Marie Radepont
email: marie.radepont@mnhn.fr

Laurianne Robinet

Céline Bonnot-Diconne

Claire Pacheco

Laurent Pichon

Quentin Lemasson

Brice Moignard

Ion beam analysis of silver leaves in gilt leather wall coverings

An analytical methodology involving Particle Induced X-ray Emission (PIXE) and Rutherford Backscattering Spectroscopy (RBS) was implemented to respectively characterize the composition and the thickness of silver leaves on gilt leather decors. These objects, ancestors of our wallpapers, are nowadays still difficult to date and their provenance is generally determined from stylistic studies. The initial aim of this study was to identify markers that could be correlated with the object provenance to help distinguishing the different gilt leathers workshops in Europe. The analytical methodology was validated on modern samples and applied to a corpus of 58 ancient gilt leathers from four countries. This study provided an assessment of the sensitivity of the ion beam techniques used, and highlighted the complexity of such analyses on thin silver leaves due to the different factors affecting them, and the composite nature of the object. Thus, the thicknesses calculated from the RBS analyses presented a great variability that seems to be related to the leaf characteristics, the manufacturing process and/or the life of the decor. Nevertheless, observations suggest that silver leaves coming from the Netherlands are thicker than the ones from Spain, Italy or France. Concerning the elemental composition, the results discarded previous hypotheses and the focus was made on gold and mercury trace elements, thus it was shown that leaves in Italian decors seem to have generally a low content of these two elements. Despite the large number of decor analyzed, the corpus should be expanded over to confirm the hypotheses raised by this research. Nevertheless the results gained from this work bring new light on the factors affecting thin metal leaves in general, which will be beneficial to all fields dealing with their analysis.

Introduction

Gilt leathers, ancestors of our wallpapers, are luxurious decorations used all over Europe between the 16 th and the 18 th century [START_REF] Koldeweij | Gilt Leather, An Important Craft and a Little Known Luxury[END_REF][START_REF] Bonnot-Diconne | Method of Use and Importance of Gilt Leather in Three Italian Palaces during the 16th and the 17th Century through the Study of Inventories, ICOM-CC Interim Meeting of the Leather Working Group[END_REF]. The manufacturing of these objects started in Spain and spread in Europe progressively, mainly in Italy, France and the Netherlands. Despite their name, gold is not present in the composition of these artifacts. Indeed, these decors were made by applying to the leather a silver leaf that is burnished with a stone, and then covered with a yellow varnish made essentially of linseed oil and pine resin to obtain a gold-like appearance [START_REF] Schulze | Goldleder zwischen 1500 und 1800 -Herstellung und Erhaltung, Arbeitsheft 17 des Landesamtes für Denkmalpflege Sachsen[END_REF]. For aesthetic effects, "gilt" and "silvered" areas can be combined (Figure 1), the latter being prepared by applying a very thin varnish or protein-based layer over the silver [START_REF] Schulze | Goldleder zwischen 1500 und 1800 -Herstellung und Erhaltung, Arbeitsheft 17 des Landesamtes für Denkmalpflege Sachsen[END_REF]. On most decors, polychromy was also added on top. Nowadays it is difficult to date and attribute gilt leathers as they are not signed, and assumptions concerning their provenance are only based on stylistic studies [START_REF] Fournet | Les cuirs dorés anciens en France[END_REF]. Identification of the constitutive materials could provide some clues regarding the decor manufacturing process or reveal physical or chemical markers specific of a period, a country or a workshop. Historical texts on the manufacture of these decors mention variation in the practices, recipes or quality of the materials depending on the countries. For example, it was reported that French workshops, to reduce the cost of the decor manufacturing, sometimes employed thinner leaves or used other metal leaves [START_REF] Fougeroux De Bondaroy | L'art de travailler les cuirs dorés et argentés, Description des arts et métiers[END_REF]. The identification and comparison of chemical elements composing a metal object is a common approach to determine its provenance [START_REF] Blet-Lemarquand | L'or et l'argent monnayés[END_REF][START_REF] Guerra | La circulation des objets en or[END_REF]. In a former study, Talland compared the silver leaf composition obtained by PIXE (Particle Induced X-ray Emission), in five gilt leather samples, two attributed to the Netherlands and three to Spain/Italy, and made the hypothesis that the differences in the chemical composition could be related to the country of origin [START_REF] Talland | The Conservation of Leather Wallhangings at the Isabella Stewart Gardner Museum, ICOM-CC Interim Meeting on the Conservation of Gilt Leather[END_REF]. These analyses showed a lower percentage of silver (close to 90 %) and a higher percentage of lead (4 to 7 %) in the Netherlandish samples compared to the Spanish/Italian ones which contained more than 95 % of silver and less than 1 % of lead. This hypothesis is based on a limited number of samples and needs to be verified over a larger corpus. Recently, an analytical methodology was developed to characterize the main components within gilt leather decors that is the leather, the silver leaf and the varnish, to increase knowledge on the manufacturing technique and degradation processes [START_REF] Bonnot-Diconne | Multi-technique analysis of giltleather wall coverings (16 th -18 th centuries)[END_REF]. For the analysis of the silver leaf, this methodology combined two ion beam analyses (IBA), first the non-invasive PIXE analyses, to characterize the elemental composition, and secondly, the RBS (Rutherford Backscattering Spectrometry) to localize the different chemical elements in the depth of the considered layer which, by assuming its density, gives access to the thickness of the metallic leaves [START_REF] Feldman | Fundamentals of surface and thin film analysis[END_REF]. Based on these measurements, it was hoped to find chemical and/or physical markers in these silver leaves that could lead historians towards the provenance of the objects. The present paper comes in the continuity of this research by applying this analytical methodology to analyze the silver leaves in a corpus of 58 historical samples originating from different geographic areas in Europe, as well as on mockup samples made according to a 18 th c. recipe [START_REF] Fougeroux De Bondaroy | L'art de travailler les cuirs dorés et argentés, Description des arts et métiers[END_REF]. The specific issues encountered during these analyses and the processes implemented in response are discussed. IBA analyses of ancient artifacts always present complicated data processing and interpretations, mainly due to the representativeness of the area analyzed, and the heterogeneity, roughness or porosity inherent to the material. Ager study recently examined the accuracy of this non-invasive technique to measure thicknesses of thin metallic layers, and concluded that the results gained from this technique on gold leaves were close to expectations [START_REF] Ager | Reconsidering the accuracy of X-ray fluorescence and ion beam based methods when used to measure the thickness of ancient gildings[END_REF]. In the case of silver leaves, we will see that several factors could play a role in the measurement of their thickness and disturb the reproducibility of the data obtained.

Materials and methods

Studied materials

The methodology was assessed with modern silver leaves bought from three suppliers: Dauvet (France) dimension 80 × 80 mm, Manetti (Italy) dimension 95 × 95 mm and Berta (Italy) "thickness 30" reference dimension 85 × 85 mm. Whilst Dauvet and Manetti leaves are manufactured by pneumatic beating, Berta leaves are made by hand beating. The leaves from the three suppliers were prepared in different ways: 1) fixed with water on a glass slide, 2) glued using rabbit skin glue on a modern vegetable leather (Azais, France) and partly burnished with an agate stone, 3) used in a gilt leather mockup prepared according to a 18 th century recipe [START_REF] Fougeroux De Bondaroy | L'art de travailler les cuirs dorés et argentés, Description des arts et métiers[END_REF]. The 58 ancient gilt leathers analyzed belong to public or private collections in France and Italy. Based on their stylistic characteristic, these were dated from the 16 th to the 18 th century and attributed to four countries: Spain, Italy, France and the Netherlands. All the decors selected had experienced no or minimal restoration. Some of these objects were assigned to two known manufacturers, André Reynier (France) and Agostino Nespola (Italy), thanks to documents present in local archives. Analyses were performed either directly on the decor or fragments of the decor, as well as on samples taken during conservation treatment.

Methodology

Ion beam analyses were directly carried out on objects without any preparation, simultaneously measuring PIXE (Particle Induced X-ray Emission) and RBS (Rutherford Backscattering Spectroscopy) at atmospheric pressure at the AGLAE facility (C2RMF). The beam was 30 µm in size and composed by protons for PIXE or alpha particles for RBS at 3 MeV for each beam [START_REF] Pichon | Development of a multidetector and a systematic imaging system on the AGLAE external beam[END_REF]. For each technique, three to six measurements per sample were performed. In few occasions, samples showed a high sensitivity to the ion beam analyses, therefore in these cases the number of measurements was reduced. PIXE spectra were collected with one SDD Low Energy X-ray (LE) detector and two SDD High Energy X-ray (HE) detectors, positioned respectively at 45° and 50° relative to the beam axis [START_REF] Pichon | Development of a multidetector and a systematic imaging system on the AGLAE external beam[END_REF]. The LE detector, that had no filter, enabled the detection of light elements thanks to a helium flow, whereas each one of the HE detectors was covered by a 50 µm thick aluminum filter. PIXE data were processed using the GUPIXWIN calculation engine [START_REF] Campbell | The Guelph PIXE software package IV[END_REF] coupled to the homemade TRAUPIXE software [START_REF] Pichon | Programs for visualization, handling and quantification of PIXE maps at the AGLAE facility[END_REF]. The composition of the target obtained from the LE and the HE detectors can be combined by using iron as the pivot element, which means that it contributes to both types of X-rays. The quantitative composition of the analyzed materials is obtained for matrix and trace elements. For the results presented here, only elements quantified as being above the detection limit were considered. RBS measurements were performed with a particle detector collecting backscattered protons set at 130° with respect to the incident beam. RBS data were processed using the SIMNRA software V6.05 [START_REF] Radepont | Towards a sharpest interpretation of analytical results assessing the uncertainty of PIXE/RBS data at the AGLAE facility[END_REF]. Imaging of the silver leaves surfaces was performed using a scanning electron microscope with a field emission gun (SEM-FEG) at 4 kV and a working distance of 6 mm. A small silver leaf was attached on carbon tape and a bridge between the leaf and the tape was made with carbon lake. Estimation of the surface occupied by microporosities was made by a pixel calculation after an image treatment using the GIMP software.

Results and discussion

Analysis of the composition of silver leaves

IBA measurements in ancient gilt leathers were carried out as much as possible away from pictorial layers, therefore the chemical elements detected are expected to come from the combination of the varnish, the silver, the glue and the leather. For each object, an analysis was also performed on the leather on its own, either from the front, if accessible, or from the back of the object, and the data were compared (Table 1). In all the decors analyzed, only silver leaves are encountered and their composition is relatively pure in silver (with over 98 at%). Variability in the quantification of chemical elements is sometimes observed in a single sample and this may be associated with the surrounding components of the decor. Many elements were detected in the leaves in different proportions but none of these could be directly correlated with the object provenance. In particular the Table 1. Chemical elements identified by PIXE in the leather alone and in the silver attached to the leather for the gilt leather wall hanging of L'Isle (Switzerland), made in The Netherlands in the 17 th c. Element quantification is provided by "X" and "x" corresponding respectively to more and less than 3 times the limit of detection and "-" indicates the absence of the element considered. 1), in varnish or in polychromy, and is related to the use of lead based products, either as dryers for the "gold varnish" [START_REF] Schulze | Goldleder zwischen 1500 und 1800 -Herstellung und Erhaltung, Arbeitsheft 17 des Landesamtes für Denkmalpflege Sachsen[END_REF][START_REF] Fougeroux De Bondaroy | L'art de travailler les cuirs dorés et argentés, Description des arts et métiers[END_REF] or pigments in the polychromy.

Element Leather Silver Na x - Mg X - Al X x Si X X P X X S X X Cl X X K X X Ca X X Ti X x Cr x - Mn X X Fe X X Ni x x Cu X X Zn X X Ga x x Ge x - As x x Se x x Br X - Rb x x Sr X X Ag - X Au - X Hg - X Pb X X
For that reason, it is not possible to rely on the lead content to make attribution.

Although copper was also suggested in previous study as a possible marker of provenance [START_REF] Talland | The Conservation of Leather Wallhangings at the Isabella Stewart Gardner Museum, ICOM-CC Interim Meeting on the Conservation of Gilt Leather[END_REF][START_REF] Bonnot-Diconne | Multi-technique analysis of giltleather wall coverings (16 th -18 th centuries)[END_REF], it was often detected by PIXE in areas where silver was not present (eg. leather only areas), therefore this element was also discarded. Thus, among all the chemical elements identified in the decors (see an example in Table 1), we focused on the two elements that seemed to be present concurrently with silver in most of the samples: gold and mercury.

Several hypotheses can explain the presence of gold and/or mercury in the silver leaves. One concerns the silver extraction process, as mercury amalgam was used to recover the gold usually present in the same ores. Another explanation can be found in ancient French regulation texts stating that the use of "sublimé corrosif", corresponding to mercury (II) chloride (HgCl 2 ), was authorized in beating workshops to pack silver bar [START_REF]Arrêté de jurisprudence sur la réglementation des batteurs français[END_REF]. Following the decor manufacture, other sources can possibly explain the presence of mercury in the silver leaves. In decors containing vermilion pigment (HgS), traces of pigment particle may have detached, contaminating the surface in other areas. In this case, it would be detected on the beam path, but would not be incorporated to the silver leaf. Also, mercury vapor either emitted from the vermilion [START_REF] Strahan | Measuring mercury emissions from cinnabar lacquer objects[END_REF] or present in the atmosphere can form amalgam with the silver [START_REF] Nowakowski | Surface mediated Ag-Hg alloy formation under ambient and vacuum conditions -AFM and XRD investigations[END_REF]. In our case, this hypothesis appears less likely since the varnish at the surface of the silver was shown to acts as a protective barrier towards the pollutants in the atmosphere [START_REF] Radepont | The impact of manufacturing and conservation on silver leaf tarnishing in gilt leather[END_REF]. Finally, adding some gold during the preparation of silver leaves was known to improve their mechanical qualities. Indeed, the formation of an Ag-Au alloy is more favorable because of the partial charge transfer from Au atoms to Ag atoms, providing significant electrostatic stabilization.

The consequence is that adding gold in silver increase the ductility of the material, making the silver leaf manufacturing process easier than with pure silver [START_REF] Lee | Geometrical and electronic structures of gold, silver, and gold-silver binary clusters: origins of ductility of gold and gold-silver alloy formation[END_REF]. Silver, gold and mercury were quantified in the silver leaves for all the objects. In the ancient gilt leather corpus, 84 % contained gold, 62 % contained both gold and mercury and only 6 % contained neither of these two elements. As a comparison, all three modern silver leaves contained gold but no mercury. The quantification of the three elements in gilt leathers attributed to two well-known manufacturers, André Reynier and Agostino Nespola are presented in Table 3, but it showed no clear correlation. Very high mercury contents were found in some decors containing mercury based pigments (from few at% up to 15 at% in one decor) although the analysis was performed far from the polychromy. The gold and mercury contents were plotted for all the gilt leather samples according to the decor firm or hypothetic provenance, with the exception of the 4 decors containing more than 0.2 at% Hg or Au (Figure 2). Although no direct correlation could be established, it seems that decors coming from Italy present in average very low gold and mercury contents compared to the other decors (Figure 2). This hypothesis would need to be confirmed by increasing the number of decor analyzed and by increasing the Spanish decor representation. If the hypothesis was verified, it could lift the uncertainty for the decors where Italy provenance was suggested.

Analysis of the thickness of silver leaves 3.2.1. Model samples

The methodology of RBS analyses was implemented first using the mockups in order to assess the impact of the leaf characteristics and preparation. For the modern leaves, the thickness of the leaf could be estimated thanks to the information given by the supplier or determined from its weight. In the case of Dauvet, one leaf composed mostly of silver weighted 0.015 g and measured 80 × 80 mm, therefore the calculated thickness was 223 nm. These values were then compared with the thickness estimated from RBS measurements of the silver leaf deposited on a glass slide. In the case of Dauvet leaves, the thickness estimated over five measurements ranged from 193 nm to 219 nm and the average value was 207 ± 26 nm. The uncertainty of 0.6 % due to the instrument and the data process of RBS analyses at the AGLAE facility is negligible next to the one due to the roughness of the sample surface, therefore only the later one will be expressed next to the thickness values in this paper. Indeed, the roughness of the surface induces a change in the profile of the RBS spectrum, increasing as a result the uncertainty of the analysis (the details about this question can be found elsewhere [START_REF] Radepont | Towards a sharpest interpretation of analytical results assessing the uncertainty of PIXE/RBS data at the AGLAE facility[END_REF]). This parameter might then be a factor playing a role in the variability of the thickness measurements. All the silver leaves analyzed by RBS displayed a variability in thicknesses more or less important over a single leaf or area. Surface roughness and silver corrosion might have a role to play in this variability [START_REF] Wu | Investigation of the foil structure and corrosion mechanisms of modern Zwischgold using advanced analysis techniques[END_REF], however another factor should be considered, the presence of microporosities greatly smaller than the beam size in the metallic layer. Considering that the RBS spectrum obtained is an average of the materials encountered by the beam in each layer, if microporosities are present, the intensity of the peaks coming from the elements present in this layer will decrease, therefore reducing the calculated thickness. In an attempt to examine this question, the silver leave surfaces were analysed by SEM-FEG, and the technique revealed the presence of microporosities in different proportions: 10.2 % of the surface occupied by microporosities for Dauvet leaves (probably combined to a significant roughness according to the SEM-FEG image), 2.0 % for Manetti leaves and 0.4 % for Berta leaves (Figure 3). The manufacturing process of the leaves (pneumatic beating for Dauvet and Manetti, hand beating for Berta) and the corrosion might affect the leaf properties and its surface appearance. At this stage, it is difficult to establish a direct correlation between the presence of these microporosities and the variability in the thickness measurements, as suggested by Ager [START_REF] Ager | Reconsidering the accuracy of X-ray fluorescence and ion beam based methods when used to measure the thickness of ancient gildings[END_REF], however more samples should be systematically observed by SEM and analyzed by RBS to examine this hypothesis. In relation to the manufacturing of gilt leather, the influence of different factors on the measurements was assessed: the nature of the supporting material, the leaf overlap that can occur when the leaf is deposited on the leather, and the burnishing step. A leaf from Dauvet was folded in two on a glass slide and its thickness was estimated by RBS measurement to be 220 ± 50 nm in the single leaf area and 415 ± 50 nm in the folded area. When the same leaf was glued on leather and analyzed by RBS, its thickness was estimated at 222 ± 44 nm in an unburnished area, but decreased to 170 nm ± 45 nm in the burnished area. From these results, it can be concluded that the supporting material has no influence on the thickness measurement. The mechanical action from the burnishing, however, seems to modify the thickness value calculated by the software, and this is an important parameter to bear in mind for the data interpretation. This effect might be explained by the slimming of the leaf or the increase of the size or the number of microporosities. Although this has not been assessed here, we may expect the thickness value calculated to vary depending on the burnishing method used or the person doing the burnishing. The influence of this factor will be investigated in the future as part of another project.

Ancient gilt leather

The thicknesses values calculated over all the ancient samples analyzed spread from 19 to 1574 nm, but most of the values are centered around 100 nm (Figure 4). For several decors, the variability in thickness values within a single sample was very high, even in a same area (Figure 5). Different factors could explain this scattering in the thickness values. As mentioned earlier, intrinsic factors linked to the silver leaf properties, such as the leaf roughness, porosity or homogeneity in the beating can induce variable values, as well as the manufacturing of the decor, and in particular the silver leaf laying and the burnishing. The high thickness values might represent a superposition or folding of silver leaves in the object, nevertheless this would appear as isolated values when several measurements are carried out. The low thickness values might result from the effect of different factors or combination of these factors that are the presence and the ratio of microporosities and the burnishing step, but also factors associated with the life of the object. In ancient gilt leather decors, the corrosion of the silver by endogenous or exogenous compounds could reduce the thickness and increase the surface roughness, and mechanical action like cleaning may cause abrasion of the leaves resulting in the removal of materials from the object surface [START_REF] Radepont | The impact of manufacturing and conservation on silver leaf tarnishing in gilt leather[END_REF]. A good illustration is provided by the measurements made on samples of the "gilt" and "silvered" areas from the "Battle of Gelboé" gilt leather wall hanging belonging to the musée national de la Renaissance in Thicknesses of silver leaves calculated using the simulation of a silver leaf glued on leather on the RBS spectra collected on two different samples of the "Battle of Gelboé" gilt leather wall hanging (made in The Netherlands, 17 th c., exhibited in the musée national de la Renaissance, Ecouen, France), one from a "gilt area" (GEL9-top) and the other from a "silvered area" (GEL10-bottom).

France (Figure 6). Silvered areas are often most exposed to both factors as they were only covered with a thin organic protective layer. Thus, the reduced thickness observed in the silvered area might originate from the silver corrosion and/or abrasion from repetitive cleaning. The manufacturing and conservation treatment can have an impact on the corrosion of silver in gilt leathers [START_REF] Radepont | The impact of manufacturing and conservation on silver leaf tarnishing in gilt leather[END_REF] therefore this evolution should also be considered when assessing the thickness of silver Despite all the variation observed in the values, the average thicknesses of silver leaves in ancient gilt leathers coming from The Netherlands are greater than the ones from the other countries (Figure 4). No tendency was observed between the thicknesses and the date of manufacture of the objects. All these results stress the necessity to expand the corpus by increasing the number of gilt leather decors analyzed, so that firmer conclusions could be drawn.

Conclusion

This project is the first study aiming at obtaining a fine measurement of the composition and the thickness of silver leaves on a large corpus of gilt leather decorations. The methodology implemented, with PIXE and RBS analyses, is adapted to the specificity of silver leaves and of these objects, as sampling, when permitted, might locally change the thickness of these thin metal leaves. However, the composite nature of this kind of artefact with the presence of different organic materials makes the analysis of the silver leaves and the interpretation of the data complex. For that reason, only chemical elements solely associated with the metallic leaf, that is silver, gold and mercury were considered although multiple elements were identified by PIXE analyses. The observation seems to indicate that the silver leaf in the from Italy are generally characterized by a low gold and mercury content, but this has to be confirmed by expanding the corpus. Regarding the thicknesses calculated from RBS analyses, the data seemed to show that silver leaves from the Netherlands are thicker than the ones from Spain, Italy or France. An important variability was seen in the thickness values, even for modern leaves coming from the same book, which can be due to different factors: surface roughness, microporosities, mechanical actions during manufacturing or cleaning, and silver corrosion. For the two former ones, an instrumental development implemented during this project, RBS cartography, will help calculating an average of the thickness of the leaf on a large surface, reducing the impact of these factors or visualizing leaf heterogeneity or overlap. This study allowed an assessment of the sensitivity of the analytical techniques used and showed the variability of the chemical composition and the thickness of the silver leaves in gilt leather decors, which are related to several factors linked to the materials, the manufacturing or the life of the objects. In the future, the corpus analyzed will be expanded to confirm the different hypotheses raised by the composition and the thickness measurements. In parallel, archives should also be studied to know if workshops always used local silver leaves or if they might have come from a different geographical area. Beyond the silver leaf, we should continue to increase our knowledge on the other materials present in gilt leathers so that all the information could be combined and may reveal clearer trend in the provenance attribution.

Figure 1 .

 1 Figure 1. Photograph of the wall hanging the "Battle of Gelboé" (made in The Netherlands, 17 th c., exhibited in the musée national de la Renaissance, Ecouen, France) and a detail of this decor showing "gilt" and "silvered" areas (© C. Bonnot-Diconne 2017).

Figure 2 .

 2 Figure 2. Elemental composition calculated from PIXE analyses for ancient gilt leathers from the corpus presented according to their country of attribution. The percentages are calculated considering only the presence of mercury and gold together with silver. The values indicated with "?" or "/" correspond to undefined provenance respectively attributed with no certainty to one country or with hesitation between two countries (Sp = Spain; It = Italy; Fr = France; Ne = The Netherlands).

Figure 3 .

 3 Figure 3. Photographs of modern silver leaves (top) from a) Dauvet, b) Manetti and c) Berta, and corresponding SEM-FEG images (bottom).

Figure 4 .

 4 Figure 4. Thicknesses calculated from RBS analyses for the corpus of ancient gilt leathers presented chronologically and according to their country of provenance. The values indicated with "?" or "/" correspond to undefined provenance (Sp = Spain; It = Italy; Fr = France; Ne = The Netherlands). In each column the black dots represent the different measurements for one single sample, and the red dash shows the average of thickness for each sample. The thin red line links all the average values.

Figure 5 .

 5 Figure 5. Simulation (blue curve) on two RBS spectra (red curve) collected from the same area in the sample (REG1.3, made in Italy, 16 th c.), giving leaf thicknesses (peak on the right of each spectrum) at 197 ± 25 nm and 159 ± 58 nm.

Figure 6 .

 6 Figure 6. Thicknesses of silver leaves calculated using the simulation of a silver leaf glued on leather on the RBS spectra collected on two different samples of the "Battle of Gelboé" gilt leather wall hanging (made in The Netherlands, 17 th c., exhibited in the musée national de la Renaissance, Ecouen, France), one from a "gilt area" (GEL9-top) and the other from a "silvered area" (GEL10-bottom).

Table 2 .

 2 Percentages of lead calculated from PIXE analyses of silver leaves in 11 gilt leathers (only considering the presence of silver, gold and mercury next to lead).

	Provenance	Date	Reference	Pb (at%)
	Spain	16 th 17 th	DEC9 CBD6	0.07 0.54
		17 th	Montalzat	0.11
	Italy	17 th	AR4.1	2.33
		18 th	Béceleuf	0.41
		17 th	Golleville	0.30
	France	17 th	DEC4.1	0.12
		17 th	EC3	0.97
		17 th	Scipion	0.28
	The Netherlands	17 th	L'Isle	0.07
		18 th	Maintenon	1.85

Table 3 .

 3 Quantification of silver, gold and mercury in gilt leathers attributed to two well-known manufacturers. Talland as a provenance marker, was compared in the 58 decors, but our results did not display a higher lead content in gilt leathers from the Netherlands, nor a correlation between the lead content and the geographical origin of the decors (Table2). In gilt leather decors, lead can be encountered in leather (Table

	Manufacturer	Provenance	Date	Reference Ag (%) Au (%) Hg (%)
			1670	AIX2	99.874	0.119	0.007
	André Reynier	France	17 th	CBD7	99.938	0.053	0.009
			17 th	AIX1	99.921	0.052	0.027
			ca. 1670	AR5	99.963	0.037	0
	Agostino Nespola	Italy	ca. 1670 ca. 1670	AR6 AR11	99.977 99.943	0.009 0.057	0.014 0
			17 th	AR3	99.912	0.073	0.015
	lead content, raised by

Acknowledgements

This study was performed under the scope of the CORDOBA project, funded by the Fondation des Sciences du Patrimoine (LabEx PATRIMA), and as part of the New AGLAE project (grant ANR-10-EQPX-22). The authors are grateful to Eric Laval for the SEM-FEG analyses at C2RMF, to Mariabianca Paris and Marcella Ioele (ISCR Roma) and to all the owners of the collections for providing the samples of gilt leathers.


MD Lorenzo Fornaro
email: lorenzo.fornaro@gmail.com

Francesco Leone

Angélique Vienot

Andrea Casadei Gardini

Caterina Vivaldi

Astrid Lièvre

Pasquale Lombardi

Emmanuele De Luca

Dewi Vernerey

Elisa Sperti

Gianna Musettini

Maria Antonietta Satolli

Julien Edeline 10

Rosella Spadi

Cindy Neuzillet

Alfredo Falcone

Giulia Pasquini

Mario Clerico

Alessandro Passardi

Paola Buscaglia

Aurélia Meurisse

Massimo Aglietta

Clémence Brac

Enrico Vasile

Francesco Montagnani

Validated nomogram predicting 6-month survival in pancreatic cancer patients receiving first-line 5-fluorouracil, oxaliplatin and irinotecan

Keywords: clinical parameters, FOLFIRINOX, laboratory parameters, prognosis, risk categories

Background: FOLFIRINOX is an option for fit patients with metastatic (MPC) and locally advanced unresectable (LAPC) pancreatic cancer. However, no criteria reliably identify patients with better outcome.

Patients and Methods:

We investigated putative prognostic factors among 137 MPC/LAPC patients treated with triplet chemotherapy. Association with 6-month survival status (primary endpoint) was assessed by multivariate logistic regression models. A nomogram predicting the risk of death at 6 months was built by assigning a numeric score to each identified variable, weighted on its level of association with survival. External validation was performed in an independent dataset of 206 patients.

Results: Four variables (performance status, liver metastases, baseline CA19.9 and neutrophil-to-lymphocyte ratio) were found associated with 6-month survival by multivariate analysis or had sufficient clinical plausibility to be included in the nomogram. Accuracy was confirmed in the validation cohort (C-index 0.762; 95%CI 0.713-0.825). After grouping all cases, four subsets with different outcomes were identified by none, 1, 2 or >2 poor prognostic features (P<0.0001).

Conclusion:

Our nomogram accurately predicts the risk of death in the first 6 months after initiation of FOLFIRINOX in MPC/LAPC patients. This tool could be useful to guide communication about prognosis and inform the design and interpretation of clinical trials.

Clinical Trial Registration:

The study is registered on ClinicalTrials.gov (NCT03590275).

MICROABSTRACT

FOLFIRINOX is an accepted standard in metastatic and locally advanced pancreatic cancer but long term prognosis is still poor. Indeed, no criteria reliably identify patients with limited, if any, chances of long-term benefit. We therefore developed and externally validated a prognostic nomogram predicting the risk of early death in pancreatic cancer patients treated with first-line triplet chemotherapy.

INTRODUCTION

Pancreatic cancer (PC) represents a major challenge, as it actually stands fourth among the leading causes of cancer death and is expected to rise up to become the second most lethal malignancy by 2030. [START_REF] Siegel | Cancer Statistics[END_REF][START_REF] Rahib | Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States[END_REF][START_REF] Ferlay | More deaths from pancreatic cancer than breast cancer in the EU by 2017[END_REF] Despite recent advances in systemic treatment, prognosis of patients with metastatic (MPC) or locally advanced, unresectable (LAPC) disease remains poor, with 5-year overall survival (OS) of less than 5%. [START_REF] Kamisawa | Pancreatic cancer[END_REF] Phase 3 trials established FOLFIRINOX (5-fluorouracil/leucovorin, oxaliplatin and irinotecan) and gemcitabine plus nabpaclitaxel (Gem-Nab) as current standards in the first-line treatment of fit patients with MPC. [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF][START_REF] Hoff | Increased survival in pancreatic cancer with nabpaclitaxel plus gemcitabine[END_REF] Both regimens proved promising efficacy also in patients with LAPC. [START_REF] Suker | FOLFIRINOX for locally advanced pancreatic cancer: a systematic review and patient-level meta-analysis[END_REF][START_REF] Hammel | Phase II LAPACT trial of nab-paclitaxel (nab-P) plus gemcitabine (G) for patients with locally advanced pancreatic cancer (LAPC)[END_REF][START_REF] Marthey | FOLFIRINOX for locally advanced pancreatic adenocarcinoma: results of an AGEO multicenter prospective observational cohort[END_REF] In particular, FOLFIRINOX is now regarded as a suitable option in LAPC cases 10,[START_REF]A randomized phase III trial comparing Folfirinox to gemcitabine in locally advanced pancreatic carcinoma (NEOPAN)[END_REF] and has been recently established as the new reference also in the adjuvant setting. [START_REF] Conroy | FOLFIRINOX or gemcitabine as adjuvant therapy for pancreatic cancer[END_REF] Nonetheless, triplet chemotherapy is burdened by potentially severe adverse events (mainly digestive and haematological toxicities, with grade 3-4 neutropenia occurring in 46% of patients treated with FOLFIRINOX, including 5.4% febrile neutropenia), and median OS barely exceeds 11 months even in selected patients enrolled in randomized studies (i.e. performance status [PS] 0-1, bilirubin level <1.5 times the upper limit of normal and age ≤75 years). [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF] In routine clinical practice, only about 25% of patients with MPC would be eligible for FOLFIRINOX. [START_REF] Peixoto | Eligibility of metastatic pancreatic cancer patients for first-line palliative intent nab-paclitaxel plus gemcitabine versus FOLFIRINOX[END_REF] Different strategies (comprehensively known as modified FOLFIRINOX) aiming at improving tolerability have been tested, and are mostly based on removing 5fluorouracil bolus and/or decreasing irinotecan dose, or on the upfront administration of growth factors support. [START_REF] Vivaldi | First-line treatment with FOLFOXIRI for advanced pancreatic cancer in clinical practice: Patients' outcome and analysis of prognostic factors[END_REF] This approach seems to reduce the rate of grade 3-4 gastrointestinal or hematologic events, with comparable results in terms of OS with the PRODIGE4-ACCORD11 trial. [START_REF] Vivaldi | First-line treatment with FOLFOXIRI for advanced pancreatic cancer in clinical practice: Patients' outcome and analysis of prognostic factors[END_REF][START_REF] Mahaseth | Modified FOLFIRINOX regimen with improved safety and maintained efficacy in pancreatic adenocarcinoma[END_REF] The Gem-Nab combination represents an accepted alternative option in first-line [START_REF] Hoff | Increased survival in pancreatic cancer with nabpaclitaxel plus gemcitabine[END_REF] .

Despite being associated with an overall similar incidence of hematologic toxicities compared to FOLFIRINOX (grade 3-4 neutropenia: 38%; febrile neutropenia: 3%), Gem-Nab results in a higher rate of grade 3 or more peripheral neuropathy (17% vs. 9%) and a lower rate of severe diarrhoea (6% vs. 12.7%) [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF][START_REF] Hoff | Increased survival in pancreatic cancer with nabpaclitaxel plus gemcitabine[END_REF] and is therefore generally regarded as a suitable option for a greater percentage of MPC patients in everyday practice. [START_REF] Peixoto | Eligibility of metastatic pancreatic cancer patients for first-line palliative intent nab-paclitaxel plus gemcitabine versus FOLFIRINOX[END_REF] With the intent to improve risk stratification and patient selection for routine clinical decision making and future trials, several authors investigated clinical and laboratory factors putatively linked with patient outcome. [START_REF] Tabernero | Prognostic factors of survival in a randomized phase III trial (MPACT) of weekly nab-paclitaxel plus gemcitabine versus gemcitabine alone in patients with metastatic pancreatic cancer[END_REF][START_REF] Vernerey | Prognostic nomogram and score to predict overall survival in locally advanced untreated pancreatic cancer (PROLAP)[END_REF] Goldstein and colleagues recently queried the MPACT study dataset, identified several variables associated with OS and developed a nomogram able to predict patient survival probability at different time points when treated with gemcitabine with or without nabpaclitaxel. [START_REF] Goldstein | Relative contribution of baseline variables in a nomogram to predict survival in patients treated with nab-paclitaxel plus gemcitabine or gemcitabine alone for metastatic pancreatic cancer[END_REF] Predictive algorithms are recently gaining momentum in clinical practice: among them, nomograms are the most frequently used tools thanks to their accuracy and ease of use. [START_REF] Shariat | Can nomograms be superior to other prediction tools[END_REF] Previous studies with modified FOLFIRINOX reported that liver metastases, PS and neutrophil-to-lymphocyte ratio (NLR) are independently associated with OS. [START_REF] Vivaldi | First-line treatment with FOLFOXIRI for advanced pancreatic cancer in clinical practice: Patients' outcome and analysis of prognostic factors[END_REF] However, no tool is available to predict single patient prognosis with the triplet regimen.

As different treatment options are available and no head-to-head comparison has been conducted so far, discussing the relative benefits and risks of FOLFIRINOX and Gem-Nab with patients is challenging. Based on these considerations, we aimed at developing and validating a simple nomogram able to predict 6-month survival probability in MPC and LAPC patients treated with first-line triplet chemotherapy (FOLFIRINOX, as per classic or modified schedule).

MATERIALS AND METHODS

Patient selection and data collection

The developing set (DS) was constituted by consecutive MPC and LAPC patients treated at a single Institution (Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy) from January 2008 to December 2014 and discussed by dedicated multidisciplinary team dealing with pancreatic malignancies. Eligible patients were identified as follows: age >18 years; cytologically or histologically confirmed pancreatic carcinoma; non resectable, stage III or IV disease according to the American Joint Committee on Cancer (AJCC) staging system; access to clinical data collected before beginning of first-line chemotherapy; availability of laboratory information before treatment initiation, objective tumour response evaluation and survival data. The FOLFOXIRI schedule used in Pisa represents an alternative to standard FOLFIRINOX, derived from the experience in colorectal cancer [START_REF] Masi | Randomized trial of two induction chemotherapy regimens in metastatic colorectal cancer: an updated analysis[END_REF] with apparently super imposable efficacy compared with FOLFIRINOX in MPC/LAPC: details about the modified regimen have been described elsewhere. [START_REF] Vivaldi | First-line treatment with FOLFOXIRI for advanced pancreatic cancer in clinical practice: Patients' outcome and analysis of prognostic factors[END_REF] The putative predictors investigated were the following: age; gender; Eastern Cooperative Group (ECOG) PS (0 vs. 1); AJCC stage (III vs. IV); tumour location (head vs. body-tail); prior surgery of primary tumour (yes vs. no); previous adjuvant chemotherapy (yes vs. no); presence of biliary drainage (yes vs. no); number of disease sites; presence (yes vs. no) of metastases at specific sites, such as liver, lung, peritoneal or bone; neutrophil, lymphocyte and platelet counts, as well as NLR and platelet-to-lymphocyte ratio (PLR) before the first cycle of treatment; pre-treatment lactate dehydrogenase (LDH), carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19.9) serum levels. Age, number of disease sites and laboratory parameters were recorded and analyzed as continuous variables.

The external validation cohort involved MPC/LAPC patients treated at different Italian and French Institutions from January 2011 to June 2017. Inclusion criteria for the validating set (VS) were the same used in the DS, as were the variables collected for analysis. All patients included in the VS received FOLFIRINOX as per PRODIGE4-ACCORD11 schedule. [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF] The analyses included in this study were performed in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of the Coordinating Centre (Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy). Written informed consent from the patients for research use of data was obtained before the investigation. The protocol is registered on ClinicalTrials.gov (NCT03590275).

Statistical analyses

An early death binary variable indicating 6-month survival status was calculated from survival times, with values "1" if an event of death occurred in the first 180 days and 0 otherwise. Association of different covariates with 6-month survival status was evaluated by building univariate unconditional logistic regressions, modelling each variable with 6-month survival status. Wald test was used to assess statistical significance, defined as a two-tailed P-value <0.05. Considering the high variability of CA19.9, this covariate was logarithmically transformed before the analyses. Statistically significant covariates were used to developed different multivariate logistic regression models. Forward and backward methods were used.

Wald test was used to assess the significance of each covariate in the multivariate model.

Global fit was evaluated with Nagelkerke's R 2 , Somer's D and model log-likelihood ratio chisquare. Collinearity was addressed using t-test, Mann-Whitney, Fisher's exact test, ANOVA, linear regressions and Variance Inflation Factors (VIF), depending on the nature of the covariates and their characteristics (binary, categorical or continuous). The same tests were also used to assess differences in clinical characteristics between patients included in the VS and DS. Decision regarding inclusion of a specific variable into the final model was addressed taking into consideration their statistical significance, the percentage of models in which it remained significant, the global fit of the model and the clinical plausibility of covariates.

Predicted probabilities were tested against the observed probabilities in the VS. Somer's D, C-index, Spiegelhalter Z-test and Brier score were used to evaluate the discrimination of the model. 95% confidence intervals (95%CIs) of the C-index were calculated with bootstrap.

Calibration plot was assessed visually. Survival analyses were performed using the Kaplan-Meier method with log-rank test and by building Cox regression models. Median follow-up times were calculated with reverse Kaplan-Meier method.

Response rate (RR) was evaluated according to RECIST v.1.1 criteria. Progression-free survival (PFS) was defined as time from start of FOLFIRINOX to clinical or radiological progression or death from any cause, whichever occurred first, or until the date of the last follow-up, at which point data were censored. OS was defined as time from start of FOLFIRINOX to death from any cause. Survival data were censored at the last follow-up.

ROC curves were used to assess the best cut-off values for categorization of continuous variables. Packages "Survival" and "rms" of R were used for all the analyses.

RESULTS

Patient characteristics and treatment outcome

Characteristics of the patients in the DS and VS are presented in Table 1. A total of 343 patients were analyzed, with 137 and 206 cases included in the DS and the VS, respectively. More patients in the VS had an ECOG PS of 1 compared to the DS (54.9% vs. 32.8%; P<0.001). NLR was also significantly higher among the patients in the VS (median, 3.2 vs.

2.3; P<0.001).

No significant differences in number and location of metastases, basal CA19.9 serum level or other known prognostic factors were observed (all P-values >0.1).

Median follow-up was 30 months for the DS and 35 months for the VS. Outcomes achieved in the two cohorts were similar. RR was 38.6% and 31.4%, while median PFS was 8.0 (95%CI 6.7-9.2) and 7.2 (95%CI 5.6-8.2) months in the DS and VS, respectively. Median OS was 11.6 (95%CI 10.5-13.9) months in the DS and 10.5 (95%CI 9.2-12.1) months in the VS. Death events were observed in the majority of patients, with only 8.8% and 9.7% of patients censored for OS in the DS and VS, respectively. Notably, there were no censored observations in the first 180 days.

Prognostic nomogram: development

All the collected variables were analyzed for association with 6-month survival (Table 2).

Four out of the considered variables were selected in the final multivariable model: ECOG PS, pre-treatment NLR, liver metastases and basal serum CA19.9 (Table 3). Collinearity analyses revealed a slight correlation between CA19.9 and presence of liver metastases and between ECOG PS and NLR. However VIF was always lower than 2, so we decided to keep the model without further modifications. On the contrary, pre-treatment PLR, number of sites involved and disease stage, although significant or borderline significant at univariate analysis, were not retained due to an excessive amount of collinearity with NLR and liver metastases. Global fit was evaluated with Nagelkerke's R 2 , Somer's D and Area Under the Curve (AUC). The model showed a good global fit with a Nagelkerke's R2 of 0.283, Somer's D of 0.592, C-index of 0.796 and a highly significant log-likelihood (P<0.0001). The resulting nomogram is showed in Figure 1.

Prognostic nomogram: validation

Probabilities predicted by the nomogram were tested against those observed in the VS.

The nomogram discriminative ability was satisfying with a Somer's D of 0.524, corresponding to a C-index of 0.762 (95%CI 0.713-0.825). Brier score resulted 0.16 and the Spiegelhalter Ztest was not significant (P=0.087). Visual inspection of the calibration plot showed a good overlap between predicted and observed probabilities, even if there was a slight underestimation for patients at very high risk of early death (Figure 2).

Survival analysis based on prognostic factors

As an ancillary analysis, we performed a categorization of the variables included in the model to assess if they could be used to stratify patients into different risk groups. In order to do that, we combined the patients in the TS and VS and designed 4 different risk categories on the basis of the number of poor prognostic features present, i.e. ECOG PS 1, presence of liver metastases, log(CA19.9) and NLR above a threshold value. ROC curves were developed for the continuous variables log(CA19.9) and NLR, and returned an AUC of 0.641 and 0.676, respectively. We therefore set a threshold of 6.75 for log(CA19.9) (which corresponds to a basal value of 845 U/mL), obtaining a sensitivity of 0.64 and a specificity of 0.62. For NLR, the threshold was set at 2.46, with a sensitivity of 0.78 and a specificity of 0.52. Median OS significantly differed among the four subgroups identified, ranging from 7.2 (95%CI 5.6-8.7) months, through 10.8 (95%CI 9.4-12.9) and 13.9 (95%CI 12.5-16.6) months and up to 18.3 (95%CI 14.5-23.5) months for patients with >2, 2, 1 and 0 risk factors, respectively (P<0.0001 for overall comparison) (Figure 3).

DISCUSSION

Life expectancy of unresectable PC patients treated with first-line chemotherapy remains poor despite the recent introduction of more active chemotherapy regimens. [START_REF] Kamisawa | Pancreatic cancer[END_REF][START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF][START_REF] Hoff | Increased survival in pancreatic cancer with nabpaclitaxel plus gemcitabine[END_REF] In light of the toxicity profile of an intensive triplet schedule such as classic or modified FOLFIRINOX, the ability to anticipate single patient prognosis is of high value. Indeed, it allows discussing the benefit-to-risk ratio of this regimen and taking a more informative decision about different firstline therapeutic options. Recently, authoritative experts advocate the need for alternative measures to understand and communicate the impact of treatment on OS. [START_REF] Saad | Understanding and communicating measures of treatment effect on survival: can we do better?[END_REF] Moreover, literature evidence demonstrates that discussion about prognosis during clinical encounters strengthens the patient-oncologist relationship, [START_REF] Fenton | Impact of prognostic discussions on the patient-physician relationship: prospective cohort study[END_REF] prompting the need for validated and easyto-use instruments to clearly communicate risks at defined time points during the course of the disease. Similar instruments have been recently proposed for second-line therapy in MPC, [START_REF] Vienot | Overall survival prediction and usefulness of secondline chemotherapy in advanced pancreatic adenocarcinoma[END_REF] but are currently lacking for the triplet chemotherapy regimen used in the first-line setting. Moreover, in case of clinical trials, prognostic nomograms could be useful tools for a better stratification of the enrolled patients and interpretation of the results in different subgroups. [START_REF] Mariotto | Cancer survival: an overview of measures, uses, and interpretation[END_REF] Our study identified easily available and measurable parameters as major determinants of prognosis in this population, such as ECOG PS, NLR, liver metastases and CA19.9 levels, making the nomogram accessible in the routine clinical setting. A large body of literature supports the prognostic importance of these variables in PC patients treated with gemcitabine-based chemotherapy, particularly for what regards PS and CA19.9 values. [START_REF] Reni | Selecting patients for resection after primary chemotherapy for non-metastatic pancreatic adenocarcinoma[END_REF][START_REF] Kou | Prognostic model for survival based on readily available pretreatment factors in patients with advanced pancreatic cancer receiving palliative chemotherapy[END_REF] Nonetheless, to the best of our knowledge, this is the first attempt to include them in a validated model able to predict early deaths of MPC/LAPC patients treated with a more modern regimen such as FOLFIRINOX. Of interest, stratifying patients for the presence of each single determinant identified different populations with distinct survival outcomes. In particular, in the most favourable risk subgroup (i.e. no poor prognostic features present) median OS was almost three-times longer than that observed for the worst-risk category (i.e. >2 poor prognostic features present), making the information retrieved by the nomogram useful for both practice and research. Currently, validated predictive biomarkers are lacking in this setting, [START_REF] Caparello | FOLFIRINOX and translational studies: towards personalized therapy in pancreatic cancer[END_REF] and prognostic stratification is thus essential to discuss alternative treatment options in single cases. Therefore, our nomogram could represent a suitable tool for the identification of different patient subgroups and prompts research on the biological basis explaining the influence of these clinical variables on survival outcome.

Variables included in the nomogram were either confirmed as independent prognostic determinants at multivariate analysis or retained due to the robust evidence of their prognostic value from available literature. Notably, tumour stage has been already demonstrated as a prognostic determinant in previous studies. [START_REF] Kamisawa | Pancreatic cancer[END_REF][START_REF] Suker | FOLFIRINOX for locally advanced pancreatic cancer: a systematic review and patient-level meta-analysis[END_REF] However, significance was not formally demonstrated in our datasets (P=0.067 at univariate analysis). This could be possibly due to the relatively low number of LAPC cases in our study, as well as the potential presence of other poor prognostic features in the LAPC cohort. We then decided not to retain it in the final nomogram. This decision was also supported by the evidence of high collinearity between disease stage and presence of liver metastases, as previously described.

Three out of four factors included in our nomogram were also included in the nomogram developed from the MPACT database (comprising PS, NLR, liver metastases, serum albumin, sum of the largest lesions, analgesic use and treatment arm), [START_REF] Goldstein | Relative contribution of baseline variables in a nomogram to predict survival in patients treated with nab-paclitaxel plus gemcitabine or gemcitabine alone for metastatic pancreatic cancer[END_REF] further underlining the external validity of our work. Notably, the relative contribution of CA19.9 to the performance of the MPACT nomogram when added to PS, NLR, liver lesions and serum albumin was limited.

On the contrary, in our study CA19.9 was the strongest predictor of OS among the analysed variables. This is in line with previous literature, which convincingly established this serum tumour marker as a main confirmed determinant of patient outcome in this population. [START_REF] Vernerey | Prognostic nomogram and score to predict overall survival in locally advanced untreated pancreatic cancer (PROLAP)[END_REF][START_REF] Reni | Selecting patients for resection after primary chemotherapy for non-metastatic pancreatic adenocarcinoma[END_REF][START_REF] Kou | Prognostic model for survival based on readily available pretreatment factors in patients with advanced pancreatic cancer receiving palliative chemotherapy[END_REF] Moreover, some limitations of the MPACT nomogram make it of relatively low immediate utility in routine practice. The lack of external validation, the few points assigned to several variables (such as analgesic use and treatment arm) and the inclusion of highly selected patients from a registrative phase 3 trial might prevent the applicability of these results to other populations and/or other treatment regimens. On the other hand, in light of the partly overlapping factors included in the two nomograms and the easy-of-use of our prognostic variables, it could be of interest to test the performance of this FOLFIRINOX nomogram among patients treated with other regimens, namely Gem-Nab. In this regard, a recently published study confirmed the role of PS, NLR and CA19.9 in a prognostic nomogram developed from a retrospective series of 210 patients treated with first-line Gem-Nab. [START_REF] Fernández | Prognostic factors for survival with nab-paclitaxel plus gemcitabine in metastatic pancreatic cancer in real-life practice: the ANICE-PaC study[END_REF] When planning the study design, we decided to build an instrument specifically addressing the risk of early death instead of general OS. This decision was taken in light of the risks of toxicity associated with FOLFIRINOX and other potential issues impacting on patient daily life, such as the need to implant a central venous catheter for prolonged infusions. Indeed, it is of little doubt that FOLFIRINOX (whichever the schedule used) remains a challenging treatment option, ideally to be used in patients able to experience the greatest benefit in terms of OS while sparing those who are likely to get little or no advantage due to very short OS probability. The choice of the 6-month period as primary outcome measure was based on the results of the PRODIGE4-ACCORD11 trial, which reported a median PFS of 6.4 months and a median OS of 11.1 months in the experimental arm. [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF] In our opinion, the probability of experiencing early death in the first 6 months after treatment initiation (i.e. less than the median PFS expected with FOLFIRINOX) can be considered an acceptable criterion to discuss with the patient treatment options alternative to triplet chemotherapy.

The main criticism of our study relies in its retrospective design and non-exhaustive nature of data collection about other potentially prognostic parameters. However, patient characteristics were generally well balanced in the DS and VS. Furthermore, when treatment activity and efficacy were investigated, no difference was reported in terms of RR, PFS and OS between the two cohorts (and so between FOLFIRINOX and FOLFOXIRI) and results were comparable with literature data. As discussed, we did not evaluate the outcome of the different risk categories with other treatment options. Therefore, it should be kept in mind that our study was not designed to validate the developed nomogram as a predictive tool to anticipate the benefit from a specific regimen (i.e. FOLFIRINOX) when compared to other options (such as Gem-Nab, single-agent chemotherapy or supportive care only). The

CLINICAL PRACTICE POINTS

• Despite recent advances, prognosis of patients with metastatic and locally advanced pancreatic cancer remains poor.

• FOLFIRINOX and gemcitabine plus nab-paclitaxel are both standards in the first-line treatment of fit patients and no head-to-head comparison has been conducted so far.

• Toxicities of triplet chemotherapy may be relevant and many patients derive limited benefit from intensive treatment: indeed, no tool is currently available to individualize the therapeutic approach in single cases.

• We therefore developed (from a single Institution experience) and validated (by external collection of cases from Italian and French referral centres) a simple nomogram able to predict 6-month survival probability in pancreatic cancer patients receiving first-line FOLFIRINOX (as per classic or modified schedule).

• Easily available, reproducible and cheap clinical and laboratory parameters confirmed their prognostic value in this population and were finally included in the model: performance status, neutrophil-to-lymphocyte ratio, liver metastases and CA19.9 levels before treatment.

• The presence of these variables also stratified our series into four risk categories with significantly different survival outcome.

• Our nomogram can be immediately implemented in clinical practice in order to improve communication about prognosis with pancreatic cancer patients.

• Moreover, this tool could be of help in clinical research, as it demonstrated to improve patient stratification.

• Validation of the nomogram in series treated with other treatment regimens such as gemcitabine plus nab-paclitaxel is warranted. iv) baseline CA19.9 >845 U/mL (see text for details).

  information retrieved from the nomogram is a more detailed prognostic assessment of the single cases, and FOLFIRINOX (or modified schedules) remains a valid option for fit MPC/LAPC patients. CONCLUSION To conclude, it is possible to accurately predict the risk of death in the first 6 months after starting FOLFIRINOX for MPC/LAPC by few easily available, reproducible and cheap clinical and laboratory parameters. Our nomogram as well as different risk categories allow immediate prognostic stratification and provide an easy-to-interpret tool for both clinicians and patients. This instrument could facilitate patient-physician communication in clinical practice and improve prognostic stratification in clinical research. Validation of this tool for other treatment regimens such as Gem-Nab is warranted.

Figure 1 .

 1 Figure 1. Nomogram predicting the risk of death at 6 months after initiation of FOLFIRINOX.

Figure 2 .

 2 Figure 2. Calibration plot for external validation of the nomogram.

Figure 3 .

 3 Figure 3. Survival curves for different risk categories.

Table 1 . Patient characteristics

 1

		Developing set		Validating set
		(N=137)		(N=206)
		N	%	N	%	P-value
	Gender
	Male	66	48.2	116	56.3	0.152
	Female	71	51.8	90	43.7
	Age (years)
	Median	60		62		0.564
	Range	33-75		41-78
	ECOG PS
	0	92	67.2	93	45.1	<0.001
	1	45	32.8	113	54.9
	Tumour site
	Head	74	54	119	57.5	0.579
	Body-tail	63	46	88	42.5
	TNM stage
	III	56	40.9	75	36.4	0.428
	IV	81	59.1	131	63.6

Abbreviations: CT, chemotherapy; ECOG PS, Eastern Cooperative Oncology Group performance status; IQR, interquartile range; N, number; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio.

Table 2 . Results of the univariate models

 2

	OR	95%CI	P-value

Table 3 . Results of the multivariable model N of patients N of events

 3

	OR	95%CI	P-value

Abbreviations: CA19.9, carbohydrate antigen 19.9; ECOG PS, Eastern Cooperative Oncology Group performance status; N, number; NLR, neutrophil-to-lymphocyte ratio; OR, odds ratio; 95%CI, 95% confidence interval.

ACKNOWLEDGEMENTS

None.

status; LDH, lactate dehydrogenase; NLR, neutrophil-to-lymphocyte ratio; OR, odds ratio; PLR, platelet-to-lymphocyte ratio.

FUNDING

This work was supported by Fondazione ARCO Onlus (Azioni Ricerche e Cure in Oncologia) (no specific grant number applies). The funding source had no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

None.