Charaf E Merzougui

Pierre Roblin

Pierre Aimar

Antoine Venault

Yung Chang

Christel Causserand

Patrice Bacchin

Pearl

Keywords: Human Serum Albumin, poly(acrylic acid), interaction, ionic strength, pH, SAXS

Introduction 3

Interactions between proteins and polymeric surfaces, whether linear (polyelectrolyte brushes) [START_REF] Senaratne | Self-Assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives[END_REF][START_REF] De Vos | Adsorption of the Protein Bovine Serum Albumin in a Planar Poly(Acrylic Acid) Brush Layer As Measured by Optical Reflectometry[END_REF] or spherical (functional nanoparticles) [START_REF] Henzler | Adsorption of Bovine Hemoglobin onto Spherical Polyelectrolyte Brushes Monitored by Small-Angle X-Ray Scattering and Fourier Transform Infrared Spectroscopy[END_REF][START_REF] Welsch | Adsorption of Proteins to Functional Polymeric Nanoparticles[END_REF][START_REF] Wang | Protein Immobilization and Separation Using Anionic/Cationic Spherical Polyelectrolyte Brushes Based on Charge Anisotropy[END_REF] , have received growing attention over the past few decades. Many processes involve this phenomenon in several fields and for versatile applications, as protein separation [START_REF] Wang | Protein Immobilization and Separation Using Anionic/Cationic Spherical Polyelectrolyte Brushes Based on Charge Anisotropy[END_REF][START_REF] Mattison | Protein-Polyelectrolyte Phase Boundaries[END_REF] , drugs delivery [START_REF] Pourjavadi | Mesoporous Silica Nanoparticles with Bilayer Coating of Poly(Acrylic Acid-Co-Itaconic Acid) and Human Serum Albumin (HSA): A PH-Sensitive Carrier for Gemcitabine Delivery[END_REF][START_REF] Das | Biofunctionalized PH-Responsive Microgels for Cancer Cell Targeting: Rational Design[END_REF] , ligands uptake [START_REF] Yu | Stability of Human Serum Albumin Structure upon Toxin Uptake Explored by Small Angle Neutron Scattering[END_REF] , enzyme immobilization [START_REF] Chen | Enzyme Immobilization on Ultrafine Cellulose Fibers via Poly(Acrylic Acid) Electrolyte Grafts[END_REF] , antibacterial encapsulation [START_REF] Rastogi | Synthesis and Characterization of Bovine Serum Albumin-Copper Nanocomposites for Antibacterial Applications[END_REF] , among many other applications in chemistry and biology [START_REF] Dubin | Macromolecular Complexes in Chemistry and Biology[END_REF] .

Biomaterials and medical devices design are among the fields in which this interaction is relevant [START_REF] Nel | Understanding Biophysicochemical Interactions at the Nano-Bio Interface[END_REF][START_REF] Wei | Protein Interactions with Polymer Coatings and Biomaterials[END_REF] . Biomaterials are often used for the treatment of diseases such as cardiac failure, but also in medical applications such as wound healing, deep skin injury repair, dentistry or orthopedics [START_REF] Bhat | Biomaterials and Bioengineering Tomorrow's Healthcare[END_REF] . In many cases, these biomaterials get into direct contact with blood. One of the fastest growing areas for biomaterials is the development of new systems for controlled and targeted drug delivery. For example, suitable membrane advanced technologies could be proposed to address several critical needs related to blood cells screening and the concentration of the growth factors stored inside the granules of platelets and expressed after their activation, and which role in the wound healing process of diabetic ulcers is predominant. For that, the architecture of membrane 4 biointerface at the nanoscale is often customized by grafting on it some functional groups such as poly acrylic acid (PAA). It is a negatively charged bioactive polymer in aqueous medium widely used to modify polymeric surfaces for drugs release and many other applications [START_REF] Tarvainen | Drug Release from Poly(Acrylic Acid) Grafted Poly(Vinylidene Fluoride) Membrane Bags in the Gastrointestinal Tract in the Rat and Dog[END_REF] . One of them is the enhancement of platelets adhesion, especially when it's conjugated to proteins such as fibrinogen. However, nonspecific interactions between other blood components and polymers used for interface modification could hamper the capture of platelets. Among those blood components, Human Serum Albumin (HSA) is an abundant plasma protein ([HSA]≈42mg.mL -1 in blood [START_REF] Leggio | About the Albumin Structure in Solution: Cigar Expanded Form versus Heart Normal Shape[END_REF] ) that maintains the blood pH and osmotic pressure and transports some molecules in the blood. This protein is able to decrease platelet adhesion and activation on a solid surface by being adsorbed onto that particular surface 18 . Therefore, in the purpose of designing functionalized membrane surfaces to selectively separate thrombocytes from other blood cells, the control of these nonspecific interactions is required.

According to the literature, a number of protein-polyelectrolyte complexes have been studied in a variety of physicochemical conditions via several analytical techniques [START_REF] Cooper | Polyelectrolyte-Protein Complexes[END_REF][START_REF] Basak Kayitmazer | Protein -Polyelectrolyte Interactions[END_REF] .

Concerning complexes of serum albumin and polymers, the interaction of bovine serum 5 albumin (BSA) with spherical polyelectrolyte brushes in solution was evidenced using Small Angle X-ray and neutron scattering (SAXS and SANS) [START_REF] Rosenfeldt | Interaction of Proteins with Spherical Polyelectrolyte Brushes in Solution as Studied by Small-Angle x-Ray Scattering[END_REF][START_REF] Wang | Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-Ray Scattering[END_REF] . Interactions between BSA and planar synthetized poly(acrylic acid) were also the object of a dedicated work [START_REF] Topuzoğulları | Molecular-Weight Distribution and Structural Transformation in Water-Soluble Complexes of Poly(Acrylic Acid) and Bovine Serum Albumin[END_REF] . Furthermore, binding of HSA with small polyelectrolyte brushes of PAA (with 25 repeating units only) [START_REF] Ran | Interaction of Human Serum Albumin with Dendritic Polyglycerol Sulfate: Rationalizing the Thermodynamics of Binding[END_REF], and with dendritic polyglycerol sulfate [START_REF] Hollmann | Characterization of a Planar Poly(Acrylic Acid) Brush as a Materials Coating for Controlled Protein Immobilization[END_REF] , has also been discussed by isothermal calorimetry (ITC). In fact, it has been proved that only one HSA is bound per chain of PAA 1.8 kDa. Then, for a pH maintained at 7.2, a study on the effect of salt concentration revealed that this binding was dominated by an attractive electrostatic potential between the two solutes [START_REF] Yu | Interaction of Human Serum Albumin with Short Polyelectrolytes: A Study by Calorimetry and Computer Simulations[END_REF] . Using Total Internal Reflection Fluorescence (TIRF) spectroscopy, it also has been proven that negatively charged BSA is strongly adsorbed on planar PAA (278 AA units ≈20kDa) at low ionic strength and pH > 5 due to electrostatic interactions [START_REF] Hollmann | Characterization of a Planar Poly(Acrylic Acid) Brush as a Materials Coating for Controlled Protein Immobilization[END_REF] . However, the current knowledge does not permit to give the exact stoichiometry of the polyelectrolyte-protein complexes for larger polymer, and if it is correlated to the size of the polyelectrolyte.

In the present study, we used SAXS combined to chromatography to assess the affinity of HSA to bind to a larger PAA (100 kDa) to check out the formation of multiprotein-6 polymer complexes compared to the 1HSA-1PAA binding reported in the literature using smaller polyelectrolyte [START_REF] Yu | Interaction of Human Serum Albumin with Short Polyelectrolytes: A Study by Calorimetry and Computer Simulations[END_REF] . First, as a starting point for further experiment with polymers, the structure of native HSA was examined in Phosphate buffer solution. Then, the HSA/PAA complexation was studied in media with different ionic strengths or pH in order to find the conditions leading to an attractive interaction. After that, a detailed study modeling the Porod volume was conducted to determine the stoichiometry of the (nHSA-PAA) complex formed. For this purpose, the influence of the HSA/PAA molar ratio on the complex size (radius of gyration and Porod volume) was appraised. Eventually, a compatible model has been found using BUNCH program [START_REF] Petoukhov | Global Rigid Body Modeling of Macromolecular Complexes against Small-Angle Scattering Data[END_REF] to fit the HSA-PAA complex scattering data. It allowed us to propose an all atoms pearl necklace model that describes the structure of complex at the ionic strength and at the pH outlined in the preceding paragraphs, and it also validated the proposed stoichiometry.

Experimental section

Materials and sample preparation

Human serum albumin (HSA, lyophilized powder, ≥96% agarose gel electrophoresis) with a molecular weight of 66,478 g.mol -1 was purchased from Sigma Aldrich and used 7 as received, as well as the poly acrylic acid (PAA) of molecular weight around 100,000 g.mol -1 (35 wt. % in H 2 O).

HSA powder was dissolved in PBS 1X (0.012M phosphate buffer solution at pH 7.4, ionic strength 0.137M) to obtain a stock solution of 50 g.L -1 HSA (750 µM), from which samples of lower concentration were prepared. The same way, the polymer solutions were prepared by dilution in PBS 1X buffer from a stock PBS solution of 100 g.L -1 PAA (1 mM).

For the investigation of native HSA structure in solution and HSA-PAA interaction, the concentration of the HSA was set at 5 g.L -1 to avoid repulsion between particles which can distort the measured intensity in small angles. The pH effect was studied using different buffer solutions at 0.1 M (TRIS base pH 8, HEPES pH 7, MES pH 6, mixture of sodium citrate di-hydrate and citric acid at pH 4-5). As for the ionic strength influence, the salts concentration was adjusted by adding the needed amounts of 4 M NaCl solution.

Then, all samples, whether protein free, polymer free or a mixture of both, were stirred using a Thermo Scientific mini centrifuge (mySPIN 12) at 10 000 rpm for 2 minutes at least, to ensure good dissolution of the solutes, before being pipetted into the samples 8 holder for the SAXS measurement. Out of use, the samples were stored at 4°C in a refrigerator.

Chromatography measurement

The chromatography unit used consisted of an Agilent 1260 infinity system connected to a UV-VIS detector set at a wavelength of 280 nm. Analyses were performed using an Agilent Bio Sec-3 (3µm, 4.6x300 mm, 300 Å) size exclusion column at room temperature.

In a typical analytical run, 50µL of a 75µM protein solution was injected, and PBS 1X was used as mobile phase for protein structure investigation at a flow rate of 0.4 ml.min -1 . As for protein-polymer complexation study, a 75µM HSA+ 50µM PAA solution was injected and the mobile phase was PBS solution containing 50 µM PAA.

SAXS measurement

SAXS measurements were performed at Laboratoire de Genie Chimique, Toulouse, on the XEUSS 2.0 bench equipped with a copper internal source (Genix3D) that produces an X-ray beam with an energy of 8 keV and a flow of 30.10 6 ph. mm².s -1 with a beam sized at 500x500 μm. The SAXS bench is equipped with an HPLC online (Agilent 1260 9 infinity) connected to the SAXS cell allowing to add a purification step by size exclusion chromatography. In direct injection mode, that is, without size exclusion chromatography step, 50 µL of sample are pipetted from the samples holder maintained at a constant temperature (20 °C) using a circulating water bath, and are injected to the measurement cell placed under vacuum to limit air absorption. Data were collected on a 150x150 mm area DECTRIS detector (Pilatus 1M) with a sample to detector distance of 1216 mm. The scattering intensity I(q) experimentally determined is expressed as a function of the scattering vector q (in Å -1 ) resulting from a photon of wavelength λ scattering off the sample at an angle 2θ.

𝑞 = 4𝜋𝑠𝑖𝑛 (𝜃) 𝜆 #(1)
Each scattering curve reported in this paper is an average of at least 6 measurements on the same sample, and each SAXS measure corresponds to a data collection time of 1800s. Scattering curves were recorded within the range 0.005 < q < 0.5 Å -1 . Finally, actual solutes scattering intensity I(q) was obtained by subtracting the buffer solution contribution from the total SAXS signal.

10

Porod volume calculation

From SAXS experiments, it is possible to obtain the Porod volume of an object from the Porod invariant Q [START_REF] Konarev | PRIMUS: A Windows PC-Based System for Small-Angle Scattering Data Analysis[END_REF] :

𝑉 𝑜𝑏𝑗 = 2𝜋 2 𝐼(0) 𝑄 * 1 -𝜙 𝑆(0) #(2)
When the dispersion is diluted the volume fraction , and the Porod 𝜙→ 0 𝑆(0)→ 1 volume can be determined by knowing the zero angle intensity I(0) and Q.

Q is called the Porod invariant because its value does not depend on the structure but on the volume fraction and the contrast between two phases :

Δ𝜌 𝑄 = ∫ ∞ 0 𝑞 2 (𝐼(𝑞) -𝐾)𝑑𝑞 = 2𝜋 2 𝜙(1 -𝜙) 𝑟 2 𝑒 (Δ𝜌) 2 #(3)
Where K is a constant defined to ensure the asymptotical intensity decay proportional to q -4 at higher angles [START_REF] Petoukhov | ATSAS 2.1towards Automated and Web-Supported Small-Angle Scattering Data Analysis[END_REF] .

The value of Q can be roughly obtained from the integration of the Kratky plot (classically with a limited integration boundary at q=0.25) by considering K=0:

𝑄 = ∫ 0.25 0 𝑞 2 𝐼(𝑞)𝑑𝑞#(4) 11
When the intensity decreases too slowly at large q (less than q -3 ) as for polymeric systems [START_REF]Scattering Methods Applied to Soft Condensed Matter[END_REF] it is well more accurate to integrate the original equation after determining the constant K. Whereby the constant K is calculated from the Porod asymptote I(q). q [START_REF] Welsch | Adsorption of Proteins to Functional Polymeric Nanoparticles[END_REF] versus q 4 at higher angles. In these calculations, the asymptote has been always determined for the same range of q between 0.3 and 0.445 with a homemade code to keep the control of the calculation. The code is given with the plots that shows the determination of K (Fig. 45) in Supplementary Information SI-2, as well as the corrected dimensionless Kratky plot obtained by subtracting K from I(q), (Fig. 6 in SI-2). It can be seen that the subtraction of the asymptote K leads to a curve that tends to zero for high q which then permit a good integration of the invariant Q.

Results and discussion

Study of single HSA

The structure of native HSA was first examined in a 0.012M phosphate buffer solution at pH 7.4, and the obtained SAXS scattering intensities were compared to those calculated from the crystal data of HSA on the Protein Data Bank (6EZQ). In order to 12 guarantee the absence of aggregates in the investigated samples, an Agilent chromatographic system is coupled upstream of the SAXS. Guinier plot (Ln I(q) vs q 2 in red line) with the residual (green curve) that allow to determine the radius gyration using Primus. The HSA 3D structure shown in cartoon representation using PyMOL software.

Figure 1 shows that the experimental SAXS spectrum from purified commercial HSA (blue line) matches very well the calculated intensity from the crystal structure (red line), evidencing that the HSA used for this study is correctly folded at 75 µM in the PBS buffer condition. These preliminary results validate the use of HSA as a template to study protein 13 polymer interaction. In these conditions we can expect that the SAXS will be precise enough to sense an interaction between HSA and PAA.

The HSA radius of gyration R g was then evaluated by fits using Foxtrot or Primus [START_REF] Konarev | PRIMUS: A Windows PC-Based System for Small-Angle Scattering Data Analysis[END_REF][START_REF] David | Combined Sampler Robot and High-Performance Liquid Chromatography: A Fully Automated System for Biological Small-Angle X-Ray Scattering Experiments at the Synchrotron SOLEIL SWING Beamline[END_REF] Both software consider the Guinier approximation that allows to determine R g from the slope of the linear fit at low q of I(q) vs q 2 (Figure 1), as it gives the forward scattering intensity I(0) from the intercept based on the following equation:

lim 𝑞→0 𝐼 = 𝐼 0 𝑒𝑥𝑝 ( -1 3 𝑞 2 𝑅 2 𝑔 ) #(5)
The result yields approximately 30 Å (30.4±0.2 Å), which is almost what was found with R g (30.2 ± 0.4 Å) in a SAXS study for HSA with the same purity >96% [START_REF] Leggio | About the Albumin Structure in Solution: Cigar Expanded Form versus Heart Normal Shape[END_REF] .

Likewise, the Porod volume and molecular weight were estimated using Primus that consider the Porod law (Supplementary Information SI-2), and found to be around 100,000 Å 3 and 64.5 kDa respectively. These results seem to be consistent with the molecular weight of HSA (66.5 kDa), which validates the use of Primus to evaluate such parameters in further experiments.

HSA-PAA interaction 14

The characterization of single protein was then followed by assessing the interactions between HSA and the polyelectrolyte chains. For that, we first studied the effect of an increase in PAA concentration on the SAXS scattering intensity for an HSA/PAA molar ratio ranging from 0 (protein alone as a reference) to a molar ratio of 3/2. As shown in Figure 2, starting from 40 µM of PAA with 75 µM HSA, a strong change in the signal at small angles is observed, which could be due to the formation of an HSA-PAA complex, while in the intermediate and high q-range the SAXS curves remain almost unchanged by the complexation. Indeed, the evolution of the slope P of the corresponding power law I(q) = q -P shows that at intermediate angles (0.1 to 0.2 Å -1 ), P slightly varies from 3.8 for single HSA and the mixture of HSA+10, 20 and 30µM PAA, to 4 when increasing the PAA concentration to 40 and 50µM (Figure 2). These results indicate a same type of surface despite the big change in the general shape shown by an increase in the slope at small angles (0.005 to 0.05 Å -1 ). In fact, the difference is more obvious, where the slope is going from almost a tray (P=0.3) for single HSA, to a slope of 1.2 when adding 50µM PAA, which corresponds to the slope of an elongated object [START_REF] Koch | Small-Angle Scattering: A View on the Properties, Structures and Structural Changes of Biological Macromolecules in Solution[END_REF] . PAA, despite being in PBS buffer, strongly influences the pH of the solution. Thus, we can suppose that the change in the SAXS curve could be correlated to a pH variation rather than to an effect of the concentration of PAA (explicitly discussed later in the paper).

In order to confirm that the change in the SAXS signal at small angles is due to the formation of a new object being an HSA-PAA complex, we compared the SAXS curves from the HSA alone at 75 µM (blue line), the PAA alone at 60 µM (green line), the mixture of both kipping the same initial concentrations (yellow line) and the sum (red line) of the SAXS signals of HSA at 75 µM and PAA at 60 µM (Figure 3A).

For the first three curves, the data were processed with GNOM [START_REF] Svergun | Determination of the Regularization Parameter in Indirect-Transform Methods Using Perceptual Criteria[END_REF] to determine biophysical parameters such as the radius of gyration R g , the maximal distance D max using the autocorrelation function P(r). This function was calculated by an indirect Fourier transformation applied to the scattering intensity I(q) using the following equation:

𝑃(𝑟) = 𝑟 2 2𝜋𝑟 2 𝑒 ∫ ∞ 0 𝑞 2 𝐼(𝑞) sin (𝑞𝑟) 𝑞𝑟 𝑑𝑞 #(6)
The pair distribution function refers to the distribution of electrons averaged over a radius r. It entirely depends on the shape and the size of the particle and vanishes at their maximum electron pair distance (D max ). The radius of gyration can be then derived from P(r) using the following expression: :

𝑅 2 𝑔 = ∫ 𝐷𝑚𝑎𝑥 0 𝑟 2 𝑃(𝑟)𝑑𝑟 2∫ 𝐷𝑚𝑎𝑥 0 𝑃(𝑟)𝑑𝑟) #(7)
This alternative estimate of R g makes use of the whole scattering curve, and is much less sensitive to interactions or to the presence of a small fraction of oligomers. The results of the radius of gyration (29.7 Å for HSA and 110.3 Å for the complex) extracted from the P(r) were almost the same compared to those obtained by fits from the Guinier plot (30.4 Å for HSA and 108.9 Å for the complex). The Porod volumes (V p ) for the HSA and the complex were also evaluated. For a flexible system such as polymer chains in solution it's difficult to evaluate the exact V p . All parameters are reported in the table attached to Figure 3A. Although the curves are calibrated in cm -1 , it is impossible to directly determine the mass concentration of the hetero-complex from the I(0) because we do neither know the association stoichiometry nor precisely the experimental specific volume and contrast of the PAA.

As observed in Figure 3A, the SAXS curve of HSA is consistent with a 3D object with a large plateau at small angles and a typical decrease at intermediate angles, whereas PAA exhibits a behavior of a flexible polymer in solution (large decrease following a power law I(q) = q -P with P close to 1.7 which corresponds to polymers with local rigidity) [START_REF] Chu | Small-Angle X-Ray Scattering of Polymers[END_REF] . The SAXS curve obtained for the complex presents a combination of properties of HSA and PAA. it shows a constant decrease at small angles and the same signal of HSA starting from 0.05 Å -1 . The direct comparison with a linear combination of HSA and PAA SAXS curves (sum curve in Figure 3A) in the same conditions of concentration reflecting the case without interactions between both partners, discloses clearly that the SAXS curve 18 observed corresponds to the creation a new object resulting from interactions between HSA and PAA. The evaluation of R g and V p shows that the complex is five times larger than HSA alone but the difficulty to evaluate correctly the Porod volume of PAA (in the complex) or its contribution in the measured I(0) does not allow to give directly the numbers of HSA and PAA forming the complex. The form of the autocorrelation function corresponding to the complex in Figure 3B shows a skewed distribution with a clear maximum at small distances corresponding to the radius of the cross-section R c (R c =39 Å from P(r) Figure 3B). This distribution is typical of elongated objects such as cylinders, rather than corresponding to amorphous aggregates [START_REF] Svergun | Small-Angle Scattering Studies of Biological Macromolecules in Solution[END_REF] . With these considerations, the length L of this cylinder can be calculated using the following relationship L=(12.R g 2 .R c 2 ) 0.5 . The diameter of the cylinder and the calculated length L and all evaluated parameters are summarized in the table attached to Figure 3B. The value of the calculated length (404 Å) is consistent with the maximal distance (390 Å) determined with P(r) calculation. One can assume that the complex of HSA/PAA-100 kDa exhibits a behavior in solution equivalent to the one of a solid cylinder measuring around 400 Å in length and 78 Å in diameter.

To validate this hypothesis predicting a formation of a defined object instead of amorphous aggregates, solutions of HSA-PAA were flowed in various mobile phases through a size exclusion chromatography column (Supplementary Information SI-1). The chromatographic profiles were then compared to that of the single HSA detected mainly at 7.74 min and as classically with a small amount of dimers at 6.9 min (Figure 4).

20

In fact, in a PBS mobile phase (without PAA) at pH 7.4, a peak at 7.7 min of retention time was obtained, corresponding to the molecular weight of HSA alone. This experiment shows that the complex can be dissociated in a dilute medium of PAA, illustrating the reversibility of the complex formation. On the other hand, if 50 µM of PAA is added to the mobile phase, the pH decreases to 5.2 and the elution profile is modified with a strong shift of the elution peak towards high molecular weights (Figure 4). This observation proves that the protein is no longer single in the solution but bound to the PAA chains.

The Gaussian shape of the peak eluted at 5.8 min corresponds to a profile of unique object in solution and not to a continuous distribution of mass such as in aggregates. 21

These experiments were run at PBS ionic strength (0.137 M) and at pH controlled by the PAA concentration, ranging from 5.2 to 7.4 as mentioned above (Figure 2). Since the pH remains higher than the HSA isoelectric point (4.7) and knowing that PAA exhibits a pKa of 4.5, we expect repulsive forces that keep them away from each other. It is because they both theoretically bear negative charges. Yet, the adsorption of HSA molecules on PAA chains is found to happen. Attractive interactions between a polyelectrolyte and a protein having the same net charge were reported by several authors [START_REF] Basak Kayitmazer | Protein -Polyelectrolyte Interactions[END_REF][START_REF] Xu | Interaction of Proteins with Polyelectrolytes: Comparison of Theory to Experiment[END_REF][START_REF] Seyrek | Ionic Strength Dependence of Protein-Polyelectrolyte Interactions[END_REF][START_REF] Xu | Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity[END_REF] . Since the proteins can carry both positive and negative charges, this binding found to occur in our study could be due to the attractive electrostatic potential between the positive charged patches on the surface of HSA and the negatively charged chains of PAA, which should be the operative force that overcome the repulsion forces. A detailed ITC study performed on the binding between HSA with negative overall charge and a negatively charged PAA [START_REF] Yu | Interaction of Human Serum Albumin with Short Polyelectrolytes: A Study by Calorimetry and Computer Simulations[END_REF] , shows the same behavior between the two solutes, where each HSA molecule has found to bind to one PAA chain causing an increase in the system entropy. It has been explained by the action of the HSA surface positive charges as a multivalent counter ion for the polyelectrolyte.

Other works doing experiments and dynamics computer simulations have addressed more deeply such phenomenon between proteins and polyelectrolytes [START_REF] Yigit | Like-Charged Protein-Polyelectrolyte Complexation Driven by Charge Patches[END_REF][START_REF] Yigit | Charged Patchy Particle Models in Explicit Salt: Ion Distributions, Electrostatic Potentials, and Effective Interactions[END_REF][START_REF] Yigit | Interaction of Charged Patchy Protein Models with Like-Charged Polyelectrolyte Brushes[END_REF] . They showed a large binding affinity between charged patchy proteins and like-charged polyelectrolyte brushes despite having the same net charge. It has been shown that the strongest attraction of typical proteins at the brush surface, is driven by multipolarity, Born energy (self-energy), and counterion-release contributions, which dominate locally over the monopolar and steric repulsions.

Ionic strength and pH effect on HSA/PAA complexation

For the study of ionic strength and pH effects, the [HSA] was set at 75 µM and the [PAA] at 50 µM (pH ~ 5 remains controlled by the polymer concentration), where the complexation was observed, while the ionic strength was varied from 0.137 M to 1 M by adding a needed amount of a 4 M NaCl solution.

Data obtained and reported below (Figure 5A) reveal a decrease in scattering intensity with the increase in ionic strength. Obviously curves are moving down especially at medium and small angles (0.005 to 0.1 Å -1 ), changing the slope, to collapse for an ionic strength of 0.75 M and 1 M with the theoretical sum (plotted as red line) of two 23 independent signals without interaction (signal of HSA 75 µM+ signal of PAA 50 µM) obtained at I=0.137 M. This finding confirms the previous assumption and bears out the fact that the binding of HSA-PAA is due to electrostatic interactions. Afterwards, salts concentration was set at 0.137 M and pH was changed from 8 to 4.6 using different buffer solutions. As shown in Figure 5B, the decrease in pH down to 5 promotes the formation of the complex whereas at pH > 5 the complex is dissociated giving the same SAXS signal as a mix of two independent contributions. These experiments show the key roles of the pH and the ionic strength on complex formation correlated to the charge distribution of amino acid forming the binding pocket of HSA and to the charge density of the PAA. The decrease in pH allows to reduce the repulsive forces until the initiation of the attractive potential between the positive charges carried on HSA and the negative polyelectrolyte chains. However, at pH lower than 5 (pH 4.6), the obtained SAXS signal (Figure 5B) is significantly different (slope P=1.5) from the one of a macromolecular complex (slope P=1.2). Since the drop of pH to 4.6 cannot sufficiently alter the net charge of the protein (isoelectric point 4.7) to create phase separation, one can consider that HSA starts being unfolded and form aggregates, because it undergoes form transition between pH 5.0 and 3.5 [START_REF] Dockal | Conformational Transitions of the Three Recombinant Domains of Human Serum Albumin Depending on PH[END_REF] .

In order to determine the best conditions of complex formation, SAXS measurements for a matrix of conditions were performed where the ionic strength was varied from 0 to 0.75 M for each pH 5, 6 and 7. As the major effects are observable at small angles due to the increase in size when the complex appears, we decided to probe the changes by evaluating the radius of gyration determined from Guinier law for each condition. Although the PAA and the complex have close R g values (R g 110 Å), the average value of R g was found around 50 Å when the HSA and the PAA do not interact, which is much lower than that of the complex. The data are plotted in Figure 6. One can note that the radius of 25 gyration of free PAA or of single HSA found to be around 110 Å and 30 Å respectively, do not depend on the pH. pH in the range 6-7 does not affect the radius of gyration of the mixture HSA+PAA, found to be approximately 50 Å for all salt concentrations investigated. This observation highlights the fact that HSA-PAA binding does not take place in these conditions. Since the radius of gyration obtained here by fits is an average of the R g of HSA and of the one of PAA, it makes sense to obtain an average proportional to the amount of each solute in

Soft Matter Accepted Manuscript

Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM.

View Article Online DOI: 10.1039/D0SM01221A

the mixture (protein/polymer ratio of 3/2). A different behavior is observed at pH 5. In fact, increasing the ionic strength from 0 to 0.75 M has led to decrease the R g from 110 Å, which is almost the polymer radius of gyration to 69.8 Å. This is due to the fact that beyond 0.15M of ionic strength the HSA-PAA complex particles begin to dissociate into free HSA molecules and free PAA chains. This dissociation is not complete at 0.75M and some complex particles are still in there since the obtained R g (69.8 Å) is still higher than the average value (50 Å) found when the HSA and PAA do not interact. This outcome sustains the previous results (Figure 5) suggesting that complete dissociation of the complex is taking place between 0.75M and 1M.

We conclude from this study that the complex is stable at pH = 5 and a salt concentration below 0.15 M. As previously, we tested the stability of the complex in HPLC with elution buffers at different pH and a constant salt concentration (I = 0.137 M) in the presence and in the absence of PAA. The results show a dissociation of the complex for pH higher than 5 (Supplementary Information SI-1). The absence of PAA in the medium also promotes the dissociation, suggesting a large value of the constant of dissociation, which requires the presence of PAA in the buffer to form the complex.

We henceforth know the conditions that lead to the assembly of HSA-PAA complexes in bulk. Therefore, this study constitutes a first knowledge brick to tune these reversible interactions between long PAA grafted on a membrane surface and HSA. Controlling the ionic strength and the pH should enable to regulate these interactions, promoting or preventing their formation. In fact, to enhance the platelets capture on PAA, low salt concentration and pH>5 should be favored to prevent the adsorption of HSA on PAA. Now that the conditions of complex formation are controlled, the stoichiometry of complexation can be investigated.

Determination of the complex stoichiometry (PAA-nHSA)

In order to determine the number of proteins that could bind to each polymer chain, SAXS experiments were performed for a concentration of PAA set at 50 µM a varying HSA concentration ranging between 75 and 600 µM. In other words, the [HSA]/[PAA] molar ratio ranged from 1.5 to 12. The experiments were performed at pH 5 and salts concentration I = 0.15 M to ensure complexation. For a HSA concentration lower than 225 µM, the SAXS curve increases without a significant change of slope at small and middle angles (0.005 to 0.1 Å -1 Figure 7). This shift of the curves corresponds to an increase in concentration of complex, considering that the contribution of PAA is negligible, compared to the one of the complex (cf. Figure 3A). Given the ratio of Porod volumes between HSA and the complex of R = 1:5, it is expected to have several HSA molecules per chain of PAA. Such behavior in solution 29 would suggest that the HSA binding mechanism to PAA is cooperative, without intermediates depending on the concentration of HSA.

When HSA concentration is close to 225 µM, the signal does not increase any more with the concentration. Beyond 225 µM, the signal changes at small angles, with a decrease in R g and I(0) values. At large angles, one observes a change in the SAXS profile. This change is due to an accumulation of free HSA in the medium and eventually the measured signal is a combination of the one of all PAA molecules involved in the complexes and of the un-bound HSA.

Table 1 summarizes the values of R g and Porod volumes for each ratio of HSA/PAA. From the values of R g reported in Table 1, it can be concluded that the attachment of proteins to the polymer chains has taken place on all the polyelectrolyte chains available for HSA/PAA molar ratios ranging between 3.6 and 4.5.

To support this hypothesis, the Porod volume was evaluated for the different HSA/PAA molar ratios. From SAXS experiments it is possible to obtain the Porod volume of an object from the Porod invariant Q as shown in the experimental section. (plots given in

𝑛 𝐻𝑆𝐴 + 1 𝑃𝐴𝐴 →1 𝑐𝑜𝑚𝑝𝑙𝑒𝑥(𝑃𝐴𝐴 -𝑛𝐻𝑆𝐴)
Where n is the number of HSA in each complex particle.

Thus, for a given initial [HSA] 0 /[PAA] 0 molar ratio, it is possible to define [HSA], [PAA] and [c] as the molar concentrations in the mixture during the complexation progress of HSA, PAA and the complex, respectively. It is also possible to determine the relative volume fraction of each solute i in the mixture as follows: (Supplementary Information SI-3)

𝜑 𝑖 = 𝑁 𝐴𝑣𝑜 .[𝑖].𝑉 𝑖 ϕ # (8)
is the volume fraction of the mixture of solutes in solution and it is expressed as:

𝜙 𝜙 = 𝑁 𝐴𝑣𝑜 ([𝑐]𝑉 𝑐 + [𝐻𝑆𝐴]𝑉 𝐻𝑆𝐴 + [𝑃𝐴𝐴]𝑉 𝑃𝐴𝐴 ) #(9)
Where V c , V HSA , V PAA are the volume of a molecule of the complex, HSA and PAA respectively. These volumes were estimated using Primus from SAXS spectra of single solutes and found to be: 500.10 3 , 100.10 3 and 50.10 3 Å 3 for the complex, HSA and PAA, respectively.

Accordingly, the Porod volume could be estimated using a theoretical model. According to the literature when the dispersion is made of n kinds of particles of volume V i and number N i , for a dilute dispersion S (0) =1, the average volume can be written as follows (O. Spalla eq. 54) [START_REF]Scattering Methods Applied to Soft Condensed Matter[END_REF] : Porod volume calculations whether from SAXS experiments or from the theoretical model were performed using a code written in Python (Supplementary Information SI-2).

𝑉 𝑀 = ∑ 𝑛 𝑖 𝑁 𝑖 𝑉
The results show that a complex model containing 4 HSA with 1 PAA gives a better fit than those run assuming 3 or 5 HSA per PAA (Figure 8), which corroborates the binding stoichiometry proposed previously. As a matter of fact, when the initial [HSA] 0 /[PAA] 0 ratio is below 4, the mixture is composed of PAA and of the HSA-PAA complex. The Porod volume is then rising up quickly when increasing HSA ratio, from the one of PAA to the one of the complex. For a [HSA] 0 /[PAA] 0 ratio of 4, one can consider that there is only the complex (PAA-4HSA) in the mixture and that the SAXS data gives access to the Porod volume of the complex. When the [HSA] 0 /[PAA] 0 is larger than 4, the mixture is composed of the complex plus some free HSA.

The complexation model (Supplementary Information SI-3) can then explain the decrease in the mean Porod volume due to the presence of non-associated HSA in addition to the HSA-PAA complex. Since the stoichiometry of the complex has been now determined, the amount of HSA that could be adsorbed on a PAA of the same size grafted onto a membrane interface can be estimated if the amount of the grafted polyelectrolyte is known.

HSA-PAA complex structure investigation

According to the analysis of the autocorrelation function P(r), we can fit the data at small and medium q (less than 0.2 Å -1 ) using a cylinder of axial length close to 400 Å and diameter equal to 78Å. Thus, the cylinder body was tested to fit the HSA-PAA complex experimental I(q) using Sasview (Supplementary Information SI-4). This model can provide information on the global shape of the complex, but provides no structural information on the internal organization of the complex. To propose a model that could be compatible with the SAXS data, we used an approach based on rigid bodies molecular modelling, completing the missing parts with dummies residues (BUNCH). The atomic structure of HSA can be used as a template for rigid bodies and the PAA polymer forms the missing part. As shown by Ballauff and all [START_REF] Yu | Interaction of Human Serum Albumin with Short Polyelectrolytes: A Study by Calorimetry and Computer Simulations[END_REF] , PAA can interact with HSA in a pocket charged positively, giving a starting point to lock a portion of PAA at the surface of HSA.

These portions of HSA-PAA complex are connected each other as a pearl necklace. The 35 rest of PAA is flexible and the conformation can be modified by the program. The program BUNCH [START_REF] Petoukhov | Global Rigid Body Modeling of Macromolecular Complexes against Small-Angle Scattering Data[END_REF] is working directly with residues of protein rather than with different types of polymer. However, the chemical structure of PAA is close to a peptides' residue and the PAA polymer can be transformed into poly-glycine chain. As both chains present a difference of mass (=1.26), the individual form factor of the dummies residues is increased in BUNCH program to compensate this default of mass. In total, the 100 kDa PAA chain can be modelled with a poly glycine chain containing 1389 residues. During the molecular modelling process against SAXS data, the position and the orientation of individual HSA is free and the conformation of polymer chain is modified in order to describe the space occupied by the PAA. The result obtained is an all atoms model compatible with SAXS data reflecting a possible organization of the complex in solution. Interestingly, the complex forms an elongated structure as a cylinder with a nonspecific orientation of the HSA. The absence of correlation peak in the data suggests that HSA does not present a periodic or partially aligned structure in the complex. This model proposes a globally flexible organization in alternative layers composed of HSA and unfold PAA without frozen structures(Figure 9). Many tests have been performed with different templates and all converge to the same organization with some variations in the position or orientation of HSA. However, the distance between two monomers of HSA always ranges between 80 and 100 Å. Besides, the distance between the extremities of the complex remains constant, found to be 414 Å (Figure 9). These results are consistent with the length of the complex previously evaluated whether from the P(r) or from the fit of the experimental I(q) of the complex performed using Sasview (Supplementary Information SI-4).

Conclusion

An assessment of the HSA protein interaction with PAA polyelectrolyte has been presented in this study combining SAXS and chromatography. The study of the HSA-PAA interactions revealed a complexation phenomenon of protein molecules with the polymer chains. Tests on the effect of the ionic strength showed that this HSA-PAA binding is chiefly an electrostatic interaction, where attractive and repulsive interactions are competing. Decreasing the pH to 5 permits to reduce the negative overall charge of the protein until the electrostatic attraction between the PAA chains and the positive patches on the protein dominates. Besides, this complexation was found to be reversible: the HSA-PAA complex tends to dissociate once put in a diluted medium (without PAA) or a pH greater than 5. That known, it is now possible to control the interactions between HSA and PAA at the membrane interface by changing the physicochemical conditions (pH, ionic strength).

An evaluation of the complex size suggests the attachment of several protein molecules to each polymer chain. The effect of the ratio of the protein concentration to the polymer concentration was also explored, and revealed that 4 HSA could be bound to each 100 kDa PAA chain. A complexation model was proposed, which allowed to describe the Porod volume of the mixture for the different HSA/PAA ratios. From this model, it could be possible to quantify the adsorbed amount of HSA on PAA of the same size grafted to a membrane surface.

A SAXS modelling study aimed at describing the complex structure has been also conducted. The cylinder body was found to fit well the experimental SAXS scattering intensities giving an idea of the global shape of the complex, but without providing any structural information. Finally, an all atom pearl necklace model was developed using BUNCH program to better describe the final structure of the complex.

It would be interesting in the future to evaluate the stoichiometry of complexation with different sizes of PAA. As it would be also interesting to assess the reproducibility of this

Figure 1 .

 1 Figure 1. Experimental SAXS scattering intensity I(q) of HSA 75 µM (blue line) compared to the SAXS

Figure 2 .

 2 Figure 2. Scattering intensities of HSA-PAA mixture and pH evolution for [HSA]=75 µM as a function of

Figure 3 .

 3 Figure 3. A) Scattering intensities of [HSA]=75 µM (blue curve), [PAA]= 60 µM (green curve), the mixture

Figure 4 .

 4 Figure 4. Comparison of chromatographic profiles for HSA 75 µM in PBS 1X mobile phase (red peak) and

Figure 5 .

 5 Figure 5. Scattering intensities of HSA-PAA mixture for [HSA]=75 µM and [PAA]=50 µM, (A) as a function

Figure 6 .

 6 Figure 6. Radius of gyration evolution as a function of both pH and ionic strength showing the threshold

Figure 7 .

 7 Figure 7. Scattering profiles of HSA-PAA mixture as a function of HSA amount ([HSA]=75 to 600 µM ;

Figure 8 .

 8 Figure 8. The Porod volume evolution as function of the initial [HSA/[PAA] ratio, using experimental SAXS

Figure 9 .

 9 Figure 9. Superimposition of SAXS data of the HSA-PAA complex and the SAXS data compatible model

Table 1 .

 1 The evolution of radius of gyration as well as the Porod volume for the HSA-PAA mixture as Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM.

	Page 29 of 50				Soft Matter
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								DOI: 10.1039/D0SM01221A
	function of the molar ratio ([PAA]=50 µM ; [HSA]=75-600 µM). [HSA]µM 75 75 150 180 nHSA/PAA Free HSA 1.5 3 3.6	225 4.5	300 6	450 9	600 12	Soft Matter Accepted Manuscript
	R g (±3Å)	30.4	108.3	107	110.1	108.6	90.6	81	74.1
	V p .10 3 (Å 3 )	99.1	428	502	500	491	400	305	239

  Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM.

	Page 31 of 50	Soft Matter
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		DOI: 10.1039/D0SM01221A
		Soft Matter Accepted Manuscript

  𝑉 𝑀 = 𝜑 𝑐 𝑉 𝑐 + 𝜑 𝐻𝑆𝐴 𝑉 𝐻𝑆𝐴 + 𝜑 𝑃𝐴𝐴 𝑉 𝑃𝐴𝐴 #(12)

					2 𝑖
		∑	𝑖 𝑁 𝑖 𝑉 𝑖 𝑛	#(10)
	The Porod volume of the mixture can be written then as a function of the relative volume
	fraction of solute, : 𝜑 𝑖
	𝑉 𝑀 =	∑ ∑ 𝑛 𝑖 𝜑 𝑖 𝑉 𝑖 𝑛 𝑖 𝜑 𝑖	=	𝑛 𝑖 ∑	𝜑 𝑖 𝑉 𝑖 #(11)

Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM. View Article Online DOI: 10.1039/D0SM01221A

Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM. View Article DOI: 10.1039/D0SM01221A

Published on 21 September 2020. Downloaded by Karolinska Institutet University Library on 9/21/2020 8:09:57 PM.View Article Online

complexation between HSA and a PAA grafted into a membrane surface for the different physiochemical conditions discussed in this article. The authors would like to acknowledge the Agence Nationale de la Recherche (MOST-ANR International Program: ANR 17-CE08-0041) for financial support, the FERMaT Federation FR3089 for providing Small Angle X-Ray Scattering laboratory facility. This work benefited from the use of the SasView application (https://www.sasview.org), originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union's Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000.

Supplementary Information SI-2)

The calculated Porod volumes (reported in Table 1) show the same behavior as that of the radius of gyration. For a HSA/PAA molar ratio of 3.6, the Porod volume is found to be around 500.10 3 Å 3 which represents the complex Porod volume (Figure 3). Then, we notice that raising the HSA concentration reduces the volume to 239.10 3 Å 3 for a molar ratio of 12. That makes sense because after saturation of all PAA chains, the excess of HSA contributes to the average of the Porod volume.

AUTHOR INFORMATION Corresponding Authors

*E-mail: merzougui@chimie.ups-tlse.fr *E-mail: roblin@chimie.ups-tlse.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest. There are no conflicts of interest to declare.

ACKNOWLEDGMENT


Dr Manuel Rodrigues
email: manuel.rodrigues@curie.fr

Khadija Ait Rais

Flore Salviat

M.D Nathalie Algret

Fatoumata Simaga

Raymond Barnhill

M.D Sophie Gardrat

M.D Vincent Servois

M.D Pascale Mariani

M.D Sophie Piperno-Neumann

M.D. Ph.D Khadija Ait-Rais

M.D Sergio Roman-Roman

Pharm.D. Ph.D Olivier Delattre

M.D. Ph.D Nathalie Cassoux

M.D. Ph.D Alexia Savignoni

M.D. Ph.D Marc-Henri Stern

M.D. Ph.D Gaëlle Pierron

Association of Partial Chromosome 3 Deletion in Uveal Melanomas With Metastasis-Free Survival

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

Setting

Monocentric study in a referral center.

Participants

Patients presenting with UMs with and without partial loss of chromosome 3.

Main Outcomes and Measures

Metastasis-free survival and overall survival at 60 months.

Results

Of the 1,088 consecutive comparative genomic hybridization arrays that were performed, 43 UMs (4%) carried partial deletions of chromosome 3. Median follow-up was 66 months. Metastasis-free survival at 60 months was 34% (95% confidence interval [CI], 15.8 to 71.4) for UMs carrying a deletion of the BAP1 (BRCA1 associated protein-1) locus (BAP1del; 24 tumors) and 81% (95% CI, 64.8 to 100) for UMs without the loss of the BAP1 locus (BAP1 normal; BAP1nl; 19 tumors; log-rank p-value = .001). Overall survival at 60 months was 65% (95% CI, 43.5 to 95.8) versus 84% (95% CI, 69.0 to 100) in the BAP1del and the BAP1nl groups, respectively (logrank p-value < .001). In these 43 cases, metastasis-free survival at 60 months was 100% for UMs without loss of the BAP1 locus or 8q gain, 70% (95% CI, 50.5 to 96.9) for UMs carrying one of these alterations and 13% for those carrying both (95% CI, 2.1 to 73.7; log-rank p-value < .001). Similarly, overall survival at 60 months was 100%, 81% (95% CI, 63.3 to 100) and 47% (95% CI, 23.3 to 93.6) in these three groups, respectively (log-rank p-value < .001).

Conclusions and Relevance

These findings suggest that partial deletion of chromosome 3 encompassing the BAP1 locus is associated with poor prognosis. A cytogenetic classification of UMs could be proposed based on the status of the BAP1 locus instead of chromosome 3, locus, while also taking chromosome 8q into account.

Introduction

Uveal melanoma (UM) is the most common primary malignant ocular tumor in adults of European ancestry 1 . Despite efficient treatment, up to 50% of the patients will eventually develop metastases [START_REF] Desjardins | Prognostic factors for malignant uveal melanoma. Retrospective study on 2,241 patients and recent contribution of monosomy-3 research[END_REF][START_REF] Singh | Uveal melanoma: trends in incidence, treatment, and survival[END_REF][START_REF] Kujala | Very long-term prognosis of patients with malignant uveal melanoma[END_REF] . Reliable prognostic assessment allows a closer monitoring of high-risk patients. Pathological prognostic factors include large tumor basal diameter, thickness, ciliary body involvement, extraocular extension, epithelioid cell histology, high mitotic rate and lymphocytic infiltration [START_REF] Brierley | The TNM Classification of Malignant Tumours[END_REF] . The gene expression profile DecisionDx-UM (GEP; Castle Biosciences, Friendswood, TX), based on the expression level of 12 genes, is frequently used in North America to complete the prognostic assessment [START_REF] Field | PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research[END_REF][START_REF] Onken | Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death[END_REF] .

In the early 1990s, recurrent cytogenetic aberrations including monosomy 3 (M3), gain of 6p and 8q were identified in UM samples [START_REF] Prescher | Nonrandom chromosomal abnormalities in primary uveal melanoma[END_REF] . In 1996, M3 was empirically shown to be a robust prognostic factor [START_REF] Prescher | Prognostic implications of monosomy 3 in uveal melanoma[END_REF] . Since then, genomic arrays have become routine tools to refine pathological prognosis along with the GEP. We previously refined the prognostic value of M3 and gain of 8q by defining three groups: (i) high-risk patients whose tumors present a M3 and an 8q gain with a 2-year metastasis-free interval (2y-MFI) of 37%; (ii) intermediate-risk with either a M3 or an 8q gain (2y-MFI: ~85%) and (iii) low-risk with neither M3 nor 8q gain (2y-MFI: ~100%) [START_REF] Cassoux | Genome-wide profiling is a clinically relevant and affordable prognostic test in posterior uveal melanoma[END_REF] .

The most common hypothesis to explain the poor prognosis of M3 tumors is the presence of one or more tumor suppressor genes (TSG) on chromosome 3. BAP1 (BRCA1 associated protein-1), a TSG located on the 3p21.1 cytoband, is now established as a main actor of UM malignant transformation as it is frequently mutated in M3 tumors and germline mutations are associated with UM predisposition [START_REF] Abdel-Rahman | Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers[END_REF][START_REF] Robertson | Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma[END_REF][START_REF] Field | Punctuated evolution of canonical genomic aberrations in uveal melanoma[END_REF][START_REF] Rai | Germline BAP1 alterations in familial uveal melanoma[END_REF][START_REF] Harbour | Frequent mutation of BAP1 in metastasizing uveal melanomas[END_REF][START_REF] Wiesner | Germline mutations in BAP1 predispose to melanocytic tumors[END_REF] . However, all or most BAP1-mutated UMs intriguingly present a M3 (or a loss of heterozygosity of the whole chromosome 3 due an isodisomy) suggesting that the role of chromosome 3 loss in UM tumorigenesis may not be restricted to BAP1 inactivation. Therefore, prognostication of UM samples with partial deletions of chromosome 3, as sometimes observed in our daily practice and by other authors, is problematic [START_REF] Abdel-Rahman | Frequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma[END_REF] . The goals of the present study were to explore these UMs with partial deletions of chromosome 3, as assessed by comparative genomic hybridization (array-CGH), in order to assess their prognosis and to determine the minimal region of deletion associated with poor prognosis.

Materials and methods

Patients

This study was approved by our institutional ethics committee. Written informed consent for the use of tissues and data for research was signed by each patient. The study complied with the principles of the Declaration of Helsinki. All patients were referred to our institution and followed up by our physicians. Clinical diagnosis of uveal melanoma was based on the presence of typical clinical findings as previously described [START_REF] Cassoux | Genome-wide profiling is a clinically relevant and affordable prognostic test in posterior uveal melanoma[END_REF] . Local treatment consisted of proton beam radiotherapy, iodine 125 brachytherapy or enucleation, depending on the size and location of tumors. Tumor samples were obtained by enucleation, endoresection or fine-needle aspiration at the time of clip or plaque positioning. Liver ultrasound, liver magnetic resonance imaging or body computed tomography were performed at diagnosis and every 6 months afterwards. Diagnosis of metastasis was systematically confirmed by a biopsy.

Genomic analysis

Tumor DNA was extracted and processed as previously described [START_REF] Cassoux | Genome-wide profiling is a clinically relevant and affordable prognostic test in posterior uveal melanoma[END_REF] . Array-CGH was performed on three different platforms according to the period when the test was performed: bacterial artificial chromosome arrays as previously described [START_REF] Trolet | Genomic profiling and identification of high-risk uveal melanoma by array CGH analysis of primary tumors and liver metastases[END_REF] , NimbleGen 4×72 K arrays (Roche NimbleGen, Madison, Wisconsin, USA) and Agilent 180K CGH/LOH custom chip (Santa Clara, California, USA). Array-CGH were interpreted by three of the authors (MR, KAR, GP). Partial deletion of chromosome 3 was defined as the loss of at least one region of chromosome 3, but not the totality, whatever its size and location. Genomic positions in this article are defined in hg18 human genome assembly.

Statistical analysis

Clinical, pathological and genomic data at diagnosis and follow-up events (local and distant recurrences, second cancers, death from UM or from any other cause) were collected. The French Death Registry was consulted for patients lost to follow-up.

The metastasis-free survival (MFS) at 60 months was defined as the proportion of patients alive and free of metastasis at 60 months of follow-up after local treatment of primary UM. The overall survival (OS) at 60 months was defined as the proportion of patients alive at 60 months of follow-up after local treatment of primary UM, whatever the cause of death. Survival distributions were estimated by the Kaplan-Meier method and compared using the log-rank test. All tests were bilateral and performed with a significant level of 5%. In order to identify variables associated with MFS, a Cox regression analysis of candidate prognostic factors was performed using a forward stepwise selection procedure. The added value of each variable to the Cox model was determined using a likelihood ratio test with a significant level of 5%.

Statistical analysis was performed using R software V3.3 (http://www.r-project.org/).

We then hypothesized that BAP1 loss was the main driver of poor prognosis in M3.

To explore this hypothesis, we compared tumors with a chromosome 3 partial deletion encompassing the BAP1 locus (24 tumors; BAP1del) and tumors with a chromosome 3 partial deletion not encompassing the BAP1 locus (19 tumors; BAP1nl). Tumors carrying a loss of the BAP1 locus frequently showed large losses of the short arm of chromosome 3 (Figure 2). MFS at 60 months was 81% (95% CI, 64.8 to 100) for the BAP1nl genomic group and 34% (95% CI, 15.8 to 71.4) for the BAP1del group (Figure 3; p=.001). OS at 60 months was 84% (95% CI, 69.0 to 100) for the BAP1nl genomic group and 65% (95% CI, 43.5 to 95.8) for the BAP1del group (p<.001). The only variables associated with MFS in univariate analysis were loss of the BAP1 locus and gain of 8q. These two variables independently contributed to MFS in multivariate analysis (Table 1).

We defined four groups depending on the BAP1 locus (lost/not lost) and 8q (gained/not gained) statuses. Prognoses of the BAP1 locus lost/8q normal and BAP1 locus not lost/8q gained were similar so we merged these two groups, as in our previous classification (eFigure 2 in the supplement) [START_REF] Cassoux | Genome-wide profiling is a clinically relevant and affordable prognostic test in posterior uveal melanoma[END_REF] . By analogy with our previous work, we defined three prognosis groups as follows: (i) a group at low risk of metastasis without loss of the BAP1 locus or 8q gain (9 cases), (ii) an intermediate risk group with tumors carrying either loss of the BAP1 locus (7 cases) or 8q gain (15 cases) and (iii) a high risk group with loss of the BAP1 locus and 8q gain (12 cases).

MFS at 60 months were 100%, 70% (95% CI, 50.5 to 96.9) and 13% (95% CI, 2.1 to 73.7) for the low-, intermediate-and high-risk groups, respectively (Figure 4; p<.001).

OS at 60 months were 100%, 81% (95% CI, 63.3 to 100) and 47% (95% CI, 23.3 to 93.6) for the low-, intermediate-and high-risk groups, respectively (p<.001).

Discussion

In this work, we explored a relatively large series of UMs with partial deletion of chromosome 3 and showed that loss of the BAP1 locus is likely to explain the poor prognosis of M3 UM. This result was obtained by two different approaches investigating indirectly the prognostic value of the most frequently deleted regions of chromosome 3 and then directly assessing the prognostic value of the loss of the BAP1 locus in this series. The first consequence is to provide a potentially more accurate estimation of the prognosis of UMs presenting a partial deletion of chromosome 3. Our classification suggested efficiency in predicting metastatic outcome, identifying a group with a very good MFS with no recurrence and a group with a high risk of 92% of recurrences with a median follow-up of more than 5 years.

Survival rates were close to what we observed in a previous series of UMs presenting either a M3 or a disomy 3, associated or not with 8q gain [START_REF] Cassoux | Genome-wide profiling is a clinically relevant and affordable prognostic test in posterior uveal melanoma[END_REF] . This hypothesis has yet to be verified in subsequent studies because direct comparison could not be done here.

Other teams are using different genomic technologies to assess UM prognosis.

Fluorescence in situ hybridization (FISH) is widely used but it may miss the loss of the BAP1 locus if the probe is not centered on this gene, as observed in several publications [START_REF] Desjardins | Prognostic factors for malignant uveal melanoma. Retrospective study on 2,241 patients and recent contribution of monosomy-3 research[END_REF][START_REF] Mensink | Chromosomal aberrations in iris melanomas[END_REF][START_REF] Worley | Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research[END_REF][START_REF] Van Gils | Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis[END_REF][START_REF] Singh | Chromosome 3 status in uveal melanoma: a comparison of fluorescence in situ hybridization and single-nucleotide polymorphism array[END_REF] . Furthermore, FISH is often performed without chromosome 8q assessment leading to suboptimal prognosis estimation. Multiplex ligation-dependent probe amplification (MLPA) assay covering the BAP1 locus is a good alternative to characterize recurrent genomic imbalances in UM but MLPA, as well as FISH and array-CGH, only evaluate copy number and, consequently does not identify isodisomic cases [START_REF] Damato | Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death[END_REF][START_REF] Larsen | MicroRNA expression analysis and Multiplex ligation-dependent probe amplification in metastatic and non-metastatic uveal melanoma[END_REF] . GEP is a transcriptomic prognosis assay that is widely used in United States [START_REF] Onken | Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death[END_REF] . This assay distinguishes two subsets of UMs either at low or high risk of metastasis by assessing the expression of 12 genes, including four that are located on the short arm of chromosome 3 (EIF1B, LMCD1, ROBO1, SATB1) and one on the 3q (FXR1). Underexpression of these genes, possibly due to M3, is associated with poor prognosis. A more accurate prediction by GEP is possible by adding the expression of PRAME, a gene located on an instable region of chromosome 22 exposed to duplication, which was correlated to the 8q status in the pivotal paper [START_REF] Field | PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research[END_REF] . To our knowledge, GEP has never been specifically tested in a large series of UMs with partial chromosome 3 deletions. Furthermore, GEP has never been compared to the combined M3/8q signature in a large cohort, impeding any conclusion on the superiority of one modality on the other. BAP1 immunohistochemistry is an alternative way to assess the prognosis of UMs [START_REF] Kalirai | Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing[END_REF][START_REF] Van Essen | Prognostic parameters in uveal melanoma and their association with BAP1 expression[END_REF] .

However, immunohistochemistry for BAP1 does not correlate in all cases to the BAP1 mutational status in UM, and is therefore not a perfect surrogate [START_REF] Kalirai | Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing[END_REF] .

In the present series, partial deletions of chromosome 3 were found in 4% of cases, which is comparable with some previous series [START_REF] Thomas | Prognostic significance of chromosome 3 alterations determined by microsatellite analysis in uveal melanoma: a long-term follow-up study[END_REF][START_REF] Tschentscher | Partial deletions of the long and short arm of chromosome 3 point to two tumor suppressor genes in uveal melanoma[END_REF][START_REF] Cross | The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro[END_REF] but lower than others [START_REF] Abdel-Rahman | Frequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma[END_REF][START_REF] Shields | Prognosis of uveal melanoma in 500 cases using genetic testing of fine-needle aspiration biopsy specimens[END_REF][START_REF] Damato | Genotypic profiling of 452 choroidal melanomas with multiplex ligation-dependent probe amplification[END_REF] .

Recruitment bias may explain part of this discrepancy but it is most probably explained by the variety of technologies, as well as the different classifications that were used. Comparison of all these studies is therefore limited. Similarly, the prognosis of these tumors was not clear as a discrepancy was observed with some series associating partial loss with good prognosis [START_REF] Abdel-Rahman | Frequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma[END_REF][START_REF] Thomas | Prognostic significance of chromosome 3 alterations determined by microsatellite analysis in uveal melanoma: a long-term follow-up study[END_REF][START_REF] Shields | Prognosis of uveal melanoma in 500 cases using genetic testing of fine-needle aspiration biopsy specimens[END_REF] while others associated it with intermediate or poor prognosis [START_REF] Damato | Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death[END_REF][START_REF] Damato | Genotypic profiling of 452 choroidal melanomas with multiplex ligation-dependent probe amplification[END_REF][START_REF] Ewens | Genomic profile of 320 uveal melanoma cases: chromosome 8p-loss and metastatic outcome[END_REF] . These differences may be explained by the absence of distinction depending on the loss of the BAP1 locus compared to other losses.

One explanation for the low MFS associated with the loss of this locus may be that the loss of one BAP1 allele contributes to the inactivation of this gene and subsequent aggressiveness of the tumor. However, the minimal region of deletion we found associated with the lowest MFS in our series (3p22.1-p14.2) includes 291 genes. Even though this region encompasses BAP1, it cannot be excluded that other important genes are present there and that haploinsufficiency of these genes affects tumorigenesis. The two alleles of a TSG are commonly inactivated in the two-hit model by a combination of different mechanisms, including total or partial loss of a chromosome, deleterious point mutations, short insertions/deletions, large-scale insertions/deletions and promoter methylations [START_REF] Knudson | Two genetic hits (more or less) to cancer[END_REF] . It is highly intriguing that, BAP1 inactivation is so frequently associated with monosomy 3 in UM, contrary to renal clear-cell carcinomas and mesotheliomas, which rather carry losses of the short arm of chromosome 3 only or deleterious mutations of both alleles [START_REF] Wiesner | Germline mutations in BAP1 predispose to melanocytic tumors[END_REF] . Furthermore, haploinsufficiency of other genes on chromosome 3, possibly on its long arm, may play a role on UM tumorigenesis. This hypothesis may be of particular interest and should be put in perspective with the recent discovery of MBD4 (3q21.3) recurrent, inactivating mutations in UM [START_REF] Rodrigues | Outlier response to anti-PD1 in uveal melanoma reveals germline MBD4 mutations in hypermutated tumors[END_REF][START_REF] Rodrigues | Evolutionary routes in metastatic uveal melanomas depend on MBD4 alterations[END_REF][START_REF] Johansson | Prolonged stable disease in a uveal melanoma patient with germline MBD4 nonsense mutation treated with pembrolizumab and ipilimumab[END_REF] .

There is, for now, no standard treatment in the metastatic setting, but new drugs are being developed in UM [START_REF] Carvajal | Metastatic disease from uveal melanoma: treatment options and future prospects[END_REF] . When an efficient treatment will be available, the following step will consist in testing this treatment in the adjuvant setting in high-risk patients [START_REF] Piperno-Neumann | A randomized multicenter phase 3 trial of adjuvant fotemustine versus surveillance in high risk uveal melanoma (UM) patients (FOTEADJ)[END_REF] .

Accurate prognosis evaluation is essential for such trials and assays able to assess the status of the BAP1 locus and 8q status may then be required. Next-generation sequencing appears to be the best option in the near future as it not only assesses copy number, heterozygosity and mutational statuses of UMs at low cost and with a lower amount of DNA, but also allow to follow circulating tumor DNA [START_REF] Smit | Combined mutation and copynumber variation detection by targeted next-generation sequencing in uveal melanoma[END_REF][START_REF] Afshar | Next-Generation Sequencing of Uveal Melanoma for Detection of Genetic Alterations Predicting Metastasis[END_REF][START_REF] Matet | Comparative Cytogenetic Abnormalities in Paired Choroidal Melanoma Samples Obtained Before and After Proton Beam Irradiation by Transscleral Fine-Needle Aspiration Biopsy and Endoresection[END_REF] . Moving towards the implementation of such technologies in our daily practice will allow ocular oncology to enter the modern age of precision medicine while reducing costs and refining UM prognosis.

Limitations

The conclusions of this work are limited by its retrospective nature, but prospective series are unrealistic given the rarity of such tumors. Instead, the present work provides evidence to refine the current UM genomic classification, which may help ophthalmologists to better predict the metastatic evolution of their patients. Before generalization, other series from different centers are required. Furthermore, one could argue that our series, composed of large tumors (median diameter of 16mm and median thickness of 10mm) is not reflecting the overall population of UM patients, particularly as larger UMs are known to host a greater frequency of genomic alterations, including 8q gains [START_REF] Rodrigues | Evolutionary routes in metastatic uveal melanomas depend on MBD4 alterations[END_REF][START_REF] Shain | The genetic evolution of metastatic uveal melanoma[END_REF] . Other centers have reported genomic studies on biopsies of smaller UMs [START_REF] Angi | Prognostic biopsy of choroidal melanoma: an optimised surgical and laboratory approach[END_REF] . However, this procedure is not consensual and must not be undertaken in inexperienced ocular oncology centers because of potential surgical complications. Multicenter collaborative studies of small UM genomics are required to address the question of partial chromosome 3 loss frequency at this stage of primary UM development. Another limitation of this study is that the array-CGH technology is not adapted to detect chromosome 3 isodisomy, an infrequent alteration in UM, probably associated with poor prognosis. SNP-array can resolve this issue but, in the future, next-generation sequencing will probably be the privileged technology to circumvent this issue. More importantly, although the BAP1 locus hypothesis is a logical hypothesis, we cannot definitely affirm that BAP1 is indeed the target of such deletions. Chromosome 3 is dense in cancer genes and the BAP1 region, for instance, encompasses the tumor-suppressor gene PBRM1, which was recently found mutated in rare UMs. To confirm the BAP1 locus hypothesis and the classification, validation series are required, ideally together with further work sequencing BAP1 to confirm the presence of a second hit.

Conclusions

These findings suggest that partial deletion of chromosome 3 encompassing the BAP1 locus is associated with poor prognosis. Consequently, a new cytogenetic classification of UMs is proposed, based on the status of the BAP1 locus instead of chromosome 3. The very frequent loss of the whole chromosome 3 in UMs raises the possibility of other genes associated with UM tumorigenesis on this chromosome. Frequencies of losses at a given position are shown at the bottom. Light gray: nonmetastatic cases (Met-; n=30); dark gray: metastatic cases (Met+; n=13).

Figure 2 .

 2 Figure 2. Copy number profiles in BAP1del cases versus BAP1nl. Frequencies

Figure 3 .

 3 Figure 3. Metastasis-free and overall survivals according to the loss of the

Figure 4 .Figure 1 .Figure 2 .

 412 Figure 4. Metastasis-free and overall survivals according to the three different

Figure 3 .

 3 Figure 3. Rodrigues et al.

Figure 4 .

 4 Figure 4. Rodrigues et al.

Tables 489 Table 1 . Univariate and multivariate analyses of risk factors for metastasis.

 4891

	490
	491	HR (95%CI): hazard ratio (95% confidence interval); mm: millimeters; n: number of
	492	cases.
		Univariate analysis
				n	HR (95%CI)	p-value
		Age	< 60 years-old ≥ 60 years-old	23 1 20 0.49 (0.17-1.4)	.17
		Gender	male female	21 1 22 0.46 (0.16-1.33)	.14
		Diameter	≤ 15 mm > 15 mm	17 1 26 1.72 (0.6-4.95)	.31
		Thickness	≤ 10 mm > 10 mm	22 1 21 1.49 (0.55-4)	.43
		Tumor location	on the equator	28 1	.07
			anterior to the equator 4	2.55 (0.69-9.36)
			posterior to the equator 10 0.36 (0.08-1.62)
		Retinal detachment No Yes	4 39 0.32 (0.09-1.11) 1	.06
		Histology	spindle cells epithelioid/mixed	10 1 13 1 (0.28-3.55)	1.00
		BAP1 locus deletion	No	24 1	.001
			Yes	19 5.91 (1.89-18.54)
		8q gain	No Yes	16 1 27 6.02 (1.36-26.61)	.007
		Multivariate analysis
				n	HR (95%CI)	p-value
		BAP1 locus deletion	No	24	.001
			Yes	19 6.65 (2.09 ; 21.18)
		8q gain	No Yes	16 27 6.88 (1.53 ; 30.86)	.01

Copy number profiles in metastatic versus non-metastatic cases.

Acknowledgments:

We thank the patients and their family members. We thank Sylvain Dureau for his help with review.

Funding/Support: This research was funded by the INCa/ITMO/AVIESAN PhD fellowship program "Formation à la recherche translationelle" (M. Rodrigues), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Ligue Nationale Contre le Cancer (Labellisation) and the Institut Curie.

Role of the Funder/Sponsor: None of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.

Results

We prospectively re-analyzed the array-CGH profiles in 1,088 UMs which had been processed between May 2006 and July 2015, and detected 43 cases (4.0%) harboring a partial deletion of chromosome 3 (eTable 1 in the supplement). Median follow-up in these 43 cases was 66 months (range: We first explored survival data in an unsupervised manner and observed three recurrently lost regions in at least eight metastatic samples: (i) from 3pter to p22.2, (ii) from 3p22.1 to p14.2 and (iii) from 3q13.2 to q24 (Figure 1). Of these, two regions were more frequently lost in metastatic cases than in non-metastatic ones: the 3pter-p22.2 region (8/13 versus 6/30 cases, respectively; p=.013; odds ratio [OR]=6.1; 95% CI, 1.2 to 34.1) and the 3p22.1-p14.2 region, which encompasses BAP1 (10/13 versus 9/30 cases, respectively; p=.007; OR=7.4; 95% CI, 1.5 to 51.8). These two regions were close and highly correlated between each other, as 8 out of 10 metastatic cases presenting a 3p22.1-p14.2 loss also presented a 3pter-p22.2 loss. The 3p22.1-p14.2 region carries 290 other genes beside BAP1, but no recurrent mutations of these 290 genes were found in public and in-house databases [START_REF] Robertson | Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma[END_REF][START_REF] Furney | SF3B1 mutations are associated with alternative splicing in uveal melanoma[END_REF][START_REF] Johansson | Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4[END_REF] .

Author Contributions: M. Rodrigues had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Conflicts of Interest:

The authors declare no conflict of interest.

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