Anthony Lethuillier
email: anthony.lethuillier@latmos.ipsl.fr

Alice Le Gall

Michel Hamelin

Sylvain Caujolle-Bert

Francis Schreiber

Nathalie Carrasco

Guy Cernogora

Cyril Szopa

Yann Brouet

Fernando Simões

F Simoes

J J Correia

G Ruffié

Electrical properties of tholins and derived constraints on the Huygens landing site composition at the surface of Titan

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

Introduction

On January 14, 2005, the Huygens probe [START_REF] Lebreton | The Huygens mission to Titan: an overview, in Titan -From Discovery to Encounter[END_REF], part of the Cassini/Huygens mission (NASA/ESA/ASI), landed on the surface of Titan, Saturn's biggest moon [START_REF] Lebreton | An overview of the descent and landing of the Huygens probe on Titan[END_REF]. After two hours and a half of descent, the probe touched a solid and possibly wet ground [START_REF] Zarnecki | A soft solid surface on Titan as revealed by the Huygens Surface Science Package[END_REF][START_REF] Atkinson | Penetrometry of granular and moist planetary surface materials: Application to the Huygens landing site on Titan[END_REF] at a latitude of 10.3 • S and a longitude of 192.4 • W [START_REF] Karkoschka | DISR imaging and the geometry of the descent of the Huygens probe within Titan's atmosphere[END_REF]. While the measurements performed by the instruments on board Huygens cannot be regarded as representative of the entire surface of Titan, they do provide an important ground truth for the understanding of the observations of the Cassini orbiter or Earth-based telescopes.

Among the instruments on board the Huygens probe, a Mutual Impedance Probe (MIP), called PWA-MIP/HASI (Permittivity Wave Altimetry-Mutual Impedance Probe/Huygens Atmospheric Structure Instrument), measured for the first time the low-frequency (namely at 45, 90 and 360 Hz) electrical properties (i.e., the dielectric constant and electrical conductivity) of the first meter of Titan's surface [START_REF] Fulchignoni | In situ measurements of the physical characteristics of Titan's environment[END_REF]. [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] recently refined the analysis of the PWA-MIP/HASI observations, accounting for new insights on the final resting position of the Huygens capsule [START_REF] Schröder | Bouncing on Titan: Motion of the Huygens probe in the seconds after landing[END_REF].

They inferred a dielectric constant of 2.5 ± 0.3 and a conductivity of 1.2 ± 0.6 nS/m at 45 Hz, values which are in agreement with previously published results but with much more reliable error bars. Following [START_REF] Grard | Electric properties and related physical characteristics of the atmosphere and surface of Titan[END_REF], [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] also reported that a sudden change in the electrical properties of the ground, namely a drop of its electrical conductivity, was recorded about 11 min after the Huygens landing. Several scenarios have been advanced to explain this event but more information on the electrical properties of materials relevant to Titan's surface at PWA-MIP/HASI frequencies are required to better understand PWA-MIP/HASI results in terms of near-surface composition and to conclude on the most plausible scenario for the observed sudden change in conductivity.

Prior to the Cassini mission, the composition of Titan's surface was thought to be that of its bulk crust i.e., dominated by water ice [START_REF] Tobie | Titan's internal structure inferred from a coupled thermal-orbital model[END_REF]. This was supported by the detection of water ice by [START_REF] Griffith | Evidence for the Exposure of Water Ice on Titan's Surface[END_REF] from telescope infrared spectroscopy measurements. However, since then, Cassini-Huygens observations have shown that water ice is only exposed at the surface of Titan at isolated locations including interdune corridors [START_REF] Barnes | Spectroscopy, morphometry, and photoclinometry of titan's dunefields from cassini/vims[END_REF]Le Gall et al., 2011] and crater rims [START_REF] Janssen | Titan's surface at 2.18-cm wavelength imaged by the Cassini RADAR radiometer: Results and interpretations through the first ten years of observation[END_REF]. In-stead, the surface seems to be covered by a layer of organic materials, by-products of the intense photochemistry activity of Titan's atmosphere [START_REF] Clark | Detection and mapping of hydrocarbon deposits on Titan[END_REF][START_REF] Janssen | Titan's surface at 2.18-cm wavelength imaged by the Cassini RADAR radiometer: Results and interpretations through the first ten years of observation[END_REF].

The atmosphere of Titan is indeed the host of a complex photochemistry that produces a wealth of solid organic matter (or aerosols) by dissociation (mainly by solar UV rays) and recombination of the molecules N 2 and CH 4 [START_REF] Lorenz | Lifting Titan's Veil: Exploring the Giant Moon of Saturn[END_REF][START_REF] Waite | The Process of Tholin Formation in Titan's Upper Atmosphere[END_REF]. The heaviest of these aerosols are eventually deposited onto the surface, progressively forming a thick sedimentary layer of organics. With time, surface processes (e.g., aeolian/pluvial/fluvial erosion, impacts, etc...) erode this layer and/or mix organics with water ice (see [START_REF] Lopes | Distribution and interplay of geologic processes on titan from cassini radar data[END_REF] for a review).

The present work aims at inferring new constraints on Titan's near-surface composition (in particular on its water-ice/organic mixture) from the electrical measurements made by PWA-MIP/HASI by comparison with laboratory measurements. While the electrical properties of water ice are well known [START_REF] Mattei | Dielectric measurements and radar attenuation estimation of ice/basalt sand mixtures as martian Polar Caps analogues[END_REF], those of Titan's aerosols have never been measured at low frequencies in laboratory. In this paper, we present the measurement bench that we have developed to fill this gap. Measurements were performed on analogs of Titan's aerosols called "tholins" at a wide range of temperatures from room temperature down to almost 90 K. Measurements made at Titan's surface temperature (93.65 ± 0.25 K [START_REF] Fulchignoni | In situ measurements of the physical characteristics of Titan's environment[END_REF]) were then used to better understand the implications of the PWA-MIP/HASI observations at the Huygens landing site (hereafter referred as to HLS).

Materials and Methods

Productions of tholins

The term "tholins" was introduced by Sagan and Khare [1979] to name the "brown, sometimes sticky, residue" they produced in laboratory at Cornell University by irradiating with various sources of energy a mixture of the cosmically abundant gases CH 4 , C 2 H 6 , NH 3 , H 2 O, HCHO, and H 2 S. The definition was later expanded to include many other components produced from different mixtures and even by irradiation of ices. It now seems that tholin-like materials are present in many bodies in the Solar System: on Pluto [START_REF] Grundy | Surface compositions across Pluto and Charon[END_REF], Charon [START_REF] Grundy | Surface compositions across Pluto and Charon[END_REF]], comets [McDonald et al., 1996;[START_REF] Stern | First extreme and far ultraviolet spectrum of a Comet Nucleus: Results from 67p/Churyumov-Gerasimenko[END_REF], Triton [McDonald et al., 1994] and, of course, Titan [START_REF] Sagan | Polycyclic aromatic hydrocarbons in the atmospheres of Titan and Jupiter[END_REF].

Several set ups have been developed to synthesize and study analogs of planetary organic aerosols in laboratory (e.g., [START_REF] Szopa | PAMPRE: A dusty plasma experiment for Titan's tholins production and study[END_REF][START_REF] Cable | Titan Tholins: Simulating Titan Organic Chemistry in the Cassini-Huygens Era[END_REF]). In particular, the PAMPRE (French acronym for Production d'Aérosols en Microgravité par Plasma REactifs -Aerosols Production in Microgravity by Reactive Plasma, [START_REF] Szopa | PAMPRE: A dusty plasma experiment for Titan's tholins production and study[END_REF]) experiment developed at LATMOS has been designed to reproduce the complex photochemistry of Titan's atmosphere. PAMPRE simulates the UV photolysis of N 2 and CH 4 and the induced photochemistry using a Capacitively Coupled Plasma (RF-CCP) discharge. This discharge is applied to a mixture of N 2 and CH 4 with a N 2 /CH 4 ratio in the range 90/10 to 99/1, consistent with Titan's atmosphere composition at different altitudes [START_REF] Waite | The Process of Tholin Formation in Titan's Upper Atmosphere[END_REF]. Inelastic electron impacts dissociate N 2 and CH 4 [START_REF] Szopa | PAMPRE: A dusty plasma experiment for Titan's tholins production and study[END_REF] and the resulting ions, neutral atoms and radicals recombine to form solid particles [START_REF] Carrasco | Volatile products controlling Titan's tholins production[END_REF]. The advantage of using a capacitive coupled RF plasma discharge is that the produced particles are in levitation between the two electrodes due to electrostatic forces and only deposit once they are heavy enough. This prevents them from interacting with the reactor walls that could act like a catalyst [START_REF] Szopa | PAMPRE: A dusty plasma experiment for Titan's tholins production and study[END_REF].

The PAMPRE-produced tholins are small spherical particles with a mean diameter between 0.093 and 1.070 µm [START_REF] Hadamcik | Laboratory light-scattering measurements with Titan's aerosols analogues produced by a dusty plasma[END_REF]; they are regarded as possible analogs of Titan's organic aerosols. They form aggregates which can be observed micro-scopically and have the macroscopic appearance of an orange powder (see Figure A.1) in agreement with the color of Titan's haze.

The PAP measurement bench

The central instrument of the PAP (French acronym for Permittivité d'Analogues Planétaires, permittivity of planetary analogs) measurement bench developed at LATMOS is a spectral analyzer (Solartron Modulab Material Test System©). The spectral analyzer uses a dipole system to generate an alternating current at frequencies in the range 1 Hz -100 kHz between two electrodes separated by the cylindrical sample of material to be analyzed. The current and potential on the electrodes are then measured giving access to the complex impedance of the sample and thereby to its complex permittivity i.e., both its dielectric constant and electrical conductivity . The PAP electrodes are cylindrical with a radius of 10 mm and a height of 8 mm. An additional module was installed in order to measure currents down to the femto-Ampere which is necessary for materials with low dielectric constant and/or conductivity.

The PAP measurement bench also includes:

1. A mechanical press to produce samples of varying bulk density. Samples are shaped as cylinder pellets of 10 mm diameter with a height of 1 mm. A pressure of up to 8.8 × 10 8 Pa (maximum tonnage of 7 tons) can be applied to them.

2. A sample holder in which the electrodes and the sample are placed.

3. A pump to create vacuum (minimum 10 -2 mbar) in the sample holder in order to prevent contamination by water ice (formed by condensation of the water in the air) of the sample when the temperature drops. 4. A cryostat to perform measurements at low temperatures. Cryogenic temperatures are reached by passive cooling, filling up with liquid nitrogen the reservoir in which the sample holder is placed. The temperature is recorded on both electrodes by PT100 temperature sensors. The temperatures recorded on the two electrodes are very close and we use the average of these two measurements to estimate the sample temperature. The lowest achievable temperature with this device is about 94 K, i.e. Titan's surface temperature at the HLS, and is obtained after ≈1 hr of cooling.

After about 10 hrs all liquid nitrogen is evaporated and the temperature increases back to room temperature within about an additional 10 hrs. The temperature range for our measurements is therefore from 94 K up to room temperature.

The PAP set-up is designed to measure the complex permittivity of solid samples.

In a harmonic regime and using complex notation, it is common to refer to the relative complex permittivity of the medium (i.e., relative to that of vacuum 0 = 8.854 × 10 -12 F/m) defined for fields and potentials in exp( jωt) as follows:

r = r -j r (1)
The real part of the permittivity r (hereafter referred to as the dielectric constant)

describes the polarizability of a material when an electrical field is applied. Its imaginary part r is related to the electrical effective conductivity σ as follows: r = σ/2π f 0 with f the working frequency. We note that while pure dielectric materials have, by definition, a zero true conductivity (i.e., no free charge), polarization mechanisms induce the heat dissipation of electrical energy, which gives rise to a non-null effective conductivity.

The derivation of the complex permittivity from PAP measurements is described in Appendix A.

Description of the samples

The tholin samples were produced by the PAMPRE experiment for 3 different compositions of the initial gas mixture: 2%, 5% and 8% of CH 4 . We investigated 15 samples i.e., 5 per composition totaling up to 300 hours of measurements most of which could be done unattended.

The material density of the tholins was measured using a helium pycnometer (Upyc-1200e-V5.04) which evaluates the volume occupied by a given sample with a known mass in a calibrated chamber by inserting helium gas in the chamber. The measured material densities (i.e. zero-porosity densities) are: ρ tholins2% = 1.45±0.02, ρ tholins5% = 1.44±0.03 and ρ tholins8% = 1.34 ± 0.03. The porosity of the compressed samples measured with PAP can be readily inferred from these material densities. We find that the porosity of our samples is in the range 10-40%. produces lower density tholins.

Futhermore, the composition of the initial gas mixture has an effect on the optical properties of the tholins; the higher the methane content the darker they appear. Additionally, the 2% samples were found to be less sticky and making pellets out of them proved to be more difficult than for the other compositions.

3 Complex permittivity of tholins We use these measurements to determine the complex permittivity of the bulk tholins (i.e., with no porosity). This can be achieved by using a mixing law to "correct" the electrical properties of the investigated tholin samples for the contribution of vacuum. More specifically, we use the Maxwell-Garnett law [START_REF] Garnett | Colours in Metal Glasses and in Metallic Films[END_REF], which is the mixing law that best fits the data points presented in Figure A.2. This law relates the complex permittivity of the sample to its porosity p as follows:

Effect of porosity

r sample = r m + 3p r m 1 -r m 1 + 2 r m -p(1 -r m ) (2)
where r m is the bulk permittivity of tholins that can then be estimated by solving the second-degree equation:

2(p -1) 2 r m (p r sample -2p + 2 r m -1) r m + r sample (1 -p) = 0 (3)
The Maxwell-Garnett mixing law assumes that the void (spherical) pores (valid for p < 0.5) are smaller than the wavelength (300 m) which is readily achieved in our case.

For example, at 94 K, 100 Hz and for a 5% methane composition the corrected complex permittivity is 4.5-j0.02.

Effect of frequency and temperature

Figure A.3 shows the complex permittivity of bulk tholins (i.e., once the correction described in section 3.1 has been applied) as a function of frequency and temperature for the three considered initial gas mixture compositions. The continuous lines and the shaded areas respectively represent the average and the standard deviation (at 2-σ) of the 5 samples measured per composition.

As expected, the dielectric constant increases with a decreasing frequency because polarization mechanisms have more time to take place when the applied electric field is slowly oscillating. Also the frequency dependence tends to disappear and the dielectric constant decreases when the temperature decreases i.e., when charge carriers become too slow to follow the applied electric field oscillations whether these oscillations are rapid or not. In particular, at Titan's surface temperature the dielectric constant seems to have reached its static limit.

The imaginary part of the complex permittivity displays a maximum at a frequency that moves towards lower values when the temperature decreases (1000 Hz at 220 K and 1

Hz at 170 K). The relaxation process at play may be a Maxwell-Wagner effect (i.e., interfacial polarization between the pores and the tholin matrix). At very low temperatures (in particular at 94 K), the relaxation peak is not visible anymore. Like for the real part, the imaginary part of the permittivity decreases with decreasing temperatures.

Effect of composition

We note a significant change in the electrical properties as a function of the composition of the gas mixture (i.e., the N 2 /CH 4 ratio) from which they have been produced.

The "5%-tholins" have the largest dielectric constant while the "8%-tholins" have the smallest (Figure A.4). This is true for all frequencies in the range 1 Hz-100 kHz.

For temperature in the range 125-225 K, the imaginary part of the permittivity of the tholins increases with an increasing methane proportion in the PAMPRE gas mixture.

Outside this temperature range, the "2%-tholins" methane proportion still has the smallest imaginary part of the permittivity but the "5% tholins" has the largest (see Figure A.4).

This is true for all frequencies in the range 1 Hz-100 kHz.

At 94 K and at a given frequency the dielectric constant varies by 0.5 and the imaginary part of the permittivity by 0.02 between the 3 studied compositions. This variation is larger than the accuracy of the spectral analyzer and higher than the dispersion of the measurements performed on multiple samples which gives us, in theory, the ability to distinguish between the different compositions.

Tholins show very low concentration of polycyclic aromatic hydrocarbons [START_REF] Derenne | New insights into the structure and chemistry of titan's tholins via 13c and 15n solid state nuclear magnetic resonance spectroscopy[END_REF][START_REF] Mahjoub | Characterization of aromaticity in analogues of titan's atmospheric aerosols with two-step laser desorption ionization mass spectrometry[END_REF], therefore, the differences observed in the electrical properties are probably not due to the presence of aromatic components. Mahjoub et al.

[2012] investigate the optical indices of tholins as a function of the methane percentage used in the initial gas mixture. They find that the 2% tholins are far more absorbant in the UV-visible than the 8% tholins and explain this difference by the quantity of nitrogen incorporated in the tholins, primarily in amines, which was found to be higher at low methane fraction in the initial gas. In amines, the nitrogen has a non-binding electrical doublet which results in a higher dielectric constant. This could explain the differences between the 8% and 5% tholins but the fact that the 2% tholins have a lower dielectric constant than the 5% suggests that additional unknown mechanisms influence the electrical properties.

The measured dielectric constant of tholins at low frequency is relatively close but different from that of water ice (3.15 at cryogenic temperatures [START_REF] Mattei | Dielectric measurements and radar attenuation estimation of ice/basalt sand mixtures as martian Polar Caps analogues[END_REF]) or of Martian analog JSC-Mars-1 (2.0-2.5 at 220 K [START_REF] Simões | Laboratory Measurements on Martian Soil Simulant JSC Mars-1 Supporting the Calibration of Instruments for Planetary Missions[END_REF]). Furthermore, at low frequencies most rocky materials present on the Earth have a dielectric constant in the range 3.4 (dry sandy soil [START_REF] Clark | Handbook of Physical Constants[END_REF]) -13.0 (sandstone [START_REF] Clark | Handbook of Physical Constants[END_REF]).

Dielectric relaxation model for tholins

One of the most commonly used models to represent the frequency dependence of the electrical properties of material is the Cole-Cole model with conduction [START_REF] Cole | Dispersion and Absorption in Dielectrics I. Alternating Current Characteristics[END_REF]:

r ( f , T) = ∞ (T) + s (T) -∞ (T) 1 + ( j2π f τ(T)) 1-α -j σ s 2π f 0 (4)
where ∞ is the relative high-frequency limit permittivity, s the static (low-frequency limit) relative permittivity, τ the relaxation time in seconds, σ s the static conductivity and α an exponent representing the broadness of the relaxation transition. It is reduced to the Debye equation when α = 0. Complementary electrical characterization of tholins are available in literature (see Table 1). In particular, [START_REF] Rodriguez | Impact of aerosols present in Titan's atmosphere on the CASSINI radar experiment[END_REF] and [START_REF] Paillou | Microwave dielectric constant of Titan-relevant materials[END_REF] measured the complex permittivity of tholins obtained from a 2% methane atmosphere in the Ku-band (namely at 10 and 13 GHz) and at 77 K to support the analysis of the Cassini Radar observations [START_REF] Elachi | Radar: The cassini titan radar mapper[END_REF]. [START_REF] Paillou | Microwave dielectric constant of Titan-relevant materials[END_REF] reported a permittivity of 1.17-j0.0033

for a "non-compacted sample" of tholins and of 2.33-j0.0206 for a "compacted sample".

These values are logically below the ones measured at lower frequencies as explained in section 3.2. It would have been useful to know the actual porosity of the samples investigated in the Ku-band in order to determine the permittivity of bulk tholins in this frequency range as we did in the 1 Hz-100 kHz domain (section 3.1). However, we do know that the "compacted samples" were obtained with a press tonnage of 15 t, which suggests that their porosity is at most 10 % [Paillou, personal communication] hence a maximum permittivity of 2.55-j0.025 based on the Maxwell-Garnett law. Interestingly, we note that the dielectric constant of tholins is larger than that of water ice at low frequencies while it is the opposite in the Ku-band.

More recently, [START_REF] Brouet | A porosity gradient in 67p/C-G nucleus suggested from CONSERT and SESAME-PP results: an interpretation based on new laboratory permittivity measurements of porous icy analogues[END_REF] measured the dielectric constant of tholins synthesized from a 5% methane atmosphere in the frequency range 50 MHz-2 GHz but at a warm temperature of 243 K.

Therefore, contrary to water ice, tholins do not seem to fully lose the frequency dependence of their dielectric constant at very low temperature. The main polarization mechanism responsible for the dielectric constant of water ice is the reorientation of H 2 O molecules. This mechanism is greatly affected by temperature and this explains why the dielectric constant of water ice loses its frequency dependence at temperature below about 150 K [START_REF] Mattei | Dielectric measurements and radar attenuation estimation of ice/basalt sand mixtures as martian Polar Caps analogues[END_REF]. In the case of tholins, the polarization mechanisms at stake must be more complex and more numerous. Their investigation is out of scope of this paper.

4 Titan's subsurface composition at the Huygens Landing site

State of art: permittivity of Titan's near-surface, case of the HLS

The first estimate of the dielectric constant of Titan's near-surface was derived from observations from the Arecibo radar system: Campbell et al. [2003] found a range of di-electric constants of 1.5-2.2 with a mean value of 1.8 for Titan's 26 • S latitude band (we recall that the Huygens landing site is at a latitude of 10.3 • S). After the arrival of the Cassini probe at Saturn, the Radar on board (operating at 13.78 GHz, [START_REF] Elachi | Radar: The cassini titan radar mapper[END_REF])

provided new and spatially resolved constraints on the dielectric constants of Titan's nearsurface both in its real-aperture active (scatterometry) and passive (radiometry) modes.

The analysis of the scatterometry data yields dielectric constants in the range 1.9-3.6 with a mean value of 2.2 [START_REF] Wye | Electrical properties of Titan's surface from Cassini RADAR scatterometer measurements[END_REF]. This analysis is based on raster scanning of large regions at a resolution of about 100 km. No comprehensive investigation of the dielectric constant of the HLS has been published yet. However, it probably exhibits a dielectric constant close to that measured in the plains of Titan, namely, 2.12 ± 0.07 [START_REF] Wye | Electrical properties of Titan's surface from Cassini RADAR scatterometer measurements[END_REF].

Radiometry insights into the surface dielectric constant come in two flavors. First, when the spacecraft is far away from Titan (25 000-100 000 km), the disk is scanned twice in two orthogonal polarizations (the spacecraft is rotated between the two scans).

These polarization pairs can be used to produce a global map of the surface dielectric constant [START_REF] Janssen | Titan's surface at 2.2-cm wavelength imaged by the Cassini RADAR radiometer: Calibration and first results[END_REF][START_REF] Janssen | Titan's surface at 2.18-cm wavelength imaged by the Cassini RADAR radiometer: Results and interpretations through the first ten years of observation[END_REF]. This map is essentially low resolution (300-500 km)

except for a a few regions that could be observed in mid-resolution in the two orthogonal polarizations during two different flybys. This is fortunately the case of the HLS region for which the dielectric map has a resolution of about 100 km and indicates a value of 1.7 ± 0.2. For comparison, the global dielectric map displays values in the range 1.0 (no polarization plus some allowance for error)-2.25 with an average of 1.54.

Second, the radiometry observations collected at all resolutions (from 5 to 500 km) can be used to build a mosaic of Titan's surface emissivity at 2.2-cm at normal incidence [START_REF] Janssen | Titan's surface at 2.2-cm wavelength imaged by the Cassini RADAR radiometer: Calibration and first results[END_REF][START_REF] Janssen | Titan's surface at 2.18-cm wavelength imaged by the Cassini RADAR radiometer: Results and interpretations through the first ten years of observation[END_REF]. The emissivity map has a resolution of about 5 km at the Huygens landing site, which has been observed several times at closest approach. It indicates an emissivity of 0.94 ± 0.01, which implies, if this region does not depart too much from a Kirchhoff surface, an effective dielectric constant of 2.7 ± 0.25.

The discrepancy between the effective relative permittivity derived from the polarized radiometry measurements (1.7 ± 0.2) and the effective relative permittivity derived from the emissivity at normal incidence (2.7 ± 0.25) suggests that Kirchhoff's approximation does not apply in general to the HLS region. Surface small-scale roughness or, more likely, volume scattering from the subsurface must be invoked in order to reconcile these two observations, although their different resolutions may play a part too. The average effective dielectric constant at 13.78 GHz of the Huygens landing site region must thus lie between 1.5 and 3.0 as also suggested by the scatterometry analysis.

The imaginary part of the complex permittivity of Titan's surface was not measured by the Cassini Radar/radiometer but the analysis of the seasonal thermal wave points to a mean 2.2-cm emission depth in the range 40-100 cm for the dominant radar-dark terrains, consistent with the value r of tholins as measured by [START_REF] Paillou | Microwave dielectric constant of Titan-relevant materials[END_REF]][Janssen et al., 2016].

Lastly, the permittivity probe PWA-MIP/HASI on board the Huygens probe measured both the real and imaginary parts of the complex permittivity of the HLS in the EVL (Extremely and Very Low) frequency range. At 45 Hz (frequency for which the PWA-MIP calibration is best), [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] found r = 2.55 ± 0.35 and r in the interval 0.2 -0.8 for the first 11 min after the landing and r = 2.35 ± 0.35 and r in the interval 0 -0.1 after. These values are consistent with the ones previously published [START_REF] Grard | Electric properties and related physical characteristics of the atmosphere and surface of Titan[END_REF] but with more reliable error bars that account for uncertainties related to the probe's resting attitude on the surface.

More importantly, we highlight that the sounding depths of the Cassini Radar/radiometer and of PWA-MIP/HASI are similar: both instruments are sensitive to the composition and structure of, roughly, the first meter below the surface [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF][START_REF] Janssen | Titan's surface at 2.18-cm wavelength imaged by the Cassini RADAR radiometer: Results and interpretations through the first ten years of observation[END_REF]. More specifically, in the case of PWA-MIP/HASI it is a weighted volume average of the first meter with a weight decreasing with depth (for a more in depth discussion see [START_REF] Lethuillier | Characterization of planetary subsurfaces with permittivity probes : analysis of the SESAME-PP/Philae and PWA-MIP/HASI/Huygens data[END_REF]). All the observations mentioned above are reported in Table 2. They are further discussed in the following section.

Constraints on the composition of the HLS from dielectric measurements

Based on the values presented in Table 2 and assuming that they are all representative of the HLS, we put some constraints on the composition of the subsurface in this region.

The range of permittivity values reported in section 4.1 is consistent with the expected dielectric constants of materials relevant to Titan (see Table 1). More specifically, permittivity values derived from microwave observations overlap those of solid and liquid hydrocarbons. They preclude solid sheets of water ice but not water ice in a porous form.

Indeed, we highlight that the values reported above are effective dielectric constants; they depend on both the composition and physical state of the surface/subsurface. In particular, they are affected by the subsurface porosity. As a consequence, microwave observations do not proscribe a fractured, porous water ice surface which would exhibit an effective dielectric constant much smaller than 3.15, the dielectric constant of bulk water ice. Likewise, while low-frequency observations preclude a compacted layer of tholin-like organics they do allow tholins as part of the near-surface composition. Assuming that the composition of the first meter below the surface is a ternary mixture of water ice, tholin dust and pores (empty, partially or fully filled with liquid methane), we use both microwave and PWA-MIP/HASI results to constrain the respective proportion of these ingredients.

For that purpose, we also assume that Titan's tholins are similar to those synthesized in laboratory from a mixture 95/5 of N 2 /CH 4 as this is the gas mixture that produces the largest abundance of solid aerosols [START_REF] Sciamma-O'brien | Titan's atmosphere: An optimal gas mixture for aerosol production?[END_REF]. Further, based on the idea that the real part of the permittivity of liquid methane at 94 K should be identical at near-DC and microwave frequencies, we use the same value of 1.67 in both frequency domains as measured by [START_REF] Leese | The Huygens surface science package (SSP): Flight performance review and lessons learned[END_REF] at 100 Hz (see Table 1). Unfortunately, this latter assumption cannot be made for the imaginary part of the permittivity for which no measurement at PWA-MIP/HASI frequencies is available in literature. As a consequence, as long as the low-frequency electrical properties of liquid hydrocarbons are not investigated in laboratory, the PWA-MIP/HASI measurements of r cannot be used to place constraints on the composition of Titan's near-surface. However, we highlight that the r value measured by PWA-MIP/HASI just after landing (see Table 2) is larger, by one or even two orders of magnitude than the expected values for tholins and water ice (see Table 1). Since liquid hydrocarbons likely also have a small loss tangent (as measured in laboratory in the microwave domain (see Table 1) and shown to be true at the surface of Titan by [START_REF] Mastrogiuseppe | The bathymetry of a titan sea[END_REF]), PWA-MIP/HASI measurements suggest that an extra conductive material is present at the HLS. The removal of this material may explain the sudden drop of conductivity recorded 11 min after the Huygens landing. This hypothesis is further discussed in section 4.3. are obtained assuming a dielectric constant of 2.2 ± 0.1 (see section 4.1). Note that our approach requires a good estimate of the dielectric constant of the HLS which is not avail-able to date, especially in the microwave domain. However, our goal here is to show that, if both the microwave and ELF dielectric constants of the ground were known with a good accuracy, we would be able to reliably constrain its composition because the constraints associated with these measurements are "perpendicular" to each other. This method could prove to be very valuable to future surface investigations of Titan.

While not very constraining, some lessons can be extracted from these diagrams.

In particular, it seems that some porosity (at least 15%) and the presence of the water ice (at least 8%) are absolutely required to explain Cassini-Huygens observations while that of tholins is not. This means that the subsurface of the HLS could, in theory, consists of a porous water ice layer (60-75% water ice and 25-40% porosity if the pores are empty).

However, the composition is most likely a combination of water ice and organic dust with some porosity, as suggested by the images taken at the surface by the Huygens probe and most of the plausible combinations displayed on Figure A.6. If the nature of the dust is tholin-like then its volumetric fraction in the first meter below the surface is at most 50-60%. Coherently, when the occupancy rate of the pores by liquid methane increases, less

water ice and/or tholins are required in the mixture. The porosity should vary in the range 15-40% for empty pores and 30-65% for saturated pores.

Constraints on the composition of the HLS from the 11-min event

This work also offers the opportunity to investigate further the implications of our favorite scenario for the sudden drop of conductivity (or r ) observed by the PWA-MIP/HASI instrument about 11 min after the Huygens landing [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] (see also Table 2).

Description of observations

The conductivity drop observed by PWA-MIP/HASI occurred in a very short amount of time (2-4 s) and concerns an area, below the Huygens lander, as wide as the distance between the MIP electrodes i.e. 1-2 m [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF]. We reiterate that the concurrent observed decrease in r is not strong enough (relative to measurement uncertainty) to be considered significant. On the other hand, the HASI temperature sensors on board the Huygens probe did measure a significant drop of 0.2 K at the same time [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] and the Huygens GCMS (Gas Chromatographer Mass Spectrometer) recorded a continuous increase in the atmospheric methane suggesting the progressive vaporization of methane in the subsurface due to heating by the probe [START_REF] Lorenz | Titan's damp ground: Constraints on titan surface thermal properties from the temperature evolution of the huygens gcms inlet[END_REF][START_REF] Niemann | Composition of Titan's lower atmosphere and simple surface volatiles as measured by the Cassini-Huygens probe gas chromatograph mass spectrometer experiment[END_REF]. Additionally, the formation of a dewdrop was observed in a camera image and could be linked to rising air with high methane humidity also due to the heating of the surface by the bottom of the probe [START_REF] Karkoschka | Rain and dewdrops on titan based on in situ imaging[END_REF]. Lastly, an unexplained attenuation was observed approximately 10 min after landing in the measurements performed by the acoustic instrument located near the bottom of the probe [START_REF] Lorenz | Silence on shangri-la: Attenuation of huygens acoustic signals suggests surface volatiles[END_REF].

Favored scenario

Based on these observations and the fact that the initially measured value of r (0.5 ± 0.3) is much higher than the values expected for water ice and tholins (see Table 1) we favor the following course of events:

1. The Huygens probe was internally heated by internal power dissipation by the primary batteries during the descent and on the surface (around 250 W, [START_REF] Lorenz | Thermal interactions of the huygens probe with the titan environment: Constraint on near-surface wind[END_REF]).

2. After landing, part of the heat was dissipated in the atmosphere by convection, the remaining being dissipated in the ground by conduction with the total heat dissipated being around 350 W [START_REF] Lorenz | Thermal interactions of the huygens probe with the titan environment: Constraint on near-surface wind[END_REF].

3. This heating leads to the vaporization of a portion of the subsurface liquid hydrocarbons filling the pores.

4. An outgassing burst occurred about 11 min after landing and removed a thin superficial layer of dust responsible for the observed conductivity.

The vaporization of liquid hydrocarbons and, above all, the removal of a superficial conductive dust layer would explain the observed drop in the near-surface electrical properties. Such removal would affect mainly the imaginary part of the subsurface permittivity, as observed. We note that the final value measured by PWA-MIP/HASI ( r in the interval 0-0.1) is compatible with a subsurface composed of any combination of water ice, tholins and vacuum.

As a further argument [START_REF] Lorenz | Thermal interactions of the huygens probe with the titan environment: Constraint on near-surface wind[END_REF] estimates that a total of 500 kJ of energy could have been transferred to the subsurface in about an hour after landing. This would be sufficient to evaporate approximately 1 cm of liquid methane (approximately 0.91 kg).

This value reduces to 0.2 kg if we take into account the possibility that the probe bounced before finding its final resting position (as suggested by [START_REF] Schröder | Bouncing on Titan: Motion of the Huygens probe in the seconds after landing[END_REF]) reducing its contact with the surface (we consider a contact area 4 times smaller than [START_REF] Lorenz | Thermal interactions of the huygens probe with the titan environment: Constraint on near-surface wind[END_REF]).

Of importance, the penetrometer on board the Surface Science Package (SSP) of the Huygens probe detected the presence of a 7-mm dust layer at the landing site [START_REF] Atkinson | Penetrometry of granular and moist planetary surface materials: Application to the Huygens landing site on Titan[END_REF]. This layer is likely composed of organic particles of low-density, therefore easily removable by the vaporizing of subsurface liquid.

Source of the conductivity

In the scenario previously mentioned the source of the conductivity is a thin layer of dust located at the surface and the removal of this layer would cause the observed drop in conductivity. Although solar UV can cause photoelectron production at planetary surfaces, in the case of Titan the UV flux is completely absorbed by the atmosphere and so this effect cannot influence the near-surface environment. Another possibility is the ionization of the near-surface atmosphere by decay of 14 C. According to [Lorenz et al., 2002] the decay rate near the surface is capable of producing 10 4 cm -2 ion pairs leading to a high air conductivity possibly detectable by PWA-MIP/Huygens. The outgassing from the subsurface could have led to the removal of the radiocarbons close to the probe therefore reducing the atmospheric conductivity in the vicinity of the instrument. This scenario could be modeled in order to evaluate the maximum conductivity of the atmosphere close to the lander and to appraise whether or not the atmospheric conductivity could explain the conductivity measured by PWA-MIP/HASI. However, the accurate modeling of this phenomenon and the addition of a highly conductive atmosphere near the Huygens probe is not a simple task and out of the scope of this work. In addition, we consider this scenario unlikely compared to the one described below.

Our favored explanation for the measured conductivity at the HLS after landing is the existence of a superficial layer of dust material with a non negligible intrinsic conductivity. In order to constrain the required characteristics, namely the thickness and conductivity, of this putative superficial conductive layer, we conducted numerical simulations with a finite element code (COMSOL Multiphysics™, see [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] for more detail) assuming a 2-layer subsurface. The numerical simulations take in account the whole of the Huygens probe (for numerical reasons the probe's shape had to be simplified, see [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] for the details of the simplification) in its two extreme attitudes at the surface (see [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF] for a description of these extreme cases). Performing numerical simulations for a set of thicknesses of the dust layer we provide estimates of the dust electrical conductivity required to explain the observed drop in electrical conductivity 11 min after landing. Figure A.7 shows the resulting upper and lower limits of the imaginary part of the effective permittivity of the conductive layer as a function of its thickness (the upper and lower limits include the uncertainty on the probe attitude at the surface, whereas the error bars account for the numerical error of the model and the measurement uncertainty of the PWA-MIP/HASI instrument). We note that if the thickness of the removed conductive layer is 7 mm (as suggested by [START_REF] Atkinson | Penetrometry of granular and moist planetary surface materials: Application to the Huygens landing site on Titan[END_REF])

then the imaginary part of its permittivity must be relatively high, namely between 2.8 and 16.0 (corresponding to a conductivity in the range 7-40 nS/m). Logically, the required imaginary part of the permittivity decreases as the thickness of the conductive layer increases; its lower limit is 0.5 ± 0.3.

Composition of the dust layer

Regarding the implications of this investigation on the composition of the putative conductive 7 mm superficial layer, we are unfortunately hampered by the lack of available information in literature on the conductivity of materials relevant to Titan's at low temperatures and low frequencies. One thing is certain: the tholins studied in this work do not have a high enough imaginary part to be a major component of this layer. Likewise, the DC conductivity of polyacetylene at 200 K is at most 10 -7 S/m -1 and shows a linear decrease with temperature [START_REF] Jones | Electronic properties of polyacetylene prepared by the durham 'photoisomer' route[END_REF], preventing it from being a major component of this layer. On the other hand, some polymers, such as polyaniline, or in general, nitrilerich organic compounds may be more conductive but their accurate electrical properties remain unknown [START_REF] Gall | Composition, seasonal change, and bathymetry of Ligeia Mare, Titan, derived from its microwave thermal emission[END_REF]. Also, nanophase iron and nanophase hematite which are present in Saturn's rings and on the surface of Iapetus [START_REF] Clark | The surface composition of iapetus: Mapping results from cassini vims[END_REF] may have relatively high conductivity at low frequencies/low temperatures: [START_REF] Nikolic | Study of dielectric behavior and electrical properties of hematite a-fe 2 o 3 doped with zn[END_REF] measured the DC electrical conductivity of hematite doped with Zn and, by extrapolating their results at Titan's surface temperature, the maximum conductivity is found to 10 -8 S/m, therefore, the possibility that hematite (doped with 10% Zn) as a major component of this layer cannot be excluded but is very low (the highest conductivity value of this component is only slightly higher than the lowest possible conductivity of this layer, additionally, this only applies to hematite with high Zn content, higher than 10%). Although no measurement of electrical properties of nanophase iron relevant to Titan were found in the literature, powdered metals tend to have a high conductivity. Therefore nanophase iron is most likely at least somewhat conductive and could be a potential component of this layer. Lastly, we note that the photochemical aging of Titan's aerosols during their sedimentation through Titan's atmosphere [START_REF] Couturier-Tamburelli | UV-Vis Light-induced Aging of Titan's Haze and Ice[END_REF] and possibly also at the surface can produce aerosols that are more conductive that the "fresh" tholins synthesized in laboratory and analyzed in this work. Future works should consider investigating the electrical properties of "processed tholins".

Conclusion and perspectives

The measurements performed by the PWA-MIP/HASI/Huygens/Cassini-Huygens instrument in 2005 provided unique insights into the electrical properties of the first meter of Titan's subsurface. However, in order to convert these measurements in term of composition, the low-frequency and low temperature electrical properties of materials possibly present at the surface of Titan need to be known. In this paper, we present the first low frequency electrical characterization of samples of tholins. Tholins are organic matter, possibly analogous to the aerosols produced in the atmosphere of Titan and then deposited onto the surface. Their electrical characterization was performed with a dedicated measurement bench developed at LATMOS. We find that the electrical properties of tholins follow predictable variations (i.e., the real and imaginary parts of the permittivity decrease with higher frequency, higher porosity and lower temperature) and show significant variations as a function of the composition of the gaseous mixture from which they were produced. Based on multi-frequency laboratory electrical characterizations and combining measurements from PWA-MIP/HASI and the Cassini Radar, we derive constraints on the composition of the Huygens Landing Site. While not very strong (a porosity between 15% and 65%, a maximum of 60% of tholins and between 20% and 75% of water ice) our results and approach can serve as a frame of reference for future studies.

We also expand the study of the sudden and significant drop of conductivity observed about 11 min after the landing of Huygens at the surface of Titan which was presented in [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF]. Our favored scenario describes how the subsurface was heated by the probe after landing enough for some subsurface methane to vaporize . This vaporizing methane may have then depleted a thin superficial conductive dust layer. Based on this scenario and numerical simulations we estimate the imaginary part of this conductive dust layer to be at least 2.8 which is very high and rules out the tholins we studied as a main component of the putative superficial dust layer. To our knowledge there is no material relevant to Titan's surface composition with such a high conductivity (more precisely, the only known possibility would be hematite with a high quantity of Zn). However, the electrical properties of a great number of possible materials remain to be investigated and we therefore emphasize the need for more laboratory characterization over wide range of frequencies.

Lastly, we note that this work and our analysis approach will be useful for future missions to Titan and, in particular, missions including a permittivity probe such as the Dragonfly mission [START_REF] Turtle | Dragonfly: Exploring Titan's Prebiotic Organic Chemistry and Habitability[END_REF], a relocatable quadcopter recently pre-selected for the New Frontiers program. If the mission is selected, Dragonfly permittivity measurements will most likely be more accurate than those performed by PWA-MIP/HASI and will therefore provide much stronger constraints on the plausible composition of Titan's subsurface and even discriminate between the different types of tholins at the surface .

We also note for future missions to Titan, that, in order to differentiate between the different composition of tholins based on their electrical properties, certain frequencies would be more useful than others. More specifically, at Titan's surface temperature, while the real part shows significant difference between compositions at all frequencies (see Figure 5 left), the most relevant frequencies to capture differences in the imaginary part due to composition are 100, 1000 and 5000 Hz (see Figure A.4a.).

A: Measurements and derivation of the sample complex permittivity

The spectrum analyzer measures the complex impedance of the sample between the electrodes sweeping the frequency range 1 Hz-100 kHz. For a given frequency, the same measurement is repeated 3 times and the average value is recorded. It takes about 22 s to measure the complex impedance over a full frequency spectrum. At the end of a typical measurement cycle which lasts about 20 h (10h for the liquid nitrogen to evaporate and 10 hours for the sample to return to room temperature), 3000 to 4000 spectra have been collected.

The measured complex impedance Z s (in Ω) is related to the capacitance C (in F)

of the parallel plate electrodes by:

C s = 1 2π f j Z s (A.1)
where f is the frequency of the input signal.

The complex capacitance of a parallel-plate capacitor is itself related to the complex relative permittivity of the medium between the parallel electrodes by:

r = C s d A 0 (A.2)
where A [m 2 ] is the surface area of the electrodes (and generally of the sample) and d [m] is the thickness of the sample.

Equation A.2 applies well if the distance between the electrodes remains much smaller than their dimensions. In addition, it assumes that the electrical field is entirely concentrated in the dielectric medium between the plates and thus neglects edge effects which induce parasitic capacitances.

In order to correct measurements performed on samples from parasitic effects, we calibrate them by measurements made with an empty cell i.e., measurements performed after removing the sample from the sample holder but without changing the distance d between the electrodes. More specifically, we measure Z void and thus C void and equation A.2 becomes:

r = C c d A 0 (A.3)
with C c the corrected complex capacitance:

C c = C s + 0 A d -C void (A.4)
The effects of the electronics were evaluated and found to be negligible. )UHTXHQF\+]

Figure A. 2

 2 Figure A.2 shows the effect of porosity on the samples electrical properties. Overall and as expected, both the real and the imaginary parts of the permittivity decrease when porosity increases, vacuum having the lowest possible values of dielectric constant and conductivity (namely r0 = 1 and r0 = 0).

Figure

  Figure A.5 shows the Cole-Cole model best fitting to our measurements in the complex plane (plotted with different scales for r and r ) at a temperature of 94 K for tholins obtained with 5% of methane. The best-fit parameters are: s = 4.24, ∞ = 4.53, τ = 2.0 × 10 -4 s, α = 0.84, σ s = 0.0183 S/m.

Figure A. 6

 6 Figure A.6 shows the lower and upper limits of the volumetric fractions of the three considered ingredients of the near-surface as derived from the microwave (red) and PWA-MIP/HASI (blue) observations using the Hashin-Shtrikman bounds of the Maxwell-Garnett mixing formula [Hashin and Shtrikman, 1962]. These limits are shown on ternary diagrams; where they intercept (gray area) is the most likely composition of the first meter below the surface of the Huygens landing site. This derivation was done assuming that the subsurface pores are saturated by liquid methane (Figure A.6a), filled with 50% of liquid methane (Figure A.6b) and empty (Figure A.6c). Constraints from microwave observations

Figure A. 3 .

 3 Figure A.3. Real and imaginary parts of the complex permittivity of bulk tholins as a function of frequency for three different temperatures and compositions (2% (a), 5% (b) and 8% (c) of methane in the initial gas mixture).

  Figure A.4. Frequency (at a temperature of 94 K, column a) and temperature (at a frequency of 100 Hz, column b) dependence and of the real and imaginary parts of the complex permittivity of bulk tholins obtained for 3 gas mixture compositions.

Figure A. 5 .

 5 Figure A.5. Complex permittivity at a temperature of 94 K in the complex plane. The best Cole-Cole fit is also represented. The scales for r and r are different in order to provide a readable figure.

Figure A. 6 .Figure A. 7 .

 67 Figure A.6. Ternary diagrams showing the constraints on the composition of the Huygens Landing Site as derived from both PWA-MIP/HASI (red) and the Cassini Radar (blue) observations and assuming that the subsurface pores are saturated with liquid methane (a), half-filled with liquid (b) or empty (c). The intersection of the constraints (gray area) represents the most likely composition of the first meter below the surface of the HLS. To help read this diagram, an example is shown (green squares) on the figure, corresponding to a mixture of 30% pores, 50% tholins, and 20% water ice.

Table A .

 A 1. Electrical properties of material relevant to Titan's surface at 94 K and at 2 frequencies, namely that of PWA-MIP/HASI and that of the Cassini Radar. Extrapolated from[START_REF] Mattei | Dielectric measurements and radar attenuation estimation of ice/basalt sand mixtures as martian Polar Caps analogues[END_REF], b[START_REF] Paillou | Microwave dielectric constant of Titan-relevant materials[END_REF] c[START_REF] Mitchell | Laboratory measurements of cryogenic liquid alkane microwave absorptivity and implications for the composition of Ligeia Mare, Titan[END_REF] , d[START_REF] Leese | The Huygens surface science package (SSP): Flight performance review and lessons learned[END_REF] at 100 Hz.

	Frequencies	45 Hz			14 GHz
		r	r × 10 -2	r	r × 10 -3
	Tholins 2%	4.00 ± 0.04 1.05 ± 0.03	1.17 (non-compacted) b 3.3 (non-compacted) b 2.33 (compacted) b 20.6 (compacted) b
	Tholins 5%	4.40 ± 0.05 1.68 ± 0.13	N/A	N/A
	Tholins 8%	3.40 ± 0.04 1.37 ± 0.04	N/A	N/A
	Water ice	3.15 a	0.40 a	3.13 ± 0.30 a	1.30 ± 0.26 a
	Liquid methane	1.67 d	N/A	1.74 ± 0.03 c	(4.93 ± 2.50) × 10 -2c
	a

Acknowledgments

The PAP measurement bench was partialy funded by the DIM-ACAV (Domaine d'Intêret Majeur-Astrophysique et Conditions d'Apparition de la Vie) program of the Région Ilede-France. The CNES (Centre National d'Etude Spatiale) also provided financial help for material resources. The data reported in this paper are freely available at: ftp:/ftp. latmos.ipsl.fr/outgoing/lethuillier/PAP_data/. The authors would like to thank Ralph Lorenz and Elizabeth Turtle for their valuable comments from which the paper greatly benefited.

cm

 [START_REF] Hamelin | The electrical properties of Titan's surface at the Huygens landing site measured with the PWA-HASI Mutual Impedance Probe[END_REF]rm &+


Patrice Cacoub
email: patrice.cacoub@aphp.fr

Si Nafa

Si Ahmed

Yasmina Ferfar

Stanislas Pol

Dominique Thabut

Christophe Hezode

Laurent Alric

Cloe Comarmond

Gafaar Ragab

Luca Quartuccio

Mohamed Hegazy

Thierry Poynard

Matthieu Resche Rigon

David Saadoun

Myers Squibb

Glaxo Smith Gilead

Kline

Merck Sharp Janssen

Roche, Servier Vifor Si Dohme

Ahmed

Bristol Myers Squibb

Janssen Gilead

Roche Laurent Alric

Christophe Hézode

Bristol Abbvie

Myers Squibb

Gilead

Merck Janssen

Dohme Sharp

Stanislas Pol

Glaxo Smith Kline

Long-term Efficacy of Interferon-Free Antiviral Treatment Regimens in Patients With Hepatitis C Virus-Associated Cryoglobulinemia Vasculitis

Novartis, Vertex, and Abbvie; has received grants from Bristol Myers Squibb, Gilead, Roche, and Merck Sharp Dohme; and has been a board member for Glaxo Smith Kline, Bristol Myers Squibb, Boehringer Ingelheim, Janssen, Gilead, Roche, Merck Sharp Dohme, Sanofi, Novartis, Vertex, and Abbvie. Thierry Poynard is the founder of BioPredictive, which markets FibroTest (patent belong to Public Organization APHP). David Saadoun has received consulting and lecturing fees from Medimmune, Abbvie, Bristol Meyer Squibb, Roche, Servier, Gilead, AstraZeneca and Glaxo Smith Kline.

Abstract:

Background & Aims: In small-size and short-term studies of hepatitis C virus-associated cryoglobulinemia vasculitis (HCV-CryoVas), patients had a higher rate of response and tolerance to direct-acting antiviral (DAA) agents than interferon-containing regimens. We collected follow-up data from a clinical trial to determine the long-term effectiveness and tolerance of all-oral, interferon-free DAA regimens in patients with CryoVas.

Methods:

We collected follow-up data from a prospective international multi-center cohort study of 148 patients with symptomatic HCV-CryoVas (53.7% with cirrhosis and 49.3% naïve to treatment with DAAs). All patients received DAA (sofosbuvir plus daclatasvir, n=53; sofosbuvir plus ribavirin, n=51; sofosbuvir plus ledipasvir, n=23; or sofosbuvir plus simeprevir, n=18), for 12 or 24 weeks, from 2014 through 2017; the median follow-up time was 15.3 months. A complete clinical response was defined as improvement of all organs involved at baseline and the absence of clinical relapse; a partial response was defined as improvement in some but not all organs involved at baseline. The primary endpoint was clinical response of CryoVas symptoms at week 12 after stopping DAA therapy.

Results: A complete response was reported for 106 patients (72.6%), a partial response for 33 patients (22.6%), and no response for 7 patients (4.8%). Cryoglobulins were no longer detected in blood samples from 53.1% of patients and 97.2% of the patients had a sustained virologic response to therapy. Premature DAA withdrawal was reported for 4.1% of patients. Factors associated with no or partial response to therapy included a severe form of CryoVas (odds ratio, 0.33, 95% CI, 0.12-0.91; P=.03) and peripheral neuropathy (odds ratio, 0.31; 95% CI 0.11-0.84; P=.02). After a median followup time of 15.3 months, 4 patients (2.8%) died. The CryoVas manifestation of purpura was cleared from 97.2% of patients, renal involvement from 91.5% of patients, arthralgia from 85.7% of patients, neuropathy from 77.1% of patients, and cryoglobulinemia from 52.2%.

Conclusions:

In a long-term follow-up analysis of data from a clinical trial, we found that more than 95% of patients with HCV-CryoVas have a full or partial response of symptoms to different DAA treatment regimens. Fewer than 5% of patients stop therapy prematurely and less than 3% die. A severe form of CryoVas and peripheral neuropathy were associated with lack of response of HCV-CryoVas to DAA therapy. KEY WORDS: DAA; VASCUVALDIC 3 study; risk; prognostic factor Introduction Symptomatic cryoglobulinemia vasculitis (CryoVas), an immune complex-mediated systemic vasculitis that mainly affects small-and medium-sized vessels, has been observed in about 15% of hepatitis C virus (HCV)-infected patients [START_REF] Ramos-Casals | The cryoglobulinaemias[END_REF][START_REF] Meltzer | Cryoglobulinemia--a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity[END_REF][START_REF] Cacoub | Cryoglobulinemia Vasculitis[END_REF][START_REF] Cacoub | Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC Group. Multidepartment Virus[END_REF][START_REF] Damoiseaux | Diagnostics and Treatment of Cryoglobulinaemia: It Takes Two to Tango[END_REF][START_REF] Kondili | Mixed cryoglobulinaemia: An important but frequently unrecognized and underestimated HCV-related condition in the real life practice[END_REF]. HCV-associated CryoVas (HCV-CryoVas) is a severe disease with an estimated 5-year mortality rate of 25% [START_REF] Ramos-Casals | The cryoglobulinaemias[END_REF][START_REF] Cacoub | Cryoglobulinemia Vasculitis[END_REF][START_REF] Ferri | Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients[END_REF]. Besides liver fibrosis, the prognosis of HCV-CryoVas is mainly dependent on vasculitis involvement of the kidney, central nervous system, heart and digestive tract [START_REF] Ferri | Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients[END_REF][START_REF] Terrier | Cryoglobulinemia vasculitis: an update[END_REF][START_REF] Fabrizi | Hepatitis C virus infection, mixed cryoglobulinemia, and kidney disease[END_REF][START_REF] Terrier | Presentation and outcome of gastrointestinal involvement in hepatitis C virus-related systemic vasculitis: a case-control study from a single-centre cohort of 163 patients[END_REF][START_REF] Terrier | Renal involvement in HCV-related vasculitis[END_REF][START_REF] Terrier | Presentation and prognosis of cardiac involvement in hepatitis C virus-related vasculitis[END_REF].

Major advances in the treatment of HCV-CryoVas have been achieved over the last decade [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Ferri | Treatment with rituximab in patients with mixed cryoglobulinemia syndrome: results of multicenter cohort study and review of the literature[END_REF][START_REF] Dammacco | Therapy for hepatitis C virus-related cryoglobulinemic vasculitis[END_REF][START_REF] Vita | A randomized controlled trial of rituximab for the treatment of severe cryoglobulinemic vasculitis[END_REF][START_REF] Sneller | A randomized controlled trial of rituximab following failure of antiviral therapy for hepatitis C virus-associated cryoglobulinemic vasculitis[END_REF][START_REF] Dammacco | Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a long-term study[END_REF]. Two prospective randomized controlled trials have demonstrated the superiority of rituximab monotherapy in comparison with conventional immunosuppressive therapy in patients with HCV-CryoVas that is refractory to antiviral therapy [START_REF] Vita | A randomized controlled trial of rituximab for the treatment of severe cryoglobulinemic vasculitis[END_REF][START_REF] Sneller | A randomized controlled trial of rituximab following failure of antiviral therapy for hepatitis C virus-associated cryoglobulinemic vasculitis[END_REF]. However, in the absence of HCV clearance, frequent relapses occurred when B cells reemerged in the peripheral blood.

Achievement of a sustained virological response (SVR) remains the main goal in HCV-CryoVas patients since clinical remission of vasculitis is closely associated with viral clearance [START_REF] Saadoun | Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis: a long-term followup study[END_REF].

Interferon alpha (IFN)-based treatment has shown a certain efficacy, although tolerance was poor [START_REF] Terrier | Cryoglobulinemia vasculitis: an update[END_REF][START_REF] Saadoun | Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis: a long-term followup study[END_REF][START_REF] Gota | Induction of clinical autoimmune disease by therapeutic interferonalpha[END_REF][START_REF] Boonyapisit | Severe exacerbation of hepatitis C-associated vasculitic neuropathy following treatment with interferon alpha: a case report and literature review[END_REF]. The recent development of direct-acting antiviral (DAA) agents with increased antiviral potency has benefited HCV-CryoVas patients. The first use of DAA (Peg-IFN/ribavirin plus boceprevir or telaprevir) in HCV-CryoVas patients resulted in a complete clinical response in 56.5% of cases [START_REF] Saadoun | PegIFNalpha/ribavirin/protease inhibitor combination in severe hepatitis C virus-associated mixed cryoglobulinemia vasculitis[END_REF][START_REF] Gragnani | Triple antiviral therapy in hepatitis C virus infection with or without mixed cryoglobulinaemia: a prospective, controlled pilot study[END_REF]. However, grade 3 or 4 adverse events were observed in up to 43.5%, and antiviral therapy discontinuation was required in one-third. More recently, all oral IFN-free DAA regimens [START_REF] Gota | Induction of clinical autoimmune disease by therapeutic interferonalpha[END_REF][START_REF] Boonyapisit | Severe exacerbation of hepatitis C-associated vasculitic neuropathy following treatment with interferon alpha: a case report and literature review[END_REF] have shown good efficacy and tolerance profile in small series of HCV-CryoVas patients. In the VASCUVALDIC 1 prospective study [START_REF] Saadoun | Sofosbuvir plus ribavirin for hepatitis C virusassociated cryoglobulinaemia vasculitis: VASCUVALDIC study[END_REF], 87.5% of twenty-four patients reported a complete response of CryoVas manifestations, 74% had an SVR 12 weeks post-treatment (SVR12) and 8% experienced serious adverse events. In a retrospective case series of 12 HCV-CryoVas patients, an SVR12 was achieved in 83% of patients, and 17% experienced serious adverse events [START_REF] Sise | Treatment of Hepatitis C Virus-Associated Mixed Cryoglobulinemia with Sofosbuvir-Based Direct-Acting Antiviral Agents[END_REF].

Sofosbuvir-based IFN-free DAA therapy was given to a cohort of 44 Italian HCV-CryoVas patients, who all showed both an SVR post-treatment and a CryoVas clinical response [START_REF] Gragnani | Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia[END_REF]. In a Spanish study, 71% of 35 HCV-CryoVas patients achieved a complete clinical response and 94% had an SVR12 after DAA treatment [START_REF] Bonacci | Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals[END_REF]. Finally, in the VASCUVALDIC 2 study, a complete clinical response was reported in 90.2% of 41 HCV-CryoVas patients and an SVR12 in all [START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF].

There are still unresolved issues in this field. Considering the very high virological and clinical response rates after the use of IFN-free DAAs, the question remains as to which HCV-CryoVas patients would still need additional immunosuppressive drugs. Also, the long-term efficacy on CryoVas manifestations and cryoglobulin production after DAA treatment remains to be seen.

Based on a large prospective cohort of HCV-CryoVas patients treated with DAA who had long-term follow-up, we aimed to 1) evaluate the effectiveness and tolerance of different IFN-free DAA combinations, and 2) search for predictive factors of complete remission of vasculitis manifestations.

Patients and Methods

Patients

The VASCUVALDIC 3 study is an international open-label, prospective, multicenter study including consecutive patients with active HCV-CryoVas who were recruited between 2014 and 2017. Patient eligibility included a minimum age of 18 years, informed consent, the presence of active HCV-vasculitis defined by skin, joint, renal, peripheral nerve, central neurological, digestive, pulmonary and/or cardiac involvement, and chronic active HCV infection (positive HCV RNA). Exclusion criteria included non-active CryoVas, human immunodeficiency virus or active hepatitis B virus infection, and current decompensated cirrhosis. Patients with a severe form of vasculitis were defined by skin necrosis, glomerulonephritis, and heart, gut and/or central nervous system involvement.

The baseline clinical evaluation included recent weight loss, neurologic involvement (peripheral and/or central nervous system), cutaneous involvement (purpura, skin necrosis), arthralgia, gastrointestinal tract involvement, renal involvement (proteinuria, hematuria and glomerular filtration rate [GFR]), and clinical signs of hepatic involvement. All patients had follow-up visits every 4 weeks until week 36, and at the end of follow-up. The diagnosis of non-Hodgkin's lymphoma was based on the World Health Organization (WHO) criteria [START_REF] Harris | World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November 1997[END_REF]. The study was performed according to the Declaration of Helsinki and was approved by the ethics committee of Pitié-Salpêtrière Hospital (Paris, France).

Immunologic and virological markers

HCV viral load was quantified using the Abbott RealTime HCV assay (Abbott, Rungis, France). HCV genotyping was performed by NS5b gene sequencing [START_REF] Laperche | Unique NS5b hepatitis C virus gene sequence consensus database is essential for standardization of genotype determinations in multicenter epidemiological studies[END_REF]. Laboratory evaluation included a complete blood count, serum chemistry profile, alanine aminotransferase (ALT), rheumatoid factor activity, C4 fraction of complement and cryoglobulin. Cryoglobulins were measured as previously described [START_REF] Musset | Characterization of cryoglobulins by immunoblotting[END_REF] and were classified according to the method described by Brouet et al. [START_REF] Brouet | Biologic and clinical significance of cryoglobulins. A report of 86 cases[END_REF]. The estimation of glomerular filtration rate (GFR) was determined by modification of diet in renal disease (MDRD) study equations. Urine collection was performed to quantify protein excretion. Liver fibrosis was evaluated (by liver biopsy and/or non-invasive tests) according to the previously validated Metavir scoring system [START_REF]Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group[END_REF].

Study Design

All patients with active HCV-CryoVas who met the inclusion criteria during the study period were prospectively included. All patients received antiviral therapy with sofosbuvir-based IFN-free DAA for 12 or 24 weeks (the duration was at the investigators' discretion). All antiviral combinations were ribavirin-free except for the sofosbuvir plus ribavirin group. Use of nonantiviral treatment, i.e. corticosteroids, immunosuppressants or plasma exchange, was at the investigators' discretion.

Endpoints

The primary endpoint was a complete clinical response of CryoVas at week 12 post-DAA treatment. A complete clinical response was defined as improvement of all organs involved at baseline and the absence of clinical relapse. Skin and joint improvement were evaluated clinically.

Renal improvement was evaluated biologically, i.e. proteinuria <0.3 g/24 h, resolution of haematuria and improvement of GFR > 20% at week 24 (if GFR < 60 ml/min/1.73 m² at diagnosis). Peripheral neurological improvement was evaluated clinically (visual analogue scales, muscle testing) and/or electrophysiologically (electromyogram abnormalities at week 12 post-DAAs compared with baseline). Patients defined as partial clinical responders at week 12 post-DAAs had improvement in some but not all organs involved at baseline. Patients with no clinical improvement at week 12 post-DAAs were defined as treatment failure.

The secondary endpoints included 1) a SVR at week 12 post-DAAs (SVR12), 2) the rate and type of side effects during the DAA treatment period and the follow-up, and 3) clinical response rates of CryoVas symptoms at the end of follow-up. A SVR was defined as the absence of detectable serum HCV RNA 12 weeks after the end of antiviral therapy. The remaining patients were classified as virological failures.

Safety assessments

Data on all adverse events were collected prospectively during the follow-up.

Statistical analysis

Quantitative variables are reported as medians and ranges. They were compared by the Wilcoxon Rank-Sum test to assess differences between responders and non-responders or by the Kruskal-Wallis test to assess differences among treatment groups. Categorical variables were described by counts and percentages and were compared between groups using the Fisher's exact test. Treatment effects were evaluated using logistic models and tested using partial Wald tests.

Odds ratios (OR) with their 95% confidence intervals (95% CI) are given. Survival was estimated using the Kaplan-Meier method. Analyses of effectiveness and tolerability were performed on the intention-to-treat population, which was defined as all enrolled patients who received at least one dose of DAA. All tests were 2-sided; P-values less than 0.05 were considered significant.

Analyses were performed using R statistical package, version 3.3.1 (www.r-project.org).

All co-authors had access to the study data and had reviewed and approved the final manuscript.

Results

Baseline characteristics of HCV-CryoVas patients

One hundred and forty-eight HCV-CryoVas patients, with a median (IQR) age of 57 (51; 67) years, were included (Table 1). The main clinical features of CryoVas at baseline included arthralgia (64.4%), neuropathy (58.1%), purpura (57.4%), glomerulonephritis (16.9%), skin necrosis (10.1%), and other visceral involvement (6.1%). A severe form of CryoVas was noted in 43 (29.1%) patients. B-cell lymphoma was present in 13 (8.8%) patients. The mean cryoglobulin level was 168 (50; 447) mg/L. Seventy-one percent of patients had type II mixed cryoglobulinemia. A low C4 serum level was noted in 70.1%. Seventy patients (53.7%) had Child-Pugh class A cirrhosis. Seventy-three (49.3%) patients were antiviral treatment-naïve, whereas seventy-five (50.7%) were virological non-responders to previous IFN-based antiviral therapy. Previous immunosuppressive treatments had been given to 21.3% of patients, i.e. corticosteroids (18.4%), plasma exchange (3.7%) and rituximab (2.2%).

Treatment efficacy and predictive factors of complete remission of vasculitis

Antiviral therapy included sofosbuvir (SOF) plus daclatasvir (n=53), SOF plus ribavirin (n=51), SOF plus ledipasvir (n=23), and SOF plus simeprevir (n=18) (Table 2). Twelve weeks following DAA discontinuation, 106 (72.6%) patients showed a complete clinical response, 33 (22.6%) a partial response and 7 (4.8%) had no response of their CryoVas manifestations. Partial responses were based on persistent renal insufficiency (n=17) and/or peripheral neuropathy (n=29). Non response was related to kidney involvement (n=3), peripheral neuropathy (n=2), and skin necrosis (n=2). Fifty-three percent of cases showed resolution of cryoglobulins. An SVR12 was obtained in 141/145 (97.2%) patients. The non-SVR12 patients were complete (n=3) or partial (n=1) responders, although three still had a detectable cryoglobulinemia. Among patients with B-cell lymphoma, 10 patients showed a complete clinical response, 2 partial response and 1 no response of CryoVas manifestations while a cryoglobulinemia was detectable in 10. During antiviral therapy, 14.3% of patients received corticosteroids, immunosuppressant or plasma exchange.

The differences in the baseline characteristics between patients who showed a complete response (n=106) versus those who had a partial or no response (n=40) of the CryoVas manifestations were the presence of neuropathy (54.3% vs. 73.3%), renal involvement (12.2% vs. 30%), arterial hypertension (25% vs. 46.7%), a severe form of CryoVas (26% vs. 35%), and previous use of immunosuppressants and/or plasma exchange (17.6% vs. 34.6%). After multivariate analysis, two baseline factors remained independently associated with a poor response of CryoVas manifestations: a severe form of CryoVas [OR 0.33, 95% CI 0.12-0.91; P=0.03] and peripheral neuropathy [OR 0.31, 95% CI 0.11-0.84; P=0.02].

Comparison of different DAA combinations for tolerance and efficacy

There was a more frequent rate of HCV genotype 3, 5 or 6 in the SOF plus daclatasvir group (Table 3). Patients in the SOF plus simeprevir group had been treated with Rituximab more frequently. Premature DAA withdrawal due to side effects was noted in 6 (4.1%) patients. Anemia was the most frequent side effect in 29% and 19% of the SOF plus ribavirin and SOF plus simeprevir groups, respectively. Skin manifestations were found in 8% and 12% of the SOF plus simeprevir and SOF plus ribavirin groups, respectively. Nausea was reported in 6% and 2% of the SOF plus simeprevir and SOF plus daclatasvir groups, respectively.

The rates of complete remission of CryoVas manifestations at week 12 post-DAA treatment and at the end of follow-up were 62% and 70% for SOF plus ribavirin, 67% and 72% for SOF plus simeprevir, 79% and 88% for SOF plus daclatasvir, and 87% and 87% for SOF plus ledipasvir, respectively. Using the SOF plus ribavirin combination as a reference group (because of its lowest CryoVas response rate), only the SOF plus ledipasvir combination showed significant superiority [OR 4.09, 95% CI 1.19-19.00; P=0.04], while there was a superiority trend for the SOF plus daclatasvir combination [OR 2.28, 95% CI 0.96-5.63; P=0.06] (Figure 1). The results were similar when the model was adjusted for the severity of vasculitis, type III mixed cryoglobulinemia and B-cell lymphoma (p=0.03 and p=0.09 for SOF plus ledipasvir and SOF plus daclatasvir, respectively).

After a median follow-up of 15.3 months, 4 (2.8%) patients had died and no CryoVas relapse was observed. The causes of death were hepatocellular carcinoma (two patients: one partial responder and one complete responder), alveolar pulmonary hemorrhage (one complete responder), and acute respiratory failure (one complete responder). The 12-month survival rate was 97% [95% CI 94, 100]. The rates of CryoVas manifestation clearance at the end of follow-up were as follows: purpura (97.2%), renal involvement (91.5%), arthralgia (85.7%), neuropathy (77.1%) and cryoglobulinemia (52.2%).

Discussion

Up to recent period, HCV-CryoVas had been a severe condition with a 10-year mortality of up to 40% (3) and an overall risk of non-Hodgkin's lymphoma that was 30 times higher than the general population [START_REF] Monti | Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias[END_REF]. The most striking results found by the present study, using different DAA combinations in a large cohort of HCV-CryoVas patients with long-term follow-up, are 1) a high rate (95.2%) of improvement in CryoVas manifestations at week 12 post-DAA treatment; 2) a low mortality rate (2.8%), with a 12-month survival rate of 97%; and 3) two baseline factors predictive of a poor response of CryoVas manifestations, i.e. a severe form of vasculitis and peripheral neuropathy.

The therapeutic approach for HCV-CryoVas has dramatically changed with all oral INFfree therapies. In published series [START_REF] Saadoun | Sofosbuvir plus ribavirin for hepatitis C virusassociated cryoglobulinaemia vasculitis: VASCUVALDIC study[END_REF][START_REF] Sise | Treatment of Hepatitis C Virus-Associated Mixed Cryoglobulinemia with Sofosbuvir-Based Direct-Acting Antiviral Agents[END_REF][START_REF] Gragnani | Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia[END_REF][START_REF] Bonacci | Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals[END_REF][START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF], the number of HCV-CryoVas patients varied from 10 to 44, and the follow-up period was less than six months after stopping DAA. The studies reported SVR rates from 74% to 100%, and complete response rates of CryoVas manifestations from 83% to100%. Such very high rates of CryoVas remission were confirmed in the present study (95.2%).

However, CryoVas patients who presented with a severe form of vasculitis or peripheral neuropathy were 3-times less likely to obtain a complete clinical response. These results are likely to be clinically useful for identifying which patients would still need additional immunosuppressive drugs. For patients with a severe form of life-threatening CryoVas, additive treatments, such as rituximab and/or plasmapheresis, should be rapidly introduced.

Immunosuppressants are not mandatory for those who present with peripheral neuropathy since there are no associated life-threatening risks, and it would take more time to achieve a complete response of the neurological manifestations. Of note, permanent damage may be irreversible such as peripheral neuropathy or renal failure, making them insensitive to treatments.

In the DAA era, greater antiviral efficacy and better tolerance of antivirals in HCV-CryoVas patients has been associated with less frequent use of non-antiviral treatments, i.e. corticosteroids, immunosuppressants or plasma exchange (14.3% in the present study). Data from the French group clearly showed a decreased rate of use of non-antiviral treatments, from the Peg-IFN-ribavirin era (43%) to the SOF plus daclatasvir era (4.8%) [START_REF] Saadoun | Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis: a long-term followup study[END_REF][START_REF] Saadoun | PegIFNalpha/ribavirin/protease inhibitor combination in severe hepatitis C virus-associated mixed cryoglobulinemia vasculitis[END_REF][START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF], which is similar to the 4.5% rate reported by Gragnani et al. (27). This was also found in a recent study where the corticosteroid doses had been reduced or treatment withdrawn in 10 out of 13 patients [START_REF] Bonacci | Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals[END_REF].

Overall, these results probably reflect the greater antiviral efficacy of more recent combinations.

HCV-CryoVas has long been a severe and challenging condition with respective estimated 5-and 10-year mortality rates of 25% and 40% [START_REF] Cacoub | Cryoglobulinemia Vasculitis[END_REF][START_REF] Terrier | Cryoglobulinemia vasculitis: an update[END_REF]. By using IFN-free DAA the mortality rates of patients with HCV-CryoVas have decreased dramatically, being less than 3% in the present study.

Use of DAA in HCV-CryoVas patients has been associated with adverse event rates of 49%-67% when ribavirin was included. When SOF plus daclatasvir was given without ribavirin, the adverse event rate was only 17% [START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF]. In the present analysis, anemia and the use of erythropoietin were seen mainly in the SOF plus ribavirin group, and to a lesser extent in the SOF plus simeprevir group. Premature DAA withdrawal due to side effects was noted in only 4.1%.

It has been recently reported that the combination of SOF plus daclatasvir is able to correct most immune abnormalities found in HCV-CryoVas patients, such as T regulatory cell deficiency and expansion of IgM + CD21 -/low memory B, T follicular helper and Th17 cells [START_REF] Comarmond | Direct-Acting Antiviral Therapy Restores Immune Tolerance to Patients With Hepatitis C Virus-Induced Cryoglobulinemia Vasculitis[END_REF].

However, despite very good virological and clinical results, all studies using DAA in HCV-CryoVas patients reported a significant proportion of patients that were still positive for cryoglobulins (50-61%), as was also found in 46.9% of the present long-term study [START_REF] Saadoun | Sofosbuvir plus ribavirin for hepatitis C virusassociated cryoglobulinaemia vasculitis: VASCUVALDIC study[END_REF][START_REF] Sise | Treatment of Hepatitis C Virus-Associated Mixed Cryoglobulinemia with Sofosbuvir-Based Direct-Acting Antiviral Agents[END_REF][START_REF] Gragnani | Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia[END_REF][START_REF] Bonacci | Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals[END_REF][START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF]. Case reports of HCV-CryoVas relapse have been described either as the reappearance or increase of cryoglobulin levels despite SVR following INF-free therapy [START_REF] Sollima | Persistent mixed cryoglobulinaemia vasculitis despite hepatitis C virus eradication after interferon-free antiviral therapy[END_REF][START_REF] Sise | Lupus-Like Immune Complex-Mediated Glomerulonephritis in Patients With Hepatitis C Virus Infection Treated With Oral, Interferon-Free, Direct-Acting Antiviral Therapy[END_REF]. This suggests that some patients continue to have B-lymphocyte clonal expansion after SVR [START_REF] Santer | Enhanced Activation of Memory, but Not Naïve, B Cells in Chronic Hepatitis C Virus-Infected Patients with Cryoglobulinemia and Advanced Liver Fibrosis[END_REF][START_REF] Vallat | Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection[END_REF]. Therefore, patients with persistently positive serum cryoglobulin results will need special attention and follow-up, particularly as they appear to be at very high risk of developing malignant lymphoproliferative disorders [START_REF] Saadoun | Increased risks of lymphoma and death among patients with non-hepatitis C virus-related mixed cryoglobulinemia[END_REF][START_REF] Landau | Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response[END_REF].

This study has some limitations, with one being that it was an open label study with no control group. Considering the very high rates of virological response and the very good safety profile that had been previously demonstrated with DAA, the use of a control group would have been unethical in a population with a severe life-threatening disease. Some patients were treated for 12 weeks and others for 24 weeks, as the duration of antiviral therapy was individually tailored. The use of non-antiviral treatment, i.e. corticosteroids, immunosuppressants or plasmapheresis, was at the investigators' discretion, although their use was fairly well distributed amongst the DAA groups.

In conclusion, the VASCUVALDIC 3 study demonstrates that in a large cohort of HCV-CryoVas patients with long-term follow-up, different combinations of all oral, interferon-and ribavirin-free, sofosbuvir-based antiviral therapy are able to induce a high rate of clinical improvement at week 12 post-DAA treatment and at the end of follow-up. The tolerance was good, with no serious adverse events and a very low rate of DAA withdrawal due to side effects. It is noteworthy that the mortality rate was very low compared to previous studies. Less than 15% of patients required the use of corticosteroids or immunosuppressants combined with antiviral therapy. Of note, a longer follow-up of HCV-CryoVas patients with viral eradication and persistent detectable cryoglobulinemia is mandatory for evaluating the remaining risk of lymphoma.

Legends of tables and figures

Riba alone

Harvoni

  Abbreviations

Figure 1 :

 1 Figure 1: Complete clinical response of HCV-associated cryoglobulinemia vasculitis according to

Table 1 :

 1 Main baseline features of 148 patients with HCV-associated mixed cryoglobulinemia vasculitis.

Table 2 :

 2 Main baseline features of patients with HCV-associated mixed cryoglobulinemia vasculitis according to the response of vasculitis manifestations after interferon-free direct-acting antiviral agents.

Table 3 :

 3 Main features of patients with HCV-associated mixed cryoglobulinemia vasculitis according to the combination of interferon-free direct-acting antivirals.

Table 1 : Main baseline features of 148 patients with HCV-associated mixed cryoglobulinemia vasculitis.

 1

	Variable		Odds Ratio		OR 95%CI	p
					Reference
					4.09 (1.19, 19.00)	0.04
	Dacla				2.28 (0.96, 5.63)	0.06
	Sime				1.23 (0.40, 4.02)	0.72
	0.5	1	2	5	10

HCV: hepatitis C virus; ALT: alanine aminotransferase; IFN: interferon alfa. Data are expressed as n (%) or mean ± SEM. * with skin necrosis, glomerulonephritis, and involvement of central nervous system, heart or gut.

Table 2 : Main baseline features of patients with HCV-associated mixed cryoglobulinemia vasculitis according to the response of vasculitis manifestations after interferon-free direct-acting antiviral treatment.

 2

						p-
		Partial or non-response	Complete response	value
		Number/mean		Number/mean
	Characteristics	[IQR]	percentage	[IQR]	percentage
	Total number	40 patients	27.4	106 patients	72.6
	Age	63 [54;68]	-	57 [51;66]	-	0.22
	Female gender	16	61.5	55	53.4	0.51
	HCV infection
	Liver fibrosis (METAVIR)
	F0-F2	13	52	45	44.1	0.51
	F3-F4	13	50	57	55.9	0.56
	HCV RNA, log IU/mL	5.84 [5.1;6.3]	-	6.08 [5.36;6.50]	-	0.40
	ALT, IU/L	41 [25;66]	-	47	-	0.69
	Serum albumin, g/L	36 [31;40]		40 [35;43]	-	0.048
	Previous IFN-based treatments
	Naïve	12	40	59	50.9	0.31
	Non-responder	18	60	57	49.1	0.31
	Mixed cryoglobulinemia-related
	Cryoglobulinemia positive	28	93.3	103	90.4	1.00
	Cryoglobulinemia type II	19	73.1	67	62	0.62
	Cryoglobulinemia type III	5	19.2	31	28.7	0.62
	Cryoglobulinemia level, mg/L	80 [44.5;377]	-	196 [72;577]	-	0.23
	Low C4 serum level	13	72.2	47	70.2	1.00
	Serum creatinine, µmol/L	70 [60;103]	-	69 [61;82]	-	0.67
	Glomerular filtration rate, mL/min	87 [46;113]	-	90 [71;111]	-	0.37
	Vasculitis
	Arthralgia	20	66.7	73	64	0.83
	Purpura	19	63.3	65	56	0.54
	Skin necrosis	3	10	11	9.5	1.00
	Neuropathy	22	73.3	63	54.3	0.065
	Renal involvement	9	30	15	12.2	0.049
	Hypertension	14	46.7	29	25	0.026
	Severe form of vasculitis*	14	35	28	26	0.31
	B-cell lymphoma	1	3.3	12	10.3	0.30
	Previous immunosuppressant
	Corticosteroids	9	34.6	15	13.9	0.021
	Rituximab	1	3.9	2	1.9	0.48
	Plasma exchange	0	0	5	4.6	0.58
	Corticosteroids, rituximab or plasma
	exchange	9	34.6	19	17.6	0.065

HCV: hepatitis C virus; ALT: alanine aminotransferase; IFN: interferon alfa. Data are expressed as n (%) or mean ± SEM. * with skin necrosis, glomerulonephritis, and involvement of central nervous system, heart or gut.

Table 3 : Main features of patients with HCV-associated mixed cryoglobulinemia vasculitis according to the combination of interferon-free direct-acting antivirals.

 3

	DAA combination		SOF/LEDI		SOF/DACLA		SOF/SIME		SOF/RIBA
			Median [IQR]		Median [IQR]		Median [IQR] or		Median
	Characteristics	N	or %	N	or %	N	%	N	[IQR] or %	p-value
	Total number	23		53		18		51

Acknowledgments

We would like to thank all the patients and their families.