Xavier Mignard

Anne-Marie Ruppert

Martine Antoine

Julie Vasseur

Nicolas Girard

Julien Mazieres

Denis Moro-Sibilot

Vincent Fallet

Nathalie Rabbe

Françoise Thivolet-Béjui

Julien Mazières

Françoise Thivolet- Bejui

Isabelle Rouquette

Sylvie Lantuejoul

Alexis Cortot

Raphaël Saffroy

Jacques Cadranel

Antoinette Lemoine

Marie Wislez
email: marie.wislez@aphp.fr.

Marie Pr

Wislez

c-MET Overexpression as a Poor Predictor of MET Amplifications or Exon 14 Mutations in Lung Sarcomatoid Carcinomas

Keywords: MET, immunohistochemistry, amplification, exon 14 mutations, lung sarcomatoid carcinoma

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

Introduction

The mesenchymal-to-epithelial transition (MET) pathway includes the tyrosine kinase receptor (TKR) c-MET, its ligand, hepatocyte growth factor (HGF), as well as downstream pathways involved in tumor growth, cell survival, invasion, and cell migration [START_REF] Cortot | Exon 14 Deleted MET Receptor as a New Biomarker and Target in Cancers[END_REF]. MET pathway activation involves different mechanisms, such as amplification of the MET gene, exon 14 splicing site mutations, activating point mutations, or HGF-dependent upregulation. Amplification of the MET gene, characterized by an increased MET gene copy number measured by FISH (fluorescence in situ hybridization), is found in 1 to 4% of non-small-cell lung cancers (NSCLCs) [START_REF] Cappuzzo | Increased MET gene copy number negatively affects survival of surgically resected non-smallcell lung cancer patients[END_REF].

Mutations in MET exon 14 and its flanking introns can induce exon 14 splicing that is thought to cause loss of the intracellular juxta-membrane domain, whilst increasing c-MET expression at the membrane. The diversity of MET exon 14 alterations, including base substitutions or indels that disrupt the branch point of intron 13, the 3 ' splice site of intron 13, or the 5 ' splice site of intron 14, requires improved technologies such as next-generation sequencing (NGS) to cover the whole c.2942-64 to c.3082+42 region [START_REF] Tong | MET Amplification and Exon 14 Splice Site Mutation Define Unique Molecular Subgroups of Non-Small Cell Lung Carcinoma with Poor Prognosis[END_REF].

Recently, MET tyrosine kinase inhibitors (TKIs), such as crizotinib and capmatinib, have proven their efficacy on tumors harboring MET mutations or amplifications, suggesting oncogene addiction, therefore rendering MET screening highly relevant (4), [START_REF] Ou | Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification[END_REF].

Lung sarcomatoid carcinomas (LSCs) are rare tumors, accounting for less than 3% of all NSCLCs. They are more prevalent among males and smokers and characterized by both high resistance to platinum-based chemotherapy and poor prognosis [START_REF] Vieira | Efficacy of first-line chemotherapy in patients with advanced lung sarcomatoid carcinoma[END_REF]. A higher frequency of MET exon 14 mutations has been reported in LSCs [START_REF] Liu | Next-Generation Sequencing of Pulmonary Sarcomatoid Carcinoma Reveals High Frequency of Actionable MET Gene Mutations[END_REF], with a prevalence ranging between 4.9% and 31.8% [START_REF] Saffroy | MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung[END_REF].

Techniques for the diagnosis of MET amplification and exon 14 mutations are not readily accessible in routine practice. FISH is expensive and time-consuming, whereas NGS panels require sufficient tumor material and high DNA quality, although samples usually are needlebiopsied formalin-fixed paraffin-embedded (FFPE) tumors. IHC could prove effective as a screening test in molecular abnormalities such as ALK rearrangements. While FISH remains the gold standard, ALK IHC is highly sensitive (100%), FISH being only performed in ambiguous IHC cases [START_REF] Mcleer-Florin | Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study[END_REF].

The aim of this study was to compare MET IHC, FISH, and exon 14 mutation detection in a series of LSC in order to evaluate IHC as a screening test.

Patients and methods

Patients and tissue tumor collection

Tissue samples were obtained from surgical lung biopsies of all consecutive patients with LSC diagnosed between 2005 and 2012 in four referral thoracic oncology centers. Clinical data and tumor characteristics were recorded as previously described [START_REF] Saffroy | MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung[END_REF]. Each patient signed an informed consent form as required by national guidelines and samples were collected in line with current legislation.

Immunohistochemistry (IHC)

In brief, c-MET protein expression was assessed by IHC on 3µm-thick sections of FFPE tumor samples, using the rabbit monoclonal primary antibody SP44 (Ventana, Arizona, USA).

The MetMab score was calculated using the method detailed in Table 1 [START_REF] Spigel | Results From the Phase III Randomized Trial of Onartuzumab Plus Erlotinib Versus Erlotinib in Previously Treated Stage IIIB or IV Non-Small-Cell Lung Cancer: METLung[END_REF]. The H-score was obtained by multiplying the intensity (from 0 to 3) by the percentage of positive cells (from 0 to 100%), thus obtaining a scale ranging between 0 and 300. The H-score positivity threshold was 150 [START_REF] Cappuzzo | Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer[END_REF]. IHC was conducted by a referent pathologist (Dr. M. Antoine).

Fluorescence in situ hybridization (FISH)

MET gene amplification was assessed using the MET/CEP7 dual color probe set from Zytovision (Clinisciences, France), according to the manufacturer's instructions. MET gene amplification was defined as a MET gene copy number ≥5 and a MET/CEP7 ratio >2 (2), performed by the same referent pathologist.

Molecular testing for MET gene sequence abnormalities

Overall, 10μm-thick sections were cut from the paraffin blocks. Tumor enrichment was performed through selection and macro-dissection of areas with at least 50% tumor cells. Total DNA was extracted and purified after paraffin removal, as previously described [START_REF] Saffroy | MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung[END_REF]. All MET alterations from c.2942-64 to c.3082+42 affecting exon 14 splice sites that are indels occurring within splice donor and acceptor sites were detected in the routine molecular testing on ISO15189 certified genetic platforms. Mutations analysis methods were previously reported [START_REF] Saffroy | MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung[END_REF]. All tumor samples were tested by a combined strategy of High Resolution Melting (HRM) assay using Lightcycler 480 system (Roche Diagnostics) in order to screen all gene sequence abnormalities of exon 14 of the MET gene c3082, c3082+1, c3082+2, and c3082+3, confirmed using MassARRAY iPLEX technology (Agena Bioscience). Samples for which material was available (n=40) were also tested by NGS using the solid tumor solution by Sophia Genetics® based on the xGen Lockdown IDT® probe-based capture technology.

Molecular screening for other mutations

Mass spectrometry was employed to test 214 mutations affecting 26 oncogenes and tumor suppressor genes (Panel Lungcarta©MassARRAY iPLEX genotyping technology [Agena Bioscience, San Diego, USA]), as previously described [START_REF] Saffroy | MET exon 14 mutations as targets in routine molecular analysis of primary sarcomatoid carcinoma of the lung[END_REF].

Statistical analyses

Continuous variables were expressed as medians with [min, max] intervals. Categorical variables were expressed as percentages. Comparisons between non-parametric continuous variables were conducted via Mann-Whitney test, and those between categorical variables with the Chi-squared test, or the Fisher's exact test when n <5. Correlation between nonnormally distributed continuous variables was calculated using Spearman's correlation coefficient. All the tests were two-sided, with results considered significant when p <0.05. Analyses were performed using GraphPad Prism (GraphPad Software Inc, California, USA).

Results

General characteristics

Between 2005 and 2012, 81 patients with LSC were included, of whom 60 (74%) were male, and 75 (94%) were smokers (Table 2). The median age was 62 years. At diagnosis, 37 patients (46%) were at stage I-II disease, and 42 (52%) Stage III-IV disease. The most common histological subtype consisted of pleomorphic carcinoma (n=63, 77.5%). The most commonly detected mutations were KRAS (n=21, 26%), and EGFR (n=11, 13.5%). Positive c-MET IHC was observed in 15 (18.5%) tumors using the H-score (median 30 [0 -260]), and in 14 (17%) using the MetMab score (median 0 [0 -3]). True MET amplification was found in six patients (8.5%) and MET exon 14 mutation in five (6%). High MET amplification (MET/CEP7 ratio ≥5) was found in two patients.

Characteristics of patients with true MET amplification (MET gene copy number ≥5 and ratio MET/CEP7 >2)

Men represented 100% of patients with MET amplification, vs 71% (n=46) of patients without amplification (p=0.18, Table SI). Tumors with MET amplification all displayed a pleomorphic histological subtype, vs 50 (77%) without amplification (p=0.33). Patients with MET amplification exhibited less KRAS mutations, yet the difference did not reach statistical significance. Patients with polysomy were not considered (n=8, 11%).

Characteristics of patients with MET exon 14 mutations

MET exon 14 mutations were detected in five patients (6%). They were more often women (80% vs 22.5%, p=0.02). No other significant differences were found according to MET mutation status (Table SII).

Correlation between IHC and MET FISH / MET exon 14 mutation

In total, 15 patients (18.5%) had a positive IHC using the H-score and 14 (17%) using the MetMab score. Among MET amplifications (n=6), three (50%) had a positive c-MET IHC. No MET exon 14 mutations were associated with MET amplification. One tumor sample out of five (20%) with exon 14 mutation exhibited c-MET positive IHC (Figure 1). Among chromosome 7 polysomies (n=8), one (12.5%) had a positive IHC, with no significant association (p=1.0). Two (25%) had a MET exon 14 mutation (versus 3% MET exon 14 mutations in patients without polysomy) with a significant association (p=0.02).

The correlation between IHC H-score and FISH proved weakly positive (R coefficient between 0 and 0.5) (MET gene copy number ≥5 regardless of MET/CEP7 ratio, i.e. including polysomies, in Figure 2, then MET gene copy number ≥5 and MET/CEP7 >2, i.e. without polysomies, in Figure 3) .

Considering FISH as the gold standard for MET amplification, IHC sensitivity was 50%, specificity 83%, positive predictive value 21.4%, and negative predictive value 94.7%. For high MET amplifications, IHC sensitivity was also 50%. IHC sensitivity for MET exon 14 mutations was 20%, with a specificity of 83%, positive predictive value of 7%, and negative predictive value of 94%.

Discussion

In this cohort of surgically resected LSCs, c-MET IHC overexpression, as well as MET amplification and MET exon 14 mutations were analyzed, to determine whether IHC could be a screening test for amplification or mutation. Altogether, 14 tumors (17%) had positive c-MET IHC, six had a MET amplification (8.5%), and five a MET exon 14 mutation (6%).

As MET amplification and exon 14 mutations are potential targets for TKIs (4) [START_REF] Ou | Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification[END_REF] in NSCLCs, and particularly in LSCs, these abnormalities must be diagnosed as efficiently as possible.

In this study, c-MET IHC could not be considered as a screening test either for MET amplification or MET exon 14 mutations, as sensitivity (50% and 20% respectively) and correlation (r=0.27) proved poor, with similar results found for MetMab and H-score. Current data regarding correlation between IHC and MET molecular alterations are discordant. Watermann et al. found a weak correlation (r=0.06, p >0.05) between MET IHC and FISH in 214 NSCLC samples [START_REF] Watermann | Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?[END_REF]. Casadevall et al. showed no association between IHC and FISH in a nonsquamous NSCLC cohort [START_REF] Casadevall | MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC)[END_REF]. Conversely, Park et al. reported a significant association between IHC score and MET amplification (p<0.001) using the Chi-squared test on a large series of 316 adenocarcinomas, with neither sensitivity nor correlation reported [START_REF] Park | MET amplification, protein expression, and mutations in pulmonary adenocarcinoma[END_REF]. Additionally, Tong et al. revealed in 687 NSCLCs a significant association between IHC and amplification, and between IHC and exon 14 mutation (p <0.001), based on Chi-squared test analyses. Nevertheless, the correlation using Spearman's test proved to be weak [START_REF] Tong | MET Amplification and Exon 14 Splice Site Mutation Define Unique Molecular Subgroups of Non-Small Cell Lung Carcinoma with Poor Prognosis[END_REF]. Furthermore, whether significant association between IHC and amplification/mutation does exist or not, sensitivity is a better parameter for diagnosis purposes.

For several other molecular abnormalities, such as ALK rearrangement, IHC proves to be an effective screening option. ALK IHC and FISH are highly correlated, with a sensitivity for IHC approaching 100% [START_REF] Mcleer-Florin | Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study[END_REF]. FISH is performed only in ambiguous IHC cases. For ROS1 rear-rangements, IHC sensitivity compared to FISH is reported close to 100% with a specificity of 97% [START_REF] Mescam-Mancini | On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas[END_REF].

As MET exon 14 mutations induce loss of ubiquitination and increased c-MET membrane presence, their lack of association with c-MET overexpression remains to understand. One explanation is that the oncogenic properties of MET exon 14 mutations involve other mechanisms (loss of serine 985 with increased kinase activity, loss of aspartate 1002 resulting in loss of pro-apoptotic signals) [START_REF] Cortot | Exon 14 Deleted MET Receptor as a New Biomarker and Target in Cancers[END_REF]. Another explanation is that LSCs have a different genomic background compared to NSCLCs, which might influence these results. Indeed, MET gene abnormalities occur in a context of other oncogenic and tumor suppressive genes abnormalities involving TP53, LKB1 or EGFR point mutations, or gene copy number variations that can influence the c-MET protein expression.

Regarding MET exon 14 mutation detection, we used a combined technique (HRM + Mas-sARRAY) for all tumor samples (n=81). This allows detection of point mutations, and indels that are deleterious with a higher sensitivity than NGS. Nevertheless, the MassARRAY technology can miss some large deletions. Therefore, NGS was performed on tumor samples with sufficient material (n=40) to increase detection sensitivity. It is worth noticing that NGS allowed detection of one exon 14 mutation that was not diagnosed by HRM + MassARRAY, but also that MassARRAY allowed diagnosis of one more exon 14 mutation, undetected by NGS (data not shown).

Few studies focus specifically on LSC. The main cohorts investigating MET pathway abnormalities in LSCs are summed up in Table SIII. Discrepancies exist in the prevalence of positive IHC, amplification or exon 14 mutations, between those cohorts. Our results tend to be on the low end of each one of those abnormalities. It is however worth noticing that patients with exon 14 mutations and negative IHC exist in other series. These discrepancies can be explained by the small number of positive cases, which also reduces the power of the given sensitivity and specificity values.

Figures and tables

5 )

 5 MET = Mesenchymal to Epithelial Transition; EGFR = Epidermal Growth Factor Receptor; TKI = tyrosine kinase inhibitor; KRAS = Kirsten rat sarcoma oncogene; BRAF = B-raf proto-oncogene; PI3KCA = Phosphatidyl-inositol-3-kinase Catalytic Subunit Alpha; IHC = immunohistochemistry; FISH = Fluorescence in situ hybridization.

Table 1 :

 1 IHC results according to the MetMab score

	≥50% strong intensity	3+
	≥50% strong or moderate intensity, but <50% strong intensity	2+	Positive IHC
	≥50% strong or moderate or weak intensity,
	but <50%

moderate or weak intensity 1+ Negative IHC No staining or <50% of any intensity 0Table 2 :

 2 Characteristics of overall population (n=81)

	Variable	All patients (n = 81)
	Gender
	Male	60 (74)
	Female	21 (26)
	Age
	Median (Range)	62 (44 -81)
	Ethnicity
	Caucasian	65 (80)
	Asian	0 (0)
	Northern African	10 (12.5)
	Sub-Saharan African	1 (1.5)
	Smoking history
	Smoker	75 (94)
	Non-smoker	5 (6)
	Clinical stage
	Stage I -II	37 (46)
	Stage III -IV	42 (52)
	Histological subtype
	Pleomorphic carcinoma	63 (77.5)
	Spindle cell carcinoma	4 (5)
	Giant cell carcinoma	6 (7.5)
	Other	8 (10)
	Mutations
	MET exon 14	5 (6)
	EGFR (TKI sensitizing)	11 (13.5)
	KRAS	21 (26)
	BRAF	2 (2.5)
	PI3KCA	4 (5)
	Positive IHC
	MetMab	14 (17)
	H-score	15 (18.5)
	Positive FISH	14 (20)
	Polysomy	8 (11)
	True MET amplification	6 (8.

Acknowledgements

The authors extend their thanks to Mrs. Gwenaëlle Pontdeme (Sorbonne Universités, UPMC Univ Paris 06, GRC n°04, Theranoscan, F-75252, Paris, France), Anita Rodenas, and Christel Daubrosse (Pathology and Cytology Laboratory, Tenon University Hospital, AP-HP) for their technical assistance regarding IHC.

Our findings suggest that MET IHC may not be employed as a screening tool. MET FISH and molecular biology techniques (NGS, WES, and fragment analysis) are recommended for the detection of amplifications or exon 14 mutations in NSCLC in routine practice.

 5) p = 0.88 p = 0.9 p = 0.9 p = 0.84


Fabienne El-Khoury Lesueur

Anne-Laure Sutter-Dallay

Lidia Panico

Elie Azria

Judith Van Der Waerden

Nolwenn Regnault Vauvillier

Marie-Aline Charles

Maria Melchior

Affiliations

The Perinatal Health of Immigrant Women in France: A Nationally Representative Study

Keywords: Migrant, women, maternal health, pregnancy, overweight, obesity, gestational diabetes mellitus, smoking, alcohol

Objective

Despite the healthy migrant effect, immigrants and descendants of immigrants face health challenges and socio-economic difficulties. We examine the health of women of migrant origin during the perinatal period.

Methods

Data come from approximately 18,000 women participating in the nation-wide French ELFE (Etude Longitudinale Française Depuis l'Enfance) study. We studied pre-pregnancy BMI, gestational diabetes mellitus (GDM), as well as tobacco, and alcohol consumption during pregnancy according to migrant status and region of origin.

Results

Women from North-Africa and Turkey had a higher risk of pre-pregnancy overweight and GDM, while women from Eastern Europe and Asia had a lower risk of pre-pregnancy overweight and obesity, but a higher risk of GDM compared to non-immigrants. Women from Sub-Saharan Africa had a higher risk of being overweight or obese pre-pregnancy.

Compared to non-immigrants, immigrants -but not descendants of immigrants-had lower levels of tobacco smoking, while descendants of immigrants were less likely to drink alcohol during pregnancy.

Conclusions

Women of migrant origin have some healthier perinatal behaviour compared to non-immigrants, but some groups are at risk of overweight and having GDM.

INTRODUCTION

Western European countries' populations comprise a considerable and growing proportion of immigrants and descendants of immigrants (OECD and UN-DESA 2013) with a sizeable proportion of births to foreign-born women (Europ-Peristat 2013). Immigrants to Europe face particular challenges, particularly in terms of socio-economic conditions since they tend to have higher rates of unemployment and work in occupations that are less advantaged than natives (OECD 2014). This disadvantage persists to Europe-born children of immigrants (OECD Centre for Opportunity and Equality 2017) and influences the health status of both generations [START_REF] Rechel | Migration and Health in the European Union[END_REF][START_REF] Missinne | Depressive symptoms among immigrants and ethnic minorities: a population based study in 23 European countries[END_REF][START_REF] Almeida | Maternal Healthcare in Migrants: A Systematic Review[END_REF].

Immigrants are also generally younger than non-immigrants (Eurostat 2017), with an increasing share of women [START_REF] Kulu | Family Dynamics Among Immigrants and Their Descendants in Europe: Current Research and Opportunities[END_REF][START_REF] Kulu | Fertility by Birth Order among the Descendants of Immigrants in Selected European Countries[END_REF].

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To date, European data on the health of immigrants and their descendants is incomplete and shows contradictory results.

On the one hand immigrants benefit from the reported 'healthy migrant effect' [START_REF] Almeida | Maternal Healthcare in Migrants: A Systematic Review[END_REF], whereby individuals who are able to migrate are healthier than their non-migrating counterparts, or non-immigrants. At the same time, because of hardship experienced during migration and in the country of arrival, as well as difficulties in access to health care and low health literacy [START_REF] Davies A, Basten | Migration: A Social Determinant of the Health of Migrants[END_REF], immigrants have elevated levels of various health problems [START_REF] Rechel | Migration and health in an increasingly diverse Europe[END_REF].

These mixed results are also observed in France: while early work showed that immigrants have an advantage in terms of mortality, more recent work indicates elevated levels of mortality, perceived health and psychological difficulties among immigrants [START_REF] Guardia | Prévalence des troubles psychiatriques et addictologiques auprès de trois générations successives de migrants : résultats d'une étude menée en population générale[END_REF][START_REF] Moullan | Why is the 'healthy immigrant effect' different between European countries?[END_REF][START_REF] Khlat | Health and Mortality Patterns Among Migrants in France[END_REF]. A reason for mixed results in the literature is that some studies pool heterogeneous groups -this is the case of studies that examine the health of 'ethnic minorities', pooling immigrants and their descendants together [START_REF] Falah-Hassani | Prevalence of postpartum depression among immigrant women: A systematic review and meta-analysis[END_REF]). Yet immigrant populations are varied not only in terms of geographic region of origin or ethnicity but also in terms of their migration history; therefore identifying populations at risk of perinatal health problems entails distinguishing specific subgroups.

Research shows that compared to non-immigrant women, immigrants who are pregnant tend to have healthier behaviours (e.g. tobacco and alcohol use) but worse physical health (e.g. BMI, gestational diabetes) and higher maternal mortality [START_REF] Gagnon | International migration and gestational diabetes mellitus: a systematic review of the literature and meta-analysis[END_REF]Saurel-Cubizolles et al. 2012;[START_REF] Delavari | Acculturation and obesity among migrant populations in high income countries -a systematic review[END_REF][START_REF] Melchior | Tobacco and alcohol use in pregnancy in France: The role of migrant status: The nationally representative ELFE study[END_REF][START_REF] Mårdby | Consumption of alcohol during pregnancy-A multinational European study[END_REF]). Yet to our knowledge, past studies have rarely examined women's region of origin and studied the health of descendants of immigrants. Pre-pregnancy BMI, gestational diabetes mellitus (GDM), alcohol and tobacco consumption are associated with adverse maternal, neonatal, and childhood health outcomes [START_REF] Metzger | Long-term Outcomes in Mothers Diagnosed With Gestational Diabetes Mellitus and Their Offspring[END_REF][START_REF] References Agrawal | The effects of maternal smoking during pregnancy on offspring outcomes[END_REF]Williams et al. 2015) that could be either prevented or treated in the case of GDM [START_REF] Crowther | Effect of Treatment of Gestational Diabetes Mellitus on Pregnancy Outcomes[END_REF]). In the present study, we test the association between women's immigrant status, their region of origin, and these maternal health behaviours and indicators using data from the French nationally representative ELFE birth cohort study. Our objective is to examine whether, after adjusting to several potential confounders, immigrants and descendants of immigrants benefit from a healthy migrant effect or are disadvantaged in terms of health outcomes and behaviours compared to non-immigrants.

METHODS

Study design and setting

ELFE (Etude Longitudinale Française depuis l'Enfance) is an ongoing longitudinal nationwide French birth cohort study.

A total of 18,041 mothers and their infants born in 2011 (n=18,329) were initially recruited using random sampling in 320 maternity wards throughout mainland France (51% participation rate). (Vandentorren et al. 2009) Women of at least 18 ! 3 years of age, planning to stay in metropolitan France in the following three years, and capable of giving informed consent in one of the study languages (French, English, Arabic, and Turkish) were included. Fifty-two women withdrew from the study, leaving a sample of 17,988 participants studied in all analyses.

Data source

At the time when women delivered, data were collected by trained midwifes via standardised face-to-face interviews at the maternity; additionally participants also completed a self-reported questionnaire on their health and health-related behaviours (e.g. smoking during pregnancy, alcohol use). Data regarding maternal gestational diabetes and birth characteristics were extracted directly from medical records. Information collected included women's sociodemographic, behavioural and medical characteristics such as age, income, region of origin, socio-professional status, pre-pregnancy body weight and height, and gestational diabetes. Partner's smoking and support during pregnancy was collected at 2 months post-partum via telephone interview by trained investigators in French or any other language requested by the participants at recruitment (response rate = 86.4%).

Study outcomes

Women's pre-pregnancy BMI (collected via medical record) was categorised into four categories (underweight, overweight, obese vs. normal). GDM was considered as diagnosed by the local health care unit (yes vs. no). Smoking status prior to pregnancy and in the last trimester of pregnancy was collected and classified into three categories (smokers, quitters during pregnancy, vs. non-smokers). Alcohol use during pregnancy was classified as follows: <1 drink per month, ≥1 drink per month vs. none.

Mother's immigrant status

Women were asked about their citizenship and birthplace, as well as those of their parents. This information was used to identify 'immigrant status' using the definition established by the French Institute for Demographic studies (INED) :

(Beauchemin et al. 2015) "non-immigrants": women born to two French-born parents, "descendants of immigrants": women born in France with at least one immigrant parent, and "immigrants": women not born in France, and without French citizenship at birth.

Region of origin

Women's geographical region of origin was defined as the region of birth for immigrant women, and parents' region of birth for descendant of immigrants (North Africa or Turkey, Sub-Saharan Africa, Eastern Europe and Asia, Other region vs. Metropolitan France/ European Union). Among descendants of immigrants, in case of discordance between maternal and paternal region of birth (5% of the cases), we used maternal region of birth. Non-immigrants' region of origin was set as 'France/EU'.

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Covariates

Several factors potentially associated with immigrant status or the studied outcomes, assessed during the birth interview, except for the partner's smoking status collected at two months post-partum via telephone, were included as potential covariates.

These included: demographic characteristics: maternal age, number of children including the ELFE child and residential area; social and economic circumstances: maternal educational level, the highest of maternal or paternal occupational grade, maternal employment status; partner characteristics: smoking status, employment status at the time of pregnancy, and educational level; pregnancy characteristics: unplanned pregnancy, and sufficient partner support during pregnancy.

Health outcomes such as tobacco use and BMI before pregnancy were also considered as covariates in some analysis when appropriate. All multivariate analysis were also adjusted for recruitment wave.

Statistical Analyses

Missing data on covariates were imputed using multiple imputations by chained equations under fully conditional specification and assuming missingness at random. Bivariate binomial and multinomial logistic regression models were implemented to identify factors potentially associated with study outcomes. All factors potentially associated with the outcome under study in bivariate analyses (p<0.20) were included in the multivariate models. Multivariate logistic regression models were implemented with a random intercept corresponding to the maternity ward, and adjusting for the study data collection wave (four waves: April; June-July; September-October; November-December). All participants with complete outcome data were included in the multivariate analyses. The non-immigrant population was considered as the reference group in all analyses. We also conducted a sensitivity analysis including the duration of stay in France (>10 years, vs. <10 years) to test for an acculturation effect.

All analyses were performed with SAS, version 9.4. Statistical analyses were conducted separately on each of the ten imputed datasets and the results (OR, CI 95%) were pooled using 'PROC MIANALYZE'.

RESULTS

Table 1 presents descriptive characteristics of women included in the analysis. [Table I1 near here] Out of 17,988 women included in the analyses, 68.4% were non-immigrant (n=12,294), 11.1% were immigrants (n=2,000), and 12.1% were descendant of immigrants (n=2,179). Immigrant status was missing for 8.4% of study women (n=1,515).

As shown in Table 2, the majority of immigrants in this sample came from North Africa or Turkey (62.8%); among descendants of immigrants a majority come from a country of the European Union other than France (53.2%). [Table II near here] Regarding pre-pregnancy BMI; 7.9% of women were underweight, 64.9% had normal BMI, 17.2% were overweight and 10% were obese (n=17,623), while 7.5% of women had GDM (n=17,045).

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Results of multivariate analyses BMI and GDM (table 3)

Maternal region of origin rather than immigrant status was associated with pre-pregnancy BMI and GDM. Compared to non-immigrants, women from Sub-Saharan Africa were more likely to be overweight (OR= 1.37 (95% CI, 1.06 -1.77)) or obese (OR=1.58 (95% CI, 1.14 -2.18)), those from North Africa or Turkey had a higher likelihood of being overweight (OR=1.39 (95% CI, 1.13 -1.71)), whereas women from Eastern Europe and Asia were less likely to be overweight (OR=0.59 (95% CI, 0.39 -0.88)), and obese (OR=0.35 (95% CI, 0.18 -0.69)). Women from North Africa or Turkey (OR=2.00 (95% CI, 1.27 -3.16)), as well as those from Eastern Europe or Asia (OR=1.68 (95% CI, 1.25 -2.25)) were more likely to have GDM compared to non-immigrant women. In complementary analysis, adjusting for the duration of stay in France did not change the direction or statistical significance of the reported associations (supplementary table S1).

[Table 3 near here] Tobacco and alcohol consumption (table 4) Immigrant women were less likely to smoke tobacco before and during pregnancy than non-immigrants, whereas descendants of immigrants had comparable smoking levels. Women from North Africa or Turkey, Sub-Saharan Africa and other countries were least likely to smoke during pregnancy. In terms of alcohol use, immigrant mothers had comparable levels to non-immigrants. Descendants of immigrants were less likely to drink less than one drink of alcohol per month compared to non-immigrants, but had comparable rates of drinking more than one alcoholic drink per month. Women from North Africa or Turkey were significantly less likely to drink alcohol than non-immigrants. These associations were not modified after adjusting for the duration of stay in France (supplementary table S2). [Table 4 near here]

DISCUSSION

Key results

In a nationwide study of women giving birth in France in 2011, we found that the region of origin, and not the immigrant status, was associated with BMI and GDM. The region was also associated with alcohol and tobacco use during pregnancy regardless of immigrant status. While immigrant women were less likely to smoke during pregnancy. These results indicate that immigrant status and the region of origin should be taken into account by health professionals aiming to prevent and address risk factors of poor pregnancy outcomes such as BMI and GDM. Given the high proportion of children born to women who are immigrant or descend from immigrants in France, as in many other European countries, addressing their health needs should be a priority.

Interpretation

BMI and GDM Explanations of higher rates of overweight and GDM among immigrants include nutritional factors, the experience of stress, discrimination and differences in screening rates compared to non-immigrants. First, according to the developmental ! 7 origins of health and disease theory, early childhood metabolic programming [START_REF] Barker | Sir Richard Doll Lecture. Developmental origins of chronic disease[END_REF]; nutritional deficiencies in foetal and early life could partly explain the higher likelihood of metabolic disorders. However our results do not fully agree with this explanation since descendants of immigrants born in France who are less likely to be exposed to early nutritional deprivation, had the same rates of GDM as immigrants. Second, the change in lifestyle habits previously documented among immigrants living in Europe, with the replacement of some healthier "native" diets by the Western diet and an increase in the consumption of energy-dense foods, could also play a role [START_REF] Gilbert | Changing dietary habits of ethnic groups in Europe and implications for health[END_REF]. Third, stress resulting from the experience of hardship as well as different forms of discrimination linked to a history of immigration could also explain unhealthy behaviours and an increase the prevalence of high BMI and GDM [START_REF] Mcewen | Central role of the brain in stress and adaptation: Links to socioeconomic status, health, and disease[END_REF]. The region of origin, essentially a proxy for ethnicity [START_REF] Gagnon | Migration and perinatal health surveillance: An international Delphi survey[END_REF], is in this case a more precise indicator of discrimination and associated stress than immigrant status [START_REF] Missinne | Depressive symptoms among immigrants and ethnic minorities: a population based study in 23 European countries[END_REF]. Importantly, our analyses accounted for multiple measures of socioeconomic position, implying that immigrant status and the region of origin influence health during the perinatal period above and beyond socioeconomic circumstances. Finally, the elevated likelihood of GDM among women of North African or Asian, but not Sub-Saharan descent, goes against findings produced by other studies [START_REF] Gagnon | International migration and gestational diabetes mellitus: a systematic review of the literature and meta-analysis[END_REF]). It could be that women of Sub-Saharan African descent are under-diagnosed, since in France screening of GDM is not universal and women belonging to low socioeconomic groups are less likely to be screened [START_REF] Dow | CA-107: Dépistage, prévalence et pronostic du diabète gestationnel en France en 2011 : l'étude ELFE[END_REF], however other studies have also observed this association [START_REF] Cosson | The Diagnostic and Prognostic Performance of a Selective Screening Strategy for Gestational Diabetes Mellitus According to Ethnicity in Europe[END_REF]). Discrepancies could be explained by the fact that some studies did not control for pre-pregnancy BMI and other potential confounding factors. Nevertheless, in additional sensitivity analysis excluding pre-pregnancy BMI from the multivariate model, women of Sub-Saharan descent were still not at increased risk of developing GDM.

Alcohol and tobacco consumption

Many immigrants originate from regions where women's levels of tobacco smoking and alcohol drinking are low (North and Sub-Saharan Africa) [START_REF] Ng | Smoking prevalence and cigarette consumption in 187 countries, 1980-2012[END_REF], which could explain lower substance use levels in immigrants from these regions. Nevertheless, a convergence of smoking behaviours towards those of the host population is observed among descendants of immigrants [START_REF] Bethel | Acculturation and Smoking Patterns Among Hispanics: A Review[END_REF][START_REF] Reeske | Changes in smoking prevalence among first-and second-generation Turkish migrants in Germany -an analysis of the 2005 Microcensus[END_REF]). It appears that women progressively adopt the behavioural norms prevalent in the host country implicating that tobacco and alcohol use levels, which are high in France, need to be monitored in women born in France to foreign parents.

Strengths and limitations

This work is one of few European investigations analysing health behaviours in immigrant women and among descendants of immigrants in a large nationwide birth cohort study, with the possibility of adjusting for multiple covariates.

Nevertheless, we have to point out potential limitations. First, we were not able to measure immigrant women's acculturation. However, associations between immigrant status and each of the studied outcomes did not significantly change in sensitivity analyses adjusted for the duration of stay in France (supplementary tables). Second, another limitation is missing data concerning study outcomes. However, the percentage of missing data did not exceed 7.7%, and it was ! 8 highest in women who did not originate from the European Union. This may have led to an underestimation of the association between immigrant status and region of origin and studied outcomes. Another limitation is the exclusion of women not planning to stay in metropolitan France in the following three years, making our sample not-representative of all immigrant women giving birth in France at the time of recruitment. Classification bias is also possible, especially concerning non-immigrant women from the French oversea territories (women born French from French parents) who were classified as originating from "EU/France". This also might have also led to an underestimation of the association between immigrant status and studied outcomes.

Conclusions

Our results do not indicate a 'healthy immigrant effect', where immigrants are healthier than non-immigrants. They do however have some healthier behaviour than natives, especially concerning tobacco and alcohol consumption. Regarding overweight/obesity and GDM, immigrant women -particularly those from North and Sub-Saharan Africa -appear at elevated risk, as are women living in their country of origin.

Our findings point to complex relations between immigrant status and health behaviours and indicators during pregnancy, which are related to nativity as well as region of origin. These results suggest that immigrant women and descendants of immigrants who are pregnant need to benefit from medical follow-up which addresses their particular needs. Given the high proportion of births that occur among immigrant women, in terms of public health, strengthening existing positive health behaviours and preventing deleterious ones during pregnancy in this population can have a substantial population impact.

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Table 1 : descriptive statistics of women participating in the ELFE (Etude Longitudinale Française Depuis l'Enfance) cohort (n=17,988, France, 2011)

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Table 2 : Region of origin of immigrant women participating in the ELFE(Etude Longitudinale Française Depuis l'Enfance) cohort study (n=17,988, France, 2011)

 2 Region of birth for immigrant women, and parents' region of birth for descendant of immigrants

	Region of origin*	Non-immigrants	Descendants of immigrants	Immigrants	Missing value
	EU/France	12294 (100%)	1064 (53.2%)	335 (15.4%)	1407 (92.9%%)
	North Turkey	-	644 (32.2%)	807 (37.0%) 15 (0.1%)
	Sub-Saharan Africa	-	157 (7.9%)	561 (25.8%)	8 (0.5%)
	Eastern Europe, Asia	-	75 (3.8%)	225 (10.3%)	4 (0.3%)
	Others, non-declared	-	51 (2.8%)	239 (11.0%) 23 (0.8%)
	Missing value		9 (0.4%)	12 (0.8%)	69 (4.5%)

*

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Table 3 : Associations between women's immigrant status and region of origin with pre-pregnancy BMI and gestational diabetes mellitus (GDM) (multivariate multinomial and binomial logistic regression models, or; 95% CI): the ELFE (Etude Longitudinale Française Depuis l'Enfance) birth cohort study, France, 2011.

 3

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Table 4 : Associations between women's immigrant status and region of origin with alcohol and tobacco consumption during pregnancy (multivariate multinomial logistic regression models, OR; 95% CI): ELFE (Etude Longitudinale Française Depuis l'Enfance) birth cohort study, France, 2011.

 4 Cubizolles M-J, Saucedo M,Drewniak N, et al (2012) Santé périnatale des femmes étrangères en France. Bull Épidémiologique Hebd 2:3-4 Vandentorren S, Bois C, Pirus C, et al (2009) Rationales, design and recruitment for the Elfe longitudinal study. BMC Pediatr 9:58. doi: 10.1186/1471-2431-9-58 Williams JF, Smith VC, Abuse the COS (2015) Fetal Alcohol Spectrum Disorders. Pediatrics 136:e1395-e1406. doi: 10.1542/peds.2015-3113

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Ethical approval:

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

The ELFE study received approval from France's consultative committee for the treatment for health information for research (CCTIRS) and the national data protection authority (CNIL).

Informed consent: Informed consent was obtained from all individual participants included in the study.