Dr Pierre Ferdinand

Dr Guillaume Laffont

Nicolas Roussel

Jonathan Boussoir

Laurent Maurin

Stéphane Rougeault

Enhancing train transportation remains a key issue since transportation means are gradually becoming saturated in capacity in modern countries. Therefore, the European Union (EU) develops initiatives to enhance the rail grid competitiveness, by means of deregulation and interoperability, and supports this process with an existing rail network planned upgrade.

In such a context, the 6 th European Framework Program (FP6) CATIEMON project (CATenary InterfacE MONitoring; 2005-2009), has been specifically devoted to the development of instrumentations and sensors able to 'control' any train entering a railway network. This was achieved by monitoring the interaction between the train pantograph and its Overhead Contact Line (OCL) at a standing location: a control gate, equipped with permanently controlled sensors.

Several solutions have been simultaneously evaluated, including different kinds of sensors and measurement systems. The electromagnetic-immune Optical Fibre Bragg Grating (FBG) sensing technology developed by CEA List has demonstrated, within this project, fully relevant, reproducible, and exploitable measurements for the infrastructure manager.

To do so, we have been working on the entire FBG instrumentation, including the development of innovative sensors, as well as the set-up of two proprietary measurements units. The first one, lightweight, small-form factor one: BraggLight, proved on field to be very handy to work in 'acrobatic' and bad weather conditions for FBG sensor lines control during their installation on the OCL. The second one, BraggFit 3 , with high-end performances, devoted to high-speed and very accurate Bragg wavelength measurements (resolution and precision better than 1 pm at 1 kHz full speed in parallel on 6 optical lines) for permanent monitoring purposes.

Such developments enable today the infrastructure managers to control the rolling stock operators' compliance with their specific regulation procedures (e.g.: authorized maximum wear on the OCL), and allow them to stop any train identified by the control gate to potentially damage their infrastructure. More generally, the FBG sensors and systems developed at CEA List can be used in many industrial sectors, including transportation (railway, marine, aeronautics) but also civil infrastructures (bridges, buildings, power plants, underground…), as well as oil & gas industry, for structure monitoring and smart structure purposes, and are particularly well-suited in harsh environments where non-intrusive high-speed sub-picometric (~ 0.5 µm/m or 5/100°C at 1 kHz) Bragg wavelength accuracy and resolution are required.

STATE-OF-THE-ART OF FBG MEASUREMENT SYSTEMS

The most straightforward mean to interrogate a FBG transducer is based on broadband source illumination (using a broadband light source such as an ASE -Amplified Spontaneous Emission -source or a SLED -Super Luminescent Diodesource) whose narrow-band reflected peak is directed into a wavelength detection system. Several methods exist to measure the wavelength shift of the sensor including a miniaturized spectrometer, a wavelength tuneable pass-band filter, an interferometer or an edge filter. Each method presents advantages and drawbacks with respect to the basic requirements of a high-performance FBG monitoring system. CEA List has already developed interrogation units using many of these techniques. Using a widely tuneable source, such a system has reached sub-picometric spectral resolution over a range of 30 nm on the telecom C-band (@ 1.55 µm). Six measurements channels were available in order to connect several sensing optical fibers (each of them incorporating several tens of wavelength-multiplexed FBG transducers) whereas two internal channels were used for referencing (absolute wavelength referencing using a gas cell, and output optical power referencing). Such a system has been deployed for field tests during a European project -called CATIEMON -that will be described in details in the forthcoming sections.

Study case: the railway industry

The railway network deregulation policy has been in progress for several years in Europe and will soon become effective. So, in this context, the interaction between the Overhead Contact Line (OCL) and the pantographs becomes the new separating interface between infrastructure managers and train operators, standing and rolling stock. Therefore, the monitoring of this interface becomes relevant for a better availability of the interoperated networks. It will contribute to a decrease of the traffic interruption and of the maintenance costs [START_REF]European transport policy for 2010: time to decide[END_REF].

At the former IWSHM conference in Stanford (2007), we presented the 5 th European Framework Program (FP5) SMITS project (Smart Monitoring of Train Systems) devoted to the contact force measurements between a train pantograph and its OCL [START_REF] Maurin | High speed realtime contact measurements between a smart train pantograph with embedded fibre Bragg grating sensors and its overhead contact line[END_REF]. SMITS project guideline was to give the rolling stock operators an onboard tool to optimize in real-time the contact force parameters during daily operation to prevent pantograph excessive wear on the OCL (to comply with standing stock specifications) and predict more accurately current collectors carbon strips lifetime to proceed to predictive maintenance. To do so, several well-suited electromagnetic-immune optical Fibre Bragg Grating (FBG) sensor lines were embedded by SMITS partners into the pantograph current collectors, turning them into two 3 points bending-based sensors in contact with the OCL. 6000 km on-line validation tests were performed during one week on a very high speed double-deck train "TGV Duplex" between Paris and Vendôme (France) at speeds up to 320 km/h. Contact forces between the pantograph and the OCL, as well as temperatures inside the current collectors, were computed and successfully compared in real time (500 Hz on 4 lines in parallel) with the existing traditional electrical gages provided by the SNCF for reference measurements. During these TGV tests, the innovative FBG measurement system developed by the CEA LIST has demonstrated its reliability and accuracy with temperature measurements, and coherence with contact force measurements in comparison with SNCF reference records.

Thanks to these good results, a second European project driven by Siemens (D) 1 , acronym CATIEMON (CATenary InterfacE MONitoring), started in 2005, supported by the 6 th European Framework Program (FP6) [3]. The motivations for such a new project are as follows: monitor and investigate the pantograph/catenary interactions directly "from the wire", measure the contact force applied by a pantograph to the OCL, minimize the infrastructure maintenance costs, prevent catenary-pantograph incidents and thus traffic interruptions, propose solutions for an automated alert system. Monitoring operations of the overhead infrastructure are regularly planned, and executed by railways companies with dedicated trains. But, such operations are complicated to realize, and imply expensive equipments as well as high-skilled people. Moreover, these methods of surveillance are only able to determine defects linked to the infrastructure, and not problems associated to trains.

In fact, badly adjusted pantographs and/or damaged current collectors may also be a source of incidents. Indeed, a too strong contact force induces an excessive wear on the OCL and cause damages to the infrastructure. On the contrary, a too weak contact force will generate electric arcing when loosing contact -damaging the pantograph carbon strips as well as the OCL -but will also induce fluctuations of electrical current, limiting train speed. So, to anticipate potential problems caused to both the infrastructure and the pantographs, a concept of inspection gate has been designed and developed by the consortium [START_REF] Bosselmann | Verfahren und Einrichtung zur Messung der Kontaktkraft eines Stromabnehmers[END_REF]. Positioned permanently and in a strategic point of a railway grid, it includes various types of sensors, and their associated instrumentations, enabling local (i.e. in well-known conditions) characterization and diagnostics at the OCLpantograph interactions [START_REF] Kusumi | Diagnosis of Overhead Contact Line based on Contact Force[END_REF][START_REF] Hyeon | Numerical simulations of the dynamic responses of railway overhead contact lines to a moving pantograph, considering a nonlinear dropper[END_REF]. Such a control gate aims not only at stopping a train presenting an immediate risk for the infrastructure (and thus reduce the risks of incident, as in tunnels) but also at estimating the impact of any train in term of infrastructure wear. In the CATIEMON project, the CEA List has been developing several FBG-based sensors 1) Uplift sensor based on displacement FBG sensors, 2) FBG-Impact sensors, and 3) distributed strain measurement of OCL with several FBG chains glued on the copper wire) and of course FBG demultiplexing units.

Railway control gate specifications

The control gate developed within the framework of the CATIEMON project is a first prototype. So it includes various measurement technologies developed by the partners: rangefinder laser for 1D uplift measurement, laser scanner coupled with a camera for a two-dimensional uplift measurement (vertical and horizontal), 3D FBG-based displacement sensors, and distributed FBG strain sensors.

Nevertheless, FBG-based metrology was the only technology capable of performing the complete range of the measurements, namely the 3D uplift (FBG triangulation sensor) of the OCL, and the distribution of strain along the contact wire. A steady arm (anchor arm used to define the contact wire position with respect to rails) was also instrumented with FBGs for hit detection. Figure 1 illustrates the location of these FBG sensors on the inspection gate.

TECHNICAL DEVELOPMENTS

Fiber Bragg Gratings (FBGs) are well known diffraction gratings photo written by UV laser interferometry within the core of singlemode fibers. An FBG consists of a submicronic modulation of the fiber core refractive index 'n', and acts as a reflector for a very narrow spectral bandwidth centred on a characteristic wavelength 'λ B ' that is directly dependent on its pitch 'Λ', through the well known relation λ B = 2.n.Λ. The Bragg wavelength depends on both temperature and strain (Fig. 2).

BraggFit 3 demultiplexing system

The BraggFit 3 system is made up of an industrial rack and a PC with a dedicated software. It enables accurate static and dynamic measurement up to the kHz, and its absolute wavelength referencing procedure.

Bragg Fit 3 performances are listed below.

Measurement performances

Measurement range: 30 nm (1.55 µm range, C band) Resolution: better than 1 pm at 1 kHz / 0.1 pm at 10 Hz Stability: over 24 h / over 20°C < 1 pm Gain 40 dB (adjustable for each channel) Frequency scan: 1 kHz (6 channels of 30 nm swept in 1 ms) Obviously, this BraggFit 3 system (Fig. 3) developed during the CATIEMON project is designed for interrogating all types of FBG optical fibre sensors (temperature, strain, pressure, force…). It is so powerful that it meets the needs of different application fields devoted to Structure Monitoring and the development/monitoring of Smart Manufacturing composite material for instance.

Measurement channels analysed in real time

BraggLight measurement system

The BraggLight system realizes the FBGs spectral analysis at a scan rate up to 200 Hz. It is made up of a compact box connected to a laptop. Communications with the PC as well as the power supply are made by means of a single USB connection. With this basic design (Fig. 4) only one fiber is connected, but multichannel versions are also possible. BraggLight performances are listed below.

Measurement performances

Measurement range: up to 80 nm (1.55 µm range) Resolution: 0.5 pm Accuracy/repeatability: 5 pm Thermal stability: 1 to 2 pm/°C (compensation available) Frequency scan: 200 Hz The working conditions (at night, bad cold weather, wind) required specific installation procedures, especially for the FBG line gluing on contact wire (Fig. 5).

Measurement channels analysed in real time

These sensing lines were fixed with special UV epoxy glue, using a UV lamp equipped with a fiber bundle. With such equipment, epoxy curing was performed relatively quickly, and continuously, while at the same time repetitive FBG spectra were acquired to control the curing process.

To perform this outdoor optical control, a dedicated FBG measurement system, small, portable and autonomous, has been developed. The BraggLight system monitors, in real time, the curing process in case of lack of power supply (it just needs an USB link to the PC for both power supply and data transfer).

Traffic monitoring with FBG sensors and high performance BraggFit 3 system

A BLS locomotive was used to calibrate the sensors versus applied contact force (50 N, 70 N, 90 N) and train speed (from 40 km/h to 100 km/h, maximal authorized speed at the gate location). The main objective was to investigate the possibility to link these two parameters with the measurements given by the optical fibre sensors (displacement, strains).

Calibrations were performed at night, and during several field tests spreading out over 18 months. During the same periods, we also collected the sensor data while the Inspection Gate was crossed by commercial trains (passengers and freights). This gave the strain distribution along a section of the OCL during the train transit along the Inspection Gate.

To do it, we simply detect the spectral Bragg lines due to local strain associated with every pantograph carbon strip(s) of a considered train. Then, we average the Bragg wavelength associated to the distributed FBG-line. With these reference data, it's now possible to identify a train out of 'standard' distribution, and potentially harmful to the infrastructure.

CONCLUSION

Several electromagnetic-immune optical FBG-based sensors and two powerful measurement units have been developed by the CEA List to equip the Inspection Gate. The picometric resolution BraggLight instrument is a small form factor unit able to scan at 200 Hz an 80 nm spectral band, i.e. some tens of FBG sensors. Just needing an USB link, this handable system provides its full potential in outdoor measurements, or where power line supply is not available. The second instrument, the BraggFit 3 system is characterized by a very high level of performances: up to 10 optical fibers spectrally scanned in real time at 1 kHz, with resolutions better than 1 pm (# 1 µm/m or # 0.01°C) , enabling to interrogate several hundreds of FBGs.

These powerful systems look like really 'universal', enabling to interrogate any FBG sensor photo-written in the 1.55 µm spectral range, and coming from any company. This may provide useful and powerful solutions in several industrial sectors (transportation, marine, aeronautics, civil engineering …), for structure monitoring & smart structure purposes.

Concerning railway industry, these new instrumentations enable to control the rolling stock operators' compliance with the infrastructure rules, and eventually to stop harmful trains identified by the control gate.

Figure 1 .

 1 Figure 1. Control gate installed in Switzerland (Lötschberg), including several FBG sensors and remote FBG measurement systems.

Figure 2 .

 2 Figure 2. FBG principle: the spectral effect of strain on Bragg wavelength.

  Number of simultaneous channels: 6 fibers (up to 10 in option) Number of sensors per channels: 30 typ. (Depending on the spectral bandwidth allocated to each FBG-based sensor) Optical connectors : E2000/APC (other in option) Processing Data logger: Autonomous system, Data acquisition / storage Client / server operations Client software: Display / Processing / Parameter set-up Data format: Binary file Interfaces: Ethernet / WiFi Electrical characteristics Voltage: 100-240 V; 50-60 Hz Power: 220 W Others Dimensions: 3U / 19'' -Weight: 10 kg

Figure 3 .

 3 Figure 3. BraggFit 3 monitoring system architecture and picture.

Figure 4 .

 4 Figure 4. BraggLight monitoring system.

Number of simultaneous channel: 1

 1 Number of sensors per channels: Typ. 30 (Depending on the spectral bandwidth allocated to each sensor FBG-based sensor) Optical connector : E2000/APC (other in option) Processing PC / software: Power supply, Driving / Parameter set-up, Data acquisition / Storage, Display / Processing Data format: Text file Interface: USB Electrical characteristics From USB link Consumption < 500 mA Others Dimensions: 165 x 173 x 56 mm 3 -Weight: 1 kg4. FIELD TESTS FBG line installation on High Voltage OCL with the BraggLight systemBoth sensors and measurement systems were installed on a site located in Switzerland, upstream to a tunnel drilled under the Alps and open in 2007 (the Lötschberg tunnel), i.e. a very critical location. To avoid perturbing the commercial traffic, the work was done at night (less traffic).

Figure 5 .

 5 Figure 5. BraggLight unit used for real-time control of FBG line epoxy curing on contact wire.

Figure 6 .

 6 Figure 6. FBG measurement units in the container to control any train passing the control gate.

CATIEMON consortium includes: Siemens (prime), BLS (CH) and ÖBB (Au) two railways companies, Furrer&Frey (CH) Cybernetix (Fr) as equipment providers, Morganite (UK) providing Carbon strips and pantoheads, plus IPHT (D) and CEA LIST (Fr) two R&D Institutes.


Philippe Kadhel

Gynécologie-Obstétrique Chu De Pointe-A-Pitre

/ Abymes

Annie Lannuzel

Résumé en français

Introduction : une perte d'expression de BRG1/SMARCA4 est retrouvée dans 5 à 10 % des carcinomes pulmonaires non à petites cellules (CPNPC), essentiellement des adénocarcinomes.

Il n'est pas clairement établi si le CPNPC SMARCA4-déficient (CPNPC-SD) représente une entité distincte parmi les CPNPC. Bien que son pronostic soit considéré comme mauvais, les résultats cliniques des inhibiteurs de points de contrôle immunitaires font l'objet de controverses.

Dans cette étude, nous rapportons les caractéristiques cliniques, pathologiques, moléculaires et immunitaires des CPNPC-SD et leur impact sur la survie globale dans une série de CPNPC-SD réséqués, ainsi que la survie globale de patients porteurs de CPNPC-SD traités par anti-PD1.

Méthodes : L'expression de BRG1 a été évaluée par immunohistochimie pour identifier les CPNPC-SD à partir de la collection de CPNPC de l'hôpital Cochin (Paris, France). Les profils moléculaires ont été analysés par séquençage à haut débit (NGS) couvrant 28 gènes.

L'environnement immunitaire a été caractérisé par une étude multiplex in situ permettant d'analyser les populations lymphocytaires T CD8+ et T régulateurs (FoxP3+), et les polynucléaires neutrophiles (CD66b+) sur 63 CPNPC SD réséqués et comparé à celui de 28 CPNPC conventionnels non-SD. Toutes les données ont été corrélées avec la survie globale. Le bénéfice d'un traitement par anti-PD-1 a été évalué chez 77 patients atteints d'un CPNPC métastatique, dont 7 avaient un CPNPC SMARCA4-déficients.

Résultats : Nous avons identifié 2 entités distinctes de CPNPC-SD selon l'expression de TTF1. Le groupe de patients atteints de CPNPC-SD/TTF1-comptait moins de patients souffrant de BPCO, avec des tumeurs de plus haut grade, plus fréquemment mutées KEAP1 et présentaient une densité intratumorale de polynucléaires neutrophiles plus élevée. Le mauvais pronostic du CPNPC-SD n'a été observé que pour ce groupe de patient. Les patients atteints de CPNPC-SD traités par des inhibiteurs anti-PD1 avaient une durée de vie significativement plus courte depuis le début de l'immunothérapie par rapport aux patients non CPNPC-SD, indépendamment de l'expression de TTF1.

Conclusions : Dans cette étude rétrospective et monocentrique de CPNPC réséqués, nous fournissons des preuves cliniques, pathologiques et de l'environnement immunitaire que le carcinome non à petites cellules déficient en SMARCA4 (CPNPC-SD) est une maladie hétérogène selon l'expression de TTF1. Nous avons montré que seuls les CPNPC-SD TTF1 négatifs avaient un comportement agressif, ce qui suggère que l'immunohistochimie BRG1 devrait être systématiquement intégrée dans la prise en charge pathologique des CPNPC TTF1 négatifs de type non-épidermoïde. L'environnement immunosuppressif particulier des CPNPC-SD suggère qu'il s'agit d'une entité particulière, différente des CPNPC conventionnel non-SD, avec un impact de l'expression de TTF1.

I. Revue bibliographique

A.

Implication du complexe SWI / SNF dans le remodelage de la chromatine L'expression génique est étroitement régulée par la structure de la chromatine. Cette dernière possède plusieurs niveaux d'organisation structurale dont le premier niveau de compaction correspond au nucléosome, unité fondamentale de la chromatine (1,2). Elle est une structure dynamique dont l'état de condensation est en perpétuel équilibre entre un état relâché et un état condensé. Cet équilibre dépend de diverses activités enzymatiques. Parmi les enzymes capables de modifier la chromatine, il est distingué celles qui vont modifier les histones, celles qui influent sur la méthylation de l'ADN et enfin celles qui utilisent l'énergie de l'ATP pour modifier directement le niveau de compaction des nucléosomes comme le complexe SWI/SNF (3). Le remodelage de la chromatine nécessite une série d'événements qui doivent être soigneusement coordonnés dans l'espace et dans le temps. Ainsi les complexes protéiques permettent d'accomplir de tels processus d'une manière beaucoup plus efficace que ne le feraient de simples protéines (4). Les complexes de remodelage de la chromatine sont regroupés en quatre grandes familles : SWI/SNF, INO80, ISWI et CHD/M-2 (5).

Plus particulièrement, le complexe mammifère non fermentable switch/saccharose (mSWI/SNF ou BAF) est un remodeleur de la chromatine qui utilise l'énergie de l'hydrolyse de l'ATP pour faire glisser, expulser, déposer ou modifier la composition des nucléosomes. Il régule ainsi l'accès de la chromatine à d'autres facteurs de liaison à l'ADN et à la machinerie transcriptionnelle (6,7). Ainsi, il joue un rôle essentiel dans le développement, la différenciation et d'autres processus cellulaires importants comme la réplication et la réparation de l'ADN (8). Le complexe est formé par l'assemblage combinatoire de deux hélicases dépendantes de l'ATP mutuellement exclusives, SMARCA2/BRM et SMARCA4/BRG1, avec de multiples sous-unités qui facilitent la liaison à l'ADN et aux histones, permettant une grande diversité de combinaisons. Outre les sarcomes, la perte de SMARCA4/BRG1 est de plus en plus rapportée dans des sous-ensembles de carcinomes peu différenciés/indifférenciés chez l'adulte survenant dans un large éventail de sites anatomiques, y compris le poumon (15)(16)(17)(18)(19)(20).

Ce n'est que ces dernières années que le carcinome pulmonaire non à petites cellules SMARCA4-déficient (CPNPC-SD) est décrit et tend à devenir une entité distincte du CPNPC conventionnel (15) probablement en raison de la reconnaissance accrue de ces altérations dans les plateformes de séquençage de routine de nouvelle génération.

C. Les carcinomes pulmonaires non à petites cellules SMARCA4déficients (CPNPC-SD)

Selon les études précédentes sur le CPNPC-SD, la perte d'expression de SMARCA4/ BRG1 se produit dans 5 à 10 % des CPNPC (15)(16)(17)(18)(19)(21)(22)(23). Les patients atteints de CPNPC-SD sont majoritairement des hommes et de gros fumeurs avec un âge moyen de 60 ans. Leur pronostic est plus mauvais que celui des patients atteints d'un CPNPC conventionnel (15)(16)(17)19,(21)(22)(23)(24)(25).

II. Objectifs de recherche

III. Original article

Statut de l'article : soumis pour une publication dans la revue Journal of Thoracic Oncology.

Title:

BRG1-loss / SMARCA4-deficient NSCLC is a heterogeneous entity

Introduction

The switch/sucrose-non-fermenting (SWI/SNF) complex regulates fundamental cellular processes by controlling access to chromatin. This complex functions as a tumor suppressor and mutations in at least one of its subunits have been demonstrated in 20% of human cancers, for over 20 years (1)(2)(3)(4). The SMARCA4 gene encodes BRG1, one of two mutually exclusive ATPase subunits of the SWI/SNF complex. SMARCA4 inactivating mutations are found in a large variety of cancers (5). Despite the publication of many reports, this entity is not yet fully known and several questions remain. The first ambiguity is related to how to identify this entity, either by the mutation of SMARCA4 gene or by the loss of expression of BRG1 protein. Indeed, the loss of BRG1 expression is often associated with SMARCA4

mutations but not all SMARCA4 mutations induce a loss of expression of BRG1. Truncating mutations (i.e. frameshift or nonsense) are most usually associated with loss of protein expression, in contrast to non-truncating mutations (i.e. missense). On the contrary the loss of BRG1 can be present without SMARCA4 mutations (6,7).

Among thoracic tumors, two entities have been described: SMARCA4-deficient undifferentiated tumor (SD-UT, formerly known as SMARCA4-deficient sarcoma) and SMARCA4-deficient non-small cell lung carcinoma (SD-NSCLC) (8)(9)(10)(11). Only SD-UT is recognized as a separate entity in the 8 th edition of the WHO classification and should be differentiated from SD-NSCLC (11). Indeed, clinical and histopathological differences have been described between these two entities. Patients with SD-UT are preferentially younger, with larger tumors of mediastinal origin, peritoneal metastases, and a worse prognosis (8,10,12). But despite these differences, a recent study suggests a biological continuum between these two tumors, SD-UT being a form of 'dedifferentiated' lung carcinoma (12). They showed that these two entities are linked at molecular level, as they share genomic alterations typical of smoking-related NSCLC (KRAS, KEAP1, STK11) and have a high tumor mutational load (8,10). Thus, this has led to the replacement of the term "sarcoma" by that of undifferentiated tumor in the 8 th WHO classification.

According to the literature, loss of expression of BRG1/SD-NSCLC occurs in 5-10% of NSCLC, predominantly male and heavy smokers with a mean age of 60 years. SD-NSCLCs are mostly TTF1/p40-negative poorly differentiated carcinomas, mainly adenocarcinomas and pleomorphic (or sarcomatoid) carcinomas (13)(14)(15)(16)(17)(18)(19)(20)(21). Many studies reported a poor prognosis of SD-NSCLC, whether by identifying them by the loss of BRG1 expression (15,16,22,23) or by the SMARCA4 mutation (24,25). In Schoenfeld's study, authors showed that although all SMARCA4 alterations are associated with poor clinical outcomes, truncating mutations which are associated with BRG1 protein loss, were associated with the poorest prognosis (6), emphasizing the importance of BRG1 immunohistochemistry to identify SD-NSCLC.

The therapeutic management of SD-NSCLC patients is not codified. These tumors do not seem to harbor actionable oncogenic driver alterations, such as EGFR, ALK, and ROS1, as in conventional NSCLC (6,7,13,20), excluding the prescription of targeted therapy. Data on the effectiveness of immunotherapy are inconsistent, some studies suggest an efficacy of immune checkpoint inhibitors (ICIs) (23,26), which may be explained by a high mutational load (8,10) ; but others report a low response to ICI (6,24,25), which may be due to low PD-L1 expression and presence of co-mutations in STK11 and KEAP1 genes (27). Consistent with the nonbenefit to ICI, a recent study using four independent cohorts of public data reported that patients with KRAS-and SMARCA4-mutant adenocarcinoma treated with ICIs consistently had the shortest progression-free survival and overall survival (25). A recent large-scale study observed similar findings, with no differences in clinical outcomes in SMARCA4-WT NSCLC patients treated with ICIs versus those with SMARCA4-mutations, except in the KRAS-mutant SMARCA4-mutated NSCLC subgroup, suggesting that co-mutations (KRAS/SMARCA4) drive poor prognosis in ICI treated patients, when compared to other subgroups. These studies suggest that SMARCA4-mutated NSCLC is a heterogeneous entity whose therapeutic sensitivity depends on additional alteration, such as KRAS. They also showed that KRASmutant NSCLC with SWI/SNF alterations, including SMARCA4 mutations, have a lower density of intratumoral PD-1+ CD8+ T cells, compared to KRAS-mutant NSCLC without SWI/SNF alterations, which may explain the unfavorable clinical outcomes in patients treated with PD-1 inhibitors (24). Indeed, studies have shown that higher level of PD-1+ CD8+ T cells and more globally CD8+ T cell is a predictive marker of response to ICI (28,29). Furthermore, we previously found, in resected NSCLC patients, that the high intratumoral density of CD8 + T cell was a favorable prognostic factor as it was associated with long-term survival, independently of treatment (30). Since we previously shown that intratumoral immune cell densities, especially CD8 +T cell, were significantly associated with molecular alterations in NSCLC (31), we hypothesized that SD-NSCLC might have a specific immune environment, which could explain its poor prognosis in untreated patients and in patients treated with ICI.

Deciphering SMARCA4/ BRG1-loss in lung cancer is still necessary as it is a heterogeneous entity, with contrasting reports in the literature on characterization and treatment. In the present study, we aimed at analyzing clinical, pathological, molecular and immune environment of 63 SD-NSCLC patients resected in the same surgical department, identified by the BRG1-loss, and we compared these tumors to non-SD NSCLC. We also reported the overall survival of 7 SD-NSCLC among 77 patients treated by PD-1 blockade within the CERTIM (Immunomodulatory Therapies Multidisciplinary Study group) network.

Material and methods

Population studied and Sample selection

We selected patients naive of treatment, operated with curative intent at Cochin Hospital,

Pathological evaluation and immunohistochemistry

Histological tumor typing was performed according to the 4 th World Health Organization criteria (34) and pathologic tumor staging was evaluated using the 8 th edition of TNM classification. All adenocarcinomas were classified into 3 grades according to their predominant architectural pattern, as previously described (32,35). Immunohistochemistry was performed using the fully automated Leica Bond III platform and antibodies against the following antigens: BRG1 (clone EPNCIR111A clone, 1:200, Abcam), TTF1 (clone SPT24, 1:100, Tebu Novocastra), P40

(clone BC28, 1:100, Zytomed), PD-L1 (clone QR1, 1:100, Diagomics/BIOCYC). SD-NSCLC was defined by a complete loss of BRG1 nuclear expression in all tumor cells.

Molecular analysis

DNA was extracted from FFPE tumor samples, selected to contain at least 30% of tumor cells, V3), and sequenced on the Ion Proton™ System (Life Technologies™). The FASTQs sequencing data are aligned to the human genome (hg19) and processed using Ion Torrent Suite V5.12.0. This package included the Torrent Variant Caller V5.12.0.2 using the built-in "Somaticlow stringency" with optimized parameters to automatically call variants with allelic ratio >5%. Samples were considered non-informative when mean coverage depth was <300X.

using

Multiplex fluorescence immunohistochemistry

Multiplexed immunofluorescence was performed on 5-µm thick serial sections from the selected tumor tissue section containing the highest density of immune cells on hematoxylineosin-safran (HES) stained slides. Staining was performed using Opal™ 4-Color IHC Kit (AKOYA Biosciences®) for CD66b (clone G10F5, BD), FoxP3 (clone 236A/E7, Abcam), pancytokeratin (clone AE1/AE3, Agilent), CD8 (clone SP16, Abcam) on Leica Bond RX (Leica Biosystem®), as previously described (37). Whole sections were scanned using multispectral imaging system Axio Scan.Z1 (Zeiss) and images were analyzed using Halo® (indica Labs).

Tumor region was selected manually, and the surface areas were calculated automatically.

Positive cells were labeled semi-automatically and counted with the software. Results are expressed as the absolute number of positive cells per square millimeter.

Statistical analysis

Statistical analyzes were conducted using R 3.3.2 (https:// www.R-project.org/). Wilcoxon-Mann-Whitney or Chi 2 test were used to find associations between clinical and molecular data.

The difference in immune cell density was estimated statistically with the Wilcoxon rank test.

For survival analysis, the clinical-end point was overall survival (OS), calculated from the date of surgical resection to the date of death or date of last follow-up. Survival was estimated using Kaplan-Meier method and compared using log-rank test. Cox proportional-hazards regression model was used for univariate analysis and all variables associated with survival with a p-value<0.05 were subsequently selected for multivariate analysis. OS was analyzed for patients treated by ICIs and measured as the time from the date of first cure of immunotherapy, to the date of death or date of last follow-up.

Results

Characteristics of SD-NSCLC

A total of 63 patients with SD-NSCLC, defined by complete loss of BRG1 expression, who underwent radical surgical excision for curative intent, are included in this study. The median age was 61 years, 51 patients (81%) were male, 59 (94%) were heavy smokers (≥15 packyears) and 43 (68%) had COPD. The major histological type was adenocarcinoma (54, 86%), mostly solid predominant pattern (36, 66%), there was only one lepidic predominant pattern and one mucinous adenocarcinoma. TTF1 expression was found in 31 (49%) tumors. Only 10 tumors (16%) had ≥ 50% PD-L1 tumor cell expression. We often found aggressive histological criteria, i.e. 45 (71%) tumors with vascular emboli, 41 (65%) with pleural infiltration and 30 (47%) with lymph node invasion (13 pN1 and 17 pN2). Pathological stages for all resected tumors were 20 (32%), 19 (30%), 22 (35%) and 2 (3%) for stage I, II, III, and IV, respectively.

Regarding the molecular characteristics, 54 of the 63 SD-NSCLC were interpretable and 42 tumors (78%) had at least one SMARCA4 mutations, composed of nonsense (n = 28), frameshift (n = 12), missense (n = 6), and splice-site (n = 5) mutations. Truncating mutations (i.e., frameshift, nonsense or splice mutations) were found in 40/42 (95%) tumors. Six tumors had 2 or 3 SMARCA4 mutations. Other mutations were identified in TP53 (63%), STK11 (27%), KEAP1 (24%), KRAS (24%), CDKN2A (17%), ARID1A (8%), PTEN (3%), and RB1 (2%) (Table 1). Concerning potential targetable driver genes, we did not identify EGFR mutation, 2 tumors had a non-V600 BRAF activating mutation, one had HER2 amplification and one had MET amplification, both confirmed by FISH, with gene-to-centromere ratio greater than 2; 5 tumors had KRAS G12C activating mutation.

We then compared clinical, pathological features and molecular alterations between SD-NSCLC (n = 63) and non-SD NSCLC (n=303) and found that SD-NSCLCs have distinct features. Indeed, SD-NSCLC patients were associated with heavier smoking history (p=0.02), more frequent COPD (p<0.001), higher tumor grade (p<0.001), more negative TTF1 expression (p<0.001), lower PD-L1 tumor cell expression (TPS) (p=0.001), more KRAS mutations (p=0.01) and absence of EGFR mutations (p=0.003). There was no significant difference according to tumor stage, TP53 and STK11 mutations (Supplementary Table 1).

Clinical Outcomes of SD-NSCLC

At the time of the study, 39 patients out of the 63 SD-NSCLC patients (71%) died. The median follow-up time was 18 months [95% CI: 0.6-0.8] for SD-NSCLC and 62 months [95% CI: 0.5-0.8] for non-SD NSCLC. SD-NSCLC patients were characterized by a poorer clinical outcome.

Overall survival (OS) rates at 2 and 5 years were respectively 58% , 37% for SD-NSCLC (N=63) and 74% [70-80], 53% [START_REF] Hasegawa | Prognostic value of peripheral and local forkhead box P3+ regulatory T cells in patients with non-small-cell lung cancer[END_REF][START_REF] Shirasawa | Impact of chemoradiotherapy on the immune-related tumour microenvironment and efficacy of anti-PD-(L)1 therapy for recurrences after chemoradiotherapy in patients with unresectable locally advanced non-small cell lung cancer[END_REF][50][51][52][53][54][55][56][57][58][59] for non-SD NSCLC (N=303) (Figure 2A).

Univariate analyses of the 63 SD-NSCLCs showed that TTF1 expression (p=0.02), KEAP1 mutations (p=0.03) and pathologic-stage (p=0.0001) were related to OS (supplementary Table 2). Cox-regression revealed that stage was the only independent prognostic factor (p=0.006, HR:3 [1.3-6.9]). We also found that TTF1-negative SD-NSCLC patients (TTF1-/BRG1loss, n=32) had shorter OS compared to TTF1-positive SD-NSCLC patients (TTF1+/BRG1loss, n=31), who had a similar OS compared to TTF1-positive non-SD NSCLC (TTF1+/BRG1retained) (Figure 2B) and as expected TTF1 expression was associated with a significant favorable outcome (p<0.0001, HR: 0,5 [0.4-0.7]) (Figure 2C). Five-year OS rates in TTF1+/BRG1loss

(53% ) and TTF1+/BRG1retained (56% [50-62]) groups were higher than those in the TTF1-/BRG1loss (10% [2-57]) and TTF1-/BRG1retained (31% ) groups (Figure 2B). This result shows that BRG1 expression appears to be a discriminating marker of clinical outcome only in TTF1-negative NSCLC. Univariate analysis of patients with BRG1loss tumors showed that in TTF1-negative tumors, only stage (p=0.01) was significantly associated to OS, whereas in TTF1-positive tumors, stage (p=0.02) and KEAP1 mutations (p=0.01) were significantly related to OS (Supplementary Table 2). None of these variables were independent in multivariate analysis.

TTF1 expression influences clinical and immune parameters in SD-NSCLC

Since the prognosis of TTF1-/BRG1loss patients was significantly different from TTF1+/BRG1loss patients, we then evaluated whether SD-NSCLC patients have distinct clinical, pathological, molecular and immune cell population characteristics according to the TTF1 expression.

Among the 63 SD-NSCLC, 31 expressed TTF1 diffusely and 32 did not. TTF1-/BRG1loss NSCLC patients were significantly associated with less COPD (p=0.03), larger tumor size (p=0.04), more large-cell and pleomorphic carcinomas (p=0.04) and higher grade adenocarcinomas (p=0.02). There was no significant difference according to smoking status, pathological stage and PD-L1 TPS. Regarding genomic alterations, TTF1-/BRG1loss NSCLC patients were associated with more KEAP1 mutations (p=0.02), without significant difference according to KRAS, TP53 and STK11 mutations (Table 1). Regarding the immune cell densities, we performed fluorescence multiplex immunohistochemistry analysis on the 63 SD-NSCLCs, according to TTF1 expression and compared these data to 28 resected non-SD NSCLCs analyzed with the same panel of multiplex fluorescence immunohistochemistry (Supplementary Table 3). Representative image of the resulting staining is shown in Figure 3.

We found no significant differences in CD8+ T cell, neutrophil and regulatory T cell densities in non-SD NSCLC, according to TTF1 expression. There was also no significant difference with CD8+ T cell density in NSCLC, according to BRG1 expression. But we found that SD-NSCLC had a significantly higher density of regulatory T cells than non-SD NSCLC, irrespective of TTF1 expression; and TTF1-/BRG1loss NSCLC had a significantly higher neutrophil density than TTF1+/BRG1loss NSCLC (Figure 3).

In the overall cohort of SD-NSCLC, neither CD8+ T-cell nor neutrophil nor regulatory T cell densities were associated with prognosis (Figure 4A). In TTF1+/BRG1loss NSCLC patients, CD8+ T-cell density was associated with an unfavorable clinical outcome (Figure 4B), while neutrophil and regulatory T cell densities, as well as the ratio of CD8+ to FoxP3+ cells, were not. In TTF1-/BRG1loss NSCLC patients, CD8+ T-cell density was not associated with prognostic value (Figure 4C), nor was the densities of neutrophils or regulatory T cells.

Overall survival of SD-NSCLC treated by anti-PD1

We then investigated the effectiveness of an anti-PD-1 immunotherapy in patients with SD-NSCLC, studying a cohort of 77 patients with metastatic-stage NSCLC receiving nivolumab or pembrolizumab, in second line or more, from the CERTIM cohort. Among these 77 NSCLC, 7

tumors were identified as SD-NSCLC with a complete BRG1-loss, including 2 TTF1-negative tumors. Among these 7 patients, there was no significant difference between the SD-NSCLC and non-SD NSCLC groups in terms of sex, smoking status, COPD, histology, TTF1 expression, PD-L1 TPS, type of anti-PD1 and KRAS mutation. Only age at diagnosis was significantly higher in non-SD NSCLC (supplementary Table 4). With a median follow-up of 5. 0.8-2.6]) (Figure 5).

Discussion

In this study, our objective was to characterize a monocentric series of SMARCA4-deficient NSCLC (SD-NSCLC) in order to participate in the dismemberment of this entity that seems to have an aggressive behavior and probably requires a dedicated therapeutic management. We defined SMARCA4-deficient NSCLC as tumors with complete loss of BRG1 expression using standard routine immunohistochemistry technic. The protein BRG1 complete loss must be distinguished from SMARCA4-mutated NSCLC because not all SMARCA4 mutations lead to a loss of BRG1 expression, which is usually found in truncating mutations (i.e. frameshift or nonsense) rather than non-truncating mutations (i.e. missense) (6,7,20,38), in agreement with our study, as 95% of BRG1-loss tumors with SMARCA4 mutation had truncating mutations. In addition, complete loss of BRG1 expression could be observed without SMARCA4 mutation, probably linked to alternative mechanisms such as mutation in other SWI/SNF protein, or epigenetic regulation effect (7,12,21,39), or even to a failure of NGS to identify SMARCA4 mutation, especially in the case of large deletion or variants involving poorly covered intronic regions. In our study, SMARCA4 mutations were not identified in 22% of BRG1-loss tumors.

We first showed that there were significant differences between SD-NSCLC and non-SD NSCLC, consistent with previous studies. SD-NSCLCs were more common in smokers and COPD patients, and their adenocarcinomas were of higher-grade, with lower PD-L1 TPS, no EGFR mutations and more KRAS mutations (6,20,24,25), including the G12C subtype, which may benefit from targeted therapy (40). We confirmed that SD-NSCLC had poorer overall survival, but this was related to TTF1-/BRG1loss NSCLC patients, as TTF1+/BRG1loss NSCLC patients had a similar outcome than TTF1+/BRG1retained NSCLC patients. This suggests that TTF1 is the main driver of negative outcome in patients with BRG1loss tumors. Similarly, in BRG1retained tumors, TTF1-negative lung adenocarcinomas were associated with poor survival, as previously reported (41), probably linked to loss of differentiation and increased metastatic potential (42). It has been proposed that loss of BRG1/SMARCA4 represents a primary genetic driver event in TTF1-/BRG1loss tumors leading to epithelial-mesenchymal transition (13), whereas the occurrence of the SMARCA4 mutation would be a later event, not related to mesenchymal transition in TTF1+/BRG1loss adenocarcinomas. In such case, TTF1+/BRG1loss NSCLC should rather be considered as a conventional adenocarcinoma, whereas TTF1-/BRG1loss NSCLC should be considered as a fully-fledged entity of poor prognosis among NSCLCs. Besides prognostic differences, we also observed that according to TTF1 expression SD-NSCLCs have distinct characteristics. Indeed, TTF1-/BRG1loss NSCLC patients were significantly associated with less COPD, larger tumor size, more large-cell and pleomorphic carcinomas, higher-grade adenocarcinomas and more KEAP1 mutation.

Regarding the immune environment, SD-NSCLC seems to be a particular entity, different from non-SD NSCLC, with an additional influence of TTF1 expression. Indeed, there was a more immunosuppressive environment, with a higher density of FoxP3 + T cells in SD-NSCLC, irrespective of TTF1 expression, and a higher density of neutrophils in TTF1-/BRG1loss NSCLC.

Interestingly, CD8+ T cell density did not differ according to BRG1 or TTF1 expression, implying that a global immune cells analysis is required to predict outcome and response to treatment. Using the Microenvironment Cell Populations-counter (MCP-counter) method ( 43), on the TCGA LUAD cohort, we were able to find, in agreement with our data, a significantly higher occurrence of neutrophils and the absence of significant difference in CD8+ T cells presence in SMARCA4 mutant (missense and nonsense) adenocarcinoma as compared to Wild-Type SMARCA4 tumors (Supplementary figure 1). Since MCP-counter does not provide regulatory T cells, we could not validate this additional result in the TCGA LUAD cohort.

We analyzed the association between survival and immune cells densities among SD-NSCLC and according to TTF1 expression. Surprisingly, we found that the positive prognostic value of CD8+ T cell (30) was lost, neutral for TTF1-/BRG1loss NSCLC and negatively associated with survival in TTF1+/BRG1loss NSCLC. This finding could have been related to the higher frequency of COPD in SD-NSCLC, as we have previously observed a loss of CD8+ T cellassociated favorable clinical outcome in COPD patients with NSCLC, related to increased exhaustion status of CD8+ T cells and a trend to a greater sensitivity to PD-1 blockade (44).

But in the context of SD-NSCLC, patients do not seem to benefit from anti-PD1 inhibitors, rather they have significantly shorter survival, compared to non-SD NSCLC patients. The influence of COPD status, KRAS mutation, and TTF1 expression could not be explored because the number of patients was too small. In contrast, the poor response to anti-PD1 was consistent with the low PD-L1 TPS in SD-NSCLC (45). The impact of a high density of FoxP3+

T cells found in SD-NSCLC, compared with non-SD NSCLC, need additional analysis in SDand non-SD NSCLC, as it might explain the resistance to immune checkpoints inhibitors (46,47). This suggests that new combination of immunotherapy including Treg-targeted therapy could be interesting in SD-NSCLC patients (46,47).

Conclusion

In this retrospective and monocentric study of resected NSCLC, we provided clinical, pathological and immune environment evidence that SMARCA4-deficient non-small cell carcinoma (SD-NSCLC) is a heterogeneous disease, mainly related to TTF1 expression. We showed that among SD-NSCLC tumors, TTF1-negative had the most aggressive behavior but all SD-NSCLC had an immune environment enriched in FoxP3+ cells and did not respond to ICI. Altogether, this suggests that BRG1 immunohistochemistry should be systematically integrated into the pathological workflow of NSCLC to help in the therapeutic management of these patients.

Table and figures legends

V. Conclusion

En résumé, nous démontrons que le carcinome pulmonaire non à petites cellules SMARCA-déficients (CPNPC-SD) est une maladie hétérogène selon l'expression de TTF1.

Nous avons montré que seuls les CPNPC-SD/TTF1négatifs avaient un comportement agressif, suggérant qu'une recherche de la perte d'expression de BRG1 par immunohistochimie devrait être systématiquement intégrée dans la prise en charge pathologique des CPNPC/TTF1négatifs de type non-épidermoïde.

Notre recherche appuie également l'importance de développer des études cliniques pour évaluer des stratégies thérapeutiques alternatives prometteuses dans les CPNPC-SD.

Figure 1 :Tableau 1 :

 11 Figure 1 : Représentation schématique de la composition du complexe SWI/SNF. Les différents noms indiqués pour la même sous-unité dans cette représentation schématique reflètent l'existence de paralogues mutuellement exclusifs. La localisation des sous-unités au sein du complexe est montrée de manière arbitraire. Certaines sous-unités sont constantes (en bleu), comme une des deux sous-unités ATPase catalytiques, SMARCA4/BRG1 et SMARCA2/BRM, présentes de manière mutuellement exclusive. Cela offre une grande diversité de combinaisons. Le complexe utilise l'énergie de l'ATP-ase pour remodeler la chromatine, régulant ainsi l'accès de la chromatine à d'autres facteurs de liaison à l'ADN et à la machinerie transcriptionnelle.

  Ils sont principalement représentés par des carcinomes peu différenciés TTF1/P40 négatifs, notamment par des adénocarcinomes et des carcinomes pléomorphes (ou sarcomatoïdes) (cf. Figures 2, 3 et Annexes (VI) -Figure supplémentaire 3) (15,16,18,19,21-23,25).

Figure 2 :

 2 Figure 2 : Image histologique d'un CPNPC-SD : adénocarcinome peu différencié d'architecture solide, HES (A), avec perte d'expression de la protéine BRG1 (témoin interne positif), étude immunohistochimique avec l'anticorps anti-BRG1 (Abcam) (B).

Figure 3 :

 3 Figure 3 : Image histologique d'un CPNPC-SD : carcinomes pléomorphes (ou sarcomatoïdes), HES.

L

  'objectif principal de cette recherche était d'élargir les connaissances sur les CPNPC-SD. Dans un premier temps, lors de mon année-recherche en master 2 effectuée à Paris, encadrée par l'équipe thoracique de Cochin, nous avons décrit les caractéristiques cliniques, pathologiques, moléculaires d'une cohorte de 87 CPNPC-SD identifiés par la perte de BRG1, à partir des données rétrospectives de notre institution. Nous avons analysé l'environnement immunitaire sur les CPNPC-SD réséqués (n=63) de notre cohorte initiale. Dans un second temps, sur la base de ces données, nous avons cherché à analyser les données clinique, pathologique, moléculaire et immunitaire des CPNPC-SD réséqués (n=63/87) de notre cohorte initiale en les comparant à des CPNPC conventionnels non-SD réséqués. Ces données ont été corrélées avec la survie globale. Nous avons également rapporté la survie globale de 7 CPNPC-SD parmi 77 patients traités par anti-PD-1 au sein d'une cohorte de CPNPC de stade métastatique (CERTIM -Immunomodulatory Therapies Multidisciplunary Study group).

Short title :

  Heterogeneity of SMARCA4-deficient NSCLCYoan Velut* a , Elise Decroix* a , Hélène Blons, PharmD, PhD b,c , Marco Alifano, MD, PhD a,d , Karen Leroy, MD, PhD a,b , Florent Petitprez, PhD e , Aurélie Boni a , Simon Garinet, PharmD b,c , Jérome Biton, PhD f , Isabelle Cremer, PhD a , Marie Wislez, MD, PhD a,g , Pascaline Boudou-Rouquette, MD h , Jennifer Arrondeau, MD h , François Goldwasser, MD, PhD h,i , Ludovic Fournel, MD, PhD d ; Diane Damotte, MD, PhD a,j , Audrey Mansuet-Lupo, MD, PhD a,j

France, betweenFigure 1 .

 1 Figure 1. We excluded squamous-cell lung carcinomas and large-cell neuroendocrine carcinomas from the study because we found no BRG1-loss among 91 squamous-cell lung carcinomas and large-cell neuroendocrine carcinomas tested. We analyzed the immune cell infiltrate of the 63 SD-NSCLC tumors, and compared with 28 similar and representative NSCLC without BRG1 loss. Finally, we assessed the benefit of immunotherapy in 7 patients with SD-NSCLC from a cohort of 77 patients with metastatic NSCLC, included in the Immunomodulatory Therapies Multidisciplinary Study group (CERTIM), who received an anti-PD-1 therapy (either nivolumab or pembrolizumab), in second line or more, between 2015-2020 (Figure 1). This study was performed with the approval of our institutional review board (CPP Ile de France II, 2008-133 and 2012 06-12, No. 2018 MS1).

2 months[

 2 95%CI: 0.2-1], we found that SD-NSCLC patients had shorter OS compared to patients with non-SD NSCLC (p=0.01, HR=3.2 [95% CI: 1.3-7.8]), regardless of TTF1 expression (p=0.4, HR=0.8 [95% CI: 0.5-1.4]) and KRAS mutation (p=0.1, HR=1.5 [95% CI:

Figure 1 :

 1 Figure 1: Description of the studied populations and the performed analyses.We identified 63 resections of SMARCA4-deficient NSCLC defined by loss of BRG1 expression, they were subjected to molecular analysis with 2 NGS panels (panel 1: The Ion AmpliSeq™ Colon-Lung Cancer Research Panel v2, Life Technologies™, France and panel 2: a custom Ion AmpliSeq™ DNA panel, including SMARCA4 and KEAP1 mutations) ; and multiplex immunohistochemistry to quantify neutrophil (CD66b+), regulatory T cell (FoxP3+) and CD8+ T cell densities. We also included a cohort of 77 metastatic NSCLC patients from the Immunomodulatory Therapies Multidisciplinary Study group (CERTIM) who received anti-PD-1 therapy, in second line or higher, in which 7 had a complete loss of BRG1 expression.Multiplex IHC: multiplex immunohistochemistry.

Figure 2 :

 2 Figure 2: Survival analysis. Comparison of overall survival for resected NSCLC according to BRG1 expression (A) combined expression of BRG1 and TTF1 expression (B) and TTF1expression alone (C). OS curves were estimated using the Kaplan-Meier method, and differences between patient groups were evaluated by log-rank test. Number at risk and percentage of alive are indicated. HR and p-value were estimated using Cox-regression. TTF1-/BRG1loss: TTF1-negative SMARCA4-deficient NSCLC, TTF1+/BRG1loss: TTF1positive SMARCA4-deficient NSCLC, TTF1-/BRG1retained: TTF1-negative non-SMARCA4 deficient NSCLC, TTF1+/BRG1retained: TTF1-positive non-SMARCA4 deficient NSCLC.

Figure 3 :

 3 Figure 3: Comparison of SMARCA4-deficient NSCLC according to TTF1 expression in resected patients. (A) TTF1-positive tubular adenocarcinoma with loss of BRG1 expression showing high infiltration of regulatory T cells (green nuclei) by fluorescent multiplex immunohistochemistry (B) TTF1-negative solid adenocarcinoma with loss of BRG1 expression, showing high infiltration of regulatory T cells (FoxP3+, green nuclei) and neutrophil (CD66b+, yellow) by fluorescent multiplex immunohistochemistry. (C) Radar plot and table showing the densities of CD8+ T, regulatory T (FoxP3+), neutrophil (CD66b+) cells among SMARCA4-deficient NSCLC and non-SMARCA4 NSCLC tumors and according to TTF1 expression. HR and p-value calculated with Cox-regression are indicated in the table beside (C). HES staining (left), chromogenic simplex immunohistochemistry (middle), fluorescent multiplex immunohistochemistry (right): CD8+ T cell (pink), CD66b+ neutrophils (yellow), FoxP3 (green in nuclei) and cytokeratin (green in membrane).

Figure 4 :

 4 Figure 4: Prognostic value of CD8+ T cell, neutrophil and regulatory T cell densities in SD-NSCLC patients (A). OS curves for CD8+ T cell density in TTF1+/BRG1loss NSCLC group (C) and TTF1-/BRG1loss NSCLC group (C). HR and p-value calculated with Cox-regression are indicated in the table below (A). OS curves were estimated by using the Kaplan-Meier method, and differences between patient groups were evaluated by log-rank test. A medianbased stratification was used to dichotomize CD8+ T-cell density as high or low, with low densities used as a reference. TTF1-/BRG1loss: TTF1-negative SMARCA4-deficient NSCLC, TTF1+/BRG1loss: TTF1-positive SMARCA4-deficient NSCLC, TTF1-/BRG1retained: TTF1-negative non-SMARCA4 deficient NSCLC, TTF1+/BRG1retained: TTF1-positive non-SMARCA4 deficient NSCLC.

Figure 5 : 1 .

 51 Figure 5: Prognostic value (OS) of anti-PD1-treated patients with SD-NSCLC (A) andaccording to TTF1 expression (B). Metastatic-stage NSCLC patients received nivolumab or pembrolizumab, in second line or more, excluding squamous-cell and large-cell neuroendocrine carcinomas (N=77, in which 7 SD-NSCLC, including 2 TTF1-negative tumors). OS curves were estimated by using the Kaplan-Meier method, and differences between patient groups were evaluated by log-rank test. HR and p-value calculated with Cox-regression are indicated in the table beside.

Figure supplémentaire 2 (

 2 Figure supplémentaire 2 (hors article) :

Figure supplémentaire 3 (

 3 Figure supplémentaire 3 (hors article) :

  AmpliSeq™ DNA panel, full gene coding sequence for SMARCA4, TP53, STK11, KEAP1, RB1, CDKN2A, ARID1A and PTEN. Briefly, the multiplex barcoded libraries are generated from 10ng of DNA following manufacturer's recommendations (Ion ampliseq library kit V2) and are normalized using the Ion Library Equalizer™ Kit. The pooled libraries are processed on Ion Chef™ System for template preparation and chip loading (Ion PI HI-Q Chef Kit, Ion PI Chip

the Maxwell® 16 FFPE Plus LEV DNA Purification Kit (Promega). All samples were characterized using 2 NGS panels:

(1)

The Ion AmpliSeq™ Colon-Lung Cancer Research Panel v2 (Life Technologies™, France), as previously described

(33,36)

, and (2) a custom Ion

Table 1 :

 1 Clinical, pathological and molecular characteristics of 63 resected SMARCA4-

	deficient NSCLC, and according to TTF1 expression. Adenocarcinoma grade corresponded to
	II (intermediate grade) for lepidic, papillary, or acinar predominant pattern and III (high grade)
	for solid, micropapillary, or solid predominant pattern and mucinous type, as previously
	described (32,35). PD-L1 TPS: tumor proportion score corresponding to the percentage of PD-
	L1 positive tumor cells. SD-NSCLC : non-SMARCA4-deficient NSCLC, COPD : chronic
	obstructive pulmonary disease.
		SD-NSCLC	SD-NSCLC TTF1(-)	SD-NSCLC TTF1 (+)	P-value
	Parameters	n=63	n= 32	n=31
	Age at diagnosis				0.5
	Median [Min, Max]	61 [38, 80]	62 [38, 80]	59 [42, 79]
	Sex number (%)				0.8
	Female	12 (19%)	7 (22%)	5 (16%)
	Male	51 (81%)	25 (78%)	26 (84%)

Acknowledgments

We thank the following clinicians and surgeons who provided follow-up information: Drs Jacky Crequit, Christos Chouaid, Marie De Torcy, Sébastien Morel, Stéphane Jouveshomme, Jean Tredaniel, Edgar Petit, Pierre Magdeleinat, Jean-François Régnard, Kais Aldabbagh, Sandrine Loutski. We also thank the following laboratory technicians: Anne Audebourg, Isabelle Hermary, Yvan Lafuente, Léa Le Lay, Nathalie Mathiot, Alice Nardetto, Phuong Lan Truong.

We thank the "Centre de Recherche des Cordeliers" core facilities for the technical and methodological help, assistance and support.

Funding information: This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Paris, Sorbonne University, Site de Recherche Intégrée sur le Cancer SIRIC CAncer Research for PErsonnalized Medicine (CARPEM), LabEx Immuno-oncology, the Institut National du Cancer (2011-PLBIO-06-INSERM 6-1).

Disclosure:

The authors declare no conflict of interest related to this manuscript.

Results : We identified 2 distinct entities among SD-NSCLC: TTF1-/BRG1loss and TTF1+/BRG1loss. The TTF1-/BRG1loss NSCLC tumors were of higher grade, more frequently KEAP1 mutated, and showed higher intratumoral neutrophil density. Altogether, these TTF1-/BRG1loss NSCLC patients had the worst prognosis. Interestingly, regardless of TTF1 expression, SD-NSCLC patients treated by anti-PD1 inhibitors had a significantly shorter OS, from the start of immunotherapy, compared to non-SD-NSCLC, highlighting the need to identify this entity.

VI. Annexes

Serment d'Hippocrate

Au moment d'être admis à exercer la médecine, en présence des maîtres de cette école et de mes condisciples, je promets et je jure d'être fidèle aux lois de l'honneur et de la probité qui la régissent.

Mon premier souci sera, de rétablir, de préserver ou de promouvoir la santé dans tous les éléments physiques et mentaux, individuels collectifs et sociaux. Je respecterai toutes les personnes, leur autonomie et leur volonté, sans aucune discrimination selon leur état ou leurs convictions.

J'interviendrai pour les protéger si elles sont affaiblies, vulnérables ou menacées dans leur intégrité ou dignité.

Même sous la contrainte, je ne ferai usage de mes connaissances contre les lois de l'humanité.

J'informerai les patients de décisions envisagées, de leurs raisons et de leurs conséquences.

Je ne tromperai jamais leur confiance et n'exploiterai pas le pouvoir hérité des circonstances pour forcer leurs consciences.

Je donnerai mes soins à l'indigent et à quiconque me les demandera.

Je ne me laisserai influencer ni par la recherche du gain ni par la recherche de la gloire.

Admis dans l'intimité des personnes, je tairai les secrets qui me sont confiés.

Reçu à l'intérieur des maisons, je respecterai les secrets des foyers.

Et ma conduite ne servira pas à corrompre les moeurs.

Je ferai tout pour soulager les souffrances, sans acharnement.

Je ne provoquerai jamais la mort délibérément.

Je préserverai l'indépendance nécessaire à l'accomplissement de ma mission.

Que je sois modéré en tout, mais insatiable de mon amour de la science.

Je n'entreprendrai rien qui ne dépasse mes compétences ; je les entretiendrai et les perfectionnerai pour assurer au mieux les services qui me seront demandés.

J'apporterai mon aide à mes confrères ainsi qu'à leurs familles dans l'adversité.

Que les hommes et mes confrères m'accordent leur estime si je suis fidèle à mes promesses,

Que je sois déshonoré et méprisé si j'y manque.

Decroix Elise

Sujet de la thèse : Carcinomes pulmonaires non à petites cellules SMARCA44-déficients : caractérisation clinico-pathologique, moléculaire et immunitaire. RESULTATS : Nous avons identifié 2 entités distinctes de CPNPC-SD selon l'expression de TTF1. Le groupe de patients atteints de CPNPC-SD dont la tumeur n'exprimait pas TTF1 comptait moins de patients souffrant de BPCO, leurs tumeurs étaient de grade plus sévère, plus fréquemment mutées en KEAP1 et présentaient une densité de neutrophiles intratumorale plus élevée. Le mauvais pronostic du CPNPC-SD n'a été observé que pour ce groupe de patient. Les patients atteints de CPNPC-SD traités par des inhibiteurs anti-PD1 avaient une durée de vie significativement plus courte depuis le début de l'immunothérapie par rapport aux patients non CPNPC-SD.

CONCLUSION : Le CPNPC-SD est une maladie hétérogène liée à l'expression de TTF1. L'évaluation de BRG1 devrait être réalisée dans les CPNPC, en particulier dans les CPNPC TTF1 négatif de type non épidermoïde, car leur pronostic est mauvais et ils ne semblent pas bénéficier de l'immunothérapie.