C Azaïs

J.-L Uzan

G Mergui

Canlorbe

Clémentine Gonthier

Cassandre Desportes

Jean-Luc Pretet

Henri Azaïs

Catherine Uzan

Jean-Luc Mergui

Geoffroy Canlorbe

Le test HPV dans le dépistage et le suivi des patientes présentant une lésion histologique de haut grade du col utérin

Keywords: papilloma virus humain, test HPV, lésion cervical intraépithélial, cancer invasif du col utérin human papilloma virus, HPV testing, cervical squamous intraepithelial lesion, cervical cancer

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

Introduction

Le cancer du col de l'utérus, qui représente 2 920 nouveaux cas et 1 117 décès en France en 2018 [START_REF]Estimations nationales de l'incidence et de la mortalité par cancer en France métropolitaine entre 1990 et[END_REF] , a pour origine une infection persistante par un papillomavirus humain haut risque (HPV HR) [START_REF] Walboomers | Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[END_REF] . Il s'agit de l'infection sexuellement transmissible la plus fréquente, la plupart des femmes et des hommes sexuellement actifs étant infectés par ces virus au cours de leur vie, généralement au début de la vie sexuelle [START_REF] Dunne | HPV and HPV-associated diseases[END_REF] . Les infections par HPV HR sont généralement transitoires, surtout chez la femme jeune, et une faible proportion des infections persistantes évoluera vers une lésion intra épithéliale de haut grade (LIEHG), lésion précurseur du cancer du col [START_REF] Khan | The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice[END_REF][START_REF] Bosch | The causal relation between human papillomavirus and cervical cancer[END_REF] . Plusieurs tests de détection des HPV HR sont actuellement disponibles sur le marché et sont plus performants que le frottis cervico-utérin (FCU) 6-8, dans le dépistage des LIEHG et le suivi après traitement de ces lésions. Leur utilisation en dépistage est pour le moment limitée en France. D'après les recommandations de 2016 de l'Institut National du Cancer (INCa), le test HPV HR n'était indiqué que dans certains cas de FCU anormaux [START_REF]Recommandations pour le dépistage du cancer du col de l'utérus en France[END_REF][START_REF]Conduite à tenir devant une femme ayant une cytologie cervico-utérine anormale, Collection recommandations et référentiels, version interactive, INCa[END_REF] , mais très récemment l'HAS propose une stratégie nationale de dépistage incluant le test HPV HR [START_REF]Dépistage du cancer du col de l'utérus : le test HPV recommandé chez les femmes de plus de 30 ans. HAS[END_REF] . Son utilisation en surveillance post-thérapeutique des LIEHG n'a pas été retenue par les dernières recommandations de l'ANAES sur le sujet datant de 2002 [START_REF]Conduite à tenir devant une patiente ayant un frottis cervico-utérin anormal[END_REF] . Des recommandations ont néanmoins été publiées en 2007 par le CNGOF [START_REF] Morcel | Utilisaion des test HPV en pratique. Extrait des mises à jour en gynécologie médicale[END_REF] et la SFCPCV [START_REF] Mergui | Guidelines for the followup of women treated for high-grade cervical neoplasia[END_REF] , introduisant la possibilité d'un test HPV HR dans les 3-6 mois après l'exérèse d'une lésion de haut grade histologique. L'objectif de cet article est de proposer un rappel sur la classification et les méthodes de détection des HPV HR, puis une revue de la littérature récente sur la place de ces tests dans le dépistage et le suivi des LIEHG après traitement.

Méthode

Une recherche sur les données de la littérature concernant les tests de détection de l'HPV HR a été réalisée en interrogeant les bases de données « Medline », « The Cochrane library », « Clinical Trials ».

Les termes de recherche utilisés étaient : « human papilloma virus », « HPV testing», «cervical squamous intraepithelial lesion », « cervical cancer ».

La recherche dans ces bases de données a été complétée par la revue des références contenues dans les méta-analyses, revues systématiques et articles originaux identifiés. Seuls les articles publiés en langue anglaise ou française entre 2000 et 2019 ont été pris en compte. Au final, nous avons retenu les revues sytématiques et les études de niveau de preuve 1 ou 2 [START_REF]Niveau de preuve et gradation des recommandations de bonne pratique[END_REF] : les méta-analyses, les essais randomisés et les études de cohorte.

HPV histoire naturelle

Les papillomavirus humains, membres de la famille des Papillomaviridae, sont des petits virus à ADN, non enveloppés qui infectent la peau et les muqueuses, plus précisément les épithéliums malpighiens. Plus de 200 types d'HPV ont été identifiés dans l'espèce humaine [START_REF] Burd | Human Papillomavirus[END_REF] . On peut les classer en fonction de leur tropisme (cutané ou muqueux) et en fonction de leur potentiel oncogénique (haut risque et bas risque). À titre d'exemple, l'HPV 1 est associé aux verrues plantaires et s'observe notamment dans les épithéliums très kératinisés de la paume ou de la plante des pieds, alors que l'HPV 16 est plus particulièrement associé à l'épithélium squameux du col de l'utérus.

Les HPV à tropisme génital, majoritairement impliqués dans le cancer du col de l'utérus, sont classés en 3 catégories : Classe 1 à haut risque (12 HPV), Classe 2 à haut risque probable (1 HPV) et haut risque possible (7 HPV) et Classe 3 à bas risque (13 HPV) (Tableau 1). Parmi les HPV à bas risque, les HPV 6 et 11 sont associés à 90% des condylomes ano-génitaux, sans potentiel d'évolution vers des lésions de haut grade [START_REF] Dunne | HPV and HPV-associated diseases[END_REF] .

Parmi les HPV HR, les HPV 16 et 18 sont responsables de 70% des LIEHG et des cancer du col invasifs (LIEHG+), mais aussi de lésions ayant d'autres localisations ano-génitales (anus, vagin, vulve, pénis) [START_REF] Burd | Human Papillomavirus[END_REF] .

Avant 30 ans près d'un quart de la population est porteur d'un HPV HR [START_REF] Heard | Favre Caractéristiques de l'infection par les papillomavirus humains dans des frottis cervicaux normaux en France en[END_REF] . Entre 30 et 40 ans le portage d'HPV HR est d'environ 10%, et après 40 ans il est de 5% [START_REF] Cuzick | Management of women who test positive for high-risk types of human papillomavirus: the HART study[END_REF] . L'incidence cumulée des LIEHG+ à 10 ans des patientes porteuses d'HPV 16 et HPV 18 sont respectivement de 17% et 14%. Pour les autres HPV HR, ce taux n'est que de 3%. [START_REF] Khan | The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice[END_REF] En accord avec ces données, dans une étude publiée en 2015 sur 18 810 femmes de plus de 30 ans ayant un test HPV HR positif et un FCU négatif, l'incidence cumulée de CIN3ou cancer invasif du col (CIN3+) à 3 ans était de 4,6%. En cas d'HPV 16 ce risque était de 10,6% et en l'absence d'HPV 16 il était de 2,4%. Les autres risques les plus élevés étaient pour l'HPV 33 (5,9%) et l'HPV 18 (5,9%). A noter que près d'un tiers des adenocarcinomes in situ étaient associés à l'HPV 18.

Groupes

Types de virus -une sensibilité clinique pour les LIEHG+ au minimum de 90% de celle du test Hybrid Capture® 2 qui est de l'ordre de 94,6 % à 97,7 %.

-une spécificité clinique pour les LIEHG+ au minimum de 98% de celle du test Hybrid Capture® 2 qui atteint 90,7 % à 94,1 %.

-une reproductibilité intra-et inter-laboratoire avec un pourcentage d'agrément des résultats d'au moins 87 % avec un test validé.

On distingue essentiellement 3 types de tests :

-Les techniques de biologie moléculaire permettant une détection de 12 à 14 HPV HR de façon simultanée et sans distinction des types présents.

-Les trousses de génotypage partiel proposant une détection combinée des principaux HPV HR avec une détection spécifique des HPV16, 18 et parfois 45.

-Les tests de génotypage développés pour identifier de façon spécifique les différents HPV (haut risque et/ou bas risque) présents dans l'échantillon.

Le suivi des patientes par génotypage plutôt que par detection simultanée des HPV HR permet de faire une distinction entre la persistance de l'infection, sa récidive précoce ou tardive, notamment en pré et post thérapeutique. Plusieurs recommandations internationales intègrent déjà le test HPV HR dans le suivi après traitement des LIEHG [START_REF]Colposcopy and programme management. Guidelines for the NHSCSP[END_REF][START_REF] Massad | updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors[END_REF] . Ces données seront reprises par les prochaines recommandations de l'INCa sur le sujet dont la publication est prévue pour les prochains mois [START_REF]Surveillance post-thérapeutique des lésions précancéreuses du col de l'utérus[END_REF] .

Conclusion

Le test HPV HR est un test très performant qui a toute sa place en première intention dans le dépistage primaire des LIEHG+. En parallèle de la vaccination, il pourrait permettre une diminution majeure de l'incidence du cancer du col de l'utérus [START_REF] Simms | Impact of scaled up human papillomavirus vaccination and cervical screening and the potential for global elimination of cervical cancer in 181 countries, 2020-99: a modelling study[END_REF]

  . Le test HPV HR possède une très bonne sensibilité permettant d'accélérer le dépistage des LIEHG+ et une excellente valeur prédictive négative permettant d'espacer le suivi des femmes ayant un test négatif (moins de 5% de risque de LIEHG+ à 6 ans). De plus, le test HPV HR permet d'identifier les femmes à risque de récidive après traitement d'une LIEHG. Il aura certainement une place centrale dans les recommandations sur les modalités du suivi des patientes traitées pour LIEHG qui seront prochainement éditées par l'INCA.

  Les performances et les indications potentielles de l'autoprélèvement constituent une question importante pour l'avenir mais ne sont pas détaillées cette mise au point. Les tests HPV sont pour la plupart des tests de biologie moléculaire, basés sur la mise en évidence de l'ADN, voire des ARN, des HPV. Ils doivent être réalisés dans des laboratoires accrédités. C'est la norme internationale ISO 15189 qui sert de référentiel qualité. Tous les tests HPV disponibles ne peuvent être utilisés pour faire du dépistage. En effet, ils doivent présenter des performances cliniques minimales définies par un groupe d'experts 23 :

	1 (Haut risque)	16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
	2A (Haut risque probable)	68
	2B (Haut risque possible)	26, 53, 66, 67, 70, 73, 82
	3 (Bas risque)	6, 11, 13, 40, 42, 43, 44, 54, 61, 70, 72, 81 89
	Tableau 1. Classification des HPV ano-génitaux selon leur potentiel oncogène. Bouvard et al.,
	Lancet Oncol, 2009 19 .
	Modalités du test HPV HR
	De très nombreux tests de détection des HPV sont commercialisés. Ils sont réalisés soit à
	partir d'un prélèvement déposé dans un milieu pour cytologie liquide ayant servi à réaliser la
	cytologie ou dans milieu dédié à la recherche d'HPV. Il est indispensable que les milieux de
	recueil des cellules aient été au préalable validés pour la recherche d'HPV par biologie
	moléculaire. Le prélèvement est classiquement réalisé par un praticien, mais certaines études
	ont montré la faisabilité d'un autoprélèvement par la patiente elle-même 20,21 . Une méta-

analyse publiée en 2018 par Arbyn et al. montre que la sensibilité d'un test HPV (à condition qu'il s'agisse d'un test basé sur amplification de cible par PCR) pour le dépistage des LIEHG+ est identique que le prélèvement soit effectué par un praticien ou par la patiente elle-même 22 .

  Le Centre National de Référence (CNR) Papillomavirus propose sur son site internet une liste des milieux liquides utilisables avec les trousses de détection des HPV (https://www.cnrhpv.fr/wp-content/uploads/2018/04/Liste-des-trousses-de-d%C3%A9tection-et-de-g%C3%A9notypage-des-HPV-valid%C3%A9es-par-les-fabricants-de-milieux-v2.pdf). Cas particulier de la grossesse : Devant l'absence de données dans la littérature sur d'utilisation du test HPV HR chez les femmes enceintes il n'est à ce jour pas recommandé pendant la grossesse. b. Données de la littérature sur les performances des tests HPV HR en dépistage Depuis 20 ans de nombreuses études ont étudié l'intérêt du test HPV HR dans le dépistage des LIEHG+, dont deux revues de la littérature avec des résultats concordants.La revue de la littérature de Whitlock et al., publiée en 2011, a évalué la performance absolue du dépistage primaire par test HPV HR comparée au FCU chez les femmes de plus de 30 ans dans les pays développés. Elle montrait que la sensibilité des tests HPV HR était de 63 % à 98 9%, 62,5% et 72,9% respectivement et les spécificités de 89,9%, 96,6%, 90,3% respectivement. Ces résultats étaient identiques avant ou après 30 ans[START_REF] Koliopoulos | Cytology versus HPV testing for cervical cancer screening in the general population[END_REF] .Ces données ont été confortées par deux méta analyses[START_REF] Murphy | HPV testing in primary cervical screening: a systematic review and meta-analysis[END_REF][START_REF] Ronco | Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials[END_REF] . La méta-analyse de Murphy et al. publiée en 2012 dans le Journal of Obstetrics and gynaecology Canada 25 , portant sur sept essais randomisés, a mis en évidence un nombre supérieur de CIN3+ diagnostiqué au cours du première examen de dépistage (RR 1,67 IC95% [1,27-2,19]) et un nombre significativement plus faible au cours du deuxième examen de dépistage (RR 0,49 IC95% [0,3 -0,66]) chez les patientes bénéficiant d'un test HPV HR plutôt que d'un FCU. La méta-analyse de Ronco et al. publiée en 2014 dans le Lancet 27 , portant sur quatre essais dépistage des LIEHG+ par test HPV HR apporte une meilleure protection contre les cancers du col de l'utérus comparé au FCU. Les performances d'un test HPV sont meilleures et permettent une détection plus précoce des LIEHG+. En actualisant ses recommandations de 2010 sur le dépistage du cancer du col de l'utérus, la HAS propose en juillet 2019 aux pouvoirs publics, une stratégie nationale de dépistage incluant le test HPV HR. Elle le recommande en première intention chez les femmes de plus de 30 ans, chez lesquelles il s'avère plus efficace que l'examen cytologique 11 .. En cas de test HPV HR négatif, un contrôle à 5 ans est recommandé. En cas de test HPV HR positif, un FCU est proposé, suivi d'un test HPV HR de contrôle à un an en l'absence de LIEHG+. A l'avenir on ne peut qu'espérer que le test HPV HR soit remboursé en dépistage, à l'instar de ce qui est fait dans d'autres pays européens 30 . Pour les femmes de moins de 30 ans, l'intérêt du test reste controversé du fait du taux élevé de portage d'HPV HR. Le taux de portage va certainement diminuer à mesure que la couverture vaccinale augmentera et l'intérêt du test HPV HR chez ces femmes pourra être réévalué. Des recommandations avaient néanmoins été publiées en 2007 par le CNGOF 13 et la SFCPCV 14 , introduisant la possibilité d'un test HPV dans les 3-6 mois après l'exérèse d'une lésion de haut grade histologique. En cas de négativité, il pouvait être proposé une surveillance cytologique à 18 mois. Ces Ces données ont été confirmées en 2017 dans l'étude anglaise rétrospective de Ceballos et al. incluant 2340 femmes ayant bénéficié d'un test Hybrid Capture® 2 dans les suites d'un traitement pour LIEHG et parmi lesquelles 19,6% avaient un test positif. Chez ces femmes avec un test HPV HR positif, 25,7% ont eu un diagnostic de LIEHG+ pendant leur suivi, Une étude prospective italienne sur 5 ans, publiée par Garutti et al. en 2017, incluant 310 patientes traitées et suivies par colposcopie plus FCU plus test HPV HR à 6 mois est en accord avec ces résultats. Un contrôle par FCU et colposcopie était réalisé à 18 mois si les 3 tests initiaux étaient négatifs, et à 12, 18 et 24 mois dans le cas contraire. Un contrôle annuel par FCU était ensuite prévu en l'absence de lésion. Aucune des patientes avec un test HPV une étude portant sur 109 patientes traitées par cryothérapie ou conisation pour des CIN 1 à 3, suivies par test HPV HR à 0, 3, 6, 9, 12, et 24 mois après traitement. La majorité des tests HPV HR étaient négatifs à 3 mois et seulement 9% des patientes avaient un test positif à un an. En cas de cryothérapie la clairance était plus faible (p<0,002) 41 . À noter que dans cette étude les effectifs étaient relativement faibles et que le taux d'HPV HR persistant après traitement était dans la fourchette basse de ce qui est décrit dans la littérature, de l'ordre de 9 à 45% 36,39,40,42-44 . Sur le même principe, Aerssens et al. ont publié en 2008 une série portant sur 122 patientes traitées par cryothérapie ou conisation pour des CIN1 (n=55) ou des CIN2-3 (n=67). Les taux de persistance d'HPV étaient de 24,9%, 20,3%, 15,3% et 8,4% à 6 semaines, 6 mois, un an et deux ans après traitement par conisation 45 . c. Propositions pour l'avenir Des données solides de la littérature montrent que le test HPV HR pourrait être d'un intérêt majeur dans le suivi post-thérapeutique des LIEHG. Un test HPV HR pourrait ainsi être proposé dans les 6 à 9 mois suivant une conisation. Un test négatif permettrait d'espacer la surveillance et à contrario un test positif devrait faire poursuivre les investigations (cytologie de triage/ colposcopie) et rapprocher la surveillance qui sera poursuivie.

	incidence significativement plus faible (p<0,01) de lésions LIEHG+ (3,6/1000 IC95% [2,6-b. Les données de la littérature
	5,1]) que les patientes du groupe Contrôle (10,0/1000 IC95% [8,2-12,3]). Ces différences
	restaient aussi significatives pour les patientes de moins de 30 ans. Les taux cumulés de Les patientes ayant eu un traitement par conisation ou laser pour des LIEHG+ nécessite une
	randomisés européens, a permis d'étudier 176 464 femmes âgées de 20 à 64 ans randomisées colposcopies n'étaient pas significativement différents entre les groupes Intervention surveillance rapprochée. Une étude danoise rétrospective publiée en 2018 a rapporté que les
	entre le test HPV HR et le FCU avec un suivi médian de 6,5 ans. Le nombre de cancers (106,2/1000 [95% CI, 100,2-112,5]) et Contrôle (101,5 [95% CI, 95,6-107,8]). Cette étude femmes (n= 59464) ayant été traitées pour une LIEHG avaient un risque augmenté de cancer contre 1,9% chez celles présentant un test HPV négatif. La valeur prédictive négative du test
	Place du test HPV HR dans le dépistage invasifs était identique dans les 2 groupes durant les 2,5 premières années de suivi (RR 0,79 montrait aussi qu'un test HPV négatif était associé à moins de 5% de risque de LIEHG+ à 6 du col même 25 ans après une conisation 32 . Les taux de récidive étaient estimés à 6,7 % en était de 98,1% (95%IC [97,4-98,7]) 39 .
	IC95% [0,46-1,36]) puis significativement plus bas dans le groupe test HPV HR vs. FCU ans. cas de traitement par électrorésection à l'anse diathermique, à 2,2 % après traitement par
	a. Les recommandations françaises actuelles (RR 0,45 IC95% [0,25-0,81]). Chez les femmes avec un test HPV HR négatif de départ ou un Pour finir Katki et al. ont publié en 2013 une étude intéressante portant sur le risque de bistouri froid et à 2,1 % après conisation laser. La taille de la lésion et la taille de la conisation
	FCU normal, l'incidence cumulée de cancers invasifs était de 8,7 pour 100 000 et 36,0 pour CIN3+ à 5 ans chez 32 374 femmes avec un test HPV HR positif et un FCU normal 29 . Cette ne semblaient pas être des facteurs de risque de récidive 33,34 contrairement au statut des
	situation a été retrouvée chez seulement 3,6 % des patientes (32 374/899 278). Leur risque de CIN3+ à 5 ans était de 4,5 % et le risque de cancer invasif était de 0,34 %, dont la moitié était berges 35,36 . En 2008 une étude française rétrospective montrait déjà l'importance du test HPV HR en post opératoire. Trois-cent-quatre-vingt-six patientes ayant bénéficié d'une conisation pour une LIEHG, suivi d'un test HPV HR dans les 3 à 6 mois suivants, étaient incluses . Le taux de positivité du test en post opératoire était de 22,5%, avec une sensibilité pour dépister une persistance ou récidive d'une LIEHG de 100% vs 72% pour le FCU, vs 67% pour le statut des berges 37 . Arbyn et al. ont proposé en 2017 une revue systématique de la littérature de 1975 à 2016, sur les facteurs associés à l'échec de traitement par exérèse d'une LIEHG (97 études, portant sur 100 000 à 5normal, étaient convoquées pour un nouveau FCU à 2 ans, puis en cas de normalité pour un 44446 patientes) 38 . L'échec de traitement était défini par la persistance de la LIEHG après
	double contrôle test HPV HR et FCU à 4 ans. À l'évaluation à 4 ans, dans le groupe des exérèse, ou bien sa récidive dans les 18 mois suivant l'exérèse. La sensibilité et la spécificité
	patientes Intervention l'incidence de LIEHG+ était significativement plus faible (p<0,01) pour prédire l'échec de traitement étaient de 55,8% (95%IC [45,8-65,5]) et 84,4% (95%IC
	(5,0/1000 IC95% [3,8-6,7]) que l'incidence dans le groupe de patientes du groupe Contrôle recommandations ont notamment été reprises par certains professionnels, notamment le [79,5-88,4]), respectivement, pour le statut des berges, et de 91,0% (95%IC [82,3-95,5]) et
	(10,6/1000 IC95% [8,7-12,9]). Ces différences restaient significatives pour les patientes de Réseau OncoRhoneAlpes en 2015 31 . 83,8% (95%IC [77,7-88,7]), respectivement, pour le résultat du test HPV HR. Un test HPV
	HR négatif dans les 3 à 9 mois après l'exérèse et des berges in sano étaient associés à un
	risque d'échec de traitement de 0,8% et 3,7%, respectivement.

En France, le dépistage des LIEHG+ pris en charge par la sécurité sociale est réalisé par FCU

[START_REF]Recommandations pour le dépistage du cancer du col de l'utérus en France[END_REF]

. Le test HPV n'est remboursé qu'en cas de FCU ASC-US. Les recommandations françaises de l'INCA de 2016, le recommandaient dans les situations suivantes

[START_REF]Conduite à tenir devant une femme ayant une cytologie cervico-utérine anormale, Collection recommandations et référentiels, version interactive, INCa[END_REF]

: -Sélection des patientes ayant un FCU ASC-US ou AGC -Contrôle à un an des patientes présentant : o Un FCU ASC-US et HPV HR positif ou FCU LSIL et colposcopie normale (possibilité de réaliser soit un FCU soit un test HPV HR). o Un FCU ASC-H avec colposcopie normale et satisfaisante (possibilité de réaliser soit un FCU soit un test HPV HR). o Un FCU AGC avec HPV HR positif et colposcopie et curetage de l'endocol négatif.

-Contrôle à 6 mois des patientes présentant un FCU ASC-H avec colposcopie non satisfaisante et curetage endocervical avec résultat histologique négatif.

-Cas particulier des patientes de moins de 30 ans : En France les recommandations sur l'utilisation du test HPV HR sont les même qu'après 30 ans, à l'exception du double immunomarquage p16/Ki67 qui est une option pour les femmes de moins de 30 ans ayant un FCU ASC-US à la place du test HPV HR.

-% alors que celle du FCU était seulement de 38 % à 65 % pour la détection des LIEHG+

[START_REF] Whitlock | Liquid-based cytology and human papillomavirus testing to screen for cervical cancer: a systematic review for the U.S. Preventive Services Task Force[END_REF]

. Il est intéressant de préciser que le taux de LIEHG+ était plus faible lors du 2 ème examen de dépistage pour les femmes suivies par test HPV HR par rapport à celles suivies par FCU (RR 0,23 IC95% [0,07-0,82]). Plus récemment une revue systématique de la Cochrane de 2017 incluant 40 études, avec plus de 140 000 femmes agées de 20 à 70 ans, a comparé le test Hybrid Capture® 2 au FCU (sur lame ou milieu liquide) dans le dépistage des LIEHG+. Les sensibiltés du test Hybrid Capture® 2, du FCU sur lame et du FCU en milieu liquide étaient de 89,,5 ans respectivement. Ces chiffres ne concernaient pas les femmes de moins de 30 ans chez qui les taux de détection de cancers invasifs du col étaient identiques entre le FCU et le test HPV HR (RR 0,98 IC95% [0,19-5,20]). Plus récemment l'étude d'Ogilvie et al., publiée en 2018 dans le JAMA 28 , ayant randomisé 16 374 patientes, a aussi montré qu'un dépistage par test HPV HR seul (groupe Intervention) permettait d'accélérer le dépistage des LIEHG+, par rapport à un dépistage classique par FCU (groupe Contrôle). La construction de l'étude était la suivante : les patientes du groupe Intervention, en cas de test HPV HR négatif, étaient convoquées 4 ans plus tard pour un double contrôle test HPV HR et FCU ; les patientes du groupe Contrôle, en cas de FCU moins de 30 ans. Concernant les patientes avec un test HPV HR négatif ou un FCU normal à l'inclusion, les patientes du groupe Intervention présentaient à l'évaluation à 4 ans une des adénocarcinomes. Parmi ces patientes HPV HR positif et FCU normal, 52 % s'avéraient HPV HR négatives au contrôle à 1 an (pourcentage stable pour tous les âges). Et chez ces patientes ayant négativé leur test HPV HR seulement 0.6% ont eu un diagnostic de LIEHG+ (n=73/11821). c. Propositions pour l'avenir On peut conclure, à la lecture de ces résultats, que des données solides de la littérature montrent que le Place du test HPV HR dans le suivi après traitement d'une LIEHG+ a. Les recommandations françaises Les modalités officielles de surveillance post-the ŕapeutique des LIEHG n'ont pas été mises à jour depuis les recommandations de l'ANAES de 2002 12 . Il y était précisé qu'une surveillance régulière associant FCU +/-colposcopie pouvait être proposée avec un premier contrôle entre 3 et 6 mois, puis en cas de normalité une répétition dans un délai de 6 mois a ̀ 1 an, avant d'envisager une surveillance cytologique annuelle. HR négatif (55%) n'a eu de diagnostic de LIEHG+ pendant le suivi. À contrario parmi les 138 patientes (45%) ayant un test HPV HR positif, 17 cas de récidive/persistance (12,3%) de LIEHG+ ont été identifiés dans les 6 à 24 mois post-conisation 40 . Dans les études suscitées, le délai à respecter entre le traitement de la LIEHG et la réalisation du test HPV HR était hétérogène et variait de 3 à 9 mois. Pour connaitre le temps de clairance des HPV HR en post-thérapeutique, Elfgren et al. ont publié en 2002, dans Obstetrics and gynecology,


PhD Thomas Dietenbeck
email: thomas.dietenbeck@sorbonne-universite.fr

Sophia Houriez-Gombaud-Saintonge

MD Etienne Charpentier

MSc Umit Gencer

PhD Alain Giron

MD Antonio Gallo

MD Samia Boussouar

MD Nicoletta Pasi

MD, PhD Gilles Soulat

MD, PhD Elie Mousseaux

PhD Sophia Houriez--Gombaud-Saintonge

MD, PhD Alban Redheuil

PhD Nadjia Kachenoura

Affiliations

Quantitative magnetic resonance imaging measures of three-dimensional aortic morphology in healthy aging and hypertension

Keywords: MRI, aorta, normal values, 3D morphology

INTRODUCTION

One of the critical features of aortic syndromes is their silent progression until a potentially lethal complication occurs [START_REF] Elefteriades | Litigation in nontraumatic aortic diseases-a tempest in the malpractice maelstrom[END_REF][START_REF] Saeyeldin | Thoracic aortic aneurysm: unlocking the "silent killer" secrets[END_REF]. Imaging, providing comprehensive coverage and high-resolution evaluation of the aorta, plays a central role in aortic disease detection, follow-up and prognostic evaluation.

Three-dimensional renderings of CT and MRI aortic angiograms are commonly used in clinical routine to identify aortic (coarctation, aneurysms, etc.) and aortic valve (regurgitation, stenosis, bicuspid aortic valve, etc.) abnormalities. Despite outstanding 3D visualization, quantitative measures for clinical decisions have been limited to local measurements of diameters at specific locations [START_REF] Goldstein | Multimodality imaging of diseases of the thoracic aorta in adults: from the American Society of Echocardiography and the European Association of Cardiovascular Imaging: endorsed by the Society of Cardiovascular Computed Tomography and Society for Cardiovascular Magnetic Resonance[END_REF][START_REF] Van Hout | How to Measure the Aorta Using MRI: A Practical Guide[END_REF]. Aortic dilation in healthy aging [START_REF] Hickson | The relationship of age with regional aortic stiffness and diameter[END_REF][START_REF] Davis | Observational study of regional aortic size referenced to body size: production of a cardiovascular magnetic resonance nomogram[END_REF], in the general population [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Turkbey | Determinants and normal values of ascending aortic diameter by age, gender, and race/ethnicity in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Casciaro | Identifying the principal modes of variation in human thoracic aorta morphology[END_REF] or in specific disease conditions [START_REF] Pape | Aortic diameter >= 5.5 cm is not a good predictor of Type A aortic dissection observations from the International Registry of Acute Aortic Dissection (IRAD)[END_REF][START_REF] Rylski | How does the ascending aorta geometry change when it dissects?[END_REF][START_REF] Kuijpers | Aortic dissection and prophylactic surgery in congenital heart disease[END_REF][START_REF] Matura | Aortic dilatation and dissection in Turner syndrome[END_REF][START_REF] Craiem | Age-related changes of thoracic aorta geometry used to predict the risk for acute type B dissection[END_REF][START_REF] Engelfriet | Beyond the root: dilatation of the distal aorta in Marfan's syndrome[END_REF][START_REF] Subramaniam | Continuous measurement of aortic dimensions in Turner syndrome: a cardiovascular magnetic resonance study[END_REF] has been reported and aortic elongation has also been studied in both CT [START_REF] Casciaro | Identifying the principal modes of variation in human thoracic aorta morphology[END_REF][START_REF] Craiem | Age-related changes of thoracic aorta geometry used to predict the risk for acute type B dissection[END_REF][START_REF] Adriaans | Aortic elongation part I: the normal aortic ageing process[END_REF][START_REF] Boufi | Morphological analysis of healthy aortic arch[END_REF][START_REF] Van Engelen | Aortic length measurements for pulse wave velocity calculation: manual 2D vs automated 3D centreline extraction[END_REF] and MRI [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Sugawara | Age-Associated Elongation of the Ascending Aorta in Adults[END_REF] using such manual or semi-automated methods. Furthermore, technical advances have been made allowing for automated 3D aortic segmentation and extraction of aortic length or diameters at specific locations from CT [START_REF] Craiem | Aging Impact on Thoracic Aorta 3D Morphometry in Intermediate-Risk Subjects: Looking Beyond Coronary Arteries with Non-Contrast Cardiac CT[END_REF][START_REF] Kurugol | Automated quantitative 3D analysis of aorta size, morphology, and mural calcification distributions[END_REF] and MR [START_REF] Volonghi | Automatic extraction of threedimensional thoracic aorta geometric model from phase contrast MRI for morphometric and hemodynamic characterization[END_REF][START_REF] Dietenbeck | 3D aortic morphology and stiffness in MRI using semi-automated cylindrical active surface provides optimized description of the vascular effects of aging and hypertension[END_REF] images. Automated 3D segmentation has two main advantages: first, it avoids errors due to the potential obliquity of imaging planes when measuring diameters, since cross-sections can be automatically placed perpendicular to the centerline at any desired location within the 3D segmented aorta; second, it is able to provide additional morphological indices, such as volume which combines both diameter and length information, and thus might be more sensitive to subtle changes in aortic morphology.

Three-dimensional aortic MRI is ionizing radiation free and has the potential to be the imaging modality of choice for patients requiring multiple scans for follow-up. While reference 2D measures of aortic morphology have been summarized by Kawel-Boehm et al. [START_REF] Kawel-Boehm | Normal values for cardiovascular magnetic resonance in adults and children[END_REF], there is a lack of normative values of segmental aortic lengths and volumes as measured by 3D MRI. Accordingly, 3D morphological indices of the thoracic aorta, including maximal diameter, volume, length, curvature and tortuosity were extracted from MR images [START_REF] Dietenbeck | 3D aortic morphology and stiffness in MRI using semi-automated cylindrical active surface provides optimized description of the vascular effects of aging and hypertension[END_REF] to: 1/ provide age-related normal values and 95% confidence interval of these indices, 2/ describe morphological changes in healthy aging and in hypertension, as the two main drivers of aortic remodeling, and 3/ identify the main determinants of aortic morphological changes.

MATERIALS AND METHODS

MRI data used in this study are part of local protocols including healthy volunteers and hypertensive subjects, with a focus on associations either between aortic stiffness and microvascular remodeling or on the non-invasive evaluation by ultrafast echo of myocardial stiffness as compared to MRI. Accordingly, the present ancillary work objectives are independent from the primary goals of these protocols.

All participants gave their written informed consent for the initial protocol and ancillary use of their data. The study protocol was approved by the local ethics committee.

Study Population

We retrospectively evaluated 201 individuals: 119 healthy controls (HCs; mean age: 48±14 years, 61 men) and 82 patients with essential hypertension (HT; 60±14 years, 43 men). Age, sex, height, weight, body surface area (BSA), body-mass index (BMI) and current smoking were collected prior to the MRI exam. Inclusion criteria for hypertensives were: absence of secondary causes of hypertension, absence of personal history of cardiovascular disease and stable regimen of antihypertensive treatment.

Patients with diabetes were not included. Healthy controls were asymptomatic and free of overt cardiovascular disease defined as a clinical history of hypertension, diabetes mellitus, dyslipidemia, renal disease, known inflammatory conditions and malignancy.

The median age of our population was 52 years; thus, a cut-off of 50 years was used to divide both groups into 2 subgroups (≤ 50 years, > 50 years) to study the effect of age.

All subjects underwent a cardiac MRI exam at either 3T (site 1, n = 61: Discovery MR750w, GE Healthcare, Chicago; site 2, n = 31: Prisma, Siemens Healthineers, Erlangen) or 1.5T (n = 109, site 3: Aera, Siemens Healthineers, Erlangen). For sites 1 and 3, gadolinium-based contrast agent was typically injected (0.1 to 0.2 mmol/kg) during aortic acquisitions. 3D data were acquired during free breathing with electrocardiographic (diastasis) and respiratory (expiration) gating in a sagittal oblique volume encompassing the thoracic aorta with the following typical scan parameters: Central blood pressures were recorded using the SphygmoCor Xcel device (AtCor Medical, Sydney) simultaneous to MRI aortic acquisitions at all sites. Three measurements were recorded during the MRI exam and their average provided central systolic (SBP) and diastolic (DBP) blood pressure as well as pulse pressure (PP), calculated as the difference between SBP and DBP.

Assessment of Aortic Morphology

The aorta was segmented using a previously described custom software (Mimosa, Sorbonne Université) [START_REF] Dietenbeck | 3D aortic morphology and stiffness in MRI using semi-automated cylindrical active surface provides optimized description of the vascular effects of aging and hypertension[END_REF]. Briefly, six anatomical landmarks were manually placed by an experienced operator (5 years) from the sino-tubular junction to the diaphragm and used to automatically initialize the aortic centerline and delimit aortic segments. The aortic lumen was then segmented using a 3D active contour [START_REF] Barbosa | Fast and Fully Automatic 3D Echocardiographic Segmentation Using B-Spline Explicit Active Surfaces: Feasibility Study and Validation in a Clinical Setting[END_REF] and a final centerline was calculated. The segmented aorta was automatically divided into 3 segments using the given landmarks (Fig. 1) according to the European Society of Cardiology guidelines [START_REF] Erbel | ESC Guidelines on the diagnosis and treatment of aortic diseases: Document covering acute and chronic aortic diseases of the thoracic and abdominal aorta of the adult The Task Force for the Diagnosis and Treatment of Aortic Diseases of the European Society of Cardiology (ESC)[END_REF]: the ascending aorta, defined as the segment between the sino-tubular junction and the brachiocephalic trunk, the aortic arch, defined as the segment comprising the supra-aortic trunks and the descending thoracic aorta, defined as the segment between the left subclavian artery and the diaphragm.

For each aortic segment, 5 morphological indices were automatically extracted: the centerline length, the maximal diameter, the volume, the total curvature [START_REF] Bullitt | Measuring tortuosity of the intracerebral vasculature from MRA images[END_REF][START_REF] Grisan | A novel method for the automatic grading of retinal vessel tortuosity[END_REF] and the tortuosity. Tortuosity was computed as

𝐿 𝑠 -𝐿 𝑑 𝐿 𝑑
, where 𝐿 𝑠 is the true segment length along the centerline and 𝐿 𝑑 is the direct length between the segment ends. Total curvature was defined as

1 𝐿 𝑠 ∫ ‖ 𝑠∧𝑠‖ ‖𝑠‖ 3 𝐿 𝑠 0
𝑑𝑠 where 𝑠̇ and 𝑠̈ are the first and second derivatives of 𝑠, the 3D coordinates of the centerline, respectively. Total curvature was normalized by 𝐿 𝑠 to account for segment length [START_REF] Grisan | A novel method for the automatic grading of retinal vessel tortuosity[END_REF].

In-plane diameters were measured at 3 specific locations (ascending aorta at the pulmonary artery level, proximal descending aorta at the pulmonary artery level and distal descending aorta at the diaphragm) to enable comparison with values commonly provided in the literature (4).

A standard computer (Intel Xeon Silver 4110 CPU, 2.1GHz and 32Go RAM) was used for the data analysis.

Statistical Analysis

All measurements were provided as median and interquartile range (IQR) as some measurement distributions were not normally distributed according to a Shapiro-Wilk normality test. To highlight effects of age and hypertension, while minimizing their confounding effect when necessary, non-parametric Wilcoxon tests were performed for continuous subjects characteristics as well as for all MRI quantitative parameters 1) between HC≤50 and HC>50 to characterize the effect of aging on thoracic aorta, and 2) between the two groups of hypertensive patients (≤ 50 and > 50 years) and the corresponding healthy control groups (≤ 50 and > 50 years, respectively) to characterize the effect of hypertension. Allometric indices such as aortic length, maximal diameter and volume were indexed to BSA to account for the effect of body size. Of note, since total curvature and tortuosity were already normalized by aortic segments length, these indices were not indexed to BSA.

Linear regression and Spearman correlation coefficients between aortic measurements

of the HC population and age were calculated and age-related normal values were considered as measurements within the 95% confidence interval for the individual response which was computed as

𝐶𝐼 95% (𝑥) = -1.65 𝜎 𝑦 √1 + 1 𝑛 + (𝑥 -𝑥̅ ) 2 (𝑛 -1)𝜎 𝑥 2
where 𝜎 𝑦 is the standard deviation of the prediction error and 𝑥̅ and 𝜎 𝑥 are respectively the mean and standard deviation of x. In this equation, x represents age.

Changes per decade correspond to 10 times the slope of the linear regression and are provided to enable comparison with values provided in the literature. The relationship of aortic morphology with age, sex, BSA, hypertension, current smoking and central SBP was studied using a multivariate regression model. A p value < 0.05 was considered as significant.

RESULTS

The whole 3D segmentation process, including data loading and results recording, took less than 5 minutes per subject. Figure 2 shows examples within each group of segmented aortas of male subjects with similar height and BSA in order to illustrate aortic morphological changes in aging and hypertension. Supplementary Figure 1 illustrates examples of aortic images obtained from the 3 acquisition sites and scanners.

Patient Characteristics

Patient characteristics, blood pressures and specific landmarks in plane aortic diameters are summarized in Table 1 for each group: healthy controls (HC≤50 and HC>50) as well as hypertensives (HT≤50 and HT>50). Central SBP and PP were significantly higher in the hypertensive groups compared to their age equivalent controls (SBP: HT≤50 (127mmHg) vs HC≤50 (109mmHg), p < 0.0001; HT>50 (126mmHg) vs HC>50 (116mmHg), p < 0.0001; PP: HT≤50 (35mmHg) vs HC≤50 (31mmHg), p = 0.0005; HT>50 (44 mmHg) vs HC>50 (36mmHg), p = 0.0006) and in the older healthy individuals compared to younger healthy controls (SBP: HC>50 vs HC≤50, p = 0.01; PP: HC>50 vs HC≤50, p < 0.0001). HT≤50 patients also had a significantly higher central DBP (p < 0.0001) and BMI (p = 0.0061), as compared to HC≤50. A significant difference in age (p=0.0012) was also observed in HT>50 patients (median age: 66 years) as compared to HC>50 (median age: 60 years).

Morphological Changes in Aging & Normal Values

Table 1 shows the median and IQR for the cross-sectional aortic diameters measured at three specific landmarks (ascending aorta, proximal and distal descending aorta). The diameters were significantly higher in HC>50 than in HC≤50 (ascending aorta: +2.4mm, p = 0.0019; proximal descending aorta: +2.3mm, p < 0.0001; and distal descending aorta: +2.5mm, p < 0.0001). These increases remained significant after diameters indexation to BSA (ascending aorta: +1.8mm.m -2 , p < 0.0001; proximal descending aorta: +2.1mm.m -2 , p < 0.0001; and distal descending aorta: +1.8mm.m -2 , p < 0.0001).

Quantitative 3D-derived measurements in the thoracic aorta (TAo), ascending aorta (AAo), aortic arch (Arch) and descending aorta (DAo) are provided in Table 2 and illustrated in Figure 3 as well as Supplementary Figure 2 for BSA-indexed measures.

A significant increase in aortic length (thoracic aorta: +21.3mm, p < 0.0001; ascending aorta: +4.3mm, p = 0.0041; aortic arch: +3.2mm, p = 0.0069; and descending aorta:

+15.1mm, p < 0.0001) and maximal diameter (thoracic aorta: +2.7mm, p = 0.0009; ascending aorta: +2.7mm, p = 0.001; aortic arch: +2.7mm, p = 0.0002; and descending aorta: +2.4mm, p < 0.0001) was observed in HC>50 individuals as compared to HC≤50, independently of the considered aortic segment. A significant increase in volume was also observed in all aortic segments between the two age groups (TAo: +29.7mL, p < 0.0001; AAo: +8.2mL, p = 0.0004; Arch: +5.6mL, p < 0.0001; and DAo: +16.5mL, p < 0.0001). Significant decreases in AAo (-0.0032mm -1 , p = 0.0024) and Arch (-0.0096mm -1 , p < 0.0001) curvatures between the two age groups were observed, while DAo curvature increased (+0.0018mm -1 , p = 0.0011), and TAo curvature was unchanged (+9.0 10 -4 mm -1 , p = 0.1382). Significant changes in tortuosity between the two age groups (HC>50 vs HC≤50) were also observed in the Arch (-0.025, p < 0.0001), DAo (+0.068, p < 0.0001) and TAo (+0.39, p < 0.0001). After indexation to BSA (Supplementary Table 1), all the above changes remained significant (p < 0.05).

Linear regressions between all MRI aortic measurements and age were summarized in Linear regressions with age for the aortic measures including length, maximal diameter, volume, total curvature and tortuosity are illustrated in Figure 4 for the AAo and DAo, along with 95% confidence intervals highlighting normal ranges. Linear regression corresponding to BSA-indexed indices are illustrated in Supplementary Figure 3.

Morphological Changes in Hypertension

Aortic morphological indices for hypertensive patients (HT≤50 and HT>50) are also provided in Tables 1 and2. No significant difference in cross-sectional diameters was found at the three anatomical landmarks between HT≤50 and HC≤50. DAo was found to be significantly shorter (-10.9mm, p = 0.0117), more tortuous (+0.035, p = 0.0057) and to have a significantly higher curvature (+0.0012mm -1 , p = 0.0153) in HT≤50 as compared to HC≤50. TAo curvature was also significantly higher in HT≤50 compared to HC≤50 (+0.0018 mm -1 , p = 0.0226).

A significant increase in cross-sectional diameter was found at the three locations between HT>50 and HC>50. Length, maximal diameter and volume were significantly higher in HT>50 as compared to HC>50 for AAo (length: +4.2mm, p = 0.0033; diameter: +2.8mm, p = 0.0002; volume: +10.5mL, p = 0.0002) and Arch (length: +3.5mm, p = 0.0141; diameter: +2.1mm, p = 0.0009; volume: +3.4mL, p = 0.0061). DAo length was significantly lower in HT>50 as compared to HC>50 (-5.7mm, p = 0.0199), while DAo maximal diameter was significantly higher (+2.8mm, p = 0.0060).

Relationship Between Aortic Morphology and Clinical Features

In multivariate analysis (Table 4) on the entire study population, age was found to be the main determinant of all aortic morphological indices except the AAo curvature and Arch tortuosity. Besides, sex and BSA were the second most common independent correlates. When indexed to BSA, aortic volumes did not vary significantly between males and females in both age groups (Figure 5, Supplementary Table 2), while there were significant differences in terms of indexed aortic length and maximal diameters.

Hypertension was the only determinant of AAo total curvature while current smoking was the only determinant of Arch tortuosity, though the model regression coefficients were low (𝑟 2 = 0.067 and 𝑟 2 = 0.109, respectively). Regression coefficients of the volume model were higher than those of other morphological indices (Table 4).

DISCUSSION

In this study, 3D aortic MRI images of 119 healthy controls and 82 hypertensive patients were automatically segmented providing segmental and global indices of aortic morphology. Cross-sectional aortic diameters at predefined anatomical landmarks as well as normal values and confidence intervals of 3D aortic measurements including length, maximal diameters, volumes, curvature and tortuosity were reported.

Significant increase in length and maximal diameters was found between young and elderly healthy controls for all aortic segments leading to a 3 to 4 times higher increase in volume between the two groups. While no marked differences were found in aortic morphology indices between hypertensives and healthy controls in the young age, we found higher diameters, length and volumes in elderly hypertensives as compared to elderly controls. Of note, younger and elderly subjects were defined according to a cuoff of 50 years which was close to the median age in this study (52 years) and was chosen according to previous aortic pathophysiology knowledge [START_REF] Redheuil | Reduced ascending aortic strain and distensibility: earliest manifestations of vascular aging in humans[END_REF] 6)) and slightly lower than those of general population studies [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Turkbey | Determinants and normal values of ascending aortic diameter by age, gender, and race/ethnicity in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF][START_REF] Mensel | Thoracic and abdominal aortic diameters in a general population: MRI-based reference values and association with age and cardiovascular risk factors[END_REF] which included patients with risk factors that are known to have an effect on the aorta.

Regarding the association of the 3D MRI-derived measures with age, a significant increase in aortic length and diameter were observed, in both ascending and descending aorta, resulting in a marked age-related increase in their volumes. These values were similar in range with those reported in other studies [START_REF] Hickson | The relationship of age with regional aortic stiffness and diameter[END_REF][START_REF] Davis | Observational study of regional aortic size referenced to body size: production of a cardiovascular magnetic resonance nomogram[END_REF][START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Adriaans | Aortic elongation part I: the normal aortic ageing process[END_REF], though a direct comparison was not possible due to differences in measurement methods (2D vs 3D), imaging modalities (CT vs. MRI), and patients cardiovascular risk.

In the ascending aorta and aortic arch, the observed increase in length and decrease in total curvature with aging suggested an unfolding of the proximal aorta, probably due to the mechanical constraints perpetually exerted on the aorta by the left ventricle combined with the subsequent and inherent aortic tissue elastic alteration [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] O'rourke | Mechanical Factors in Arterial Aging: A Clinical Perspective[END_REF]. In the descending aorta, the elongation and increased total curvature and tortuosity reflected the transition from a straight to a more tortuous segment. This is consistent with the unbound nature of the ascending aorta within the anterior mediastinum whereas the descending aorta is tied to the spine through the posterior thoracic arteries. Our findings also indicated the gradual transition from a roundish aortic arch to an unfolded shape, which has been previously described [START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF][START_REF] Adriaans | Aortic elongation part I: the normal aortic ageing process[END_REF][START_REF] Ostrevy | The apex of the aortic arch backshifts with aging[END_REF][START_REF] Tawfik | The effect of age and gender on tortuosity of the descending thoracic Aorta[END_REF]. Of note, these significant decreases in AAo and Arch curvatures and increase in DAo curvature with age resulted in an overall unchanged TAo curvature.

While no marked differences in aortic morphological measures could be observed between healthy subjects and hypertensive patients considering subjects ≤ 50 years, significant length, maximal diameter and volume increases at the level of the ascending aorta and aortic arch were observed between hypertensive patients over 50 years compared to their matched healthy controls. Indeed, as they comprehensively assess the aortic morphology, these 3D indices could provide a better means of detecting subclinical changes than local cross-sectional diameter measurements since they overcome most of the 2D-measures drawbacks such as the subjective positioning in the most dilated or narrow section as well as possible slice obliquity. Furthermore, differences in aortic morphology under the effect of hypertension in the older individuals and their absence in younger groups might reflect the aggravating and integrative effects of hypertension through a lifespan, rather than acute effects on aortic morphology. Indeed, it is well known that higher-pressure conditions within the aorta in hypertensive subjects progressively damage the elastin fibers of the aortic wall inducing a higher stiffness [START_REF] O'rourke | Mechanical Factors in Arterial Aging: A Clinical Perspective[END_REF][START_REF] Redheuil | Age-related changes in aortic arch geometry: relationship with proximal aortic function and left ventricular mass and remodeling[END_REF]. This increased stiffness can be associated with an aortic dilation to reduce the blood pressure and consequently the load exerted on the left ventricle. The differences in aortic morphology measures found in our elderly hypertensive patients as compared to the healthy controls reflects this geometrical remodeling of the aorta as it adapts to the higher blood pressure regimen. This aortic remodeling remains however within normal range as no diameter > 40mm have been measured.

The multivariate analysis performed on the entire study group revealed that age, sex and BSA were the major determinants of aortic morphology as previously reported [START_REF] Davis | Observational study of regional aortic size referenced to body size: production of a cardiovascular magnetic resonance nomogram[END_REF][START_REF] Turkbey | Determinants and normal values of ascending aortic diameter by age, gender, and race/ethnicity in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]. Although women had smaller aorta than men in terms of length, diameter and volume, they had similar tortuosity and curvature. Moreover, after indexation to BSA, no significant differences in volumes were observed between men and women.

Limitations

One limitation of our study is the unbalanced and relatively small samples especially in the elderly healthy controls and young hypertensives groups. However, one might emphasize that our distributions are consistent with those in the general population as elderly healthy subjects without cardiovascular comorbidities were difficult to enroll and prevalence of hypertension is known to considerably increase with age [START_REF] Guo | Trends in Prevalence, Awareness, Management, and Control of Hypertension Among United States Adults, 1999 to 2010[END_REF].

Furthermore, a large proportion of our data were acquired after contrast agent injection during a standardized clinical procedure. This ultimately enhances the signal to noise ratio in the resulting aortic images but might limit the usefulness in patients with counterindication to contrast agents. However, one might note that our segmentation technique was shown to be robust to low image quality, as it was able to reliably segment 4D flow MRI-derived aortic angiograms [START_REF] Bouaou | Analysis of aortic pressure fields from 4D flow MRI in healthy volunteers: Associations with age and left ventricular remodeling[END_REF][START_REF] Houriez--Gombaud-Saintonge | Comparison of different methods for the estimation of aortic pulse wave velocity from 4D flow cardiovascular magnetic resonance[END_REF], which are known to have lower signal to noise ratio than conventional anatomical images. Our MRI data were acquired from three different sites using different field strengths (1.5T and 3T) and sequences (SPGR and SSFP). This might have slightly altered the distribution of our normal but reflects the real-life conditions with multi-center inclusions. Finally, our study population did not include patients with a higher risk of aortic dissection to fully demonstrate the clinical usefulness of the 3D measures against a 2D measure of diameters. However, such studies would need normative values of the 3D morphology which was the primary objective of the present study. Besides, we believe that a 3D rendering of the aorta with precise and local geometric measures, resulting from our segmentation, may help in the planning of aortic surgery especially in complex, tortuous geometry or excessive dilation where the aortic pathway would not be easy to track.

Conclusion

Normal values of global and segmental aortic length, volume, curvature and tortuosity as well as diameters at specific landmarks were automatically measured from 3D MRI to serve as reference measures of 3D aortic morphology. Such 3D aortic indices increased significantly with age and hypertension among the elderly subjects. Further longitudinal studies on larger cohort including patient with cardiovascular diseases are required to evaluate the full benefit of this 3D morphometric analysis. Pairwise Wilcoxon test: * p < 0.05, ** p < 0.01, *** p < 0.001 for comparisons of healthy controls > 50y and hypertensives ≤ 50y against healthy controls ≤ 50y, † p < 0.05, † † p < 0.01, † † † p < 0.001 for comparisons of hypertensives > 50y against healthy controls > 50y.

FIGURES

Figure 1 3D aortic segmentation and partition into segments. Green dots: anatomical landmarks; purple line: aortic centerline; red segment: ascending aorta; green segment: aortic arch; blue segment: descending thoracic aorta

Examples of aortic 3D morphology in each group: (a) Healthy control (HC) male ≤ 50y, (b) Healthy male > 50y, (c) Hypertensive (HT) male ≤ 50y and (d) Hypertensive male > 50y. These subjects had similar height and body surface area with ranges of 173 -175 cm and 1.78 -1.87 m², respectively. Normal morphological measures in the ascending aorta (AAo, top row) as well as in the descending aorta (DAo, bottom row).

Red line: linear regression; grey area: 95% confidence interval for the individual response; blue dots: healthy controls. 𝜌: Spearman correlation coefficient.

(site 1 )

 1 spoiled gradient echo (SPGR), voxel size = 0.67×0.67×3.19 mm 3 , echo time = 1.3ms, repetition time = 3.1ms and flip angle = 24°; (site 2) steady state free precession (SSFP), voxel size = 0.98×0.98×1mm 3 , echo time = 1.3ms, repetition time = 311ms and flip angle = 19°; and (site 3) SSFP, voxel size = 0.66×0.66×1.13mm 3 , echo time = 1.5ms, repetition time = 283ms and flip angle = 90°.

Figure 3

 3 Figure 3Boxplot representation of morphological changes between groups in the ascending

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5Boxplot representation of ascending aorta (AAo), aortic arch (Arch) and descending

Table 3

 3 DAo: ρ = 0.462) in all aortic regions. The slopes of the linear regression summarized in Table3revealed an increase per decade in aortic length (TAo:

	revealing significant (p < 0.0001) age-related associations for all indices except
	for TAo curvature (p = 0.283) and AAo tortuosity (p = 0.389). Associations between
	age and volume (TAo: ρ = 0.605; AAo: ρ = 0.526; Arch: ρ = 0.536; DAo: ρ = 0.602)

were stronger than those with maximal diameter (TAo: ρ = 0.485; AAo: ρ = 0.478; Arch: ρ = 0.528; DAo: ρ = 0.571) or length (TAo: ρ = 0.542; AAo: ρ = 0.431; Arch: ρ = 0.335;

Table 2 -Global and segmental aortic morphology quantitative measures

 2

		Healthy controls ≤ 50y	Healthy controls > 50y	Hypertensives ≤ 50y	Hypertensives > 50y
	Thoracic aorta
	Length (mm)	235.5 [217.0; 254.8]	256.8 [239.1; 276.6] ***	225.6 [211.6; 238.3]	256.4 [239.7; 270.3]
			p < 0.0001	p = 0.1354	p = 0.6606
	Maximal diameter (mm)	27.3 [24.9; 29.6]	29.9 [27.0; 32.6] ***	28.7 [25.8; 30.7]	32.8 [30.2; 35.5] † † †
			p = 0.0009	p = 0.3068	p = 0.0002
	Volume (mL)	84.5 [70.8; 111.9]	114.1 [98.1; 134.6] ***	98.8 [73.3; 113.6]	139.3 [110.1; 178.2] † †
			p < 0.0001	p = 0.6343	p = 0.0032
	Total curvature (mm -1 )	0.023 [0.022; 0.025]	0.023 [0.022; 0.025]	0.025 [0.023; 0.027] *	0.025 [0.023; 0.027] † †
			p = 0.1382	p = 0.0226	p = 0.0067
	Tortuosity	1.50 [1.28; 1.69]	1.89 [1.68; 2.17] ***	1.57 [1.46; 1.88]	2.04 [1.82; 2.37] †
			p < 0.0001	p = 0.0628	p = 0.0299
	Ascending aorta
	Length (mm)	51.9 [45.7; 58.8]	56.2 [52.3; 63.3] **	53.9 [50.4; 57.4]	60.4 [55.8; 66.0] † †
			p = 0.0041	p = 0.5310	p = 0.0033
	Maximal diameter (mm)	27.3 [24.9; 29.6]	29.9 [26.9; 32.6] ***	28.7 [25.8; 30.7]	32.8 [30.2; 35.4] † † †
			p = 0.0010	p = 0.2936	p = 0.0002
	Volume (mL)	26.5 [21.4; 34.7]	34.7 [28.5; 43.5] ***	33.3 [24.4; 36.9]	45.2 [35.9; 53.2] † † †
			p = 0.0004	p = 0.1857	p = 0.0002
	Total curvature (mm -1 )	0.037 [0.032; 0.042]	0.033 [0.030; 0.037] **	0.037 [0.034; 0.039]	0.035 [0.030; 0.038]

Acknowledgements

Not applicable.

Funding

We would like to acknowledge the FRM project ING20150532487 for funding part of the technical aspects of this study, the FRHTA for funding part of the acquisitions and of clinical data generation and the ECOS-SUD exchange project number A15S04 (France-Argentina).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Association between age and aortic measurements for healthy individuals (n = 119).

𝜌: Spearman correlation coefficient.

Competing interests

The authors declare that they have no competing interests Authors' contributions AR, NK and TD designed the study. AGa, AR, EM, GS, NP and SB carried out the data acquisition. SHGS, TD and UG carried out the experiments. AGi, AR, NK and TD interpreted the results. NK and TD drafted the manuscript. All authors edited and revised the manuscript. All authors read and approved the final manuscript.

Competing Interests

The authors declare that they have no competing interests Illustration of images and segmentation quality in representative data from our 3 acquisition sites. Site 1: SPGR sequence acquired at 3T (Discovery MR750w, GE Healthcare, Chicago); site 2: SSFP sequence acquired at 3T (Prisma, Siemens Healthineers, Erlangen); site 3: SSFP sequence acquired at 1.5 T (Aera, Siemens Healthineers, Erlangen).

TABLES

Supplementary Figure 2

Morphological changes between groups in the ascending aorta (AAo), aortic arch (Arch) and descending aorta (DAo).

Blue shades: healthy controls (HC) ≤ and > 50 years, red shades: hypertensives (HT) ≤ and > 50 years. * p < 0.05, ** p < 0.01, *** p < 0.001.

Supplementary Figure 3

Normal morphological measures indexed to BSA in the ascending aorta (AAo, top row) as well as in the descending aorta (DAo, bottom row).

Red line: linear regression; grey area: 95% confidence interval for the individual response; blue dots: healthy controls. 𝜌: Spearman correlation coefficient.


PhD Charlotte Lecerf
email: charlotte.lecerf@curie.fr

PhD Maud Kamal
email: maud.kamal@curie.fr

Sophie Vacher
email: sophie.vacher@curie.fr

Walid Chemlali
email: walid.chemlali@curie.fr

Anne Schnitzler
email: anne.schnitzler@curie.fr

Claire Morel
email: claire.morel@curie.fr

Coraline Dubot
email: coraline.dubot@curie.fr

Emmanuelle Jeannot
email: emmanuelle.jeannot@curie.fr

Didier Meseure
email: didier.meseure@curie.fr

Jerzy Klijanienko
email: jerzy.klijanienko@curie.fr

Odette Mariani
email: odette.mariani@curie.fr

Edith Borcoman
email: edith.borcoman@curie.fr

Valentin Calugaru
email: valentin.calugaru@curie.fr

Nathalie Badois
email: nathalie.badois@curie.fr

Anne Chilles
email: anne.chilles@curie.fr

Maria Lesnik
email: maria.lesnik@curie.fr

Samar Krhili
email: samar.krhili@curie.fr

Olivier Choussy
email: olivier.choussy@curie.fr

Caroline Hoffmann
email: caroline.hoffmann@curie.fr

Eliane Piaggio
email: eliane.piaggio@curie.fr

Ivan Bieche
email: ivan.bieche@curie.fr

Christophe Le Tourneau
email: christophe.letourneau@curie.fr

Christophe Le

Immune gene expression in head and neck squamous cell carcinoma patients

Keywords: head and neck squamous cell carcinoma, immune checkpoints, prognostic biomarker, gene expression

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

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the seventh cause of cancer with a yearly 40-50% mortality [START_REF]Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN[END_REF]. Classical risk factors for HNSCC include tobacco and alcohol consumption, as well as human papillomavirus (HPV) infection that has been demonstrated to have a prognostic impact [START_REF] Fakhry | Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial[END_REF].

The Cancer Genome Atlas reported the genomic landscape of more than 270 primary HNSCC [START_REF] Genome | Comprehensive genomic characterization of head and neck squamous cell carcinomas[END_REF] with mutations in several oncogenes including PIK3CA (21%) and HRAS (4%), as well as in tumor suppressor genes including TP53 (72%), CDKN2A (22%), FBXW7 (5%), KMT2D (MLL2) (18%), and PTEN (2%) [START_REF] Genome | Comprehensive genomic characterization of head and neck squamous cell carcinomas[END_REF][START_REF] Sablin | Identification of new candidate therapeutic target genes in head and neck squamous cell carcinomas[END_REF]. Genomic alterations involving the cell cycle (TP53, CCND1, CDKN2A), as well as FGFR1 amplifications, and tumor genomic alterations burden were shown to be prognostic and potential therapeutic targets for patients with HNSCC [START_REF] Dubot | Comprehensive genomic profiling of head and neck squamous cell carcinoma reveals FGFR1 amplifications and tumour genomic alterations burden as prognostic biomarkers of survival[END_REF]. No relevant biomarkers for tailored therapeutic strategies have been identified in HNSCC to date.

Beside surgery, radiotherapy and chemotherapy, HNSCC treatment includes targeted therapy and immunotherapy. Cetuximab, a monoclonal antibody that targets EGFR (epidermal growth factor receptor), has been the first targeted therapy approved in HNSCC, both in the locally advanced setting combined with radiotherapy [START_REF] Bonner | Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck[END_REF] and in the first-line recurrent and/or metastatic setting in combination with chemotherapy [START_REF] Vermorken | Platinumbased chemotherapy plus cetuximab in head and neck cancer[END_REF]. Two anti-PD-1 immune checkpoint inhibitors have been approved for the treatment of recurrent and/or metastatic HNSCC refractory to platinum therapy in 2016 [START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF][START_REF] Seiwert | Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial[END_REF]. These agents are better tolerated than chemotherapy and demonstrated durable responses in a minority of patients [START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF][START_REF] Seiwert | Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial[END_REF].

At the tumor microenvironment (TME) level, the infiltration of HNSCC by innate and adaptive immune cells has been well documented. Several studies have identified immune cells with a prognostic value, such as CD8+ T cells, Foxp3+ regulatory T cells. The presence of tertiary lymphoid structures were also reported to affect prognosis [START_REF] Badoual | Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF][START_REF] Pretscher | Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+ B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oroand hypopharyngeal carcinoma[END_REF][START_REF] Hartmann | Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer[END_REF][START_REF] Kerrebijn | Macrophage and dendritic cell infiltration in head and neck squamous-cell carcinoma; an immunohistochemical study[END_REF][START_REF] Germain | Tertiary Lymphoid Structure-Associated B Cells are Key Players in Anti-Tumor Immunity[END_REF]. OX40, PD-1 and CTLA-4

5
were shown to have a significantly higher expression in T-cell subsets isolated from tumors of HNSCC patients [START_REF] Montler | OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer[END_REF]. Few integrative studies reported the prognostic value of immune genes in HNSCC.

We aimed in this study to assess the expression of immune genes and to evaluate their prognostic value in untreated HNSCC patients. 6

Patients and Methods

Patients

We retrieved samples from HNSCC patients who underwent upfront surgery at the Institut Curie between 1990 and 2006. We selected 96 patients with complete clinical, histological and biological data and long-term follow-up.

This study was approved by the internal review board of Institut Curie and was conducted in accordance with the ethics principles of the Declaration of Helsinki. In accordance with the French regulations, all patients were informed that analyzes were to be performed on the biological specimens obtained during their treatment, and they did not express their opposition.

Gene selection

Fourty-six genes involved in the immune process were selected, including 30 genes defined as immune-related genes and 16 genes that were defined as immune-cell subpopulation genes (Supplementay Table 1). We chose TBP (Genbank accession number NM_003194) which encodes the TATA box binding protein as an RNA control gene.

DNA sequencing & mutation assessment

Targeted DNA sequencing of a selection of 100 genes corresponding to the most frequently altered genes in HNSCC and potential therapeutic targets was performed on Illumina HiSeq2500 sequencer and then annotated in the COSMIC and 1000 genome databases [START_REF] Forbes | The Catalogue of Somatic Mutations in Cancer (COSMIC)[END_REF] ,as described in [START_REF] Dubot | Comprehensive genomic profiling of head and neck squamous cell carcinoma reveals FGFR1 amplifications and tumour genomic alterations burden as prognostic biomarkers of survival[END_REF] Tumor genomic alterations burden was assessed by summing the number of recurrent deleterious genomic alterations (SNV+CNV) per sample.Sanger sequencing was also performed to confirm PIK3CA, KRAS, HRAS and NRAS mutations.

HPV genotyping

HPV status was assessed at the Pathology Department of the Institut Curie. HPV typing was conducted using total DNA isolated from formalin-fixed tissue blocks. Real-time PCR was performed with Sybr®Green and specific primers for HPV16 and 18 using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

Real time quantitative RT-PCR

PCR consumables, RNA extraction, ctDNA synthesis and PCR reaction conditions were previously described in detail [START_REF] Pignot | Large-scale realtime reverse transcription-PCR approach of angiogenic pathways in human transitional cell carcinoma of the bladder: identification of VEGFA as a major independent prognostic marker[END_REF]. Primers are described in Supplementary Table 2. For each investigated gene, mRNA values ≥3 were considered as overexpressions and ≤0.33 as underexpressions. We previously used the same cut-off value for altered tumor gene expression [START_REF] Pignot | Large-scale realtime reverse transcription-PCR approach of angiogenic pathways in human transitional cell carcinoma of the bladder: identification of VEGFA as a major independent prognostic marker[END_REF].

Immunohistochemistry/OX40L protein expression

We performed IHC assay by using OX40L (Cell Signaling Technology, rabbit, 59036, 1/100, pH9) antibody in a series of 20 HNSCC among the 96 HNSCC patients corresponding to 10 patients with high OX40L mRNA level and 10 with low OX40L mRNA level (supplementary material and methods). We performed immunohistochemical study of the tumor microenvironment by using OX40L immunostaining in tumor cells, stromal cancer associated fibroblasts (CAFs) and mononuclear inflammatory cells (MICs). All quantifications were performed with blinding to patient status by 2 expert pathologists.

Statistical analyses 8

The clinicopathological features were tested for association with disease-free interval (DFI) by using Log-rank test. DFI was determined from the time of initial diagnosis to the time of the first event among locoregional recurrence, metastatic recurrence or second cancer. The clinicopathological and biological characteristics were tested for association with transcript level expression by using chi-square tests for categorical variables. The association between clinical variables and RNA levels was tested using Kruskal-Wallis H tests. Cox proportional hazards regression was used to estimate hazard ratio (HR) and their 95% confidence intervals (95% CI) for covariates associated with DFI showing significance at p<0.1 on univariate analysis. Differences between two populations were judged significant at confidence levels greater than 95% (p<0.05) [START_REF] Cox | Regression Models and Life-Tables[END_REF]. Unsupervised hierarchical cluster analyses were performed using Morpheus algorithm to identify homogenous genes and tumor groups regarding molecular data. 9

Results

Patient characteristics

The characteristics of the 96 untreated HNSCC patients are listed in Table 1. Median age was 56 years [range: 35-78]. Most patients were males with tobacco and alcohol consumption. Twelve patients (13%) had an HPV-positive with a majority in oropharyngeal cancer. Pathological staging showed a high proportion of stage IV. The main tumor location was oral cavity (45%), followed by oropharynx (21%), larynx (18%) and hypopharynx (17%).

Most patients had less than three tumor genomic alterations as previously determined [START_REF] Dubot | Comprehensive genomic profiling of head and neck squamous cell carcinoma reveals FGFR1 amplifications and tumour genomic alterations burden as prognostic biomarkers of survival[END_REF].

HPV infection was the only characteristic that significantly impacted disease free interval (DFI) (p=0.032, Log-rank test), with a higher DFI reported for HPV-positive patients.

mRNA expression of immune genes

mRNA expression of the immune-related genes

Among the 30 immune-related genes analyzed, 18 genes were significantly deregulated in HNSCC tumors as compared to normal head and neck tissue (p<0.05), all being overexpressed except ICOSLG and TNFRSF14 (Table 2). Seven genes were overexpressed in more than 50% of tumors (4-1BB, GITR, ICOS, OX40L, CD70, IDO1 and CD80). OX40L had the highest mRNA level with a median of 6.52 fold and was overexpressed in 74% of tumors. Expression of PVRIG was significantly higher in HPV-positive tumors, whereas GITR, NT5E and CD276 expressions were significantly lower in HPV-positive tumors (Table 3). All immune-related genes except ICOSLG showed no modification in the expression profile in HNSCC who harbored oncogenes mutations (NRAS, HRAS, KRAS or PIK3CA) as 10 compared to not mutated. Regarding tumor genomic alterations burden, 20 out of the 30 immune-related genes (67%) were significantly overexpressed in tumors with less than three tumor genomic alterations except for GITR, which showed a lower mRNA level (Table 3).

Unsupervised hierarchical clustering analyses of 96 HNSCC samples with the 30 immunerelated genes showed that the majority of genes coding a receptor protein clustered together as compared to genes coding a ligand protein (Supplementary Figure 1).

mRNA expression of the immune-cell subpopulation genes

Among the 16 immune-cell subpopulation genes analyzed, 7 genes (44%) were significantly deregulated in HNSCC tumors as compared to normal head and neck tissue (p<0.05).

FOXP3 was overexpressed, while only NCAM1 was underexpressed in more than 50% of the tumors as compared to normal head and neck tissue (Table 4).

Expressions of PDGFRB and NCAM1 were significantly different according to HPV status (Table 5). All immune-cell subpopulation genes except PDGFRB and FUT4 were significantly underexpressed in HNSCC with more than three tumor genomic alterations.

A similar unsupervised hierarchical clustering analysis of 96 HNSCC samples with the 16 immune-cell subpopulation genes showed that the lymphocytes specific genes also clustered together (Supplementary Figure 2).

OX40L protein expression using immunochemistry

Using IHC, OX40L protein was expressed in epithelial cancer cells for most tumor samples (H Score: 1-2.5) with a predominantly cytoplasmic location in various populations of the TME, including mononuclear inflammatory cells (MICs), fibroblasts and muscle (Supplementary Figure 3).

Prognostic value of immune gene expression 11

Twelve out of the 30 immune-related genes (40%) were associated with a short DFI in univariate analysis, including four genes with a high mRNA level and eight genes with a low mRNA level (Supplementary Table 3). High OX40L mRNA level (p=0.0009) and low PD-1 mRNA levels (p=0.004) were associated with the highest risk of recurrence (Figure 2).

Eight out of the 16 immune-cell subpopulation genes (50%) were associated with a short DFI, including two genes with a high mRNA level and six genes with a low mRNA level (Supplementary Table 4). High PDGFRB mRNA level (p<0.0001) and low CD3E or CD8A mRNA levels (p=0.0009 and p=0.004, respectively) were associated with the highest risk of recurrence (Figure 3). OX40L, PD-1, PDGFRB, CD3E and CD8A immune genes were significantly associated with a short DFI (OX40L: p=0.005, PD-1: p=0.019, PDGFRB: p=0.0004, CD3E: p=0.011, and CD8A: p=0.016) (Supplementary Table 5) in a multivariate analysis taking into account all clinical parameters associated with a short DFI with a p-value <0.1 (Table 1).

Expression of actionable immune genes

PD-1/PD-L1, TIGIT, OX40/OX40L, and CTLA4 are currently major actionable genes in the context of immunotherapy strategies. Among the 96 tumors analyzed, 82 tumors (85%) overexpressed at least one of these six genes. Thirty three tumors (34%) overexpressed simultaneously TIGIT, CTLA4, PD-1/PD-L1 and OX40/OX40L. No tumor exclusively overexpressed PD-1 or OX40. PD-L1 was exclusively overexpressed in only one sample (1%). TIGIT was exclusively overexpressed in only two samples (2%) and CLTA4 in three samples (3%) whereas OX40L was exclusively overexpressed in 29 samples (30%) (Figure 4A).

Discussion

We assessed the prognostic value of selected immune genes expression in a retrospective analysis of 96 HNSCC patients who underwent primary surgery at Institut Curie. Our results show that most significantly overexpressed genes were 4-1BB (77%), IDO1 (75%), OX40L (74%) and GITR (74%) immune-related genes, and FOXP3 (62%) immune-cell subpopulation genes. Eighty five percent of tumors analyzed overexpressed actionable immunity genes, including PD-1/PD-L1, TIGIT, OX40/OX40L and/or CTLA4. High OX40L mRNA level (p=0.0009) and low PD-1 mRNA level (p=0.004) were associated with the highest risk of recurrence. Patients with high PDGFRB mRNA levels and low CD3E or CD8A mRNA levels also were at the highest risk of recurrence. Overall, around half of immune genes had a deregulated mRNA level in tumor cells as compared to normal head and neck tissue. Tumors with a low number of genomic alterations had higher mRNA levels of immune genes.

More specifically, our results show that among immune-cell subpopulation genes, antigen presenting cells, dendritic cells and B and T cells had a higher mRNA level in tumors whereas natural killer cells specific genes (i.e. NCAM1) were underexpressed. Furthermore we observed a high FOXP3 mRNA level not correlated to recurrence. This suggests a high numbers of T regulatory cells that was also reported in HNSCC patients [START_REF] Badoual | Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF]. Positive associations were observed between OX40L and FOXP3 mRNA levels and between OX40L and CD4 mRNA levels using the Spearman rank correlation test (r=+0.27, p=0.0083 and r=+0.46, p<0.0001 respectively).

OX40L, PD-1, PDGFRB, CD3E and CD8A were associated with a poor prognosis in our study, especially a high mRNA level of OX40L. The prognostic value of OX40L expression is controversial in the literature and depends on the type of cancer [START_REF] Goux | mRNA Expression levels of genes involved in antitumor immunity: Identification of a 3gene signature associated with prognosis of muscle-invasive bladder cancer[END_REF][START_REF] Shibahara | OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy[END_REF]. The overexpression of OX40L was significantly associated with a higher risk of recurrence in bladder cancer [START_REF] Goux | mRNA Expression levels of genes involved in antitumor immunity: Identification of a 3gene signature associated with prognosis of muscle-invasive bladder cancer[END_REF], whereas, it was associated with prolonged progression-free survival in glioblastoma [START_REF] Shibahara | OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy[END_REF].

13

In our HNSCC patients, OX40L was expressed in the microenvironment of the tumor cell notably in fibroblasts at the protein level. In this regard, we observed a marked positive association between OX40L and PDGFRB mRNA levels using the Spearman rank correlation test (r=+0.47 and p<0.0001, data not shown). Similarly the expression of OX40L was detected in a subset of carcinoma associated fibroblasts characterized by an immunosuppressive environment in triple negative breast cancer patients [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF]. No clear cell location of OX40L protein was reported in the literature for HNSCC.

Low mRNA level of PD-1 correlated with a poor prognosis in our series. The absence of PD-L1 mRNA overexpression in circulating tumor cells following anti-PD1 treatment was strongly associated with an objective response in HNSCC patients [START_REF] Strati | Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma[END_REF]. The prognostic value of PD-1 mRNA level was also reported in high grade serous ovarian carcinoma [START_REF] Kunze | Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma[END_REF] and in non-small cell lung cancer [START_REF] Lafuente-Sanchis | Association of PD-1, PD-L1, and CTLA-4 Gene Expression and Clinicopathologic Characteristics in Patients With Non-Small-Cell Lung Cancer[END_REF]. Low mRNA level of PD-L1 had also a poor prognosis although less significant than PD-1 which is consistent to KEYNOTE-048 trial interim findings who reported an improved overall survival and duration of response versus standard therapy in patients with PD-L1-positive recurrent or metastatic HNSCC [START_REF] Burtness | LBA8_PRKEYNOTE-048: Phase III study of first-line pembrolizumab (P) for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC)[END_REF].

Our results also demonstrated that high mRNA level of PDGFRB, a major fibroblast specific gene, was associated with a poor prognosis. Similar results were reported in prostate cancer [START_REF] Singh | Gene expression correlates of clinical prostate cancer behavior[END_REF]. A high PDGFRB protein expression was also reported to correlate with a short survival in renal cell carcinoma [START_REF] Frödin | Perivascular PDGFR-β is an independent marker for prognosis in renal cell carcinoma[END_REF] and pancreatic adenocarcinoma [START_REF] Yuzawa | PDGFRβ expression in tumor stroma of pancreatic adenocarcinoma as a reliable prognostic marker[END_REF] patients.

HNSCC patients with low mRNA levels of CD3E or CD8A had a poor prognosis. Similarly, high CD3 and CD8 mRNA expression was associated with a decreased risk of relapse in early breast cancer patients [START_REF] Tsiatas | Abstract P1-07-03: Evaluation of the prognostic value of CD3, CD8 and FOXP3 mRNA expression in early breast cancer patients treated with anthracycline-based adjuvant chemotherapy[END_REF]. CD8 overexpression correlated with prolonged overall survival and recurrence-free survival in bladder cancer patients [START_REF] Goux | mRNA Expression levels of genes involved in antitumor immunity: Identification of a 3gene signature associated with prognosis of muscle-invasive bladder cancer[END_REF].

Eighty-five percent of HNSCC tumors had at least an actionable immune gene overexpression (PD-1/PD-L1, TIGIT, OX40L/OX40, and CTLA4) in our series. OX40L mRNA level was overexpressed in 74% of tumors and had the worst prognostic value, suggesting that OX40L is a relevant therapeutic target for HNSCC patients.

14

Several clinical trials evaluate OX40L agonists either as single agent or in combination with anti-PD-1/PD-L1 agents (NCT02315066, NCT02923349, NCT02410512, NCT02221960, NCT02705482). A phase 1 trial evaluating an anti-OX40 agonistic monoclonal antibody (9B12) in 30 patients with refractory metastatic solid malignancies showed a favorable safety profile and tumor shrinkage in 12 patients [START_REF] Curti | OX40 is a potent immune-stimulating target in late-stage cancer patients[END_REF].

In addition in our series, thirty four percent of HNSCC tumors co-expressed PD-1/PD-L1, TIGIT, OX40/OX40L, and CTLA4 together suggesting that a combination of immune checkpoint inhibitors may be a relevant therapeutic strategy in HNSCC patients. Several clinical trials are ongoing with other immunotherapy drugs such as GITR (e.g. NCT01239134 or NCT02697591) or 4-1BB (e.g. NCT03364348) generally administered in combination in advanced solid malignancies. 15

Conclusions

OX40L and PDGFRB overexpression was associated with poor outcome in HNSCC patients treated with primary surgery. On the contrary PD-1 overexpression was associated to good prognostic. These results suggest their relevance as potential prognostic biomarkers to be validated in an independent cohort. Immunotherapy was recently demonstrated to improve overall survival in the recurrent and/or metastatic setting of HNSCC patients with pembrolizumab [START_REF] Seiwert | Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial[END_REF] and nivolumab [START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF] that target PD-1. However, only a minority of patients benefit from these agents [START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF][START_REF] Seiwert | Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial[END_REF], highlighting the urgent need to identify other relevant immune targets.

Tables

7

 7

Figure 1

 1 Figure 1 illustrates mRNA expression of the 46 immune genes according to clinical and molecular characteristics of the 96 HNSCC patients.

Figure

  Figure 4B describes the clinical and molecular characteristics of the 82 HNSCC patients with

12

 12

Figure 1 .

 1 Figure 1. Gene expression versus molecular alterations and clinical patient characteristics

Figure 2 .Figure 3 .Figure 4 .Figure 1

 2341 Figure 2. Relationship between disease-free interval and (A) OX40L and (B) PD-1 mRNA

Table 1 .

 1 Clinical, biological and pathological characteristics of the 96 HNSCC patients in relation with disease-free interval (DFI)

					Patients (%)	Events	a (%)	DFI b
	Total				96 (100)	45 (47)
	Age
	<56				46 (48)	20 (44)	0.89 (NS)
	≥56				50 (52)	25 (50)
	Gender
	Female				19 (20)	8 (42)	0.71 (NS)
	Male Alcool c				77 (80)	37 (48)
	Yes				50 (70)	24 (48)	0.17 (NS)
	No Tobacco consumption	d			21 (30)	8 (38)
	Yes				58 (73)	28 (48)	0.075 (NS)
	No				22 (27)	7 (32)
	HPV status
	Negative				84 (87)	42 (50)	0.032
	Positive				12 (13)	3 (25)
	UICC stage
	Stage I				10 (10)	5 (50)	0.68 (NS)
	Stage II				15 (16)	6 (40)
	Stage III				12 (13)	4 (33)
	Stage IV				59 (62)	30 (51)
	Tumor location
	Oral cavity				43 (45)	22 (51)	0.053 (NS)
	Oropharynx				20 (21)	5 (25)
	Larynx				17 (18)	8 (47)
	Hypopharynx Oncogenes' mutational status	e		16 (17)	10 (63)
	Not mutated				78 (81)	36 (46)	0.079 (NS)
	At least one mutated Number of molecular alterations	f	18 (19)	9 (50)
	<3				56 (60)	27 (48)	0.082 (NS)
	≥3				37 (40)	17 (46)
	a Events: locoregional and/or metastatic recurrence, second cancer
	b Log-rank test
	c Alcohol use was considered at 10 gr/day or more (i.e. alcohol unit). Information was available for 71 patients.
	d Tobacco use was considered at 10 pack years or more. Information was available for 80 patients.
	e PIK3CA, NRAS, HRAS or KRAS oncogenes
	f Number of molecular alterations among a selection of 100 genes as previously determined in Dubot et al., 2018. Information
	was available for 93 patients.
	DFI: disease-free interval; NS: not significant; HNSCC: head and neck squamous cell carcinoma; UICC: Union for International
	Cancer Control

Table 2 .

 2 mRNA expression of 30 immune-related genes relative to normal tissue mRNA level

	Gene	Protein	Alias	Head and neck normal tissue a	Head and neck squamous cell carcinomas	b p-value	% under expression	% normal expression	% over expression
				(n=27)	(n=96)
	HLA-DRA	Ligand		1.0 (0.01-5.87)	0.74 (0.01-9.95)	0.91 (NS)	41%	47%	13%
	LAG3	Receptor		1.0 (0.29-4.96)	2.01 (0.2-16.7)	0.001	6%	58%	35%
	PVR	Ligand	CD155	1.0 (0.66-3.93)	1.22 (0.47-4.78)	0.28 (NS)	0%	95%	5%
	PVRIG	Receptor	CD112R	1.0 (0.23-7.45)	1.45 (0.17-8.13)	0.24 (NS)	5%	77%	18%
	TIGIT	Receptor		1.0 (0.04-9.66)	2.77 (0.09-23.4)	<0.0001	2%	51%	47%
	CD96	Receptor		1.0 (0.14-4.74)	1.12 (0.12-7.28)	0.59 (NS)	8%	79%	13%
	CD226	Receptor		1.0 (0.49-4.16)	0.85 (0.08-4.3)	0.05 (NS)	10%	88%	2%
	TNFSF9	Ligand	CD137L, 4-1BBL	1.0 (0.17-5.4)	1.54 (0.14-10.1)	0.011	4%	78%	18%
	TNFRSF9	Receptor	CD137, 4-1BB	1.0 (0.23-8.39)	5.05 (0.61-29.9)	<0.0001	0%	23%	77%
	TNFSF18	Ligand	GITRL	1.0 (0-36.1)	1.81 (0.05-58.4)	0.063 (NS)	9%	55%	35%
	TNFRSF18	Receptor	GITR	1.0 (0.02-4.21)	4.13 (0.57-17.4)	<0.0001	0%	26%	74%
	ICOSLG	Ligand	ICOSL, B7H2	1.0 (0.46-2.52)	0.64 (0.1-7.98)	0.0003	8%	89%	3%
	ICOS	Receptor		1.0 (0.11-6.99)	3.45 (0.27-24.6)	<0.0001	1%	44%	55%
	OX40L	Ligand	TNFSF4, CD134L, CD252	1.0 (0-9.94)	6.52 (0.24-48.0)	<0.0001	1%	25%	74%
	OX40	Receptor	TNFRSF4, CD134	1.0 (0.29-2.73)	2.12 (0.17-13.0)	<0.0001	1%	74%	25%
	CD70	Ligand	TNFSF7	1.0 (0.25-4.32)	4.21 (0.22-92.0)	<0.0001	1%	34%	65%
	CD27	Receptor	TNFRSF7	1.0 (0.05-18.6)	1.92 (0.04-14.3)	0.11 (NS)	7%	58%	34%
	TIM-3	Ligand	HAVCR2	1.0 (0.35-2.03)	1.57 (0.24-8.3)	0.0006	1%	78%	21%
	LGALS9	Receptor	GALECTINE-9	1.0 (0.34-3.02)	1.21 (0.1-10.2)	0.31 (NS)	5%	84%	10%
	PD-L1	Ligand	CD274	1.0 (0.31-2.79)	1.26 (0.16-38.7)	0.056 (NS)	6%	70%	24%
	PD-L2	Ligand	PDCD1LG2	1.0 (0.29-2.75)	1.45 (0.27-13.6)	0.035	4%	77%	19%
	PD-1	Receptor	PDCD1, CD279	1.0 (0.22-8.09)	1.97 (0.11-14.2)	0.019	6%	62%	32%
	ENTPD1	Ligand	CD39	1.0 (0.42-2.48)	1.02 (0.2-3.26)	0.93 (NS)	2%	97%	1%
	IDO1	Ligand		1.0 (0.09-8.68)	6.41 (0.14-184.4)	<0.0001	2%	23%	75%
	NT5E	Ligand	CD73	1.0 (0.32-2.35)	1.03 (0.06-6.35)	0.79 (NS)	6%	82%	12%
	TNFRSF14	Receptor	HVEM	1.0 (0.42-1.6)	0.61 (0.07-1.75)	0.0006	18%	82%	0%
	CD276	Receptor	B7H3	1.0 (0.34-1.79)	2.05 (0.26-7.61)	<0.0001	1%	77%	22%
	CD80	Ligand	B7-1	1.0 (0.16-5.5)	5.35 (0.67-23.0)	<0.0001	0%	20%	80%
	CD86	Ligand	B7-2	1.0 (0.37-2.61)	1.81 (0.27-5.98)	<0.0001	1%	78%	21%
	CD28	Receptor		1.0 (0.31-6.49)	1.32 (0.1-8.16)	0.36 (NS)	5%	79%	16%
	a Median (range) of gene mRNA levels; the mRNA values of the samples were normalized such that the median of the 27 head and neck normal tissues mRNA values was equal to 1
	b Kruskal Wallis's H Test

Table 3 .

 3 mRNA expression of 30 immune-related genes in HNSCC according to HPV status and the number of molecular alterations

	HPV-versus HPV+	Not mutated versus mutated oncogenes	Number of molecular alterations <3 versus ≥3

Table 4 .

 4 mRNA expression of 16 immune-cell subpopulation genes relative to normal tissue mRNA level

	Gene	Cellular specificity	Alias	Head and neck normal tissue a	Head and neck squamous cell carcinoma	b p-value	% under expression	% normal expression	% over expression
				(n=27)	(n=96)
	ITGAX	dendritic cells		1.0 (0.22-3.23) 2.04 (0.52-13.0)	<0.0001	0%	74%	26%
	PDGFRB	fibroblast		1.0 (0.22-5.89)	1.1 (0.17-5.58)	0.87 (NS)	3%	93%	4%
	PTPRC	hematopoietic cells	CD45	1.0 (0.39-6.41)	1.24 (0-6.1)	0.63 (NS)	5%	83%	12%
	MS4A1	LB	CD20	1.0 (0.06-161.9) 1.57 (0.02-51.2)	0.14 (NS)	22%	39%	40%
	CTLA4	LT	CD152	1.0 (0.03-5.05)	3 (0.21-15.1)	<0.0001	1%	49%	50%
	PRF1	LT		1.0 (0.43-3.34) 1.35 (0.12-8.62)	0.13 (NS)	8%	72%	20%
	CD3E	LT		1.0 (0.26-7.59) 1.58 (0.13-11.2)	0.18 (NS)	6%	68%	26%
	CD2	LT		1.0 (0.19-6.73) 1.62 (0.14-13.2)	0.20 (NS)	8%	70%	22%
	FOXP3	LTc		1.0 (0-5.16)	3.98 (0.33-18.2)	<0.0001	0%	39%	62%
	CD8A	LTc		1.0 (0.21-6.54) 1.46 (0.08-17.4)	0.65 (NS)	10%	62%	28%
	CD4	LT helper		1.0 (0.56-2.71) 1.04 (0.11-5.33)	0.62 (NS)	6%	90%	4%
	GZMA	LT/NK		1.0 (0.35-6.15) 2.16 (0.11-18.9)	0.011	8%	55%	37%
	GZMB	LT/NK		1.0 (0.2-18.4)	2.16 (0.09-22.2)	0.0002	2%	57%	41%
	NCAM1	NK	CD56	1.0 (0.1-7.6)	0.15 (0.01-7.56)	<0.0001	66%	27%	7%
	CD14	macrophages/monocytes		1.0 (0.39-3.54) 1.17 (0.17-3.41)	0.19 (NS)	3%	95%	2%
	FUT4	neutrophils	CD15	1.0 (0.35-4.18)	0.7 (0.13-3.37)	0.002	12%	88%	1%
	a Median (range) of gene mRNA levels; the mRNA values of the samples were normalized such that the median of the 27 head and neck normal tissues mRNA values was equal to 1
	b Kruskal Wallis's H Test

LT: T cell; LB: B cell; NK: Natural killer, LTc: Cytotoxic T lymphocyte 22

Table 5 .

 5 mRNA expression of immune-cell subpopulation genes in HNSCC according to HPV status and the number of molecular alterations Median (range) of gene mRNA levels; the mRNA values of the samples were normalized such that the median of the 27 head and neck normal tissues mRNA values was equal to 1 b Kruskal Wallis's H Test

	HPV-verus HPV+	Not mutated versus mutated oncogenes	Total number of alterations
	(84 versus 12)	(78 versus 18)	<3 versus ≥3
			(56 vs 37)

LT: T cell; LB: B cell; NK: Natural killer, LTc: Cytotoxic T lymphocyte

Acknowledgements

We would like to acknowledge the help of the NR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043 and CIC IGR-Curie 1428 (Center Of Clinical Investigation) for their support.

This work was supported by the Fondation ARC pour la recherche sur le cancer (ARC) and ICGEx project ANR-10-EQPX-03 (Equipement de biologie intégrative du cancer pour une médecine personnalisée).

Conflict of interest statement

Pr. Le Tourneau has participated in advisory boards of MSD, BMS, Merck Serono, Astra Zeneca, Novartis, Roche and Nanobiotix.

Supplementary material

Supplementary Figures

Supplementary Figure 1. Heat map of the 30 immune related genes mRNA levels in our series of 96 HNSCC Supplementary Figure 2. Heat map of the 16 immune-cell subpopulation genes mRNA levels in our series of 96 HNSCC Supplementary Figure 3. OX40L protein expression in two HNSCC patients Supplementary Tables Supplementary Table 1. Description of immune-related genes and immune-cell Supplementary Table 2. Oligonucleotide primer sequences used for qRT-PCR genes subpopulation genes Supplementary Table 3. Relationship between mRNA expression of immune-related genes and disease-free interval (DFI) in the 96 HNSCC Supplementary Table 4. Relationship between mRNA expression of immune-cell subpopulation genes and disease-free interval (DFI) in the 96 HNSCC Supplementary Table 5. Multivariate COX analysis of disease-free interval (DFI) in our series