Léon Serre-Fredj

Franck Jacqueline

Maxime Navon

Guillaume Izabel

Léo Chasselin

Orianne Jolly

Michel Repecaud

Pascal Claquin
email: pascal.claquin@unicaen.fr

Coupling High frequency monitoring and bioassay experiments to investigate a Harmful algal bloom in the Bay of Seine (French-English Channel)

Keywords: Lepidodinium chlorophorum, eutrophication, FRRf, , Transparent exopolymeric particles, flow cytometry

Coastal ecosystems are increasingly threatened by eutrophication and dystrophy. In this context, the full pattern of a bloom dominated by the dinoflagellate, Lepidodinium chlorophorum, was investigated by a high frequency monitoring buoy equipped with sensors allowing nutrients and photosynthesis measurements. An increase of the N/P ratio affected phytoplankton physiology leading to bloom collapse with a slight oxygen depletion. In parallel, enrichment experiments were performed on the natural bloom population. After 5 days of incubation the community structure, using flow cytometry and several physiological parameters were analysed. The data reveal a potential N and P co-limitation and a decoupling between primary production and productivity in fully enriched conditions. Under unbalanced N/P inputs, high level of alkaline phosphatase activity and transparent exopolymeric particle production, which favour phytoplankton sedimentation, were observed. Nutrient inputs and their stoichiometry control phytoplankton growth, the community structure, physiological regulations, the fate of the bloom and consequences.

Introduction

As the ocean's first frontier, coastal ecosystems are increasingly under threats that affect many essential benthic and pelagic habitats of marine species [START_REF] Barbier | The value of estuarine and coastal ecosystem services[END_REF]. Eutrophication, which is due to excess nutrient inputs in coastal systems caused by human activities is one of the major environmental problems affecting coastal ecosystem in the world [START_REF] Rabalais | Global change and eutrophication of coastal waters[END_REF]. These inputs affect both the concentration of nutrients and their stoichiometry [START_REF] Martin | Fresh water influence on nutrient stoichiometry in a tropical estuary, Southwest coast of India[END_REF][START_REF] Watanabe | Estuarine circulation-driven entrainment of oceanic nutrients fuels coastal phytoplankton in an open coastal system in Japan[END_REF]. The growth and diversity of primary producers, like phytoplankton, which are at the base of the marine food web, are largely controlled by these inputs [START_REF] Smith | Responses of estuarine and coastal marine phytoplankton to nitrogen and phosphorus enrichment[END_REF].

Unbalanced nutrient inputs and dystrophy, both frequently linked with eutrophication, have several effects on phytoplankton: modification of communities [START_REF] Leruste | Physiological and behavioral responses of phytoplankton communities to nutrient availability in a disturbed Mediterranean coastal lagoon[END_REF][START_REF] Shen | Historical Changes in Nutrient Structure and its Influences on Phytoplantkon Composition in Jiaozhou Bay[END_REF], alter the growth rate [START_REF] Nwankwegu | Nutrient addition bioassay and phytoplankton community structure monitored during autumn in Xiangxi Bay of Three Gorges Reservoir, China[END_REF], produce allochemical-like toxins which, in turn, favour monospecific blooms [START_REF] Granéli | Harmful algal blooms of allelopathic microalgal species: The role of eutrophication[END_REF]. Associated with other environmental parameters including light, temperature, water mass resistance time, or river discharges, nutrient inputs can lead to phytoplankton blooms [START_REF] Heisler | Eutrophication and harmful algal blooms: A scientific consensus[END_REF] that may be classified as harmful algal blooms (HAB) [START_REF] Anderson | Approaches to monitoring, control and management of harmful algal blooms (HABs). Ocean Coast. Manag., Safer Coasts, Living with Risks: Selected Papers from the East Asian Seas Congress 2006[END_REF]. HAB species are usually split into two groups, one of which is able to produce toxins or harmful metabolites that damage wildlife, or result in poisoning of human seafood, and the second in which high densities of non-toxic cells can harm the environment by producing scums or foams which lead to oxygen depletion [START_REF] Anderson | Harmful algal blooms and eutrophication: Nutrient sources, composition, and consequences[END_REF].

Nutrient inputs into the Bay of Seine (France) are mainly controlled by inputs from the Seine River [START_REF] Aminot | Nutrients and phytoplanktonic growth in the Bay of Seine, France[END_REF] although smaller rivers inputs also have local impacts [START_REF] Lemesle | Impact of seaweed beachings on dynamics of δ15N isotopic signatures in marine macroalgae[END_REF]. Over the last decades, many programmes for the management of nutrient inputs have reduced nutrient discharges but have mainly had an impact on phosphorus inputs, whereas N inputs remain high [START_REF] Garnier | Managing the Agri-Food System of Watersheds to Combat Coastal Eutrophication: A Land-to-Sea Modelling Approach to the French Coastal English Channel[END_REF]. The stoichiometry of nutrient inputs have been broadly modified over the last decade and very high N/P ratio inputs are regularly measured in the Seine River and Seine estuary [START_REF] Meybeck | Trajectories of river chemical quality issues over the Longue Durée: the Seine River (1900S-2010)[END_REF].

Although chlorophyll biomass has decreased in recent years in the English Channel [START_REF] Gohin | Twenty years of satellite and in situ observations of surface chlorophyll-a from the northern Bay of Biscay to the eastern English Channel. Is the water quality improving? Remote Sens[END_REF], the relative proportion of the dinoflagellate group has increased [START_REF] Hernández-Fariñas | Temporal changes in the phytoplankton community along the French coast of the eastern English Channel and the southern Bight of the North Sea[END_REF][START_REF] Widdicombe | Long-term phytoplankton community dynamics in the Western English Channel[END_REF]. In the Bay of Seine, several species of dinoflagellate like Karenia mikimotoi and Lepidodinium chlorophorum are able to form large harmful algal bloom (HAB) [START_REF] Napoléon | Dynamics of phytoplankton diversity structure and primary productivity in the English Channel[END_REF]. Lepidodinium chlorophorum is known to produce large quantities of transparent exopolymeric particles (TEP) [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF] and to be responsible for the mortality of marine organisms due to oxygen depletion induced by decomposition of the population [START_REF] Sournia | The repetitive and expanding occurrence of a green, bloom-forming dinoflagellate (Dinophyceae) on the coasts of France[END_REF].

Many studies have shown that phytoplankton communities can respond within a few hours after an environmental change [START_REF] Lefort | Short-time scale coupling of picoplankton community structure and single-cell heterotrophic activity in winter in coastal NW Mediterranean Sea waters[END_REF][START_REF] Thyssen | The emergence of automated high-frequency flow cytometry: revealing temporal and spatial phytoplankton variability[END_REF] which stresses out the necessity of implementing a high frequency sampling [START_REF] Bouman | Dependence of light-saturated photosynthesis on temperature and community structure[END_REF]. In the present work, a late summer L. chlorophorum bloom event was studied in detail using high frequency measurements made by an instrumented buoy called SMILE (0° 19.68', 49° 21.23'). In addition to standard sensors (temperature, salinity, turbidity, oxygen, light and fluorescence), the SMILE buoy is equipped with a fast repetition rate fluorimeter (FRRf), which enables photobiological and physiological measurements of the phytoplankton communities, and two types of nutrient sensors, which together provide new and more realistic pictures of bloom dynamics.

In addition to monitoring the bloom event, enrichment bioassay experiments were performed on samples collected during the bloom in order to understand how nutrient inputs influence the structure and physiological parameters of the communities. The specific objectives of our studies were to: 1) Characterise bloom dynamics from an original point of view by including physiological, productivity, and nutrients measurements at a high frequency.

2) investigate phytoplankton dynamics and the ecophysiology of a community dominated by L. chlorophorum as a function of nutrient inputs.

Material and Methods

High frequency measurements using an instrumented buoy

The bloom event was monitored in situ using the SMILE buoy in the Bay of Seine (Fig. 1)

measuring since 2016 which belong the COAST-HF network (French Coastal ocean observing system -High frequency). Different types of measurements were performed at different frequencies by the buoy. were measured by the chemical probe Wiz (SYSTEA S.p.A, Italy) once a day while an optical sensor, OPUS (Trios, Germany), also measured NO 3 -at 20-min intervals. All measurements were performed at one-meter depth.

List of measurements performed at high frequency by the buoy

High-frequency measurements of inorganic nutrients

The Wiz probe uses micro loop flow analysis (µLFA) for autonomous measurements of four nutrients (NO 3 -, PO 4 2-, NO2 -) using colourimetric methods and NH3 with a fluorimetric method [START_REF] Azzaro | Automated Nutrients Analysis for Buoys in Sea-Water and Intercalibration[END_REF][START_REF] Moscetta | Instrumentation for continuous monitoring in marine environments[END_REF]. As the buoy has limited energy, measurements were only performed once a day. The detection limits were 0.35 µmol.l -1 for NO 3 -and 9.2.10 -2 µmol.l -1 for PO 4 2-

. NO 3 -was also measured using an optical sensor (OPUS, Trios, Germany). The OPUS UV is a spectral sensor equipped with a xenon flash lamp as light source and a high-end miniature spectrometer (MMS, Zeiss, Germany) with 256 channels covering the spectral range from 200 to 360 nm as detector. This sensor measures NO 3 -at high frequency with low energy consumption. A 20-mm optical path allowed measurements from 1 to 357 µmol.l -1 with a precision of 0.18 µmol.l -1 .

High frequency photosynthetic measurements

FLCs (fluorescent light curve) were measured in situ by the buoy using a FRRf-ACT2. The samples were analysed after a 5 min period of dark incubation for the oxidation of the Quinone A (QA). At the end of the dark period F0 (minimum fluorescence) was measured by using non actinic weak light. A single-turnover (ST) protocol consisting of 100 flashlets of 1 µs with 2 µs of interval was applied for measuring Fm (maximum fluorescence in dark) [START_REF] Kolber | Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols[END_REF]. The maximum quantum efficiency of PSII (Fv/Fm) was calculated as [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF]):

= ( -)
Samples were then exposed to 10 light steps of increasing PAR (from 0 to 1,500 µmol photon.s -1 .m - 2 ) for 30-sec each step. The effective quantum efficiency of PSII (Fq'/Fm') was measured at each light step as [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF]:

= ( -′)
Where Fm' is the maximum fluorescence under light and F' the steady state fluorescence under light.

The relative electron transport rate (rETR, relative unit) was calculated for each irradiance (E) as:

( ) = ×
The maximum relative electron transport rate (rETRmax) was estimated by fitting the FLC data to the model of [START_REF] Webb | Carbon dioxide exchange of Alnus rubra : A mathematical model[END_REF] modified by [START_REF] Boatman | Improving the Accuracy of Single Turnover Active Fluorometry (STAF) for the Estimation of Phytoplankton Primary Productivity (PhytoPP)[END_REF] using ACT2RUN (Chelsea Technologies, UK) software to estimate α and Ek with α, the initial slope of the FLC, and Ek the light saturation index:

( ) = × × 1 - -× × (1 - )
rETRmax is calculated as:

= ×
Using the absorption algorithm of [START_REF] Oxborough | Direct estimation of functional PSII reaction center concentration and PSII electron flux on a volume basis: a new approach to the analysis of Fast Repetition Rate fluorometry (FRRf) data[END_REF], the PSII electrons flux per unit volume is calculated as follows:

! "#$$ = × - × % × 10 '
where Ka is an inherent constant of the FRRf. Finally, the PSII flux per unit volume (JVIImax) is calculated as:

() $$ = ! "#$$ × × 3600 24 × 10 .
where JVIImax is expressed in mmol e -.m -3 .h -1 The electron transport rate (ETRIImax) is calculated as:

// = () $$ [1ℎ3!]
where ETRIImax is expressed in mmol e -.mg chla -1 .h -1

Enrichment experiment

Bioassays were designed to assess the impacts of enrichment on phytoplankton assemblages and on physiological responses. The design was modified from [START_REF] Ly | Phosphorus limitation during a phytoplankton spring bloom in the western Dutch Wadden Sea[END_REF] to fit our specific requirements. Seawater was sampled on the 29 th of August 2019 at the SMILE buoy site during the bloom event. The seawater was filtered just after sampling on a 100-µm mesh to remove grazers.

One hour after sampling, enrichments were applied to 500-ml sub-samples in polycarbonate bottles to perform the bioassays. All the bioassays were incubated in a water bath incubator under natural sunlight for 5 days. The water bath incubator was continuously fuelled with seawater pumped directly from the sea to maintain the temperature. The seawater temperature and PAR were recorded at 5-min intervals by respectively, a RBRsolo T logger and a RBR solo 3 PAR logger connected to a

Li-COR "Underwater Quantum Sensor" LI-192. Five types of enrichments, each with five replicates, were performed in the incubator: C (control, no enrichment), P (+P), N (+N), NSI (+N+Si) and PNSI (+P+N+Si). The enrichments applied to the bioassays were defined by the maximum value of N, P and Si measured over the year 2018 in the Bay of Seine by the National Network for Environmental Survey SOMLIT (https://www.somlit.fr/ since 2007 at this site) as respectively 50 µmol.l -1 for N, 3 µmol.l -1 for P and 50 µmol.l -1 for Si.

After 5 days, after homogenisation, 25 ml were sampled from all the bioassay bottles to measure phtosynthetic parameters, and phytoplankton functional types and alkaline phosphatase activity (APA). At day 5, supplementary measurements of TEP, nutrients and Chla concentration were performed. Only the data obtained at day 5 are shown here. All the measurements were also made just before enrichment at day 0 (D0).

Photosynthetic measurements

Photosynthetic measurements were performed with a second FRRf-ACT2. The same protocol as the one described above for in-situ measurements was applied to the samples for incubation. A 5-ml aliquot of sample was placed in the measuring chamber of the FRRf-ACT2 and FLC were performed after a 5-min period of dark adaption.

Flow cytometry

. Unfixed sample were analysed within one hour of sampling with the CytoSense (Cytobuoy b.v., Netherland) equipped with a blue laser (488 nm, 50 mW) and a green laser (552 nm, 50 mW). This produces pulse shapes based on the inherent optical properties of the particle when they cross the laser: sideward angle scatter (SWS), forward scatter (FWS), red (FLR, 668-734 nm), orange (FLO, 601-668 nm) and yellow fluorescence (FLY, 536-601 nm). The threshold was set at 16 mV to reduce data acquisition concerning non-photosynthetic particles triggered on FLR, for each sample

(5 per enrichment) 380 µl were analysed with a speed of 2.0 µl.s -1 . The CytoSense can analyse chains, cells, or colonies between 1 and 800 µm in diameter, microspheres measuring between 1 µm (Yellow-Green fluorescent, FluoSpheres®) and 1.6 µm (non-fluorescent, provided by Cytobuoy)

were used to calibrate size recording (daily use). In order to improve the clustering a mix of bead were analysed with the 1 µm and 1.6 µm and supplementary beads after this experiment (2 µm, 6 µm, 10 µm, 20 µm, Fluoresbrite® YG microsphere, Polyscience) to establish size regression (2-6 µm and 6-20 µm) and discriminate picoeukaryotes, nanoeukaryotes and microphytoplankton. To distinguish the phytoplankton (Fig. , 2), five clusters were determined using the cells' optical properties and attributed to Synechococcus spp., picoeukaryotes, nanoeukaryotes and microphytoplankton, Cryptophytes and Lepidodinium chlorophorum [START_REF] Elbrächter | Gymnodinium chlorophorum, a new, green, bloom-forming dinoflagellate (Gymnodiniales, Dinophyceae) with a vestigial prasinophyte endosymbiont[END_REF][START_REF] Hansen | Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)[END_REF]. The Synechococcus spp. cluster has the smallest FWS signal and a high orange fluorescence (FLO) signal which matches very small cells with a high concentration of phycoerythrin.

To accurately define the cytometric signature of L. chlorophorum, this species was isolated from the water sample during the bloom event. After a few days in culture, the isolated strains of L.

chlorophorum were analysed on the CytoSense to match the signature. Picoeukarotic cells are small cells (< 2 µm) and produce low FLR and FWS signals. Nanoeukaryotics and microphytoplankton cells were differentiated from picoeukaryotic cells using the amplitude of the FLR signal and the bead signal. Cryptophyte clusters have higher FLO than Synechoccocus due to the high concentrations of phycoerythrin in their cells and an FWS equivalent to the nanoeukaryotic and microphytoplankton cells [START_REF] Olson | Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry[END_REF][START_REF] Thyssen | Onset of the spring bloom in the northwestern Mediterranean Sea: influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure[END_REF].

Measurements of inorganic nutrients (NO 3 -, PO 4 3-, Si(OH)4)

Water samples were collected and filtered through a cellulose acetate filter (ClearLine, CA, 33 mm, 0.45 µm) in 50 ml falcon tubes and immediately frozen (-20 °C), with the exception of Si(OH)4 (4 °C). Analyses were conducted using a Seal Analytical AA-3 system [START_REF] Aminot | Dosage automatique des nutriments dans les eaux marines: méthodes en flux continu[END_REF].

The limits of quantifications were 0.02 µmol.l -1 for PO 4 3and 0.05 µmol.l -1 for NO 3 -, NO2 -, Si(OH)4.

N/P ratio was calculated as (NO2 -+ NO 3 -)/ PO 4 3-.

Chlorophyll-a measurements

Water samples (250 ml) were filtered through a glass-fiber filter (Whatman, GF/F, 47 mm) and immediately frozen (-20 °C) until analysis. Ten ml of 90% acetone (v/v) were added to extract the pigment and the samples were then left in the dark at 4 °C for 12 h. After being centrifuged for 5 min at 1,700 g twice, the Chl a concentration of the extracts was measured using a Trilogy fluorimeter (Turner Designs, Sunnyvale, USA) according to the method of [START_REF] Strickland | A Practical Handbook of Seawater Analysis[END_REF].

Transparent exopolymeric particles (TEP)

Water samples (150-200 ml) were filtered through polycarbonate membrane filters (Millipore, 0.4 µm) and immediately frozen (-20 °C) until analysis. According to [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF] adapted from [START_REF] Passow | A dye-binding assay for the spectrophotometric measurement of transparent exopolymer particles (TEP)[END_REF], the filters were stained with a solution 0.02%

Alcian blue (Sigma) with 0.06% acetic acid (pH: 2.5). Excess dye was removed by adding water before centrifugation at 3,000 g, 19 °C for 15 min. This washing cycle was repeated twice, then 6 ml of 80% H2SO4 were added. After 2 h, measurements were conducted using a spectrometer (Ultrospec 1000, Pharmacia Biotech) at 787 nm. Calibration were done using a xanthan gum (10-700 µg) as standard, as described by [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF]. After being divided by the Chla concentration, TEP concentrations are expressed in µg Xanthan eq.µg chla -1 .

Alkaline phosphatase activity (APA)

APA, as the potential maximum activity per chlorophyll unit,was measured according to [START_REF] Hrustic | Exploring the distance between nitrogen and phosphorus limitation in mesotrophic surface waters using a sensitive bioassay[END_REF]. Samples (3920-µl) were placed in an UV-cuvette and 80 µl of 4-Methylumbelliferyl phosphatase (MUF-P) 500 µM substrate were added. While incubating at room temperature, the samples were measured at hourly intervals over a total period of 7 h.

Measurements were taken with a RF-6000 spectrofluorophotometer (Shimadzu, Japan). APA was calculated as the slope of the linear regression of the evolution of concentration while incubating.

Using a MUF standard curve, the results are expressed in concentration units per hour divided by the concentration in Chl a to normalize (nM.h -1 .µg chla -1 .l -1 ).

Statistical and data analysis

Analyses were conducted using R software (R-project, CRAN) version 3.6.1. As the incubation data did not follow a normal law, a Kruskall-Wallis test was performed using "stats" package and a Conover-Iman pairwise test was performed using the "conover.test" package. Only significant tests with a p-value < 0.05 were accepted.

Results

Bloom time series

High frequency measurements provided by the SMILE buoy are presented in Fig. 3. An increase in FFU and oxygen saturation on the 24 th of August indicated the beginning of the bloom and remained high until the end of the bloom event on the 30 th of August, 2019 (Fig. 3 A,B). The highest dissolved O2 concentration and FFU were measured on the 27 th of August with respectively 16.5 mg O2 .l -1 and 165 FFU. This highest value of FFU was also reached the 28 th , 29 th and 30 th

August because of a saturation of the FFU sensor. The lowest oxygen concentration ever recorded in 2 years (data available) was measured the 31 th August with a value of 5.9 mg.l -1 . Based on the variation in O2 and FFU, the bloom lasted six days. The temperature increased from 19. 3 °C on the 20 th of August to reach a maximum of 23.0 °C on the 27 th of August and decreased to 19.6 °C on the 3 rd of September (Fig. 3 C). This short rise in temperature occurred after a drop in temperature starting on the 27 th of August. Turbidity increased on the 27 th of August, three days after the beginning of the bloom, to reach 120 NTU before dropping on the 29 th of August (Fig. 3 D).

Between the 25 and 30 th of August, high freshwater input events were observed, leading to a drop in salinity from 33.7 to 31.4 on the 29 th of August (Fig. 3 E). The light pattern appeared to be quite stable between days (with a maximum of 1,600 µmol photons.m -2 .s -1 ) with a decrease at mid-bloom (Fig. 3 F).

Nutrients N, P, Si followed the typical summer trend observed at this site in the English Channel (see Data SOMLIT, the French Coastal Monitoring Network; https://www.somlit.fr/ since 2007 at this site) with low stocks of nutrients at the beginning of August followed by nutrient inputs at the end of the month (Napoleon et al., 2012). An increase in PO 4 3-and NO 3 -concentrations was observed on the 21 st of August just before the bloom began (Fig. 4 B,C,D). After the beginning of the bloom, NH3 increased from 2.3 µmol.l -1 to 5.5 µmol.l -1 on the 4 th of September (Fig. 4 A).

During the bloom, a decrease was observed in the concentration of PO 4 3-from 1.7.10 -1 to 0.2.10 -1 µmol.l -1 (Fig. 4 D) whereas there was an increase in NO 3 -which reached 1.5 µmol.l -1 and increased the N/P ratio to 71 (Fig. 4 E) on the 26 th of August. The OPUS sensor enabled nitrate concentrations to be monitored at a higher frequency (20-min intervals) than the WIZ sensor (24-h intervals). Both sensors showed the same general trend for NO 3 -with an increase at the beginning of the bloom event followed by a decrease on the 29 th of August (Fig. 4 B,C). The high frequency data provided by the OPUS sensor revealed a dramatic drop in NO 3 -concentrations on the 26 th of August, two days after the beginning of the bloom (Fig. 4 C). The concentration on Si(OH)4 on the 30 th of August was 13.16 µmol.l -1 according to the SOMLIT database and the Si/N and Si/P ratios were respectively 8.7 and 329.0.

Fv/Fm displayed a daily cyclic pattern, the overall decrease of Fv/Fm the first day (from 20 th to 26 th August) is less than 0.03. Daily variation display more variation than this overall decrease, the mean value for this 6-days period is 0.57 ± 0.01 (n=60). The Fv/Fm dropped on the 26 th of August and reached a minimum value of 0.30 on the 28 th of August. Finally, Fv/Fm increased to reach a value of 0.55 on the 1 st of September (Fig. 5 A). JVIImax showed a daily cycle based on the daily light cycle. JVIImax values showed a steadily rising trend that reached a peak of 89.51 mmol e - .m -3 .h -1 on the 28 th of August. After this peak, a dramatic drop in JVIImax values was observed. The JVIImax value reached a value of 89.5 mmol e -.m -3 .h -1 on the 27 th of August (Fig. 5 B). ETRIImax values also showed a daily cycle, in which the values increased steadily before a dramatic drop on the 28 th of August. The ETRIImax reached a value of 29.4 mmol e -.mg chla -1 .h -1 on the 3 rd of September (Fig. 5 C).

Incubation results

During the 5-day incubation period, the mean value of the water temperature was 19.8 °C and the maximum value of the PAR was 2112 µmol photon.m -2 .s -1 with an overall mean value during daily light periods of 441 µmol photon.m -2 .s -1 .

The initial inorganic nutrient pool was low in the D0 sample with 3.0 µmol.l -1 of nitrate and a phosphate value below the detection limit (< 0.02 µmol.l -1 ). After incubation, nitrate concentrations in N and NSi enrichments decreased from 53 µmol.l -1 (D0 value + enrichment) to 31.5 ± 7.65 µmol.l -1 in each enrichment (Fig 6 . A), silica was not consumed in the NSi enrichment (mean = 54.28 µmol.l -1 ) (Fig 6 . C). In the P enrichment, the phosphate concentration decreased from 3 µmol.l -1 (D0 value + enrichment) to 1.9 ± 0.15 µmol.l -1 (Fig 6 . D). Finally, in the NPSi enrichment, both nitrate and phosphate were nearly fully consumed with 3 ± 0.07 µmol.l -1 of nitrate and 0.09 ± 0.05 µmol.l -1 of phosphate left while only 19 ± 2.14 µmol.l -1 of silica remained at the end of the incubation period. After 5 days of incubation, the concentration of Chl a decreased in the control, P, N, and NSi enrichments compared to the D0 value of 8.51 ± 1.04 (n=3) whereas in the NPSi Chl a concentration (Fig. 7 A) increased significantly (mean = 22.16 µg.l -1 ,Kruskall-Wallis: χ 2 = 13.6; pvalue = 8.6.10 -3 , Conover: mean pvalue = 1.9 10 -3 ).

Some common patterns regarding cells concentration were observed in the cytometric phytoplankton groups in all bioassays with enrichment. In the group of smaller cells:

Synechoccocus, the number of cells dropped after 5 days of incubation with no significant difference between enrichments (Fig. 7 B). Regarding picoeukaryotes, the mean concentration of cell in the P enrichment (2.0.10 4 ± 3.8.10 3 cells.cm -3 ) was higher than the control, and the N and NSi enrichments, with mean values of respectively, 1.8.10 4 , 8.6.10 3 ± 2.8.10 3 and 7.3.10 3 ± 2.3.10 3 cells.cm -3 . NPSi enrichment showed the highest concentration of picoeukaryotes (mean = 5.4.10 4 ± 1.5.10 4 cells.cm -3 ) (Kruskall-Wallis: χ 2 = 19.0, pvalue = 7.9.10 -4 ) (Fig. 7 C). Nanoeukaryotes also showed the highest concentration for NPSi enrichment with 9.9.10 4 ± 3.4.10 4 cells.cm -3 (Kruskall-Wallis: χ 2 = 13.3, pvalue = 1.2.10 -2 , Conover: mean pvalue = 1.7.10 -3 ). Regarding other treatments, controls contained higher concentrations of nanoeukaryotes (2.4.10 3 ± 8.8.10 2 cells.cm -3 ) than other enrichments with respectively 2.1.10 3 ± 4.5.10 2 ,1.8.10 3 ± 1.0.10 3 and 1.9.10 3 ± 9.1.10 2 cells.cm -3 with P, N and NSi enrichments (Fig. 7 D). Cryptophytes exhibited the same trend as nanoeukaryotes with a higher concentration in the control than in P, N and NSi enrichments (Fig. 7 E) while NPSi enrichment showed the significant higher concentration (χ 2 = 17.5, pvalue = 1.4.10 -3 , Conover: mean pvalue= 4.2.10 -3 ), but the value may be too low to draw conclusion. Finally, with an initial concentration of 389 ± 166 cells.cm -3 L. chlorophorum decreased in all clusters except for the NPSi enrichment. The lowest concentration of 127 ± 26 cells.cm -3 was measured in controls while the highest was observed in NPSi enrichment with 1,876 ± 1,496 cells.cm -3 . The difference in NPSi enrichment observed in this species was highly significant (Kruskall-wallis: χ 2 = 18.5, pvalue = 9.7.10 -4 , Conover: mean = 2.2.10 -4 ) compared to in all the other enrichments (Fig. 7 Low values of APA 1.2.10 -3 ± 1.5.10 -4 nM.h -1 .mg chla -1 .l -1 were measured at D0 just after sampling. After incubation, APA increased in all enrichments by a factor of 15, 19; 24.8; 44.6; 4.4 respectively, in the control, P, N, NSi and NPSi enrichments compared to at D0. The minimum value of 6.6.10 -3 ± 3.9.10 -3 nM.h -1 .mg chla -1 .l -1 was observed in the NPSi cluster (Fig. 7 G) but no significant differences were found among enrichments (Kruskall-Wallis test). As observed for APA, the concentration of TEPincreased in all treatments except in the NPSi enrichment where TEP decreased. The concentration of TEP increased by a factor of 3 in the P enrichments (0.3 ± 0.05 µg Xanthan equ.µg chla -1 ), by a factor 1.9 in both the N and NSi enrichments (0.5 ± 0.21 µg Xanthan equ.µg chla -1 ). NPSi enrichment presented the lowest concentration (0.043 ± 0.03 µg Xanthan equ.µg chla -1 ) but no significant differences were found among the treatments because of the high variability of the results between replications (Fig. 7 H).

Fv/Fm had a significantly high value in the NPSi enrichment with a mean of 0.46 ± 0.03 (Kruskall-Wallis: χ 2 = 10.6, pvalue = 3.0.10 -2 , Conover: mean pvalue= 1.2.10 -2 ) (Fig 8 . A) which is higher than the D0 value (0.41). The highest maximum production capacity (JVIImax) was also measured in the NPSi enrichments (Fig 8 . B) with a value of 162 ± 77 mmol e -.m -3 .h -1 revealing a significant difference from that in the other treatments (Kruskall-Wallis : χ 2 = 9.7, pvalue = 4.0.10 -2 , Conover: mean pvalue= 3.6.10 -2 ) and again higher than the D0 value (6.1 mmol e -.m -3 .h -1 ). The highest electron transport rate (ETRIImax) was measured in the P and NSi enrichments (Fig 8. B) with values of respectively, 15.5 ± 5.2, 15.3 ± 6.5 mmol e -.mg chla -1 .h -1 . A value of 8.1 ± 3.1 mmol e -.mg chla -1 .h -1 was measured in the NPSi enrichment but no significant differences were found between this and the other treatments (Kruskall-Wallis : χ 2 = 10.6, pvalue = 3.0.10 -2 ), Conover: mean pvalue = 7.6.10 -2 ) (Fig 8 . C), the value is however lower than the D0 value (21.7 mmol e -.mg chla - 1 .h -1 ).

Discussion

The high frequency data provided by the instrumented buoy SMILE alerted us to the occurrence of a HAB bloom dominated by the Dinophyceae Lepidodinium chlorophorum in late August 2019, in the Bay of Seine. L. chlorophorum contained green chloroplasts probably derived from a prasinophyte endosymbiont [START_REF] Hansen | Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)[END_REF]. Dinophyceae species with green chloroplasts are rare [START_REF] Hansen | Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)[END_REF] and to our knowledge L. chlorophorum is the only "green

Dinophyceae" that has been identified in the English Channel [START_REF] Sournia | The repetitive and expanding occurrence of a green, bloom-forming dinoflagellate (Dinophyceae) on the coasts of France[END_REF] and REPHY data base), so we were able to define a specific cytometric signature to characterise this species in our samples. The cytometric signature was confirmed in a strain isolated during the bloom thus allowing us to be extremely confident in our determination and the counting of L. chlorophorum in the present study. Although L. chlorophorum was first described in the English Channel in 1982 [START_REF] Sournia | The repetitive and expanding occurrence of a green, bloom-forming dinoflagellate (Dinophyceae) on the coasts of France[END_REF], its ecology is still poorly documented. It is described as an euryhaline species, able to produce dinocysts [START_REF] Elbrächter | Gymnodinium chlorophorum, a new, green, bloom-forming dinoflagellate (Gymnodiniales, Dinophyceae) with a vestigial prasinophyte endosymbiont[END_REF][START_REF] Honsell | Gymnodinium chlorophorum (Dinophyceae) in the Adriatic Sea: electron microscopical observations[END_REF], with an optimum growth temperature of 22 °C established by [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF] using a L. chlorophorum strain isolated in the English Channel. The conditions at the end of August 2019 were suitable to trigger a major L. chlorophorum bloom. The monitoring data from the SMILE buoy revealed the influence of nearby river outflows which, in summer, in the Bay of Seine, are defined as warmer and more turbid freshwater inputs enriched in nutrients [START_REF] Morelle | Annual Phytoplankton Primary Production Estimation in a Temperate Estuary by Coupling PAM and Carbon Incorporation Methods[END_REF][START_REF] Napoléon | Spatiotemporal dynamics of physicochemical and photosynthetic parameters in the central English Channel[END_REF]. As already reported in many studies, these factors favour Dinophyceae blooms [START_REF] Glibert | Margalef revisited: A new phytoplankton mandala incorporating twelve dimensions, including nutritional physiology[END_REF], whose L. chlorophorum [START_REF] Pozdnyakov | Investigation of Harmful/Nuisance Algae Blooms in Marine Environments[END_REF] underlined the importance of rises in temperature while [START_REF] Karasiewicz | Harmful algae niche responses to environmental and community variation along the French coast[END_REF][START_REF] Siano | Citizen participation in monitoring phytoplankton seawater discolorations[END_REF] pointed to the role of a drop in salinity during L. chlorophorum bloom events. Beside this general behaviour, which is widely described in the literature, our high-frequency data allowed us to monitor both the bloom and nutrient dynamics and the physiological state of the community in detail. The combination of a rise in temperature, a drop in salinity and nutrient inputs characteristic of estuarine waters triggered a bloom event in the bay which lasted six days on the monitoring site. After six days, the level of fluorescence dropped to its original value, marking the end of the bloom. The end of a bloom event can be triggered by different processes, such as a predatory [START_REF] Tiselius | Growth and decline of a diatom spring bloom phytoplankton species composition, formation of marine snow and the role of heterotrophic dinoflagellates[END_REF], virus event [START_REF] Schroeder | Virus Succession Observed during an Emiliania huxleyi Bloom[END_REF] or nutrient depletion [START_REF] Walve | Blooms of Baltic Sea Aphanizomenon sp. (Cyanobacteria) collapse after internal phosphorus depletion[END_REF]. In the present study, the high N/P ratio points to phosphate depletion. Thus, P appears to be a limiting nutrient and can be assumed to be the main driver of the decline in the bloom. Previous studies demonstrated that a bloom collapse can be explained by phosphate limitation [START_REF] Walve | Blooms of Baltic Sea Aphanizomenon sp. (Cyanobacteria) collapse after internal phosphorus depletion[END_REF]. The hypothesis of a P-limited bloom is supported in this study by the Fv/Fm value, which underwent a dramatic drop associated with a decline in biomass estimated by FFU one day after this drop. A value of 0.56 was measured for the Fv/Fm during the first three days of the bloom event, then decreased to 0.29 at mid bloom. This decrease could be partly due to a non-photochemical quenching mechanism [START_REF] Harrison | Effects of nutrients and irradiance on PSII variable fluorescence of lake phytoplankton assemblages[END_REF] but in our case, the minimum value appeared to be tightly linked to the increase in the N:P ratio. In the literature, a drop in the maximum quantum yield has been associated with nutrient limitation [START_REF] Behrenfeld | In Search of a Physiological Basis for Covariations in Light-Limited and Light-Saturated Photosynthesis1[END_REF][START_REF] Claquin | Effects of simulated benthic fluxes on phytoplankton dynamic and photosynthetic parameters in a mesocosm experiment (Bay of Brest, France)[END_REF], rapidly rising temperatures [START_REF] Zhang | Photochemical responses of phytoplankton to rapid increasing-temperature process[END_REF], a change in salinity (D'ors et al., 2016), or even a parasitic event [START_REF] Park | Effects of two strains of the parasitic dinoflagellate Amoebophrya on growth, photosynthesis, light absorption, and quantum yield of bloom-forming dinoflagellates[END_REF]. As L. chlorophorum tolerate a rise in sea temperature and a drop in salinity [START_REF] Pozdnyakov | Investigation of Harmful/Nuisance Algae Blooms in Marine Environments[END_REF][START_REF] Siano | Citizen participation in monitoring phytoplankton seawater discolorations[END_REF], in our case, we assume that the decline in the maximum quantum yield was most likely caused by the phosphate limitation. P limitation has been shown to significantly reduce the Fv/Fm in natural communities, in the field [START_REF] Napoléon | Spatiotemporal dynamics of physicochemical and photosynthetic parameters in the central English Channel[END_REF], in bioassays [START_REF] Ly | Phosphorus limitation during a phytoplankton spring bloom in the western Dutch Wadden Sea[END_REF], and in lab cultures [START_REF] Napoléon | Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species[END_REF][START_REF] Tan | The feasibility of Fv/Fm on judging nutrient limitation of marine algae through indoor simulation and in situ experiment[END_REF][START_REF] Wongsnansilp | Effects of phosphorus on the growth and chlorophyll fluorescence of a Dunaliella salina strain isolated from saline soil under nitrate limitation[END_REF][START_REF] Wu | Physiological regulation of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) in response to inorganic phosphorus limitation[END_REF]. Besides, the maximum quantum yield decreased at mid bloom; the JVIImax remained high because of the high level of biomass, and then dropped dramatically with the collapse of the bloom.

High-frequency sampling enabled by the SMILE buoy provided evidence for a link between P limitation and the collapse of a HAB bloom of L. chlorophorum. In a more general context, dinoflagellates have been associated with eutrophication since industrial development [START_REF] Kim | Decadal-scale variations of sedimentary dinoflagellate cyst records from the Yellow Sea over the last 400 years[END_REF]) and a recent shift has been observed [START_REF] Fischer | Return of the "age of dinoflagellates" in Monterey Bay: Drivers of dinoflagellate dominance examined using automated imaging flow cytometry and long-term time series analysis[END_REF][START_REF] Klais | Spring phytoplankton communities shaped by interannual weather variability and dispersal limitation: Mechanisms of climate change effects on key coastal primary producers[END_REF][START_REF] Spilling | Shifting Diatom-Dinoflagellate Dominance During Spring Bloom in the Baltic Sea and its Potential Effects on Biogeochemical Cycling[END_REF] at global scale comprising an increase in the dinoflagellate/diatom ratio. [START_REF] Wasmund | The Diatom/Dinoflagellate Index as an Indicator of Ecosystem Changes in the Baltic Sea. 2. Historical Data for Use in Determination of Good Environmental Status[END_REF] pointed to the relationship between an increase in the dinoflagellate/diatom ratio and a decrease in the quality indicator of environmental status assessment. The same trend has been described in the western English channel [START_REF] Widdicombe | Long-term phytoplankton community dynamics in the Western English Channel[END_REF] and in the Bay of Somme [START_REF] Hernández-Fariñas | Temporal changes in the phytoplankton community along the French coast of the eastern English Channel and the southern Bight of the North Sea[END_REF]. High-frequency monitoring allowed to understand and characterise short term processes and the factors which can trigger a large-scale bloom event and its fate. A bioassay enrichment experiment was performed during the bloom event to more deeply explore the behaviour and possible, control of bloom dynamics. Studying enrichments in a phytoplankton community involved in a natural bloom event is quite rare, and this enrichment bioassay enabled us to test other outcomes and their effects on phytoplankton communities as well as on several physiological indicators. However, the results obtained during the incubation have to be carefully consider in comparison with in situ data because the daily dynamics was not followed during the five days and many factors like hydrodynamics or grazing for example were removed. Beyond drawing a parallel between our microcosm experiments and our observations of a natural bloom, this second step of our study allowed us to investigate experimentally shifts in phytoplankton diversity during a HAB of L. chlorophorum and its effect on ecological functions such as primary productivity as a function of nutrient inputs. The relationship between primary productivity and biodiversity drives ecosystem biomass production and is a therefore a major question of ecological functioning and ecosystem management [START_REF] Hodapp | Unifying" the Concept of Resource Use Efficiency in Ecology[END_REF].

The first outcome of this experiment was the remarkable behaviour of NPSi enrichment in comparison with other treatments. NPSi enrichment resulted in higher concentrations of almost all cytometric populations apart from Synechococcus. Bloom communities were not sustained by partial enrichment in N, or P or NSi. Only NPSi enrichment was able to trigger growth of the phytoplankton community. Unfortunately, an NP enrichment could not be performed because only five conditions could be tested into our incubator. Despite the absence of NP enrichment conditions, the experiment highlighted an apparent N, P co-limitation in this bioassay. N and P had to be added to significantly stimulate growth. Furthermore, it is important to note that in the NPSi enrichment, unlike Si, N and P were depleted. Such co-limitations have been extensively studied and discussed in coastal ecosystems [START_REF] Davidson | Nutrients in synergy[END_REF] and NP inputs are known to promote dinoflagellate growth [START_REF] Reynolds | The Ecology of Phytoplankton, Ecology, Biodiversity and Conservation[END_REF]. During our experiments, the growth of all cytometric eukaryotic phytoplankton communities was stimulated under NPSi enrichment. Our results showed that the growth of L .chlorophorum was only stimulated in the fully replete condition.

A higher primary production capacity (JVIImax) and a higher physiological status (Fv/Fm) were observed in the NPSi enrichment while lower productivity capacity (ETRIImax) was measured in comparison with other treatments. The decoupling of production (JVIImax) and productivity (ETRIImax) in the NPSi enrichment was unexpected. This decoupling can be attributed to the size community structure, as differences in productivity between picoeucarykote, nanoeukaryote and microphytoplankton have been reported [START_REF] Lefebvre | Spatial and temporal dynamics of size-structured photosynthetic parameters (PAM) and primary production (13C) of pico-and nano-phytoplankton in an atoll lagoon[END_REF][START_REF] Li | Size-dependent photosynthetic variability in the North Pacific Subtropical Gyre[END_REF][START_REF] Zhu | Primary Productivity Dynamics in the Summer Arctic Ocean Confirms Broad Regulation of the Electron Requirement for Carbon Fixation by Light-Phytoplankton Community Interaction[END_REF] and may be one consequence of photoacclimatation processes. The increase in biomass in the NPSi enrichment reduced light penetration and increased self-shading [START_REF] Huisman | Light-Limited Growth and Competition for Light in Well-Mixed Aquatic Environments: An Elementary Model[END_REF] leading to an increase in the concentration of Chla in the photosystem antenna [START_REF] Falkowski | Acclimation to Spectral Irradiance in Algae[END_REF][START_REF] Macintyre | Photoacclimation of Photosynthesis Irradiance Response Curves and Photosynthetic Pigments in Microalgae and Cyanobacteria1[END_REF]. Such regulation has been associated with an internal increase in pigment self-shading, thereby reducing the pigment-specific light absorption coefficient (σPSII) [START_REF] Finkel | Resource limitation alters the 3/4 size scaling of metabolic rates in phytoplankton[END_REF]. In our study, the lowest specific light absorption coefficient (σPSII) at 440 nm [START_REF] Kolber | Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols[END_REF] was measured in the NPSi enrichment (See supplementary data).

Therefore, the self-shading mechanism makes it possible to explain the uncoupling observed between JVIImax and ETRIImax in these optimal growth conditions.

APA (as the potential maximum activity per chlorophyll unit) was quantified in order to characterise the P limitation during our experiment [START_REF] Kuenzler | Phosphatases of marine algae[END_REF][START_REF] Lin | Phosphorus physiological ecology and molecular mechanisms in marine phytoplankton[END_REF][START_REF] Tanaka | Specific Affinity for Phosphate Uptake and Specific Alkaline Phosphatase Activity as Diagnostic Tools for Detecting Phosphorus-Limited Phytoplankton and Bacteria[END_REF]. Alkaline phosphatase enables cells to access organic phosphate but the process is energy consuming [START_REF] Falkowski | Review of Aquatic Photosynthesis[END_REF][START_REF] Lin | Phosphorus physiological ecology and molecular mechanisms in marine phytoplankton[END_REF]. Dinoflagellates can make efficient use of dissolved organic phosphate [START_REF] Wang | Utilization of dissolved organic phosphorus by different groups of phytoplankton taxa[END_REF], and quantifying APA is one way to asses this use when P is limited. According to the APA, the enrichment conditions applied in this bioassay can be gathered in three groups: (i) high P limitation for N and NSi, enrichments; (ii) medium P limitation for control and P and finally (iii) low P limitation for NPSi enrichment. The lack of any significant difference between controls and P enrichment despite the addition of P is surprising. P enrichment is N limited, which notably affects cell physiology. Previous studies showed that other limitations, like N [START_REF] Kuenzler | Phosphatases of marine algae[END_REF] or Si [START_REF] Fuentes | Silicon Deficiency Induces Alkaline Phosphatase Enzyme Activity in Cultures of Four Marine Diatoms[END_REF], can lead to an increase in APA. Changes in the community structure may also influence APA [START_REF] Lin | Rapidly diverging evolution of an atypical alkaline phosphatase (PhoAaty) in marine phytoplankton: insights from dinoflagellate alkaline phosphatases[END_REF][START_REF] Yuan | Phytoplankton communities determine the spatio-temporal heterogeneity of alkaline phosphatase activity: evidence from a tributary of the Three Gorges Reservoir[END_REF]. The observed differences in the picoeukaryote community under P replete conditions could alter nutrient uptake behaviour because picoeukaryotes are known to be very efficient in assimilating phosphorus. We hypothesise that this community produces more alkaline phosphatase than the other communities observed in the other enrichments. In addition, picoeukaryotes appeared to be favoured by P enrichment. This result showed that picoeukaryote was able to grow under N limitations, which are frequently reported in oligotrophic areas [START_REF] Mackey | Picophytoplankton responses to changing nutrient and light regimes during a bloom[END_REF][START_REF] Siokou-Frangou | Plankton in the open Mediterranean Sea: a review[END_REF] but rarely in eutrophic areas like the Bay of Seine where N limitation is only observed in summer [START_REF] Napoléon | Spatiotemporal dynamics of physicochemical and photosynthetic parameters in the central English Channel[END_REF][START_REF] Thorel | Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France)[END_REF]. Our results also showed that Synechococcus was unable to grow under any of the conditions we tested, which is probably a bias introduced by our incubation methodology. The closed system could create greater predatory pressure leading to a higher death rate of Synechococcus [START_REF] Agawin | Nutrient and temperature control of the contribution of picoplankton to phytoplankton biomass and production[END_REF].

Si depletion did not occur either during the bloom event or during the bioassay, but even if Si was not limited during the course of this experiment, diatoms were not able to take over domination from dinoflagellates. In the literature, dinoflagellate dominance over diatoms is frequently reported to be driven by Si depletion [START_REF] Liang | Nutrient-limitation induced diatom-dinoflagellate shift of spring phytoplankton community in an offshore shellfish farming area[END_REF][START_REF] Zhou | Nutrients structure changes impact the competition and succession between diatom and dinoflagellate in the East China Sea[END_REF]. Moreover, previous works showed that both in a natural bay [START_REF] Lie | Changes in the nutrient ratios and phytoplankton community after declines in nutrient concentrations in a semi-enclosed bay in Hong Kong[END_REF] and in a model [START_REF] Tett | N:Si ratios and the 'balance of organisms': PROWQM simulations of the northern North Sea[END_REF], when the N:Si ratio increases, the dinoflagellate/diatom ratio also increases. This supports our field measurements, which showed that environmental status (temperature and salinity)

combined with an input of N and P triggered the dinoflagellate bloom even though Si was not limiting. Inputs of unbalanced nutrient water would not have triggered the bloom, as shown by the absence of growth in the experiment when only P or N were added.

TEP is widely associated with carbon excretion by phytoplankton [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF][START_REF] Klein | Dynamics of soluble extracellular polymeric -substances and transparent exopolymer particle pools in coastal ecosystems[END_REF] even if bacteria can also promote TEP precursors [START_REF] Li | Marine bacterial transparent exopolymer particles (TEP) and TEP precursors: Characterization and RO fouling potential[END_REF][START_REF] Li | Compositional similarities and differences between transparent exopolymer particles (TEPs) from two marine bacteria and two marine algae: Significance to surface biofouling[END_REF].

Moreover, in the literature, TEP production is associated with N limitation [START_REF] Beauvais | Transparent exopolymer particle (TEP) dynamics in relation to trophic and hydrological conditions in the NW Mediterranean Sea[END_REF][START_REF] Corzo | Production of transparent exopolymer particles (TEP) in cultures of Chaetoceros calcitrans under nitrogen limitation[END_REF][START_REF] Deng | Effects of nutrient limitation on cell growth, TEP production and aggregate formation of marine Synechococcus[END_REF] or high N/P ratio [START_REF] Mari | Dynamics of transparent exopolymeric particles (TEP) production by Phaeocystis globosa under N-or P-limitation: a controlling factor of the retention/export balance. Harmful Algae, Bloom Dynamics and Biological Control of Phaeocystis: a HAB Species in[END_REF]. In our study, P, N, NSi enrichments displayed higher TEP concentrations per Chla unit. As mentioned previously, in all these experimental conditions, phytoplankton growth was limited in P or in N. Conversely, under non-limited growth conditions, i.e., the NPSi treatment, TEP production was reduced, which is physiologically coherent. Indeed, TEP can be considered as an overflow of carbon. As nutrient stresses do not allow proper carbon assimilation in the different metabolic pathways, carbon is accumulated as carbohydrates and part is excreted as TEP precursors. L. chlorophorum is described in the literature as a high TEP producer [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF][START_REF] Sournia | The repetitive and expanding occurrence of a green, bloom-forming dinoflagellate (Dinophyceae) on the coasts of France[END_REF]. [START_REF] Claquin | Effects of temperature on photosynthetic parameters and TEP production in eight species of marine microalgae[END_REF] showed under replete conditions that L. chlorophorum was able to produce 10 times more TEP than other seven species tested which belonged to the main phytoplankton phyla. Such high TEP production favours the formation of aggregates and the sinking rate [START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF] which can lead to anoxia events that affect the benthos community [START_REF] Passow | Transparent exopolymer particles (TEP) in aquatic environments[END_REF]. In the present study, an O2 depletion was observed at the end of the bloom; but although rare, this event was far from anoxia.

Conclusions

In this study, the entire temporal pattern of a HAB species, L. chlorophorum, identified by microscopy and flow cytometry, was monitored. We showed that the high-frequency measurements of physical-chemical and biological parameters provided by the SMILE buoy allowed to detect the triggers of the bloom and its fate. There were not flow cytometer on the buoy, but our study pointed out that such a measurement associated with the others parameter available at high frequency would represent a great opportunity to deeper explorer phytoplankton dynamics and manage HAB events.

The bioassay experiments performed during this bloom event allowed to explore physiological regulation at a community scale. The regulation of photosynthetic parameters was investigated as a function of nutrient inputs and of community structure, which made it possible to characterise uncoupling of production and productivity in some circumstances. The bioassay experiments also provided evidence of potential P and N co-limitation in this ecosystem and revealed that unbalanced inputs favour high TEP production with a population dominated by L.chlorophorum, which, in turn, can lead to massive bloom, sedimentation, and local oxygen depletion. Management of the nutrient inputs ratio has to be considered both in connection with the triggering -but also with the fate of blooms.

-

  Conductivity and temperature (tetracon sensor, WTW TM , Germany), turbidity (Seapoint turbidity meter, Seapoint Sensor, USA), oxygen (AADI Oxygen optode, Aanderraa, Norway) and in-vivo fluorescence (Cyclops-6K, Turner Design, USA) sensors that measure fluorescent in fluorescein in fluorescent units (FFU) are grouped in a multiparameter probe NKE instrument (MP7, NKE Instrumentation ®, France), PAR (Photosynthetically active radiation) is measured with a Satlantics sensor (Satlantics, Italy). All these parameters were measured at 20-min intervals (data available doi.org/10.17882/53689). Photosynthetic parameters were determined at 2-h intervals using the FastOcean FRRf3 sensor with the Act2 system (Chelsea Technologies, UK) with an excitation light provided by a blue light-emitting diode (450 nm). Inorganic nutrients (NO 3

  F). The values measured for L. chlorophorum in the incubation are consistent with the maximum concentration value observed in august 2010, in the Bay of Seine by the French Observation and Monitoring program for Phytoplankton and Hydrology in coastal waters (REPHY , DOI: 10.17882/47248) with 418.3 cells.cm -3 .

Acknowledgements

We thank Bertrand Le Roy from BOREA for L. chlorophorum isolation, Tania Hernández-Fariñas from Ifremer for her comments and advices and the COAST-HF network for technical support. This work was funded by the SMILE 2 and RIN ECUME projects respectively supported by l'Agence de l'Eau Seine Normandie,the European Regional Development Fund of Normandie, and La Région Normandie and by the PLEASE PhD project supported by l'Agence de l'Eau Seine Normandie and La Région Normandie


Prof Florence Duffaud
email: florence.duffaud@ap-hm.fr

Antoine Italiano

Emannuelle Bompas

MD Maria Rios

MD Nicolas Penel

MD Olivier Mir

Sophie Piperno-Neumann

MD Christine Chevreau

MD Corinne Delcambre

MD François Bertucci

Pascaline Boudou-Rouquette

MD Mathilde Cancel

Christophe Perrin

Esma Saada-Bouzid

Laure Monard

Camille Schiffler

MD Loic Chaigneau

Alice Hervieu

MD Olivier Collard

MD Corinne Bouvier

MD Vincent Vidal

Sylvie Chabaud

MD Jean-Yves Blay

French Sarcoma

MD Group Florence Duffaud

Sophie Piperno

Pascaline Boudou

Esma Saada

Efficacy and safety of regorafenib in patients with metastatic or locally advanced chondrosarcoma: Results of a non-comparative, randomised, double-blind, placebo controlled, multicentre phase II study

Keywords: metastatic chondrosarcoma, progression after standard chemotherapy, regorafenib

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

Introduction:

Chondrosarcomas are a heterogeneous group of mesenchymal tumors representing the second most common primary bone tumor in adults, and accounting for 20% of new primary bone cancer cases. In the past three decades, there has been no significant improvement in the survival of patients with metastatic chondrosarcoma, which has no standard systemic therapy of any proven efficacy; nothing beyond surgical resection has proven benefit, [START_REF] Gelderblom | The clinical approach towards chondrosarcoma[END_REF][START_REF]NCCN Clinical Practice Guidelines in Oncology: Bone Cancer, Version 1[END_REF] with only anecdotal activity reported with a variety of sarcoma-focused cytotoxic regimens [START_REF]NCCN Clinical Practice Guidelines in Oncology: Bone Cancer, Version 1[END_REF][START_REF]Bone sarcomas: ESMO-PaedCan-EURACAN Clinical practice Guidelines for diagmosis, treatment and follow-up[END_REF][START_REF] Italiano | Advanced chondrosarcomas: role of chemotherapy and survival[END_REF][START_REF] Fox | Phase II study of sequential gemcitabine followed by docetaxel for recurrent Ewing sarcoma, osteosarcoma, or unresectable or locally recurrent chondrosarcoma: results of Sarcoma Alliance for Research Through Collaboration Study 003[END_REF]. Consequently, there is a clear need for more effective systemic treatment for patients with metastatic and/or unresectable advanced chondrosarcoma.

Early clinical data suggest activity of VEGFR kinase inhibitors such as sorafenib and sunitinib in patients with bone sarcomas [START_REF] Dubois | Phase I and pharmacokinetic study of sunitinib in pediatric patients with refractory solid tumors: a Children's Oncology Group Study[END_REF][START_REF] Pacey | Efficacy and safety of sorafenib in a subset of patients with soft tissue sarcoma from a phase II randomized discontinuation trial[END_REF]. Regorafenib demonstrated antitumour activity in pretreated metastatic non-adipocytic soft tissue sarcoma [START_REF] Mir | Safety of regorafenib in patients with advanced soft tissue sarcoma (REGOSARC):a randomised, double-blind, placebo-controlled, phase 2 trial[END_REF], a population for which pazopanib has also demonstrated activity in prolonging PFS [START_REF] Van Der Graaf | Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind placebo-controlled phase 3 trial[END_REF]. Furthermore, compared with placebo, regorafenib also improved PFS in chemotherapy-refractory metastatic osteosarcoma (10) (4.0 versus 1.0 months) in the REGOBONE osteosarcoma cohort. The objective of the present study, was to explore the antitumour activity of regorafenib in patients with progressive metastatic and/or recurrent chondrosarcoma after failure of conventional chemotherapy.

Patients and methods

Study design and participants

REGOBONE, an investigator-initiated signal-seeking trial, is a basket study of five parallel independent cohorts of different metastatic bone sarcoma histopathologic subtypes. Parallel cohorts assessed the activity and safety of regorafenib or placebo, using a randomised, non-comparative, double-blind, placebo-controlled phase 2 trial design. We present now the results of the chondrosarcoma cohort.

The study was approved by an ethical and regulatory committee (French Ethical Committee, Comité de Protection des Personnes Sud Méditerrannée 1, approved on March 26, 2014). All patients provided written informed consent before enrolment, and one study amendment (protocol V6, June 29, 2016) expanded enrollment to include paediatric patients aged > 10 years (although ultimately no children were enrolled). The trial is registered in the European Clinical Trials Register database (EudraCT N°: 2013-003910-42) and at ClinicalTrials.gov (NCT02389244). Eligible patients were required to have histological diagnosis of chondrosarcoma, and objective disease progression within 6 months prior to study entry measured by RECIST v1.1, both confirmed by a centralised review, as well as measurable disease by RECIST v1.1 not amenable to curative-intent, and previously treated with 1-2 previous lines of chemotherapy for locally-advanced or metastatic disease. The complete list of other eligibility criteria, along with the protocol are described in the Appendix A1 are available on line (http://www.unicancer.fr/protocole-regobone). Patients were randomly assigned (2:1) to receive either oral regorafenib or matched placebo. After centrally confirmed disease progression (according to RECIST 1.1), patients initially randomised to placebo were offered crossover to open-label regorafenib. Central pathological review was done by an expert bone sarcoma pathologist from the "Réseau de Relecture en Pathologie des Sarcomes Osseux" in France [START_REF] Perrier | The cost-saving effect of centralized histological reviews with soft-tissue and visceral sarcomas, GIST, and desmoid tumors: the experiences of the pathologists of the French Sarcoma Group[END_REF] Registration and randomisation (2:1) were centralised via a web-based system (IWRS) using permuted blocks design provided by an independent partner (ATLANSAT). Patients, pharmacists, investigators, site study teams, and sponsor were all blinded to the allocated-treatment. Treatment allocation was masked until centrally confirmed disease progression. Patients were randomly assigned to receive best supportive care combined with either regorafenib 160 mg orally (four tablets of 40 mg once daily, three weeks on and 1 week off), or matched placebo tablets. Best supportive care included any method to preserve the comfort and dignity of the patients and excluded any disease-specific anti-neoplastic agents. Dose interruptions and/or dose reductions recommendations have been previously described [START_REF] Duffaud | Efficacy and safety of regorafenib in adult patients with metastatic osteosarcoma: a non-comparative, randomised, double-blind, placebo-controlled, phase 2 study[END_REF].

The primary endpoint was the progression-free rate (PFR) at 12 weeks, defined as the proportion of patients without disease progression at 12 weeks, after confirmation by central radiological review according to RECIST1.1. Secondary endpoints included: progression-free survival (PFS) per modified RECISTv1.1, objective response rate (ORR), overall survival (OS), duration of overall response (DoR), and safety/tolerability. PFS was measured from the date of randomisation until the date of confirmed radiological progression or death from any cause, whichever occurred first.

For patients who were event-free at the time of the analysis, PFS was censored at the time of the final adequate tumour assessment. Centrally assessed progression was used for the analysis. OS was defined as the time from randomisation to the date of death from any cause and censored at the date of final contact for patients alive. Objective response to treatment corresponded to proportion of patients with complete or partial response as best response from randomisation. Duration of response, which applies only to responders was measured from the time of first documented response (complete response or partial response) until the first documented disease progression or death. Patients who died from causes other than progression were censored at the date of death.

Statistical analysis

When the REGOBONE study was designed there was a paucity of published data regarding PFS of patients with metastatic chondrosarcoma following failure of standard treatments. The literature reported the median of PFS with an inactive drug was about 12 weeks for metastatic chondrosarcoma [START_REF] Fox | Phase II study of sequential gemcitabine followed by docetaxel for recurrent Ewing sarcoma, osteosarcoma, or unresectable or locally recurrent chondrosarcoma: results of Sarcoma Alliance for Research Through Collaboration Study 003[END_REF][START_REF] Navid | Combination of gemcitabine and docetaxel in the treatment of children and young adults with refractory bone sarcoma[END_REF], and that an active drug should at least double this median to be considered of interest. At the same time, a large retrospective study (4) that analysed the benefit of first-line chemotherapy in 180 metastatic chondrosarcoma patients reported a median PFS for the overall group of 4.7 months [95% confidence interval (CI) 3-6.5]. Therefore, we chose progression-free rate at 12 weeks as the primary endpoint. We calculated the sample size by A'Hern single stage design for phase 2 trials similar to a Fleming phase II design but assuming an exact binomial distribution [START_REF] 'hern | Sample size tables for exact single-stage phase II designs[END_REF]. These hypotheses on median PFS translated into a 75% progression-free rate at 12 weeks, defined as the expected efficacy in the experimental arm. A limit of 50% or less would mean that the regorafenib did not warrant further investigation. A sample size of 23 patients provided 80% power to reject the null hypothesis with a one-sided, type 1 error of 5%, with 16 successful patients being the lower cutoff point of decision making. To account for a possible non-assessable patient rate of 5%, an additional patient was required in the experimental arm (total 24 patients). A sample size of 12 evaluable patients was required in the placebo arm. No comparative hypothesis was formulated and no statistical comparison between the control and experimental arms was planned.

Thereby, the primary endpoint and all other efficacy outcomes were analysed by modified intention to treat, including all patients who initiated blinded study drug treatment, with no major protocol violation. Major protocol violations were defined as deviations that could potentially affect efficacy analysis, including patients not meeting important inclusion or exclusion criteria.

The occurrence of adverse events was analysed in the safety population, defined as all confirmed CS patients who received at least one dose of the intended treatment. The severity of the adverse events was graded according to the NCI-CTCv4.0. The percentage of progression-free patients at 12 weeks was calculated in each arm with their respective 95% confidence interval. PFS and OS were estimated using the Kaplan-Meier method. We used SAS (version9.4) for all analyses.

Results

From September 24, 2014 to February 04, 2019, forty-six adult patients were accrued and randomised in this CS cohort, representing the population for safety analysis (Figure 1). Five patients were excluded from safety and efficacy analyses, all in the regorafenib arm, because the CS diagnosis was not histologically confirmed by central review (2 patients with diagnosis changed to osteosarcoma were included in the REGOBONE osteosarcoma cohort (29), 1 patient with chordoma, and 2 patients with soft tissue sarcomas). One CS was excluded from the efficacy analysis, in the regorafenib arm, due to lack of confirmed progressive disease at study entry. In total, 40 patients with histologically confirmed advanced chondrosarcoma with confirmed disease progression constitute the population for efficacy analysis: 24 patients randomised to regorafenib, and 16 initially on placebo. Two patients remain on therapy at the time of analysis, 1 on blinded treatment, and 1 on regorafenib after crossover.

As described precisely in Table 1, the baseline characteristics of 40 patients were well balanced between the two arms except for a small imbalance in age, ECOG Performance Status, and in histological subtype. Histological subtypes other than conventional CS were slightly more frequent in the regorafenib arm (21%), than on placebo (13%). Seventy-two percent of patients received only one previous chemotherapy regimen for metastatic/recurrent disease prior to study entry. All patients had metastatic disease except two with locally advanced disease in the placebo arm. The majority of patients had previously received doxorubicin, cisplatin and ifosfamide.

At the time of the analysis, the median follow-up of surviving patients was 35.9 months (IQR 29-46.8). Efficacy endpoints are reported in Table 2. Thirteen patients (54% one-sided 95% CI 35.8%-[) were non progressive at 12 weeks in the regorafenib group while 5 (31% one-sided 95% CI 13.2%-[) were progression-free on placebo. Two durable partial responses were observed in the regorafenib arm of 6.6 and 16.6 months respectively. At the time of the analysis, 18 of 24 patients (75%) progressed after randomisation to the regorafenib arm, versus 15 of 16 patients (94%) initially randomised to placebo; 18 deaths in 24 patients (75%) were reported in the regorafenib arm versus 11/16 (69%) deaths on placebo. All causes of death except one were disease progression.

Median PFS was 19.9 weeks (95%CI 11.4-34.9) in the regorafenib arm, and 8.0 weeks (95%CI 4.3-23.4) on placebo. PFS rate at 12 weeks was 61% (95%CI 39-78), and 43% (95%CI 23-62) at 24 weeks, for patients on regorafenib, versus 31% (95%CI 11-54) and 25% (95%CI 8-47) on placebo, respectively. Following centralised confirmation of progressive disease, 15 of 16 patients randomised to placebo crossed over to regorafenib. One placebo patient did not crossover due to major decline of performance status. The median treatment duration was 3.4 months (95%CI 1.6-8.3) on regorafenib, 1.7 months (95%CI 1.6-1.7) on placebo. Transient discontinuation, occurred in 11 (46%) of 24 patients in the regorafenib arm, and in 7 (44%) of 16 patients on placebo. Dose reductions were reported in 15 (63%) of 24 patients in the regorafenib arm versus 1 (6%) of 16 patients on placebo. Regorafenib was reduced to 120mg/ day for 10 patients (41.7%) and to 80 mg/ day for 5 patients (20.8%), placebo was reduced to 80 mg/day for one patient (6.3%).

Dose reductions for toxicity were reported in 12 (50%) of 24 patients in the regorafenib arm versus 0 (0%) of 16 patients on placebo, and were due to haematological toxicity in 1 patient (thrombocytopenia), and to non-haematological toxicities in 12 patients, mainly hand-foot syndrome (N=4), asthenia (N=3), diarrhea (N=3), weight loss (N=2) and arthralgia (N=2). Four treatmentrelated serious adverse events occurred (16%) in 25 patients in the regorafenib arm vs 0 (0%) of 16 patients on placebo. All were at least grade 2, including arrhythmia (N=1), ulcerative keratitis (N=1), hepatocellular injury/liver cytolysis (N=1), and headache (N=1). Safety data are shown in Table 3 for the two groups until optional crossover. The most common grade  3 treatment-related AEs during the double-blind period in the regorafenib arm included pain (20%), hypertension (12%), asthenia (12%), thrombocytopenia (8%), diarrhoea (8%), and hypokaliema (8%). There was one toxic death reported on regorafenib linked to Grade V hepatic toxicity.

Discussion

Prior data from the osteosarcoma cohort of this multi-cohort trial indicated activity of regorafenib in delaying progression of that form of bone sarcomas. However, the data from this cohort study did not meet the primary endpoint to be consider a success. According to the study design criteria for success, 16/24 non progressive patients at 12 weeks in the regorafenib arm would have been necessary for a positive criterion. This number is not reached with only 13 successes (54.2%). However, considering placebo results: with only 5/16 (31.3%) non progressive patients at 12 weeks, widely below P0 hypothesis, and with a median PFS of 19.9 weeks with regorafenib, versus of 8 weeks on placebo, or 12.0 weeks in the placebo patients who crossed-over to open-label regorafenib, our study suggest that regorafenib might nonetheless have modest activity in this setting. It might slow disease progression in patients with progressive metastatic CS after failure of prior chemotherapy Furthermore, our study showed a meaningful result with 47% of these metastatic patients remaining progression-free at 6 months on regorafenib, while only 25% on placebo remained with stable disease. Surprisingly in our regorafenib arm, our results may appear inconsistent with those reported with pazopanib ( 14) in a single-arm phase 2 study, with a median PFS of 19.9 weeks (less than 5 months) with regorafenib versus 7.9 months with pazopanib, and with PFR at 12 weeks of 54.2% with regorafenib versus a DCR (disease control rate) at 16 weeks (primary endpoint) of 43% with pazopanib. However, the 6-month PFS rates with kinase inhibitors are quite similar with a 6-months (24-weeks) PFS rates of 47% with regorafenib, 47% with dasatinib [START_REF] Schuetze | Phase 2 study of dasatinib in patients with alveolar soft part sarcoma, chondrosarcoma, chordoma, epithelioid sarcoma, or solitary fibrous tumor[END_REF], and 55% with pazopanib [START_REF] Chow | Results of a Prospective Phase 2 Study of Pazopanib in Patients With Surgically Unresectable or Metastatic Chondrosarcoma[END_REF] respectively.

Although the median OS is 11.7 months (95% CI 7.2-18.8) for patients randomised to regorafenib and 19.9 months (95% CI 6.2-40) for those on placebo, the confidence intervals are overlapping in this small study and, virtually all the placebo-treated patients crossed over to receive open-label regorafenib.

This study also confirms that metastatic chondrosarcoma is a very aggressive disease, with median PFS of 8 weeks on placebo, worse than our null hypothesis of 12 weeks in the study design. Our null hypothesis was probably overestimated. The possible inappropriate choice of our H0 hypothesis might have contributed to the fact that our primary objective was not achieved statistically.

We note that regorafenib may not be tolerable when initiated at full dose (160 mg per day) in a large fraction of patients, and our data suggest that patient-specific dose modifications to deliver a dose tailored to individual patient tolerance may nonetheless represent a clinically beneficial dosing strategy. The overall safety profile of regorafenib was as previously published and generally amenable to dose modifications, although we did note one episode of fatal hepatic toxicity.

The present study has other limitations, since it was statistically non-comparative, done in only one country, and included a relatively small number of patients.

Although this trial did not meet the statistical plan to be judjed positive, this exploratory randomized trial confirms the aggressive behavior of metastatic CS overall. The data from this cohort will help investigators design other trials to study novel strategies which might eventually improve outcomes for patients with advanced chondrosarcomas. For example, although activity from single agent immune checkpoint inhibitor therapy has been only rarely noted in chondrosarcomas [START_REF] Tawbi | Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial[END_REF][START_REF] Paoluzzi | Response to anti-PD1 therapy with nivolumab in metastatic sarcomas[END_REF][START_REF] Wagner Mj Riciotti | Response to PD1 inhibition in conventional chondrosarcoma[END_REF], regorafenib in combination with anti-PD1 therapy has recently demonstrated very encouraging antitumour activity in patients with advanced gastrointestinal cancers [START_REF] Fukuoka | Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and dose-expansion phase Ib trial (REGONIVO, EPOC1603)[END_REF]. Future studies should extrapolate from the carcinomas to determine if these two therapies (immune activation and targeted therapy) can be combined in a synergistic way to improve chondrosarcoma outcomes.

Additionally, new insights might evolve as to whether molecularly distinct subsets of chondrosarcomas (e.g. CS with IDH1 or IDH2 mutations, or CS with aberrant DNA damage/repair genes) might have different responses to multi-kinase inhibition with regorafenib or other agents. Future trials of new agents will need to take validated targets and explore them for clinical effect in patients with chondrosarcomas in order to make progress against this mesenchymal malignancy.

grants form Pfizer, Takeda, JYB receives reserch support and honoraria from Eisei, Eli Lilly, MSD, BMS, GSK, Ignyta, Novartis, Pharmamar and Roche un related to this work, and AI, CP, NP, OC, LC, CD, MC, LM, MR, EB, SC, FB, CS, SPN, CB, VV, AH, ESB, have declared no conflicts of interest .

PFS according to RECIST and central radiological review

PFS, according to RECIST, be measured from the date of randomization until the date of radiological progression or death whatever the cause (if death occurs before progression). The date of disease progression will be the date of first observation of progression according to RECIST 1.1 guidelines and central radiological review. Patients who have not progressed or died at the time of analysis, or who die after three or more missed visits, will be censored at the time of the latest date of assessment from their last evaluable RECIST assessment.

Median PFS was 8 weeks (95%CI [4.3-23.4]) in placebo arm vs 19.4 weeks [11.4; 38] in regorafenib arm.

The progression-free survival rate at 12 weeks is 31% in the placebo arm and 61% in the regorafenib arm. The rate of regorafenib arm is different from the non-progression rate at 12 weeks (primary endpoint), because for the non-progression rates at 12 weeks we used the tumor assessment at 12 weeks +/-2 weeks. 1 patient progressed in this acceptable delay (11006 at 13 weeks) and 1 patient ( 02018) is censored at his last tumor evaluation because he stopped treatment for SUSAR without progression, but for the primary endpoint it's a failure.

PFS, according to RECIST, be measured from the date of randomization until the date of radiological progression or death whatever the cause (if death occurs before progression). The date of disease progression will be the date of first observation of progression according to RECIST 1.1 guidelines and central radiological review. Patients who have not progressed or died at the time of analysis, or who die after three or more missed visits, will be censored at the time of the latest date of assessment from their last evaluable RECIST assessment.

Overall survival

The follow-up of alive patients is at median of 35.9 months (min: 9.5, max: 52.9).

At the time of the analysis, 29 deaths were notified, 11 under placebo (69%) and 18 under regorafenib (75%).

One patient died of toxicity probably due to regorafenib (hepatitis).

3 reasons for death are missing in the database, because they occurred after the end of patients' follow-up. 2 patients died of progression and 1 reason was still missing at the time of analysis.

OS Median was 19.9 months (95%CI [6.2-40.0]) in placebo arm vs 11.7 months [7.2-18.8] in regorafenib arm.

Figure 2

 2 Figure2shows the PFS curves per blinded central review. Figure 3 shows the Overall Survival (OS) curves, including 15 of 16 (94%) of placebo patients who crossed over to open-label regorafenib. The median OS is 11.7 months (95% CI 7.2-18.8) for patients randomised to regorafenib and 19.9 months (95% CI 6.2-40) for those randomised to placebo. The swimmer plots on Figure 4 show the initial PFS and PFS after cross over for the 15 patients initially randomised to placebo who subsequently received open-label regorafenib. The waterfall plots on Figures 5 show the tumour responses obtained on blinded treatment, and on open-label regorafenib.

Figure 1 :

 1 Figure 1: Consort diagram -study population

Figure 2 :

 2 Figure 2: Progression free survival (primary endpoint per blinded central review)

Figure 3 :

 3 Figure 3: Overall survival including crossed over of 83% of placebo patients

Table 15 : Study withdrawal

 15

	LIBTRAIT	All
	Placebo Regorafeni b
	N=16	N=24	N=40

Figure 5: Waterfall plots of beast responses (regorafenib arm and placebo arm)

Table 3 :

 3 Adverse events in >= 10% of patients per treatment group before crossover 20

		Regorafenib	Placebo
		N=25		N=16
		Any grade		>= 3	Any grade		>=3
	BLOOD AND LYMPHATIC SYSTEM DISORDERS
	ANAEMIA	4 (12.0%) 1 (4.0%)	1 (6.3%)	0	(0.0%)
	THROMBOCYTOPENIA	4 (16.0%) 2 (8.0%)	1 (6.3%)	0	(0.0%)
	LYMPHOPENIA	3 (12.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)
	GASTROINTESTINAL DISORDERS
	DIARRHOEA	13 (52.0%) 2 (8.0%)	5 (31.3%)	0	(0.0%)
	NAUSEA	11 (44.0%) 1 (4.0%)	5 (31.3%)	0	(0.0%)
	CONSTIPATION	8 (32.0%) 0 (0.0%)	2 (12.5%)	0	(0.0%)
	STOMATITIS	8 (32.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	VOMITING	6 (24.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)
	DRY MOUTH	6 (24.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	ABDOMINAL PAIN	3 (12.0%) 0 (0.0%)	2 (12.5%)	0	(0.0%)
	HAEMORRHOIDS	4 (16.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS
	PAIN	20 (80.0%)	5 *	(20.0%)	11 (68.7%)	1	(6.3%)
	ASTHENIA/FATIGUE	18 (72.0%) 3 (12.0%)	6 (37.5%)	0	(0.0%)
	MUCOSITIS	8 (32.0%) 1 (4.0%)	1 (6.3%)	0	(0.0%)
	FEVER	3 (12.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	INFECTION	4 (16.0%) 1 (4.0%)	3 (18.7%)	1	(6.3%)
	EYE DISORDERS
	ULCERATIVE KERATITIS	0 (0.0%)	1 *	(4.0%)	0 (0.0%)	0	(0.0%)
	INVESTIGATIONS
	WEIGHT DECREASED	12 (48.0%) 1 (4.0%)	1 (6.3%)	0	(0.0%)
	HEPATOCELLULAR INJURY	0 (0.0%)	1 * (4.0%)	0 (0.0%)	0	(0.0%)
	WEIGHT INCREASED	0 (0.0%) 0 (0.0%)	2 (12.5%)	0	(0.0%)
	METABOLISM AND NUTRITION DISORDERS
	ANOREXIA	11 (44.0%) 1 (4.0%)	2 (12.5%)	0	(0.0%)
	HYPOALBUMINAEMIA	3 (12.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	NERVOUS SYSTEM DISORDERS
	DYSGEUSIA	5 (20.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)
	NEURALGIA	4 (16.0%) 2 (8.0%)	1 (6.3%)	0	(0.0%)
	RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS
	DYSPHONIA	10 (40.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)
	DYSPNOEA	4 (16.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)
	COUGH	4 (16.0%) 0 (0.0%)	0 (0.0%)	0	(0.0%)
	SKIN AND SUBCUTANEOUS TISSUE DISORDERS
	OTHER SKIN TOXICITY	15 (60.0%) 1 (4.0%)	1 (6.3%)	1	(6.3%)
	HAND AND FOOT SKIN REACTION	12 (48.0%) 1 (4.0%)	3 (18.8%)	0	(0.0%)
	PRURITUS	3 (12.0%) 0 (0.0%)	1 (6.3%)	0	(0.0%)

* related serious adverse event

Acknowledgments

We thank the patients and their family for their participation in the study. We thank all the UNICANCER sarcoma staff members involved in the trial management. The data management was conducted by UNICANCER, the analysis was done by Centre Léon Bérard. We thank the patient advocacy group InfoSarcomes. We thank the French National Cancer Institute for funding the labelled networks for management of sarcoma (e.g, NetSarc, RRePS, RESOS, and InterSarc).

Funding

Bayer Healthcare SAS (Loos, France) supplied regorafenib and placebo tablets and provided funding to UNICANCER. The funder had no role in study design, data collection, monitoring, analysis, and interpretation or writing of the report. Once the trial has been designed, UNICANCER, as the Sponsor for the study, in collaboration with the French Sarcoma Group was responsible for all aspects of the trial. This study was not funded by NIH.

Author contributions

FD and SC designed and supervised the trial and wrote the article ; FD, SC, and CS, analysed the data and contributed to the trial design. All other authors supervised clinical patient management, reviewed and approved the article.

Conflict of interest statement

FD received travel grants from Pharmamar, and Leo Pharma, attended advisory boards for Bayer, Lilly. CC attended advisory boards for Leo Pharma, OM attended advisory boards and chaired meetings with Amgen, Astra Zeneca, Bayer, Bleuprint, Eli Lilly, Roche, Servier, PBR received travel