IC95% : 0,02-1,2) par comparaison au bras hypofractionné de 40 Gy dans l'ensemble du sein (1,1% : 95% CI 0,5-2,3). La réduction des effets indésirables notamment de l'apparence du sein mais également de la fibrose est significativement en faveur de l'irradiation partielle du sein. Cette technique d'irradiation partielle du sein est nettement mieux tolérée que celle proposée dans les programmes hypofractionnés accélérés à deux séances par jour[START_REF] Olivotto | Interim cosmetic and toxicity results from RAPID: a randomized trial of accelerated partial breast irradiation using threedimensional conformal external beam radiation therapy[END_REF].

  d'apoptose radio-induit lymphocytaire nous semble une indication pertinente, surtout si le volume d'irradiation nécessite une couverture importante. Comme discuté précédemment, les patientes jeunes atteints d'un cancer avec une composante intracanalaire associée à la tumeur infiltrante, pourraient bénéficier de cette technique pour recevoir un boost intensifié si l'étude Young boost montre un avantage et à condition d'être identifiée comme à bas risque de toxicité.

  de plus de 60 ans, une tumeur de moins de 2 cm, sans atteinte ganglionnaire, de faible indice pathologique de prolifération (si possible associé un score génomique faible), avec une forte expression des récepteurs hormonaux sans surexpression de HER2. Ces paramètres, associés au résultats du test d'apoptose radio-induit lymphocytaire afin de caractériser le risque de toxicité, permettent de proposer une modification thérapeutique en selon les modalités des essais de phase 3 publiés. Au couple « risques faibles de récidive et de toxicité » est associé un traitement hypofractionnement modéré de l'ensemble du sein selon les schémas canadiens ou anglais. Au couple « risque faible de récidive et élevé de toxicité » est associé une réduction du volume d'irradiation en hypofractionnement modéré selon le protocole anglais Import Low. Enfin, au couple « risques élevés de récidive et de toxicité », est associé une technique d'irradiation conformationnelle avec modulation d'intensité, en protraction, sans accélération, avec une couverture optimale des volumes cibles (du sein ou de la paroi et des aires ganglionnaires).

Tests de radiosensibilité individuelle pour un traitement personnalisé : évaluation du risque de toxicité

  La deuxième question est de savoir si une dose totale augmentée localement et dans un volume réduit (boost) peut être utile en termes de réduction du risque de récidive locale. Une première réponse avait été donnée avec les résultats de l'essai EORTC 22881-10882 montrant un risque réduit de 41% à Le grade élevé n'étant un facteur de récidive locale que dans les premières années[START_REF] Vrieling | Prognostic factors for local control in breast cancer after long-term follow-up in the EORTC Boost vs No Boost Trial: a randomized clinical trial[END_REF]. Ainsi, cette sous-population de femmes jeunes traitées pour une tumeur infiltrante avec composante intra-canalaire pourrait bénéficier d'un boost intensifié. C'était l'objectif de l'essai « M03RBC-young boost » qui a comparé deux types de boost, de 26 Gy et de 16 Gy, chez les patientes jeunes (moins de 50 ans) prises en charge par une association radiochirurgicale conservatrice. Seuls les résultats cosmétiques à 4 ans sont disponibles, montrant un effet délétère du boost intensifié[START_REF] Brouwers | Predictors for poor cosmetic outcome in patients with early stage breast cancer treated with breast conserving therapy: Results of the Young boost trial[END_REF]. Afin de pallier les problèmes que posent les tests réalisés sur des fibroblastes, un grand nombre d'études se sont intéressées au tissu sanguin. En effet, le sang est un matériel biologique facilement Un boost de 10 à 16 Gy a été délivré à 449 patientes (98,5%) et une hormonothérapie adjuvante a été administrée à 349 patientes (76,5%). Avec un suivi médian de 38.6 mois, le taux de fibrose de grade 2 ou plus était de 14% (n=64). Une diminution significative de l'incidence de la fibrose de grade 2 ou plus était observée pour des taux élevés d'apoptose radio-induit lymphocytaire

	(p=0,012). Aucune fibrose de grade 3 ou plus n'a été observée lorsque le taux d'apoptose radio-induit
	lymphocytaire était supérieur ou égal à 12%. Une réduction significative de la toxicité a été
	observée en l'absence d'hormonothérapie (subdistribution hazard ratio [sHR]=0,31, p=0,007) et
	lorsque le test d'apoptose radio-induit lymphocytaire était supérieur ou égal à 12% (sHR=0,45,
	p=0,002).
	Au dernier congrès de l' European Society for Radiotherapy and Oncology (ESTRO) (avril
	2019), ces résultats ont été confirmés par les données de l'étude européenne Requite montrant un
	risque significativement de fibrose lorsque le taux d'apoptose radio-induit lymphocytaire est bas [22].
	lymphocytaire bas (inférieur à 16 %) était significativement corrélé avec le pourcentage de toxicité
	tardive de grade supérieur à 2 et qu'un taux d'apoptose radio-induit lymphocytaire élevé (supérieur à
	24 %) permettait de prédire avec 99% d'exactitude l'absence de risque majoré de séquelles après
	radiothérapie à visée curative (excellente sensibilité) [19-21]. Dernièrement, ces résultats ont pu être
	confirmés de manière indépendante par l'étude prospective multicentrique française [6]. Ainsi, 502
	patientes ont été traitées par association radiochirurgicale conservatrice dans dix centres français. Le
	test d'apoptose radio-induit lymphocytaire a été réalisé par cytométrie de flux avant le traitement.

généralement le volume mammaire restant ou la paroi après mastectomie, les aires sous-et sus-claviculaires, avec un prolongement axillaire pour une couverture optimale de l'étage supérieur de Berg et également la chaîne mammaire interne depuis les résultats positifs de l'étude de l'European Organisation for Research and Treatment of Cancer (EORTC)

[START_REF] Poortmans | Internal mammary and medial supraclavicular irradiation in breast cancer[END_REF]

. La question posée n'est plus d'indiquer ou pas la radiothérapie adjuvante mais d'améliorer sa délivrance avec une technique optimisée permettant de couvrir un volume cible important en protégeant au mieux les tissus normaux environnants.

10 ans avec un boost de 16 Gy après traitement conservateur, mais sans allongement de la survie globale

[START_REF] Bartelink | Impact of a higher radiation dose on local control and survival in breast-conserving therapy of early breast cancer: 10-year results of the randomized boost versus no boost EORTC 22881-10882 trial[END_REF]

. L'analyse récente montre que la sous-population jeune (moins de 50 ans) atteints d'une tumeur invasive mais avec une composante intracanalaire bénéficie du boost de manière incontestable et à long terme.

3.

accessible, disponible en grande quantité, et qui demande généralement une manutention moins importante que d'autres fluides biologiques ou prélèvements tissulaires. Les premiers travaux se sont principalement basés sur l'étude du lymphocyte, cellule sanguine connue pour son importante radiosensibilité. Si certains tests lymphocytaires se basent toujours sur les tests de clonogénicité

[START_REF] West | A comparison of the radiosensitivity of lymphocytes from normal donors, cancer patients, individuals with ataxia-telangiectasia (A-T) and A-T heterozygotes[END_REF]

, d'autres techniques ont été développées. A la fin des années 1990, Ozsahin et al. ont proposé pour la première fois un nouveau test basé cette fois sur l'apoptose lymphocytaire

[START_REF] Ozsahin | Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes[END_REF]

. L'apoptose est une forme de mort cellulaire radio-induite dont l'importance dépend plus du type cellulaire que de la dose délivrée. Elle est, par exemple, particulièrement importante dans les tissus lymphoïdes et représente la principale forme de cytotoxicité des lymphocytes. Cette réponse est rapide (6 à 72h après l'irradiation) et peut facilement être mesurée par une diminution de fluorescence de l'ADN nucléaire, détectable en cytométrie en flux

[START_REF] Zamai | Optimal detection of apoptosis by flow cytometry depends on cell morphology[END_REF]

. Le principe de ce test est simple : deux tubes de sang sont prélevés avant le traitement et un d'entre eux est irradié ex-vivo à 8 Gy pendant que l'autre sert de contrôle (0 Gy); 48h plus tard, le taux d'apoptose radio-induite des lymphocytes CD8, plus spécifique que celui des CD4, est calculé par rapport à leur taux d'apoptose basal (contrôle). Deux études prospectives , respectivement chez 399 patients et 150 patients ont ainsi montré qu'un taux d'apoptose radio-induit L'objectif principal était l'impact du taux d'apoptose radio-induit lymphocytaire sur le risque de toxicité cutanée radio-induite (fibrose et télangiectasie). Dans l'analyse définitive, 456 patientes ont été analysées (90,8%) dont 108 (23,7%) ont reçu une irradiation de l'ensemble du sein et des aires ganglionnaires.

Remerciements

Les auteurs remercient le Siric Montpellier Cancer pour leur soutien financier et logistique du programme « radiobiologie ».

Financement

Ce travail a reçu le soutien financier du Siric Montpellier Cancer Grant INCa_Inserm_DGOS_12553.

Liens d'intérêts

D. Azria est cofondateur de la société Novagray. Les autres auteurs déclarent l'absence de conflit d'intérêts.

Rôle des auteurs