PhD Laure Dossus

Agnès Fournier

Marc J Gunter

Sabina Rinaldi

Anne-Sophie Navionis

Isabelle Romieu

Carine Biessy

Bertrand Hemon

Elisabete Weiderpass

Gianluca Severi

Marie Al Rahmoun

Neil Murphy

Augustin Scalbert

Mathilde His

Michèle Matta

Sally Moldan Thank

Antoine Pariente

Romieu I, Biessy C Navionis As

THIS THESIS HAS BEEN PREPARED IN THE FOLLOWING INSTITUE

Keywords: Breast Cancer, Inflammation, Interleukins, Adipokines, Nonsteroidal Anti-Inflammatory Drugs, Glucocorticoids, Antiplatelet Drugs, Aspirin, Epidemiology. adventure

Chronic inflammation might promote breast cancer development. Therefore, the possibility of stemming tumorigenic inflammatory effects with pharmacological compounds has drawn significant interest.

The objective of my doctoral thesis was to better understand the role of inflammation on breast cancer development.

First, I evaluated the associations of eleven inflammatory biomarkers (cytokines and adipokines) with breast cancer risk in about 1,600 case-control pairs from the EPIC cohort.

The results suggested that a higher leptin-to-adiponectin ratio decreased the risk of peri/premenopausal breast cancer, while high levels of TNF-α increased postmenopausal breast cancer risk.

Then, I evaluated the associations of several anti-inflammatory and antiplatelet drugs with breast cancer risk using self-reported (EPIC cohort) and drug reimbursement (E3N cohort) data. Nonsteroidal anti-inflammatory drugs were not associated with breast cancer risk. However, at antiplatelet dosage, aspirin use was associated with a transient higher breast cancer risk few years after treatment start, followed by a lower breast cancer risk. Another antiplatelet drug, clopidogrel, was associated with a higher breast cancer risk, regardless the duration of use. Glucocorticoid use was associated with a lower risk of invasive breast cancer, that seemed restricted to oestrogen-dependant and stage 1 or 2 tumours and was associated with a higher risk of in situ and stage 3/4 breast cancers.

The findings of this thesis suggest that inflammation plays a modest role in breast cancer development and that its impact on breast cancer could be limited to certain sub-populations or certain breast cancer subtypes.

RESUME

Le cancer du sein pourrait être favorisé par l'inflammation chronique et par conséquent être prévenu par l'utilisation de médicaments agissant sur l'inflammation.

L'objectif de ma thèse était de mieux comprendre le rôle de l'inflammation sur le développement du cancer du sein.

J'ai d'abord évalué les associations entre onze biomarqueurs de l'inflammation (cytokines et adipokines) et le risque de cancer du sein chez environ 1600 paires cas-témoin de la cohorte EPIC. Le ratio leptine/adiponectine était associé à une diminution du risque de cancer du sein chez les femmes avant la ménopause alors que des niveaux élevés de TNF-α étaient associés à un risque accru de cancer du sein chez les femmes ménopausées.

Ensuite, j'ai évalué les associations entre plusieurs médicaments anti-inflammatoires et antiagrégants plaquettaires et le risque de cancer du sein en utilisant des données autorapportées (cohorte EPIC) ou de remboursement de médicaments (cohorte E3N).

Globalement, l'utilisation d'anti-inflammatoires non stéroïdiens n'était pas associée au risque de cancer du sein alors, alors qu'une durée d'utilisation longue d'aspirine, en tant qu'antiagrégant plaquettaire et donc à faible dose (≤325 mg), était associée à une diminution du risque de ce cancer. En revanche, un autre antiagrégant plaquettaire, le clopidogrel, était associé à une augmentation du risque de ce cancer, indépendamment de sa durée d'utilisation.

L'utilisation de glucocorticoïdes était associée à une diminution du risque de cancer du sein infiltrants, dépendants aux oestrogènes et de stades 1 et 2 mais à une augmentation du risque de cancer in situ et de stades 3/4.

Les résultats de cette thèse suggèrent que l'inflammation joue un rôle mineur dans le développement du cancer du sein.

Mots-clés

Cancer du sein, inflammation, interleukines, adipokines, anti-inflammatoires non stéroïdiens, antiagrégants plaquettaires, glucocorticoïdes, épidémiologie.

RESUME SUBSTANTIEL

Au cours des dernières décennies, les résultats des études expérimentales et épidémiologiques ont suggéré que l'inflammation chronique pourrait favoriser le développement du cancer du sein. Ces observations ont conduit à l'hypothèse d'une possible prévention de ce cancer grâce à l'utilisation de médicaments agissant sur l'inflammation tels que les anti-inflammatoires non stéroïdiens (AINS), l'aspirine à faible dose et les glucocorticoïdes. Cependant, les études épidémiologiques sur les biomarqueurs d'inflammation ou sur les médicaments antiinflammatoires et le risque de cancer du sein sont soit rares, soit hétérogènes.

L'objectif de ma thèse était de mieux comprendre le rôle de l'inflammation sur le développement du cancer du sein grâce à deux approches complémentaires :

1) En évaluant les associations entre 11 cytokines et adipokines et le risque de cancer du sein.

2) En évaluant les associations entre plusieurs médicaments anti-inflammatoires et antiagrégants plaquettaires et le risque de cancer du sein, par le biais de données autorapportées ou de remboursement de médicaments.

Différents projets ont été menés dans l'étude prospective européenne sur le cancer et la nutrition (cohorte EPIC), et sa composante française, l'étude épidémiologique auprès de femmes de la mutuelle générale de l'éducation nationale (cohorte E3N).

La cohorte européenne EPIC regroupe plus de 500 000 participants dans 10 pays européens, suivis depuis 1990. Les informations relatives au mode de vie et aux facteurs reproductifs ont été recueillis par auto-questionnaire à l'inclusion. Cinq pays (France, Allemagne, Pays-Bas, Royaume-Uni et Danemark) ont aussi collecté des données auto-rapportées sur l'utilisation régulière d'AINS. La plupart des participants ont également fourni un échantillon sanguin.

L'identification des cas de cancers incidents pendant le suivi repose sur le croisement de registres de cancers, des dossiers d'assurance et hospitaliers, et d'un suivi actif par le contact des participants et de leurs proches.

L'étude E3N s'appuie sur une cohorte de 98 995 femmes volontaires françaises, adhérentes à la Mutuelle Générale de l'Education Nationale (MGEN), suivies depuis 1990. Les informations relatives au mode de vie, aux facteurs reproductifs et aux antécédents médicaux ont été recueillies tous les deux à trois ans par auto-questionnaires. Les cancers auto-déclarés sont validés par obtention des comptes rendus anatomopathologiques. Depuis 2004, les données de remboursement de médicaments issues de la MGEN sont aussi disponibles Dans une première partie, j'ai évalué les associations entre des niveaux circulants de biomarqueurs inflammatoires (cytokines et adipokines) et le risque de cancer du sein, globalement et selon le sous-type tumoral, le statut ménopausique, l'adiposité et l'utilisation d'hormones exogènes chez environ 1 600 paires cas-témoin sélectionnés dans la cohorte EPIC. Les résultats de cette étude ont montré que le ratio leptine/adiponectine étaient associé à une diminution du risque de cancer du sein chez les femmes avant la ménopause alors que des niveaux élevés de TNF-α étaient associés à un risque accru de cancer du sein chez les femmes ménopausées.

Dans une seconde partie, j'ai évalué les associations entre l'utilisation d'AINS et le risque de cancer, globalement et selon le sous-type tumoral, le statut ménopausique et les principaux facteurs de risque de cancer du sein. Durant un suivi moyen de 13 ans, 7 379 cas de cancer du sein incidents ont été diagnostiqués chez les 140 981 participantes incluses (âge moyen au début du suivi : 53 ans). L'utilisation régulière d'AINS n'était pas associée au risque de cancer du sein, globalement et par sous-type de cancer du sein. Cependant, une interaction a été observée entre l'utilisation régulière d'AINS et l'utilisation de traitement hormonaux de la ménopause (THM) chez les femmes ménopausées suggérant une diminution du risque de cancer du sein chez celles ayant déjà utilisées des THMs.

Ensuite, j'ai évalué les associations entre l'utilisation d'AINS, d'aspirine faible dose et d'un autre antiagrégant plaquettaire, le clopidogrel, et de glucocorticoides avec le risque de cancer du sein dans la cohorte E3N. Durant un suivi moyen de 9 ans, 2 887 cas de cancer du sein ont été identifiés chez les 62 512 femmes ménopausées suivies (âge moyen au début du suivi : 63 ans). Globalement, l'utilisation d'AINS n'était pas associée au risque de cancer du sein.

Cependant, l'utilisation d'AINS était associée à une diminution du risque de cancer du sein chez les femmes ayant aussi utilisées des inhibiteurs de la pompe à protons.

Une longue durée d'utilisation d'aspirine à faible dose (≤ 325 mg et donc en tant qu'antiagrégant plaquettaire) était associée à une diminution du risque de cancer du sein tandis qu'un autre antiagrégant plaquettaire, le clopidogrel, était associé à une augmentation du risque de ce cancer, plus particulièrement pour le cancer du sein non-dépendant aux oestrogènes, et ce quelle que soit sa durée d'utilisation. L'utilisation de glucocorticoïdes était associée à une augmentation du risque de cancer du sein in situ et une diminution du risque de cancer infiltrants. Parmi les cancers du sein infiltrants, les glucocorticoïdes étaient associés à une diminution des cancers du sein de stades 1, 2 et dépendants aux oestrogènes, et associés à une augmentation du risque des cancers du sein de stages 3 et 4.

Les résultats de cette thèse suggèrent que :

-L'inflammation joue un rôle mineur dans le développement du cancer du sein.

Cependant, elle pourrait être impliquée dans certains sous-groupes, et plus particulièrement chez les femmes ménopausées.

-Les effets des médicaments antiagrégants plaquettaires/anti-inflammatoires sur le risque de cancer du sein chez les femmes ménopausées semblent complexes et dépendre des sous-types et facteurs de risque de cancer du sein, de la durée d'utilisation ainsi que de l'utilisation d'autres médicaments.

Le rôle de l'inflammation ainsi que celui des médicaments anti-inflammatoires/anti-agrégants plaquettaires sur le développement du cancer du sein doit être confirmé dans des études supplémentaires. Les prochaines études épidémiologiques doivent prendre en compte les sous-types et facteurs de risque de cancer du sein, et pour les médicaments, la dose et la durée d'utilisation ainsi que l'utilisation d'autres médicaments.

to Béatrice Fervers and Florence Menegaux for actively participating to the thesis follow-up committee and giving me precious feedbacks.

To my closest friends, Aricia, Benji, Marina, Ben, Vincent (known as GG), David, Helène, Candice, and Morgane. Because student life was much more fun at your side.

To all members of my family for contributing to my personal growth and for your constant support. I would like to particularly thank my parents, Pierre-Henri, my sisters, and Léo. cancer risk: results from the EPIC study. Annual meeting of the NCI cohort consortium, 28 th -30 th November 2018, Rockville (USA). Presented by Laure Dossus.

Cairat M, Rinaldi S, Navionis AS, Romieu I, Biessy C, Weiderpass E, Gunter M, Dossus L, on behalf of EPIC. Circulating inflammatory biomarkers and breast cancer risk: results from the EPIC study. Forum du Cancéropôle Lyon Auvergne Rhône-Alpes, 4 th -5 th April 2019, Lyon (France).

Cairat M, Al Rahmoun M, Gunter M, Severi G, Dossus L, Fournier A. Utilisation d'aspirine faible dose et risque de cancer du sein dans une cohorte prospective de femmes ménopausées. Colloque AFCROs, 18 th June 2019, Paris (France). This poster was the subject of a despatch by APMnews (https://www.apmnews.com).

Cairat M, Al Rahmoun M, Gunter M, Severi G, Dossus L, Fournier A. Utilisation d'anti-inflammatoires non stéroïdiens et risque de cancer du sein dans une cohorte prospective de femmes ménopausées. Colloque AFCROs, 18 th June 2019, Paris (France). This poster was the subject of a despatch by APMnews (https://www.apmnews.com). Although breast cancer has been intensively studied and many breast cancer risk factors have been identified, there are still more than two million breast cancer cases diagnosed each year worldwide [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians[END_REF]. A better understanding of the disease aetiology as well as new prevention strategies are urgently needed.

LIST OF ABBREVIATIONS

In the past decades, experimental and observational studies have accumulated evidence that chronic inflammation could promote breast cancer development [START_REF] Trinchieri | Cancer and inflammation: an old intuition with rapidly evolving new concepts[END_REF]. The possibility of stemming tumorigenic inflammatory effects with pharmacological compounds has drawn significant interest. Consequently, nonsteroidal anti-inflammatory drugs (NSAIDs), antiplatelet drugs and glucocorticoids are seen as potential candidates of cancer preventive medications [START_REF] Crusz | Inflammation and cancer: advances and new agents[END_REF][START_REF] Vainio | Non-steroidal anti-inflammatory drugs and chemoprevention of cancer[END_REF].

In this first chapter, I will highlight the need for a better understanding of breast cancer development by presenting an overview of its aetiology and epidemiology. In addition, I will describe the potential role of inflammation in breast carcinogenesis and summarize results from epidemiological studies. I will then proceed to explain why NSAIDS, antiplatelet drugs and glucocorticoids are potential cancer preventive medications and highlight the necessity for more epidemiological studies.

Breast cancer 1.The breast: anatomy and functions

The breast is the tissue overlying the chest muscles. Each breast is composed of a mammary gland surrounded by connective, glandular, and adipose tissue. The mammary gland is organized into 15 to 20 sections, called lobes. Within each lobe are smaller structures, called lobules, where milk is produced. The lobes and lobules are linked by thin tubes called ducts.

During lactation, each mammary gland drains milk towards the nipples via the ducts. Lymph nodes, located near the breast in the axilla (armpit), filter the lymphatic fluid (also called lymph) and store lymphocytes. Blood vessels and lymph vessels are present in each breast and respectively drain blood and lymph. The development of mammary glands is influenced from puberty by steroid hormones produced by the ovaries, such as oestrogen and progesterone [START_REF] Hassiotou | Anatomy of the human mammary gland: Current status of knowledge[END_REF].

An overview of carcinogenesis

Cancers are characterized by the uncontrollable growth of abnormal cells which go beyond their usual boundaries to invade adjoining parts of the body and/or spread to other organs.

Carcinogenesis, the transformation of normal cells into cancerous cells, is a multistep process that develops through a latency period of several years before the clinical onset of the disease.

It is the result of an accumulation of detrimental variations in the genome (mutations). These mutations might be inherited/transmitted during cell division, or acquired by random replication errors or exposure to carcinogens (e.g. smoking) [START_REF] Pitot | The molecular biology of carcinogenesis[END_REF]. These successive alterations confer a selective advantage to the affected cells, leading to an intense proliferation. These cancerous cells have some biological properties in common which allow them to evolve, subsist, proliferate, and invade other tissue. These properties, also called the hallmarks of cancer, were proposed by Douglas Hanahan and Robert Weinberg [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF][START_REF] Hanahan | The hallmarks of cancer[END_REF] and are as follow:

sustaining proliferative signalling

Classification according to their invasiveness

When cancer cells stay within their emerging location (e.g. ducts or lobules), the cancer is called in situ. In contrast, when cancer cells infiltrate neighbouring tissue, this is called invasive breast cancer.

Tumour stage

The stage of the cancer is defined by the Tumour, Node, Metastasis (TNM) system established by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) [START_REF] Sobin | International Union Against Cancer (UICC). TNM classification of malignant tumours[END_REF]. The stage is generally determined at the time of diagnosis and describe the extent of the disease. The TNM system is based on the assessment of three components: the tumour size (T), the absence/presence and extent of regional lymph nodes metastasis (N), and the absence/presence of distant metastasis (M). Each of these components are characterized and combined to define the five stages of breast cancer ranging from stage 0 for carcinoma in situ to stage IV for metastatic cancers.

Histological grade

Histological grade, as defined by the Scarff-Bloom-Richardson grading system (SBR), involves the evaluation of three morphological features: the percentage of tubule formations, the change in cell size, and the uniformity and frequency of cell mitosis/division. A score between 1 and 3 is assigned to these three categories. The sum of the three scores defines the SBR grade ranging from 1 to 3.

This grading system represents how closely cancer cells look like healthy breast cells. The higher the grade is, the more the appearance and growth of cancer cells differs from healthy breast cells. Histological grades provide important prognostic information: the lower the grade is, the better the survival [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF][START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF].

Molecular subtypes

Hormones play an important role in breast cancer progression because they influence the cell function and contribute to the proliferation of epithelial tumour cells [START_REF] Key | The role of oestrogens and progestagens in the epidemiology and prevention of breast cancer[END_REF]. Breast cancer can be defined by the presence on cancer cells of receptors for oestrogen (ER) and progesterone (PR).

Breast cancers are classified as oestrogen-receptor positive (ER+) when the proportion of cells expressing ER is above 1%, and progesterone-positive (PR+) when the proportion of cells expressing PR is above 1%. On the contrary, breast cancers are classified as oestrogenreceptor negative (ER-) or progesterone-receptor negative (PR-) when the proportion of cells expressing ER or PR is less than 1% [START_REF] Yip | Estrogen and progesterone receptors in breast cancer[END_REF]. The hormone receptor status on all invasive breast cancers is determined at diagnosis as it is an important indicator for the potential response to endocrine therapy [START_REF] Hammond | American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer[END_REF]. Breast cancer can also be classified by the overexpression of the human epidermal growth factor receptor 2 (HER2), an oncogene promoting cancer growth [START_REF] Gutierrez | HER2: biology, detection, and clinical implications[END_REF]. The HER2 status is determined on all invasive breast cancers as targeted therapy (e.g. trastuzumab) is needed to treat HER2+ tumours [START_REF] Wolff | Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update[END_REF]. Women with ER+, PR+ or HER2+ breast cancers generally have a better survival rate than those with ER-, PR-or HER2-breast cancers [START_REF] Arciero | ER(+)/HER2(+) Breast Cancer Has Different Metastatic Patterns and Better Survival Than ER(-)/HER2(+) Breast Cancer[END_REF][START_REF] Hwang | Impact of Breast Cancer Subtypes on Prognosis of Women with Operable Invasive Breast Cancer: A Population-based Study Using SEER Database. Clinical cancer research : an official journal of the American Association for[END_REF]. 1.4. Descriptive epidemiology 1.4.1. Incidence In both sexes combined, breast cancer is the second most diagnosed cancer, with approximately 2.09 million new cases diagnosed worldwide in 2018 (11.6% of the total cases). Among women, breast cancer is the most diagnosed cancer in the world (accounting for almost 1 in 4 cancer cases). In 2018, the highest incidence rates of breast cancer were observed in developed countries (North American, Australia and Western Europe) (Figure 1). Elevated incidence rates in developed countries are attributed to a higher prevalence of known lifestyle risk factors, which are described in the following part (1.5. Aetiology). In Europe, it is the most diagnosed cancer, with 552,513 new cases diagnosed (12.4% of all cases) in 2018. In France, 56,162 new cases (12.3% of all cases) were diagnosed in 2018 [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians[END_REF][START_REF] Ferlay | Global Cancer Observatory: Cancer Today[END_REF].

Mortality

In both sexes combined, breast cancer is the fifth leading cause of cancer death, with 626,679 deaths by breast cancer (6.6 % of the total cancer deaths) worldwide in 2018 [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians[END_REF]. Among of the total cancer deaths, the highest mortality rates were observed in developing countries compared to developed countries (Figure 2). These variations in mortality rates across countries have been attributed to differences in access to early diagnosis and optimum treatment [START_REF] Allemani | Global surveillance of cancer survival 1995-2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2)[END_REF]. In Europe, breast cancer is the third leading cause of cancer death, with 137,707 deaths by breast cancer (7.1% of the total cancer deaths) in 2018. In France, 13,353 women (7.3% of the total cancer deaths) died of breast cancer in 2018 [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians[END_REF][START_REF] Ferlay | Global Cancer Observatory: Cancer Today[END_REF].

Aetiology

Breast cancer is a multifactorial disease with several well identified risk factors, including genetic, medical, hormonal/reproductive, anthropometric, lifestyle, and environmental factors. IARC and the French national institute on cancer ("Institut National du Cancer", INCa) suggested that alcohol consumption, obesity, low levels of physical activity and, to a lesser extent, smoking and unhealthy diet, were responsible for around 40% of new breast cancer cases in France in 2015 [START_REF] Soerjomataram | Cancers related to lifestyle and environmental factors in France in 2015[END_REF]. However, the aetiology of breast cancer remains in part unknown, and among all the identified risk factors, few of them might be modifiable. Below, I describe the different risk factors of breast cancer.

Age and sex

Sex is the major risk factor for breast cancer. Breast cancer affects both men and women.

However, the incidence is much higher in women and breast cancer in men represents less than 1% of all breast cancers worldwide [START_REF] Korde | Multidisciplinary meeting on male breast cancer: summary and research recommendations[END_REF]. Breast cancer incidence rates increase with age, exponentially before menopause and slowly after. Around 50% of breast cancers are diagnosed between 50 and 69 years old and around 28% after 69 years old [START_REF] Fidler | Cancer incidence and mortality among young adults aged 20-39 years worldwide in 2012: a population-based study[END_REF].

Genetic/familial factors

About 5% to 10% of breast cancers are thought to be hereditary [START_REF] Kwong | Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries[END_REF]. A woman's risk for breast cancer is around 80% higher if she has a first-degree relative who had breast cancer compared to women without family history [START_REF]Familial breast cancer: collaborative reanalysis of individual data from 52 epidemiological studies including 58,209 women with breast cancer and 101,986 women without the disease[END_REF]. The risk goes up if more family members are affected and varies according to their age at diagnosis. This increased risk from family history of breast cancer might be explained in part by genetic variants which have been previously identified such as mutations in genes BRCA1 and BRCA2 [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF]. However, these mutations would contribute to only 25% of familial breast cancers and other genes might be involved such as PTEN or TP53 [START_REF] Mavaddat | Genetic susceptibility to breast cancer[END_REF]. A large number of low-penetrance risk alleles identified in Genome-Wide Association Studies may also explain part of the breast cancer heritability [START_REF] Skol | The genetics of breast cancer risk in the post-genome era: thoughts on study design to move past BRCA and towards clinical relevance[END_REF].

Medical factors

Proliferative benign breast diseases, with or without atypia, are associated with a significant increase in the risk of developing breast cancer. Compared to women without proliferative benign breast diseases, breast cancer risk increases by 58% for women with proliferative benign breast diseases without atypia, and is multiplied by four for women with proliferative benign breast diseases with atypia [START_REF] Salamat | Subtypes of Benign Breast Disease as a Risk Factor of Breast Cancer: A Systematic Review and Meta Analyses[END_REF].

Breast density, which reflects the proportion of fibrous and glandular tissues in the breast, is a strong risk factor for breast cancer [START_REF] Mccormack | Breast density and parenchymal patterns as markers of breast cancer risk: a meta-analysis. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Boyd | Mammographic density and the risk and detection of breast cancer[END_REF]. Women with a breast density ≥ 75% have their breast cancer risk multiplied by five compared to women with a breast density <5% [START_REF] Mccormack | Breast density and parenchymal patterns as markers of breast cancer risk: a meta-analysis. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF].

Hormonal and reproductive factors

Breast cancer is a hormone-dependant cancer, which appears only after puberty among women. There is strong evidence that sex steroid hormones, in particular oestrogens, both endogenous and exogenous, are involved in breast cancer development [START_REF] Key | The role of oestrogens and progestagens in the epidemiology and prevention of breast cancer[END_REF]. A variety of other hormonal and reproductive factors, which increase lifetime exposure to oestrogen and progesterone, are identified as breast cancer risk factors. Overall, these factors are supposed to have a stronger effect on ER+ or PR+ breast cancers compared to ER-or PR-breast cancers [START_REF] Althuis | Etiology of hormone receptor-defined breast cancer: a systematic review of the literature. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF].

Menstrual factors including early age at menarche and late menopause expose women to a longer time of ovulatory menstrual cycles and consequently hormones, raising their risk of getting breast cancer. Breast cancer risk increases by 5% for every year younger at menarche, and independently by 3% for every year older at menopause [START_REF] Menarche | menopause, and breast cancer risk: individual participant meta-analysis, including 118 964 women with breast cancer from 117 epidemiological studies[END_REF].

Additionally, pregnancy-related factors, including age at first full-term pregnancy, number of children and breastfeeding, are also known to influence breast cancer risk. Just after delivery, parous women have an elevated breast cancer risk that peaks around five years after childbirth and persists for more than twenty years. After, this risk decreases until conferring a protective effect 34 years after childbirth [START_REF] Nichols | Breast Cancer Risk After Recent Childbirth: A Pooled Analysis of 15 Prospective Studies[END_REF]. After this long period, breast cancer decreases by 3% for every year younger at age at first full term pregnancy and by 7% for each birth [START_REF] Nichols | Breast Cancer Risk After Recent Childbirth: A Pooled Analysis of 15 Prospective Studies[END_REF]. Pregnancies that end as a spontaneous or induced abortion do not increase a woman's risk of developing breast cancer [START_REF] Beral | Breast cancer and abortion: collaborative reanalysis of data from 53 epidemiological studies, including 83,000 women with breast cancer from 16 countries[END_REF]. Breastfeeding is an established protective factor of breast cancer among pre and postmenopausal women, with breast cancer risk decreasing by 4% for each year of breastfeeding, independently of the number of births [START_REF]Breast cancer and breastfeeding: collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries, including 50302 women with breast cancer and 96973 women without the disease[END_REF].

In 1999, IARC concluded in a monograph that there was sufficient evidence in humans for the carcinogenicity of oral contraceptives containing oestrogen in combination with a progestogen [START_REF]evaluation of carcinogenic risks to humans :combined estrogen-progestogen contraceptives and combined estrogen-progestogen menopausal therapy. A review of human carcinogens[END_REF]. Compared to never users, breast cancer risk increased by 24% for current oral contraceptive users. However, the effect of oral contraceptives on the risk of breast cancer decreases gradually after cessation of use, returning to that of never-users within 10 years after cessation [START_REF]Breast cancer and hormonal contraceptives: collaborative reanalysis of individual data on 53 297 women with breast cancer and 100 239 women without breast cancer from 54 epidemiological studies[END_REF]. Use of menopausal hormone therapies (MHT) has been convincingly shown as a risk factor of postmenopausal breast cancer [START_REF]Type and timing of menopausal hormone therapy and breast cancer risk: individual participant meta-analysis of the worldwide epidemiological evidence[END_REF]. Every MHT type, except vaginal oestrogens, is associated with excess breast cancer risk, even though the increased risk is stronger for oestrogen-progestogen than oestrogen-only preparations. The risk increases substantially with duration of use. In addition, other exogenous hormones such as diethylstilboestrol, a synthetic oestrogen used by pregnant women to prevent miscarriage from 1940 to 1970, are known to increase breast cancer risk among users and their offspring [START_REF]on the Evaluation of Carcinogenic Risks to Humans. A review of human carcinogens[END_REF].

Body fatness and physical activity

The effect of body fatness on breast cancer development differs according to menopausal status. In 2017, the World Cancer Research Fund International (WCRF), which updated its previous analysis of all published prospective studies on diet, nutrition and physical activity and the risk of developing breast cancer [START_REF]Diet, Nutrition, Physical activity an Breast Cancer[END_REF], concluded that there was strong evidence that:

-being overweight or obese in young adulthood (between the ages of about 18 and 30 years) or in adulthood before the menopause decreases the risk of premenopausal breast cancer -being overweight or obese in young adulthood decreases the risk of postmenopausal breast cancer while being overweight/obese or gaining weight throughout adulthood increases the risk of postmenopausal breast cancer -being physically active decreases the risk of premenopausal (only vigorous activity) and postmenopausal breast cancer (any activity including vigorous, occupational, recreational, walking and household activity) -being tall increases the risk of premenopausal and postmenopausal breast cancer.

Alcohol consumption and tobacco

The WCRF concluded that the consumption of alcoholic drinks is probably a cause of premenopausal breast cancer and a convincing cause of postmenopausal breast cancer [START_REF]Diet, Nutrition, Physical activity an Breast Cancer[END_REF].

In their meta-analyses, the risk of premenopausal breast cancer increases by 5% per 10 grams of alcohol per day while the risk of postmenopausal breast cancer increases by 9% per 10 grams of alcohol per day.

Hundreds of epidemiological studies have been published on the relationship between breast cancer and active or passive smoking. However, there is still no consensus on whether tobacco affects breast cancer development. In a monograph of 2004, IARC concluded that there was plausible biological evidence on the detrimental effect of tobacco smoke on breast cancer development. However, there was no association between active/passive tobacco smoking and breast cancer incidence in epidemiological studies [START_REF]IARC monographs on the evaluation of carcinogenic risks to humans[END_REF]. Since then, new epidemiological studies have consistently observed a positive association between current smoking and breast cancer risk, with relative risk estimates ranging from 1.10 to 1.30 [START_REF]A review of human carcinogens. Part E: Personal habits and indoor combustions / IARC Working Group on the Evaluation of Carcinogenic Risks to Humans[END_REF].

Diet

Currently, no specific dietary habits are identified as risk factors of breast cancer. However, in 2017, the WCRF suggested that there was some evidence that:

-consuming foods containing carotenoids and diets high in calcium might decrease the risk of breast cancer, -non-starchy vegetables might decrease the risk of ER-breast cancer, -the consumption of dairy products might decrease the risk of breast cancer among premenopausal women.

The potential influence of a specific dietary pattern (e.g. Mediterranean diet) on breast cancer development is unclear [START_REF]Diet, Nutrition, Physical activity an Breast Cancer[END_REF].

Environmental and occupational factors

Few environmental and occupational exposure are identified as breast cancer risk factors except for ionising radiation from medical treatments such as X-ray, which increases the risk of breast cancer particularly when the patient is exposed during puberty [START_REF] Ronckers | Radiation and breast cancer: a review of current evidence[END_REF]. In a monograph published in 2020 [START_REF] Iarc | Night shift work[END_REF], IARC concluded that there was limited evidence in humans for the breast carcinogenicity of night shift work.

Take-home messages

Breast cancer is the most diagnosed cancer and the most common cause of cancer death among women in the world is a heterogeneous and multifactorial disease, for which many risk factors are identified, such as age, height, ionizing radiation, genetic factors, a wide list of reproductive/hormonal factors, obesity, or alcohol consumption. However, only a few of these risk factors are modifiable.

A better understanding of the aetiology of breast cancer as well as new strategies to prevent breast cancer occurrence are urgently needed to strengthen primary prevention of this disease.

Inflammation

Principles and functions

Inflammation is a component of a complex biological response by the immune system to stimulants (e.g. microbial infection or tissue injury) [START_REF] Ahmed | An overview of inflammation: Mechanism and consequences[END_REF]. Acute inflammation is well-known to restore cellular homeostasis after any detrimental disorder. The classical symptoms of acute inflammation include redness, pain, swelling and heat.

The major immune cells contributing to inflammation are antigen-presenting cells (macrophages and dendritic cells), leukocytes including granulocytes (neutrophils, eosinophils, and basophils), mast cells, and lymphocytes including natural killer (NK) cells.

Non-immune cells such as platelets, epithelial cells, endothelial cells, and fibroblasts also participate to inflammatory processes. A wide range of inflammatory mediators are released by these cells such as leukotrienes, vasoactive amines, peptides, eicosanoids, proinflammatory cytokines, and acute-phase proteins.

The inflammatory response depends on the nature of stimulants and on the level of damage.

For instance, a bacterial infection induces the production of inflammatory mediators such as inflammatory cytokines [e.g. tumour necrosis factor (TNF), interleukin-1 (IL-1) and IL -6] and chemokines. In most cases, this inflammatory response extends toward systemic effects through the secretion of acute phase proteins [e.g. c-reactive protein (CRP) and coagulation factors] by the liver cells [START_REF] Ahmed | An overview of inflammation: Mechanism and consequences[END_REF]. TNF-α and IL-1β also induce the translocation of the cellular transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to the nucleus where it activates genes for immune and inflammatory response [START_REF] Tergaonkar | NFkappaB pathway: a good signaling paradigm and therapeutic target[END_REF]. NF-κB activation leads to cyclooxygenase-2 (COX-2) expression and inflammatory cytokine release (e.g. IL-6). COX-2 induces the biosynthesis of prostaglandins (PGs) and other eicosanoids (Figure 3) [START_REF] Gomes | Eicosanoids and cancer[END_REF]. On the other hand, a viral infection leads to a distinct signalling pathway through the production of another class of cytokines such as type-1 interferons (IFNs) and involves cytotoxic lymphocytes. A parasitic infection as well as allergens induce the production of IL-4, IL-5, IL-13, and histamine.

Once the potential danger is eradicated, the inflammatory response is immediately followed by the production of anti-inflammatory mediators such as IL-10, TGF-β, or endogenous cortisol and therefore inflammation is terminated. The role of each mediator on the resolution of inflammation is not completely known.

Chronic inflammation

If inflammation fails to end, the acute inflammation turns into a chronic stage. For instance, it is recognized that inflammation might persist in the case of chronic infection (e.g.

Helicobacter Pylori or sexually transmitted infections), auto-immune diseases, or among overweight or obese people [START_REF] Engin | Adipocyte-Macrophage Cross-Talk in Obesity[END_REF][START_REF] Fain | Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells[END_REF][START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance[END_REF][START_REF] Kershaw | Adipose tissue as an endocrine organ[END_REF][START_REF] Schaffler | Role of adipose tissue as an inflammatory organ in human diseases[END_REF][START_REF] Galic | Adipose tissue as an endocrine organ[END_REF]. However, chronic inflammation is not well understood, and other triggers remain to be identified. Contrary to acute inflammation, chronic inflammation might be deleterious and is involved in many diseases such as atherosclerosis, obesity, type 2 diabetes, asthma, in inflammatory bowel diseases, neurodegenerative diseases, rheumatoid arthritis and cancers [START_REF] Ahmed | An overview of inflammation: Mechanism and consequences[END_REF][START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF].

Inflammation and carcinogenesis

Since the discovery, by Rudolf Virchow in 1863, of the infiltration of leukocytes into tumour tissue, many experiments have confirmed the role of the immune system and inflammation in tumour proliferation, immunosuppression, angiogenesis, invasion, and metastasis [START_REF] Piotrowski | Reports of practical oncology and radiotherapy[END_REF]. In 2011, tumour-promoting inflammation and avoiding immune destruction were identified by Hanahan and Weinberg as hallmarks of cancer [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. Indeed, the tumour microenvironment contains many different inflammatory mediators including pro-inflammatory cytokines, chemokines, and eicosanoids [such as prostaglandins (PGs), thromboxane (TX), and leukotrienes (LTs)] that might generate an immunosuppressive microenvironment [START_REF] Wang | Immunosuppression associated with chronic inflammation in the tumor microenvironment[END_REF].

Inflammation has been hypothetized to recruit and change the function of various immune cells [START_REF] Wang | Immunosuppression associated with chronic inflammation in the tumor microenvironment[END_REF] and to supply bioactive molecules to the tumor microenvironment including growth factors (sustaining proliferative signaling), survival factors (regulating cell death), proangiogenic factors and extracellular matrix-modifying enzymes (accelerating angiogenesis, invasion, and metastasis), signals that initate the epithelial-to-mesenchymal transition [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF], and reactive oxygen/nitrogen species (promoting the evolution of transformed/initiated cells) [START_REF] Trinchieri | Cancer and inflammation: an old intuition with rapidly evolving new concepts[END_REF].

Inflammation might be particularly relevant for breast cancer because cytokines that are elevated in the event of chronic inflammation, such as IL-6 and TNF-α, have been shown to upregulate angiogenesis and to stimulate aromatase activity within breast tissues (leading to oestrogen synthesis) [START_REF] Hong | Interleukin-6 and its receptor in cancer: implications for Translational Therapeutics[END_REF][START_REF] Knupfer | Significance of interleukin-6 (IL-6) in breast cancer (review)[END_REF][START_REF] Zhao | Tumor necrosis factoralpha stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter 1.4[END_REF]. Experimental data suggest that TNF-α could be involved in carcinogenesis by activating NF-қB and consequently inducing the expression of genes associated with cell proliferation, apoptosis, angiogenesis, and metastasis [START_REF]NF-kappaB and cancer: mechanisms and targets[END_REF]. TNF-α has also been shown to induce insulin resistance [START_REF] Mraz | The role of adipose tissue immune cells in obesity and low-grade inflammation[END_REF], which is potentially involved in breast cancer development [START_REF] Hernandez | Association between insulin resistance and breast carcinoma: a systematic review and meta-analysis[END_REF]. Also, leptin, an adipokine (a hormone produced mainly by adipocytes) could promote breast cancer by stimulating the conversion of androgen into oestradiol or inducing a metabolic dysregulation leading to high levels of insulin and IGF-1 [START_REF] Jarde | Molecular mechanisms of leptin and adiponectin in breast cancer[END_REF], which are hypothesized to increase breast cancer risk development [START_REF] Hernandez | Association between insulin resistance and breast carcinoma: a systematic review and meta-analysis[END_REF][START_REF] Key | Insulin-like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: pooled individual data analysis of 17 prospective studies[END_REF]. On the contrary, adiponectin, an adipokine with anti-inflammatory properties, might prevent cancer development by inhibiting angiogenesis and promoting apoptosis [START_REF] Sugiyama | Adiponectin inhibits colorectal cancer cell growth through the AMPK/mTOR pathway[END_REF][START_REF] Bråkenhielm | Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis[END_REF]. The adiponectin to leptin ratio has been shown to decrease proliferation of ER+ breast cancer cell lines and increase proliferation of ER-breast cancer cells lines (70).

Inflammation and breast cancer risk: up-to-date epidemiological evidence

Prospective studies have shown that not only local inflammation but also systemic inflammation could be associated with carcinogenesis. CRP is the most studied inflammatory biomarker within the framework of breast cancer risk. In a meta-analysis of twelve prospective studies conducted mainly on postmenopausal women (published in 2015), a doubling of serum concentrations of CRP was associated with a significant 7% increase in breast cancer risk [START_REF] Chan | Circulating C-Reactive Protein and Breast Cancer Risk-Systematic Literature Review and Meta-analysis of Prospective Cohort Studies. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. Since then, five prospective studies reported the CRP-breast cancer associations mainly among postmenopausal women [START_REF] Wang | Plasma C-Reactive Protein and Risk of Breast Cancer in Two Prospective Studies and a Metaanalysis[END_REF][START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Gunter | Circulating Adipokines and Inflammatory Markers and Postmenopausal Breast Cancer Risk[END_REF][START_REF] Dossus | C-reactive protein and postmenopausal breast cancer risk: results from the E3N cohort study[END_REF]. Among them, two studies in a joint publication noted no association between CRP and breast cancer risk [START_REF] Wang | Plasma C-Reactive Protein and Risk of Breast Cancer in Two Prospective Studies and a Metaanalysis[END_REF], while others confirmed the positive association between CRP and breast cancer risk [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF], or suggested that the association was confined to non-MHT users [START_REF] Gunter | Circulating Adipokines and Inflammatory Markers and Postmenopausal Breast Cancer Risk[END_REF] or overweight women [START_REF] Dossus | C-reactive protein and postmenopausal breast cancer risk: results from the E3N cohort study[END_REF]. One prospective study reported no association between CRP and breast cancer risk among premenopausal women [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF]. The CRP-breast cancer associations warrant further investigation since most epidemiological studies were not able to adjust/stratify by breast cancer subtypes, menopausal status, or risk factors such as adiposity or use of exogenous hormones.

With respect to adipokines, a meta-analysis of six prospective and nine case-control studies on adiponectin and breast cancer risk (published in 2014) concluded that low levels of adiponectin were positively associated with risk of postmenopausal breast cancer [START_REF] Macis | Circulating adiponectin and breast cancer risk: a systematic review and meta-analysis[END_REF].

However, among the studies in which blood samples were taken before breast cancer diagnosis, none of them found a significant association between pre-diagnostic levels of adiponectin and postmenopausal breast cancer risk [START_REF] Tworoger | Plasma adiponectin concentrations and risk of incident breast cancer[END_REF][START_REF] Gaudet | Do adipokines underlie the association between known risk factors and breast cancer among a cohort of United States women?[END_REF][START_REF] Touvier | Association between prediagnostic biomarkers of inflammation and endothelial function and cancer risk: a nested case-control study[END_REF][START_REF] Cust | The influence of overweight and insulin resistance on breast cancer risk and tumour stage at diagnosis: a prospective study[END_REF]. Elevated leptin levels have been observed in serum of breast cancer patients compared to control subjects [START_REF] Pan | Association between serum leptin levels and breast cancer risk: An updated systematic review and meta-analysis[END_REF]. However, few prospective studies evaluated the associations between pre-diagnostic levels of leptin and breast cancer risk [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Gunter | Circulating Adipokines and Inflammatory Markers and Postmenopausal Breast Cancer Risk[END_REF][START_REF] Gross | Adipocytokines, inflammation, and breast cancer risk in postmenopausal women: a prospective study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Harris | Plasma leptin levels and risk of breast cancer in premenopausal women[END_REF]. Among them, one study noted a significant lower premenopausal breast cancer risk with high levels of leptin [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF], while other suggested no significant association between levels of leptin and breast cancer risk among premenopausal [START_REF] Harris | Plasma leptin levels and risk of breast cancer in premenopausal women[END_REF] or postmenopausal women [START_REF] Gunter | Circulating Adipokines and Inflammatory Markers and Postmenopausal Breast Cancer Risk[END_REF][START_REF] Gross | Adipocytokines, inflammation, and breast cancer risk in postmenopausal women: a prospective study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. The leptin to adiponectin ratio, which is a marker of insulin resistance [START_REF] Finucane | Correlation of the leptin:adiponectin ratio with measures of insulin resistance in non-diabetic individuals[END_REF], has been positively associated with postmenopausal breast cancer diagnosis [START_REF] Korner | Total and high-molecular-weight adiponectin in breast cancer: in vitro and in vivo studies[END_REF][START_REF] Chen | Serum adiponectin and leptin levels in Taiwanese breast cancer patients[END_REF], but no study evaluated it prospectively. The associations between adipokines and breast cancer risk, overall and by breast cancer subtypes or risk factors need to be evaluate in prospective studies.

TNF-α levels were not associated with breast cancer risk in several prospective studies [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Gross | Adipocytokines, inflammation, and breast cancer risk in postmenopausal women: a prospective study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Heikkila | Associations of circulating C-reactive protein and interleukin-6 with cancer risk: findings from two prospective cohorts and a meta-analysis[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Krajcik | No association between serum levels of tumor necrosis factor-alpha (TNF-alpha) or the soluble receptors sTNFR1 and sTNFR2 and breast cancer risk. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. One prospective study found a lower breast cancer risk with high levels of TNF-α [START_REF] Dias | Low-Grade Inflammation, Oxidative Stress and Risk of Invasive Post-Menopausal Breast Cancer -A Nested Case-Control Study from the Malmo Diet and Cancer Cohort[END_REF].

To my knowledge, very few prospective studies have investigated the role of other proinflammatory cytokines on breast cancer development. It is also unknown whether chronic inflammation in general is relevant to breast cancer risk, or whether specific pathways are involved.

Take-home messages

Inflammation is an essential protective part of the immune system's response to infection or injuries.

However, when it persists over time, it might become deleterious. is a recognized hallmark of cancer could be involved in breast cancer development. However, epidemiological findings on inflammatory biomarkers and breast cancer risk are limited.

Further well-designed prospective studies should evaluate the associations between inflammatory biomarkers and breast cancer risk by menopausal status and breast

cancer subtypes.

Nonsteroidal anti-inflammatory drugs

Definition and classification

Acetylsalicylic acid is the first NSAID manufactured following the extraction of its ancestor, salicilin, from bark of willow trees. Acetylsalicylic acid was introduced into the market under the commercial name Aspirin® in 1899 [START_REF] Montinari | The first 3500 years of aspirin history from its roots -A concise summary[END_REF]. Indomethacin and ibuprofen were among the first non-aspirin NSAIDs to be commercialized, respectively in 1964 and 1965 (93). Since then, a large number of other NSAIDs, with antipyretic, analgesic and anti-inflammatory properties, have been developed (Annex 1). In France, most NSAIDs are only available through prescriptions, except for aspirin, ibuprofen, throat pastilles of flurbiprofen and dermal diclofenac, which are also available over the counter (OTC) [START_REF] Ameli | le site de l'Assurance Maladie en ligne. Bien utiliser les anti-inflammatoires non stéroïdiens (AINS)[END_REF]. The society which maintains and develops the Anatomical, therapeutic, chemical (ATC) classification system, the WHO Collaborating Centre for Drug Statistics Methodology (https://www.whocc.no/), classifies preparations referring to aspirin (intended for analgesic or anti-inflammatory use)

into "Salicylic Acid and Derivatives" (ATC code: N02BA) and preparations referring to other NSAIDs into "Anti-Inflammatory and Anti-Rheumatic Products, Non-Steroids" (M01A). Of note, aspirin at low dose (≤325 mg per pill) is not a NSAID (4. Antiplatelet drugs).

Pharmacological properties and therapeutic use

Pharmacological actions

The mechanism of action of NSAIDs was first described during the early 1970's by John Vane. He demonstrated that NSAIDs disrupt the biosynthesis of PGs, which are responsible for pain, inflammation and fever (Figure 3) [START_REF] Vane | Inhibition of prostaglandin synthesis as a mechanism of action for aspirinlike drugs[END_REF][START_REF] Flower | Inhibition of prostaglandin biosynthesis[END_REF]. It was initially believed that a single COX was responsible for the biosynthesis of PGs and TxA2 [START_REF] Hemler | Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme[END_REF]. After the identification of COX-1 (in 1976) and COX-2 (in 1991), NSAIDs appeared to cause adverse gastro-intestinal toxicities and bleeding via COX-1 inhibition [START_REF] Vane | Cyclooxygenases 1 and 2[END_REF]. Soon after, NSAIDs selectively inhibiting the COX-2 (known as selective COX-2 inhibitors or coxibs) were developed in the hope for a reduction of gastrointestinal toxicities and an enhancement of anti-inflammatory properties [START_REF] Vane | Mechanism of action of nonsteroidal anti-inflammatory drugs[END_REF]. However, emerging concerns about cardiovascular toxicities of these new NSAIDs resulted in the withdrawal of some of them in the early 2000s (100). It is now well understood that NSAIDs block the synthesis of PGs and TxA2 by irreversible acetylation of a serine in both COXs, limiting the access of arachidonic acid to the catalytic active site of the enzymes (Figure 3). NSAIDs inhibit both COXs to a different extent leading to different benefits and harms (101). All NSAIDs reversibly inhibit the COXs, except aspirin which permanently inhibits them (102).

Indications

NSAIDs can be used for the temporary relief of minor aches and pains (for instance due to headache, toothache, backache, muscle pain, or menstrual cramps) and to reduce fever.

Prescription-strength NSAID therapy is generally used to relieve chronic musculoskeletal pain and inflammation in conditions such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, or other rheumatologic conditions. Prescription-strength NSAIDs can also be used for the temporary relief of post-surgical pain or traumatic injury (103).

Main side effects

NSAIDs are associated with a wide well-recognized range of side effects including nervous system disorders (e.g. amnesia, seizure, or insomnia), gastrointestinal troubles (e.g. gastrointestinal bleeding, gastritis, or ulcers), liver and bilious disorders (e.g. hepatitis or liver failure), cardiovascular troubles (myocardial infarction, stroke, or hypertension), kidney problems (e.g. renal failure), respiratory problems (e.g. dyspnoea), blood disorders (e.g. haemorrhage), or allergies (e.g. pruritus or dermatitis) (103, 104). The side effects vary according to the molecule, dose, and duration of use. In response to a wide variety of stimuli, arachidonic acid, an unsaturated fatty acid embedded in cell membranes, is released into the cells by the phospholipase A2 (PLA2). This free arachidonic acid is subsequently converted via COX-1 or COX-2, lipoxygenases (LOXs) and P450 monooxygenase pathways into eicosanoids. COX-1 is constitutively expressed in many human organs (e.g. blood vessels, lung, kidneys, stomach, or colon) and tissues (e.g. smooth muscle cells, endothelial cells, or platelets). COX-1 induce the biosynthesis of prostaglandins (PGs) mainly involved in the maintenance of normal renal function in the kidneys (PGE2, PGI2), mucosal protection in the gastrointestinal tract (PGE2), vasodilatation (PGI2), vasoconstriction and induction of platelet aggregation (Thromboxane A2, Tx). COX-2 is constitutively found in kidneys, brain, endometrium, and ovary. However, when induced by proinflammatory cytokines or growth factors, COX-2 might be expressed in different cells including fibroblasts, endothelial cells, or inflammatory cells such as macrophages. COX-2 promote the biosynthesis of PGs, especially PGE2 which is particularly involved in inflammation. Different LOXs were identified including 5-LOX, 12-LOX, or 15-LOX. LOXs are expressed in leucocytes and are involved in the biosynthesis of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). 5-LOX is generally absent in normal epithelia but is induced by proinflammatory stimuli. P450 metabolizes arachidonic acid into epoxyeicosatrienoic acids (EETs), HETEs and hydroperoxyeicosatetraenoic acids (HPETEs). Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX-1 and COX2. Low-dose aspirin inhibits COX-1. Glucococorticoids inhibit PLA2. In France, the concomitant use of proton pump inhibitors (PPI) is highly recommended to prevent or treat NSAID-induced gastrointestinal adverse events among people:

-Older than 65 years old,

-Or with a history of peptic ulcer disease, -Or concomitantly using antiplatelet drugs, anticoagulants, or corticoids.

However, a French study published in 2020 reported that 80% of co-prescriptions of PPI with NSAIDs were not in line with these guidelines (106).

Antitumor effects

Since Bennet and Del Tacca found in 1975 that PGE 2 were overexpressed in human colonic cancer tissues, compared to normal tissues, high PG and Tx levels have been also found in several human malignancies, such as colon, lung, breast, head and neck cancers [START_REF] Gomes | Eicosanoids and cancer[END_REF](107)(108)(109). These discoveries gave rise to the hypothesis that certain PGs and Tx were involved in the growth and spread of cancer cells and that NSAIDs, by inhibiting PG synthesis through COX-1 and COX-2 inhibition, could prevent or inhibit tumour growth (110,111).

This hypothesis has been supported by the fact that abnormal levels of COX- interacting directly with leukocytes and affecting the immune system, and suppresses tumour growth and migration (182, 183). However, the mechanism of action of low-dose aspirin in the prevention of cancer has not been elucidated: both COX-dependent and COX-independent pathways could be involved. Aspirin has been hypothesized to decrease breast cancer by reducing eicosanoid biosynthesis/release through the inhibition of COX-1 (Figure 3). This hypothesis is supported by the fact that COX-1 and TXA 2 were found to be overexpressed in several cancers including breast cancers (108,109,(117)(118)(119)184). In addition, the inhibition of the TxA 2 biosynthesis was shown to suppress mammary tumorigenesis or breast cancer metastasis in animal models (107).

-
Aspirin could regulate other pathophysiological events in breast carcinogenesis, such as reprogramming the mesenchymal to epithelial transition (183, 185), inhibiting the Wnt/bcatenin and NF-kB signalling and the acetylation of extra-COX proteins (186).

There is also evidence that thienopyridines might have anti-cancer properties (187).

Ticagrelor was found to inhibit platelet-tumour cell interactions and metastasis in human and murine breast cancer (188), and to inhibit lung, liver, and bonne marrow cancer metastasis in mouse models (189). The inhibition by cangrelor (the in vitro equivalent of clopidogrel) of platelet secretion of vascular endothelial growth factor, thrombospondin, and transforming growth factor-β1 was stronger in breast cancer patients compared to healthy patients (190). In mice models, clopidogrel prevented the binding of cancer cell-derived microparticles to fibrinogen-platelets aggregates at the site of thrombosis, thereby suppressing the development of the tumour and reducing metastasis (191).

Breast cancer prevention: up-to-date epidemiological evidence

Low-dose aspirin has been intensively evaluated as preventive cancer medication (192)(193)(194)(195)(196) and consistently associated with a lower colorectal cancer incidence in observational studies and randomized controlled trials (196,197) 200). In the ASPREE trial, low-dose aspirin was associated with an increased risk of any solid cancer at stage 4. However, the authors did not present the associations between low-dose aspirin and stages of breast cancer (200). A meta-analysis of 22 cohort studies and 16 case-control studies (published in 2019) suggested that aspirin use was associated with a 10% decreased risk of breast cancer, that seemed restricted to in situ or ER/PR+ tumours and postmenopausal women (201). However, the significant benefit for aspirin users was only found in case-control studies. In a meta-analysis of 13 cohort studies (published in 2017), a duration-response relationship between aspirin use and breast cancer risk was observed (202). The authors of these two meta-analyses detected substantial heterogeneity of results among studies as well as publication bias and highlighted the need for more epidemiological studies with well-controlled confounding and more accurate assessment of exposure to aspirin. Of note, the fact that most studies did not distinguish between low-dose and standard-dose formulations may have contributed to the reported heterogeneity of results.

Results on the associations between use of other antiplatelet drugs and breast cancer risk could help to further elucidate whether use of aspirin would impact breast cancer risk through platelet inhibition or through other mechanisms [START_REF] Lakhani | WHO Classification of Tumours of the Breast[END_REF][START_REF] Sobin | International Union Against Cancer (UICC). TNM classification of malignant tumours[END_REF][START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF]. A meta-analysis of 6 randomized controlled trials and 3 cohort studies published in 2017 concluded that there was no association between clopidogrel or prasugrel use and cancer risk (203). Since then, recent observational studies noted that clopidogrel use, alone or in combination with lowdose aspirin, might reduce the risk of several cancers (204-206) including colorectal, other gastro-intestinal, non-gastrointestinal, or haematological cancers (206). However, no epidemiological study evaluated the associations between antiplatelet drugs other than lowdose aspirin and breast cancer risk.

Take-home messages

Low-dose aspirin has been intensively investigated as a potential anti-cancer medication. However, epidemiological evidence is currently insufficient to draw conclusions on the impact of low-dose aspirin use on breast cancer risk.

Other antiplatelet drugs

have not been explored as potential breast cancer preventive medications in epidemiological studies.

More epidemiological studies are needed and should distinguish between low-dose and standard-dose aspirin formulations. Results on other antiplatelet drugs could help to further elucidate whether aspirin would impact breast cancer risk through platelet inhibition rather than through its potential anti-inflammatory properties. Glucocorticoids are corticosteroids that bind to the glucocorticoid receptor (GR). GR is a member of the nuclear receptor superfamily of transcription factors expressed in various tissues. When glucocorticoids bind to the GR, the receptor becomes activated and the GRglucocorticoid complex translocates into the cell nucleus (208). Once inside the nucleus, the complex can:

-Binds directly to DNA elements called glucocorticoid response elements to stimulate target gene expression and activate gene transcription. For instance, glucocorticoids induce the formation of lipocortin-1, an important inhibitor of the phospholipase A 2 , thereby blocking eicosanoid production including PGs and leukotrienes (Figure 3), Likewise, high doses of glucocorticoids (depending on the molecules) bind to mineralocorticoid receptors influencing the electrolytes and water balance. 4). Approval for the study was obtained from the relevant ethical review boards of the participating institutions as well as from the IARC ethics committee. -"Are you currently taking anti-inflammatory drugs at least three times per week?" in

France.

-"Have you ever taken aspirin continuously for 3 months or more?" in the United Kingdom.

-"During the past 4 weeks, did you take any regular medications? If yes, please specify the medication." in Germany.

-"Do you take aspirin daily?" in the Netherlands.

-"Have you used more than one painkilling tablet/month during the last year? If yes, how many pills containing aspirin and how many pills containing ibuprofen: none; 2-3/month; 1-2/week; 3-6/week; 1-3/day; 4-5/day; 6+/day?" in Denmark.

1.5. Co-variables

Education level

Educational level was collected at baseline for each centre and the replies were then categorized into a common classification using three categories (none or primary education, technical/professional/secondary school, and longer education).

Familial history of breast cancer

Only in France, Spain, Utrecht, Norway and Bilthoven, participants were asked to report whether their mother or sister(s) were diagnosed with a breast cancer. The age at diagnosis was also asked.

Medical conditions

In all centres, participants were asked to report previous illnesses and age of onset of each of these events. Most centres included information on heart disease, diabetes, stroke, hypertension, hyperlipidaemia, gallstones, polyps of the large bowl, hysterectomy, oophorectomy, and breast surgery.

Hormonal and reproductive factors

Age at menarche

In all EPIC countries, women were asked to either report their age at menarche or to choose a predefined category of age at menarche. Age at menarche was then dichotomized (≤13 years and >13 years).

Reproductive life

In all EPIC countries, women were asked to report whether they had ever been pregnant and whether they had ever had any live born children or still births (miscarriage or abortion).

They could also report the number of live births and their age at first delivery (second, third and last delivery). Women were considered to have had a full-term pregnancy if they reported at least one live birth or still birth. In Norway and Umeå, women were asked to directly give the number of full-term pregnancies. For the other countries, the number of full-term pregnancies was calculated from the number of live births and the number of still births. Age at first full term pregnancy was considered as the minimum age between the two types of full-term pregnancies (stillbirths or live births). This information was missing for Norway, Bilthoven, Malmö and Umeå. In all centres except Bilthoven and Umeå, women were asked whether they had ever breastfed one on their three first and last delivery. Women who reported to have breast fed at least one of her children was considered as having ever breastfed.

Menopausal status

A combination of different factors was used to determine the menopausal status at baseline.

Women were considered premenopausal when they reported they were still menstruating or had at least nine menstrual periods over the previous 12 months. Women who had missing or incomplete questionnaire data or who had had a hysterectomy, were considered premenopausal when they were younger than 42 years (because among the female EPIC participants who had complete questionnaire data, almost all women who were younger than 42 years were premenopausal). Women were considered postmenopausal when they reported fewer than four menses in the past year, or when they reported a bilateral ovariectomy.

Women who had missing or incomplete questionnaire data, were considered postmenopausal when they were older than 55 years (because among the female EPIC participants who had complete questionnaire data, almost all women who were older than 55 years were postmenopausal). Women between 42 and 55 years old who had had a hysterectomy and using exogenous hormones were considered perimenopausal women.

Phase of menstrual cycle among premenopausal women at the time of blood donation

For all women classified as premenopausal at the time of blood donation, phases of menstrual cycle (early follicular, late follicular, peri-ovulatory, midluteal, and other luteal) were determined using two different methods: "forward' dating" (counted forward from the woman's reported date of the start of her last menses) and "backward dating" (counted backward from the date of the start of her next menses after blood donation, which the woman reported on a prepaid postcard and sent back to the recruitment centre afterwards).

Preference was given to backward dating because the length of the second half of the cycle (luteal phase) is generally more constant than the first half (follicular phase).

Exogenous hormone use

All EPIC centres had information on oral contraceptive use including ever use, age at first use and duration of use. In all EPIC centres except Umeå, women were asked to report whether they were currently using oral contraceptives.

Alcohol consumption

Alcohol consumption at baseline was collected for the whole EPIC cohort. Participants reported the number of standard glasses of wine, beer, cider, sweet liquor, distilled spirits, or fortified wines they consumed daily or weekly during the 12 months before recruitment.

These replies were used to estimate the quantity of ethanol consumption per day. Alcohol consumption was also assessed from 24-hour dietary recalls in a subset of the cohort, to standardise the dietary information received from all centres. Lifetime alcohol consumption was also collected for 76% of participants.

Tobacco smoking

The following information on cigarette smoking was collected through baseline questionnaires: smoking status (current, former, or never), age at which they started and quit smoking, number of cigarettes per day currently smoked, and number of cigarettes smoked per day during different periods of life. Information on passive smoking was available in 6 countries (France, Italy, the Netherlands, Germany, Denmark, and Norway). All centres (except those in the Netherlands and Norway) also have information on current and past cigar and pipe smoking.

Recruitment and baseline information

In 1990, 499,668 French women born between 1925and 1950, living , living Q11 also included information on quality of life and sleep patterns.

While Q3 and Q4 were only sent to women who responded to the previous questionnaire, Q2, Q5, Q6, Q7, Q8, Q9, Q10, and Q11 were sent to the whole cohort. The response rates were varied from 77% to 92%.

Data from the MGEN database

Every three months, the MGEN provides the E3N team with updated information on vital status and address of the participants that are still affiliated with this health scheme.

Additionally, the MGEN provides, for each cohort participant, data on all outpatient reimbursements for health expenditure issued since January 1, 2004.

Biological data

Between 1994 and 1999, blood samples were collected among around 25,000 participants.

Between 2009 and 2011, saliva samples were collected among around 45,000 women who did not previously provide blood samples. As mentioned previously, the MGEN provides updated information on vital status of the participants that are still affiliated with this health scheme. Deaths can also be reported by family members or by the postal services (when the questionnaire is returned). When information on vital status is missing, the National Death Index is screened to check whether the participant is still alive. In the event of the death of a participant, cause of death is retrieved through the French National Causes of Death Registry (Cépi-DC Inserm).

Cancer events

As shown in Figure 5, each questionnaire included a specific section on health status including cancer occurrence. Participants were asked to report cancer site and date of diagnosis and to provide a copy of pathology reports. The contact details of the participant's physician were also requested in each follow-up questionnaire. When participants could not provide the pathology reports, physicians were contacted to confirm the reported cancers and were asked to send a copy of the pathology reports. The proportion of false-positive selfreports was low (<5%) for breast cancers. Death certificate information, mentioning the underlying cause of death, the chain of events that led to death, as well as all other diseases or conditions contributing to death, was also taken in consideration to identify any cancer cases which were not self-reported in the follow-up questionnaire. However, when a cancer event was identified through this means, it was impossible to investigate the event if the participant did not report the contact details of her physician(s) in any follow-up questionnaire. In addition, even with death certificates some cancer events might have been missed. When a cancer case was identified through death certificates and when no further information could be retrieved, including date of diagnosis, women were censored at the date of the last questionnaire returned and were considered as non-cases at that date.

Breast cancer characteristics were retrieved from the pathology reports. This includes exact localisation, morphology, grade and stage at diagnosis, and molecular status (ER, PR or HER2 status).

Breast cancer cases are coded by a staff member of the E3N team according to different international tables from the WHO and the UICC:

-the ICD-10 to define the exact localisation of the tumour (239).

-The ICD-0 2/3 to determine the tumour morphology and grade (234).

-The TNM system to describe the stage [START_REF] Sobin | International Union Against Cancer (UICC). TNM classification of malignant tumours[END_REF].

2,734 prevalent breast cancers were diagnosed in Q1 and 8,238 incident primary breast cancer were diagnosed between Q1 and Q11.

Exposure to anti-inflammatory and antiplatelet drugs

The MGEN provides, for each E3N participant, data on all drug reimbursements issued since January 1st, 2004. The available data for each reimbursement are date of drug purchase and national code number (Code Identifiant de Presentation, CIP). The national code number enabled us to retrieve the following information from Thériaque® (a French-drug database:

www.theriaque.org): ATC code, molecule, route of administration, number of pills/ampules per package, dose per pill/ampule, and DDD. In Q4, Q6, and Q7, women were asked to report whether they were currently using anti-inflammatory drugs at least three times per week (Figure 6). In Q5 women could also report (as free text) any other drug that they were currently using at least three times per week.

Co-variables

The co-variables of interest for the presented work are described below.

Educational level

Educational level was recorded in Q1. Participants could tick boxes corresponding to their educational level: 1) no schooling, 2) school certificates, 3) vocational education, 4) from high-school to two years of higher education, 5) from three to four years of higher education, 6) at least five years higher education.

Familial history of breast cancer

Familial history of cancer was collected in Q1 and Q6. In Q1, participants could tick boxes to declare whether their grand-parents, mother, father, sisters, brothers, uncles, aunts, or children had ever had breast, bowel, or other cancers. In Q6, participants could report whether a member of their family had ever had a cancer and specify the exact cancer site and the age at diagnosis.

Medical factors

In each questionnaire from Q3, participants could note in a free-text section any disease from which they were suffering. Additionally, participants were asked to specify if they had been diagnosed with specific diseases such as the following:

History of benign breast diseases

As mentioned above (2.3. Follow-up) information on lifetime personal history of benign breast diseases (such as adenoma and fibro-adenoma, mastitis, and fibrocystic diseases) was first collected in Q2. The age at diagnosis, diagnostic procedures and treatment was reported.

This information was then updated in several questionnaires from Q3 to Q9.

Other medical events

Information on lifetime personal history of other medical events was first collected in Q1 (or Q2 for some of them) and then updated in several subsequent questionnaires. Indeed, women were asked to report, together with the corresponding age at / date of diagnosis (mm/yyyy), if they had had diabetes (all questionnaires), hypertension (all questionnaires except Q10), hypercholesterolemia (Q1, Q2, Q6, Q7, Q8, Q9, Q10 and Q11), angina pectoris (all questionnaires), myocardial infarction (all questionnaires), stroke, pulmonary embolism (all questionnaires), deep vein thrombosis (all questionnaires from Q2), arteritis of the lower limbs (all questionnaires from Q2), cardiac dysrhythmia (Q2-Q10), artificial if prompted by bilateral oophorectomy, radiotherapy, or chemotherapy and to be natural otherwise. When menopausal status could not be determined, women were considered premenopausal if they had not reached the age of 51 years (the median age at natural menopause in the cohort) at end of follow-up and menopausal otherwise. After Q8, all E3N participants were at least 55 years old and therefore most of them had reached menopause.

Age at menopause was self-reported in Q1, Q6, and Q7.

Exogenous hormone use

Detailed information on lifetime use of oral contraceptives and MHT were collected through Q2. Participants were asked to report brand names (from which type and route of administration could be deduced for MHT), together with the start date and duration of each episode of hormone use. This information was then updated in each follow-up questionnaire until Q9. Since 1 st January 2004, the MGEN provides, for each E3N participant, data on MHT reimbursements.

Anthropometric factors and physical activity

Weight was available in each questionnaire. Height was available in Q1, Q4 and from Q6 to Q11. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Waist and hip circumferences were collected in Q4 and in all questionnaires from Q7.

In Q1, Q3, Q5, Q7, Q8, and Q11, women were asked to report their current physical activity habits including recreational and household activities and to specify the frequency and duration of these activities. For each physical activity, the MET-hours was estimated based on the Compendium of Physical Activities (236).

CHAPTER 3: CIRCULATING INFLAMMATORY BIOMARKERS AND BREAST CANCER RISK IN THE EPIC STUDY

Objective

The objective of this chapter was to conduct a case-control study nested within the EPIC cohort to evaluate the associations of pre-diagnostic circulating concentrations of 11 inflammatory biomarkers (TNF-α, IFN-γ, IL-6, IL-8, IL-10, IL-13, IL-17D, IL-1RA, CRP, leptin and adiponectin) with breast cancer risk, overall and by tumour receptor status, menopausal status, adiposity and exogenous hormone use.

Materials and methods

Selection of cases and controls

EPIC participants were eligible for inclusion in the current study if, at recruitment, they:

-donated their blood -were cancer-free (other than non-melanoma skin cancer)

-were not pregnant.

Cases were selected among all EPIC eligible women with a first primary invasive breast cancer diagnosis at least two years after blood collection and before December 2012 and with known information on hormone receptor status. For each breast cancer case, one control was matched. Controls were selected randomly among all eligible EPIC participants who were alive and without cancer diagnosis (except non-melanoma skin cancer) at the time of cancer diagnosis of his matched case (incident density sampling). Controls were matched to cases on centre of recruitment, age, menopausal status, phase of the menstrual cycle for premenopausal women and fasting status at blood collection.

Inflammatory biomarker assessment

Pre-diagnostic plasma levels of cytokines and adipokines were measured using commercially available immunoassays provided by Meso Scale Discovery, a highly sensitive immunoassay platform, in the laboratories of the Biomarkers Group at IARC.

Statistical analysis

Geometric means were used to describe biomarker concentrations among cases and controls.

Log-transformed biomarker concentrations were used in all following analyses. Partial

Pearson's correlations between biomarkers and with anthropometric factors, adjusted for age at blood collection and laboratory batch, were estimated among overall controls and by menopausal status. We used conditional logistic regression to estimate the ORs and 95% CI of breast cancer, overall and by menopausal status, per one standard deviation (SD) increase in biomarker concentration. All biomarkers, except IL-13, were considered as continuous variables and categorized into quartiles based on the distribution of the concentrations among controls. Most of the IL-13 measurements were below the LOQ and IL-13 was therefore dichotomized into value higher and lower than the LOQ.

All analyses were conditioned on the matching variables. Models were also adjusted either for BMI or waist circumference (WC).

Heterogeneity was investigated by use of exogenous hormones at blood collection, age at diagnosis, breast cancer subtypes, time between blood collection and diagnosis, WC, BMI, and country, by introducing interaction terms in the models. For WC and BMI unconditional logistic regressions adjusted for matching factors were used.

Main findings

Cases were diagnosed on average 8.4 years after blood collection at a median age of 61.4

years. Most tumours were ER-positive (80.4%), PR-positive (68.2%), and HER2-negative (78.6%). Characteristics and mean concentrations of the biomarkers were similar in cases and controls. All biomarkers were moderately correlated with each other, except for IL-17D which was not correlated with any other biomarkers and IL-13 which was only moderately correlated with IL-8 and IL-10. BMI and WC showed the highest correlations with leptin (r> 0.5) and were also positively correlated with IL-6, IL-1RA and CRP (r>0.3) and negatively correlated with adiponectin (r<-0.25). Similar correlations were observed among women before or after menopause.

Overall, no statistically significant association was observed between the inflammatory markers and breast cancer risk. Among pre/perimenopausal women (720 cases), the leptin-toadiponectin ratio was inversely associated with breast cancer [WC-adjusted: OR 1SD =0.87 (0.76-0.99)]. Among postmenopausal women (838 cases), no statistically significant associations were found between biomarkers on a continuous scale and breast cancer risk.

However, in categorical models, higher TNF-α levels were positively associated with breast cancer risk [WC-adjusted: OR Q4 versus Q1= 1.44 (1.01 -2.05)].

Conclusions

These results suggest that the ratio of leptin-to-adiponectin might have relevance for peri/premenopausal breast cancer development, while higher levels of TNF-α could be involved in postmenopausal breast cancer development. Whether leptin-to-adiponectin ratio and TNF-α are associated with breast cancer risk independently of anthropometry deserves replication.

Paper

Status: In preparation.

Contribution: First author, conducted statistical analyses and wrote the first draft of the manuscript.

Statistical analysis

Multivariable Cox regression models with age as the time scale were used to estimate HRs for the association of "regular' NSAID exposure with BC incidence, overall and by BC subtypes. Models were adjusted for BMI, educational level, MHT use, age at first full term pregnancy, and alcohol intake. Effect modification by main breast cancer risk factors was evaluated by using likelihood ratio tests to compare models with and without cross-product interaction terms. Heterogeneity between breast cancer subtypes was evaluated by competing risk analyses, with women who developed the competing breast cancer subtypes censored at the time of occurrence and cases with missing information on the studied subtype excluded from the corresponding analysis (240).

Main findings

Of the 140,981 women included in the current analysis, 9,907 (7%) were "regular" NSAID users at baseline. During a median follow-up of 13 years, 7,379 incident breast cancer cases were diagnosed (816 in situ, and 6,563 invasive).

Compared to women who were not "regular" NSAID users, "regular" users were older, more often overweight, or obese, less educated, and had less frequent exposure to oral contraceptive and more frequent exposure to MHT. "Regular" NSAID use was most frequent among women from Denmark and the United Kingdom than other EPIC countries.

In multivariable models, there was no statistically significant association between "regular" NSAID use and breast cancer risk overall. Compared with women who were not "regular" users at baseline, the HR for "regular" users of NSAIDs was 0.99 (95% CI= 0.90 to 1.08).

There was a borderline significant heterogeneity between in situ and invasive breast cancers NSAID and ever MHT use was observed for all other invasive breast cancer subtypes examined.

Conclusions

In this large prospective European cohort study, we found a decreased risk of invasive breast cancer with "regular" NSAID use only in postmenopausal women who ever used MHT. No association was found between NSAID use and breast cancer risk in non-MHT users.

Additional investigation is needed to better understand interactions between MHT use, NSAIDs and breast cancer risk. The objectives of this chapter were, using data from the prospective E3N cohort, to evaluate the associations between the use of NSAIDs, antiplatelet drug and glucocorticoids with breast cancer incidence, overall and by breast cancer subtypes, risk factors, and use of other drugs.

Materials and Methods

Study Population and Follow-up

Postmenopausal E3N participants were eligible for inclusion if they:

-had no history of cancer (except basal cell carcinoma and in situ colorectal tumours) before A total of 62,512 postmenopausal women, followed during a median time of 9 years, were included in the following projects (including 2,864 incident breast cancer cases diagnosed).

Exposure assessment

For each delivery of medicines containing the drugs of interest, we extracted data on date of purchase, molecule, number of pills per package, and dose per pill.

We defined as "recurrent" users of the drug of interest women with at least two reimbursements during any previous three-month period since January When studying the risk of different breast cancers characterized by their invasiveness, molecular, histological or stage/grade subtype, competing risk analysis was performed using the cause-specific hazards approach (240, 243). Cases with missing information on a given tumour characteristic were excluded from the corresponding analyses.

3. Nonsteroidal anti-inflammatory drugs and breast cancer risk

Main findings

At the end of follow-up, 38,493 (62%) women had been recurrently exposed to NSAIDs.

Compared to women who were occasionally or never exposed to NSAIDs, recurrent users were more often overweight or obese, had more frequent medical follow-up, exposure to other drugs (paracetamol, anti-arthritics, glucocorticoid, PPI and MHT), and comorbidities such as migraine and arthrosis. There were no other major differences.

Overall

Conclusions

In this large, prospective cohort of postmenopausal women with up to 10 years of follow-up, we observed a decreased risk of breast cancer with NSAID use only among women who previously used PPI. Our study is the first one to evaluate the modifying effect of PPI on the NSAIDs-breast cancer associations. Therefore, our finding of an interaction between NSAID and PPI use regarding the risk of breast cancer deserves replication in other settings.

Paper

Status: Published.

Contribution: First author, conducted statistical analyses, wrote the first draft of the manuscript, submitted it to the journal, and replied to reviewers' comments.

Conclusions

In this cohort of French postmenopausal women, a transient increase in breast cancer risk was observed during the 3 rd year of low-dose aspirin use followed by a decrease in risk.

Clopidogrel use was associated with increased breast cancer risk that seemed restricted to ER-tumours, with no clear trend according to duration of use.

Conclusions

This study suggests that breast cancer subtypes should be considered when evaluating the associations between glucocorticoid use and breast cancer risk.

CHAPTER 6: DISCUSSION

My doctoral thesis aimed to provide additional epidemiological evidence on the potential role of inflammation on breast cancer development by i) identifying inflammatory biomarkers associated with breast cancer risk and ii) investigating whether drugs with anti-inflammatory properties might prevent breast cancer occurrence. Overall, results from this thesis suggested that inflammation plays a modest role in the development of breast cancer and that its impact on breast cancer risk could be limited to certain sub-populations or certain breast cancer subtypes.

Inflammatory biomarkers and breast cancer risk

In the EPIC cohort, we found no overall association between pre-diagnostic blood levels of eleven inflammatory markers and breast cancer risk. However, an inverse association was observed with the leptin to adiponectin ratio among pre/perimenopausal women and a positive association with TNF-α levels among postmenopausal women.

No previous epidemiological study evaluated the leptin to adiponectin ratio regarding breast cancer risk. However, a few prospective epidemiological studies reported that high levels of leptin and low levels of adiponectin were associated with a decreased risk of breast cancer among premenopausal women [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Tworoger | Plasma adiponectin concentrations and risk of incident breast cancer[END_REF]244), even though the associations did not reach statistical significance in some of them [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Tworoger | Plasma adiponectin concentrations and risk of incident breast cancer[END_REF]. The plausibility of the associations between leptin to adiponectin ratio and premenopausal breast cancer risk needs to be further addressed in experimental studies. In premenopausal women, high levels of leptin have been hypothesized to reduce breast cancer risk through regulation of ovarian folliculogenesis (245) and reduction of follicular oestradiol secretion (246). Because the leptin to adiponectin ratio is a marker of insulin resistance [START_REF] Finucane | Correlation of the leptin:adiponectin ratio with measures of insulin resistance in non-diabetic individuals[END_REF], the inverse association found between this ratio and breast cancer risk might not be due to inflammation.

Our finding on TNF-α is not in line with previous prospective epidemiological studies. Most of them reported no significant associations between TNF-α levels and breast cancer risk [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Gross | Adipocytokines, inflammation, and breast cancer risk in postmenopausal women: a prospective study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Heikkila | Associations of circulating C-reactive protein and interleukin-6 with cancer risk: findings from two prospective cohorts and a meta-analysis[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Krajcik | No association between serum levels of tumor necrosis factor-alpha (TNF-alpha) or the soluble receptors sTNFR1 and sTNFR2 and breast cancer risk. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. However, because the number of cases included was smaller than in our study, most of the previous studies were not able to categorize TNF-α into quartiles [START_REF] Agnoli | Biomarkers of inflammation and breast cancer risk: a case-control study nested in the EPIC-Varese cohort[END_REF][START_REF] Gross | Adipocytokines, inflammation, and breast cancer risk in postmenopausal women: a prospective study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Krajcik | No association between serum levels of tumor necrosis factor-alpha (TNF-alpha) or the soluble receptors sTNFR1 and sTNFR2 and breast cancer risk. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF].

In contrast to our results, one prospective study reported an inverse association between high levels of TNF-α and breast cancer risk [START_REF] Dias | Low-Grade Inflammation, Oxidative Stress and Risk of Invasive Post-Menopausal Breast Cancer -A Nested Case-Control Study from the Malmo Diet and Cancer Cohort[END_REF]. In EPIC, results from a previous study suggested also an increased risk of endometrial cancer in the highest quartile of TNF-α, compared to the lowest (247). The positive association between TNF-α and breast cancer is highly plausible since experimental data suggested that TNF-α could be involved in carcinogenesis by activating NF-қB and consequently inducing the expression of genes associated with cell proliferation, apoptosis, metastasis and angiogenesis [START_REF]NF-kappaB and cancer: mechanisms and targets[END_REF], or by stimulating aromatase activity in breast tissue leading to oestrogen synthesis [START_REF] Zhao | Tumor necrosis factoralpha stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter 1.4[END_REF].

Our study, which was the largest population-based prospective cohort examining the inflammatory biomarkers-breast cancer associations, supports that menopausal status needs to be considered when assessing these associations. In our study, we did not find any heterogeneity according to molecular subtypes of breast cancer. However, some results from subgroup analyses were based on relatively small numbers of cases. Since breast cancer is a heterogeneous disease, there is a need to robustly evaluate the role of inflammation according to histologic subtypes and provide data on less common but more aggressive tumour types e.g. ER-PR-or triple negative breast cancers. Results from our study suggested that anthropometry factors were strong confounders of the associations between some inflammatory biomarkers with postmenopausal breast cancer. Indeed, obesity is an established risk factor of postmenopausal breast cancer and contribute to chronic inflammation [START_REF] Fain | Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells[END_REF][START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance[END_REF][START_REF] Kershaw | Adipose tissue as an endocrine organ[END_REF][START_REF] Schaffler | Role of adipose tissue as an inflammatory organ in human diseases[END_REF][START_REF] Galic | Adipose tissue as an endocrine organ[END_REF]. It is important that further studies evaluate whether anthropometry confounds or modifies the inflammation-breast cancer associations. It would also be interesting to evaluate to what extend inflammation may mediate the obesity-breast cancer association. Lastly, the inflammation pathways are correlated to the oestrogen and insulin pathways, and there is also a need to disentangle the effect on breast cancer development of inflammation pathways from the two others.

Drugs with anti-inflammatory properties and breast cancer risk

Results from our pharmacoepidemiologic studies conducted in the EPIC or E3N cohorts suggested that NSAIDs, low-dose aspirin and glucocorticoids might have different effects on breast cancer development and that these effects may differ according to the type and duration of drug use but also according to the breast cancer subtypes considered. These associations may also vary depending on subgroups of the population or on the use of other drugs. Indeed, in the EPIC and E3N cohorts, no overall association was observed between NSAID use and breast cancer risk. However, in the EPIC cohort (where NSAID use was selfreported), there was a borderline significant interaction by MHT use suggesting an inverse association between NSAID use and breast cancer risk only among postmenopausal women who had ever used MHT. In the E3N cohort, using drug reimbursement data updated during follow-up, there was a significant interaction by PPI use suggesting an inverse association between NSAID use and breast cancer risk only among postmenopausal women who previously used PPI.

In the E3N cohort, use of aspirin at antiplatelet dosage was associated with a higher breast cancer risk few year after treatment star, followed by a lower breast cancer risk. In the study on NSAIDs, overall, aspirin at anti-inflammatory dosage was not associated with breast cancer risk. However, because it is mainly used sporadically, it was not possible to capture a long duration of use. Therefore, comparison of results across different dosage of aspirin was difficult. We also evaluated the association between clopidogrel, another antiplatelet drug, and breast cancer risk to test whether low-dose aspirin and clopidogrel showed similar associations with breast cancer risk, which was not the case, suggesting that the decreased risk of breast cancer found with a long duration of low-dose aspirin might be due to other properties than its antiplatelet properties. Indeed, clopidogrel use was associated with increased breast cancer risk that seemed restricted to ER-tumours, with no clear trend according to duration of use.

In the E3N cohort, glucocorticoid use was associated with an increased risk of in situ breast cancer and a decreased risk of invasive breast cancer. Among invasive cases, glucocorticoid use was associated with a decreased risk only for ER+ breast cancer, stage 1 and 2 breast cancer, or with the highest categories of cumulative number of reimbursements, and was associated with an increased risk of stage 3/4 breast cancers.

Our results on NSAIDs are consistent with the majority of results from prospective studies, suggesting no reduced risk of breast cancer with NSAID use (142,147,148,150,155,160,(248)(249)(250)(251)(252)(253)(254)(255). To our knowledge, most of the studies evaluating whether the NSAID-breast cancer associations differed by MHT use did not observe any significant effect modification (142,148,149,250,252,256,257) and only one study reported an inverse association between NSAID use for at least five years and breast cancer risk among MHT users (148). Another study showed a statistically significant positive association between standard dose aspirin use and breast cancer risk among MHT users for ten years or more (151). Nevertheless, this result should be considered cautiously because the number of exposed cases in that category was very low (n=25). In all the other studies, risk estimates associated with NSAID use among MHT users were not reported. In addition, the interaction with MHT use was not confirmed in the E3N cohort, which has more accurate data on both MHT and NSAID use than the EPIC cohort. The modifying effect of PPI on the NSAIDsbreast cancer associations we observed within the E3N cohort was not examined in previous epidemiological studies.

Our results on low-dose aspirin are consistent with two meta-analyses which suggested a duration-response relationship between aspirin use and breast cancer risk (194,202). In the latest randomized double-blind placebo-controlled trial (ASPREE: participants aged ≥ 65 years old, median follow-up of 4.7 years), daily use of low-dose aspirin (100 mg) had no effect on breast cancer incidence (199). This lack of effect could be due to the fact that follow-up as well as duration of aspirin use were not long enough to expect a benefit of aspirin (34) (35) (200)(201). However, our study shows close estimates compared to the ones found in the latest meta-analysis of randomized controlled trials on any cancer incidence and aspirin use, with a transient increase in risk in the three first years of use, followed by a decrease in risk (200). It therefore seems important to be able to assess the low-dose aspirinbreast cancer risk associations according to duration of use. In our analyses, the number of cases was too limited to evaluate the low-dose aspirin-breast cancer associations for a duration longer than five years of use. No previous epidemiological studies evaluated the clopidogrel-breast cancer association.

Our results on glucocorticoids are not in line with the only epidemiological study specifically designed to evaluate the glucocorticoid-breast cancer associations, which reported a null association between glucocorticoid use and invasive breast cancer risk (230). However, the authors were not able to stratify their analyses by breast cancer subtypes. Our results The prospective design of our studies minimizes recall bias and the large number of participants and incident breast cancer cases allowed us to examine interactions of drug use with breast cancer risk factors and to estimate associations with different breast cancer subtypes. While many medico-administrative databases do not have data on breast cancer risk factors, our studies showed that breast cancer risk factors are not major confounders in the associations between the studied drugs and breast cancer risk. Our results suggested that future pharmacoepidemiologic studies should consider breast cancer subtypes, risk factors, and use of other drugs because, in our analyses, they were found to modify the associations between anti-inflammatory/antiplatelet drug use and breast cancer risk. However, some of our subgroup analyses were based on low numbers of cases and, because of the large number of comparisons evaluated, they need to be interpreted cautiously before confirmation in other prospective studies. In the EPIC cohort, NSAID exposure was self-reported in a baseline questionnaire and there was a lack of data on characteristics of NSAID use, such as duration, dose, or frequency of use. Thus, we were not able to examine a potential dose-or durationresponse relationship. In the E3N cohort, drug exposure was identified through a drug reimbursement database, which avoids differential recall bias between cases and non-cases and allowed us to consider precise information on exposure (including duration, dose, and timing of use). However, our statistical power remained limited to evaluate breast cancer risk associated with a long duration of use and future studies should be able to do so. We might be able to do so in the E3N cohort when the follow-up will be longer. In the EPIC cohort the NSAIDs-breast cancer associations did not differ according to menopausal status. In the E3N cohort, we only evaluated the effect of drugs on breast cancer development among postmenopausal women. The associations between NSAIDs, antiplatelet drugs, glucocorticoids and breast cancer risk among premenopausal is undervalued and further studies should consider menopausal status. In conclusion, there is a real need to perform large, well-powered analyses where less common tumour subtypes or subgroups can be addressed, and a long duration of use can be captured.

Perspectives

Additional evidence on the role of inflammation/anti-inflammatory drugs on breast cancer development are needed. Therefore, we planned several additional analyses (listed below).

First, in the EPIC cohort, we will use mediation analysis to evaluate and quantify:

-the role of leptin to adiponectin ratio as a mediator in the association between obesity and pre/perimenopausal breast cancer,

-and the role of TNF-α as a mediator in the association between obesity and postmenopausal breast cancer.

Then Lastly, to provide more definitive evidence on the association of inflammation with breast cancer development, data from prospective cohort studies collaborating within the National Cancer Institute (NCI) Cohort Consortium will be pooled (project approved by the NCI cohort consortium steering committee and pending funding). Data from 22 prospective cohorts (including more than 44,000 breast cancer cases, among over 1.4 million study participants) will be pooled to evaluate the association between aspirin/NSAIDs and breast cancer risk, according to breast cancer subtypes and risk factors. Data from 14 case-control studies nested within prospective cohorts (including more than 7,200 breast cancer cases and 15,000 matched controls) will be pooled to evaluate the associations between CRP, leptin, and adiponectin with breast cancer risk, according to breast cancer subtypes and risk factors. ≠ "In the ATC classification system, the active substances are classified in a hierarchy with five different levels. The system has fourteen main anatomical/pharmacological groups or 1st levels. Each ATC main group is divided into 2nd levels which could be either pharmacological or therapeutic groups. The 3rd and 4th levels are chemical, pharmacological or therapeutic subgroups and the 5th level is the chemical substance". ≠ "In the ATC classification system, the active substances are classified in a hierarchy with five different levels. The system has fourteen main anatomical/pharmacological groups or 1st levels. Each ATC main group is divided into 2nd levels which could be either pharmacological or therapeutic groups. The 3rd and 4th levels are chemical, pharmacological or therapeutic subgroups and the 5th level is the chemical substance".

ANNEXES

ATC codes

ATC codes

  Cairat M, Al Rahmoun M, Gunter M, Severi G, Dossus L, Fournier A. Nonsteroidal anti-inflammatory drug use and breast cancer risk in a prospective cohort study of postmenopausal women. 35 th International Conference on Pharmacoepidemiology & Therapeutic Risk Management, 24 th -28 th August 2019, Philadelphia (USA). Cairat M, Rinaldi S, Navionis AS, Romieu I, Biessy C, Weiderpass E, Gunter M, Dossus L, on behalf of EPIC. Circulating inflammatory biomarkers and breast cancer risk: results from the EPIC study. IARC Scientific Council, 5 th February 2020, Lyon (France). Presented by Sabina Rinaldi. Cairat M, Al Rahmoun M, Gunter M, Severi G, Dossus L, Fournier A. Antiplatelet drug use and breast cancer risk in a prospective cohort study of postmenopausal women. 36 th International Conference on Pharmacoepidemiology & Therapeutic Risk Management, 16t h -17 th September 2020, virtual event. Cairat M, Al Rahmoun M, Gunter M, Severi G, Dossus L, Fournier A. Glucocorticoid use and breast cancer risk in a prospective cohort study of postmenopausal women. 36 th International Conference on Pharmacoepidemiology & Therapeutic Risk Management, 16t h -17 th September 2020, virtual event.

Figure 1 :

 1 Figure 1: Age standardized incidence rates of breast cancer. . .............................................................Figure 2: Age standardized mortality rates of breast cancer. ................................................................ Figure 3: An overview of eicosanoid synthesis pathways. ...................................................................Figure 4: EPIC countries and centres. ...................................................................................................Figure 5: Timeline of data collected between 1990 and 2014 (E3N Cohort). ......................................Figure 6: Photo of one questionnaire including question on the use of anti-inflammatory drugs ........

Figure 4 :

 4 Figure 1: Age standardized incidence rates of breast cancer. . .............................................................Figure 2: Age standardized mortality rates of breast cancer. ................................................................ Figure 3: An overview of eicosanoid synthesis pathways. ...................................................................Figure 4: EPIC countries and centres. ...................................................................................................Figure 5: Timeline of data collected between 1990 and 2014 (E3N Cohort). ......................................Figure 6: Photo of one questionnaire including question on the use of anti-inflammatory drugs ........

Figure 5 :

 5 Figure 1: Age standardized incidence rates of breast cancer. . .............................................................Figure 2: Age standardized mortality rates of breast cancer. ................................................................ Figure 3: An overview of eicosanoid synthesis pathways. ...................................................................Figure 4: EPIC countries and centres. ...................................................................................................Figure 5: Timeline of data collected between 1990 and 2014 (E3N Cohort). ......................................Figure 6: Photo of one questionnaire including question on the use of anti-inflammatory drugs ........

Figure 6 :

 6 Figure 1: Age standardized incidence rates of breast cancer. . .............................................................Figure 2: Age standardized mortality rates of breast cancer. ................................................................ Figure 3: An overview of eicosanoid synthesis pathways. ...................................................................Figure 4: EPIC countries and centres. ...................................................................................................Figure 5: Timeline of data collected between 1990 and 2014 (E3N Cohort). ......................................Figure 6: Photo of one questionnaire including question on the use of anti-inflammatory drugs ........

Table 1 :

 1 Data collected on NSAIDs in EPIC ........................................................................................ LIST OF ANNEXES Annex 1: Different types of nonsteroidal anti-inflammatory drugs. ................................................... Annex 2: Different types of antiplatelet drugs. ................................................................................... Annex 3: Different types of systemic glucocorticoids. ....................................................................... Annex 4: Description of the SIDIAP database and study population. ................................................

-

  deregulating cellular energetics resisting cell death genome instability and mutation inducing angiogenesis evading growth suppressor enabling replicative immortality avoiding immune destruction -tumour-promoting inflammation activating invasion and metastasis. 1.3. Tumour characteristics Breast cancer comprises multiple entities that have different histological and biological features, clinical presentations, and behaviours. 1.3.1. Histological classification Breast cancers are distinguished according to their tissue origin. The most frequent types of breast cancer begin in the cells of the ducts (called ductal breast cancer and representing 40 to 75% of all breast cancers) or in the cells of the lobules (called lobular breast cancer and representing 5 to 15% of all breast cancers). Other breast cancers including tubular, medullary, papillary, and mucinous are less frequent (each representing less than 2% of all breast cancers) (9).

Figure 1 :

 1 Figure 1: Age standardized incidence rates of breast cancer.

Figure 2 :

 2 Figure 2: Age standardized mortality rates of breast cancer.

  NSAIDs can be further classified in the following therapeutic subgroups (Annex 1): Acetylsalicylic acid (>325 mg per pill) inflammatory and anti-rheumatic agents, non-steroids (M01AX).Preparations containing combinations of NSAIDs with opioids, muscle relaxants, antibacterials, or other anti-inflammatory drugs are classified in N02AJ, M03B, J01, or M01B, respectively. The ATC codes for topical use of NSAIDs preparations starts by M02AA.

Figure 3 :

 3 Figure 3: An overview of eicosanoid synthesis pathways.

  secreting their granule components: cytokines, chemokines, platelet-derived growth factors, vascular endothelial growth factors, basic fibroblast growth factors and insulin growth factors, producing and releasing TxA2 (which might accelerate tumour growth, invasion and promote angiogenesis), gathering around cancer cells, assisting tumour cells in evading immune destruction, and facilitating the circulation of cancer cells, inducing an invasive epithelial-mesenchymal transition phenotype of tumour cells, and influencing pathways that activate cell-bound protease-activated receptors, leading to the activation of inflammatory and angiogenic responses. The possible roles of activated platelet in inflammation and cancer growth support the hypothesis that antiplatelet agents might reduce cancer development. Preclinical studies have demonstrated that aspirin inhibits the proliferation of breast tumour cells, promotes apoptosis

  . Recent US guidelines even recommend low-dose aspirin for the prevention of colorectal cancer in a subset of patients (aged between 50 and 69 years old) with a favourable risk-benefit profile(198). However, findings for breast cancer are limited with few published results from randomized controlled trials. In a randomized controlled trial published in 2013 (Women's Health Study: participants aged ≥45 years old, median follow-up of 17.5 years), low-dose aspirin (100 mg) used every other day for an average of 10 years had no impact on breast cancer risk compared to placebo (Hazard Ratio[HR]=1.02, 95% confidence interval [CI]= 0.89 -1.18, n exposed cases=385) (196). This could be because low-dose aspirin was used less frequently than in usual prescriptions for cardiovascular prevention (i.e. daily), which could be less effective in terms of cancer prevention. The latest randomized double-blind placebo-controlled trial of daily low-dose aspirin (100 mg) (ASPREE: participants aged ≥65 years old, median follow-up of 4.7 years), showed no effect of low-dose aspirin on breast cancer incidence [HR=1.03 (0.80 -1.32), n exposed cases=127] (199). This lack of effect could be due to the fact that follow-up as well as duration of aspirin use were not long enough to expect a benefit of aspirin. Indeed, in a meta-analysis of randomized controlled trials published in 2018, among participants aged ≥ 70 years, an increased risk of any cancer was observed during the first 3 years of follow-up], followed by a reduced incidence of any cancer after 5 years of follow up] (

-

  repress transcription of genes containing a negative glucocorticoid response element, or bind to transcription factors (such as the NF-қB and activator protein-1), leading to the inhibition of various pro-inflammatory molecules including IL-1, IL-2, IL-4, IL

Figure 4 :

 4 Figure 4: EPIC countries and centres.

1 .

 1 Presentation of the cohort The French component of EPIC, E3N (Etude Epidémiologique auprès de femmes de l'Education Nationale, https://www.e3n.fr/), is an ongoing prospective cohort, which initially aimed to investigate the relationship between lifestyle, diet, hormones, environment and female cancers. Nowadays, other major chronic diseases such as diabetes and cardiovascular diseases are also being studied (238). The E3N study was approved by the French National Commission for Data Protection and Privacy (Commission Nationale Informatique et Libertés, CNIL).

Figure 5 :

 5 Figure 5: Timeline of data collected between 1990 and 2014 (E3N Cohort).

Figure 6 :

 6 Figure 6: Photo of one questionnaire including question on the use of anti-inflammatory drugs

  author, conducted statistical analyses, wrote the first draft of the manuscript, submitted it to the journal and replied to reviewers' comments.

  study baseline (July 1 st , 2004), -had healthcare reimbursement during year 2004, -answered before the beginning of follow-up for the current study to the last follow-up questionnaire sent in 2002, -and no missing date of breast cancer diagnosis or censoring. Follow-up started on July 1 st , 2004 and ended at the date of diagnosis of any cancer (with the exception of basal cell carcinoma and in situ colorectal tumour), latest completed questionnaire, or November 17 th , 2014 (date at which the last considered questionnaire was sent to participants), whichever occurred first.

  suggesting a decreased risk of invasive breast cancer especially for ER+ tumour are supported by experiments showing that glucocorticoids might stimulate the expression of the sulfotransferase SULT1E1 (which plays a role in deactivating oestrogen) (213, 216), affect angiogenesis (215), or inhibit inflammatory and growth factors (214, 258). Our findings supporting an increased risk of in situ breast cancer could be explained by the fact that glucocorticoids might slow down tumour growth(259, 260). An increased risk of stage 3/4 breast cancers with glucocorticoid exposure is also in line with experiments suggesting that glucocorticoids promote breast cancer metastasis (221), induce the epithelial-to-mesenchymal transition (261), or activate the TEA domain transcription factor 4 (262). In addition, in the 1960's, a study among breast cancer patients reported that the use of adrenal steroids (including glucocorticoids), compared to non-use, increased the risk of metastasis (222).

  , we will use data from the Information System for the Development of Research on Primary Care (SIDIAP) database, in which data on drug prescription are available from 2006 and 2018, to evaluate the associations between NSAIDs use and breast cancer risk, overall and by menopausal status. During my thesis, I spent 1 month at the Institut Universitari d'Investigació en Atenció Primaria (IDIAP) Jordi Gol (in Barcelona), after being awarded a French Mobility Scholarship from the Cancéropôle Lyon Auvergne Rhône-Alpes, to investigate the feasibility of this study. The data source and the selection of the study population are presented in the Annex 4.

  3.2.4. Main recommendationsGuidelines have been developed to reduce the risk of NSAID complications. In brief, NSAIDs should be used one at a time, at the minimal efficient dose, and during the shortest possible duration. NSAIDs should be used with caution among the elderly who run a far greater risk of side effects. NSAIDs are not recommended, or should be used with caution,

	among people with a history of peptic ulcer, stomach bleeding, any history of haemorrhage,
	any gastrointestinal problems, severe hepatocellular damage, sever renal impairment, severe
	heart failure, hyperlipidaemia, diabetes, hypertension, asthma, hypersensitivity to the
	medication or history of allergies. NSAIDs are not recommended during pregnancy,
	particularly during the third trimester (105). The previous guidelines vary according to the
	molecule.

Further epidemiologic data, with accurate assessment of NSAID use, well-controlled confounders and long follow-up, are needed to confirm the role of NSAIDs and identify potential modalities of use (molecules, doses, duration, populations) that could be beneficial towards breast cancer prevention.

  The authors of this meta-analysis highlighted a high heterogeneity among the included studies. The major differences of the available epidemiological studies were in the accuracy of the exposure assessment (measured at baseline or updated during follow-up), in the exposure definition (ever use, regular use, long-term use, etc.), or in the types of NSAIDs considered (all NSAIDs, all NSAIDs but aspirin, aspirin, ibuprofen, COX-2 inhibitors, etc.). Main side effects A wide range of side effects associated with antiplatelet drugs is recorded (103). The side effects associated with low-dose aspirin or clopidogrel include asthenia, fever, dizziness, nervous system disorders (e.g. amnesia, somnolence), immune system disorders (e.g. In addition, platelet count has been positively correlated with serum levels of CRP and multiple cytokines including IL-1RA, IL-4, IL-6, IL-7, IL-8, IL-12, and IFN-γ in colorectal cancer patients (179). Platelets might modulate inflammation and tumour progression by(107, 168, 169, 180, 181):

	receptor pathways (121, 122), production of angiogenic factors (123), induction of antiapoptotic protein expression (124), or the activation of NF-kB transcriptional activity (125). Furthermore, PGE 2 might induce the massive infiltration of immune cells into the tumour microenvironment, which in turn results in the establishment of a chronic inflammatory microenvironment (120). There is also accumulating evidence that PGE 2 4. Antiplatelet drugs 4.1. Definition and classification Around 40 years after the commercialization of aspirin, an increased risk of bleeding was reported among patients treated with this molecule (92). Thereafter, aspirin had been 4.2.2. Indications Antiplatelet drugs are widely used in primary or secondary cardio/cerebrovascular prevention. In 2010, the French National Authority for Health (Haute Autorité de Santé, HAS) and the French Medicine Agency (Agence Nationale de Sécurité du Médicament et des produits de santé, ANSM) recommended that the prescription of an antiplatelet treatment 4.2.3. allergies, oedema, or hives), skin disorders (e.g. eczema or pruritus), blood and lymphatic
	induces aromatase activity leading to oestrogen synthesis (111). Thus, NSAIDs might prevent In addition, some studies reported heterogeneity of the NSAIDs-breast cancer risk suggested to have antithrombotic properties and to treat cardiovascular diseases (161). It is should be based on risk factors (assessed through a risk scale) and clinical characteristics system disorders (e.g. haemorrhage or gastrointestinal bleeding), gastro-intestinal disorders
	breast cancer development (especially ER+ breast cancers) by inhibiting COX-2 and associations according to BMI (147, 148), alcohol consumption (149), age at menarche (147), now recognized that aspirin decreases platelet aggregation and thrombus formation. At low such as risk of bleeding or history of peptic ulcer (163). (e.g. ulcers or gastritis), hepatic injuries (e.g. hepatitis or hepatic failure), renal and urinary
	consequently reducing the levels of PGE 2 and oestrogen. The inhibition of COX-1 could also age at first full term pregnancy (148), menopausal status (147, 150, 151), or MHT use (151). dose (≤325 mg per pill), aspirin is considered as an antiplatelet drug. Several other troubles (renal failure or gynecomastia), musculoskeletal disorders (e.g. arthritis or myalgia),
	partly explain the probable role of NSAIDs on the prevention of breast cancer. In brief, by While studies with self-reported data on NSAID use had limited data on exposure and were antiplatelet drugs are available such as ticlopidine and clopidogrel (Annex 2). The same In primary prevention, the need for platelet inhibition is calculated according to a specific respiratory (e.g. asthma or dyspnoea), and cardio-vascular problems (e.g. vasculitis,
	inhibiting COX-1 in platelets, NSAIDs prevent the release of pro-inflammatory/growth prone to exposure misclassification, studies using medico-administrative healthcare databases molecule can exist in different dosages and administration routes (oral or parenteral). In scale, called systematic coronary risk evaluation (SCORE), that was initiated by the European pneumopathy).
	Ticlopidine was the first agent of a new class of antiplatelet drugs, thienopyridines, which
	inhibit adenosine diphosphate (ADP). ADP, by binding the P2Y12 receptor on platelets, -co-administration of aspirin (75 to 160 mg/day) with clopidogrel (75 mg/day) is 4.3. Antitumor effects
	plays a central role in platelet activation and aggregation. Because ticlopidine was associated recommended after a stent placement or balloon angioplasty among people without history of Platelets, which are anucleate cell fragments generated by megakaryocytes in the bone
	epidemiological studies that NSAIDs reduce the risk of colorectal cancer (144, 145), but with significant toxicities, it has been substituted by other thienopyridines including myocardial infarction, marrow, are essentials in maintaining haemostasis and preventing bleeding (166). However,
	1 and COX-2 results for breast cancer are still not conclusive. A meta-analysis of 21 case-control and 12 clopidogrel, ticagrelor, cangrelor and prasugrel. Other antiplatelet drugs, more recent and less -co-administration of aspirin (75 to 160 mg/day) with prasugrel (75 mg/day) or with uncontrolled progression of platelets could lead to excessive thrombus formation, vascular
	were found in several human cancer tissues including breast tissues (112-119). Pro-cohort studies published in 2015 suggested that NSAIDs, especially aspirin (regardless of the often used, include glycoprotein platelet inhibitors (e.g., abciximab, eptifibatide, tirofiban), ticagrelor (180 mg/day) is recommended after an acute coronary syndrome, occlusion, and subsequently vascular diseases (167). Emerging evidence suggested that
	inflammatory PGs such as PGE2 might promote cancer cell growth, proliferation and dose) and selective COX-2 inhibitors, reduced breast cancer risk by about 10% (146). The protease-activated receptor-1 antagonists (e.g., vorapaxar) or adenosine reuptake inhibitor -administration of clopidogrel alone (75mg/day) is recommended when aspirin cannot be platelet could contribute to inflammation and to the pathophysiology of cancer (107, 168,
	(e.g., dipyridamole) (Annex 2). taken.

angiogenesis, boost cell migration and invasion, and facilitate tumour cells to escape from immune system's attacks

[START_REF] Gomes | Eicosanoids and cancer[END_REF]

. PGE2 has been shown to alter several pathways involved in cancer development (120) such as the activation the RAS-MAPK or epidermal growth factor mediators by platelets, which are supposed to be involved in breast cancer development [more details in the section on antiplatelet drugs (4.3. Antitumor effects)]. Furthermore, NSAIDs might alter other COX independent pathways that are involved in breast cancer development such as generation of reactive oxygen species, inhibition of NF-κB-mediated signals, decreasing insulin resistance, regulation of 15-LOX-1, or other apoptotic genes (103, 126-131).

3.4. Breast cancer prevention: up-to-date epidemiological evidence

In 1997, the IARC published its first handbook of cancer prevention focusing on NSAIDs

(132)

. In this first volume, an international working group of experts evaluated the evidence of aspirin (regardless of the dose), sulindac, piroxicam and indomethacin as potential cancer preventive drugs. Although there was sufficient evidence for the cancer preventive activities of these compounds in experimental studies

(126,(133)(134)(135)(136)(137)(138)

, the experts concluded that epidemiological studies provided limited or inadequate evidence. At this time, epidemiological studies about the hypothetical benefits of NSAIDs were mainly based on colorectal, oesophageal, and gastric cancers. Only a few epidemiological studies on NSAIDs and breast cancer risk were published (139-143). Today, there is good evidence from authors also suggested that the protective effect of NSAIDs might be restricted to ER/PR+ tumours. However, the benefits of aspirin or other NSAIDs was only statistically significant in case-control but not in cohort studies, where exposure was assessed prior to breast cancer onset.

(149,(152)(153)(154)(155)(156)(157)(158)(159)(160)

had very limited data on potential confounding factors and were not able to evaluate whether the associations differed by specific breast cancer subtypes or risk factors.

Furthermore, there is a lack of epidemiological studies considering the use of other drugs as potential confounders/effect modifiers. For all these reasons, further studies are needed with well-controlled confounding factors, available data on breast cancer subtypes, and more accurate assessment of NSAID use, including types, dose, and duration of use.

Take-home messages

NSAIDs have anti-cancer properties have been intensively investigated as cancer preventive agents in epidemiological studies and consistently associated with a decreased risk of colorectal cancer are hypothesized to decrease breast cancer risk but epidemiological findings are still inconclusive.

France, antiplatelet drugs other than low-dose aspirin are only available on prescription (103).

The WHO Collaborating Centre for Drug Statistics Methodology classifies antiplatelet drugs in the following group: "Platelet Aggregation Inhibitors Excl. Heparin" (ATC code: B01AC).

Preparations containing combinations of low-dose aspirin with statins or angiotensinconverting-enzyme inhibitors are classified in C10BX. Preparations containing combinations of low-dose aspirin with beta blocking agents are classified in C07FX.

4.2. Pharmacological properties and therapeutic use

4.2.1. Pharmacological actions

At low-dose, aspirin irreversibly inhibits COX-1 (particularly in platelets) and therefore the biosynthesis of TxA 2 (Figure

3

). The irreversible inactivation of COX-1 by low-dose aspirin leads to the virtually complete inhibition of TXA 2 (162). Of note, NSAIDs which reversibly inhibit COX-isozymes do not have this feature.

Society of Cardiology (https://www.heartscore.org/). The SCORE risk scale, which estimates the risk of fatal cardiovascular disease events over a ten-year period, is based on the following risk factors: gender, age, smoking, systolic blood pressure, and total cholesterol (164). Aspirin at a dose of 75-160 mg/day is recommended when cardiovascular risk is higher than 5%. Among people with diabetes, the cardiovascular risk is calculated according to the United Kingdom Prospective Diabetes Study (UKPDS) risk engine (http://www.dtu.ox.ac.uk/riskengine/index.php) (165).

In secondary prevention:

-administration of aspirin alone (at a dosage between 75 and 325 mg/day) is recommended after a stroke, transient ischemic attack or among people with a stable coronary heart disease (including after a coronary bypass) or peripheral arterial obstructive disease, -co-administration of aspirin (75 to 160 mg/day) with clopidogrel (75 mg/day), prasugrel (10 mg/day) or ticagrelor (180 mg/day) is recommended during the year following a myocardial infarction (with or without stent). Then, low-dose aspirin will be taken alone,

4.2.4. Main recommendations

Antiplatelet drugs should be used with caution among the elderly who run a far greater risk of side effects. Before starting antiplatelet agents, the patient should undergo assessment for bleeding risk. Temporary cessation of antiplatelet drugs increases the risk of cardiovascular events. Therefore, cessation must be professionally managed and based on a risk-benefit assessment. If the antiplatelet is an essential therapy, the medications should be resumed as quickly and safely as possible. Most common contraindications of antiplatelet agents include severe heart, hepatic or renal impairment, asthma, oesophagus or gastro-intestinal ulcers, stomach bleeding, any history of gastrointestinal problems, any haemorrhage, coagulation or haemostasis disorders, haemophilia, significant thrombocytopenia, recent chirurgical intervention, allergic or chronic rhinitis, and hypersensitivity to the medication. Antiplatelet drugs are not recommended during pregnancy (only during the third trimester for low-dose aspirin). 169). Indeed, thrombocytosis, defined as a platelet count of >400 K/μL of blood, was observed in cancer patients and associated with poor prognosis (170) in various cancers such as colorectal

(171, 172), breast (173), lung (174, 175)

, renal (176), cervical (177), and ovarian (178).

designed epidemiological studies on the associations between glucocorticoid use and breast cancer risk, with data on potential confounders and breast cancer subtypes, are needed to address current knowledge gaps.

	5.2.2. Indications colitis, hepatic or renal insufficiency, hypertension, severe myasthenia, hypokalaemia, there was no association between at least 3 prescriptions of systemic or inhaled 6. Thesis objectives CHAPTER 2: DATABASES
	Synthetic glucocorticoids are used to treat a wide range of diseases (103). Systemic sodium retention, lactase deficiency, or osteoporosis will need particular attention from glucocorticoids and invasive breast cancer risk [inhaled glucocorticoids: odds ratio
	glucocorticoids are used as hormone replacement therapy in case of adrenal insufficiency medical services. A long glucocorticoid therapy could be complemented by a diet low in (OR)=1.00 (0.95 -1.10), n exposed cases=593; systemic glucocorticoids: OR=1.00 (0.96 -My doctoral thesis aimed to better understand the role of inflammation on breast cancer 1. The EPIC study
	(e.g., in Addison's disease [a rare condition which is primarily caused by autoimmune sodium, low in sugar, high in protein, and in some cases, using vitamin D supplements and 1.10), n exposed cases= 908)] (230). No associations were found when the authors development using two complementary approaches:
	destruction of the adrenals, leading to deficiency of cortisol, aldosterone and adrenal PPI. categorized the glucocorticoid exposure into recent and former use, according to intensity or 1.1. Presentation of the cohort
	androgens]), rheumatologic conditions (e.g. arthrosis, tendinitis, sprain, bursitis, arthritis, duration of use, or stratified the analysis by menopausal status (pre, peri, postmenopausal). 1) to evaluate the associations between breast cancer risk and a panel of 11 inflammatory The European Prospective Investigation on Cancer and Nutrition (EPIC, https://epic.iarc.fr/)
	rheumatoid arthritis, or talalgia), autoimmune diseases (e.g. multiple sclerosis, inflammatory 5.3. Antitumor effects As mentioned by the authors themselves, they were not able to stratify their analyses by biomarkers (TNF-α, IFN-γ, IL-6, IL-8, IL-10, IL-13, IL-17D, IL-1RA, CRP, leptin study is a multi-centre ongoing prospective cohort study, mainly designed to investigate the
	bowel disease, ulcerative colitis, psoriasis, autoimmune haemolytic anaemia, or systemic The action of glucocorticoids in tumor progression is controversial and might have diverse breast cancer subtypes. However, glucocorticoids might have a different impact on breast and adiponectin) in about 1600 case-control pairs in the European Prospective aetiology of cancers (233). This study enrolled more than 521,000 participants, aged between
	lupus erythematosus), nephrology disorders (e.g. nephrotic syndrome), allergies (e.g. allergic influences depending on the type of cancer (213). Regarding breast cancer, potential cancer development according to breast cancer molecular status. Three other epidemiological Investigation into Cancer and Nutrition (EPIC) study 25 and 70, recruited between 1992 and 2000 from 23 centres across 10 Western European
	rhinitis), or respiratory diseases (e.g. chronic obstructive pulmonary disease). Systemic beneficial and harmful effects of glucocorticoids have been reported. On the one hand, studies suggested that "corticoid" or "steroid" use was associated with a decreased risk of countries including Spain, Italy, France, the United Kingdom, Greece, Germany, the
	glucocorticoids might prevent breast cancer development by affecting angiogenesis (214, breast cancer (160, 231, 232). However, there was no detailed information on exposure 2) to evaluate the associations between breast cancer risk and NSAIDs, antiplatelet drugs
	215), inhibiting growth and inflammatory factors (214), stimulating the expression of the and glucocorticoids using self-reported and drug reimbursement data in the EPIC
	sulfotransferase SULT1E1 (which plays a role in deactivating oestrogen) (213, 216), and study and its French component, the E3N (Étude Épidémiologique auprès de femmes
	suppressing cell migration/invasion (217, 218). In addition, glucocorticoids might exert some de la Mutuelle Générale de l'Éducation Nationale) cohort.
	Thus, whether glucocorticoids promote or inhibit breast cancer growth is still under
	dermatitis or skin rash) (209, 210). The side effects of other routes of administration of investigation.
	glucocorticoids are mainly the same as those seen with systemic glucocorticoids (211). The
	most frequent adverse effects of dermal glucocorticoids include atrophy, striae, rosacea, 5.4. Breast cancer prevention: up-to-date epidemiological evidence
	perioral dermatitis, acne, and purpura (212). Previous epidemiological studies suggested that use of systemic glucocorticoids increases the
	risk of non-Hodgkin lymphoma (223), skin (223-225), bladder (226), and prostate (227, 228)
	5.2.4. Main recommendations cancers.
	As many glucocorticoid-related adverse effects depend on dose and duration of treatment, it

glucocorticoids can also be used at high doses to treat lymphomas, leukaemia, or multiple myeloma and to mitigate the side effects of anticancer drugs. Topical glucocorticoids are used to treat conditions like dermatoses, eye inflammations, or ear infections. Inhaled glucocorticoids are used to treat asthma (103).

5.2.3. Main side effects

Systemic glucocorticoids are known to increase blood sugar levels (which can lead to diabetes), suppress the body's ability to absorb calcium (which can lead to osteoporosis), increase cholesterol and triglyceride levels, suppress the immune system (by decreasing leukocytes and cytokines production), induce a loss of muscle tissue, and resorb sodium leading to fluid retention. Glucocorticoids could also lead to cardiovascular problems (e.g. arrythmia, pulmonary embolism, heart failure, or hypertension), gastrointestinal troubles (e.g. gastrointestinal bleeding, or ulcers), kidney damages, metabolic troubles (e.g. weight gain or oedema), musculoskeletal disorders (arthritis or arthralgia), and skin disorders (acne, purpura, is recommended to use glucocorticoids during the shortest duration possible. Glucocorticoids must be mainly avoided in case of hypersensitivity, infection, psychiatric conditions, or surgical intervention. People with diabetes, history of peptic ulcer disease or ulcerative anti-tumour effects by reducing eicosanoid production through the inhibition of phospholipase A 2 (Figure

3

). On the other hand, glucocorticoid might promote breast cancer growth by facilitating tumour cells to escape from immune surveillance and inducing insulin resistance (219, 220). Recent experiment using mice models suggested that glucocorticoids might promote breast cancer metastases (221). This hypothesis is not new: results from a study published in the 1960's indicated that, compared to non-use, the use of adrenal steroids (including glucocorticoids) increased the risk of metastasis among breast cancer patients (222).

To my knowledge, only two epidemiological studies have been previously published, both based on data from nationwide medico-administrative databases from Northern Denmark

(229, 230)

. The latest study, which was an extension of the previous one (229) suggested that assessment and the number of exposed cases was small in two studies (n ≤ 22). There is thus a lack of epidemiological studies on the relation between glucocorticoid use and breast cancer risk. Further well-designed epidemiological studies should evaluate the glucocorticoidsbreast cancer associations according to breast cancer subtypes or risk factors as well as dose, duration, and timing of glucocorticoid use.

Take-home messages

Glucocorticoids play a complex/dual role in breast cancer initiation and progression in experimental studies are not associated with breast cancer risk in the only epidemiological study with detailed data on glucocorticoid exposure Further well-Netherlands, Denmark, Sweden, and Norway (Figure

  Information on MHT use covered ever and current use, age at first use, duration of use and brand name of current MHT use. Umeå did not have information of current MHT use and Malmö did not have information on age at first MHT use. From the MHT brand name, the type of hormone and the route of administration Sitting height was measured in France, Italy, Spain, Utrecht, Greece, Germany and Denmark (235). BMI was calculated for each participant and split into four categories:

	underweight (<18.5 kg/m²), normoweight (≥18.5 -<25 k g/m²), overweight (≥25 -<30) and
	obese (≥ 30 kg/m²).
	Physical activity
	The baseline questionnaire assessed past-year physical activity including occupational,
	leisure/recreational and household activities. For recreational and household activities,
	participants reported the duration of activities during a typical week during the past summer
	and winter. Household activities included housework, home repair, gardening and stair
	could be deduced.
	1.5.5. Anthropometric factors, physical activity, and energy intake
	Anthropometry
	All EPIC centres except Umeå and Norway, have either self-reported (France and part of the
	UK) or measured information on weight, height, hip circumference, and waist circumference

(WC)

. In Norway and Umeå, only self-reported information of height and weight were collected. In Oxford and in France anthropometric factors were measured only for a restricted number of participants, but self-reported weight and height were obtained from all individuals.

climbing. Recreational activities included walking, cycling and sports activities. A metabolic equivalent of task (MET) value was assigned to each reported activity according to the Compendium of Physical Activities (236). The mean numbers of hours per week of summer and winter household and recreational activities were estimated and then multiplied by the appropriate MET values to obtain MET-hours per week of activity. A four-level physical activity index was derived by combining occupational physical activity together with time participating in cycling and other physical exercise (such as keep fit, aerobics, swimming, and jogging). Participants were categorized into inactive, moderately inactive, moderately active and active (237).

1.5.6. Alcohol consumption and tobacco

  The second questionnaire (Q2) asked detailed information on menstrual factors (age at menarche, menstrual cycle length, menopausal status, and age at menopause), reproductive history (treatments for infertility and for each pregnancy: age at pregnancy, duration and outcome and breastfeeding), lifetime history of use of oral contraceptives, MHT, or other hormonal treatments (for each episode of use: drug name, age at first use, and duration of use), lifetime

	personal history of benign gynaecological diseases (for each disease: type of disease, age at
	diagnosis, diagnostic procedures, and treatment), lifetime personal history of other diseases
	(diabetes, stroke, cancer, etc.), and medical follow-up (including information on recent
	mammogram). The third (Q3) and the eighth (Q8) questionnaires also included a diet history
	questionnaire, and the fourth questionnaire (Q4) focused on specific anthropometric
	measures. Q2 to Q9 updated information on menopausal status and use of hormonal
	treatments. Q2 to Q11 updated information on health status as well as data such as weight
	and tobacco consumption. Q3 to Q9 included questions on current use of drugs. Q10 included
	questions on individual's autonomy and well-being, oral health, visual, and hearing ability.
	included questions on socio-demographic factors (educational level and current employment
	status), anthropometric measures (current and at different times in life), menstrual and
	reproductive factors (age at menarche, parity, age at first pregnancy, breastfeeding, and
	menopausal status), lifetime medical and surgical history, family history of cancer,
	gynaecological follow-up (pap smear frequency and date of last mammogram), lifetime
	tobacco consumption, and current physical activity.
	2.3. Follow-up

in metropolitan France and insured by a specific national health scheme covering mainly teachers (Mutuelle Générale de l'Education Nationale, MGEN), were invited to participate in the study. Twenty percent

(98,995 women)

agreed. Age and region of residence were not different between participants and non-participants (238). At baseline, participants signed an informed consent and replied to the first questionnaire (Q1), which was common to all EPIC centres, and 2.3.1. Self-reported data Every 2-3 years after recruitment, participants completed self-administered questionnaires to update previous information or to collect new information, as summarized in Figure

5

.

  For menopausal women, age at menopause was defined as age at the latest menstruation (unless due to hysterectomy and if the last menstrual period occurred before MHT use) or as age at bilateral oophorectomy, if relevant. If this information was not available, other information was used to determine age at menopause, in deceasing order of priority: self-reported age at menopause, age at first MHT use, and age at start of menopausal symptoms. When age at menopause could not be determined, women were imputed a menopausal age of 51 years for natural menopause or unknown type of menopause and 47 years in case of artificial menopause, ages which correspond to the median ages for natural and artificial menopause in the cohort, respectively.

Table 1 :

 1 Data collected on NSAIDs in EPIC.

	Definition of "regular" NSAID use
							Questions asked to participants
	Countries	Type		Frequency	Duration	Period
	The	Aspirin		Daily		Current	"Do you take aspirin daily?"
	Netherlands
	United	Aspirin			≥ 3 months	Current	"Have you ever taken aspirin continuously
	Kingdom						for 3 months or more?"
	Denmark	Aspirin		≥3 times/week		Last year "Have you used more than one painkilling
		Ibuprofen					tablet/month during the last year? If yes,
							how many pills containing aspirin and how
							many pills containing ibuprofen: none; 2-
							3/month; 1-2/week; 3-6/week; 1-3/day; 4-
							5/day; 6+/day?"
	France	Any	anti-	≥3 times/week		Current	"Are you currently taking anti-inflammatory
		inflammatory				drugs at least three times per week?"
		drugs
	Germany	Any NSAIDs			Current	"During the past 4 weeks, did you take any
							"regular" medications? If yes, please specify
							the medication."

  [HR insitu=1.27 (0.98 -1.65); HR invasive =0.[START_REF] Flower | Inhibition of prostaglandin biosynthesis[END_REF] (0.87 -1.05); P heterogeneity =0.05]. Risk estimates did not vary significantly by other breast cancer subtypes (P homogeneity ≥0.36), breast cancer risk factors (all P interactions between 0.10 and 0.96) or country (P heterogeneity =0.95). However, there was a borderline significant interaction by MHT use. "Regular" NSAID use was inversely associated with overall breast cancer risk among postmenopausal women who had ever used MHT [HR MHT =0.87 (0.76 -0.99)] but not among never MHT users [HR noMHT = 1.09 (0.95 -1.26); P interaction =0.06]. The test for interaction was stronger for invasive breast cancer [HR MHT =0.84 (0.73 -0.96); HR noMHT = 1.08 (0.93 -1.25); P interaction = 0.05], but far from significance for in situ cases [HR MHT =1.25 (0.82 -1.92); HR noMHT = 1.18 (0.81 -1.73); P interaction =0.99]. A similar pattern of associations by "regular"

  1, 2004. For NSAIDs, drug reimbursement data were complemented by self-reports before follow-up start. Women who self-reported NSAID current use in the 2000 or 2002 questionnaires were considered NSAID "recurrent" users from follow-up start. We also classified exposure according to time since last and first use, age at first use, cumulative duration of use, and cumulative number of defined daily doses (DDD). The DDD is the assumed average daily maintenance dose for a molecule used for its main indication in adults (241).Multivariable Cox regression models with age as the time scale and stratified by birth cohort (in 5-year categories) were used to estimate HRs for the association of exposure with breast cancer incidence, overall and by breast cancer subtypes. Exposure as well as other factors issued from the reimbursement database and covariates updated during follow-up were considered as time-varying parameters. Consequently, for a given drug, participants contributed follow-up as non-exposed until purchasing the drug for the second time in a three-month period. Cumulative duration/dose and time since last and first use were also updated during follow-up. Exposure, as well as other variables coming from the reimbursement database such as use of MHT, number of consultations and use of other drugs were, therefore, lagged by 6 months to minimize reverse causation bias due to early symptoms(242).

	Statistical analysis
	Educational level, recent mammogram, BMI, physical activity level, lifetime history of
	benign breast disease, family history of breast cancer, age at menarche, age at menopause,
	parity and age at first full-term pregnancy, lifetime use of oral contraceptives, lifetime use of
	MHT and alcohol consumption were systematically included in the multivariable models.
	Breastfeeding, number of consultations with the doctor during the preceding six months,
	smoking status, lifetime histories of comorbidities of interest and "recurrent" use of other
	drugs since January 1, 2004 were tested as potential confounding factors.
	Effect modification by age, BMI, MHT use, comorbidities, and other drugs (all considered as
	time-varying parameters) was evaluated by including cross-product interaction terms in the
	Cox models.

  , recurrent NSAID use was not associated with breast cancer risk [HR= 1.00 (0.92 -1.08)]. The NSAID-breast cancer associations did not differ by NSAID types, breast cancer subtypes, risk factors and comorbidities, nor by duration and dose of use. However, there was a statistically significant interaction by PPI use. Recurrent NSAID use was inversely associated with overall breast cancer risk among women who had ever recurrently used PPIs [HR =0.86 (0.74 -0.99)] but not among never/occasional PPI users [HR = 1.07 (0.97 -1.18);

	P interaction =0.01].

  Annex 1: Different types of nonsteroidal anti-inflammatory drugs ∞ . In the ATC classification system, the active substances are classified in a hierarchy with five different levels. The system has fourteen main anatomical/pharmacological groups or 1st levels. Each ATC main group is divided into 2nd levels which could be either pharmacological or therapeutic groups. The 3rd and 4th levels are chemical, pharmacological or therapeutic subgroups and the 5th level is the chemical substance".§ I reported only preparations containing acetylsalicylic acid. ∞ Excluding topical preparations.

	ATC codes ≠ SALICYLIC ACID AND DERIVATIVES (N02BA) § N02BA01 BUTYLPYRAZOLIDINES (M01AA) M01AA01 M01AA02 M01AA03 M01AA05 M01AA06 ACETIC ACID DERIVATIVES AND RELATED SUBSTANCES (M01AB) Molecules Acetylsalicylic acid Phenylbutazone Mofebutazone Oxyphenbutazone Clofezone Kebuzone M01AB01 Indometacin M01AB02 Sulindac M01AB03 Tolmetin M01AB04 Zomepirac M01AB05 Diclofenac M01AB06 Alclofenac M01AB07 Bumadizone M01AB08 Etodolac M01AB09 lonazolac M01AG04 Meclofenamic acid COXIBS (M01AH) M01AH01 Celecoxib M01AH02 Rofecoxib M01AH03 Valdecoxib M01AH04 Parecoxib M01AH05 Etoricoxib M01AH06 Lumiracoxib M01AH07 Polmacoxib OTHER ANTI-INFLAMMATORY AND ANTI-RHEUMATIC AGENTS, NON-STEROIDS (M01AX) M01AX01 Nabumetone M01AX02 Niflumic acid M01AX04 Azapropazone M01AX07 Benzydamine M01AX13 Proquazone M01AX14 Orgotein M01AX17 Nimesulide M01AX18 Feprazone M01AX22 Morniflumate M01AX23 Tenidap M01AX68 Combinations of Feprazone ≠ "M01AG02 Tolfenamic acid M01AG03 Fufenamic acid Annex 2: Different types of antiplatelet drugs.
	M01AB10	Fentiazac
	M01AB11	Acemetacin
	M01AB12	Difenpiramide
	M01AB13	Oxametacin
	M01AB14	Proglumetacin
	M01AB15	Ketorolac
	M01AB16	Aceclofenac
	M01AB17	Bufexamac
	M01AB51	Combinations of indometacin
	M01AB55	Combinations of diclofenac
	OXICAMS (M01AC)
	M01AC01	Piroxicam
	M01AC02	Tenoxicam
	M01AC04	Droxicam
	M01AC05	Iornoxicam
	M01AC06	Meloxicam
	M01AC56	Combinations of Meloxicam
	PROPIONIC ACID DERIVATIVES (M01AE)
	M01AE01	Ibuprofen
	M01AE02	Naproxen
	M01AE03	Ketoprofen
	M01AE04	Fenoprofen
	M01AE05	Fenbufen
	M01AE06	Benoxaprofen
	M01AE07	Suprofen
	M01AE08	Pirprofen
	M01AE09	Flurbiprofen
	M01AE10	Indoprofen
	M01AE11	Tiaprofenic acid
	M01AE12	Oxaprozin
	M01AE13	Ibuproxam
	M01AE14	Dexibuprofen
	M01AE15	Flunocaprofen
	M01AE16	Alminoprofen
	M01AE17	Dexketoprofen
	M01AE18	Naproxcinod
	M01AE51	Combinations of ibuprofen
	M01AE52	Naproxen and esomeprazole
	M01AE53	Combinations of Ketoprofen
	M01AE56	Naproxen and misoprostol
	FENAMATES (M01AG)
	M01AG01	Mefenamic acid

ACKNOWLEDGMENTS

I would like to express my grateful acknowledgments to all the people who make this 4.3. Paper Status: Accepted. Contribution: First author, conducted statistical analyses, wrote the first draft of the manuscript, submitted it to the journal, and replied to reviewers' comments.

Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article Agency for Research on Cancer / World Health Organization. The Ligue National Contre le Cancer for funding this PhD, the International Agency for Research on Cancer for completing my doctoral grant, as well as the Institut national du cancer and World Cancer Research Fund for funding the study on inflammatory biomarkers and breast cancer I was fortunate to work on.

INFLAMMATION AND BREAST CANCER RISK IN THE EPIC COHORT

consumption, physical activity, education level and diet), history of previous illnesses, reproductive factors, and anthropometric factors. Extensive details on the standardized recruitment procedures have been published previously (233). Below, I describe briefly the collected variables used in the present projects. 1.2.2. Biological data At baseline, blood samples were collected from 387,889 individuals including 246,000 women from all countries. Blood was collected according to a standardized protocol in France, Germany, Greece, Italy, the Netherlands, Norway, Spain, and the United Kingdom (233). Serum (except in Norway), plasma, erythrocytes (except in Norway), and buffy coat were separated and aliquoted in 28 plastic straws of 0.5 ml each. These samples were then split into two mirror halves of 14 aliquots each and one was stored locally and the other half in a centralized biobank at the IARC in liquid nitrogen (- 196 °C). In Norway, blood fractions were collected in 20 plastic straws of 0.5 ml each (16 plasma and 4 buffy coat), half of which (8 plasma et 2 buffy coat) were shipped to IARC for storage in the central repository. In Denmark and Sweden, blood samples were stored in tubes (not in plastic straws) in local repositories (IARC is not suitable for storing tubes). In Sweden, the samples are kept in deep freezers at -70°C, and in Denmark in nitrogen vapor (-150°C).

Cancer events

Cancer cases were identified during follow-up based on population cancer registries in Denmark, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom, and on a combination of methods, including health insurance records, contacts with cancer and pathology registries, and active follow-up of participants and their next of kin in France, Germany, and Greece. In the present work, first primary cases of breast cancer were polyarthritis, Horton's disease (Q9-Q11), rheumatism (Q1), spondylarthritis ankylosing (Q10-Q11), arthrosis (Q8, Q10), arthritis (Q8), migraine (all questionnaires from Q2 except Q8), asthma (all questionnaires except Q7), chronic bronchitis (all questionnaires), hay fever (Q2, Q3, Q7, and Q8), and chronic inflammatory bowel diseases (all questionnaires except Q11). We considered that a woman has a history of one of the previous diseases when she reported it for the first time in a questionnaire.

Gynaecological follow-up

In Q1, women were asked to report if they had already undergone a mammogram together with the date of their last mammogram. In every other questionnaire, women reported whether they had undergone a mammogram since their answer to the previous questionnaire.

In Q3, Q9, Q10, and Q11, the date of the last mammogram was asked.

Hormonal and reproductive factors

Age at menarche

In the first two questionnaires, women were asked to report the age at which they had their first menstrual period.

Reproductive life

Number of pregnancies was recorded through Q1 and Q2. In Q2, other information was collected such as age at the beginning of each pregnancy (up to 12) and the duration of each pregnancy. For each birth, women were also asked to report if they breastfed their child and to specify the breastfeeding duration.

Menopausal status and age at menopause

Menopausal status was updated throughout the follow-up, based on self-reports of being postmenopausal (Q1, Q6, Q7, and Q8) as well as information on MHT use (Q2-Q8), oral contraceptives (Q2-Q8), menstrual cycles (Q2-Q8), date of last menses (Q2, Q3, Q4, Q6, and Q7), history of menopausal symptoms (Q2-Q7), history of hysterectomy and age at surgery (Q1, Q2, Q5, Q6, Q7, and Q8), history of simple or bilateral oophorectomy and age at surgery (Q2, Q5, and Q8), history of radiotherapy and / or chemotherapy (leading to definitive menses cessation; Q2). Women were considered postmenopausal if they reported 12 consecutive months of amenorrhea (unless due to hysterectomy), bilateral oophorectomy, ever use of MHT, or if they self-reported to be menopausal. Menopause was considered to be 2.6.7. Alcohol consumption and tobacco In Q3, Q5, and Q8, women were asked to report their current consumption of alcohol. The type, frequency and quantity of alcohol consumption were collected. These data were used to estimate the quantity of ethanol consumption per day.

In all questionnaires except Q9, women were asked to report their smoking status. They were asked to report whether they were a current regular smoker (at least 1 cigarette per day), current occasional smoker, past regular smoker, past occasional smoker, or never smoker.

Other information was collected in some questionnaires such as the type of tobacco, frequency, or duration of smoking.

CHAPTER 4: NONSTEROIDAL ANTI-INFLAMMATORY DRUGS AND BREAST CANCER RISK IN THE EPIC STUDY

Objective

The objective of this chapter was, using data from the EPIC study, to investigate the associations between "regular" NSAID use and breast cancer risk, overall and by breast cancer subtypes and according to various breast cancer risk factors.

Materials and methods

Study population

Female participants in EPIC were eligible for inclusion in the current study if they had no history of cancer prior to recruitment, no missing date of cancer diagnosis or censoring and no missing data on exposure. A total of 140,981 women were therefore included in the current study.

Follow-up

Women were followed from study entry (around 1990) until the date of last known contact, cancer diagnosis, death, emigration, or the end of the follow-up period (between 2008 and 2014 depending on the centre), whichever came first.

Exposure assessment

The baseline questions asked to the participants are noted in Table 1. In EPIC, "regular" use of NSAIDs was defined by combining participants responding "yes" to the question in France, in the United Kingdom, and in the Netherlands, those who cited NSAID and/or aspirin as "regular" medications in Germany, and people responding at least "3 pills per week " for using aspirin or ibuprofen in Denmark.

Take-home messages

The results of this thesis suggest that:

Inflammation plays a modest role in breast cancer development and its role might be limited to postmenopausal women.

The relationships between the drugs with anti-inflammatory properties and breast cancer risk are complexes. In addition, because the studied drugs have several pharmacological properties, we do not know whether the drugs might impact breast cancer development through their anti-inflammatory properties or through other ones.

Since breast cancer is a heterogeneous disease, there is a need to robustly evaluate the role of inflammation or anti-inflammatory drugs according to breast cancer subtypes within large and well-powered analyses where less common tumour subtypes or subgroups can be addressed, and, for medications, a long duration of use can be captured.

Annex 4: Description of the SIDIAP database and study population.

The Information System for the Development of Research in Primary Care database

SIDIAP (http://www.sidiap.org/index.php/en) is a large prospective population-based database in Catalonia. In Spain, general practitioners (GP)s and nurses play an essential role in the public health care system as they are responsible for primary health care, long-term prescriptions, and specialist/hospital referrals. The Spanish public healthcare system covers more than 98% of the population, and more than two thirds of the Catalan population see their GP at least once a year ( 263

Case-control selection

In the following, we have defined SIDIAP0 as the date of entry into the SIDIAP database. In a first step, we extracted data from women with at least 1.5 years of medical history in the SIDIAP database (i.e. not deceased, not lost to-follow-up, not reaching the end of data availability into SIDIAP within 1.5 years after SIDIAP0). Women with any cancer (except basal cell carcinoma) within 1.5 years after SIDIAP0 are excluded.

Within the cohort defined above, we will perform a nested case-control study using risk set sampling (cases are eligible for sampling as controls before their diagnosis date).

Cases are women with breast cancer diagnosed after SIDIAP0+1.5 years. For cases, the date of breast cancer diagnosis defines the index date, and T0 is equal to SIDIAP0. We further define T1 as T0 + one year. Controls (up to 10 per case) are women with no cancer (any cancer except basal cell carcinoma) diagnosed between SIDIAP0 and the time of the case's breast cancer diagnosis, alive and still registered into SIDIAP at that date. They are matched on year of birth (±1 year), primary care centre, menopausal status, and must have a medical history duration (time between SIDIAP0 and the time of the matched case's breast cancer diagnosis) >= the medical history duration of the matched case. Controls are assigned the same index date, the same T0, and the same T1 as those of the matched case (since medical history of the control is longer than the one of the case, a new T0 is defined, equal to the T0 of the case). For latency considerations and in order to minimize reverse causality, we define a lagged index date which is the index date minus 6 months (242). Any exposure or adjustment data recorded after the lagged index date (likely not causal) is disregarded.

Index date = date of each case's breast cancer diagnosis

Lagged index date = Index date minus 6 months

The T0-T1 period is used to identify prevalent NSAID exposure. It is a period with no cancer diagnoses (see Figure below). Finally, 24,697 cases will be included and 117,916 matched controls. The median age of the study population at T0 is 53 years old. In the study population 67% of women had at least 1 prescription of NSAID or aspirin and 34% had at least 2 prescriptions in any three-month period.