Keywords: Power Soccer, Thermolysis, Skin temperature

activation appears to be insufficient to produce a local thermogenesis and does not make up for the laminar effect of the wind created by the players' movements on the pitch. Widely used, the technique of heating by a hair dryer could be a quick and effective solution but its effect is not lasting over the period of play. These results would be supported by the study of a larger number of subjects.

1

RESUME :

Introduction. Le foot-fauteuil (FF) est un sport collectif d'opposition pour personnes ayant un handicap moteur lourd qui se pratique en salle avec un fauteuil électrique spécifique permettant la conduite et la frappe du ballon. Pour les pratiquants de FF, la chaleur d'activation musculaire est limitée. Matériel et méthode. L'étude s'est intéressée aux conditions de pratique du FF dans les clubs français pour évaluer l'impact de la température ambiante sur la performance des joueurs. A partir d'études de cas, nous avons vérifié si l'activation musculaire du joueur en situation de match produit une thermogenèse périphérique cutanée permettant d'envisager des mécanismes d'homéostasie. Les informations recueillies par questionnaire ont montré que la température ambiante à l'entraînement est insuffisante (17,7±1,7°C) et ne correspond pas à celle souhaitée (21,1±2,1°C). Quelle que soit leur classification, 81% des joueurs considèrent que le froid impacte négativement leur performance et plusieurs techniques de réchauffement sont utilisées. Au cours de matchs simulés, les enregistrements de la température cutanée par thermo-bouton installé sur le dos de la main de 4 joueurs ont montré une tendance à la thermolyse locale. L'utilisation du sèche-cheveux apporte un rapide bénéfice à court terme (+13,9 et 16,4°C en 4-5min) qui ne couvre pas le temps d'une mi-temps de jeu. En conclusion, la température ambiante des lieux de pratique trop fraiche serait un facteur limitant pour le contrôle du fauteuil et la sensibilité des joueurs de FF. L'activité musculaire semble insuffisante pour produire une thermogénèse locale et ne permet de compenser l'effet laminaire du vent créé par les déplacements des joueurs sur le terrain. Largement répandue, la technique de réchauffement par un sèche-cheveux pourrait-être une solution rapide et efficace mais son effet n'est pas durable sur une période de jeu. Ces résultats doivent être confortés par l'étude d'un effectif plus important.

Mots clés : Power soccer, thermolyse, température cutanée.

SUMMARY :

Background. Powerchair football is a team sport for people with a heavy motor disability. It is played in a sports hall with a specific power wheelchair, which makes it easier to move around and kick the ball. For powerchair football players, muscle activation heat is limited. Method. The study looked into the playing conditions of powerchair football in French clubs to assess the impact of the hall temperature on the players' performances. From case studies, we checked if the player's muscle activation during a game produces peripheral cutaneous thermogenesis that would allow mechanisms of homeostasis. Survey information showed that the ambient temperature at practice is not sufficient (17,71,7°C) compared to that expected (21,12,1°C). No matter their classification, 81% of players consider that the cold has a negative impact on their performance and many warming techniques have to be used. During simulated games, recordings of skin temperature with the use of temperature recorders on the back of the hand of 4 players showed a tendency to local thermolysis. Using a hair dryer a quick short-term temperature increase, less than a half-time on the game (+13,9 and 16,4°C in 4-5min), was obtained. Conclusion. It is concluded that the ambient temperature of sports halls that are too cold could a limiting factor to control the wheelchair control and player's sensibility. Muscle

Introduction

Le foot-fauteuil électrique est un sport collectif d'opposition permettant à des personnes atteintes de handicap moteur lourd de pratiquer en compétition. Le mouvement du fauteuil se fait avec un joystick, généralement manié à la main mais aussi avec le pied ou le menton selon les capacités fonctionnelles de l'utilisateur Compte-tenu de l'hétérogénéité des limitations fonctionnelles des joueurs et du respect d'équité sportive, un système de classification est régi par la Fédération Internationale de Powerchair Football Association (www.fipfa.org) sous l'égide de l'International Paralympic Committee. En foot-fauteuil, la performance et la qualité du jeu reposent sur les vitesses de déplacement (limitées à 2,8 m.s - dans les 2 sens) et sur des passes décisives et stratégiques médiées par l'utilisation du fauteuil par les joueurs [1]. En match, la dépense énergétique a été mesurée à 34% au-dessus de celle repos pour un effort perçu comme un peu dur [2]. En fonction de l'environnement thermique ambiant et de l'activité du sujet, l'homéostasie de la température corporelle se régule à partir d'un système dissipateur ou producteur de chaleur. Les tissus (nerveux, viscères et musculaires) produisent de la chaleur et interviennent dans la thermogenèse. La chaleur d'activation musculaire se trouve limitée pour les personnes lourdement atteintes sur le plan moteur comme ceux pratiquants du foot-fauteuil [3]. Le but de cette note est d'étudier la température ambiante des lieux de pratique du footfauteuil afin d'en connaitre l'impact sur les joueurs de manière générale et en fonction de leur classification. De plus, à partir d'études de cas, nous avons vérifié si les actions de joueurs en situation de match produisent une thermogenèse périphérique cutanée permettant d'envisager des mécanismes d'homéostasie chez ces joueurs.

Méthodes

Un recueil d'informations a été effectué par questionnaires auprès des responsables de club pour recenser les conditions ambiantes des lieux de pratique mais aussi chez les joueurs pour identifier l'impact du froid (échelle de sensation et confort thermique) sur les performances et les techniques de réchauffement utilisées. Les résultats d'échelle de sensation et confort thermique entre PF1 et PF2 ont été comparé à l'aide du test non-paramétrique de Mann-Whitney. En pratique, un enregistrement en continu de la température cutanée de la main a été réalisé au cours de matchs simulés à l'entraînement. Les variations de température ont été mesurées avec un thermo bouton (Thermochron « ibutton » DS1922L) installé sur le dos de la main contrôlant le joystick chez 4 joueurs de l'Equipe de France de foot-fauteuil (classification PF1 et PF2). Les données enregistrées ont été comparés à la valeur seuil de l'inconfort digitale de 23°C proposé par Brajkovic [4]. La fréquence d'échantillonnage d'enregistrement était de 10 secondes puis moyennée toutes les minutes.

Pour cette étude, les clubs devaient être affiliés à un club et les joueurs licenciés à la Fédération Française Handisport pour la compétition du foot-fauteuil pour la saison 2019-2020. Chaque participant volontaire a signé un consentement éclairé.

Population

Treize responsables de club (sur 42) représentant quatre niveaux de pratique et vingt-sept joueurs (24 hommes ; 3 femmes) âgés de 24 ± 7,5 ans ont répondu au questionnaire. Trois niveaux de pratique sont représentés : international (10), division 1 (8) et division 2 (9) sont répartis en 2 classifications différentes selon les limitations fonctionnelles, PF1 (n=17) pour les limitations élevées, PF2 (n=10) pour les limitations modérées (www.fipfa.org). Le type de pathologie des joueurs est hétérogène et représentatif de la discipline : myopathies (9), pathologies neuromusculaires (7), tétraplégies (4), paraplégie (1), paralysies cérébrales (2), autres pathologies telles que l'arthrogrypose, l'ostéogenèse imparfaite, la dysplasie de Kniest (4). Parmi les répondants au questionnaire, la température cutanée de la main de 4 membres de l'Équipe de France de foot-fauteuil (hommes âgés de 22,7 ± 2,9 ans) présentant différentes pathologies (myopathie, neuromusculaire, tétraplégie, autre pathologie) a été mesurée en continu pendant un match d'entraînement.

Résultats

Pour les représentants, la température ambiante du gymnase est considérée comme froide (54%). La température ambiante du gymnase est connue (pour 62% des clubs) ; celle-ci est en moyenne déclarée par les représentants à 17,7 ± 1,7°C alors qu'elle est souhaitée par 85% des 13 responsables de 21,1 ± 2,1°C. La température du gymnase est un facteur limitant au bon déroulement des entrainements pour la totalité des répondants avec une influence plus importante en fonction de la saison : 92% en hiver, 46% en automne, 8% au printemps et en été. 85% des clubs mettent en oeuvre des techniques de réchauffement (sèche-cheveux, 85% ; chaufferettes, 31% ; boissons chaudes, 31% ; ventilateur, 15% ; autres techniques, 23% : chauffage d'appoint, plaids, …). Pour les joueurs, le froid est ressenti principalement aux extrémités (55% mains, 44% pieds, 29% jambes, 11% à la tête, 0% bras). Le gymnase n'est pas assez chauffé pour 63% des joueurs. Aucune différence significative n'a été trouvée en fonction de la classification (PF1-PF2) (Tableau 1). 81% des joueurs ont déclaré que le froid impacte négativement leur performance (notamment en déficit de mobilité et de précision). Pour 73% des joueurs, une méthode de réchauffement (69% classifiés PF1 contre 31% en PF2). Elle est jugée satisfaisante pour 75% des joueurs, l'insatisfaction concerne que les PF1.

Une étude de cas sur l'évolution des températures cutanées au cours d'un match de 4 joueurs repartis en 2 groupes (contrôle-PF2 et utilisant la technique de chauffage du sèche-cheveux-PF1) est représentée dans la figure 1. La température moyenne pour la durée du match est inférieure pour les témoins 23,8 ± 1,2°C comparativement au chauffage 32,1 ± 3,6°C. Pour les joueurs contrôles (J3 et J4), la température cutanée diminue respectivement de 10,3% et 11,3% pendant les phases jouées malgré une légère tendance au réchauffement à la mitemps. Tout au long du match, le joueur J4 a une température cutanée supérieure à la valeur seuil alors que le joueur J3 n'y qu'à 15,8%.

Les variations fluctuent de -23,1 à +18,1% pour J1 et pour J2, la température cutanée maximale a été de 43.8°C pour une valeur minimale de 27.4°C. L'usage du sèche-cheveux permet une augmentation rapide et nette de la température cutanée (+ 13,9°C pour J1 et +16,4°C pour J2 en 4-5min) mais cet effet reste temporaire et nécessite d'être réitéré à chaque remplacement. Ces deux joueurs ont une température cutanée de la valeur seuil durant la totalité du match.

Discussion

Même si l'échantillon de réponses est faible, la température ambiante des lieux de pratique est jugée trop froide par les joueurs et les dirigeants de club de foot-fauteuil électrique. Néanmoins une organisation est mise en place pour limiter les effets jugés néfastes à la performance en foot-fauteuil. Nous n'avons pas constaté de différence significative de perceptions entre la catégorie de joueurs PF1 et PF2. L'échelle générique utilisée n'est peutêtre pas assez sensible chez des sujets ayant, en raison de leur pathologie, une possible inhibition des capteurs cutanés, thermiques et vasculaires. L'échantillon restreint de joueurs (n=27) ne permet pas de repérer de différences physiopathologiques ni de seuil d'inconfort thermique liées au type de pathologie. Parmi les répondants, les joueurs PF1 s'organisent en nombre par rapport au PF2 en adaptant des techniques de réchauffement. La production d'un flux d'air chaud aurait un effet positif immédiat. Cependant l'efficacité de cette méthode s'estomperait dès son arrêt, ne produisant qu'un bénéfice pic (figure 1). Elle pourrait être anticipée avant de rentrer sur le terrain mais ne couvre pas la totalité d'une période de match. Pour les joueurs témoins n'utilisant aucun moyen particulier, on observe à chaque période de jeu, une tendance à la thermolyse cutanée de la main. Cette baisse de température pourrait s'expliquer par l'écoulement laminaire d'air frais produit par les déplacements des joueurs. Ainsi, le vent apparent pourrait induire par convection une baisse de la température cutanée, non compensée par la thermogénèse de la musculature du haut corps limitée par l'atteinte neuromusculaire. Cette hypothèse expliquerait les légères augmentations mesurées pendant les 5 min de pause de mi-temps. Si le port du gant pouvant générer une perte de mobilité chez certains joueurs n'est pas souhaité, une protection carénée autour du joystick pourrait être une solution technique pour limiter la déperdition calorique au niveau de la main.

Conclusion

Les acteurs du foot-fauteuil jugent la température ambiante des lieux de pratique trop froide. Des actions collectives et individuelles permettent d'en limiter les effets néfastes sur la performance. Sur plan cutané, les cinétiques de la température de la main « joystick » varient au cours d'un match, montrant une tendance à la thermolyse locale, indépendamment de la classification. La valeur seuil de 23°C semble trop élevée pour les joueurs PF2 et trop faible pour les joueurs PF1. La technique de réchauffement avec un sèche-cheveux ne serait pas satisfaisante sur la durée d'un match bien qu'elle soit la plus répandue au sein des clubs.

Déclaration de liens d'intérêts.

Les auteurs déclarent ne pas avoir de liens d'intérêts. Arch Phys Med Rehabil 2016 Nov;97(11):1938-1944. [3] Guttmann L, Silver J, Wyndham. Thermoregulation in spinal man. J Physiol 1958;142:406-419.

Références

[4] Brajkovic D, Ducharme MB, and Frim J. Influence of localized auxiliary heating on hand comfort during cold exposure. J Appl Physiol. 1998;85: 2054-2065. Perte de mobilité correspond à une perte de l'amplitude du geste. Perte de précision, 8 gestuelle moins précise. Perte de sensation, perte de la sensibilité. Autres, perte de 9 concentration ou augmentation de la fatigue ou perte de réactivité. 10

  [1] Kumar A, Karmarkar AM, Collins DM, et al. Pilot study for quantifying driving characteristics during power wheelchair soccer. J Rehabil Res Dev 2012;49:75-82.[2] Barfield JP, Newsome L, Malone LA. Exercise intensity during power wheelchair soccer.


Mario Campone

Thomas Bachelot

Isabelle Treilleux

Barbara Pistilli

Julia Salleron

Valérie Seegers

Monica Arnedos

Delphine Loussouarn

Qing Wang

Laurence Vanlemmens

Marta Jimenez

Maria Rios

Véronique Diéras

Agnès Leroux

Gilles Paintaud

Keyvan Rezai

Fabrice André

Maëva Lion

Jean-Louis Merlin
email: jl.merlin@nancy.unicancer.fr

A phase II randomised study of preoperative trastuzumab alone or combined with everolimus in patients with early HER2-positive breast cancer and predictive biomarkers (RADHER trial)

Keywords: biomarker, everolimus, HER2+ breast cancer, PI3K/AKT/mTOR, trastuzumab

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

INTRODUCTION

Trastuzumab has considerably improved the outcome in HER2-positive breast cancer in both adjuvant and metastatic settings, with a clear survival benefit even more pronounced when combined with chemotherapy [START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF][START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF][START_REF] Piccart-Gebhart | Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer[END_REF] . Nonetheless, 10% of operated patients treated with trastuzumab plus adjuvant chemotherapy develop resistance within 4 years, while almost all patients with metastatic disease ultimately progress under trastuzumab [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF] .

Targeting the extracellular domain of HER2, trastuzumab, initially reported to trigger HER2 internalization and degradation, blocks downstream signaling pathways, mainly PI3K/AKT/mTOR and MAP kinase (MAPK) pathways which regulate cell proliferation and survival [START_REF] Lewis | Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies[END_REF] . Trastuzumab was later proved to induce antibody-dependent cell cytotoxicity (ADCC) [START_REF] Gennari | Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2[END_REF] . Resistance to trastuzumab can derive from dysregulation of multiple factors including increased signaling of upstream growth factor receptors, alterations of the HER2 receptor, mutation-induced repression of PTEN and/or activation of PI3K [START_REF] Vernieri | Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: Current knowledge, new research directions and therapeutic perspectives[END_REF] . mTOR inhibitors targeting the PI3K/AKT/mTOR signaling pathway, address several of these mechanisms and have been proposed to overcome resistance to trastuzumab. Everolimus is an oral mTOR inhibitor approved for use in advanced hormone receptor-positive HER2-negative breast cancer. In preclinical studies, everolimus overcame PI3K/AKT/mTOR signaling-mediated trastuzumab resistance in PTEN-deficient breast cancer cells [START_REF] Lu | Preclinical testing of clinically applicable strategies for overcoming trastuzumab resistance caused by PTEN deficiency[END_REF] , and acted synergistically with trastuzumab [START_REF] Zhu | Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo[END_REF] , restoring inhibition of cell proliferation and induction of apoptosis. Some clinical trials reported that these preclinical benefits translate to the clinical setting; indeed, treatment with everolimus alone or with chemotherapy in trastuzumab-resistant HER2positive metastatic breast cancer patients restored sensitivity to trastuzumab [START_REF] Andre | Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial[END_REF] .

A pilot study in 11 patients reported 1 pathologic complete response (pCR) and 4 clinical partial responses, suggesting the value of trastuzumab as monotherapy in the preoperative setting [START_REF] Gennari | Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2[END_REF] . Further, the results of this study suggest that trastuzumab acts through ADCC more than to down-modulation of HER2 pathways. We confirmed these findings in HER2-positive breast cancer cell lines and in tumor specimens of patients receiving trastuzumab as preoperative monotherapy [START_REF] Lion | Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer[END_REF] .

Subsequent studies of trastuzumab in combination with chemotherapy in neoadjuvant setting, presented pCR rates from 43% to 65% [START_REF] Coudert | Multicenter phase II trial of neoadjuvant therapy with trastuzumab, docetaxel, and carboplatin for human epidermal growth factor receptor-2-overexpressing stage II or III breast cancer: results of the GETN(A)-1 trial[END_REF][START_REF] Coudert | Preoperative systemic (neo-adjuvant) therapy with trastuzumab and docetaxel for HER2overexpressing stage II or III breast cancer: results of a multicenter phase II trial[END_REF][START_REF] Buzdar | Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer[END_REF] .

The current study evaluated the addition of everolimus to preoperative trastuzumab in HER2positive primary breast cancer in terms of clinical response at 6 weeks. Furthermore, this "window-of-opportunity" study allowed for multiple translational research on both pre and post-treatment samples. This could help understanding the pharmacodynamic activity of both treatments and identifying potential molecular predictive biomarkers for efficacy of trastuzumab, alone or combined with everolimus.

PATIENTS AND METHODS

Study Design

This phase II, randomized, open-label study was performed in 6 French comprehensive cancer centers. One hundred and twenty patients were planned to be centrally randomized (1:1) to receive trastuzumab (weekly; 4 mg/kg intravenous loading dose then 2 mg/kg for 5 weeks), alone (Arm T) or combined with everolimus (10 mg/day for 6 weeks; last dose <24 hours prior to surgery) (Arm T+E), with stratification on primary tumor, regional lymph nodes, and estrogen receptor status. Two everolimus dose reductions for toxicity were permitted after recovery to grade 1. All patients underwent breast-conserving surgery after 6 weeks.

Adjuvant chemotherapy, radiotherapy, and hormone therapy were left to the physician's decision according to local guidelines. The study was approved by a national ethics committee and the French Competent Health Authority, and was conducted in accordance with the Declaration of Helsinki.

Patients

Patients had to be 18 years or older, have histologically-confirmed non-metastatic breast cancer with no prior treatment, HER2-positive (3+ immunohistochemistry (IHC) score or 2+ with gene amplification using in situ hybridization), and be candidate for breast-conserving surgery (T0-3, N0-2, ≥15 mm measured with ultrasonography). Patients had an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate hematologic, renal, hepatic, cardiac and pulmonary function. An amendment allowed the inclusion of patients with bilateral disease and with multifocal disease for whom a breast conserving surgery was not possible. Patients with inflammatory breast cancer, cardiac disease or other serious illness were not eligible. All patients gave written informed consent prior to enrolment.

Tumor assessments

Tumor response was assessed by clinical examination and echography at baseline, then after 3 and 6 weeks (immediately before surgery). Three core needle biopsies were collected at baseline and 2 punch samplings were done on the surgery specimen for biomarker analyses. At each time point, one specimen was formalin-fixed paraffin-embedded (FFPE) for IHC and the others were snap-frozen in liquid nitrogen and stored at -80°C until analyzed.

Initially included as mandatory in the study protocol, the biopsy during pre-operative treatment e.g. 3 weeks after treatment initiation was amended as optional since it was a source of refusal to participate to the study by many patients.

Translational studies

We selected candidate biomarkers to be tested before and after exposition to treatment for their potential interest in assessing efficacy and predicting sensitivity to trastuzumab and/or everolimus + trastuzumab. We hypothesized that trastuzumab and everolimus + trastuzumab combination could induce an arrest of proliferation, induction of apoptosis, inhibition of PI3K/AKT/mTOR and MAPK signaling pathways. Finally, anti-tumor immune reaction was assessed before and after treatment through evaluation of lymphocyte and macrophage infiltration. The pharmacokinetics and the metabolomic analyses associated to this study were already published [START_REF] Bernadou | Influence of tumour burden on trastuzumab pharmacokinetics in HER2 positive nonmetastatic breast cancer[END_REF][START_REF] Jobard | Longitudinal serum metabolomics evaluation of trastuzumab and everolimus combination as pre-operative treatment for HER-2 positive breast cancer patients[END_REF] .

PIK3CA alterations analysis

As routinely used at the time of this study, PIK3CA gene (NM_006218.2) exon 9 and 20 hotspot pathogenic variants were analyzed by targeted next generation sequencing of DNA isolated from 63 FFPE tumor specimens (58/63) and frozen specimens (5/63), upon standard protocol with Ion Torrent PGM (ThermoFisher Scientific) following the manufacturer's instruction with an entire coverage of the two exons and an average depth of more than 1000X. The NestGENe software (SoftGenetics) was used for sequence alignment and variation calling.

Immunohistochemistry

All biomarkers but PTEN were assessed on FFPE specimens from baseline core needle biopsies and surgical residues. PTEN IHC was performed either on FFPE specimen from baseline biopsies or from FFPE surgical residues. FFPE blocks were cut at 4 µm thickness.

Deparaffinization was performed on Discovery XT automate (Roche). After heat induced antigen retrieval in CC1 buffer (pH8.4) and blockage of the endogenous peroxidases in H2O2, the slides were incubated with anti-phospho-4EBP1 (p4EBP1) (Ser65), anti-phospho-S6 Ribosomal Protein (pS6RP) (Ser235/236) and anti-PTEN (138G6) (Cell Signaling), anti-LKB1 and anti-phospho-AKT1 (pAKT1) (Ser473) (Abcam), or with anti-Ki67 (SP6) (Thermo Scientific). Antibody binding was revealed using OmniMap anti-rabbit HRP kit (Roche) for LKB1, p4EBP1, pS6RP, Ki67, and PTEN antibodies or UltraMap anti-rabbit HRP kit (Roche) for pAKT1 antibody followed by ChromoMap DAB kit (Roche). Sections were then counterstained with hematoxylin and bluing (Roche).

For eIF4E and Caspase 3, deparaffinization and antigen retrieval were performed on a PTLink (Dako) using the target retrieval solution High pH (Dako). The slides were then incubated in Autostainer (Dako) for blockade of the endogenous peroxidases in H2O2 followed by anti-eIF4E (Cell Signaling) or anti-caspase 3 (BD Biosciences). Antibody binding was revealed using Flex Envison kit (Dako). Sections were counterstained with hematoxylin (Dako).

Blinded to the clinical data, the slides were analyzed for both the percentage and the intensity (1+ =low, 2+ =intermediate, 3+ =high) of cytoplasmic staining in the infiltrative carcinomatous cells for pS6RP, caspase3 and eIF4E antibodies or nuclear staining for p4EBP1, PTEN and Ki67 antibodies and both cytoplasmic and nuclear staining for LKB1 and pAKT1 antibodies. A single score (from 0 to 6) was derived from intensity and percentage parameters of pAKT1, pS6RB, p4EBP1, eIF4E, LKB1 and PTEN staining, according to Allred et al. [START_REF] Allred | Immunocytochemical analysis of estrogen receptors in human breast carcinomas. Evaluation of 130 cases and review of the literature regarding concordance with biochemical assay and clinical relevance[END_REF] . Allred scores at baseline and at surgery were compared. PTEN repression was considered when less than 50% of the cells were stained.

Multiplex Bead Immunoassay

The expression of pMEK1 and pERK1/2 was quantitatively analyzed using the Bio-Plex ® Multiplex Immunoassay (Bio-Rad), as described by Chergui et al. [START_REF] Chergui | Validation of a phosphoprotein array assay for characterization of human tyrosine kinase receptor downstream signaling in breast cancer[END_REF] . Protein extracts from baseline and surgical frozen tumor samples (15-20 mg with at least 50% of tumor cells) were incubated overnight at 37°C with fluorescent capturing beads bearing antibodies against the target phosphoproteins (Bio-Rad), then exposed to streptavidin-phycoerythrin and detected with fluorescein-conjugated biotinylated antibodies. Results were recorded as mean fluorescence intensity of triplicated analyses.

Immunological study

Tumor slides from the baseline and surgical FFPE tumor samples were assessed for the infiltration by CD8+ lymphocytes (Spring Bioscience, Roche) and CD68+ macrophages (Dako) performed on a BenchMark XT immunostainer (Ventana). Antigen retrieval was performed by incubating slides on CC1 buffer for 36 minutes. Then the antibody was incubated for 1 hour at 37°C and revealed by ultraView Universal DAB Detection Kit (Roche).

Scoring was performed as previously described [START_REF] Ladoire | In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival[END_REF] .

Endpoints

The primary endpoint was the clinical response rate at 6 weeks (WHO criteria), analyzed in all randomized patients (intent to treat [ITT]). Secondary endpoints included the pCR rate at 6 weeks, safety graded by NCI-CTCAE v3.0, identification of candidate biomarkers predictive of response, pharmacodynamic and pharmacokinetic parameters of trastuzumab and everolimus plus trastuzumab combination. Using Sataloff classification [START_REF] Sataloff | Pathologic response to induction chemotherapy in locally advanced carcinoma of the breast: a determinant of outcome[END_REF] , pathologic response was centrally reviewed on the complete set of slides from the surgical specimen, and defined as pCR if total or near total therapeutic effect was established, or partial pathological response if a decrease in tumor cellularity of at least 50% was observed, excluding nodes.

Statistical Analyses

With P0=10% (clinical response at 6 weeks with trastuzumab alone [START_REF] Menard | HER-2positive breast carcinomas as a particular subset with peculiar clinical behaviors[END_REF] ) and using α=0.05 and β=0.05, 60 patients per arm were required to demonstrate a true improvement in clinical response to 40% (P1) with combined trastuzumab and everolimus. For missing clinical response data at 6 weeks, data from 3 weeks treatment were used. Clinical and pathologic responses were compared between treatment arms using Chi² tests. Non-parametric Wilcoxon tests were used for associations between baseline biomarkers and response.

Cochran-Mantel-Haenszel tests were used to compare trastuzumab alone arm (arm T) and trastuzumab plus everolimus arm (arm T+E) parameters. Analyses were performed with SAS software (version 9.3) and R (version 3.3.2), using a 5% significance threshold.

RESULTS

Due to slow recruitment rate, an independent data monitoring committee recommended stopping the study after randomization of 82 patients (41 per arm, Figure 1) between July 2008 and April 2012. Patient and tumor characteristics were well balanced between the two arms (Table 1).

Antitumor Activity

The overall clinical rates were 34.1% and 43.9% and the pathological response rates were 43.6% and 47.5%, for arm T and arm T+E, respectively. No statistically significant difference was observed between both arms (Table 2). No significant difference in clinical (P=0.82) and pathological response rate (P=0.36) was observed between hormone receptor positive (HR+) and negative (HR-) tumors in both treatment arm (Table S1)

Safety

Broadly speaking, the incidence of the adverse events (AEs) associated with trastuzumab treatment increased with the addition of everolimus (Table S2). Nonetheless, toxicity was largely mild to moderate, grade 3-4 AEs were only reported in arm T+E: Seven patients (17.5%) experienced mucositis, 2 (5.0%) hyperglycemia, 1 (2.5%) neutropenia, and 1 (2.5%) transaminitis. The occurrence rate of grade 3-4 AEs was significantly higher in patients in arm T+E with 22.5% of the patients having at least one grade 3-4 AE versus none in arm T (P=0.002, Fisher test). In arm T+E, 2 patients stopped both agents early, 1 due to grade 4 mucositis and the other one due to nausea/vomiting, transaminase elevations, and headache (all grade 2), and 3 patients stopped everolimus only due to grade 2-3 mucositis. In addition, 7 patients had everolimus dose reduction exclusively for grade 1 to 3 mucositis.

Translational Research results

Proliferation and apoptosis

Ki67 and caspase-3 were assessed on 55 matched baseline and surgery tumor samples. As a surrogate marker for proliferation, Ki67 significantly decreased between baseline and surgery in both arms (P<0.001 in each am). No difference was observed between the two arms (Table 3). While no difference was observed in arm T between clinical complete responders and non-complete responders, a significant difference was observed in arm T+E (P=0.036) (Figure 2).

No difference in apoptosis induction, analyzed through activated caspase-3 expression, was observed at baseline and at surgery between arm T and arm T+E. Additionally, no treatmentinduced variation was observed in arm T and in arm T+E. Furthermore, no relationship was observed with clinical response in both arms.

HER2 downstream signaling biomarkers

PTEN repression and PIK3CA alteration

From 59 interpretable NGS, 14 were positive for a PIK3CA hot-spot pathogenic variant (23.7%). PTEN was obtained from 21 baseline biopsies or 38 surgery samples. Considering the entire patients population, no significant variation in PIK3CA mutation (P=0.991) and in PTEN repression rates was observed between baseline and surgery (P=0.300) (Table S3).

There was no inter-arm significant difference in the variation in PIK3CA mutation and PTEN expression rates, considered either alone or combined (PTEN loss or PIK3CA mutation).

Moreover, PIK3CA mutational status was found to be related to clinical response (P=0.029) with a higher proportion of responders (complete and partial responses) observed in patients with wild-type (92%) versus mutated PIK3CA tumors (8%) (Table 4). No variation in Ki67 labelled nuclei rate was observed between PIK3CA mutated and wild type tumors (P=0.95)

PI3-kinase -AKT -mTOR pathway

pS6RP was assessed by IHC on 55 matched baseline and surgery tumor samples, pAKT1, p4EBP1, eIF4E on 54, and LKB1 on 53. No difference was observed at baseline and at surgery in PI3K/AKT/mTOR signaling biomarker expression between arm T and arm T+E. A significant decrease in pAKT1 (cytoplasmic and nuclear), pS6RP and nuclear LKB1 was observed between baseline and surgery (Table 3) while no variation was observed for cytoplasmic LKB1, eIF4E, and p4EBP1 in both arms. When PTEN repression was observed, no consequent decrease in pAKT expression was observed. Furthermore, no relationship was observed between any biomarker and clinical complete response in both treatment arms (Figure S2).

MAP kinase signaling pathway

pMEK1 and pERK1/2 expression were obtained from 37 matched baseline and surgery tumor samples. Within arm T no variation in pMEK1 and pERK1/2 was observed between baseline and surgery, while within arm T+E, significant inductions of pMEK1 (P=0.005) and pERK1/2 (P=0.030) were observed, leading to a significant difference between the two treatment arms (P=0.014 and 0.017 respectively) (Table 3).

Lymphocyte and macrophage infiltration

Lymphocyte and macrophage infiltration was assessed in 47 matched baseline and surgery tumor samples. On the 3 points scale, CD8 score increased significantly in both arms between baseline and surgery (mean increase 0.63 (SD=0.76), P=.007 and 0.37 (SD=0.76), P=0.018 for arm A and B, respectively) with no difference in the mean increase rate between arm A and B (P=0.32). (Table 3). Furthermore, no difference was observed between the two arms (Figure S1). No difference was observed in CD68 tumor infiltrating macrophages rate between baseline and surgery in arm T and T+E, and between the two arms (Table 3). No relationship was found with complete clinical response (Figure S1).

Relationship with pathological response

For all biomarkers, no relationship was observed with complete clinical or overall pathological response (data not shown).

DISCUSSION

PI3K/AKT/mTOR signaling represents a potential resistance mechanism to trastuzumab. We thus evaluated the clinical and biologic effects of a first line pre-operative mTOR and HER2 co-targeting therapy in HER2-positive early operable breast cancer patients. Our results confirm the clinical efficacy of trastuzumab given alone for a short period. The addition of everolimus proved a trend but not a significant increase in clinical response rate. In addition, pathologic response rates were similar for patients treated with or without everolimus.

Although the premature termination of this study weakened the statistical power, the 95% level aimed in the hypothesis was ambitious. The 82 patients included still represents an acceptable 83% power, but our results are inconclusive on that point.

In this preoperative setting, none of the candidate biomarkers tested but PIK3CA mutational status was predictive of trastuzumab efficacy. Patients with wild type PIK3CA tumor had a higher clinical response rate than patients with PIK3CA mutated tumor in the overall population, potentially predicting trastuzumab efficacy. Andre et al. [START_REF] Andre | Molecular Alterations and Everolimus Efficacy in Human Epidermal Growth Factor Receptor 2-Overexpressing Metastatic Breast Cancers: Combined Exploratory Biomarker Analysis From BOLERO-1 and BOLERO-3[END_REF] showed that in HER2positive advanced breast cancer, PIK3CA mutations may be a predictive biomarker for everolimus efficacy. However, sub-population analysis showed that only patients with hormone receptor-negative tumors had statistically significant progression free survival benefit with everolimus treatment. In the present study, only 17 out of the 41 patients randomized in the everolimus arm had hormone receptor-negative tumors. Given the low number of samples in this subgroup of patients, it was futile to draw conclusion for the predictive value of PIK3CA mutations for everolimus efficacy in this setting.

In the present trial, patients with HER2-positive breast cancers received trastuzumab alone or combined with everolimus in pre-operative setting. This drastically differs from other trials considering the impact of alteration of PTEN/PI3-kinase/AKT pathway of the clinical efficacy of drugs targeting this pathway, but mostly performed in HER-negative advanced breast cancers resistant to mTOR inhibitors [START_REF] Leo | Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, doubleblind, placebo-controlled, phase 3 trial[END_REF] , in HER2-negative metastatic breast cancers resistant to aromatase inhibitors [START_REF] Kornblum | Randomized Phase II Trial of Fulvestrant Plus Everolimus or Placebo in Postmenopausal Women With Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer Resistant to Aromatase Inhibitor Therapy: Results of PrE0102[END_REF][START_REF] Jones | Fulvestrant plus capivasertib versus placebo after relapse or progression on an aromatase inhibitor in metastatic, oestrogen receptor-positive breast cancer (FAKTION): a multicentre, randomised, controlled, phase 2 trial[END_REF] , in metastatic triple negative breast cancers [START_REF] Kim | LOTUS investigators. Ipatasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer (LOTUS): a multicentre, randomised, doubleblind, placebo-controlled, phase 2 trial[END_REF][START_REF] Schmid | Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial[END_REF] .

In hormone receptor-positive, HER2-negative advanced breast cancers, data from BOLERO-2 trial [START_REF] Hortobagyi | Correlative Analysis of Genetic Alterations and Everolimus Benefit in Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results From BOLERO-2[END_REF] , showed that the PFS benefit with everolimus was maintained regardless of the hyperactivation status of PI3-kinase/mTOR pathway i.e. in tumor bearing at least one mutation in PIK3CA, PTEN, AKT1, or PIK3R1 genes or low PTEN expression.

Further, even if the clinical benefit of PI3-kinase inhibitors [START_REF] Leo | Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, doubleblind, placebo-controlled, phase 3 trial[END_REF][START_REF] André | Alpelisib for PIK3CA-Mutated, Hormone Receptor-Positive Advanced Breast Cancer[END_REF] and AKT inhibitors [START_REF] Jones | Fulvestrant plus capivasertib versus placebo after relapse or progression on an aromatase inhibitor in metastatic, oestrogen receptor-positive breast cancer (FAKTION): a multicentre, randomised, controlled, phase 2 trial[END_REF][START_REF] Kim | LOTUS investigators. Ipatasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer (LOTUS): a multicentre, randomised, doubleblind, placebo-controlled, phase 2 trial[END_REF][START_REF] Schmid | Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial[END_REF] , compared to placebo, was observed in both PTEN/PI3-kinase/AKT altered and unaltered tumor bearing patients, no direct statistical comparison of the two groups was assessed to ascertain the higher benefit in the PTEN/PI3-kinase/AKT altered subgroup, making any extrapolation very hazardous.

In PrE0102 trial [START_REF] Kornblum | Randomized Phase II Trial of Fulvestrant Plus Everolimus or Placebo in Postmenopausal Women With Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer Resistant to Aromatase Inhibitor Therapy: Results of PrE0102[END_REF] , as previously in BOLERO-2 and TAMRAD trials [START_REF] Piccart | Everolimus plus exemestane for hormone-receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: overall survival results from BOLERO-2 †[END_REF][START_REF] Bachelot | Randomized phase II trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study[END_REF] , the improved PFS achieved by adding everolimus to fulvestrant in aromatase inhibitors-resistant tumors could be related to the development of aromatase inhibitors resistance that may select tumors being highly dependent on PI3K/AKT/mTOR pathway and more sensitive to mTOR inhibition.

In BELLE-3 trial [START_REF] Leo | Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, doubleblind, placebo-controlled, phase 3 trial[END_REF] , it was suggested that the higher efficacy of buparlisib in PIK3CA mutated tumors could be due to pretreatment of all patients with an mTOR inhibitor which may upregulate the PI3-kinase pathway via feedback activation. In LOTUS trial [START_REF] Kim | LOTUS investigators. Ipatasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer (LOTUS): a multicentre, randomised, doubleblind, placebo-controlled, phase 2 trial[END_REF] , the differential effect of ipatasertib and paclitaxel in PIK3CA/AKT1/PTEN altered tumors was not statistically demonstrated with median PFS values of 9.0 (95% CI 4.6-NA) and 5.3 (95% CI 3.6-7.3) months, respectively in altered and non-altered tumors. In PAKT trial [START_REF] Schmid | Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial[END_REF] , it was hypothetized that the benefit of capivasertib and paclitaxel might be limited to the PIK3CA/AKT1/PTEN altered tumors subgroup although an overall survival benefit cannot be excluded in patient with nonaltered tumors. In SOLAR-1 trial [START_REF] André | Alpelisib for PIK3CA-Mutated, Hormone Receptor-Positive Advanced Breast Cancer[END_REF] , clinically relevant PFS benefit was initially reported in patients with hormone-refractory, HER2-negative advanced breast cancers with a PIK3CA mutation, but, more recently, updated data [START_REF] André | Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1[END_REF] reported no significant difference in OS with 39.3 months (95% CI, 34.1-44.9) in the alpelisib plus fulvestrant arm versus 31.4 months (95% CI, 26.8-41.3) in the placebo plus fulvestrant arm (HR = 0.86; 95% CI, 0.64-1.15; P = 0.15).

In FAKTION phase 2 trial [START_REF] Jones | Fulvestrant plus capivasertib versus placebo after relapse or progression on an aromatase inhibitor in metastatic, oestrogen receptor-positive breast cancer (FAKTION): a multicentre, randomised, controlled, phase 2 trial[END_REF] , the significant improvement of PFS observed with capicasertib and fulvestrant versus placebo observed in the overall population was preserved in the PI3kinase/PTEN nonaltered group (HR 0.56, 0.33-0.96, P=0.035) but not in the altered group (HR 0.59, 0.34-1.03, P=0.064).

As compared to everolimus, using PI3-kinase inhibitors are associated with higher incidence of adverse events and higher cost. Therefore, the combination of everolimus to endocrine therapy could remain an effective and reasonably well-tolerated option in HR+ HER2negative breast cancers progressing after endocrine therapy, independently of PIK3CA mutational status [START_REF] Andre | Molecular Alterations and Everolimus Efficacy in Human Epidermal Growth Factor Receptor 2-Overexpressing Metastatic Breast Cancers: Combined Exploratory Biomarker Analysis From BOLERO-1 and BOLERO-3[END_REF][START_REF] Vernieri | Everolimus versus alpelisib in advanced hormone receptorpositive HER2-negative breast cancer: targeting different nodes of the PI3K/AKT/mTORC1 pathway with different clinical implications[END_REF] We reported here a rapid decrease of the PI3K/AKT/mTOR activity and tumor cell proliferation in response to trastuzumab treatment. Such pharmacodynamics of downstream pathways of HER2 were expected following trastuzumab therapy [START_REF] Nahta | Pharmacological strategies to overcome HER2 cross-talk and Trastuzumab resistance[END_REF] . The addition of everolimus to trastuzumab did not synergized the PI3K/AKT/mTOR inhibition, but resulted in the activation of the MAPK pathway. PI3K/AKT/mTOR and MAPK pathways are interconnected with multiple crosstalk and feedback loops. Indeed, tumor biopsies obtained from patient with melanoma, breast, and colon cancer treated in the neoadjuvant setting with everolimus displayed ERK activation [START_REF] Saini | Targeting the PI3K/AKT/mTOR and Raf/MEK/ERK pathways in the treatment of breast cancer[END_REF][START_REF] Carracedo | Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer[END_REF] . The negative feedback loop suppression by mTOR inhibitor can explain the activation of MEK-ERK pathway in the combination arm suggesting the potential interest of combining mTOR with MEK inhibitors to allow full blockade of HER2 downstream signaling.

On the other hand, we detected rapid decrease of PTEN protein expression. PTEN, a negative regulator of the PI3K signaling, is mutated or lost in various cancers and loss of PTEN causes resistance to trastuzumab [START_REF] Nagata | PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients[END_REF] . In our study, no variation in PIK3CA gene mutation and PTEN repression rates was detected between baseline and surgery and did not suggest any early induction of resistance to HER2 inhibition through the alteration of PI3kinase signaling pathway.

There is increased evidence that ADCC is an important mechanism of action of trastuzumab. Furthermore, preclinical and clinical studies demonstrated that tumor-infiltrating lymphocytes can predict outcome of trastuzumab-containing treatment [START_REF] Loi | Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial[END_REF][START_REF] Varadan | Immune Signatures Following Single Dose Trastuzumab Predict Pathologic Response to PreoperativeTrastuzumab and Chemotherapy in HER2-Positive Early Breast Cancer[END_REF] . Our analyses showed an increase of tumor-associated lymphocytes infiltrates following only 6 weeks of trastuzumab confirming its efficacy to induce immune reactions, independently of everolimus treatment. This provide strong rationale for combining trastuzumab with enhancer of immunity such as checkpoint inhibitors. No relationship was observed between PIK3CA mutations and lymphocyte infiltration either at diagnosis (P=.099) or at surgery (P=.043). Our findings contrast from those achieved in post-operative setting in the ShortHER trial [START_REF] Guarneri | PIK3CA Mutation in the ShortHER Randomized Adjuvant Trial for Patients with Early HER2+[END_REF] with a better DFS within the HER2-enriched subtype, in PIK3CA mutated tumors, hypothetically in relation with an upregulation of immune-related genes. However our study differs from ShortHER trial regarding the duration of treatment by trastuzumab (6 weeks in our study vs 9 weeks or 1 year in ShortHER) and the combination of trastuzumab with chemotherapy in ShortHER trial.

In conclusion, combining everolimus with trastuzumab, altered the regulation of both PI3K and MAPK pathways, with evidence of early cell adaptation to treatment. Moreover, the inability to identify predictive biomarkers of activity suggests that response to trastuzumab alone or combined with everolimus may be driven by additional mechanisms. members (Jean-Luc Canon, Stefan Michiels, Serge Evrard), Sarah MacKenzie (funded by Unicancer, France) and Lilian Amrein from Unicancer for medical writing and editing.

Breast Cancer: Association with Prognosis and Integration with PAM50 Subtype. Clin Cancer Res. 2020 Nov 15;26 (22):5843-5851.

Figure 1: Flow-Chart of the study

Figure 2 : Relative Ki67 (A), pMEK1 (B) and pERK1/2 (C) variation calculated between initial biopsy and surgery following pre-operative treatment by trastuzumab alone (arm T) and trastuzumab + everolimus (arm T+E).

Light grey boxes are calculated from all patients' data, dark grey boxes from complete clinical responders and white boxes from non-complete responders. WHO, World Health Organization a. Complete response + partial response at 6 weeks or after 3 weeks in the absence of 6-week data, in the ITT population. b. Note that for the 2 bilateral patients, one breast was identified before initiating the treatment for the response assessment (one patient was HER2-positive on both sides and presented a stable disease and a complete pathologic response on the selected breast, while the contralateral tumor did not respond; the other patient was HER2-positive on one side, selected for response assessment, and had an undetermined HER2 status on contralateral tumor) c. Three patients were not evaluable; two not treated (one per arm), one patient progressed and was treated with chemotherapy before surgery (trastuzumab arm).

TABLES

Table 1 . Patient, tumor and treatment characteristics, ITT population

 1

	Arm T	Arm T+E
	Trastuzumab	Trastuzumab +
	alone	Everolimus
	N=41	N=41

Table 2 . Summary of efficacy outcomes

 2

	Arm T	Arm T+E	P-value
	Trastuzumab	Trastuzumab +
	alone	Everolimus

Table 3 : Ki67 and signaling biomarkers analysis.

 3 Relative variations (∆) were calculated from baseline and surgery specimens and compared d within arm T and arm T+E and e between arms. Data are presented as f mean variation (SD, %).

		∆ arm T d,f	P value	∆ arm T+E d,f	P	T vs T+E e
					value
	Ki67 a	-12.4 (14.5), N=23	<0.001	-20.8 (23.2), N=32	<0.001	NS
	n_pAKT1 b -2.09 (1.90), N=23 <0.001	-2.26 (1.98), N=31	<0.001	NS
	c_pAKT1 b -1.35 (2.25), N=23	0.009	-2.26 (2.41), N=31	<0.001	NS
	pS6RP b	-0.78 (1.65), N=23	0.033	-1.50 (2.05), N=32	<0.001	NS
	n_LKB1 b	-1.26 (2.28), N=23	0.015	-1.97 (2.50), N=30	<0.001	NS
	c_LKB1 b	0.39 (1.50), N=23	NS	-0.40 (1.89), N=30	NS	NS
	eIF4E b	0.04 (1.72), N=23	NS	0.42 (2.51), N=31	NS	NS
	p4EBP1 b	0.00 (0.67), N=23	NS	0.03 (0.55), N=31	NS	NS
	pMEK1 c	-1.3 (52.6), N=19	NS	115.2 (195.1), N=18	0.005	0.014
	pERK1/2 c 0.04 (39.8), N=19	NS	48.3 (103.8), N=18	0.030	0.017
	CD8	0.63 (0.76), N=19	0.007	0.37 (0.76), N=30	0.018	NS
	CD68	0.33 (0.69), N=18	NS	0.10 (0.77), N=29	NS	NS
	n_pAKT1: nuclear phosphorylated AKT1, c_pAKT1 : cytoplasmic phosphorylated AKT1, pS6RP : phosphorylated
	S6 ribosomal protein, n_LKB1 nuclear LKB1, c_LKB1: cytoplasmic LKB1

a % stained nuclei from IHC.

b

Allred scoring from IHC. c normalized fluorescence intensities from multiplex immunoassays.

Table 3 : Ki67 and signaling biomarkers analysis.

 3 Allred scoring from IHC. c normalized fluorescence intensities from multiplex immunoassays. Relative variations (∆) were calculated from baseline and surgery specimens and compared d within arm T and arm T+E and e between arms. Data are presented as f mean variation (SD, %).

		∆ arm T d,f	P value	∆ arm T+E d,f	P	T vs T+E e
					value
	Ki67 a	-12.4 (14.5), N=23	<0.001	-20.8 (23.2), N=32	<0.001	0.201
	n_pAKT1 b -2.09 (1.90), N=23 <0.001	-2.26 (1.98), N=31	<0.001	0.742
	c_pAKT1 b -1.35 (2.25), N=23	0.009	-2.26 (2.41), N=31	<0.001	0.269
	pS6RP b	-0.78 (1.65), N=23	0.033	-1.50 (2.05), N=32	<0.001	0.211
	n_LKB1 b	-1.26 (2.28), N=23	0.015	-1.97 (2.50), N=30	<0.001	0.293
	c_LKB1 b	0.39 (1.50), N=23	0.278	-0.40 (1.89), N=30	0.304	0.228
	eIF4E b	0.04 (1.72), N=23	0.539	0.42 (2.51), N=31	0.265	0.654
	p4EBP1 b	0.00 (0.67), N=23	0.999	0.03 (0.55), N=31	0.790	0.663
	pMEK1 c	-1.3 (52.6), N=19	0.953	115.2 (195.1), N=18	0.005	0.014
	pERK1/2 c 0.04 (39.8), N=19	0.709	48.3 (103.8), N=18	0.030	0.017
	CD8	0.63 (0.76), N=19	0.007	0.37 (0.76), N=30	0.018	0.317
	CD68	0.33 (0.69), N=18	0.066	0.10 (0.77), N=29	0.488	0.248
	n_pAKT1: nuclear phosphorylated AKT1, c_pAKT1 : cytoplasmic phosphorylated AKT1, pS6RP : phosphorylated
	S6 ribosomal protein, n_LKB1 nuclear LKB1, c_LKB1: cytoplasmic LKB1

a % stained nuclei from IHC. b

Table 4 : PIK3CA mutation and PTEN expression loss rates, considered either alone or combined in trastuzumab alone (arm T) and trastuzumab + everolimus (arm T+E) and relationship with clinical response rate.

 4 PTEN expression was analysed using IHC, b PIK3CA mutations using NGS. c calculated from data collected from the entire patient population (arm T and arm T+E)

					P value	Entire	Clinical	P value	Complete	P value
					(T vs	population	response	(No vs	clinical	(No vs
					T+E)		rate	Yes)	response	Yes)
			Arm T	Arm T+E			(CR+PR) c		rate (CR) c
	PIK3CA mutation b No	23 (85.2%) 22 (68.8%) 0.242 45 (76.3%) 22 (91.7%) 0.029 10 (90.9%) 0.269
	N=59	Yes	4 (14.8%) 10 (31.2%)		14 (23.7%) 2 (8.3%)		1 (9.1%)
	PTEN loss a	No	7 (29.2%)	14 (40%) 0.393 21 (35.6%) 10 (38.4%) 0.683 5 (41.7%) 0.623
	N=59	Yes	17 (70.8%) 21 (60%)		38 (64.4%)( 16 (61.5%)		7 (58.3%)
	PTEN loss or	No	5 (21.7%)	7 (21.9%)	0.990 12 (21.8%)	7 (30.4%)	0.190	3 (30%)	0.673
	PIK3CA mutation N=55	Yes	18 (78.3%)	25 (78.1%)		43 (78.2%)	16 (69.6%)		7 (70%)

a

ACKNOWLEDGEMENTS

We thank the patients and their families who participated in this study, the study sites for trial management, Céline Mahier Aït-Oukhatar from Unicancer for study management, EuraxiPharma for study management support, independent data monitoring committee

Financial support

Jean-Louis Merlin received grant from the French Health Ministry (PHRC 2008/17-2).

Unicancer as the study sponsor received support from Novartis, and Roche.

Conflicts of interest statements

MC received research fundings and served as speaker / advisor (compensated to the hospital) for Astra Zeneca, Novartis, Abbvie, Sanofi, Lilly, Pfizer, Sandoz, Accord, G1


Elham Jelodari Mamaghani

Khaled Medini

Sustainable, resilient, and agile manufacturing and service operations: Lessons from COVID-19 Resilience, agility and risk management in production ramp-up

Keywords: Ramp-up production, agility, resilience, risk management, production uncertainties

Production ramp-up is challenged by several hurdles such as unpredicted events to achieve targets like providing on-time service in critical situations and under budget constraints. The interrelated concepts of resilience, agility and risk management increase the ability of the system to handle the changes effectively. Therefore, in order to successfully manage production ramp-up particularly in high variety environments, resilience, risk, and agility should be considered in a holistic way. This will help address complexity and uncertainty underlying manufacturing and service operations. This paper aims to fill this gap by reviewing the literature and reporting on the basic concepts and interrelationships of resilience, agility, and risk management in the ramp-up phase. Ultimately, the paper aims to lay some foundations for further research in the crossing of these areas.

Introduction

Nowadays, companies to win the market competition have to raise the variety of their offering to satisfy a larger panel of customers by presenting more individualized products [START_REF] Elmaraghy | Product Variety Management[END_REF]. Among the critical steps of the marketing of individualized products is the ramp-up phase, particularly in terms of when and how it should be conducted [START_REF] Lanza | Simulation of Personnel Requirements during Production Ramp-up[END_REF]. However, production ramp-up management is confronted with several challenges such as uncertainty on both demands and on capacity [START_REF] Vda | FAST 2025 -Massiver Wandel in der automobilen Wertschöpfungsstruktur. Europa bleibt starker Industriestandort und behält F&E-Vormachtstellung[END_REF]. For instance, these uncertainties may lead to problems in product quality and therefore the associated cost should be taken into account in the definition of ramp-up budget. Agility and robustness play a major role in handling uncertainties on time in production systems [START_REF] Herrmann | Ramp-up Failure Rate. Einführung einer Qualitätskennzahl für den Produktanlauf[END_REF]. An important consequence of agility is a quick response to environmental changes like customers' demands, supplier changes, pricing pressure, etc. In contrast, the aim of a robust process is the reduction or avoidance of any destructions. The concept of resilience is propounded to combine both agility and robustness to increase the ability of the system to handle the changes effectively [START_REF] Bhamra | Resilience: The concept, a literature review and future directions[END_REF][START_REF] Ivanov | Adaptation-based supply chain resilience[END_REF].

Resilience is closely related to and relying on risk management. Authors such as [START_REF] Dewaelheyns | Legal reform and aggregate small and micro business bankruptcy rates: evidence from the 1997 Belgian bankruptcy code[END_REF] explored bankruptcy situations in the construction industry of Belgian companies and revealed that the lack of effective handling of uncertainty by risk management was one of the major reasons of the bankruptcies. Risk management supports the identification of risk factors and their impacts during the ramp-up as well as suitable solutions [START_REF] Schatteman | Methodology for integrated risk management and proactive scheduling of construction projects[END_REF].

In order to successfully manage production ramp-up particularly in high variety environments, resilience, risk, and agility should be considered in a holistic way. This will help address complexity and uncertainty underlying manufacturing and service operations. According to the authors' best knowledge, no review paper is available to cover production ramp-up considering these three concepts altogether. This paper aims to fill this gap by reviewing the literature and reporting on the main basic concepts and interrelationships of resilience, agility, and risk management in the ramp-up phase.

The remainder of the paper is organised as follows, Section 2 describes the research methodology. Section 3 reports on the results of literature review. Section 4 discusses the identified papers out of the literature review. Finally, Section 5 provides conclusion and propositions for future work.

Research methodology

The aim is to conduct a systematic literature review to shed more light on resilience, risk and agility in ramp-up management. The approach proposed by [START_REF] Govindan | Reverse logistics and closed-Loop supply chain: A Comprehensive Review to Explore the Future[END_REF] is identified as appropriate for the current research. They described that each systematic literature review has four steps: material collection, descriptive analysis, category selection and material evaluation. The literature search approach used to extract the relevant articles is iterative to refine the search parameters (e.g. key words, date of publication, etc.). The different steps of the procedure to extract the articles are presented as follows and the articles are summarized in table 1:

1. Definition of the scope: The databases and the journals are chosen such that they are representative of the research. The applied databases and search engines are Science Direct, Scopus, and Google Scholar.

Paper selection:

The keywords used include the following risk, agility, resilience, ramp-up. The first filtering criterion of the papers is the time span; only papers published between 2004 and 2021 were selected. These include journal and conference papers. An initial search of the databases resulted in a total number of papers of 38 papers. 7 papers out of these are associated with risk analysis, risk management, challenges of risks in ramp-up production. 20 papers are concerned with the resilience concept (not only in ramp-up management due to the lack of studies). 13 papers of the studied articles are related to agility and its challenges in ramp-up management (see Figure 1). The papers cover fundamental studies or applications to industries such as automotive or electronics.

Results from literature review

Resilience

In today's complex manufacturing environment, failure and uncertainties become more frequent. On the one hand, including some approaches to handling these uncertainties is vital and on the other hand, having a resilient system is important. Resilience deals with unexpected challenges by presenting solutions to adversities. The resilience concept is identified by what, why, and how a system reacts when facing failures [START_REF] Zhang | Toward a resilient manufacturing system[END_REF]. Hence, recognizing its characteristics was a challenge for researchers [START_REF] Coutu | How Resilience Works[END_REF]. This concept has been applied in various areas; some researchers have included resilience as an attribute to deal with personal stress in enterprises [START_REF] Sheffi | The Resilient Enterprise: Overcoming Vulnerability for Competitive Advantage[END_REF] while [START_REF] Christopher | Building the Resilient Supply Chain[END_REF][START_REF] Rice | Building a Secure and Resilient Supply Network[END_REF][START_REF] Heinicke | Implementation of resilient production systems by production control[END_REF] investigated through the topic of supply chain and controlling natural disasters. By exploring in the literature, it was seen that the resilience concept was addressed not only in 18% 33% 50% Risk Agility Resilience ramp-up but also in production systems at large. Therefore, the remainder of this section will focus on the large area of production systems.

Resilience of production systems relies on drivers such as closed loop of the tasks in production control, functional map of production control tasks, implementation of robust processes. An interesting point in [START_REF] Heinicke | Implementation of resilient production systems by production control[END_REF] was the combination of two opposite concepts namely agility and robustness in resilience. The authors found that creating standard information in production ramp-up about the process of material flows has an effective role to set resilience system. Other authors studied the impact of several factors on system resilience [START_REF] Heinicke | Influence of shifts in production programs on the resilience of production systems[END_REF][START_REF] Dinh | Resilience engineering of industrial processes: principles and contributing factors[END_REF]. Among the growing orientations in production systems is the combination of resilience and lean production strategies. The impact of this combination on the maintenance and performance of the whole system was reported on in [START_REF] Zarrin | Simulation optimization of lean production strategy by considering resilience engineering in a production system with maintenance policies[END_REF][START_REF] Azadeh | Performance optimization of integrated resilience engineering and lean production principles[END_REF].

Resilience is one of the drivers to achieve sustainability in the industry [START_REF] Perrings | Resilience and sustainable development[END_REF]. To assess this driver, [START_REF] Aleksić | An assessment of organizational resilience potential in SMEs of the process industry, a fuzzy approach[END_REF] proposed a fuzzy model to reach the value of resilience factors. The authors determined that the suggested mathematical model could not give appropriate results while using a conceptual model could be more suitable. [START_REF] Ouedraogo | How to learn from the resilience of Human-Machine Systems?[END_REF] studied the resilience of human machines during perturbations. Some series of possible learning scenarios were proposed to improve the resiliency of the system.

Risk Management

Customers' requirements are increasing day to day which lead to tough competition and frequent product development and ramp-up projects [START_REF] Elmaraghy | Product Variety Management[END_REF]. This results in larger number of product variants and recurring changes [START_REF] Vda | FAST 2025 -Massiver Wandel in der automobilen Wertschöpfungsstruktur. Europa bleibt starker Industriestandort und behält F&E-Vormachtstellung[END_REF]. Therefore, manufacturers have to cope with products that are more complex in less time with less money and make sure to production (SOP) on time.

Risk management is a key factor in the ramp-up of production to reduce uncertainties and disruption in the manufacturing systems. Early identification and minimization of the risks of ramp-up can lead to effective actions and suitable response strategies during the transition step [START_REF] Elstner | Methodical approach for consideration of rampup risks in the product development of complex products[END_REF]. In production systems such as in the automotive sector, if a price for each product is considered 25.000€ and 25000 cars are produced per month, 44€ million will be lost if the manufacture is undertaken a risk of SOP reduction to obtain a return on the sale of 7% besides reduction of the product life cycle in one month [START_REF] Cube | Execution of ramp-up projects in day-today operations through a quantitative risk management approach[END_REF].

There are different approaches to risk management that depend on the applied sector; for example in car manufacturing top-down risk analysis is commonly used to model the risks [START_REF] Cube | Execution of ramp-up projects in day-today operations through a quantitative risk management approach[END_REF]. Risk management approaches (top-down) in ramp-up projects of car manufacturing can be categorized into four models [START_REF] Cube | Execution of ramp-up projects in day-today operations through a quantitative risk management approach[END_REF], 1) Planning and organizational models, 2) coordination models, 3) knowledge management models, and 4) central defects and need for action were considered in the paper. In automotive sector, surveys can be used to determine and model risks in the sub-process of pre-series logistics [START_REF] Filla | Risk profiles for the pre-series logistics in automotive ramp-up processes[END_REF]. In hybrid manufacturing technologies, ramp-up production is more challengeable than single products. [START_REF] Nau | Risk assessment of hybrid manufacturing technologies for ramp-up projects[END_REF] proposed a systematic design approach to hybrid manufacturing and software a method was suggested to evaluate the risks in the ramp-up production.

Agility

Unpredicted events in the ramp-up phase prevent the system from obtaining targets on time. Agility is likely to help companies deal with unpredicted events and reach time-tovolume without exceeding the ramp-up budgets [START_REF] Bergs | Agile ramp-up production as an advantage of highly iterative product development[END_REF]. Furthermore, agility can provide the right product at the right time [START_REF] Agarwal | Modeling agility of supply chain[END_REF]. [START_REF] Kremsmayr | On the application of agility principles in ramp-up management: Approaching the challenges in the high-end powder metallurgy industry[END_REF] studied agility application in the metallurgy industry. It was shown that early and reliable information on product and process maturity has an important impact on rampup production.

The papers report on several approaches to improve the agility in production ramp-up. Analysis of the manufacturing context and proposing data system implementation is one of the approaches applied in electronics manufacturing systems studied by [START_REF] Helo | Managing agility and productivity in the electronics industry[END_REF]. [START_REF] Shi | Rapid production ramp-up capability: a collaborative supply network perspective[END_REF] proposed creating a relation among critical parts of supply networks and operational activities for production ramp-up through a conceptual experimental framework. They argued that systematic ramp-up production is very important to achieve time-to-volume. In the ramp-up phase of physical products for the agile product development, [START_REF] Schuh | Enabling agility in product development through an adaptive engineering change management[END_REF] proposed an adaptive engineering change management (ECM). This approach is guided by the following initial questions: What is the data structure? How processes are implemented and what are the roles? Finally, what are the communication tools? Based on these challenges a three-layer model with a responsive design was proposed [START_REF] Schuh | Enabling agility in product development through an adaptive engineering change management[END_REF]. Collecting real-time data via approaches of industry 4.0 can support the learning and decision-making process to improve the agility in ramp-up production [START_REF] Schmitt | On the future of ramp-up management[END_REF]. [START_REF] Bergs | Agile ramp-up production as an advantage of highly iterative product development[END_REF] mentioned two challenges in agile production ramp-up; the first one is the identification of adjustment levers to manage the uncertainties in production ramp-up and the second one is to develop a concept to stop a predetermined design in the contract.

Discussion

The previous sections shows some commonalities as well as complementarities between the various concepts. For example, what can be observed in the literature of agility is the focus on timely response to change and change management to survive and dealing with diversities [START_REF] Herbane | Rethinking organizational resilience and strategic renewal in SMEs[END_REF] to satisfy the market needs. Indeed, in agility, the speed of responding to the changes is more important than resilience [START_REF] Shekarian | An examination of the impact of flexibility and agility on mitigating supply chaindisruptions[END_REF][START_REF] Miceli | Thriving, not just surviving in changing times: How sustainability, agility and digitalization intertwine with organizational resilience[END_REF]. [START_REF] Gligor | Distinguishing between the concepts of supply chain agility and resilience[END_REF] clarified different and common features in agility and resilience concepts in the supply chain that interested readers can refer to. In Figure 2 the common and distinctive features of the two concepts are illustrated.

Figure 2. Common and distinctive features of agility and resilience [START_REF] Miceli | Thriving, not just surviving in changing times: How sustainability, agility and digitalization intertwine with organizational resilience[END_REF] Manufacturing and service operations are becoming exponentially more complex. This adds to the challenges ahead of academics and practitioners in the broad field of industrial management. More specifically, ensuring the resilience of the system, be it a production system or a supply network, is confronted with more difficulties such as the uncertainty induced by changing enterprise operating environment and evolving customer requirements. Therefore, a holistic approach integrating resilience, agility, and risk criteria is required for manufacturing and service companies. The ramp-up phase as a driver of product introduction in the market should integrate these criteria.

To this end, the interrelationships between resilience, agility, and risk should be explicitly defined to unleash their synergies. From a more practical point of view, these criteria should be taken into account at various levels during ramp-up, organizational, information system, and levels. This perspective could benefit from existing works such as [START_REF] Nau | Risk assessment of hybrid manufacturing technologies for ramp-up projects[END_REF][START_REF] Mubarik | Resilience and cleaner production in industry 4.0: Role of supply chain mapping and visibility[END_REF].

The literature review presented in this paper, uncovered also some thematic areas, which might need further investigation; this includes learning and human factors, risk, and sustainability. The learning process has a prominent impact on production ramp-up particularly from an agility point of view. The learning process affects heavily the adaptation of personnel when unpredictable events occur during ramp-up. Moreover, in the ramp-up phase, to handle risks one should have a comprehensive view of various risk factors to provide appropriate mitigation plans limiting temporal and financial changes. Furthermore, sustainability in ramp-up production has not been investigated extensively in the literature. Hence, there is a research potential in the literature. Among the questions to be addressed at this point, can be cited the following, how sustainability can be still improved during ramp-up? How sustainability assessment can be integrated within the triptych resilience, agility, and risk? [START_REF] Mubarik | Resilience and cleaner production in industry 4.0: Role of supply chain mapping and visibility[END_REF].

Finally, most of the addressed case studies are in electronics or automotive sectors. The critical situation due to COVID-19 showed that agility, risk, and resilience in ramp-up production should be included in new industries (ex. ventilators). It is an important lack in the literature that can be addressed in future research work.

Conclusion

This paper presented and discussed a literature review of resilience, agility, and risk in production ramp-up. The vast majority of the reviewed studies support that all three concepts are a key for ensuring enterprise competitiveness, particularly under critical conditions. However, the further investigation involves addressing the interrelationships of these concepts to unleash their synergies and to adapt to changing environments.

Figure 1 .

 1 Figure 1. Covering papers of three subjects

Table 1 .

 1 Summary of the identified papers

	Reference	Risk	Agility	Resilience	Ramp-up
	[1]				*
	[2]				*
	[3]		*		*
	[4]		*		*
	[5]		*		*
	[6]		*		*
	[7]	*		*	*
	[8]	*			*
	[9]				*
	[10]	*			*
	[11]	*			*
	[12]	*			*
	[13]	*			*
	[14]		*		*
	[15]		*		*
	[16]		*		*
	[17]		*		*
	[18]		*		*
	[19]		*		*
	[20]			*	*
	[21]			*
	[22]			*
	[23]			*
	[24]			*
	[25]			*
	[26]			*
	[27]			*
	[28]			*
	[29]			*
	[30]			*
	[31]			*
	[32]			*
	[33]			*
	[34]		*	*
	[35]			*
	[36]			*
	[37]		*	*
	[38]	*	*	*

Acknowledgements

This work is partly supported by the German-French Academy for the Industry of the Future through RAMP-UP Seed project (https://www.future-industry.org/research/advancedmanufacturing/ramp-up/).