Laurence Galoisy
email: laurence.galoisy@sorbonne-universite.fr

Georges Calas

Role of alkali field strength on the speciation of Ni 2+ in alkali borate glasses: comparison with crystalline Ni-borates

Keywords: borate glasses, glass structure, UV-visible spectra, XANES spectra, crystalline borates, Mg coordination

This work presents a comparative spectroscopic study of Ni speciation in binary alkali borate glasses, with up to 35 mol% alkali oxides, using optical absorption spectroscopy and Ni K-edge X-ray Absorption Near Edge Structure (XANES)-and taking advantage of comparison with previous structural information obtained with Ni K-edge Extended X-ray Absorption Spectroscopy (EXAFS) on the same samples. Nickel-bearing K, Na or Li borate glasses exhibit an exceptional diversity of coloration (from green to yellow, brown, and purple). This is due to a progressive coordination change of Ni 2+ with increasing alkali content, from 6-, to 5-and 4-Nicoordination, each coordination state having a peculiar structural significance. [6] Ni 2+ is predominant in low alkali borate glasses, where Ni octahedra cluster in ordered Ni-rich domains. [4] Ni 2+ is formed at low alkali cation field strength: it predominates in high potassic glasses but it is only a minor coordination state in Na-borate glasses and it is absent in Li-borate glasses, in which, by contrast, [5] Ni 2+ prevails. For these three coordination states, the comparison with the structure of crystalline borates suggests a diversity of linkages between Ni 2+ and the borate polymeric network. Among the alkali borate glasses investigated, each Ni-coordination state shows similar spectroscopic properties. This implies that the modification of Ni speciation with glass composition arises only from a variation of the relative contribution of 6-, 5-and 4-coordinated Ni 2+ without significant modification of the spectroscopic parameters characteristic of each site. The coordination number of Ni 2+ is correlated with the field strength of the modifier cations present in the glass: higher the field strength of the modifier cations, higher the nickel coordination number. High-K borate glasses favor the formation of [4] Ni 2+ , as

Introduction

Alkali borate glasses exhibit an original structure based on the complex interplay between three-and fourcoordinated boron (referred to as [3] B and [4] B, respectively). Alkali elements are able to charge compensate the charge deficit of [4] B or to depolymerize the borate network by generating non-bridging oxygen atoms. By contrast to most glasses, which are considered to have only a short range ordered structure, these glasses show a crystal-like medium range order in alkali-poor compositions, due to the presence of rigid super-structural units based on an interlinkage between [3] B and [4] B sites [START_REF] Wright | My borate life: an enigmatic journey[END_REF]. The coordination change from [3] B to [4] B with increasing alkali content modifies the structural properties of the other glass components, as alkali ions associated to B turn from a modifying to a charge-compensating role. As a consequence, alkali borate glasses exhibit important modifications of their structure, properties and dynamics as a function of the cation content and field strength.

It has long been observed that the presence of transition elements in glasses causes spectacular color changes with or without any modification of their oxidation state [START_REF] Weyl | Coloured Glasses[END_REF][START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]: in alkali borate glasses, transition elements are at the origin of one of the most noticeable chemical dependence of the color of oxide glasses. For instance, Ni 2+ in potassium borate glasses gives rise to colors that vary from green to yellow to brown and to purple with increasing potassium content. This color change corresponds to a decrease of the coordination number of Ni 2+ from 6 to 5 and 4 with increasing alkali levels. A low field strength of alkalis plays a major role in enhancing the conversion to [4] Ni 2+ as also observed in silicate glasses [START_REF] Galoisy | Overview of the environment of Ni in oxide glasses in relation to the glass colouration[END_REF]. A similar behavior has been shown in cobalt-bearing alkali borate glasses [START_REF] Hunault | Effect of cation field strength on Co2+ speciation in alkali-borate glasses[END_REF], with a similar decrease in the coordination number of Co 2+ from 6 to 5 and 4 with increasing alkali levels and a low cation field strength favoring [4] Co 2+ . Similarly, the coordination number of uranyl groups in Li borate glasses decreases in 30Li2O-70B2O3 borate glasses relative to 10Li2O-90B2O3 glasses, with 5 and 6 equatorial oxygen ligands, respectively [START_REF] Hunault | Speciation change of uranyl in lithium borate glasses[END_REF].

Alkali borate glasses show a more complex evolution of Ni 2+ coordination with glass composition than alkali silicate glasses [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF]: at low alkali content, borate glasses are the only Ni-bearing oxide glasses to exhibit a green color, indicative of the octahedral coordination of Ni 2+ . Extended X-ray Absorption Spectroscopy (EXAFS) demonstrated that these octahedral sites occur in ordered defective domains [START_REF] Cormier | Evidence of Ni-containing ordered domains in low alkali borate glasses[END_REF][START_REF] Galoisy | Environment of Ni, Co and Zn in low alkali borate glasses: information from EXAFS and XANES spectra[END_REF]. With increasing alkali content, these nanodomains progressively disappear, in relation with a coordination change of [6] Ni 2+ to [5] Ni 2+ and [4] Ni 2+ [START_REF] Galoisy | Chemical stability of Ni-enriched nanodomains in alkali borate glasses[END_REF]. Until now, most studies have discussed Ni 2+ -coordination in alkali borate glasses on the only basis of Ni 2+ crystal field spectra [START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF][START_REF] Terczyńska-Madej | Coordination and valence state of transition metal ions in alkali-borate glasses[END_REF]. The present study provides a new interpretation of optical absorption spectroscopic properties of Ni 2+ in alkali (Li, Na, K) borate glasses, with an alkali content ranging from 10 to 35 mol%. These data are compared to Ni-bearing crystalline borates. Complementary structural information is obtained from Ni K-edge X-ray absorption near edge structure (XANES) spectroscopy. We also use previous EXAFS data obtained on the same glasses [START_REF] Galoisy | Chemical stability of Ni-enriched nanodomains in alkali borate glasses[END_REF]. We confirm the presence of [5] Ni 2+ as in most oxide glasses [START_REF] Galoisy | Overview of the environment of Ni in oxide glasses in relation to the glass colouration[END_REF], in addition to [4] -and [6] Ni 2+ , the proportion between these different coordination states being dependent on glass composition. The use of these complementary techniques allows reject some exotic coordination states invoked in the past, such as square-planar, 8-or 12-fold coordinated Ni 2+ . The relative proportion of [4] -, [5] -and [6] Ni 2+ is composition dependent and is responsible for the coloration of Ni 2+ -bearing alkali borate glasses.

Experimental methods

Glass synthesis

Li, Na and K borate glasses have been prepared by melting appropriate amounts of dried high purity grade K2CO3, Na2CO3, Li2CO3 and H3BO3. Starting reagents were Li2CO3 (Merck, 99%), Na2CO3 (Merck, 99.5%), K2CO3 (Merck 99%) and B2O3 (Aldrich, 99.98%). Nickel was introduced as NiO (Merck, 99.9%). After decarbonation at 750°C, the compositions were melted in a Pt crucible at 1100°C during 4 hours to limit alkali losses. The glasses were quenched by pouring onto a metallic plate to obtain high quench speed giving plates with parallel faces of about 0.5 mm thickness and a few cm diameter. They were not annealed. Low alkali borate glasses were kept in vaseline oil to prevent glass hydration during exposure to air, particularly with less than 20 mol% alkali oxides. To minimize the effect due to the hydroscopic nature of these samples, the optical spectra data were recorded as quickly as possible after synthesis. Special care was given to the synthesis of samples low in network modifiers, which may show the presence of some unfused NiO due to the low solubility of NiO causing light scattering in these compositions [START_REF] Hecht | Study of the structure of nickel in soda-boric oxide glasses[END_REF]. The absence of light scattering effects in the optical absorption spectra demonstrates that NiO is well dissolved of our samples.

The amorphous nature of the glasses was checked by x-ray diffraction. The actual composition of the glasses was obtained by flame Atomic Absorption Spectroscopy for boron and alkalis (within ± 1 %) and by electron microprobe for Ni (within 0.05% after averaging 10 analytical points) using the Cameca microbeam Microprobe of the Camparis analytical facility of Sorbonne University. Glass composition, normalized to 100 mol%, is given in

Crystalline references

Crystalline CaNiSi2O 6 was used as a reference for slightly distorted [6] Ni 2+ [START_REF] Galoisy | Spectroscopic evidence for five-coordinated Ni in CaNiSi2O6 glass[END_REF] and NiCr2O 4 and Ni-bearing ß-Al2O3 for [4] Ni 2+ [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF]. KNiPO 4 and α-Ni2P2O7 were used as a reference for [5] Ni 2+ in a square pyramid geometry and coexisting [6] -and [5] Ni 2+ in a trigonal bipyramid symmetry, respectively [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF]. The synthesis conditions of these crystalline references and their spectroscopic properties have already been reported [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Galoisy | Spectroscopic evidence for five-coordinated Ni in CaNiSi2O6 glass[END_REF][START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF].

Optical absorption spectroscopy

Crystal field transmission spectra of parallel polished plates of glasses have been recorded at room temperature between 250 to 2500 nm, with an UV-Visible-NIR computerized 2300 CARY spectrophotometer.

After conversion of wavelengths into wavenumbers, a baseline correction was performed to account for the physical processes superimposed to electronic intraconfigurational transitions (coherent light scattering, ligandto-metal charge transfer transitions).

Diffuse reflectance spectroscopy was used for the crystalline references, using a 9-inch-diameter integrating sphere coated with halon. These samples were prepared as finely ground powders deposited on Al sample holders. Diffuse reflectance values were obtained by reference to halon. Reflectance values (R) are converted into a remission function F(R) using the Kubelka-Munk formalism:

F(R) = (1 -R 2 )/2R.
F(R) is a good approximation of the absorbance of a light scatterer of infinite thickness [START_REF] Manceau | Nickel-bearing clay minerals: 1. Optical spectroscopic study of nickel crystal chemistry[END_REF]. We first removed the background of the crystal field spectra obtained either in the diffuse reflectance mode or in transmission. We fitted the spectra (in a wavenumber scale, proportional to energy) into Gaussian components using a home made software to determine the transitions responsible for the various absorption bands, as already discussed for Ni-bearing minerals [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Manceau | Nickel-bearing clay minerals: 1. Optical spectroscopic study of nickel crystal chemistry[END_REF].

X-ray absorption spectroscopy

X-ray Absorption Spectroscopy (XAS) experiments at the Ni K-edge were performed on the D44 station at the LURE-DCI synchrotron source (Orsay, France). The storage ring was operating at 1.85 GeV positron energy and 200mA positron current. XAS spectra were collected in transmission mode and sample thickness was optimized to get an absorption step between 0.5 and 1. The X-ray detectors were ionizing chambers filled with air.

XANES (X-ray absorption near edge structure) spectra were recorded at room temperature and collected from 8200 to 8600 eV using a Si (311) double-crystal monochromator, which provides a spectral resolution of 1.9 eV. The energy steps were 0.15eV for the pre-edge region and 0.5 eV for the main part of the edge, with 3sec accumulation time. Energy calibration was repeatedly checked by recording metallic Cu reference spectra after every three measurements and is accurate within 0.3 eV. The XANES spectra have been normalized to the atomic absorption above the absorption threshold, extracted by a polynomial fit. First and second derivative functions were used to determine the exact position of the various XANES features. Pre-edge features have been extracted after main-edge background removal, using an arctangent function.

Results

XANES

Crystalline references

Fig. 1. Normalized Ni-K edge XANES spectra of crystalline references: [6] NiSO4, 6H2O and Ca [6] NiSi2O6, K [5] NiPO4 and [4] NiCr2O4. Ni 2+ occurs in 6-and 5 coordination in α-Ni2P2O7. The two regions A and B represent the pre-edge and main edge resonance, respectively. The inset represents the XANES spectrum of a Ni 2+ square planar complex, which shows the characteristic signature of this coordination state (arrow) (after [START_REF] Keene | Solvent-modified dynamic porosity in chiral 3D kagome frameworks[END_REF],

modified). This diagnostic feature is not observed in the XANES spectra of alkali borate glasses.

Reference compounds were chosen to correspond to the most probable coordination numbers of Ni 2+ in borate glasses: regular and distorted octahedral site in NiSO4, 6H2O and CaNiSi2O6, respectively; square pyramid site in KNiPO4 (C 4v site symmetry) and tetrahedral site in [4] NiCr2O4. Ni 2+ occurs in both 6-and 5 coordination (trigonal bipyramid site with a D 3h site symmetry) in α-Ni2P2O7. The two regions of the XANES spectra, the pre-edge (A feature) and the main edge (B feature), show an evolution of their relative intensity and lineshape as a function of Ni 2+ coordination geometry (Fig. 1). The A feature corresponds to electronic transitions from the 1s core-level to the 3d empty orbitals and is quadrupolar and parity forbidden. As a consequence, it is enhanced by an increased p character of the final state, such hybridization being favored in the absence of an inversion center [START_REF] Brown | X-ray absorption spectroscopy and its applications in mineralogy and geochemistry[END_REF]. The pre-edge intensity increases from 6-to 5-and 4-coordinated Ni. The B Feature is ascribed to dipolar transitions to empty 4p states superimposed to multiple scattering resonances.

As it results from the transition to the p empty density of state of the absorber, it is influenced by the sp hybridization of the oxygen neighbors and an increased hybridization decreases the p character of the final state.

This causes the intensity of the B feature to decrease from 6-to 5-and 4-coordinated Ni 2+ .

The sharpest B feature occurs in the spectrum of [6] NiSO4, 6H2O, in agreement with a regular geometry of the octahedral Ni-site. It is split by 2.8 eV in Ca [6] NiSi2O6, consistent with a distortion of the Ni 2+ site. The lowest intensity of the B feature is observed in the spectrum of NiCr2O4, the spectra of the [5] Ni 2+ compounds

showing an intermediate intensity. The lowest intensity of the A feature is found in a pure octahedral symmetry as in NiSO4, 6H2O and it increases with the hybridization of the Ni 2+ 3d and 4p orbitals in non-centrosymmetric sites, which is observed for [4] Ni 2+ in NiCr2O4. As for the B feature, the intensity of the A feature for [5] Ni is intermediate relative to that of [4] -and [6] Ni 2+ compounds. Finally, a square planar environment gives rise to a characteristic Ni (1s → 4pz) transition superimposed to the rising main edge (inset, Fig. 1). The absence of this peculiar feature allows reject such Ni coordination state in alkali borate glasses (see 3.1.2). ( [6] Ni 2+ ) and KB35 ( [5]-and [4] Ni 2+ ) glasses.

Borate glasses

The XANES spectra of LiB10, NaB10 and KB10 glasses (Fig. 2a) are almost identical. The low intensity of the pre-edge feature (A) indicates a regular octahedral coordination, as in nickel sulfate, NiSO4, 6H2O (Fig. 1). These spectra are different from the XANES spectrum of NiO [START_REF] Gougoussis | Intrinsic charge transfer gap in NiO from Ni K-edge x-ray absorption spectroscopy[END_REF]. It exhibits a characteristic main edge (B)

rising at lower energy than in the glasses due to O to Ni charge-transfer processes, the appearance of new features at 8337 eV and 8352 eV and a shift of the multiple scattering XANES resonance (C) from 8358 to 8362 eV. These differences in the XANES spectra are a confirmation that the ordered defective NiO-like domains evidenced by EXAFS in these glasses [START_REF] Cormier | Evidence of Ni-containing ordered domains in low alkali borate glasses[END_REF][START_REF] Galoisy | Environment of Ni, Co and Zn in low alkali borate glasses: information from EXAFS and XANES spectra[END_REF] do not correspond to unreacted NiO crystallites.

From KB10 to KB27 and KB35 (Fig. 2b), the intensity of the pre-edge (A) increases and that of the main edge (B) decreases, as observed in [4] Ni 2+ vs. [6] Ni 2+ references. The XANES spectra of NaB30 show a pre-edge intensity close to that of KB35. This is consistent with a transformation of Ni 2+ sites from a regular octahedral coordination to non-centrosymmetric sites at increasing alkali content. Features B and C are well structured for KB10 but become broader for KB27 and KB35, as a result of Ni 2+ -site distribution. Finally, none of the investigated spectra shows the presence of the Ni (1s → 4p z) transition characteristic of a square-planar geometry as observed in the XANES spectra of Ni 2+ square-planar complexes ( Fig. 1) [START_REF] Keene | Solvent-modified dynamic porosity in chiral 3D kagome frameworks[END_REF].

UV-visible-near IR absorption spectroscopy

Crystalline references

Fig. 3. Remission function F(R) of Ni-bearing crystalline references: Ca [6] NiSi2O6, α-[5+6] Ni2P2O7, K [5] NiPO4 and ß-Al2O3: [4] Ni. This function is a good approximation of sample absorbance in the Kubelka-Munk formalism

(see 2.3).
Octahedral Ni 2+ is characterized by three spin allowed transitions at 8280 cm -1 (crystal field transition), 13170 cm -1 and 24450 cm -1 in CaNiSi2O6 (Fig. 3). These transitions correspond to a crystal field splitting 10Dq = 8280 cm -1 and a Racah parameter B = 890 cm -1 (the free ion value is B=1030 cm -1 ) [START_REF] Galoisy | Spectroscopic evidence for five-coordinated Ni in CaNiSi2O6 glass[END_REF]. The crystal field transition bears a Gaussian shape with a full linewidth of 2700 cm -1 , due to a low distortion of the [6] Ni 2+ -site.

Spin-orbit coupling effects enhance the intensity of the spin-forbidden transitions to 1 E g (D) and 1 A1g (G) levels at 14900 and 19670 cm -1 , respectively.

The absorption bands of [4] Ni 2+ observed in ß-Al 2 O 3 are shifted to lower wavenumber values relative to [6] Ni 2+ . They occur near 5000, 8500 and 15500 cm -1 [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF]. Site distortion (Jahn-Teller effect) and spin-orbit coupling cause the splitting of the 15500 cm -1 transition. The characteristic field-independent spin forbidden transitions at 13000 and 13900 cm -1 retain a narrow lineshape in the optical spectra of [4] Ni 2+ .

The crystal field spectra of [5] Ni 2+ present a higher number of allowed optical transitions (five and six in Trigonal bipyramid (TBP) and Square pyramid (SP) configurations, respectively) than in octahedral or tetrahedral coordination [START_REF] Jazouli | Structure and colour of diphosphate pigments with square pyramid environment around chromophore ions (Co 2+ , Ni 2+ , Cu 2+ )[END_REF]. In KNiPO4, the broad band near 10000 cm -1 arises from partly overlapping absorption bands. As a consequence, the two absorption bands occurring at low wavenumbers have different linewidths in [5] Ni 2+ by contrast to [6] Ni 2+ in which the linewidths are similar. The most intense absorption band occurs at a position intermediate between that in [6] -and [4] Ni 2+ , near 22000 cm -1 with a shoulder near 19000 cm - The position of the characteristic transitions of octahedral and tetrahedral Ni 2+ is represented by a full and a dashed line, respectively, as a guide for the eye, to appreciate the modification of the position of the main absorption bands. The position of the main band for [5] Ni 2+ occurs at lower wavenumbers (22000-23000 cm -1 ) as compared to [6] Ni 2+ (24000-25000 cm -1 ).

The spectra of the KB10 (Fig. 4a), NaB10 (Fig. 4b) and LiB10 (Fig. 4c) glasses are almost identical and show similar decomposition into Gaussian components (Fig. A1). These spectra characterize the presence of [6] Ni 2+ as recognized in previous studies [START_REF] Weyl | Coloured Glasses[END_REF][START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]. The green color of these glasses is a visual indication that the [6] Ni 2+ sites are not significantly distorted. Indeed, distorted [6] Ni 2+ sites are known to impart a yellow color in crystalline materials, due to a splitting of the main band that causes a modification of the transmission window )! [START_REF] Rossman | Origin of the yellow color of complex nickel oxides[END_REF]. The spectra do not show the presence of light scattering, assigned in other studies (see 2.1) to unreacted NiO [START_REF] Hecht | Study of the structure of nickel in soda-boric oxide glasses[END_REF].

In K-borate glasses (Fig. 4a), the optical spectra show dramatic changes in relation with the appearance of different Ni-coordination sites. The dashed vertical line corresponds to the contribution of tetrahedral Ni 2+, showing that the position of the main [4] Ni 2+ absorption band remains stationary. The absorbance value at this position dashed line allows assess the progressive increase of the proportion of [4] Ni 2+ with increasing K-content from KB27 to KB35. The whole absorption spectrum is affected by the progressive domination of the tetrahedral Ni 2+ transitions at increasing potassium content. In the KB27 glass, showing a deep brown color, the most intense transition occurs near 22000 cm -1 . However, in addition to this absorption band characteristic of [5] Ni 2+ , the spectrum is characterized by the presence of a band near 15600 cm -1 , diagnostic of the 3 T1(F) → 3 T1(P) transition of [4] Ni 2+ . The band near 5000 cm -1 has a Gaussian shape (Fig. A3) and corresponds to the [4] Ni 2+ crystal field transition. The color of the KB31 and KB35 glasses is purple: the transmission window is redshifted towards lower wavenumbers, and another transmission window appears in the 23000-24000 cm -1 range (violet domain). These spectra show two intense bands near 15500cm -1 and 17900 cm -1 (Fig. 4) that correspond to the 3 T1(F) → 3 T1(P) transition of [4] Ni 2+ , split by the distortion of the [4] Ni 2+ site (Jahn Teller effect). The band near 22000 cm -1 due to [5] Ni 2+ , shows a dramatic decrease (in KB31 vs. KB27), in relation with the disappearance of this coordination in KB35. The two bands located near 4800 and 8700 cm -1 are redshifted and more intense than in the other borate glasses. The tetrahedral coordination of Ni 2+ is confirmed by the presence of the weak field-independent spin-forbidden transitions, 3 T1(F) → 1 T2(D) and 3 T1(F) → 1 E(D) at 13000 cm -1 and 13900cm -1 , respectively [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF].

The spectra of Na-borate glasses are depicted on Fig. 4b. Though presenting a similar spectral shape and position of the Gaussian components as in the LiB10, NaB10 and KB10 glasses (Fig. A1), the NaB20 glass shows an absorption coefficient twice larger, as well as a modification of the absorption features in the near infrared domain. This indicates the contribution of a new, non-resolved, Ni 2+ speciation. The same trend towards lower wavenumbers is observed in LiB30 and NaB30 glasses, with the main band shifted from 24000 to 22700 cm -1 . The most intense absorption band peaks outside the values usually found for [6] Ni 2+ . This characterizes the formation of [5] Ni 2+ sites. Together with the appearance of a contribution at 20000 cm -1 (on the low wavenumber side of the main absorption band), this shift causes the transmission window to shift from 17500 to 15000 cm -1 .

Consequently, the color turns to yellow/brown hues. In the spectrum of NaB30, the absorbance at the position of the dashed vertical line indicates the appearance of minority tetrahedral Ni 2+ . For Li-borate glasses (Fig. 4c)

there is also a red shift of the transitions with increasing alkali content, indicating a transformation from [6] Ni 2+

to [5] Ni 2+ . By contrast to Na-borate glasses, the spectra do not show any contribution of tetrahedral Ni 2+ even at high Li-content: the position of the dashed line corresponding to this coordination state falls in a transmission window. The spectrum of LiB30 may then be considered as typical of [5] Ni 2+ and is described in more detail in 4.4. As for the crystalline references, the intensity of the two features A and B shows an opposite behavior as a function of the Ni-site geometry (Fig. 5). The relative intensity of the pre-edge varies by a factor 5 with Nicoordination. The pre-edge and shape resonance intensity of [5] Ni 2+ is intermediate between [6] Ni 2+ and [4] Ni 2+ . In octahedral symmetry, as in NiSO4, 6H2O, the pre-edge shows the lowest intensity. This intensity increases from [5] -to [4] Ni 2+ due to the hybridization of the Ni 2+ 3d and 4p orbitals in non-centrosymmetric sites. Such hybridization explains also the decreasing intensity of the main edge, as it arises from transitions to 4p-type levels. As depicted in Fig. 5, the XANES parameters of low alkali borate glasses correspond to what is expected for octahedral Ni 2+ , as those of the K-rich KB31 and KB35 glasses correspond to majority tetrahedral Ni 2+ . The XANES parameters of NaB30 and LiB30 glasses remain at values intermediate between [6] Ni 2+ and [4] Ni 2+ , consistent with the presence of [5] Ni 2+ .

Discussion

Discussion of the EXAFS-derived average coordination number of Ni 2+

Previous EXAFS data on alkali borate glasses have shown the presence of Ni 2+ in 5-and 6-fold coordination in sodium borate glasses [START_REF] Xu | Local structure around Ni 2+ ions in sodium borate glasses[END_REF] and of 4-, 5-and 6 coordinated Ni 2+ in lithium, sodium and potassium borate glasses (in the same samples as those here investigated) [START_REF] Galoisy | Chemical stability of Ni-enriched nanodomains in alkali borate glasses[END_REF]. Both studies provide a similar picture of Ni-O distances and Ni-coordination number (Fig. 6a), depicting a decrease of the Ni-O distances with increasing alkali content. The EXAFS-derived Ni-O distances exclude the presence of "exotic" coordination sites mentioned in earlier studies [START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF][START_REF] Berkes | The optical spectra of nickel in alkali tetraborate glasses[END_REF] and further discussed in 4.1.3.

The average Ni-O distance and Ni coordination number decrease with increasing alkali content. The LiB10, NaB10 and KB10 glasses exhibit the same Ni-O distance at 2.04-2.05Å. This distance is smaller than expected for a regular octahedral site, typically 2.08Å in NiO. However, a decrease of the Ni-O distance by 0.02-0.04Å has been observed in NiO thin films [START_REF] Anspoks | Atomic structure relaxation in nanocrystalline NiO studied by EXAFS spectroscopy: role of nickel vacancies[END_REF] and some crystalline Ni borates may show [6] Ni-O distances as low as 2.05-2.06Å [START_REF] Schmitt | Ni6B22O39•H2O -extending the field of nickel borates[END_REF]. The EXAFS parameters are consistent with the disappearance of [6] Ni 2+ in alkali-rich borate glasses, with Ni-O distances smaller in K-bearing glasses. This indicates a higher concentration of tetrahedral nickel in K-borate glasses, as confirmed by the average Ni coordination number (Fig. 6b).

Optical spectroscopic signatures of Ni 2+

Optical spectroscopic properties of Ni 2+ -bearing borate glasses are characterized by a well-marked chemical dependence, interpreted as indicating a Ni coordination change with glass composition [START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF].

However, in the literature, Ni 2+ coordination has been mostly determined from a qualitative discussion of crystal field spectra, interpreted as arising from [6] Ni 2+ and [4] Ni 2+ . "Exotic" coordination numbers have also been evocated in the literature. The presence of 12-coordinated Ni 2+ was mentioned in Na-borate glasses [START_REF] Berkes | The optical spectra of nickel in alkali tetraborate glasses[END_REF], despite such Ni 2+ coordination is unknown [START_REF] Shannon | Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides[END_REF]. Square-planar coordination or 8-coordinated Ni 2+ were also evoked in alkali borate glasses [START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF][START_REF] Sarkar | Optical absorption of nickel(II) and mixed-alkali effect in K2O-Li2O-B2O3 glasses[END_REF]. The optical properties of [8] Ni 2+ in garnets [START_REF] Ross | Structure and crystal-field spectra of Co3Al2(SiO4)3 and (Mg,Ni)3Al2(SiO4)3 garnet[END_REF] with optical transitions at 4000, 6000 and 20000 cm -1 characteristic of 8-coordinated Ni 2+ do not correspond to the spectra observed in borate glasses. Moreover, EXAFS-derived Ni-O distances and XANES data, discussed in 3.1 for the square-planar coordination, exclude the presence of these unusual environments, which are packing-inefficient in oxide glasses.

The signature of three Ni 2+ coordination states can be observed in the optical spectra of KB10, LiB30 and KB35 for [6] Ni 2+ , [5] Ni 2+ and [4] Ni 2+ , respectively (Fig. 7). With increasing alkali content, the apparent maximum of the main transition is redshifted, indicating a decreasing Ni 2+ coordination (and hence decreasing crystal field intensity), as observed in crystalline references. As the optical transitions that characterize a Ni 2+ coordination state do not vary much as a function of medium range surrounding [START_REF] Rossman | Origin of the yellow color of complex nickel oxides[END_REF], they can be used to determine Ni 2+ coordination in glasses. This is especially true for the narrow [4] Ni 2+ transition at 15600 cm -1 , identified by its small linewidth and located in a spectral domain, in which there is no optical transition caused by other Ni 2+ speciations. for the main coordination states. From top to bottom: [4] Ni 2+ , [5] Ni 2+ in trigonal bipyramid (TBP) and in square pyramid geometry (SP) and [6] Ni 2+ .

Chemical dependence of Ni 2+ coordination in alkali borate glasses.

The main absorption band occurs in the visible region at a position that depends on Ni 2+ coordination. As this band is relatively sharp, its position determines the transmission window and hence the color of the glass.

As shown on Fig. 8a, this band is redshifted from [6] Ni 2+ to [5] Ni 2+ , defining a transmission window in the green and orange, respectively. In the case of [4] Ni 2+ the intense characteristic transition near 15600 cm -1 causes a major change in the transmission window, which occurs on the high wavenumber side of this band, leading to a purple color.

The molar extinction coefficient ε of the most intense band is also chemically dependent, as it increases with increasing alkali content, with the largest variations observed for potassium glasses (Fig. 8b). Five coordinated and tetrahedral Ni 2+ sites do not possess an inversion center and consequently display similar molar extinction coefficient (ε) values, which are higher than those of octahedral [6] Ni 2+ . This explains the limited increase of ε at high alkali content as already observed in Ni-bearing borate glasses [START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]. As for the other spectroscopic parameters, ε values exhibit a continuous variation with glass composition, an additional evidence of the mixing of various coordination states of Ni 2+ in alkali borate glasses. Although not observed in silicate and aluminosilicate glasses, 6-coordinated Ni is the prevailing coordination state in low-alkali borate glasses, whatever the nature of the alkali. The optical absorption spectra of low alkali borate glasses are the same for the three alkalis, indicating that crystal field splitting is not influenced by the field strength of the alkali. The spectra can be fitted in six Gaussian components (Fig. A1).

The position of the absorption bands, with a crystal field splitting of 7500-8000 cm -1 , characterizes [6] Ni 2+ [START_REF] Manceau | Nickel-bearing clay minerals: 1. Optical spectroscopic study of nickel crystal chemistry[END_REF][START_REF] Rossman | Origin of the yellow color of complex nickel oxides[END_REF][START_REF] White | Optical spectra of chromium, nickel, and cobalt-containing pyroxenes[END_REF]. However, the optical absorption bands (Fig. A1) cannot be fitted using a single Gaussian component and the crystal field band shows a splitting of 1500 cm -1 , indicating a slight distortion of the Ni 2+ site. These spectra confirm the absence of NiO: NiO thin films show an absorption spectrum with the main bands at 28500, 21700 and 13500 cm -1 [START_REF] Niedermeier | Band gap bowing in NixMg1-xO[END_REF], these values being different from those observed in glasses near 24100, 13200 and 7500 cm -1 . This confirms the XANES results presented in 3.1.2.

A peculiar property: the clustering of Ni 2+ octahedra in low-alkali borate glasses.

In low alkali glasses, alkali cations are consumed by the coordination change of boron: their chargecompensating role for [4] B hinders their ability to play a similar role for Ni. This may explain the presence of 6coordinated Ni in ordered clusters [START_REF] Cormier | Evidence of Ni-containing ordered domains in low alkali borate glasses[END_REF][START_REF] Galoisy | Environment of Ni, Co and Zn in low alkali borate glasses: information from EXAFS and XANES spectra[END_REF][START_REF] Galoisy | Chemical stability of Ni-enriched nanodomains in alkali borate glasses[END_REF], which do not require further charge compensation to be stabilized in the glass structure. This behavior explains the independence of the spectroscopic properties of [6] Ni 2+ on the nature of the alkali for low-alkali borate glasses. These Ni-rich ordered domains have optical properties and a local structure distinct from NiO [START_REF] Cormier | Evidence of Ni-containing ordered domains in low alkali borate glasses[END_REF][START_REF] Galoisy | Environment of Ni, Co and Zn in low alkali borate glasses: information from EXAFS and XANES spectra[END_REF]. Their formation may be related to the presence of rigid units such as boroxol, pentaborate or tetraborate groups, which show only a limited coordination change of boron [START_REF] Wright | My borate life: an enigmatic journey[END_REF]. The contribution of [6] Ni decreases with increasing alkali content as borate superstructural units are transformed into smaller, less rigid units. This clustering is based on edge-sharing octahedra, an intersite linkage that is observed in various nickel borates and Ni-substituted Mg borates, including ludwigite Mg2Ni(BO3)O2, Ni2B2O5 pyroborate, kotoite Ni3(BO3)2 or M 2+ 2(M 3+ )(BO3)O2 hulsite with two-dimensional Ni-domains [START_REF] Medrano | Magnetic properties of Ni5Sn(O2BO3)2 ludwigite[END_REF]. In these structures, divalent ions occur in distorted octahedra interlinked by edge-sharing and edge-linked to BO3 units (Fig. 9a). These associations form 1D or 2D cationic domains, ribbons or ladders, such as in Ni2B2O5 [START_REF] Busche | Synthesis and Crystal Structure of the first zinc containing pyroborates Ni1.5Zn0.5(B2O5) and Co1.5Zn0.5(B2O5)[END_REF],

though not as extended as the domains determined by EXAFS in low alkali (Li, Na, K) borate glasses [START_REF] Galoisy | Environment of Ni, Co and Zn in low alkali borate glasses: information from EXAFS and XANES spectra[END_REF]. showing a preferential association with [3] B (after [START_REF] Strauss | Synthesis, structure, and electrochemical properties of Na3MB5O10 (M= Fe, Co) containing M 2+ in tetrahedral coordination[END_REF], modified)

Links with physical properties of borate glasses

The presence of Ni-clustering may explain some properties of Ni-bearing low alkali (Li, Na, K) borate glasses: (i) the low solubility of NiO in these glasses at low alkali content [START_REF] Paul | Activity of nickel oxide in alkali borate melts[END_REF] may be due to the difficulty of accommodating Ni-rich domains in the disordered structure of borate melts; this solubility increases with the alkali content, as these domains progressively disappear above 20 mol% alkali oxides [START_REF] Galoisy | Chemical stability of Ni-enriched nanodomains in alkali borate glasses[END_REF]; (ii) the Ni 2+ Electron Paramagnetic Resonance (EPR) signal vanishes below 200K in Na-borate glasses [START_REF] Hecht | Study of the structure of nickel in soda-boric oxide glasses[END_REF], indicating a transition at low temperature from a superparamagnetic to an (anti)ferromagnetic state, compatible with the presence of NiO-based clusters; such transition from a paramagnetic to an antiferromagnetic state at Neel temperature has also been observed by EPR in nano-NiO particles [START_REF] Rubinstein | Electron spin resonance study of NiO antiferromagnetic nanoparticles[END_REF]; (iii) the magnetic moment increases with alkali a [4] M 2+ O [4] B b [3] B Na +

[3] B [6] M 2+ O nb concentration [START_REF] Juza | Color centers in alkali borate glasses containing cobalt, nickel, or copper[END_REF], which has been interpreted as indicating a decreasing contribution of [6] Ni 2+ up to an alkali concentration of about 20 mol%, above which Ni 2+ occurs in lower coordinated sites.

Structural significance of tetrahedral Ni 2+

Spectroscopic properties of tetrahedral Ni 2+ in borate glasses.

There is a major influence of the nature of the alkali on the proportion of [4] Ni 2+ , a coordination state which is not detected in Li-borate glasses and remains a minority in Na-borate glasses. The presence of [4] Ni 2+ in K2O-rich borate glasses has been observed since decades by optical spectroscopy [START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]. The distortion of the [4] Ni 2+ site, arising from a static Jahn-Teller effect, is shown by optical spectroscopy (see 3.2.2). The tetrahedral coordination of Ni 2+ is confirmed by XANES (see 4.1.2) and by the EXAFS-derived [START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF] Ni-O distances, around 1.95Å [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF]. The low value of the EXAFS Debye-Waller factor found in these glasses [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF] indicates that the site distortion detected by optical spectroscopy results from angular distortion of the Ni-site rather than a distribution of Ni-O distances. The spectroscopic properties of [4] Ni 2+ are similar to those encountered in silicate and aluminosilicate glasses [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF][START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. II. Geochemical implications[END_REF][START_REF] Kado | Structural significance of nickel sites in aluminosilicate glasses[END_REF], showing that [4] Ni 2+ sites in oxide glasses are independent on the type of glass. In silicate glasses, these sites are consistent with a network-former position, provided two alkali ions are present for charge compensation [START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF]. However, the small intensity of a Ni-B contribution prevents the direct detection by EXAFS of a linkage with the borate network, contrary to the Ni-Si contribution detected around [4] Ni 2+ in silicate and aluminosilicate glasses [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. II. Geochemical implications[END_REF][START_REF] Kado | Structural significance of nickel sites in aluminosilicate glasses[END_REF].

The structure of borate crystals is of interest to suggest various geometries of the linkage between tetrahedral divalent cations (Zn 2+ , Mg 2+ …) and the borate framework. For instance, tetrahedral Zn is preferentially linked by corner-sharing with BO3 triangles in Na3ZnB5O10 [START_REF] Chen | Synthesis and crystal structure of a novel pentaborate Na3ZnB5O10[END_REF] and Li0.48Na0.52ZnBO3 [START_REF] Chen | Syntheses and characterization of two alkali-metal zinc borates, α-LiZnBO3 and Li0.48Na0.52ZnBO3[END_REF].

Sodium pentaborates Na3MB5O10 (M=Co, Fe 2+ ) also show a linkage of tetrahedral divalent cations with BO3 triangles (Fig. 9b). High temperature favors also tetrahedral coordination of small divalent cations, as shown by the coordination of Zn 2+ in the LiZnBO3 polymorphs: as ZnO4 tetrahedra exist in the high temperature phase, the low temperature polymorph shows trigonal bipyramidal Zn [START_REF] Tamilarasan | Developing intense blue and magenta colors in α-LiZnBO3: the role of 3d-metal substitution and coordination[END_REF]. A preferential bonding to BO3 triangles may be rationalized by considering bond valence constraints [START_REF] Galoisy | Spectroscopic evidence for five-coordinated Ni in CaNiSi2O6 glass[END_REF]: trigonal B brings a charge compensation of 1 for the oxygen atoms, as compared to 0.75 for tetrahedral B, which minimizes the charge deficit of the oxygen involved in the bond to a tetrahedral cation.

Links with physical properties of borate glasses.

The presence of [4] Ni 2+ sites requires the charge deficit be compensated by alkalis. This means a competition with boron for charge compensation, as was observed in Zr alumino-borosilicates where it was possible to determine the relative preference for charge compensation between various cations [START_REF] Cormier | Competition for charge compensation in borosilicate glasses: Wide-angle x-ray scattering and molecular dynamics calculations[END_REF]. This competition may explain the chemical dependence of the proportion of [4] Ni 2+ observed in some K-and Naborate glasses (there is no measurable [4] Ni 2+ in Li-borate glasses and in alkali-poor borate glasses compositions). By contrast to borate glasses, the [4] Ni 2+ proportion remains almost independent on alkali concentration in silicate glasses [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Turner | Ligand-Field spectra and structure of nickel in silicate glasses[END_REF]. The competition for charge compensation may be responsible for the decrease of Ni-solubility above about 30 mol% alkalis in borate glasses, as the formation of stable borate superunits will require alkalis to stabilize the [4] B sites they contain instead of Ni-sites. Finally, an increasing proportion of [4] Ni 2+ at increasing alkali content may explain the continuous increase of the magnetic moment [START_REF] Juza | Color centers in alkali borate glasses containing cobalt, nickel, or copper[END_REF].

Structural significance of five-coordinated Ni 2+

Several optical absorption spectra of [5] Ni 2+ in crystalline borates, phosphates and silicates have been published recently because of the interest for environmentally-friendly brown pigments: LiMgBO3:Ni [START_REF] Tamilarasan | Exploring the colour of 3d transition-metal ions in trigonal bipyramidal coordination: identification of purple-blue (CoO5) and beige-red (NiO5) chromophores in LiMgBO3 host[END_REF],

NiB4O7 [START_REF] Knyrim | High-pressure syntheses and characterization of the transition metal borates beta-MB4O7 (M = Mn 2+ , Ni 2+ , Cu 2+ )[END_REF], Ni2P2O7 [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Tena | Co2P2O7-Ni2P2O7 solid solutions: structural characterization and color[END_REF], SrNiP2O7 [START_REF] Jazouli | Structure and colour of diphosphate pigments with square pyramid environment around chromophore ions (Co 2+ , Ni 2+ , Cu 2+ )[END_REF], KNiPO4 [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Livitska | The double phosphates M I M II PO4 (M I -Na, K; M II -Mg, Mn, Co, Ni, Zn)-synthesis from chloride melts and characterization[END_REF] or BaZn2-xNixSi2O7 and BaMg2-xNixSi2O7

sorosilicates [START_REF] Thieme | Lilac ceramic pigments based on Ba0.5Sr0.5Zn2xNixSi2O7 solid solutions[END_REF]. The local structure of [5] Ni 2+ in two borate crystals is depicted on Fig. 10, showing that this coordination state may be associated to either triangular or tetrahedral borate groups. The presence of [5] Ni 2+ in borate glasses at medium Na-and K content and medium to high Li content is shown by three kinds of spectroscopic evidences, which are exemplified in the LiB30 glass. (i) The XANES features such as pre-edge and shape resonance intensity are intermediate between those of [4] -and [6] Ni. (ii)

Similarly, EXAFS data indicate a mixture of [4] Ni 2+ and [5] Ni 2+ at medium Na-and K-concentrations, with EXAFS-derived Ni-O mean distances of about 1.98Å and a Ni mean coordination number around 5. (iii) Crystal field spectra are similar to those first described in brown Ni-bearing silicate and aluminosilicate glasses [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Kado | Structural significance of nickel sites in aluminosilicate glasses[END_REF].

These spectra may be fitted with 6 Gaussian components (Fig. A2), which are located at energy positions characteristic of [5] Ni 2+ (Fig. 11a andb). The main band near 23400 cm -1 shows a Gaussian shape on its high wavenumber side, an indication of the absence of [6] Ni 2+ expected at or above 25000 cm -1 .

Decreasing crystal field intensity arises from a number of ligands smaller in 5-coordination relative to an octahedral site. However, the shorter Ni-O distances in 5-vs. 6-coordination may partly compensate lower crystal field splitting values. The result is a red shift of about 1000-2000 cm -1 of the [5] Ni 2+ optical transitions relative to those of [6] Ni 2+ (Fig. 11b). This shift can be considered as a spectroscopic signature of 5-coordinated levels, the position of which depends on covalence effects. Their position is intermediate between that measured in spectra of [4] Ni 2+ and [6] Ni 2+ , a nice illustration of the intermediate covalence of [5] Ni 2+ -O bonds (Fig. 7).

Fig. 11. a. Gaussian fit of the optical absorption spectrum of LiB30 glass corresponding to [5] Ni 2+ in a square pyramid geometry. b. Energy levels of [6] Ni 2+ in regular octahedral coordination and [5] Ni 2+ , showing the redshift of the main optical transitions from octahedral to square pyramid geometry. The assignments are based on [START_REF] Gerloch | Single-crystal polarized spectrum and paramagnetic anisotropy of five-co-ordinate square pyramidal nickel(II) complexes[END_REF].

The driving mechanism of Ni 2+ coordination change in borate glasses

The coordination number of a cation is correlated with the field strength of the other cations. If these cations have a higher field strength, defined as the ratio of the net charge Z and the square of the average bond distance to oxygen d (Z/d 2 ), they will take more of the electron density from the O atoms and they will have a higher acidic character. A larger Ni coordination number is then required to balance the Ni 2+ charge. The investigation of a large number of Ni-bearing alkali silicate and aluminosilicate glasses [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Kado | Structural significance of nickel sites in aluminosilicate glasses[END_REF] shows that Ni 2+ coordination changes as a function of cation field strength, low field strength K + stabilizing [4] Ni 2+ as higher field strength Na + rather stabilizes [5] Ni 2+ .

Octahedral coordination of Ni 2+ is observed in alkali-poor borate glasses, the structure of which is dominated by superstructural units [START_REF] Wright | My borate life: an enigmatic journey[END_REF]. In these glasses, the addition of alkalis favors BO4 tetrahedral structural units, rather than the formation of nonbridging oxygen atoms (NBO). The 6-fold coordination state of Ni 2+ , may bonds [START_REF] Wong | Glass structure by spectroscopy[END_REF]. In intermediate, more basic compositions, for which not all the ligands are strongly bound to other cations, the optical spectra indicate a band at greater wavenumbers than that of [4] Ni 2+ . The present work assigns this band to a signature of [5] Ni 2+ , on the basis of optical absorption, XANES and EXAFS spectra. Finally, in the most alkali-rich glasses, which show the highest concentration of [4] B, the bond valence model predicts that the connection of [4] Ni 2+ to a bridging oxygen between two B tetrahedra satisfies the charge deficit of this bridging oxygen. The predominance of [4] Ni 2+ in high K-borate glasses shows that the remaining deficit of this tetrahedral divalent cation can only be ensured by low field strength cations. These cations also stabilize [4] Ni 2+ in silicate and aluminosilicate glasses [START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF][START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. I. Spectroscopic determination of coordination states[END_REF][START_REF] Kado | Structural significance of nickel sites in aluminosilicate glasses[END_REF][START_REF] Turner | Ligand-Field spectra and structure of nickel in silicate glasses[END_REF].

[5]

Ni 2+ may be seen as a reactive intermediate in reactions involving ligand exchange, such as those operating during viscous flow (Fig. 12). This would be equivalent to the presence of a minority five-coordinated Al or Si species in silicate and aluminosilicate glasses, corresponding to local bond-breaking and rearrangement that characterizes the dynamics of the molten state [START_REF] Stebbins | Glass structure, melt structure, and dynamics: some concepts for petrology[END_REF]. At the difference of the directional, covalent B-O bonds, the weaker Ni-O bonds are more prone to be broken because of their ionic and hence non-directional nature, inducing that Ni 2+ is mostly found in 5-fold coordination. Even if Ni 2+ has the highest crystal field stabilization energy (CFSE) in 6-fold coordination, the relative concentration of these transition states is thus expected to be more important than for network former cations that are only marginally affected by this transient coordination change. It is interesting that, in hydrothermal solutions, experimental data and first principles calculations show also the presence of 5-coordinated Ni 2+ forming [5] Ni 2+ -triborate complexes [START_REF] Graff | Complexation of nickel ions by boric acid or (poly)borates[END_REF]. Crystal field stabilization energy (CFSE) considerations also favor the abundance of [5] Ni 2+ . CFSE is 12Dq and 10Dq for [6] Ni 2+ and [5] Ni 2+ , respectively, Dq being the crystal field splitting parameter. The smaller number of ligands results in smaller Dq values for [5] Ni 2+ relative to [6] Ni 2+ . However, [5] Ni 2+ -O distances are smaller than [6] Ni 2+ -O distances, which increases CFSE values. Finally, the difference between the CFSE of [6] and [5] Ni 2+ , remains small, about 1.5 kJ.mole -1 [START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. II. Geochemical implications[END_REF]. This explains the potential stability of 5-coordinated Ni 2+ in oxide glasses.

Fig. 13. Chemical dependence of Ni coordination in alkali borate glasses. The [4] Ni 2+ fraction is derived from the intensity of the [4] Ni 2+ absorption band near 17000 cm -1 .

An estimate of the relative proportion of [4] Ni 2+ and [5] Ni 2+ can be derived from the intensity of the 17000 cm -1 absorption band of [4] Ni 2+ , under the assumption that the molar absorption coefficient ε of [4] Ni 2+ remains constant among the glasses. The chemical dependence may be quantified through the average B-coordination, defined by the N4 parameter: N4 = [4] B/( [3] B+ [4] B). It has been shown that the N4 value corresponds to the molar ratio of alkali to boron [START_REF] Clarida | Dependence of N4 upon alkali modifier in binary borate glasses[END_REF]. The proportion of [4] Ni 2+ ranges between almost 100% to less than a few %, in the K-rich and alkali poor borate glasses, respectively (Fig. 13)

At higher network-modifier fractions, the depolymerization of the borate network favors the formation of NBO's. In the presence of Ni 2+ , alkalis play a dual role in charge compensating [4] Ni 2+ and interacting with the borate network. The relatively large field strength of lithium favors the formation of [4] B [START_REF] Michaelis | Probing alkali coordination environments in alkali borate glasses by multinuclear magnetic resonance[END_REF], which explains the absence of [4] Ni 2+ units. By contrast, larger alkalis generate higher proportions of NBO's because their lower field strength, which explains that alkalis may be involved in charge compensating [4] Ni 2+ units. Figure 13 demonstrates that there is no direct relation between the chemical dependence of Ni and B coordination numbers: the structure of the polymeric network does not provide an objective estimate of Ni 2+ speciation.

As pointed out by Adrian Wright [START_REF] Wright | My borate life: an enigmatic journey[END_REF], the network-modifying cations play a critical role in the structure of borate glasses, although this role is frequently overlooked. The high temperature data of Lin and Angell [START_REF] Lin | Electronic spectra and coordination of Ni 2+ in potassium borate glass and melt to 1000°C[END_REF] provide an enlightening illustration of this behavior, demonstrating that [4] Ni 2+ concentration in borate glasses and melts increases from the glassy to the molten state. This variation is related to a modification of the role of alkalis associated to the coordination change of boron during the melting of borate glasses [START_REF] Cormier | Temperature-induced structural modifications between alkali borate glasses and melts[END_REF], according to the relation describing the change of the role of the alkali M:

BO4 + Mcomp→BO3 + Onb + Mmod
where Mcomp and Mmod refer to a charge-compensating or modifying role of the alkali and Onb designates a nonbridging oxygen. This means that alkali "activity" increases in the melt, making them available for chargecompensating [4] Ni 2+ .

Comparison with the role of Mg 2+ in borate glasses

Divalent nickel often mimics the structural behavior of Mg 2+ due to the similarity in their ionic radius. In alkali silicate and aluminosilicate glasses and melts, Mg 2+ is present in 4-, 5-and minority 6-fold coordination, the relative proportion of which follows a similar dependence as Ni 2+ as a function of glass composition [START_REF] Fiske | The structural role of Mg in silicate liquids: a high-temperature 25 Mg, 23 Na and 29 Si NMR study[END_REF][START_REF] Cormier | Structural study of Ca-Mg and K-Mg mixing in silicate glasses by neutron diffraction[END_REF]. The investigation of Mg coordination in Na-Mg borate glasses [START_REF] Kamitsos | Vibrational spectra of magnesium-sodium-borate glasses. 2. Raman and mid-infrared investigation of the network structure[END_REF] and K-Mg borate glasses [START_REF] Kawazoe | Coordination of Mg2+ in oxide glasses determined by X-ray emission spectroscopy[END_REF] has shown the presence of [6] Mg or [4] Mg in alkali-poor and alkali-rich compositions, respectively. This observation is in line with the present results deduced from the spectroscopic properties of Ni in these glasses. In alkaline earth borate glasses, the proportion of tetrahedral boron, N4, increases with the concentration of alkalis and alkaline earths [START_REF] Greaves | Inorganic glasses, glass-forming liquids and amorphizing solids[END_REF]. However, the N4 value decreases from Ba to Mg. This dependence on the cation field strength is reverse to what is observed in alkali borate glasses [START_REF] Yiannopoulos | Structure and properties of alkaline earth borate glasses[END_REF]. This observation is consistent with the structure of crystalline Mg-borates, showing a preferential association of Mg sites with triangular borate units.

As discussed above, this structural property is expected from the bond valence model and may drive the local structure around Mg in borate glasses.

Finally, a recent study on borosilicate glasses [START_REF] Bisbrouck | Impact of magnesium on the structure of aluminoborosilicate glasses: A solid-state NMR and Raman spectroscopy study[END_REF] demonstrated that the most salient structural effect of the incorporation of Mg is an increase in the [3] B population. This effect can be attributed to the larger cation field strength of Mg 2+ relative to that of other network-modifying cations. This was ascribed to the poor capability of Mg 2+ for charge-compensating the charge deficit of [4] B units due to relatively high field strength.

The local structure around Ni is then similar to that of Mg, with a specific linkage of [4] Ni 2+ with triangular [3] B units. Such association may help in rationalizing the structure of borate glasses, as the building blocks used to describe the topology of crystalline borates [START_REF] Hawthorne | A bond-topological approach to borate minerals: A brief review[END_REF] may give some insight on the local structure of borate glasses.

Conclusion

A detailed picture of the speciation of Ni 2+ in alkali borate glasses was obtained by coupling optical absorption spectroscopy with Ni K-edge XANES and previous EXAFS data. These glasses show a broad range of colors of as a function of alkali field strength, from yellow and brown to purple. The absorptivity of tetrahedral and five-coordinated Ni 2+ is similar, a property that gives all intermediate alkalinity borate glasses the same brown color as silicate and aluminosilicate glasses containing an alkali with the same field strength.

Our results update previous descriptions of the optical spectra of Ni 2+ in alkali borate glasses based on the coexistence of only octahedral and tetrahedral Ni 2+ . By using complementary spectroscopic methods, we show that nickel speciation in borate glasses corresponds to the coexistence of [4] -, [5] -and [6] Ni 2+ , each coordination state having a specific structural position: clusters of [6] Ni edge-sharing octahedra at low alkali content, [4] Ni tetrahedra connected to the borate network in presence of low field strength elements as charge compensators and [5] Ni sites corresponding to reactive species that inherit from molten state dynamics in presence of medium field strength elements. Cation field strength defines the capacity of alkalis to charge compensate [4] B and [4] Ni 2+ , and plays a crucial role in controlling the local structure around Ni 2+ in these glasses. This causes [4] Ni 2+ to be stabilized in high K-borate glasses. The quantification of the proportion of tetrahedral Ni 2+ shows a nonlinear dependence on alkali concentration. This results from a competition for charge compensation between Ni and B, as already observed in other glasses. The inefficiency of Li to charge compensate [4] Ni 2+ explains the cm -1 corresponds to the main contribution of [5] Ni 2+ in the KB27 glass. It progressively decreases at higher Kcontent.

Fig. 2 .

 2 Fig. 2. Normalized Ni-K edge XANES spectra of alkali borate glasses. a. Comparison between glasses with 10 mol% alkali (LiB10, NaB10, KB10) and NiO; b. Comparison between glasses with 10%, 27% and 31%

Fig. 4 .

 4 Fig. 4. Optical absorption spectra of Ni 2+ in a. K-borate glasses, b. Na-borate glasses, c. Li-borate glasses.

1 .

 1 M olar extinction C oefficient (L! .m! o! l! -

4. 1 Fig. 5 .

 15 Fig. 5. Evolution of the intensity of the pre-edge (A feature) and of the main edge (B feature) for the XANES spectra of crystalline references and alkali borate glasses (see Fig. 1 and Fig.2).

Fig. 6

 6 Fig. 6 Evolution of the EXAFS parameters of Ni 2+ in alkali borate glasses from the data of [11] and [24]. Left: Ni-O distances; right: Ni-coordination number. The value of the Ni-O distances allows separate the contribution of 4-, 5-and 6-coordinated Ni.

Fig. 7 .

 7 Fig. 7. Optical absorption spectra normalized to the most intense absorption band of each spectrum and corresponding to a dominant Ni 2+ coordination. The top of the figure represents the optical transitions expected

Fig. 8 .

 8 Fig. 8. a. Evolution of the apparent maximum of the most intense absorption band of Ni 2+ in alkali borate glasses (in l/mol.cm). b. Evolution of the molar extinction coefficient ε with the alkali content.

4. 2 .

 2 Structural significance of octahedral Ni 2+ 4.2.1. Spectroscopic properties of octahedral Ni 2+ in borate glasses.

Fig. 9 .

 9 Fig. 9. a. Edge-sharing M 2+ octahedra in the structure of M2B2O5 pyroborate (after the data of [35]). Bridging oxygens bound to Ni are shown in red. Other oxygen atoms have been omitted. b. Tetrahedral M 2+ in the structure of sodium transition-metal pentaborate Na3MB5O10 showing a preferential association with[3] B (after

Fig. 10 .

 10 Fig. 10. The two main geometries of five-coordinated Ni: a. trigonal bipyramid in LiMgBO3:Ni 2+ (after the data of [45], modified) with linkages to triangular B units; b. square pyramid in ß-NiB4O7 (after the data from [46], modified), with linkages to tetrahedral B units.

  competition for available ligands, due to the strength of the B-O bonds relative to the weaker Ni-O

Fig. 12 .

 12 Fig. 12. Generation of a 5-coordinated site, trigonal bipyramid (left) or square pyramid (right) during atomic motion and bond breaking associated to the viscous flow of a melt.

  absence of this coordination in Li-borate glasses. Due to the many similarities between Ni 2+ and Mg 2+ , the diversity of the structural scenarios around Ni 2+ in alkali borate glasses may shed light on the structural behavior of Mg in the same glasses, underlining the diversity of local structures around these divalent cations, as a function of glass composition.

Figure A2 .

 A2 Figure A2. Optical absorption spectra of Ni 2+ in 20 and 30 mol% alkali borate glasses: the spectra of NaB20 and LiB20 are similar to those of 10 mol% alkali, with an increasing non-Gaussian character of the components in the near IR region, showing a higher distortion of the[6] Ni 2+ site. The optical spectrum of NaB30 shows the appearance of tetrahedral Ni 2+ with a characteristic transition near 17000 cm -1 . The spectrum of LiB30 is typical for 5-coordinated Ni (see Fig.11a and b).

Figure A3 .

 A3 Figure A3. Optical absorption spectra of Ni 2+ in high K borate glasses. With increasing K-content, the

Table 1 :

 1 Table 1 with nominal composition in brackets. The glasses appear visually homogeneous, Chemical composition of the alkali borate glasses investigated (mol%). The nominal compositions are given in brackets.

	transparent, with a constant coloration.
		B2O3	Li2O	Na2O	K2O	NiO
	LiB10	88.1 (90)	10 (10)			1.9 (2)
	LiB20	79 (80)	19.3 (20)			1.7 (2)
	LiB30	69 (70)	29.2 (30)			1.8 (2)
	NaB10	89.8 (90)		8.8 (10)		1.4 (2)
	NaB20	79.5 (80)		20 (20)		1.5 (2)
	NaB30	69.5 (70)		28.8 (30)		1.7 (2)
	KB10	87.1 (90)			11.4 (10)	1.5 (2)
	KB20	80.3 (80)			18 (20)	1.7 (2)

. A characteristic narrow spin-forbidden transition is observed at 13900 cm -1 as in crystalline phosphates and borates[13, 19,

[START_REF] Galoisy | Structural environment of nickel in silicate glass/melt systems. II. Geochemical implications[END_REF]. The spectrum of α-Ni2P2O7[START_REF] Galoisy | Network forming nickel in silicate glasses[END_REF] is remarkably similar to that of MgNiP2O7[START_REF] Moutataouia | Crystal structure and optical properties of a new nickel magnesium diphosphate[END_REF]. The position of the main absorption bands is shifted relative to the spectrum of KNiPO4, due to a different geometry of the[START_REF] Galoisy | Overview of the environment of Ni in oxide glasses in relation to the glass colouration[END_REF] Ni 2+ square pyramidal site. However, square pyramid and trigonal bipyramid sites may mutually transform easily, which makes it difficult to decipher among these two geometries. An important spectroscopic property of Ni 2+ is that the most intense absorption bands are redshifted with decreasing Ni-coordination number. This is consistent with a crystal field intensity decreasing with the number of ligands.3.2.2. Borate glassesThe optical spectra of alkali borate glasses (Fig.4) show dramatic differences as a function of the alkali content, at the origin of well-known spectacular color variations[START_REF] Weyl | Coloured Glasses[END_REF][START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]. All spectra lie on a horizontal background. The values of the molar extinction coefficient ε found in this study are similar to those of the literature[START_REF] Weyl | Coloured Glasses[END_REF][START_REF] Wong | Glass structure by spectroscopy[END_REF][START_REF] Paul | Co-ordination equilibria of nickel (II) in alkali borate glasses[END_REF]. The nature of the alkali directly influences the chemical dependence of glass color and hence the shape of the optical absorption spectra, an indication of a coordination change of Ni 2+ . Glasses with low and intermediate Li or Na content are green and brown, respectively. Glasses with high potassium content are purple, while glasses with a high Li or Na content remain brown.

Acknowledgements

We gratefully acknowledge the support of Institut Universitaire de France. We also thank Franck Hawthorne for sharing his knowledge on the structural logic of borate minerals.

Appendix:

Gaussian fits of the investigated glasses


Sophie Cousin

MD Coralie Cantarel

MSc Jean-Philippe Guegan

Thibault Mazard

MD Carlos Gomez-Roca

MD; Jean-Philippe Metges

MD Carine Bellera

PhD; Antoine Adenis

MD; Iphigenie Korakis

MD; Pierre- Guillaume Poureau

MD Kevin Bourcier

MD Maud Toulmonde

MD Michèle Kind

MD Christophe Rey

PhD Céline Auzanneau

Alban Bessede

PhD Isabelle Soubeyran

MD Antoine Italiano
email: a.italiano@bordeaux.unicancer.fr

Pr Antoine

Regorafenib-avelumab combination in patients with biliary tract cancer (REGOMUNE): a single-arm, open-label, phase II trial

Keywords: regorafenib, avelumab, biliary tract cancer, immunotherapy, cholangiocarcinoma

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

Introduction

Biliary tract cancers (BTCs) represent a group of aggressive malignancies classified based on their site of origin across the biliary tree as intrahepatic (iCCA), extrahepatic (eCCA), and gallbladder cancer.

Patients diagnosed with BTC have a poor prognosis because the majority of these patients are not eligible for curative surgery. [1] In the advanced disease setting, systemic therapy combining cisplatin with gemcitabine represents the current standard of care. [2] After the failure of first-line therapy, therapeutic options are limited. [2] Given the importance of angiogenesis in BTC tumorigenesis, several anti-angiogenic agents have been investigated in this setting. [3] Regorafenib, a small molecule that targets several protein kinases including PDGFR, VEGFR, FGFR, Flt-3, RET, and c-Kit is the sole anti-angiogenic agent that has shown significantly improved clinical benefit compared to placebo in the context of a randomized study. [START_REF] Demols | Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial[END_REF] Indeed, regorafenib significantly improved PFS and tumor control in comparison to place in a randomized phase II study including patients with previously treated metastatic/unresectable biliary tract cancer in the second-or third-line setting. [START_REF] Demols | Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial[END_REF] Inflammation and immune modulation have also been shown to play a crucial role in BTCs. [START_REF] Sia | Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes[END_REF][START_REF] Montal | Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma[END_REF] Clinical trials investigating immune checkpoint inhibitors in patients with BTC have reported only modest results, [START_REF] Piha-Paul | Efficacy and safety of pembrolizumab for the treatment of advanced biliary cancer: Results from the KEYNOTE-158 and KEYNOTE-028 studies[END_REF] underscoring the need for novel approaches including combination therapies and the identification of efficacy biomarkers.

There are several lines of evidence indicating that targeting vascular endothelial growth factor (VEGF) and its receptor (VEGFR) may be synergistic with immune checkpoint inhibition in human tumors. [START_REF] Yi | Synergistic effect of immune checkpoint blockade and anti-angiogenesis in cancer treatment[END_REF][START_REF] Rahma | The Intersection between Tumor Angiogenesis and Immune Suppression[END_REF] Therefore, we hypothesized that combining an anti-angiogenic agent with anti-programmed cell death 1 (PD-1)/anti-programmed cell death ligand 1 (PD-L1) antibodies may be associated with significant clinical benefit in patients with advanced BTC that has not responded to previous standard chemotherapy regimens.

Here, we report the clinical and biomarker results of a phase II study investigating the combination of regorafenib plus the anti-PD-L1 antibody, avelumab, in patients with advanced BTC.

METHODS

Study design and participants

REGOMUNE is a single-arm, multicenter phase II basket study for which patients were recruited from four French sites. In the BTC cohort, patients were eligible if they were at least 18 years old and had histologically proven advanced or metastatic BTC, an Eastern Cooperative Oncology Group performance status of 0-1; measurable disease according to RECIST 1.1; [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF] at least one previous line of systemic treatment; and adequate hematological, renal, metabolic, and hepatic functions (see study protocol for a full list of eligibility criteria). Blood tests included the assessment of blood cell count, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, bilirubin, lipase, creatinine phosphokinase, coagulation test, creatinine, and urea nitrogen. The main exclusion criteria included previous treatment with avelumab or regorafenib, previous treatment with anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody, and are detailed in the protocol. As required by French regulations, the protocol was approved by a central institutional review board (Comité de Protection des Personnes Sud-Est II, Lyon, France), which reviewed the appropriateness of the clinical trial protocol and the risks and benefits to study participants. All patients provided written informed consent.

Procedures

After assessing eligibility, the patients received regorafenib, 160 mg per day on a 3 week on/1 week off schedule, in cycles of 28 days. Avelumab treatment began on Cycle 1 Day 15, by intravenous infusion once every 2 weeks at a dose of 10 mg/kg.

Treatment was continued until disease progression, unacceptable toxicity, the investigator's decision to discontinue, or withdrawal of patient consent. Participants were monitored for adverse events at every follow-up assessment. Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. Laboratory assessments were conducted at baseline, and every 2 weeks thereafter until treatment discontinuation. Regorafenib dose modifications to manage adverse events were allowed (see study protocol).

The regorafenib dose could be reduced to 120 mg and then to 80 mg. Dose interruptions were allowed based on the clinical situation. Patients requiring a delay of > 4 weeks since the last dose of regorafenib had to permanently discontinue regorafenib but could continue avelumab if it was deemed appropriate. No dose reduction of avelumab was allowed. Dose interruptions were allowed based on the severity of immune-related adverse events. Patients requiring two or more consecutive cancellations of avelumab injection had to permanently discontinue avelumab but were allowed to continue regorafenib. Tumor lesions were assessed according to RECIST version 1.1 at baseline (within 4 weeks prior to Cycle 1 Day 1) and every 8 weeks until disease progression or the start of another treatment. Tumor samples were collected at baseline and on Cycle 2 Day 1 for all consenting patients to assess the impact of treatment on the tumor microenvironment and to identify potential biomarkers associated with outcomes.

Outcomes

The primary endpoint was the objective response rate defined as the proportion of patients with objective response (confirmed or unconfirmed) under treatment based on adapted RECIST 1.1 after centralized radiological review.

Secondary objectives included best overall response, 1-year progression-free survival (PFS), 1-year overall survival (OS), and safety. Best overall response was defined as the best response across all time points. Durable clinical benefit was defined as the proportion of patients with objective response and/or stable disease lasting more than 6 months. PFS was defined as the time from study treatment initiation to death from any cause or the first occurrence of disease progression based on RECIST 1.1 after centralized radiological review. OS was defined as the time from study treatment initiation to death from any cause. Safety was graded as per the common toxicity criteria stated in NCI CTC-AE v5.0.

Statistical analysis

This study was based on a Bayesian adaptive phase 2 design approach and followed an adaptive trial design. The primary endpoint was the objective response rate under treatment. The probability of success was estimated from a beta-binomial model. The maximum response probability threshold and minimum response probability threshold were defined as 40% and 20%, respectively. [START_REF] Zohar | Bayesian design and conduct of phase II single-arm clinical trials with binary outcomes: a tutorial[END_REF] The maximum sample size was set at 50 patients. The analysis of the primary endpoint was carried out sequentially, with interim analyses planned after a 16-week follow-up for the first ten patients and then every five patients.

At each interim analysis, stopping rules recommended that the trial be stopped due to inefficacy (if there was a high predictive probability [>80%] that the objective response rate was less than or equal to 20%, the minimum response probability threshold) or efficacy (if there was a high predictive probability [>80%] that the objective response rate was greater than or equal to 40%, the maximum response probability threshold).

The efficacy population included all participants who met the eligibility criteria and who received at least one complete or two incomplete treatment cycles. All enrolled patients who initiated the study treatment were included in the safety analysis.

The median follow-up was calculated using the reverse Kaplan-Meier method, and survival endpoints were described using the Kaplan-Meier method. Data for patients who were alive and event-free were censored at the date of the last follow-up.

Quantitative variables were described using the median and range. Qualitative variables were described using frequency, rates, and the 95% confidence interval (95%CI) (binomial law). Estimated parameters are reported with two-sided 95% CIs. P values less than 0.05 (typically < 0.05) were considered to be statistically significant. The statistical analyses were performed using SAS software (version 9.4).

This study is registered with ClinicalTrials.gov, number NCT03475953.

Tissue sample analysis

Tumor biopsies were collected at baseline and on Cycle 2 Day 1. These samples were analyzed to characterize the impact of regorafenib combined with avelumab on the tumor microenvironment and to identify potential predictive biomarkers of clinical benefit. Immunohistofluorescence analysis was performed on the automated Ventana Discovery XT staining platform (Ventana Medical Systems). Slides of tumor tissue were deparaffinized by heating to 69°C combined with the application of Discovery Wash solution during 8 minutes. This operation was repeated 3 times.

The slides were incubated with the following primary antibodies: anti-CD8 (clone C8/144B, Dako), anti-CD163 (clone 10D6, Leica), anti-PD-L1 (clone QR1, Diagomics), and anti-cytokeratine 19 (clone A53-B/A2.26, Ventana). Bound primary antibodies were detected using either OmniMap anti-Ms or Rb-HRP with the Opal detection kit (Akoya). The slides were counterstained with spectral DAPI (Akoya) and cover-slipped. The stained slides were imaged on a multispectral imaging system (Vectra Polans, Akoya) and analyzed using Inform image analysis software (Akoya, version 2.4.1) to segment the tissue into tumor and stroma and to phenotype the cells.

Role of the funding source

The study was sponsored by the Institut Bergonié, Comprehensive Cancer Center (Bordeaux, France). The data were collected with the sponsor data management system and were analyzed and interpreted by representatives of the sponsor in collaboration with the investigators. SC, CC, CB, and AI had access to the raw data.

The funders of the study (Bayer and Merck) had no role in the study design, data collection, data interpretation, or writing of the report. The corresponding author had full access to all of the data in the study and had the final responsibility for deciding to submit the manuscript for publication.

RESULTS

A total of 34 patients were recruited for the study between November 21, 2018 and November 13, 2019. According to the Bayesian adaptive design, inclusion was stopped prematurely, as there was a high predictive probability (98.6%) that the objective response rate was less than or equal to 20%. Twenty-nine patients were eligible and included in the efficacy endpoint. Five patients were excluded from efficacy analysis for reasons prespecified in the protocol: one due to protocol deviation regarding the inclusion criteria and four because of early discontinuation without any tumor assessment (Figure 1). The patient characteristics are summarized in Table 1. The median age was 63 years (range 36-80), and 47.1% of the patients were women. All of the patients had already received systemic treatment for advanced disease, with a median of two (range 1-4) previous lines of treatment.

Out of the 29 patients included in the efficacy assessment, four patients (13.8%) achieved a partial response; as such, the primary efficacy criterion was not reached.

Regarding the best overall response according to RECIST, 11 (37.9%) patients demonstrated stable disease, including 10 patients (34.5%) with tumor shrinkage (range from -1.2% to -85.6%) (Figure 2). Fourteen patients (48.3%) had progressive disease. Among 15 patients with objective response or stable disease at 6 months, 14 had an iCCA intra-hepatic cholangiocarcinoma. The median duration of response was 10.4 months.

The median follow-up was 9.8 months (95%CI 6.6-12.4). The median PFS was 2.5 months (95%CI 1.9-5.5). The 6-and 12-month PFS were 27.6% (95%CI 13.1-44.3) and 6.9% (95%CI 0.6-24.3), respectively.

At the time of analysis, 24 patients discontinued treatment. Reasons for treatment discontinuation were disease progression for 23 patients (95.8%) and alteration of general status for 1 patient (4.2%). Seventeen patients (58.6%) patients were still alive, with 5 (17.2%) still under treatment. The median OS was 11.9 months (95%CI 6.2-NA) (Figure 3). The 6-and 12-month OS rates were 75.7% (95%CI 53.5-88.3) and 44.4% (95%CI 19.0-67.3), respectively. Thirty-four patients received at least one dose of regorafenib and/or avelumab and were therefore evaluated for safety. The treatment was generally well-tolerated.

Treatment-related adverse events and laboratory abnormalities that were reported in more than 5% of patients for grades 1-2 and any for grades 3 and 4 are shown in Table 2. The most common clinical treatment-related adverse events were fatigue, infusion-related reaction, palmo-plantar erythrodysesthesia, diarrhea, dysphonia, and mucositis. The most common treatment-related lab abnormalities were transaminitis and thyroid-stimulating hormone (TSH) increase. At least one serious adverse event was reported in 21 patients (62%). Twenty-six (76.5%) and 19 (55.9%) patients experienced treatment modifications with regorafenib (19 patients with temporary discontinuation, 12 patients with dose reduction, and 3 patients with permanent discontinuation) and avelumab (12 patients with perfusion interruption, 10 patients with administration cancellation but not permanent discontinuation), respectively. No patient died from drug-related toxicity.

Analysis of the tumor biopsies revealed that high expression by tumor cells of PD-L1

and indoleamine 2,3-dioxygenase 1 (IDO1) were associated with a better durable clinical benefit rate and improved PFS (46.15% for high PD-L1 expression versus 7.14% for low PD-L1 expression, 85.7% for high IDO1 expression versus 7.14% for low IDO1 expression; 5.45 (1.68-NA) months for high PD-L1 expression versus 2.28 (1.87-5.78) months for low PD-L1 expression, 5.78 (2.00-NA) months for high IDO1 expression versus 1.91 (1.84-5.45) months for low IDO1 expression) (Figure 4). A comparison of the tumor microenvironment between iCCA and eCCA revealed a higher infiltration of eCCA by tumor-associated macrophages (TAMs) (p=0.052) (Supplementary Figure 1).

DISCUSSION

Biliary tract cancers are a heterogeneous group of adenocarcinomas for which nextgeneration sequencing has revealed several targetable alterations including fibroblast growth factor receptor (FGFR) fusion, isocitrate dehydrogenase (IDH) mutations, the human epidermal growth factor receptor (HER) family, DNA damage repair (DDR) pathways, and BRAF mutations with promising clinical results. [START_REF] Valle | New Horizons for Precision Medicine in Biliary Tract Cancers[END_REF] Although the first objective of our study was not reached, nearly 50% of patients experienced clinical benefit resulting in objective response (13.8%) or stable disease with tumor shrinkage (34.5%).

Several studies have shown that molecules such as VEGF and fibroblast growth factor that play a crucial role in tumor angiogenesis are upregulated in BTC. [3,[START_REF] Moeini | Molecular Pathogenesis and Targeted Therapies for Intrahepatic Cholangiocarcinoma[END_REF] Interestingly, preclinical experiments investigating anti-angiogenic drugs such as bevacizumab, [START_REF] Vaeteewoottacharn | Inhibition of carbonic anhydrase potentiates bevacizumab treatment in cholangiocarcinoma[END_REF] sorafenib, [START_REF] Sugiyama | Potent in vitro and in vivo antitumor activity of sorafenib against human intrahepatic cholangiocarcinoma cells[END_REF] vandatenib, [START_REF] Yoshikawa | Vandetanib (ZD6474), an inhibitor of VEGFR and EGFR signalling, as a novel molecular-targeted therapy against cholangiocarcinoma[END_REF] and axitinib [START_REF] Takahashi | Axitinib (AG-013736), an oral specific VEGFR TKI, shows potential therapeutic utility against cholangiocarcinoma[END_REF] have shown that such agents can reduce both tumor growth and neo-angiogenesis in xenograft models of BTC.

Regorafenib is the most-investigated antiangiogenic drug in patients with advanced BTC who have had progression despite standard therapy. [START_REF] Demols | Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial[END_REF][START_REF] Kim | A multi-institutional phase 2 trial of regorafenib in refractory advanced biliary tract cancer[END_REF][START_REF] Sun | A phase 2 trial of regorafenib as a single agent in patients with chemotherapy-refractory, advanced, and metastatic biliary tract adenocarcinoma[END_REF] Demols et al.

reported the first double-blind, placebo-controlled clinical trial investigating regorafenib in this setting. A total of 66 patients with refractory BTC were randomized to receive regorafenib or a placebo. [START_REF] Demols | Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial[END_REF] The study met its endpoint by showing an improvement from 1.5 to 3 months (HR, 0.49; 95%CI, 0.29-0.81; p=0.005). Despite demonstrating some level of regorafenib activity in patients with refractory BTC, these studies showed that such patients have a very poor outcome, with a median PFS and OS ranging from 3-3.9 months and 5.3-7.9 months, respectively. [START_REF] Demols | Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial[END_REF][START_REF] Kim | A multi-institutional phase 2 trial of regorafenib in refractory advanced biliary tract cancer[END_REF][START_REF] Sun | A phase 2 trial of regorafenib as a single agent in patients with chemotherapy-refractory, advanced, and metastatic biliary tract adenocarcinoma[END_REF] Monoclonal antibodies targeting PD-1 and PD-L1 represent one of the most important breakthroughs in cancer treatment for several tumor types. Studies that have evaluated these agents in patients with BTC report activity in a subset of patients, with response rates ranging from 3-11%, median PFS of 1.4-3.7 months, and median OS of 5.2-14.2 months. Interestingly, when responses occurred, they were durable-all lasting more than 6 months. [START_REF] Piha-Paul | Efficacy and safety of pembrolizumab for the treatment of advanced biliary cancer: Results from the KEYNOTE-158 and KEYNOTE-028 studies[END_REF][START_REF] Kim | A Phase 2 Multi-institutional Study of Nivolumab for Patients With Advanced Refractory Biliary Tract Cancer[END_REF] Anti-angiogenic agents can reduce immunosuppression.

Conversely, immunotherapies can impact the vasculature and have antivascular effects; thus, immunotherapy and anti-angiogenic drugs have the potential to create a virtuous cycle of immunostimulation and vascular remodeling within tumors. [START_REF] Yi | Synergistic effect of immune checkpoint blockade and anti-angiogenesis in cancer treatment[END_REF][START_REF] Rahma | The Intersection between Tumor Angiogenesis and Immune Suppression[END_REF] This potential synergy has been shown in several preclinical models and in the clinical setting, as illustrated by the recent approval of axitinib plus avelumab or pembrolizumab as a first-line treatment for advanced or metastatic renal cell carcinoma. [START_REF] Motzer | Avelumab plus Axitinib versus Sunitinib for Advanced Renal-Cell Carcinoma[END_REF] Little is known about the tumor microenvironment of BTC. Recent data have shown that the immunological profile of BTC is not dependent on the IDH1/2 mutational status [START_REF] Makawita | IDH1 and IDH2 Driven Intrahepatic Cholangiocarcinoma (IHCC): A comprehensive genomic and immune profiling study[END_REF] . In iCCA, two subgroups have been defined based on transcriptomic analysis; one of which includes up to 40% of cases and is characterized by an immunological signature resulting from strong T lymphocyte infiltration and the activation of immune checkpoints. [START_REF] Sia | Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes[END_REF] Integrative molecular characterization of eCCA has also revealed that only a minority of BTCs (11%) are characterized by an inflammation signature. [START_REF] Montal | Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma[END_REF] Interestingly, in our study, all but one patient with clinical benefit (objective response or stable disease) had an iCCA. Moreover, we found that eCCA had higher levels of TAM infiltration. Several lines of evidence suggest the important role of TAMs in cholangiocarcinoma progression, and their presence has been associated with poor outcomes. [START_REF] Loeuillard | Immunobiology of cholangiocarcinoma[END_REF] In our cohort, patients with high PD-L1 expression were more likely to benefit from the treatment. These results agree with those of previous studies showing that high PD-L1 expression was associated with improved outcomes in patients with BTC treated with immune checkpoint inhibitors [START_REF] Ahn | High PD-L1 expression is associated with therapeutic response to pembrolizumab in patients with advanced biliary tract cancer[END_REF] . We also observed that high IDO1 expression was significantly associated with improved outcomes. IDO1 is a ratelimiting enzyme involved in the metabolism of the essential amino acid tryptophan and plays a crucial role in modulating the tumor microenvironment [25] . Our results are also in line with those of previous studies revealing that certain subsets of patients with solid tumors exhibiting IDO1 overexpression had favorable outcomes on anti-PD-1 therapy. A possible explanation is that IDO1 overexpression is, in this context, a surrogate of the presence of tumor-reactive T cells within the tumor microenvironment. For instance, Hamid, et al. found that high IDO1 expression correlated with an improved efficacy of ipilimumab in patients with metastatic melanoma. [26] Analyses of other patient cohorts, including patients with non-small cell lung cancer, have shown similar results. [27] Most of the patients required dose reductions of regorafenib due to adverse events.

A recent open-label trial that included 116 evaluable patients with metastatic colorectal cancer showed that a regorafenib dose-escalation strategy (starting dose of 80 mg/day with weekly escalation in 40 mg increments to 160 mg/day if no significant drug-related adverse events occurred) may represent an alternative approach for optimizing regorafenib dosing with comparable activity and a lower incidence of adverse events compared with a standard dose strategy (160 mg/day for 21 days of a 28-day cycle). [28] Such a strategy may be worth investigating when combining regorafenib with immune-oncology agents.

Although one limitation of our study is the limited sample size in relation to the heterogeneity of BTC, our results demonstrated clinical activity of the combined regorafenib and avelumab therapy in a subset of patients with advanced BTC.

Further studies should investigate such a regimen in a population selected based on a favorable tumor microenvironment signature (high PD-L1 and/or high IDO1 expression).

Funding:

The

Figure 2 .Figure 3 .

 23 Figure 2. Waterfall plot (A) and spider plot (B) of best overall response in biliary tract cancer patients treated with regorafenib plus avelumab (n=29, Response based on central review assessment according to RECIST 1.1)

Figure 4 .

 4 Figure 4. IDO1 and PDL1 expressing tumors are associated with a favorable clinical outcome in patients with BTC treated with regorafenib combined with velumab. Illustration of IDO1 and Cytokeratin 19 (CK19) stainings in tumor samples -collected before treatment onset -from a) Patient #1 with negative/low IDO1 expression and a poor clinical outcome (PFS: 1,8 mo) and b) Patient#2 with high IDO1 expression and a favorable clinical outcome (PFS: 7,5 mo). c) Rates of durable clinical benefit according to the expression level of IDO1 within CK19 positive tumor cells. d) Kaplan-Meier curves of progression-free survival according to the expression level of IDO1 in CK19 positive cells; blue curve: low IDO1 expression and red curve: high IDO1 expression. Illustration of PDL1 and Cytokeratin 19 (CK19) stainings in tumor samples -collected before treatment onset -from e) Patient #3 with low tumor expression of PDL1 and a poor clinical outcome (PFS: 3,8 months) and f) Patient#4 with high PDL1 expression by tumor cells and a favorable clinical outcome (PFS: 14,9 months). g) Rate of clinical benefit according to the expression level of PDL1 within tumor cells. h) Kaplan-Meier curves of progression-free survival according to the expression level of PDL1 in tumor cells; blue curve: low PDL1 expression and red curve: high PDL1 expression.

  study was sponsored by the Institut Bergonié, Comprehensive Cancer Center (Bordeaux, France). The funders of the study (Bayer and Merck) had no role in the study design, data collection, data interpretation, or writing of the report.

	TABLES
	Tables 1. Patients characteristics
		Eligible patients for safety
		N=34
	Median age (years, range)	63.1 (36 -80)
	Gender
	Female	16 (47.1%)
	Male	18 (52.9%)
	Performans status ECOG
	0	16 (47.1%)
	1	17 (50.0%)
	Unknown	1 (2.9%)
	Tumor location
	Intra-hepatic	26 (76.5%)
	Extra-hepatic	7 (20.6%)
	Gallbladder	1 (2.9%)
	Number of metastatic sites
	Single	6 (17.6%)
	Multiple	28 (82.4%)
	Metastatic sites
	Liver	27 (79.4%)
	Lung	20 (58.8%)
	Peritoneum	14 (41.2%)
	Node	18 (52.9%)
	Other	11 (32.4%)
	Previous chemotherapy treatment
	Platinum-based	34 (100%)
	Gemcitabine-based	34 (100%)
	Topoisomerase I or II inhibitor	13 (38.2%)
	Taxanes	3 (8.8%)
	Previous lines of treatment for
	advanced disease
	1	14 (41.2%)
	2	12 (35.3%)
	>2	8 (23.5%)