Frédéric Foucher
email: frederic.foucher@cnrs-orleans.fr

Frances Westall

Franz Brandstätter

René Demets

John Parnell

Charles S Cockell

Howell G M Edwards

Jean-Michel Bény

André Brack

Testing the survival of microfossils in artificial martian sedimentary meteorites during entry into Earth's atmosphere: the STONE 6 experiment

Keywords: Meteorites, Thermal histories, Astrobiology, Mineralogy, Mars

Introduction

Research into the oldest traces of life

Research into the origin of life and the most ancient traces of life is hampered by the fact that suitable rocks dating back to the first billion years of Earth's history are lacking due to alteration by metamorphism and destruction by plate tectonics. The oldest known sedimentary rocks in the Isua Greenstone Belt and on Akilia island, in south-western Greenland (3.85-3.7 Ga, Nutman et al., 1997;[START_REF] Blichert-Toft | The Nd and Hf isotopic evolution of the mantle through the Archean. Results from the Isua supracrustals, West Greenland, and from the Birimian terranes of West Africa[END_REF] consist of cherts (i.e. silicified volcanic silts and sands), banded iron formation (BIF) and minor strata such as conglomerates [START_REF] Fedo | Metasomatic Origin of Quartz-Pyroxene Rock, Akilia, Greenland, and Implications for Earth's Earliest Life[END_REF], although the amphibolite to granulite-grade metamorphism makes interpretation of the sedimentary protoliths difficult [START_REF] Mojzsis | Evidence for life on Earth before 3.800 million years ago[END_REF][START_REF] Rosing | 13C-Depleted Carbon Microparticles in >3700-Ma Sea-Floor Sedimentary Rocks from West Greenland[END_REF][START_REF] Van Zuilen | Reassessing the evidence for the earliest traces of life[END_REF] [START_REF] Westall | Exogenous carbonaceous microstructures in Early Archaean cherts and BIFs from the Isua Greenstone Belt: implications for the search for life in ancient rocks[END_REF]. The oldest unambiguous traces of life occur in 3.5-3.3 Ga-old Early Archean silicified sediments (cherts) from the Pilbara (Australia) and Barberton (South Africa) greenstone belts.

The cherts contain a variety of traces of life, including colonies of small coccoidal chemo(litho)trophic microorganisms on volcanic rock/particle surfaces [START_REF] Furnes | Early Life Recorded in Archean Pillow Lavas[END_REF]Westall et al., 2006b) and anoxygenic photosynthetic mats formed in sub-aerial shallow water environments [START_REF] Allwood | Stromatolite reef from the Early Archaean era of Australia[END_REF][START_REF] Allwood | Controls on development and diversity of Early Archean stromatolites[END_REF][START_REF] Walsh | Evaluation of Early Archean Volcaniclastic and Volcanic Flow Rocks as Possible Sites for Carbonaceous Fossil Microbes[END_REF]Westall et al., 2006a;[START_REF] Tice | Photosynthetic microbial mats in the 3,416-Myr-old ocean[END_REF]. From the relatively advanced level of evolution of these microorganisms Westall and Southam (2006) concluded that life had to have appeared much earlier, before ≈4.0 Ga. The presence of microorganisms in shallow water to littoral environments show that the relatively inhospitable conditions on the primitive Earth (high flux of UV radiation, moderate to relatively high seawater temperatures, high seawater salinity, anoxygenic atmosphere, meteorite bombardment, etc.) did not inhibit life.

The presence of liquid water on Mars during the Noachian period (-4.5 to 3.5 Ga) is now well established [START_REF] Bibring | Mars Surface Diversity as Revealed by the OMEGA/Mars Express Observations[END_REF][START_REF] Carr | The Surface of Mars[END_REF]) and the hydrological system of Noachian Mars as been described by Fasset and [START_REF] Fassett | Valley network-fed, open-basin lakes on Mars: Distribution and implications for Noachian surface and subsurface hydrology[END_REF]. Environmental conditions on early Mars during this period were habitable and, in some respects, similar to those existing on the primitive Earth (presence of liquid water, moderate to relatively high water temperatures in the vicinity of hydrothermal vents and volcanic activity, probably high salinity, probably slightly acidic pH due to the CO2 atmosphere, high flux of UV radiation, meteorite bombardment, etc.) [START_REF] Westall | Life on the Early Earth: A Sedimentary View[END_REF]. Since Mars had the same ingredients for life (liquid water in contact with mineral surfaces, carbon and other essential elements, and sources of energy), it is widely believed that life could have appeared on that planet [START_REF] Brack | Why exobiology on Mars?[END_REF][START_REF] Mckay | An Approach to Searching for Life on Mars, Europa, and Enceladus[END_REF][START_REF] Sephton | Extraterrestrial Organic Matter and the Detection of Life[END_REF]Southam et al., 2007;Southam and Westall, 2007;[START_REF] Walter | Preservation of Biological Information in Thermal Spring Deposits: Developing a Strategy for the Search for Fossil Life on Mars[END_REF][START_REF] Wentworth | Antarctic Dry Valleys and indigenous weathering in Mars meteorites: Implications for water and life on Mars[END_REF]. Given the lack of terrestrial rocks old enough to contain traces of the first living cells, the discovery of traces of primitive life in ancient rocks on Mars would help understand the origin of life in general [START_REF] Brack | Life on Mars: a clue to life on Earth[END_REF][START_REF] Brack | An Integrated Exobiology Package for the Search for Life On Mars[END_REF][START_REF] Westall | An ESA study for the search for life on Mars[END_REF].

Present and future martian missions are dedicated to studies of the hab-itable potential of the martian surface (rocks and regolith), as well as to the search for past and present traces of life. The lander Phoenix looked for water on the surface of Mars and tried to determine if life could still exist in the surface materials [START_REF] Renno | Physical and Thermodynamical Evidence for Liquid Water on Mars[END_REF]. However, the probability of finding living microorganisms in the uppermost centimetres of regolith is very small. Nevertheless, one of our goals was to test the hypothesis that living endolithic organisms (i.e. microorganisms that live within rocks) could survive ejection from the host planet, transport through space, and entry into the atmosphere of another planet, such as Earth. With this objective in mind, we subjected living endolithic organisms, Chroococcidiopsis, protected by a 2 cm thick layer of rock to the heat shock of entry into the Earth's atmosphere.

Other missions to Mars will investigate the habitability of the surface (the NASA-led Mars Science Laboratory (MSL), named Curiosity, 2011 mission) and search for traces of present or past life (the ESA led 2018 ExoMars mission). The search for traces of life in situ promises to be challenging since it is already difficult to unambiguously identify traces of fossil life in ≈3.5

Ga-old terrestrial rocks, even with the availability of highly sophisticated instrumentation capable of high resolution observation and analysis. Thus, the most promising method of studying martian materials to detect traces of life will be the international Mars Sample Return mission planned for 2023 (The MEPAG-ND-SAG, 2008).

An alternative method of searching for traces of martian microbial re-mains (if life appeared on Mars) is to analyse sedimentary meteorites. This solution has two advantages: it is cheaper than space exploration and very accurate analyses can be made in the laboratory. The SNC (Shergottite, Nakhlite, Chassignite) meteorites form a small group of rare meteorites originating from Mars. All the known 52 SNC meteorites so far found1 consist of igneous rocks, mainly basaltic, that are a priori incompatible with the presence of biogenic remains (except as endoliths). However, sediments deposited in bodies of standing water would constitute suitable materials for hosting traces of life. The STONE 6 experiment was therefore carried out in order to test the survival of martian analogue sediments and microfossils embedded in the sediments during entry into the Earth's atmosphere. The survival of living endolithic microorganisms was also investigated.

STONE experiments

STONE 6 is the latest in a series of experiments whose objectives are to test the effect of entry into the Earth's atmosphere on different sedimentary meteorite analogues [START_REF] Brack | Do meteoroids of sedimentary origin survive terrestrial atmospheric entry? The ESA artificial meteorite experiment STONE[END_REF]. The rock samples are fixed around the stagnation point of the heat shield of spherical FOTON capsules, 2250 kg in weight and 2.3 m in diameter, used to carry out experiments in lower Earth orbit.

Previous successful STONE experiments include the STONE 1 experi-ment which tested a carbonate (dolostone, i.e. dolomite, fossil calcareous shells, quartz and feldspar), and an artificial sediment composed of basalt (80%) and gypsum (20%), and a reference sample of dolerite (a variety of basalt) [START_REF] Brack | Do meteoroids of sedimentary origin survive terrestrial atmospheric entry? The ESA artificial meteorite experiment STONE[END_REF]. Only the dolostone survived. Although it did not exhibit a fusion crust, its composition changed due to high temperature processes; the carbonate decomposed into CaO and MgO (periclase). All the samples in the 2005 STONE 5 experiment, dolerite, carbonate cemented sandstone, and gneissic impactite, survived atmospheric entry [START_REF] Brandstätter | Mineralogical alteration of artificial meteorites during atmospheric entry. The STONE-5 experiment[END_REF][START_REF] Parnell | Response of sandstone to atmospheric heating during the STONE 5 experiment: Implications for the palaeofluid record in meteorites[END_REF]. A fusion crust formed on the dolerite suggesting that the temperatures attained at the heat shield of the capsule were comparable to those experienced by meteoroids. This STONE experiment also tested the survival of living bacterial and fungal spores, as well as dried vegetative endoliths that were placed in tiny wells drilled into the 1 cm-thick rock discs. However, the shield constituted by the rocks (1 cm) was too thin and the microorganisms were carbonized during atmospheric entry [START_REF] Cockell | Interplanetary Transfer of Photosynthesis: An Experimental Demonstration of A Selective Dispersal Filter in Planetary Island Biogeography[END_REF]. Since endolithic photosynthesising microorganisms require cannot live at great depths within a rock, it is unlikely that they could have been imported from another planet, therfore [START_REF] Cockell | Interplanetary Transfer of Photosynthesis: An Experimental Demonstration of A Selective Dispersal Filter in Planetary Island Biogeography[END_REF] concluded that photosynthesis had to have evolved on Earth.

Our experiment, STONE 6, tested the effect of the atmospheric entry on three samples, the reference sample of dolerite and two sedimentary rocks samples. The latter included Devonian lake sediments ≈400 My-old from the Orkney Islands, Scotland and a ≈3.5 billion years old volcanic sandstone, the

ACCEPTED MANUSCRIPT

Kitty's Gap Chert, from the Pilbara region of Australia. This paper concentrates on the observations and analysis carried out on the latter sample. [A second experiment that flew on the same mission, LITHOPANSPERMIA, consisted of a rock coated with fungal species and is not part of our experiment, de la Torre et al. ( 2009).]

Materials and methods

The Kitty's Gap Chert

The 3.446 Ga-old Kitty's Gap Chert (Fig. 1) forms part of the Coppin Gap Greenstone Belt, in the Pilbara, north-western Australia. it consists of layers of silicified volcanic silts and sands deposited in an intertidal environment (Westall et al., 2006b;[START_REF] De Vries | Stratigraphic continuity and early deformation of the central part of the Coppin Gap Greenstone Belt, Pilbara, Western Australia[END_REF]. The volcanic precursors consisted of K-feldspars, Ti-bearing biotites, volcanic glass shards, amphiboles, and ghost spherulites that were replaced by K-phyllosilicates (hydromuscovite, KAl 2 [(OH,F) 2 |AlSi 3 O 10 ]-H 2 O) [START_REF] Orberger | Microfacies and origin of some Archean cherts (Pilbara, Australia)[END_REF]. Early diagenetic silicification of the volcaniclastic sediments was due to silica-rich hydrothermal fluids as well as silica-saturated seawater (van den [START_REF] Van Den Boorn | Dual role of seawater and hydrothermal fluids in Early Archean chert formation: Evidence from silicon isotopes[END_REF]. The rock was subjected to regional low grade metamorphism (prehnite-pumpellyite grade, Westall et al., 2006b). Westall et al. (2006b) documented traces of primitive small, anaerobic fossil microorganisms in these sediments, including silicified biofilms and colonies of coccoidal, rodshaped, filamentous microorganisms. Although remnants of photosynthetic mats (photosynthesis is a relatively advanced metabolism) occur in the sed-iments, the majority of the microorganisms were probably lithotrophic (a very early metabolism), obtaining their carbon and energy from inorganic sources, in this case the rock/mineral surfaces. The carbonaceous microfossils are well preserved owing to their precocious silicification and the lowgrade metamorphism of the rocks, although the carbon in the microfossils is thermally mature as is to be expected in rocks of this age and metamorphic grade.

The shallow water volcanic environmental setting of the Kitty's Gap Chert is one that could have existed on Noachian Mars [START_REF] Westall | Life on the Early Earth: A Sedimentary View[END_REF]Fasset and Head-III, 2008;Greenwood and Blake, 2006), as demonstrated by recent orbital and in situ geochemical studies [START_REF] Poulet | Phyllosilicates on Mars and implications for early martian climate[END_REF][START_REF] Bibring | Mars Surface Diversity as Revealed by the OMEGA/Mars Express Observations[END_REF][START_REF] Squyres | The Spirit Rover's Athena Science Investigation at Gusev Crater[END_REF][START_REF] Bishop | Phyllosilicate Diversity and Past Aqueous Activity Revealed at Mawrth Vallis[END_REF][START_REF] Milliken | Opaline silica in young deposits on Mars[END_REF][START_REF] Squyres | Detection of Silica-Rich Deposits on Mars[END_REF]. These sediments were, moreover, deposited in an epoch (or soon after) when Mars could have hosted life at its surface [START_REF] Westall | Life on the Early Earth: A Sedimentary View[END_REF]Southam and Westall, 2007). The primitive chemo(litho)trophic organisms that lived on the Early Archaean Earth could have inhabited the Noachian martian surface. For this reason, the small, primitive cells that characterise the Kitty's Gap sediments can be considered as analogies for putative early martian microorganisms. The sediments with their fossilised microorganisms therefore constitute useful analogues for martian rocks of Noachian age.

Sample preparation before launch

In the previous STONE experiments, the rocks were cut into discs 7 cm diameter and 1 cm thickness. One of the objectives of the STONE 6 mission was to test one aspect of the hypothesis of panspermia by evaluating the survival of microbial spores protected by the thickness of the samples. The STONE 5 experiment demonstrated that 1 cm of rock (cut as flat discs) was not sufficient and it was thus decided to increase the thickness. For practical reasons, the samples being now thicker than the sample holders, the rocks were milled into a flanged dome shape that was 2 cm thick at its apex in order to provide more protection for the living microorganisms. The 3.5 Ga-old Kitty's Gap Chert was, however, too fractured to be able to be cut into the required shape and was therefore crushed into ≈3 mm-sized fragments and mixed with space-qualified cement (Sauereisen Electrotemp cement No.8 ) before being poured into a mould of the required form. The space cement is a magnesium phosphate (aMgH x (PO y ) z .bH 2 O) in a zircon (ZrSiO 4 ) and silica (SiO 2 ) base. The final shape of the sample after machining before its fixation on the capsule is shown in Fig. 2. The sample holder consists of an annular disk of phenolic silica, i.e. the same material used for the ablative capsule heat shield. The exposed part of the sample was 5 cm in diameter.

The holder was fixed onto the FOTON-M3 capsule near the stagnation point by carbon-carbon screws.

A live, hydrated culture of the photosynthetic endolithic microorganism, Chroococcidiopsis, was painted on the back of the rock before it was screwed into the sample holder.

Flight details

The FOTON-M3 capsule was launched by a Soyuz-U launcher rocket from Baikonur (Kazakhstan) on 14 September 2007. On the 26 th September after 12 days in lower Earth orbit, the velocity of the capsule was slightly decreased from 7.7 km/sec to 7.6 km/sec for atmospheric entry. The re-entry module underwent thermal shock as it entered the stratosphere. Mineralogical changes detailed below show that the temperature on the ablation surface started from a few o C, increased up to 2000 o C at the apex and decreased to less than 0 o C within a few tens of seconds. The speed of the capsule was reduced by parachute and brake rocket for landing in a field in Kazakhstan.

Two of the three STONE samples survived the experiment, as did the LITHO-PANSPERMIA sample; the reference dolerite sample was lost. A photograph of the capsule after landing is shown in Fig. 3. The Kitty's Gap Chert sample exhibited a bright white fusion crust across the entire exposed surface (see insert in Fig. 3). The final thickness of the sample ranged from 9 to 10 mm, corresponding to a loss of ≈50% of the initial thickness by ablation.

After landing, the samples were carefully and rapidly removed from the capsule in relatively sterile conditions using glove-covered hands and stored in a protective holder (Fig. 4) for transport to a clean room at the European Space & Technology Centre (ESTEC, Noordwijk, The Netherlands), from where they were collected for analysis. The transport containers had been specifically constructed to protect the samples during transport after the disintegration of one of the rocks (sandstone) used in the STONE 5 experiment.

Sample preparation after landing

In order to study changes in the Kitty's Gap Chert fragments through the thickness of the reconstituted rock, 30 µm and 500 µm thick polished sections were prepared (Fig. 5). Some of the sections were etched by hydrofluoric acid (HF) in order to dissolve the chert and to reveal the embedded organic matter. The method chosen consisted of etching portions of the 500 µm thick sections with HF droplets (24% in concentration) deposited for 45 min, followed by abundant rinsing with milliQ water. Slightly etched surfaces of 30 µm thick thin sections were also observed (HF 2.5% and HF 5% during 30 min).

Analytical methods

Several methods were used to observe, analyse and characterize the sample. An optical microscope (Olympus BX51, CBM-Orléans) was used to make preliminary observations and to localize specific areas of interest. Atomic force microscopy (AFM, Veeco D3100, CBM-Orléans) and Scanning Electron Microscopy (FEG SEM, Hitachi S4200, Centre de Microscopie Électronique, Orléans) were used both on etched and un-etched surfaces to make high magnification observations of specific structures. Elemental analyses were made with an Energy Dispersive X-ray spectrometer (EDX) attached to the SEM, as well a Cameca SX 50 microprobe (BRGM-Orléans). Raman spectroscopy was made with a Dilor XY800 (ISTO-Orléans), and WITec Alpha 500RA (CBM-Orléans) to make compositional analysis and to study the changes in the organic matter and in the minerals with reference to distance from the fusion crust.

Results

General optical aspect

The postflight rock exhibits a flat bright white vitreous surface, in comparison to the matt variegated cream-coloured cupola of the preflown reconstituted rock. The sample is 9 to 10 mm thick, whereas the preflown rock had a maximum thickness of 20 mm at its apex.

In cross section, the postflight sample can be divided into three zones parallel to the fusion crust (Fig. 5). The fusion crust itself is ≈0.8 mm thick and characterised by a bright white colour and glassy reflection. The lower ≈5 mm of the sample appears blackened to the naked eye although in thin section it is only the cement that has become black. In between the fusion crust and the lower blackened layer the sample shows a dull creamy white colour. In the following, mineralogical and morphological changes in the flown rock will be described for each constituent with respect to distance from the exposed surface, labelled x. Postflight sample. In the fusion crust (i.e. for x < 0.8 mm), the chert fragments are completely fused to a glassy texture in which no volcanic protoliths can be distinguished (Fig. 6b). Several bubbles consisting of trapped air (Raman analyses) are also observed (Fig. 6b), the air being incorporated during rapid cooling of the melted material after atmospheric entry. Broadening of the 465 cm -1 peak of quartz in the Raman spectrum (Fig. 6a) taken within the fusion crust indicates that the quartz phase is completely amorphous. As can be observed in the optical micrograph and the Raman map of the 465 cm -1 quartz peak (Figs. 6b,c), the transition between the fusion crust and the underlying non-fused sample is relatively sharp with only a narrow band of mixed amorphous and crystalline quartz between 0.7 mm < x < 0.8 mm (Fig. 6a). Quartz below 0.8 mm from the sample surface is well preserved (Fig. 6a) although some fractures traverse the chert fragments (Fig. 7a).

In this zone fractures in the chert matrix also radiate out from embedded volcanic protolith particles and are commonly lined with bubbles (Fig. 7b).

There is no change in the size and shape of the fluid inclusions in the chert in the zone furthest away from the fusion crust but, in the middle of the sample (between 0.8 mm and 3 mm), the H 2 O-filled inclusions have rounded shapes and can be up to 50 µm in size.

Volcanic protoliths

Preflight sample. The silicified hydromuscovite-replaced volcanic protoliths in the original rock exhibit irregular angular to euhedral shapes, depending on the original composition of the protolith (feldspar, volcanic glass, pyroxene; [START_REF] Orberger | Microfacies and origin of some Archean cherts (Pilbara, Australia)[END_REF]. Raman spectra of the hydromuscovite are characterised by a weak but highly fluorescent signal, the weakness of the signal being due to strong silicification of the original rock. Ti oxides, anatase and rutile, are associated with the volcanic protoliths and are common throughout the sample. Anatase is the dominant phase and occurs in aggregates dispersed throughout the chert fragments, as well as along the edges of the volcanic protholiths. Rutile occurs as tiny (≈1 µm) sparse isolated crystals within the anatase, especially inside the protoliths.

Carbon is frequently associated with anatase.

Postflight sample. In the fusion crust (top 0.8 mm) where the chert fragments are glassy, there is no trace of the protoliths due to complete melting of the minerals (Fig. 6b). In the middle of the section (between 0.8 mm and 5 mm), the volcanic protoliths have an inhomogeneous dark appearance (Fig. 8a) compared to their transparent brownish colour at the back of the sample (Fig. 8b). They are characterised by water-filled spherical inclusions (Fig. 9) although the concentration of the inclusions within the hydromuscovite is variable owing to local variations in the degree of silica replacement of the phyllosilicate.

Cement

Preflight sample. The relatively porous cement has a whitish colour and contains zircon and MgO crystals up to 100 µm in size mixed in a matrix consisting of hydrated magnesium phosphate and silica.

Postflight sample. The most immediately apparent change in the cement is the dark colouration in the lower ≈5 mm of the flown sample (Fig. 5) and fusion with the chert fragments in the fusion crust (top 0.8 mm) leading to the incorporation of Mg in the fused chert (Fig. 10e). As in the chert fragments, air bubbles occur in the cement in the fusion crust and up to 2 mm away from the sample surface. Fractures occur in the space cement throughout the thickness of the sample and are common around the chert particles.

Zircon crystals in the cement are well preserved below the fusion crust (up to 2 mm from the surface) (Fig. 10d) but adjacent to the fusion crust (between 0.8 and 2 mm) the mineral occurs in three forms: as well preserved crystals, as aggregates of tiny (≈1 µm) spherical crystals (Fig. 10c), and as dendritic zircon crystals (Fig. 10b). The tiny spherical crystals are associ-ated with a new mineralogical phase that could not be identified by Raman spectroscopy (Fig. 10a). Electron microprobe analyses show that it contains zircon and magnesium oxide. The distinct change in the morphology, and size of the crystals in the fusion crust (Fig. 10), indicates that temperatures exceeded the maximum service temperature of the cement (1426 o C, manufacturer's data).

Microfossils

Preflight sample. Fossilised microbial cells in the form of colonies of coccoids, chains of coccoids, filaments and associated EPS were identified by HR-SEM microscopy in the original rock (Fig. 11a,b) (cf. Westall et al., 2006b). The fossil cells are individually too small (< 1 µm) to be distinguished in the fine grained chert matrix by optical microscopy. HR-SEM documented the close relationship between the fossilised colonies of coccoids and the volcanic protoliths (Westall et al., 2006b, Fig. 11a,b). Optical microscopy shows that aggregates of dark spots are commonly associated with the volcanic protoliths and Raman spectroscopy confirmed their carbonaceous composition (Fig. 12). [START_REF] Orberger | Microfacies and origin of some Archean cherts (Pilbara, Australia)[END_REF] also noted a close association between carbon and the surfaces of the volcanic protoliths. D and G peak intensities in the Raman spectra of the spots of carbon in the aggregates indicate that the kerogen has a maturity that is consistent with the low grade metamorphism experienced by rocks in the region (prehnite-pumpellyite to lowermost greenschist). We interpret the aggregates observed by optical microscopy in thin section to be the same colonies of coccoids observed by HR-SEM.

Postflight sample. HR-SEM observation of unetched and etched surfaces of thin sections of the flown samples document the presence of a variety of carbonaceous structures including aggregates of tiny carbonaceous spots (Figs.

13, 14), coccoidal structures, filaments and EPS (Figs. 11c,d,13), interpreted to be the same types of microfossils described by Westall et al. (2006b) in a sawn, etched surface of the Kitty's Gap Chert. The Raman analyses of large aggregates of spots of carbonaceous matter demonstrated the maturation of the kerogen towards the fusion crust (see Fig. 15).

Our observations of thin section surfaces brought to light another type of carbonaceous structure associated with the volcanic protolith surfaces. Some of the protoliths exhibit tunnel-like cavities at their edges (Fig. 16a) filled with smooth, globular or filamentous filmy carbonaceous matter (Fig. 16bd), probably representing microbial corrosion pits filled with EPS and the degradation products of chemolithotrophic microorganisms (cf. [START_REF] Furnes | Early Life Recorded in Archean Pillow Lavas[END_REF][START_REF] Banerjee | Direct dating of Archean microbial ichnofossils[END_REF].

Modern Chroococcidiopsis biofilm

The back of the sample was inoculated with a live culture of Chroococcidiopsis microorganisms using a brush. These ≈5 µm sized photosynthetic microorganisms (Fig. 17a) occur naturally as endolithic organisms within cracks in rock surfaces [START_REF] Cockell | Interplanetary Transfer of Photosynthesis: An Experimental Demonstration of A Selective Dispersal Filter in Planetary Island Biogeography[END_REF].

No live microorganisms were found on the back of the flown rock. HR-SEM revealed carbonaceous oval cell-like structures occurring either singly (Fig. 17b), or attached to each other in apparent cell division, associated with a carbonaceous film. These structures have a limited size range from 4-5 µm. Given their carbonaceous composition and morphological similarity with the original microorganisms, we believe that they represent the carbonised remains of Chroococcidiopsis. Had the carbonaceous spheroids been produced simply from the soot of the burnt organisms, they would not exhibit the regularity of size and shape that is characteristic of these microorganisms.

Raman analysis of the burnt carbon showed D and G band components characteristic of trigonal and tetrahedral carbon bonding structures, referred to as the graphite and diamond structural types. In order to determine the thermal diffusion experienced during atmospheric entry throughout the Kitty's Gap Chert sample, we constructed a new model based on the previous models used by [START_REF] Brandstätter | Mineralogical alteration of artificial meteorites during atmospheric entry. The STONE-5 experiment[END_REF] and [START_REF] Parnell | Response of sandstone to atmospheric heating during the STONE 5 experiment: Implications for the palaeofluid record in meteorites[END_REF]. The details of our model are explained in Annex 1.

Discussion

Mineralogical changes

The model was calibrated using the quartz melting temperature of 1723 o C (Fig. 6) which allowed us to determine that the temperature of the exposed The white colour of the fusion crust may be related more to the fusion of the space cement than to that of the embedded chert fragments. The dark colour of the cement in the lower part of the sample is similar to that noted by [START_REF] Brandstätter | Mineralogical alteration of artificial meteorites during atmospheric entry. The STONE-5 experiment[END_REF] for the carbonate cement of the sandstone in the STONE 5 experiment, which was attributed to thermal alteration. As in the STONE 5 experiment, it appears that flames and heat penetrated behind the sample holder through gaps between the carbon screws and the siliceous sample holder. However, the phase changes observed within the sample show that alteration due to heat from the trailing side was not a significant contribution. This explains the good correlation between the analytical model and the observations. conditions [START_REF] Bustin | Mechanisms of graphite formation from kerogen: experimental evidence[END_REF]. In conclusion, in spite of the mineralogical alteration during atmospheric entry and the maturation of the kerogen, the silicified microorganisms and EPS in the un-melted part of the sample were physically well preserved. We conclude that microfossils from Mars (if martian life existed) could be found in martian sedimentary meteorites.

Survival of microfossils

Panspermia

Despite the fact that flames may have penetrated to the back of the rock and burnt the Chroococcidiopsis biofilm, mineralogical transformations due to the transfer of heat from the surface of the rock indicate that temperatures at the back of the sample were about 630 o C (Fig. 20). This is too high for survival of living microorganisms. Thus even without flames reaching the back of the sample, the 2 cm of rock protection would not have been sufficient to protect endolithic microorganisms. At the surface of the Earth, phototosynthetic endoliths, such as Chroococcidiopsis, need to have minimum access to light and therefore never penetrate at depths greater than ≈ 5 mm into the rock. These results are thus in agreement with the conclusion of [START_REF] Cockell | Interplanetary Transfer of Photosynthesis: An Experimental Demonstration of A Selective Dispersal Filter in Planetary Island Biogeography[END_REF] that it is unlikely that photosynthetic organisms can survive transport between planets and, thus, that photosynthesis must have appeared independently on Earth.

What about chemo(litho)trophic organisms? Lithotrophic microorganisms are not dependent on light, obtaining their carbon and energy from inorganic sources. They are common colonisers of rocks and have been found at great depths in deep mines, such as the 3 km deep gold mines of South Africa [START_REF] Lin | Long-Term Sustainability of a High-Energy, Low-Diversity Crustal Biome[END_REF]. Knowing that rocks ejected during impact can originate from some depth in the crust (≤ 100 m, [START_REF] Melosh | Swapping rocks: Exchange of surface material among the planets[END_REF][START_REF] Melosh | Exchange of meteorites (and life?) between stellar systems[END_REF], it is possible that rocks containing lithotrophic microorganisms in their fractures could be ejected by impact. Indeed, if life is still present on Mars, it will occur in protected subsurface environments (McKay et al., 1992). [START_REF] Horneck | Protection of bacterial spores in space, a contribution to the discussion on panspermia[END_REF] have shown that microbial spores can resist space conditions when protected by a rock/mineral coating but resistance of the living microorganisms to atmospheric entry had not been tested until the STONE 5 and 6 experiments.

The heat flux model (Annex 1, Fig. 19) can be used to determine the minimal thickness of rock necessary to ensure the survival of living organisms.

Using the thermal diffusivity of the basalt, the most common rock type that could contain endolithic organisms (κ basalt = 8.10 -7 m 2 .s -1 ), the maximal temperature reached within the sample has been plotted in Fig. 20. It is shown that the thickness needs to be higher than ≈5 cm in order to keep a maximal temperature at the bottom of the meteorite that does not exceed 113 o C, the maximal temperature for (hyperthermophilic) life. However, it is worth noting that this model is based on the heating profile experienced by the FOTON capsule. In the case of true meteorites it has been shown that the velocity is higher in such a way that the temperatures generated by frictional heating will be higher but will last less time [START_REF] Parnell | Response of sandstone to atmospheric heating during the STONE 5 experiment: Implications for the palaeofluid record in meteorites[END_REF].

As a consequence, the temperature reached within the rock will be probably lower than the estimated one in the STONE 6 experiment. The increase of temperature at the surface will be associated with higher ablation of matter but this effect will be counterbalanced by the rotation of the meteorites and by a decrease in the heating time. The quantity of matter ablated during this experiment can thus be considered to be relatively similar to that for true meteorites.

It should be noted that, although the Mars analogue sediment and its microfossils survived the thermal shock of atmospheric entry this experiment does not show if they could have survived the mechanical pressures exerted during the ejection of rocks from Mars. The martian meteorites show moderate levels of shock, typically about 30 GPa [START_REF] Head | Martian Meteorite Launch: High-Speed Ejecta from Small Craters[END_REF]. Temperatures can be up to 10,000 o C near the impact point leading to vaporization of the rock, but are typically between 500-3000 o C for the surrounding rock. 1 compares the compressive strengths of a variety of naturally occurring lithologies. These values provide some indication of the resistance of different rock types to pressure, thus providing an approximation of their reaction to impact pressure. Chert has a higher compressive strength (¿250 MPa) than limestone (50-100 MPa) or highly weathered rock (1-5 MPa). Because of their compactness, the silicified volcanic sediments from the 3.446 Ga-old Kitty's Gap better resist these temperatures than less consolidated porous materials [START_REF] Stöffler | Glasses formed by hypervelocity impact[END_REF][START_REF] French | Traces of Catastrophe, A handbook of shockmetamorphic effects in terrestrial meteorite impact structures[END_REF]. Moreover, despite the intense effects of impacts on the country rock, [START_REF] Melosh | Impact cratering mechanics: Relationship between the shock wave and excavation flow[END_REF] determined that intact rock fragments can be ejected from planetary surfaces without suffering too much petrological damage if they were protected by stress wave interference close to the free surface. Indeed, the existence of intact (but shocked) volcanic rocks from Mars is testimony to this process. O'Keefe and Ahrens (1985) calculated that rock fragments about 22 m in diameter could be launched to escape velocity from a 50 km crater on Mars.

Conclusion

The objectives of the STONE 6 experiment were to determine the effects of thermal alteration during atmospheric entry of martian analogue sediments and the survival of extant and fossil microorganisms during atmospheric entry. The admixture of fragments of ≈3.5 Ga-old, silicified volcanic sediments and space cement survived the thermal shock well, forming a white fusion crust. This contrasts strongly with the black crust typical of basaltic martian meteorites (although the colour of the fused material in our experiment may be an artefact of the space cement mixture).

A number of critical mineralogical phase changes and alterations occurred in the chert fragments at different depths in the thickness of the flown rock, including melting of the quartz and hydromuscovite, transition of quartz into quartz and back to quartz, and degassing of OH in the hydromuscovite.

The evolution of the temperature within the sample could be thus modelled,

showing that the maximum temperature reached at the surface of the sample was 1775 o C and at the back of the sample it was ≈630 o C.

Microfossils embedded in the chert fragments survived structurally, except in the fusion crust and only a slight maturation of the kerogen was observed. By analogy, it is concluded that microfossils dating back from the Noachian period could also survive the atmospheric entry and that they could be found in martian sedimentary meteorites, assuming survival during the initial impact leading to ejection. Although our model of the thermal gradient within the sample shows that 2 cm of rock thickness would not be sufficient to protect live microorganisms from the high temperatures experienced at the back of the sample, it may still be possible for endolithic lithotrophs in martian crustal rocks to survive ejection, space transport and atmospheric entry in larger meteorites.

Annex 1

Modelling temperature distribution

We constructed an analytical model in order to determine the evolution of the temperature within the sample during atmospheric entry. Previously [START_REF] Kimura | Back-transformation of high-pressure phases in a shock melt vein of an H-chondrite during atmospheric passage: Implications for the survival of high-pressure phases after decompression[END_REF] and [START_REF] Parnell | Response of sandstone to atmospheric heating during the STONE 5 experiment: Implications for the palaeofluid record in meteorites[END_REF] used a Heviside step function (i.e. T (t) = 0 for t < 0, T (t) = T max for 0 < t < τ and T (t) = 0 for t > τ )

to model the thermal conditions undergone by meteorites. This model is associated with very rapid heating, temperature retention for a short period of time and then very rapid cooling. However, the experimental data of the heat stream measured on the capsule shows that heating, and thus the temperature, at the surface of the sample was progressive (Fig. 18). In order to take this evolution into account as well as possible in the model, the curve was decomposed into several sections of constant temperature (Fig. 18). Adopting an initial temperature of 0 o C, which is approximately the temperature at the top of the stratosphere (stratopause), the temperature at the heating surface at the time t is thus given by:

T (0, t) =                  T 0 for t ≤ τ 0 T 1 for τ 0 < t ≤ τ 1 . . . T n for τ n-1 < t ≤ τ n (1)
The global function T (x, t) is thus expressed for each step n as T n (x, t).

Time evolution of the temperature distribution can be obtained by solving the following diffusion equation for each step:

∂T ∂t = κ ∂ 2 T ∂x 2 . ( 2
)
Where κ is the thermal diffusivity of the material. Since the sample is initially in thermal equilibrium, for the first step the solution is given by:

T 0 (x, t) = T 0 . (3)
Mathematical induction can be used to prove that the statement

T n (x, t) = T n-1 (x, t) + (T n -T n-1 ) 1 -erf x 4κ(t -τ n-1 ) (4)
holds for all natural numbers n, with erf the error function given by :

erf(z) = 2 √ π z 0 e -ζ 2 dζ. (5) ∂ erf(z) ∂z = 2 √ π e -z 2 . ( 6
)
It is worth noting that for x = 0, the condition T n (0, t) = T n (Eq. 1) is satisfied.

Basis:

In the following, we define:

χ n = x 4κ(t -τ n-1 ) (7)
and thus:

∂χ n ∂x = 1 4κ(t -τ n-1 ) , ( 8
)
∂χ n ∂t = - x 2(t -τ i-1 ) 4κ(t -τ n-1 ) . (9)
For n = 1:

T 1 (χ 1 ) = T 0 + (T 1 -T 0 )(1 -erf χ 1 ) (10)
This leads to:

∂T 1 (x, t) ∂t = (T 1 -T 0 ) 2 √ π e -χ 2 1 χ 1 2(t -τ 0 ) , (11)
∂ 2 T 1 (x, t) ∂x 2 = (T 1 -T 0 ) 2 √ π e -χ 2 1 χ 1 2κ(t -τ 0 ) . ( 12
)
The two sides of the diffusion equation (Eq. 2) are thus equal for n = 1, so the statement (Eq. 4) is true for n = 1. Thus it has been shown that T 1 (x, t) holds.

Inductive step:

Assuming T n (x, t) holds, it must be shown that then T n+1 (x, t) holds, that is:

T n+1 (x, t) = T n (x, t) + (T n+1 -T n ) 1 -erf x 4κ(t -τ n ) . ( 13
)
The two parts of the Eq. 2 are given by:

∂T n+1 (x, t) ∂t = ∂T n (x, t) ∂t + (T n+1 -T n ) 2 √ π e -χ 2 n+1 χ n+1 2(t -τ n ) , (14)
∂ 2 T n+1 (x, t) ∂x 2 = ∂ 2 T n (x, t) ∂x 2 + (T n+1 -T n ) 2 √ π e -χ 2 n+1 χ n+1 2κ(t -τ n ) . (15) Since ∂T n (x, t) ∂t = κ ∂ 2 T n (x, t) ∂x 2 , (16)
it is deduced that

∂T n+1 (x, t) ∂t = κ ∂ 2 T n+1 (x, t) ∂x 2 . ( 17
)
therefore T n+1 (x, t) holds.

Since both the basis and the inductive step have been proved, it has now been proved Eq. 4 is solution for all natural n Q.E.D.

Numerical application

In order to fit the "stairs" curve used in the model (Fig. 18), a well known temperature is needed. The observations show that the quartz particles located at the exposed surface melted in the first ≈0.8 mm (Fig. 6); the temperature reached at this depth is thus equal to the melting point of quartz,

i.e. 1723 o C [START_REF] Shchipalov | Thermodynamic analysis of melting, vitrification, and crystallization in the SiO 2 system, Glass and Ceramics[END_REF]. Using the thermal diffusivity of the quartz (κ Qz = 5.10 -6 m 2 .s -1 ), Eq. ( 4 • T quartz f = 1723 o C, the melting point of quartz [START_REF] Shchipalov | Thermodynamic analysis of melting, vitrification, and crystallization in the SiO 2 system, Glass and Ceramics[END_REF]),

• T cement = 1426 o C, the maximum service temperature of the cement,

• T muscovite f = 1320 o C, the melting point of muscovite,

• T ana→rut = 610 o C, the phase transition temperature of anatase into rutile [START_REF] Jamieson | Pressure-Temperature Studies of Anatase, Brookite Rutile, and TiO 2 (II): a Discussion[END_REF]),

• T quartz α→β = 573 o C, the phase transition temperature from α quartz into β quartz [START_REF] Shchipalov | Thermodynamic analysis of melting, vitrification, and crystallization in the SiO 2 system, Glass and Ceramics[END_REF][START_REF] Mosbah | Use of the α → β quartz transition to monitor the temperature increase produced by a proton microbeam[END_REF]),

• T hydro = 250 o C, the lower temperature of loss of hydroxyl water in hydromuscovite [START_REF] Threadgold | A Hydromuscovite with the 2M 2 Structure, from Mount Lyell, Tasmania[END_REF].

Thus, according to the model, the quartz melted in the first 0.4 mm, the cement underwent temperatures exceeding the maximum service temperature in the first 2 mm and the muscovite melted in the first 3 mm. Throughout the sample, anatase transforme into rutile, α quartz turned into β quartz and devolatilization occurred in the hydromuscovite. These results are in good accordance with the observed mineral alteration despite the approximation on the thermal diffusivity of the sample.

Figure captions

  3.2. Rock fragments 3.2.1. Chert matrix Preflight sample. The original rock consists of volcanic protoliths replaced by hydromuscovite within a microcrystalline (≤ 1 µm) matrix of α quartz (quartz) that, in places, has been slightly recrystallised into euhedral crystals 5-10 µm in size. Small (≈1 µm) and irregular-shaped fluid inclusions occur in the chert matrix of the original rock. Sample preparation for flight produced no mechanical modifications, such as fracturing (N.B. the original rock is moderately fractured with the fractures coloured by Fe oxide deposits).

  Mineralogical changes observed in the flown Kitty's Gap Chert include (1) melting of quartz (1723 o C, Shchipalov, 2002), hydromuscovite (1320 o C) and cement (1426 o C, manufacturer's data) in the fusion crust, (2) the change in quartz phase from α quartz into β quartz at 573 o C (Mosbah et al., 1997), (3) an increase in the size of fluid inclusions in the chert, and (4) the devolatilization of the hydromuscovite (250 o C, Threadgold, 1959). The change in phase from α to β is accompanied by a corresponding change in crystal lattice size from (a = b = 4.913 Å, c = 5.405 Å for the α quartz and a = b = 4.9977 Å and c = 5.4601 Å for the β quartz; Mosbah et al., 1997). Rapid quenching of the rock brings about a retrograde phase change back to α quartz and another change in lattice dimension. The dilatation and relaxation of the lattice are probably the causes of the fractures observed in the quartz.

  surface was 1775 o C. Since the rock particles are embedded in cement, the thermal diffusivity of the cement was used in the model to estimate the temperatures within the sample. Since diffusivity in the cement is lower than in the chert, the curves plotted in Fig. 19 correspond to minimal calculated temperatures. They show that the estimated minimal temperature at the back of the sample is 630 o C.

Figure 11

 11 Figure 11 is a comparison of pre-flight and post-flight microstructures (from Westall et al., 2006b) showing that both the filamentous and the coccoid structures are well preserved in the flown rock. The ratio of the graphite (G) and disorder (D) peak intensity (G/D) is plotted versus the distance from the exposed surface in Fig. 15. The G/D ratio increases with temperature increase and decreasing distance from the fusion crust, thus demonstrating thermal maturation of the kerogen during atmospheric entry. Graphitization occurs typically at a temperature of about 900 o C, depending on pressure

  ) leads to the maximum temperature reached at the surface: 1775 o C. This temperature is then used as a reference to estimate the temperature deeper within the sample Since the rock particles are embedded in the cement, the thermal diffusivity of the cement (κ cement = 6.10 -7 m 2 .s -1 ) is used to determine the temperature distribution within the sample. Since the cement is the least diffusive component, the temperatures obtained in the model therefore correspond to minimal values. The temperature versus time is plotted for various depths in Fig. 19. The curves have been smoothed in order to erase the stepped aspect associated with the model. The following relevant mineral alteration temperatures have been added to Fig. 19 in order to compare the model with the experimental observations:

Figure 1 :

 1 Figure 1: Field view of the original rock from the Kitty's Gap Chert, Pilbara, Australia.

Figure 2 :

 2 Figure 2: Final shape of the reconstituted sample consisting of 3 mmsized fragments of chert mixed in space cement (a); top (b) and bottom (c) views of the Kitty's Gap Chert sample. The fragments of the original rock mixed in cement are clearly visible.

Figure 3 :

 3 Figure 3: FOTON-M3 capsule after landing showing emplacement of the rock sample around the stagnation point. Detail of the Kitty's Gap Chert sample, showing the presence of a white fusion crust over the whole of the exposed surface and streaming of solidified molten rock and heat shield material (insert).

Figure 4 :

 4 Figure 4: (a) Sketch of transportation container and (b) photograph of the sample in the container in the clean room at ESA-ESTEC.

Figure 5 :

 5 Figure 5: Polished thin section 30 µm thick of the Kitty's Gap Chert sample after atmospheric re-entry. The fusion crust is at the top, denoted by a thin (< 1 mm) bright white layer. Note the dark colour of the cement in the lower part of the image.

Figure 6 :

 6 Figure 6: Analysis of the quartz in the fusion crust. (a) Raman spectra of quartz (i) spectrum at 0.4 mm from the exposed surface (glassy state), (ii) spectrum at 0.7 mm from the exposed surface (mixed state), and (iii) spectrum at 1.2 mm from the exposed surface. (b) The optical image clearly shows the presence of bubbles in the glassy fusion crust. (c) Raman mapping associated with the optical image represents the evolution of the width of the 465 cm -1 peak of the quartz, the dark area being associated with fused quartz.

Figure 7 :

 7 Figure 7: (a) SEM image of cracks in and around a quartz particle after slight etching (HF 5%, 30 min, distance from the exposed surface 7.6 mm). (b) Hydromuscovite grains after loss of OH, surrounded by cracks in the quartz matrix (distance from the exposed surface 4.6 mm).

Figure 8 :

 8 Figure 8: Optical view of the volcanic protoliths (a) in the middle of the section (distance from the exposed surface 4 mm) with a dark appearance and (b) at the back of the sample (distance from the exposed surface 8 mm).

Figure 9 :

 9 Figure 9: Optical and Raman images of a melted hydromuscovite grain located at 1.9 mm from the exposed surface. The H 2 O-filled bubbles are clearly highlighted by Raman spectroscopy (colour coding: brown for quartz, blue for melted hydromuscovite and green for water).

Figure 10 :

 10 Figure 10: (a) Raman spectra of the cement showing the transformation of zircon into new phases: (i) spectrum of the small circular grains (b) observed in the fusion crust (distance from exposed surface 1 mm), (ii) spectrum of the dendritic structures (c) observed near the fusion crust crust (distance from exposed surface 1.5 mm) and (iii) spectrum of the well preserved zircon particles (d) at the back of the sample (distance from exposed surface 8 mm). (e) SEM image showing the intermixing of the fused Mg-rich cement (lighter coloured areas with dendritic crystals of zircon, arrow) and chert fragments in the fusion crust (un-etched surface, distance from the exposed surface 0.8 mm). Note the bubbles of trapped air.

Figure 11 :

 11 Figure 11: Comparison between (a) pre-flight and (b) post-flight microfossils. (a) and (c) filaments (distance from exposed surface 7.3 mm) and (b) and (d) coccoid structures (distance from exposed surface 7.2 mm).

Figure 12 :

 12 Figure 12: (a) Optical and (b) Raman analysis of organic matter on the surface of a hydromuscovite grain. The Raman image clearly shows the distribution of anatase and carbon around the grain (colour encoding: carbon in yellow, anatase in pink, quartz in brown, muscovite in blue and rutile in green).

Figure 13 :

 13 Figure 13: (a) Optical transmitted light image of spots of organic matter forming a dark aggregate (probably a fossilised colony of microorganisms, cf.

  around a hydromuscovite grain (distance from the fusion crust 9.8 mm). (b) and (c) HRSEM images of the tunnel structures located in (a). A thick film of silicified EPS has filled the tunnels (arrows). Filamentous structure observed in the tunnel (c).is shown by AFM in (d) (80×80 µm 2 in size, AFM image processing using W SxM,[START_REF] Horcas | W SxM: A software for scanning probe microscopy and a tool for nanotechnology[END_REF].

Figure 17 :

 17 Figure 17: (a) Optical view of Chroococcidiopsis bacteria. (b) HRSEM image of the carbonized bacteria at the back of the sample after the experiment.

Figure 18 :

 18 Figure 18: Variation in temperature at the exposed surface of the flown sample during entry into the Earth's atmosphere as a function of heating time. The "stairs" curve used for the model have been reported.

Figure 19 :

 19 Figure 19: Temperature distribution as a function of heating time and depth in the sample, after smoothing of the curves. The temperatures of relevant mineral alteration have been added to the figure.

Figure 20 :

 20 Figure 20: Maximum temperature versus depth in a basalt sample estimated from the analytical model. The maximal temperature for life has been reported (113 o C).

Figures

  Figures

  Figure 1:

  Figure 2:

  Figure 5:

  Figure 6:

  Figure 10:

  Figure 12:

  Figure 13:

  Figure 14:

  Figure 15:

  Figure 16:

Figure 17 :

 17 Figure 17:

Table 1 :

 1 Comparison of the compressive strengths of different rock types

As the writing of this paper (November

2009) the number of martian meteorites found is 53, 52 SNC meteorites and ALH84001[START_REF] Papike | Silicate mineralogy of martian meteorites[END_REF], http://www.imca.cc/mars/martian-meteorites-list.htm).

Acknowledgments

We thank the European Space Agency for providing the flight opportunity on the FOTON M3. CNES supported the experiment financially. We thank the Museum of Natural History of Vienna for preparation of the reconstituted Kitty's Gap Chert sample.

ACCEPTED MANUSCRIPT

 Westall et al., 2006b)


MD Mario Airoldi

Pietro Gabriele

MD Anna Maria Gabriele

MD Massimiliano Garzaro

Luca Raimondo

Fulvia Pedani

Fabio Beatrice

Giancarlo Pecorari

Carlo Giordano

Induction chemotherapy with carboplatin and taxol followed by radiotherapy and concurrent weekly carboplatin + taxol in locally advanced nasopharyngeal carcinoma

Keywords: Nasopharyngeal carcinoma, neoadjuvant chemotherapy, chemoradiation, carboplatin, taxol

Introduction

In nasopharyngeal carcinoma (NPC) the place of chemotherapy (CHT), which is not discussed in metastatic disease, is controversial for the initial management of the disease.

Recent trials and meta-analyses highlight the need to associate chemotherapy with radiotherapy (RT): concomitant chemo-radiotherapy (CRT) appears to be now the standard treatment for locally advanced (T2B and more) and/or node positive (N+) patients [START_REF] Guigay | Nasopharyngeal carcinoma and therapeutic management: the place of chemotherapy[END_REF].

Despite its more questionable role, the addition of induction CHT remains attractive in locoregionally advanced NPC patients, partly with the purpose of shrinking down the primary tumour before irradiation and partly in order to eradicate micrometastases without delay. However, phase III trials, comparing induction chemotherapy with radiotherapy alone [START_REF]VUMCA I trial: preliminary results of a randomized trial comparing neoadjuvant chemotherapy (cisplatin, epirubicin, belomycin) plus radiotherapy vs. radiotherapy alone in stage IV (N2, M0) undifferentiated nasopharyngeal carcinoma: a positive effect on progression-free survival[END_REF][START_REF] Chua | Preliminary report of the Asian-Oceanian Clinical Oncology Association randomized trial comparing cisplatin and epirubicin followed by radiotherapy versus radiotherapy alone in the treatment of patients with locoregionally advanced nasopharyngeal arcinoma[END_REF][START_REF] Ma | Results of a prospective randomized trial comparing neoadjuvant chemotherapy plus rsdiotherapy with radiotherapy alone in patients with locoregionally advanced nasopharyngeal carcinoma[END_REF][START_REF] Haeryama | A prospective randomized trial comparing neoadjuvant chemotherapy with radiotherapy alone in advanced nasopharyngeal carcinoma[END_REF], failed to show an improvement in overall survival, despite a significant reduction in local and distant failures.

Four recent published studies [START_REF] Oh | Induction chemotherapy followed by concomitant chemoradiotherapy in the treatment of locoregionally advanced nasopharyngeal cancer[END_REF][START_REF] Rischin | Excellent disease control and survival in patients with advanced nasopharyngeal cancer treated with chemoradiation[END_REF][START_REF] Al-Amro | Neoadiuvant chemotherapy followed by concurrent chemoradiation therapy in locally advanced nasopharyngeal carcinoma[END_REF], performed in extra-European countries, shown that induction CHT followed by CRT obtained high responses in patients coming from both endemic and non endemic areas: in two trials the authors proposed a three-drugs induction scheme, while in the others cisplatin + epirubicin or paclitaxel and carboplatin were respectively administered.

We have published our previous experience with induction cisplatin plus epirubicin followed by conventional radiotherapy plus every 3 week cisplatin administration [START_REF] Airoldi | Induction Chemotherapy with Cisplatin and Epirubicin followed by radiotherapy and concurrent Cisplatin in locally advanced nasopharyngeal carcinoma observed in a non-endemic population[END_REF]; our results confirm the high activity of this approach in a non-endemic population.

No phase II trials have investigated a two-drug combination during conventional, non-splitted, radiotherapy after a full course of induction chemotherapy.

When the study was designed the association carboplatin + taxol was known as a feasible and effective combination in advanced-metastatic nasopharyngeal tumours. [START_REF] Airoldi | Carboplatin plus taxol is an effective third-line regimen in recurrent undifferentiated nasopharyngeal carcinoma[END_REF][START_REF] Tan | Phase II trial of a paclitaxel and carboplatin combination in Asian patients with metastatic nasopharyngeal carcinoma[END_REF][START_REF] Yeo | A phase II study combination paclitaxel and carboplatin in advanced nasopharyngeal carcinoma[END_REF] These two drugs were known as radiosensitizers and weekly taxol infusion was indicated as an ideal way to give dose-density with enhanced therapeutic index. [START_REF] Seidman | One-hour paclitaxel via weekly infusion: dose-density with enhanced therapeutic index[END_REF] In this paper we report our experience with neoadjuvant chemotherapy with carboplatin + taxol followed by radiotherapy with weekly carboplatintaxol combination in locally advanced NPC, observed in a non-endemic population.

Methods and Materials

Eligibility

In the present multicentric retrospective analysis patients having histological confirmed NPC, stage III-IVB according to 2010 AJCC stage classification (7 th ed.), who had received no previous CHT and/or RT were included. Individuals aged >18 presenting measurable disease and WHO performance status (WHO-PS) of 0 or 1 were also considered eligible for our study. Clinical introduction of this two-drug association was approved by the Ethics Committee of each participating centre and all patients provided written informed consent.

Patients were excluded in case of inadequate organ function, as indicated by an absolute neutrophil count ≤1.5 x 10 9 /L, platelet count ≤100 x 10 9 /L, serum creatinine ≥1.5 times the upper limit of normal or 24-hour creatinine clearance ≤ 50 ml/min, serum bilirubin > 1.5 times the upper limit of normal (ULN), ALT, AST and alkaline phosphatase levels ≥ 2 times the ULN. Moreover we considered not eligible patients presenting prior malignancy apart from non-melanoma skin cancer or carcinoma in situ of the uterine cervix, pre-existing motor or sensory neurotoxicity ≥ WHO grade 2, significant hearing impairment unless due to NPC, active uncontrolled infection, unstable cardiac disease and pregnancy or lactation. Written informed consent was obtained from all patients.

Evaluation and follow-up

The main contents of pre-treatment evaluation have been the following: physical examination, assessment of PS, urine analysis, complete blood cell count (CBC) with differential, serum electrolytes, liver function tests, magnetic resonance, and measurement of index lesions. Other tests included electrocardiography (ECG) and 24-hour creatinine clearance. Baseline imaging was composed by bone scan, contrast-enhanced computed tomography (CT) scan of thorax and abdomen, CT and/or magnetic resonance imaging (MRI) of the nasopharynx and neck.

During the treatment period, patients underwent weekly physical examination and toxicity assessment. Laboratory exams and 24-hour creatinine clearance had to be repeated every 3 weeks, while CBC count had to be performed on days 1 and 8 of each cycle of neoadiuvant chemotherapy and once a week during RT.

Assessment of tumour response by clinical examination and head and neck MRI or CT was performed after neoadjuvant CHT. Once completed CHT and CRT, endoscopy, head and neck MRI or CT, thoracic and abdominal CT scan could be performed.

Six to eight weeks after the completion of CRT, complete physical examination, nasopharyngoscopy, thoracic and abdominal CT scan, MRI of nasopharynx, biochemistry and blood cell count could be performed. Patients had to be followed up by clinical examination every 2 months during the first year, every 3 months for the subsequent 2 years and every 6 months thereafter. Nasopharyngoscopy and imaging (CT, MRI or ultrasound investigation) were disposed for patients developing signs or symptoms suggestive of disease recurrence. Endoscopic or ultrasound guided biopsy had to be performed, if deemed necessary.

Tumour response was assessed according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria. Systemic toxicity from treatment was graded according to WHO and Radiation Therapy Oncology Group (RTOG) criteria.

Induction Chemotherapy

Neoadjuvant CHT consisted of 3 cycles of paclitaxel 175 mg/m2 administered over 3 hours followed by carboplatin area under the concentration-time curve 6 (carboplatin dosing to the area under the curve of 6 was calculated using Calvert Formula) administered over 30 to 60 minutes , repeated on day 22. Paclitaxel was preceded by premedication with dexametasone (20 mg) , diphenidramine (25 mg) and ranitidine (50 mg). If the absolute neutrophil count was less 1,200/mm3 or platelets was less than 100,000/mm3 on day 22, treatment was held until counts returned to this level. If the cycle was held more than 3 weeks, patients proceeded to tumor response evaluation and started chemo-radiotherapy. Prophylactic use of recombinant granulocyte colony stimulating factor was not allowed.

Concurrent chemo-radiotherapy

Chemotherapy consisted of 60 mg/m2 of paclitaxel given over 1 hour intravenously every week.

Patients were pretreated with dexametasone 20 mg given orally on the evening prior to and the morning of paclitaxel infusion. Diphenidramine 25 mg and ranitidine 50 mg were given iv prior to paclitaxel infusion. Carboplatin was given after paclitaxel infusion, for 30 to 60 minutes.

Carboplatin dosing to the area under the curve of 1 was calculated using Calvert Formula.

Antiemetics were administered as the routine practice. Colony stimulating factors were not used.

Dose modification for toxicity

Toxicity was graded according to the WHO Criteria. Chemotherapy dose modifications were made for grade 3-4 mucositis or mucositis requiring hospitalization for hydratation/pain management or for grade 3-4 hematologic toxicity. If grade 4 mucositis occurred, chemotherapy was withheld and resumed after the mucositis improved to ≤ grade 2. Carboplatin was withheld if a second episode occurred , and both paclitaxel and carboplatin were withheld if a third episode occurred.

For hematologic toxicity, the dose of paclitaxel and carboplatin was reduced to 50% for granulocyte count of 500 to 1000/µL and/or platelet count of 50,000 to 75,000/µL. Both drugs were withheld if the granulocyte and/or platelet levels dropped below 500/µL and 50,000/µL, respectively. Colony stimulating factors were not used. If the weekly granulocyte count was < 500/µL, or platelet was < 50,000/µL, radiotherapy was withheld until counts, measured twice weekly , recovered to above that level.

Enteral tube feeding was used following physician's prescriptions.

Radiotherapy

Radiation treatment was started 3 weeks after the three cycles of Neo Adjuvant Chemotherapy (NACT). Three-dimensional conformal radiotherapy (3D-CRT) was performed on all patients.

Treatment was done using 6 MV photons on a Siemens Primus Linear Accelerator with a multileaf collimator.

For head immobilization in the supine position, a thermoplastic facial mask (MED-TEC) was used.

CT-scans were performed for treatment planning.

The clinical target volume (CTV) and organs at risk (OaRs) were outlined on the axial images and were determined according to the CT/RMI findings of the gross tumour and microscopic extension.

A 10-mm expansion margin was applied to the CTV to obtain the Planning Target Volume (PTV).

The CT images were transferred to the PLATO-Nucletron Planning System, which allowed to obtain Digitally Reconstructed Radiographs (DRR) from the digital image set.

Planned RT consisted of 70 Gy, in 35 fractions, over 7 weeks to all known sites of disease and 50 Gy to sites of potential spread, including the uninvolved neck.

Residual cervical lymphoadenopathy was supplemented with electron beams.

For the first 40 Gy, the nasopharyngeal area and the upper neck were irradiated in one volume with conformal lateral opposing fields. An anterior field was used for the lower neck and supraclavicolar fossa with a laryngeal block.

Thereafter, in 18 cases the radiation beams were rearranged to shrink the field size progressively, after every 10 Gy, to establish gradually a 100% isodose level that covered the PTV with a total actual dose of 70 Gy.

In 12 patients, in order to reach maximal conformity, after the first 40 Gy of treatment, we employed 5 to 7 fields for the nasopharyngeal region.

The beam arrangements were determined depending on the anatomical features of the tumour and its relationship with surrounding structures. Wedges were used to improve the conformity of the isodose curves, when necessary.

Maximal doses to the optic pathways, brain stem and spinal cord were limited to 50 Gy, 54 and 45Gy, respectively. For all patients and treatment plans, dose-volume histograms (DVHs) were calculated for the PTV and OaRs.

Treatment verification was obtained with portal imaging for each new field, and repeated weekly.

Treatment interruptions were only allowed for severe normal tissue reactions, such as confluent mucositis.

Statistical methods

Objective response rate (ORR) represented the primary endpoint and it could be defined as the proportion of patients whose best response was either partial or complete (PR+CR).

Secondary endpoints included disease control rate (DCR), defined as the proportion of patients whose best response was either PR or CR or stable disease (SD), occurrence of grade 3-4 adverse events, as well as progression free survival (PFS) and overall survival (OS).

PFS definition was as follows: the time from the date of the study entry up to the date of first progression, second primary malignancy or death from any causes, whichever came first. Subjects not progressed or died at the time of the analysis were censored at the last disease assessment date. OS was defined as the time from the date of the study entry to the date of death from any cause. Subjects who were not reported as dead at the time of the analysis were censored at the date they were last known to be alive. Survival curves were estimated using the Kaplan-Meier method.

All enrolled patients meeting the above described eligibility criteria and not presenting major violations within the duration of our study became subjects of the analysis. Such analysis, indeed, was restricted to patients who received at least one cycle of either induction CHT or CRT.

Results are expressed as point estimates and their 95% confidence intervals (95% CIs). Analysis were carried out using SAS Software, version 9.1 (SAS Institute, Cary, NC).

Results

From 2002 to 2007, 62 patients, affected by NPC, have been observed in the participating centres; 30 patients were enrolled in this study protocol. Twenty-two (73.3 %) patients were males and 8 patients were females (26.7%) with a median age of 54 years (range 29-69). ECOG performance status was 0 in 27 patients (90%) and 1 in 3 patients (10%). WHO histology was as follows: type 2 histology in 3 patients (10%), type 3 in 27 patients (90%). Positive tissue EBV DNA was observed in 23 patients (76%). According to the 2010 AJCC staging system 14 patients had stage III (47%), 13 patients stage IVa (43%) and 3 patients stage IV b (10%) disease; T 3-4 lesions were 25/30 (83.3%) while N2/3 lesions were 21/30 (70%) (tab 1). Local extension outside nasopharyngeal cavity was as follows: bone lysis in 7 patients (23.3%), paranasal sinuses involvement in 3 patients (10%), infratemporal fossa extension in 6 patients (20%) and intracranial extension in 9 cases (30%).

Activity

All 30 patients completed the planned treatment without protocol violations, and therefore they all were considered assessable for response. After two cycles of CHT no clinical progression was registered and all patients completed the third cycle. After three cycles of neoadjuvant CHT, we registered the following results: 10 patients (33%) achieved clinical and imaging complete response (CR); eighteen patients (60%) had a partial response (PR); 6 patients (20%) achieved PR on the tumour site (T) and no change in regional lymph nodes (N), twelve patients (40%) achieved a PR on T and N; two patients (6.6%) had no change neither in T nor in N site. Objective response rate was 93% (95% CI 78.4 -98.8%), while disease control rate was 100% (95% CI 94.1% -100%).

At the end of CRT, 26 patients (87%) achieved a clinical CR at both T and regional node and 4 patients (13%) were in PR (2 patients on T and N , 1 patient on T only and 1 patient on N only) for an overall response rate of 100 % patients. Objective response rate was 100% (95% CI 89.1%-100%).

Follow-up at 12 weeks confirmed that 26 patients (87%) had reached a CR and that 4 patients (13 %) were in PR according to imaging techniques. One patient with partial response in the neck was submitted to bilateral neck dissection achieving a complete remission.

At a median follow-up time of 49.5 months (range 16-89 months), 6 (20%) patients progressed and 4 (13.3%) died for tumour. The 3 and 5 year PFS was 80% (95% CI 69% -99%) and 75.0% (95% CI 55%-85%) respectively and the 3 and 5 year OS was 85.% (95% CI 69%-92%) and 80.0% (95% CI 66%-91%) respectively (fig. 1). There was a non significant PFS and OS difference between stage III and IV lesions (p>0.05).

Five year loco-regional control was 90.3% (95% CI 69%-99%) and five year distant metastases free survival was 85% (95% CI 72%-97%)(Fig 2).

In the follow-up a progression was found in 6 patients and, in particular, two patients had T relapse, one had T and N recurrence, three developed distant metastases. All patients (3 cases; median time to metastases = 30 months ; range 18-36) with distant metastases (lung 2, lung + bone 1) died after a median period of 27 months (range 21-39 months); they were treated with a median of 2 lines of chemotherapy (range 1-4). T relapses (median time to relapse= 19 months) were treated with reirradiation plus chemotherapy achieving a persistent complete remission. One patient with T and N relapse was treated with chemotherapy achieving a PR. The second-line chemotherapy was cisplatin plus epirubicin in all cases. Toxicity A list of acute toxicities with reference to WHO criteria is reported in table 2 and 3.

The most clinical relevant side effect of NACT was hematologic toxicity; grade 3-4 neutropenia was observed in 25 cases (83 %) with grade 4 in 15 patients (50.%) without any episode of febrile neutropenia .G-CSF was administered to 6 patients (20%). Grade 3 thrombocytopenia occurred in 4 patients (13.3%) without any hemorrhagic episode while grade 3 anaemia was observed in 3 patients (10%).

The G 3 non-hematologic toxicity was represented only by oropharyngeal mucositis in 2 patients (6.6%). All patient received 3 full-dose cycles of CHT.

The most frequent toxicity during CRT was oropharyngeal mucositis occurring in all cases; 16 patients (53.3 %) developed G3 toxicity and 5 (16.6%) had G4 toxicity. Five of them (16.6%) required admission to hospital for intravenous hydration and nasal gastric tube feeding. Grade 3-4 haematological toxicity during CRT was as follows: neutropenia in 19 patients (63.3%; grade 4 in 5 cases -16.6%), thrombocytopenia in 5 (16.6%) and anaemia in 5 (16.6%). No febrile neutropenia or hemorrhagic episode was seen. During CRT renal toxicity and neurotoxicity were mild and only 3 patients (10%) developed grade 3 neuropathy, which was treated with gabapentin and resolved in three months. In 5 patients (16.6 %) weight loss was significant and they lost ≥ 10% of their body weight. No toxic deaths were registered during or immediately after treatment.

Twenty-seven patients (90%) received a dose between 66 and 70 Gy; only 3 patients (10 %) interrupted radiation therapy for more than 5 days.

During radiotherapy 189 out of 210 scheduled cycles (90%) were administered : 21 patients (70%) received 7 cycles, 2 patients (6.6%) 6 cycles, 2 patients (6.6 %) 5 cycles and 5 patients (16.6%) 4 cycles; the relative dose intensity of taxol and carboplatin were 80.1% and 74.4% , respectively.

We registered mild late side effects and sequelae due to CRT according to RTOG criteria. Neck fibrosis was present in 15 patients (50%) after 6 months of follow-up but it did not take to a relevant clinical problem. RTOG grade 2 xerostomy and disgeusia persisted in 6 patients (20%) during the first year of follow-up and in 3 patients (10%) thereafter. Transient swallowing disorders were reported in 3 (10 %) patients. We observed sensorineural hearing loss in 2 (6.6 %) patients from the beginning of CRT. Such side effect improved slowly after one year from the end of CRT in 1 patient, while it was irreversible in one patient.

Discussion

In NPC despite the advances made in clinical therapies of curative RT, with or without concurrent CHT, the 5-year overall survival rates of ~ 75% underscore opportunities for improvement, in particular for patients with advanced disease when survival rates decline to < 60% [START_REF] Lee | Treatment results for nasopharyngeal carcinoma in the modern era : the Hong Kong experience[END_REF].

The results of the last meta-analysis [START_REF] Baujat | Chemotherapy in locally advanced nasopharyngeal carcinoma: an individual patient data meta-analysis of eight randomized clinical trials and 1753 patients[END_REF] confirm the role of concurrent chemoradiotherapy as a standard treatment for locoregionally advanced NPC.

A significant benefit was found for overall survival (6% at 5 years) and event-free survival (10% at 5 years) with the addition of chemotherapy. The benefit on survival was essentially observed when chemotherapy was administered concomitantly with radiotherapy.

Induction chemotherapy delivered before radiotherapy is an attractive strategy in NPC patients.

However, phase III trials, comparing induction chemotherapy with radiotherapy alone [START_REF]VUMCA I trial: preliminary results of a randomized trial comparing neoadjuvant chemotherapy (cisplatin, epirubicin, belomycin) plus radiotherapy vs. radiotherapy alone in stage IV (N2, M0) undifferentiated nasopharyngeal carcinoma: a positive effect on progression-free survival[END_REF][START_REF] Chua | Preliminary report of the Asian-Oceanian Clinical Oncology Association randomized trial comparing cisplatin and epirubicin followed by radiotherapy versus radiotherapy alone in the treatment of patients with locoregionally advanced nasopharyngeal arcinoma[END_REF][START_REF] Ma | Results of a prospective randomized trial comparing neoadjuvant chemotherapy plus rsdiotherapy with radiotherapy alone in patients with locoregionally advanced nasopharyngeal carcinoma[END_REF][START_REF] Haeryama | A prospective randomized trial comparing neoadjuvant chemotherapy with radiotherapy alone in advanced nasopharyngeal carcinoma[END_REF], failed to show an improvement in overall survival, despite a significant reduction in local and distant failures.

In a report of pooled data of two prospective randomized trials [START_REF] Chua | Long-term survival after cisplatin-based induction chemotherapy and radiotherapy for nasopharyngeal carcinoma : a pooled data analysis of two phase III trials[END_REF], the use of induction CHT resulted in a 5.4% improvement in disease specific survival rate (p= 0.029) at 5 years, while no significant difference was found in the overall survival rates.

Two randomized studies on the use of NACT showed positive results. The VUMCA I study [START_REF]VUMCA I trial: preliminary results of a randomized trial comparing neoadjuvant chemotherapy (cisplatin, epirubicin, belomycin) plus radiotherapy vs. radiotherapy alone in stage IV (N2, M0) undifferentiated nasopharyngeal carcinoma: a positive effect on progression-free survival[END_REF] showed significant reduction of both local and distant failures after chemotherapy. Ma et al. [4] also demonstrated significant improvement in local control after NACT.

Hareyama noted that the use of NACT did not result in a significant improvement in disease-free or overall survival, but there was a positive tendency in favour of the NACT for distant metastasis free survival [START_REF] Haeryama | A prospective randomized trial comparing neoadjuvant chemotherapy with radiotherapy alone in advanced nasopharyngeal carcinoma[END_REF]. The negative study by Chan et al. [START_REF] Chan | A prospective randomized study of chemotherapy adjunctive to definitive radiotherapy in advanced nasopharyngeal carcinoma[END_REF] is limited by its low power, since it included only 77 patients. Although the overall results of the Asian-Oceanian Clinical Oncology Association (AOCOA) study is negative [START_REF] Chua | Preliminary report of the Asian-Oceanian Clinical Oncology Association randomized trial comparing cisplatin and epirubicin followed by radiotherapy versus radiotherapy alone in the treatment of patients with locoregionally advanced nasopharyngeal arcinoma[END_REF], an unplanned subgroup analysis showed significant improvement in local control among 53 patients with very large nodes.

Five recently published studies [START_REF] Oh | Induction chemotherapy followed by concomitant chemoradiotherapy in the treatment of locoregionally advanced nasopharyngeal cancer[END_REF][START_REF] Rischin | Excellent disease control and survival in patients with advanced nasopharyngeal cancer treated with chemoradiation[END_REF][START_REF] Al-Amro | Neoadiuvant chemotherapy followed by concurrent chemoradiation therapy in locally advanced nasopharyngeal carcinoma[END_REF][START_REF] Airoldi | Induction Chemotherapy with Cisplatin and Epirubicin followed by radiotherapy and concurrent Cisplatin in locally advanced nasopharyngeal carcinoma observed in a non-endemic population[END_REF] have shown that induction CHT followed by CRT obtained high responses in patients coming from both endemic and non endemic areas: in two trials the authors proposed a three-drugs induction scheme, while in the others cisplatin + epirubicin or paclitaxel and carboplatin were respectively administered.

In four out of five trials the drug administered during radiotherapy was cisplatin alone; only in Oh paper [START_REF] Oh | Induction chemotherapy followed by concomitant chemoradiotherapy in the treatment of locoregionally advanced nasopharyngeal cancer[END_REF] a two drug combination of 5-fluorouracil and hydroxyurea is administered with once-daily radiotherapy on a week-on week-off schedule.

Our clinical post-CRT CR rate (87%) is good even if it is probably overestimated unless the radiologists examined serial CT images or the ENT performed multiple biopsies from the tumour area. Our PFS and OS results are superimpose able to the most favourable of these four papers [START_REF] Rischin | Excellent disease control and survival in patients with advanced nasopharyngeal cancer treated with chemoradiation[END_REF][START_REF] Al-Amro | Neoadiuvant chemotherapy followed by concurrent chemoradiation therapy in locally advanced nasopharyngeal carcinoma[END_REF][START_REF] Chan | Phase II study of neoadjuvant carboplatin and paclitaxel followed by radiotherapy and concurrent cisplatin in patients with locoregionally advanced nasopharyngeal carcinoma: therapeutic monitoring with plasma Epstein-Barr Virus DNA[END_REF] conforming the high activity of NACT + CRT even in a non-endemic population.

Our neoadjuvant scheme has achieved a percentage of CR (33%) similar to the one reported by our group with a cisplatin+epirubicin combination [START_REF] Airoldi | Induction Chemotherapy with Cisplatin and Epirubicin followed by radiotherapy and concurrent Cisplatin in locally advanced nasopharyngeal carcinoma observed in a non-endemic population[END_REF] in a superimpose able group of patients.

Probably the two drug administration during RT has given more myelotoxicity and mucositis even if a direct comparison cannot be done. We chose carboplatin instead of cisplatin with the aim of reducing acute toxicity. In a recent paper weekly carboplatin gave less anaemia, renal toxicity, mucositis, weight loss in comparison to 3-weekly cisplatin administration. [START_REF] Chitapanarux | Chemoradiation comparing cisplatin versus carboplatin in locally advanced nasopharyngeal cancer: randomised, non inferiority, open trial[END_REF] Severe nausea/vomiting was less frequent in carboplatin+taxol group (0% vs 12.5%). Late toxicity was superimpose able in our two series.

Our percentage of distant metastases is quite low (10%) even if it is possible to suppose that an induction CHT with three-drugs scheme or a molecular targeted therapy integration may improve these results, as reported in other head and neck tumours, different from nasopharyngeal region; however such observation needs to be evidenced by mean of future randomized trials.

According to our previously reported experience in a very similar series [START_REF] Airoldi | Induction Chemotherapy with Cisplatin and Epirubicin followed by radiotherapy and concurrent Cisplatin in locally advanced nasopharyngeal carcinoma observed in a non-endemic population[END_REF] we can draw the following conclusions: 1) NACT with carboplatin and taxol is very well tolerated with a clinical impact superimpose able to cisplatin + epirubicin;

2) A two drug combination during conventional radiotherapy is a feasible treatment with a good compliance, an high relative dose intensity and no significant life-threatening toxicity;

3) A two drug combination during radiotherapy has an higher acute severe toxicity (neutropenia, anemia, oropharyngeal mucositis, weight loss) compared to cisplatin every three weeks ; 4) Tumour recurrence can be effectively treated with re-irradiation + CHT, obtaining long lasting complete remissions: this is in accordance with recent reports [START_REF] Kramer | Toxicity and outcome analysis of patients with recurrent head and neck cancer treated with hyperfractionated split-course reirradiation and concurrent cisplatin and paclitaxel chemotherapy from two prospective phase I and II studies[END_REF][START_REF] Spencer | Final report of RTOG 9610, a multi-institutional trial of reirradiation and chemotherapy for unresectable recurrent squamous cell carcinoma of the head and neck[END_REF][START_REF] Langer | Phase II study of low dose paclitaxel and cisplatin in combination with split course concomitant twice-daily reirradiation in recurrent squamous cell carcinoma of the head and neck: results of Radiation Therapy Oncology Group Protocol 9911[END_REF].

Although the small series hereby presented, NACT with carboplatin + taxol followed by concomitant weely carboplatin + taxol + conventional radiotherapy seems to be a safe and effective treatment for a non endemic population affected by locoregional advanced NPC; however this approach needs further phase 3 trials.

Fig. 1

 1 Fig.1Overall Survival (OS) and Disease Free Survival (PFS) -30 patients with a median follow-

Fig. 2

 2 Fig. 2 Local Recurrence Disease Free Survival (LRDFS) and Distant Metastases Disease Free

Table 1 -

 1 Tumour stage versus Node stage (AJCC Staging 2010)

		T1	T2	T3	T4	Total
	N0	--	--	1	8	9
	N1	--	--	0	0	0
	N2	3	1	9	5	18
	N3 *	1	--	--	2	3
	Total	3	2	10	15	30

* All patients were N3a

Table 2 -

 2 Acute toxicity (WHO criteria), maximum toxicity per patient during induction CHT

	WHO Grade	0	1-2	3	4
	Neutropenia	0 (0%)	5 (16.7 %) 10 (33.3%)	15 (50%)
	Thrombocytopenia	2 (6.6%)	24 (80.0%) 4 (13.3%)	0 (0%)
	Anemia	2 (6.6%).	15 (50.0%) 3 (10.0%)	0 (0%)
	Nausea/Vomiting	15 (50.0%) 15 (50.0%) 0 (0%)	0 (0%)
	Anorexia	23 (76.7%) 7 (23.3%)	0 (0%)	0 (0%)
	Oropharyngeal Mucositis	21 (70.0%) 7 (23.3%)	2 (6.6 %)	0 (0%)
	Alopecia	6 (20%)	24 (80.0%)
	Skin	0 (0%)	--	--	--
	Cardiotoxicity	0 (0%)	--	--	--
	Renal toxicity	0 (0%)	--	--	--
	Neurotoxicity	0 (0%)	--	--	--
	Weight loss	36 (87.7%) 4 (13.3%)	--	--

*Febrile neutropenia in no patient .

Table 3 -

 3 Acute toxicity (WHO criteria), maximum toxicity per patient, during CRT

	WHO Grade	0	1-2	3	4
	Neutropenia	0 (.0%)	11 (36.6%)	16 (53.3%)	3 (10%)
	Thrombocytopenia	10 (33.3%) 17 (56.6%)	3 (10%)	0 (0%)
	Anaemia	8 (26.6%) 17 (56.6%)	5 (16.6%)	0 (0%)
	Nausea/Vomiting	18 (60.0%) 12 (40.0%)	0 (0%)	0 (0%)
	Oropharyngeal Mucositis	0 (0 %)	9 (30%)	16 (53.3%)	5 (16.6%)
	Alopecia	6 (20%)	24 (80%)
	Skin	2 (6.6%)	21 (70.0%)	7 (23.3%)	0 (0%)
	Cardiotoxicity	30 (100%)	--	--
	Renal toxicity	28 (93.3%) 2 (6.6%)	--
	Neurotoxicity	12 (40%)	15 (50%)	3 (10%)	0 (0%)
	Weight loss	32 (80.0%) 8 (20.0%)	5 (16.6%)	0 (0%)
	NGT	yes 5 pts (16.6%)

Conflict of Interest Statement

All Authors disclose any financial and personal relationships with people or organisations that could influence their work.