Manon Thierry

Susana Boluda

Benoît Delatour

Serge Marty

Danielle Seilhean

Brainbank Neuro- Ceb

Neuropathology Network

Marie-Claude Potier

Prof Charles Duyckaerts
email: charles.duyckaerts@aphp.fr

Valérie Rigau

Serge Milin

Human subiculo-fornico-mamillary system in Alzheimer disease: Tau seeding by the pillar of the fornix

Keywords: Alzheimer disease, Tau, Abeta, subiculo-fornico-mamillary system, seeding, propagation, biosensor, microcryodissection, CLARITY, electron microscopy, axon propagation

In Alzheimer's disease (AD), Tau and Aβ aggregates involve sequentially connected regions, sometimes distantly separated. These alterations were studied in the pillar of the fornix (PoF), an axonal tract, to analyze the role of axons in their propagation. The PoF axons mainly originate from the subicular neurons and project to the mamillary body. Forty-seven post-mortem cases at various Braak stages (Tau) and Thal phases (Aβ) were analyzed by immunohistochemistry. The distribution of the lesions showed that the subiculum was affected before the mamillary body, but neither Tau aggregation nor Aβ deposition was consistently first. The subiculum and the mamillary body contained Gallyas positive neurofibrillary tangles, immunolabeled by AT8, TG3, PHF1, Alz50 and C3 Tau antibodies. In the PoF, only thin and fragmented threads were observed, exclusively in the cases with neurofibrillary tangles in the subiculum. The threads were made of Gallyas negative, AT8 and TG3 positive Tau. They were intra-axonal and devoid of paired helical filaments at electron microscopy. We tested PoF homogenates containing Tau AT8 positive axons in a Tau P301S biosensor HEK cell line and found a seeding activity. There was no Aβ immunoreactivity detected in the PoF. We could follow microcryodissected AT8 positive axons entering the mamillary body; contacts between Tau positive endings and Aβ positive diffuse or focal deposits were observed in CLARITY-cleared mamillary body. In conclusion, we show that non-fibrillary, hyperphosphorylated Tau is transported by the axons of the PoF, from the subiculum to the mamillary body, and has a seeding activity. Either Tau aggregation or Aβ accumulation may occur first in this system: this inconstant order is incompatible with a cause and effect relationship. However, both pathologies were correlated and intimately associated, indicating an interaction of the two processes, once initiated.

Introduction

Alzheimer's disease (AD) is characterized by extracellular accumulation of β-amyloid peptides (Aβ) and intracellular aggregation of hyperphosphorylated Tau proteins (hp-Tau). Aβ peptides accumulate in different types of extracellular deposits (diffuse, focal, neuritic, stellate and subpial [START_REF] Delaère | Subtypes and differential laminar distributions of beta A4 deposits in Alzheimer's disease: relationship with the intellectual status of 26 cases[END_REF]), while hp-Tau aggregates in the neuronal soma, leading to the formation of neurofibrillary tangles (NFTs) [START_REF] Brion | Mise en évidence immunologique de la protéine Tau au niveau des lésions de dégénérescence neurofibrillaire de la maladie d'Alzheimer[END_REF][START_REF] Grundke-Iqbal | Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology[END_REF]. Accumulation of hp-Tau in neurites leads to the formation of neuropil threads, thought to be mainly dendritic [START_REF] Braak | Occurrence of neuropil threads in the senile human brain and in Alzheimer's disease: a third location of paired helical filaments outside of neurofibrillary tangles and neuritic plaques[END_REF], while the hp-Tau positive neurites of the corona of the senile plaques are thought to be mainly axonal [START_REF] Schmidt | Comparative epitope analysis of neuronal cytoskeletal proteins in Alzheimer's disease senile plaque neurites and neuropil threads[END_REF]. Thal phases (from 0 to 5) evaluate the progression of Aβ deposits [START_REF] Thal | Phases of Aβ-deposition in the human brain and its relevance for the development of AD[END_REF] and Braak neurofibrillary stages (from 0 to VI), of hp-Tau accumulation [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF].

Misfolded Tau proteins injected in Tau transgenic mice which are too young to develop lesions induce early development of Tau aggregates that apparently propagate like a prion, following neural circuits [START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF]. Aβ pathology is also able to propagate in a prion-like way [START_REF] Meyer-Luehmann | Exogenous induction of cerebral β-amyloidogenesis is governed by agent and host[END_REF] hence the term propagon that has been suggested for these protein assemblies [START_REF] Eisele | Propagation of Aß pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies[END_REF]. The mechanism and kinetics of the initiation and propagation of Tau and Aβ lesions are still poorly understood, both in experimental animals and in humans. The amyloid cascade hypothesis [START_REF] Hardy | Alzheimer's disease : the amyloid cascade hypothesis[END_REF][START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF] posits that Aβ peptide aggregation is an early pathological process driving Tau phosphorylation, neurofibrillary tangle formation, synaptic loss and neuronal death.

We studied the subiculo-fornico-mamillary system in the human brain, to analyze the progression of Tau and Aβ pathologies, their timing and their relationship. The fornix, localized below the corpus callosum, is laterally limited on each side by the pillar of the fornix (PoF), a large intrahemispheric associative bundle, 4.5 cm long and 0.75 cm in diameter. That bundle, a key element of the Papez circuit, links the hippocampal formation (including the subiculum) with the mamillary body, the septal and the accumbens nuclei [START_REF] Vann | The mammillary bodies: two memory systems in one?[END_REF] (Fig. 1a).

The PoF is exclusively made of axons that can thus be studied in isolation, while they are difficult to identify in the neuropil where they are mixed with dendrites. The PoF degenerates during AD [START_REF] Copenhaver | The fornix and mammillary bodies in older adults with Alzheimer's disease, mild cognitive impairment, and cognitive complaints: A volumetric MRI study[END_REF]. The abundance of fibers of hippocampal origin in the PoF is illustrated by fornico-mamillary atrophy, in which neuronal loss in the hippocampus causes atrophy of the PoF and of the mamillary body (Fig. 1b). The majority of the axons travelling in the PoF originate from the neurons of the subiculum, where NFTs form as early as Braak stage III and amyloid deposits are seen at Thal phase 2. Those axons project to the mamillary body, where NFTs appear from Braak stage IV and Aβ peptides accumulate at Thal phase 3 [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF][START_REF] Thal | Phases of Aβ-deposition in the human brain and its relevance for the development of AD[END_REF]. The hippocampo-fornico-mamillary system is vascularized by different arteries: the posterior cerebral artery for the subiculum, the anterior communicating artery for the mamillary body [START_REF] Hauw | Vascular pathology[END_REF]; diffusion through the vascular system is thus implausible. As both structures are wide apart, passive diffusion from one to the other is also unlikely. This system is thus well suited to investigate the propagation of Tau and Aβ lesions through defined axonal connections.

Materials and methods

Cases

Forty-seven post-mortem brains were investigated. All the brains had been removed and collected at Pitié-Salpêtrière hospital (Paris). They were stored in 4% formalin. In all the cases, the diagnostic procedure included Aβ (6F3D, Dako), Tau (AT8, Thermo-Fisher), α-synuclein (5G4, Sigma-Aldrich) and TDP-43 (Proteintech Group) immunohistochemistry. The hippocampus, the PoF and the mamillary body were analyzed for each case. Among the 47 cases that we studied, 38 brains had been collected in a brain donation project and stored in the French national brain biobank Neuro-CEB (Bioresource Research Impact Factor number = BRIF BB-0033-00011). The cohort was composed of 15 women and 32 men, aged 51 to 96 (Table 1). The cases included 28 patients with no or asymptomatic AD lesions, deceased of other causes (Braak 0-IV) and 19 patients with established AD (Braak V-VI). Among the 28 asymptomatic patients, 17 had mild Tau alterations associated with no or only little Aβ accumulation (Thal phase 1 or 2). It is common to observe cases with Tau pathology in the entorhinal area and hippocampus and no Aβ accumulation in the brain ( [START_REF] Braak | Frequency of stages of Alzheimer-related lesions in different age categories[END_REF], see also the prevalence curves obtained from the data of [START_REF] Braak | Frequency of stages of Alzheimer-related lesions in different age categories[END_REF] in the companion paper [START_REF] Duyckaerts | Prevalence, incidence and duration of Braak's stages in the general population: can we know?[END_REF]). Those cases have been recently isolated under the name of PART [START_REF] Crary | Primary age-related tauopathy (PART): a common pathology associated with human aging[END_REF], a questionable construct [START_REF] Duyckaerts | PART is part of Alzheimer disease[END_REF]. Fourteen cases had Lewy bodies. Cases diagnosed with primary tauopathies were excluded from the study (i.e. progressive supranuclear palsy, corticobasal degeneration, MAPT mutation, argyrophilic grain disease, Pick's disease). The eight cases at Braak 0/I/II corresponded to control brains with no lesion in the hippocampus. Some Tau pathology was present in the subiculum in the 20 cases at Braak III/IV. The 19 cases at Braak V/VI had extensive Tau pathology in the subiculum but also in the neocortex. Thal phases ranged from 0 to 5 (Table 1). Six additional cases (also listed in Table 1) were used for CLARITY, microcryodissection, electron microscopy analysis and cell culture seeding assays. The project was submitted to the "committee for the protection of persons" (ethical committee) Ile de France VI which did no object to the study (april 20 th 2016 session).

Histology analysis

The hippocampus, PoF and mamillary body were sampled after a fixation of at least two months in a 10% formalin solution (containing 4% formaldehyde). The samples were embedded in paraffin. The PoF was dissected and embedded parallel to its long axis over a distance of at least 1 cm. The blocks were cut at a thickness of 3 µm.

Immunohistochemistry

Immunohistochemistry was performed with the Nexes station automated system (Ventana Medical System Inc., Roche). Deparaffinized and rehydrated sections were pretreated and blocked, then incubated with the primary antibodies listed in supplementary Table 1, online resource. The AT8, TG3, PHF1, Alz50, C3 and anti-myelin basic proteins (MBP) primary antibodies were detected with the ultraView Universal DAB Detection Kit (Ventana Medical System Inc., Roche) or OptiView DAB immunohistochemistry Detection Kit (Ventana Medical System Inc., Roche), according to the automated procedure summarized in supplementary Table 1, online resource. The kits include secondary antibodies (goat anti-mouse IgG, anti-mouse IgM and antirabbit) conjugated with horseradish peroxidase (HRP). The addition of H202 and diaminobenzidine leads to the formation of a brown precipitate in presence of HRP. The anti-Aβ 6F3D immunohistochemistry was performed manually following formic acid pretreatment (98% formic acid for five minutes); the primary antibody was incubated overnight. The MBP/Neurofilament and Aβ/Tau double immunostainings made use of the same protocol as previously described for the first primary antibody (anti-MBP and anti-Aβ 6F3D, respectively). The second primary antibody (anti-Neurofilament and AT8 anti-Tau, respectively) was revealed with the ultraView Universal Alkaline Phosphatase Red Detection Kit (Ventana Medical System Inc., Roche), according to the automated procedure summarized in supplementary Table 1, online resource. The detection kit includes secondary antibodies (goat anti-mouse IgG, anti-mouse IgM and anti-rabbit) conjugated with alkaline phosphatase (AP) and reagents (naphtol and Fast Red), leading to the formation of a red precipitate in presence of AP. The slides were counterstained with Harris haematoxylin and dehydrated. Coverslips were mounted in Pertex (Histolab, Labelians).

Gallyas silver impregnation

Gallyas method [START_REF] Gallyas | Silver staining of Alzheimer's neurofibrillary changes by means of physical development[END_REF] was applied with the modifications proposed by Uchihara et al. [START_REF] Uchihara | Alzheimer-type pathology in melanin-bleached sections of substantia nigra[END_REF], that result in the bleaching of the undesired myelin staining. The slides were counterstained with 0.1% Nuclear Fast Red and dehydrated. Coverslips were mounted in Pertex (Histolab, Labelians).

Quantifications

An area covering the whole thickness of the subiculum was systematically observed with a x20 objective after AT8, TG3, Alz50, PHF1, and C3 anti-Tau immunohistochemistry and Gallyas staining. One hundred neuronal profiles and ghost tangles were examined. The proportion of neuronal profiles (total number of neurons as denominator) with pre-tangles, NFTs and ghost NFTs was assessed. A pilot study [START_REF] Gundersen | Optimizing sampling efficiency of stereological studies in biology: or "do more less well![END_REF] had determined that the error coefficient improved only slowly when the number of assessed profiles was above 100.

The percentages of positive profiles were converted into grades from 0 to 3 (0: 1% or less; 1: from 2% to 34%; 2: from 35% to 67%; 3: 68% or more). The volume density of Aβ deposits in the subiculum was estimated by point counting after anti-Aβ 6F3D immunohistochemistry [START_REF] Weibel | practical methods for biological morphometry[END_REF]. A video camera transmitted the picture of the subiculum to a screen on which a grid of points was superimposed. The points were regularly spaced at 80 µm intervals (9 rows x 12 columns). The proportion of points falling onto Aβ immunoreactive material over a total of 100 points (± 10, depending on the size of the subiculum) was counted to estimate the areal density AA [number of points falling onto Aβ immunoreactive material]/[total number of points]. AA is an unbiased estimator of the volume ratio VV [volume of Aβ deposits]/[total volume], according to Delesse principle (AA = VV) [START_REF] Weibel | practical methods for biological morphometry[END_REF]. As Aβ was heterogeneously distributed, we chose to evaluate the areas in which the density appeared the highest: the assessment thus concerned the highest (rather than the mean) proportion of volume. The numerical densities of AT8, TG3, Alz50, PHF1, C3 and Gallyas positive axons were semi-quantitatively assessed on longitudinal sections of the PoF after adequate immunohistochemistry or staining. The following scale was applied: 0: no staining (1% and less); 1: rare and patchy staining (from 2% to 34%); 2: staining with intermediate density (from 35% to 67%); 3: staining with high density (68% or more). In the mamillary body, the density of AT8, TG3, Alz50, PHF1, C3 and Gallyas positive pre-tangles, NFTs and ghost NFTs was semiquantitatively assessed as previously (from grade 0 to 3), as well as the volume proportion of Aβ deposits.

Density maps of the lesions in the mamillary body

Voronoi (or "Dirichlet") tessellation was used to draw quantitative maps of the numerical density of the lesions in the mamillary body [START_REF] Duyckaerts | Evaluation of neuronal numerical density by Dirichlet tessellation[END_REF]. The maps were drawn with an apparatus made of a video system, of linear transducers fastened to the moving stage of the microscope, and of a computer with a mouse. The microscopic picture was visible on the computer screen. The mouse allowed drawing the contour of the studied region and pointing the Tau AT8 positive NFT or pre-tangles and the Aβ 6F3D positive deposits. The borders of the medial and lateral nuclei of the mamillary body were manually drawn on the video screen with the mouse. The transducers associated the points that had been drawn with X and Y coordinates [START_REF] Grignon | Cytoarchitectonic alterations in the supramarginal gyrus of late onset Alzheimer ' s disease[END_REF] and guaranteed that they followed the movement of the microscope stage. Maps of Aβ deposits and AT8 Tau positive neuronal profiles were obtained. Coordinates of the borders of the medial and lateral nuclei of the mamillary body, and of the pointed lesions were stored in a file at the end of the acquisition step. These coordinates were used to analyze the data with a specially devised tessellation program. The region was divided into polygons limited by sides located at mid-distance of two neighboring lesions and perpendicular to the line that joined them. Each polygon contained only one labeled lesion (Aβ deposit or neurofibrillary tangle or pretangle). The polygons covered the entire area of the region without overlaps or interstices, i.e. "tessellated" the plane. The area of each individual polygon is inversely proportional to the numerical density of the pointed lesions that it contains. When the density is high, the lesions are close to each other and the polygons are small. Assigning a color to a range of polygon areas allowed drawing a 'heat map' of the numerical density of the lesions [START_REF] Duyckaerts | Voronoi tessellation to study the numerical density and the spatial distribution of neurones[END_REF][START_REF] Duyckaerts | Evaluation of neuronal numerical density by Dirichlet tessellation[END_REF].

In vitro seeding experiments

Preparation of PoF homogenates. Approximately 100 mg of frozen PoF sampled from cases 49, 50 and 53, listed in Table 1, were thawed on ice. The tissue was mixed with ice-cold phosphate-buffered saline (PBS) containing protease and phosphatase inhibitor cocktail (Thermo Scientific), to obtain a final solution of 10% weight/volume. Tissue was homogenized by 15-20 strokes in a 1 mL glass homogenizer (Fisher Scientific), briefly sonicated on ice with a Misonix Q125 sonicator (Q-sonica) and clarified by centrifugation (3000 g, 5 min, 4°C), as previously described [START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF]. Supernatants were collected, aliquoted and stored at -80°C.

Preparation and treatment of biosensor cells sensitive to Tau seeding. Tau P301S biosensor HEK cells (#CRL-3275, ATCC, LGC Standards [START_REF] Holmes | Proteopathic tau seeding predicts tauopathy in vivo[END_REF]) were grown in complete culture medium (DMEM with 10% FBS and 1% penicillin/streptavidin, Gibco, Thermo Fisher Scientific). They were treated with clarified PoF homogenates as in [START_REF] Furman | Sensitive detection of proteopathic seeding activity with FRET flow cytometry[END_REF][START_REF] Holmes | Proteopathic tau seeding predicts tauopathy in vivo[END_REF], with minor modifications. Approximately 170, 000 cells contained in 650 µL of complete medium were plated, per well, in a 24-well plate and left overnight in the incubator (37 °C, 5% CO2 and ≥ 80% relative humidity). At around 60% of confluence, 100 µL of a transduction mix containing the PoF homogenate was added per well, in duplicate. The transduction mix was obtained by combining 43.75 µL Opti-MEM (Gibco, Thermo Fisher Scientific) and 6.25 µL Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific), with 12.5 µL of PoF homogenate added to 37.5 µL Opti-MEM. The transduction mix was briefly spun, then incubated for 1 hour at room temperature, before being added to the cells. Treated cells were left in the incubator during 48h.

The experiment was replicated three times.

Analysis. After treatment, cells were rinsed with PBS and re-plated on 13 mm diameter coverslips coated with poly-D-lysine (Sigma-Aldrich). The coverslips were left overnight in the incubator. They were rinsed with PBS and fixed for 10 min in 4% PFA. The cells were counterstained for 10 min with DAPI in 0.05% Triton-X100. The coverslips were mounted with Fluoromount (Sigma-Aldrich). Slides were stored in the dark at 4°C until observation with an inverted confocal Leica TCS SP8 DLS (Leica Microsystems). The CFP-positive cells were manually counted on the confocal images.

Aβ immunoassay

Aβ immunoassay of the clarified PoF homogenates, prepared as described above, was performed with the V-PLEX Plus Aβ Peptide Panel 1 (6E10) kit (Meso Scale Discovery), following the instructions of the manufacturer. A clarified frontal cortex homogenate derived from an AD brain (Braak VI, Thal 5) was used as positive control.

Electron microscopy

The aim was to observe, with electron microscopy, AT8 positive axonal threads present in the PoF. The subiculum and the PoF were sampled from two AD formalin-fixed brains (cases 50 and 52). An additional PoF was sampled from a formalin-fixed brain devoid of any AD lesions and was used as control (case 48). The samples were sliced into 200 µm thick sections using a vibratome VT 1000S (Leica Microsystems). The PoF was cut along its longitudinal axis and the subiculum perpendicularly to its long axis. No AT8 Tau labeling was observed with the immunogold method that appeared unsuitable for post-mortem formalin-fixed material. We thus used the direct visualization of diaminobenzidine precipitates after AT8 Tau immunohistochemistry performed without pretreatment and without detergent on the vibratome slices. Regions of interest (with an area of 1 mm 2 ) were dissected on the slices. The samples were washed in phosphate buffer and transferred into a 1% osmium solution for 1h. After several washes in water, they were stained "en bloc" with a 5% uranyl acetate solution for 30 min. The samples were dehydrated in graded ethanol (from 50% to 100%) followed by acetone.

They were incubated in 50% acetone -50% Epon overnight, followed by pure Epon for 2h, before being cured at 60°C for 48h in a dry oven. Semi-thin (0.5 µm) and ultrathin (70 nm) sections were cut using an EM UC7 (Leica Microsystems). Ultrathin sections were collected on copper grids and analyzed with a Hitachi HT700 electron microscope (Hitachi, Milexia) operating at 100 kV. Pictures were taken with 2048 X 2048 AMT R41-B CCD camera (AMT, Milexia).

Microcryodissection

One formalin-fixed mamillary body was sampled from an additional AD case (case 51). The sample was microcryodissected following the procedure previously described [START_REF] Thierry | Alzheimer's senile plaque as shown by microcryodissection, a new technique for dissociating tissue structures[END_REF]. Briefly, cycles of freezing and thawing induce changes in the water volume contained in the sample, ice occupying more space than water. Repeated cycles separate the elements of the tissue. The sample was rinsed and left in water on a Peltier module.

Three-hundred and twenty freezing and thawing cycles were achieved (program settings: 700 seconds 'on', freezing; 300 seconds 'off', thawing). Microcryodissected mamillary body was embedded in paraffin and analyzed by immunohistochemistry as described above.

CLARITY

A 3 mm thick slice of a formalin-fixed mamillary body was taken from an AD case (< 1 year fixation; Braak VI, Thal 5; Case 52) and processed by the CLARITY technique adapted to post-mortem tissue [START_REF] Ando | Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D[END_REF]. The sample was embedded in an acrylamide hydrogel constituted of 4% acrylamide and 0.25% temperaturetriggering initiator VA-044, in PBS. The sample was thoroughly washed in the clearing solution (200 mM boric acid, 4% SDS, pH 8.5) and placed in the X-CLARITY tissue clearing system (Logos Biosystems Inc.). A direct current (1.5 A) and a constant flow of clearing solution heated to 40°C were applied during a total of 96 h. The clearing solution was renewed every 24h. After clearing, the sample was pretreated with formic acid 98% for 15 minutes. The tissue was blocked with 10% of goat and donkey sera (with a 1:1 ratio). The sample was incubated with a mouse monoclonal antibody anti-Aβ (4G8, BioLegend) and a rabbit polyclonal antibody anti-total-Tau (B19, gift from J.-P. Brion, Free University of Brussels, Belgium). Donkey anti-mouse Alexa 488 and goat antirabbit Alexa 555 (Life Technologies, Thermo Fisher Scientific) were used as secondary antibodies. The sample was post-fixed in 4% paraformaldehyde and autofluorescence was quenched with 0.2 M glycine. The day before observation, the tissue was mounted with RapidClear®. The microscope analyses were made with an upright multiphoton Zeiss 710 NLO (Carl Zeiss Microscopy). The images were post-analyzed with Fiji (Fiji Is Just ImageJ, National Institutes of Health) and Imaris (Bitplane, Oxford Instruments).

Statistical analyses

All the data were analyzed with GraphPad Prism 6, at a significance level of 5%. For discrete neuropathological data, nonparametric statistical tests were performed: Spearman test was used for analysis of correlation, Kruskal-Wallis and Friedman tests were used for one-way analysis of variance (respectively for untied or tied data), followed by Dunn post-hoc multiple comparisons when the null hypothesis was rejected.

The means of groups with or without Lewy bodies were compared with Mann-Whitney. To illustrate the relationship between the data, we also drew a regression line with the hp-Tau semi-quantitative measures (Supplementary Fig. 2 and3, online resource), with the hypothesis that the discrete data describing a severity gradient could be interpolated by the regression between the grades and that the grades had a binomialtending to a Gaussiandistribution. In the seeding experiments, the number of CFP-positive HEK cells was expressed as a percentage of the total cell number counted. The mean percentages were compared using a Student t-test, performed after an arcsine square root transformation (as recommended, [START_REF] Snedecor | Statistical methods[END_REF] to approach a normal distribution).

Results

Distribution of Tau and Aβ accumulation in the subiculo-fornico-mamillary system

Aβ deposition in the subiculo-fornico-mamillary system Aβ deposits were observed in the subiculum and in the mamillary body (Fig. 2a and2c). No Aβ immunoreactivity was detected in or around the axons of the PoF (Fig. 2b). This result was confirmed by Aβ40 and Aβ42 immunoassays in PoF homogenates from three cases at Braak stages 0 (negative control), IV and VI.

In the PoF homogenates, Aβ40 and Aβ42 concentrations were below 2% and 8.5% of the positive control values, respectively. The volume proportions of Aβ deposits in the subiculum and in the mamillary body were positively correlated (Spearman test, ρS = 0.720, p < 0.0001). Aβ deposits were observed in the subiculum in 27 cases, in the absence of deposit in the mamillary body in two cases, while Aβ deposits in the mamillary body (25 cases) were systematically associated with Aβ deposits in the subiculum (Fig. 2g and2i; supplementary Fig. 1, online resource). Lewy pathology did not modify Aβ accumulation (Mann-Whitney test, p > 0.05).

Hp-Tau accumulation in the subiculo-fornico-mamillary system

Pre-tangles, neurofibrillary and ghost tangles were observed in the subiculum and mamillary body (Fig. 2d and2f). Only axonal threads, made of thin and fragmented AT8 positive axons, were found in the PoF (Fig. 2e). Three groups of cases (control, intermediate, severe) were then considered according to Braak neurofibrillary stages (Fig. 2j). In the eight control cases (Braak 0, I, II), no Tau AT8 immunoreactivity was, by definition, observed in the subiculum; Tau positive accumulation was also absent in the PoF and in the mamillary body. In the 20 intermediate cases (Braak III, IV), hp-Tau accumulations were detected in the subiculum and PoF but were scarce in the mamillary body. In the 19 severe cases (Braak V, VI), they were abundant in the subiculum, PoF and to a lesser extent, in the mamillary body. Hp-Tau accumulation in the PoF was correlated both with hp-Tau accumulation in the subiculum, its area of origin (Spearman test, ρS = 0.885, p < 0.0001) and in the mamillary body, its target area (Spearman test, ρS = 0.724, p < 0.0001). The cases with hp-Tau accumulation in the subiculum, PoF, and mamillary body could be hierarchically ranked. Tangles in the subiculum were associated with axonal threads in the PoF in all but one case. Axonal threads in the PoF were systematically associated with tangles in the subiculum. Similarly, axonal threads in the PoF were observed in the absence of tangles in the mamillary body, while tangles in the mamillary body were always associated with axonal threads in the PoF (Fig. 2h; supplementary Fig. 2, online resource). Lewy pathology had no influence on the abundance and topography of hp-Tau accumulation (Mann-Whitney test, p > 0.05).

Topography of Aβ deposits and hp-Tau accumulation in the mamillary body

Aß distributed peripherally, centrally or both peripherally and centrally in the mamillary body. The peripherally located deposits occupied a circular zone at the border of the medial nucleus of the mamillary body; they were stellate or irregular and diffuse. The central deposits were spherical, diffuse or focal (Fig. 3a, 3c and3e). Most of the peripheral distributions were observed in the least affected cases (5 cases at Braak < 5 and Thal < 4 for a total of 6 peripheral distributions). The central, or central and peripheral distributions, were observed in the most advanced cases (14 cases among 19 with those distributions).

The topography of intraneuronal hp-Tau aggregation in the medial nucleus of the mamillary body started and predominated laterally close to the arrival of the PoF. The severity of the involvement of the lateral nucleus was in striking contrast with the relatively moderate changes observed in the medial nucleus (Fig. 3b,3d and 3f). To illustrate the heterogeneity of distribution of Aβ accumulation and hp-Tau positive pre-NFTs and NFTs, we drew density maps in six cases at various degrees of severity (Fig. 4).

Seeding properties of axonal Tau species in the PoF

The distribution of Tau pathology in the subiculo-fornico-mamillary system suggested an axonal progression of the pathology from the subiculum to the mamillary bodies through the PoF. To ascertain that possibility, we determined wether the AT8 positive accumulation observed in the PoF had seeding properties in Tau P301S biosensor HEK cells, co-expressing Tau P301S-CFP (cyan fluorescent protein) and Tau P301S-YFP (yellow fluorescent protein). PoF homogenates, containing AT8 positive axons (i.e. enriched in hp-Tau), indeed induced the development of numerous CFP positive inclusions of Tau P301S in the biosensor cells (Fig. 5b,5c and 5d), contrarily to the homogenate obtained from a control PoF, devoid of any hp-Tau AT8 positive accumulations (Fig. 5a and5d). CFP and YFP signals were colocalized.

Characterization of PoF Tau molecular species, associated with a seeding activity Phosphorylation and conformation features of Tau axonal threads in the PoF

To identify the molecular Tau species associated with the PoF seeding properties, TG3, PHF1, Alz50, C3 Tau immunohistochemistry and Gallyas silver impregnation were performed in a subset of 19 cases comprising four control cases (Braak 0, I, II), eight intermediate AD cases (Braak III, IV) and seven advanced AD cases at Braak VI (Fig. 6a to 6d). Tau axonal pathology observed in intermediate cases (Braak III-IV) was associated only with AT8, TG3 and to a lesser extent PHF1 positive axonal threads. A few Alz50 and C3 positive dots were additionally observed in the severe cases (respectively 6 and 5 cases among 7 severe cases).

Only two severe cases had a few Gallyas positive threads (Fig. 7). Since the seeding activity was detected even in the case of intermediate severity, in which axons of the PoF were found to be only AT8, TG3 and PHF1 positive, we concluded that the seeding activity could still be present in the absence of misfolded, fibrillary or truncated Tau (Alz50, Gallyas and C3 negative).

Electron microscopy analysis of axonal Tau accumulations

Paired helical filaments (PHFs) were frequently observed in the subiculum at electron microscopy, but not within the axons of the PoF derived from the same AD case (Fig. 8a and8b). Tau accumulation in the PoF was located in the axoplasm and did not involve the myelin sheath. DAB electron-dense precipitates did not permit to identify cytoskeleton structures or specific transport organelles in the PoF (Fig. 8b). We concluded that, as already shown by immunohistochemistry and histology, Tau accumulation in the PoF was generally made of non-fibrillary hp-Tau. Tau species found in the axons of the PoF, despite the absence of fibrillary forms of the molecule or of PHF, had a seeding activity in the HEK biosensor cells.

the PoF, principally issued from the subicular neurons, were, by contrast, preferentially immunolabeled by AT8 and TG3 antibodies, less extensively by PHF1 antibody, and were devoid of any PHF except in two severe cases.

We were able to show that these non-fibrillary molecular species had a seeding activity on the Tau P301S HEK biosensor cells. The seeding activity was independent from N-truncated Tau (no C3 positive threads), aggregated Tau (no Gallyas positive threads) or PHF (no PHF at electron microscopy) and was associated with a high concentration of Tau phosphorylated at the specific sites Ser202/Thr205 and Thr231, recognized by AT8 and TG3 antibodies. This suggested that the axons of the PoF, containing but a subset of the Tau species detected in the cell body of the subicular neurons, could transmit pathology to the neurons of the mamillary body that secondarily acquired the same, and complete, immunolabeling and staining profile as those of the subiculum. We could not find any influence of α-synuclein pathology on Tau aggregation and Aβ deposition in the subiculofornico-mamillary system.

The NFT seen in the subiculum preceded the appearance of threads in the PoF. The presence of AT8 positive threads in the PoF has already been described [START_REF] Plowey | Hippocampal phospho-tau/MAPT neuropathology in the fornix in Alzheimer disease: an immunohistochemical autopsy study[END_REF]. The authors also mentioned AT8 positive astrocytes in the mamillary body. We considered, as they did, that these were changes characteristic of Aging Related Tau Astrogliopathy (ARTAG). We completed the study of Plowey and Ziskin [START_REF] Plowey | Hippocampal phospho-tau/MAPT neuropathology in the fornix in Alzheimer disease: an immunohistochemical autopsy study[END_REF] by studying the PoF tangentially to its direction: this procedure allowed us to have a better view on the Tau positive threads. As mentioned, we also characterized the immunological profile of the threads and showed that they had a seeding activitydespite the absence of PHF demonstrated by electron microscopy.

Tau hyperphosphorylation has been shown to strongly contribute to the seeding efficiency and propagation capacity of Tau [START_REF] Hu | Hyperphosphorylation determines both the spread and the morphology of tau pathology[END_REF]. Recent evidence, however, indicates that the conformation of Tau protein, independently of its phosphorylation state, is crucial [START_REF] Mirbaha | Inert and seedcompetent tau monomers suggest structural origins of aggregation[END_REF]. The abnormal conformations of Tau in the PHF of AD [START_REF] Falcon | Tau filaments from multiple cases of sporadic and inherited Alzheimer's disease adopt a common fold[END_REF][START_REF] Fitzpatrick | Cryo-EM structures of tau filaments from Alzheimer's disease[END_REF] and of chronic traumatic encephalopathy [START_REF] Falcon | Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules[END_REF], and in Pick's bodies [START_REF] Falcon | Structures of filaments from Pick's disease reveal a novel tau protein fold[END_REF] have been described at the molecular level with cryo-EM, but PHF could be a final change that occurs late, while the seeding properties may be present early in the course of Tau alteration. It has been shown that misfolded monomers actually play an essential role in the seeding of Tau pathology [START_REF] Mirbaha | Inert and seedcompetent tau monomers suggest structural origins of aggregation[END_REF][START_REF] Sharma | Tau monomer encodes strains[END_REF], but their conformation has not been elucidated. It could be similar or different from the conformation described in PHF. The sequence (VQIINK/VQIVYK) that is exposed in the seeding monomers and is thought important for their aggregation [START_REF] Von Bergen | Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming beta structure[END_REF][START_REF] Mirbaha | Inert and seedcompetent tau monomers suggest structural origins of aggregation[END_REF] could be engaged in the fibrillation, after the initial seeding.

In all the cases with accumulation of Aβ in the mamillary body, Aβ had also accumulated in the subiculum while two cases with Aβ accumulation in the subiculum had none in the mamillary body. This regional distribution is compatible with a transmission of Aβ pathology from the subiculum to the mamillary body (as already suggested by Thal et al. [START_REF] Thal | Phases of Aβ-deposition in the human brain and its relevance for the development of AD[END_REF], see their Fig. 6). However, we could not find any histological or biochemical evidence of a transport of Aβ through the PoF. This negative result does not preclude the possibility of a transfer of soluble species, such as oligomers. As far as we know, there is currently no biosensor yet available to detect Aβ aggregation seeding activity. It has been repeatedly emphasized that the progression of Aβ pathology through the brain was not only related to connections but to other possible pathways such as diffusion through extracellular space or through basal membrane [START_REF] Eisele | Propagation of Aß pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies[END_REF]: in the human, Tau pathology was absent fromand Aβ accumulated ina disconnected piece of cortex in a patient with AD [START_REF] Duyckaerts | Dissociation of Alzheimer type pathology in a disconnected piece of cortex[END_REF]; transmission of Aβ pathology may occur through contaminated dural graft [START_REF] Hervé | Fatal Aβ cerebral amyloid angiopathy 4 decades after a dural graft at the age of 2 years[END_REF][START_REF] Kovacs | Dura mater is a potential source of Aβ seeds[END_REF] or injection of contaminated cadaver derived growth hormone [START_REF] Cali | Iatrogenic Creutzfeldt-Jakob disease with Amyloid-β pathology: an international study[END_REF][START_REF] Duyckaerts | Neuropathology of iatrogenic Creutzfeldt-Jakob disease and immunoassay of French cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of Aβ pathology[END_REF][START_REF] Jaunmuktane | Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy[END_REF][START_REF] Jaunmuktane | Evidence of amyloid-β cerebral amyloid angiopathy transmission through neurosurgery[END_REF][START_REF] Ritchie | Amyloid-β accumulation in the CNS in human growth hormone recipients in the UK[END_REF]. In transgenic mice, Aβ deposits may develop in the brain after intraperitoneal injection of Aß-containing brain extracts [START_REF] Eisele | Peripherally applied Aβ-containing inoculates induce cerebral β-amyloidosis[END_REF]. Because the mamillary body is not contiguous with the subiculum, passive diffusion through the subiculo-mamillary system is less probable than axonal transport through the PoF.

We attempted to find a link between Tau aggregation and Aβ accumulation in the subiculo-fornicomamillary system. Although there was indeed a strong correlation between the volume densities of Aβ deposits and the semi-quantitative score of Tau in the subiculum, fornix and mamillary body, we were unable to demonstrate a hierarchical order that would suggest a cause-and-effect relationship: in three cases, the presence of Tau NFTs in the mamillary body was not associated with Aβ deposits; in 5 cases in which Aβ deposits were seen, there was no Tau aggregates in the mamillary body. A cause-and-effect relationship between Aβ accumulation and Tau aggregates has been sought for decades [START_REF] Braak | Amyloid-β may be released from non-junctional varicosities of axons generated from abnormal tau-containing brainstem nuclei in sporadic Alzheimer's disease: a hypothesis[END_REF][START_REF] Mann | Amyloid or tau: the chicken or the egg?[END_REF]: in the cascade hypothesis Aβ accumulation causes Tau aggregates [START_REF] Hardy | Alzheimer's disease : the amyloid cascade hypothesis[END_REF]. It should, however, be emphasized that Tau aggregation has never been observed in APP or APPxPS1 transgenic mice even in animals expressing human Tau. In the opposite scenario, only tangle-bearing neurons are able to produce fibrillary Aβ [START_REF] Braak | Amyloid-β may be released from non-junctional varicosities of axons generated from abnormal tau-containing brainstem nuclei in sporadic Alzheimer's disease: a hypothesis[END_REF] but aggregation of abnormal Tau, sharing the immunohistochemical properties of AD Tau, is not always associated with Aβ accumulation [START_REF] Braak | Frequency of stages of Alzheimer-related lesions in different age categories[END_REF], see figure in [START_REF] Duyckaerts | Prevalence, incidence and duration of Braak's stages in the general population: can we know?[END_REF]. A third scenario remains to be discussed: the relationship between Aβ deposition and Tau aggregation could be a promotion of the second by the first rather than a link of cause and effect. Aβ deposition, indeed, promotes Tau aggregation and diffusion [START_REF] He | Amyloid-β plaques enhance Alzheimer's brain tau-seeded pathologies by facilitating neuritic plaque tau aggregation[END_REF][START_REF] Saito | Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation[END_REF][START_REF] Vergara | Amyloid-β pathology enhances pathological fibrillary tau seeding induced by Alzheimer PHF in vivo[END_REF], possibly through an interaction with the prion protein [START_REF] Gomes | Aβinduced acceleration of Alzheimer-related τ-pathology spreading and its association with prion protein[END_REF], but, by contrast, Tau aggregation does not promote Aβ deposition [START_REF] Héraud | Increased misfolding and truncation of tau in APP/PS1/tau transgenic mice compared to mutant tau mice[END_REF][START_REF] Lewis | Enhanced neurofibrillary degeneration in transgenic mice expressing mutant Tau and APP[END_REF][START_REF] Pooler | Amyloid accelerates tau propagation and toxicity in a model of early Alzheimer's disease[END_REF]. In the subiculofornico-mamillary system, the distribution of the lesions strongly suggests that pathology is initiated by Tau aggregation in the subiculum. Tau aggregation appears to follow the anterograde direction, affecting first the PoF and reaching secondarily the mamillary body. It is correlated with Aβ accumulation, but this correlation is not a consequence of a cause-and-effect relationship: there is at least one case of Aβ accumulation that is not associated with Tau pathology and there are several cases of Tau aggregation without Aβ accumulation. In agreement with this absence of causality, experimental evidence suggests that the correlation is rather due to a promotion of Tau aggregation by Aβ: when Aβ accumulation, independently initiated, keeps progressing, it tends to promote Tau pathology. The simultaneous increase of Tau and Aβ pathology explains the positive correlation. The interaction between Tau aggregation and Aβ accumulation probably takes place in the senile plaque: microcryodissection and CLARITY indeed show a physical interaction of both pathologies at that level.

Acknowledgements. This project received financial support from France Alzheimer (project "Propagation of Tau and Aβ aggregation through axons in the subiculo-fornico-mamillary system: tracking propagons" and Ibisa (Project "Neuro-CEB better"). Manon Thierry was funded by a doctoral scholarship from the Ministry of Research and Higher Education, attributed by the doctoral school "Physiology, Physiopathology and Therapeutics -ED394" (Sorbonne University). The human samples were obtained from the Neuro-CEB brain bank (BRIF number 0033-00011), partly funded by the patients' associations ARSEP, ARSLA, "Connaître les Syndromes Cérébelleux", France-DFT, France Parkinson, and by Vaincre Alzheimer Fondation, to which we express our gratitude. We are also grateful to the patients and their families. We thank Sabrina Leclère-Turbant and Marie-Claire Artaud for their technical and managerial assistance with post-mortem human samples. The brain bank is hosted by the Biological Resources Platform of Pitié-Salpêtrière Hospital, APHP. Brain clarification with CLARITY was performed on the ICM HISTOMICS platform. Cell culture experiments were performed on the ICM CELIS platform. Biphoton, Confocal and electronic microscopy pictures were acquired on the ICM.quant imaging platform. We thank the technical team of the Raymond Escourolle neuropathology department, as well as the technical staff involved at ICM, including Valérie Thuries and Annick Prigent for their help and technical advices in histology, Julien Dumont for his assistance and expertise in CLARITYcleared tissue imaging and analysis, and Dominique Langui and Asha Baskaran for their help and knowledge in electron microscopy analysis. We also thank Jean-Pierre Brion and Kunie Ando for their valuable advices and discussion. density maps obtained with a Voronoï (or "Dirichlet") tessellation [START_REF] Duyckaerts | Evaluation of neuronal numerical density by Dirichlet tessellation[END_REF]. Polygons cover the mamillary body without interstices or overlap. Each polygon contains only one dot (each dot corresponding to a plotted lesion) and is limited by borders located at mid-distance between the enclosed dot and its neighbours. Each point of the plane enclosed in a polygon is closer to the dot it contains than to any other dots of the map. The numerical density is inversely proportional to the polygon area (polygons are large when the lesions are wide part and small when they are close to each other) Aβ (row 2) or Tau (row 4) lesions. Color scales: local density scales in unit/mm². Note that in case 40 the distribution of Aβ deposits was mainly central while it was peripheral and central in cases 43 and 47. The numerical density of the centrally located deposits was lower (in blue) than the density of peripherally distributed ones (red).

  as a function of Braak stage, in the subiculum, pillar of the fornix and mamillary body (Kruskal-Wallis and Dunn post-test in comparison with the control group which included cases at Braak stages 0/I/II: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001). NFTs: neurofibrillary tangles, ATs: axonal threads. Error bars: Standard error of the mean.

Fig. 3 e

 3 Fig. 3 Aβ extracellular deposits and hp-Tau intraneuronal accumulation in the mamillary body. a Aß deposits in the mamillary bodies, anti-Aß 6F3D immunohistochemistry (scale bar: 400 µm). b Tau pathology (pre-tangles, tangles and threads) in the mamillary body after AT8 anti-hp-Tau immunohistochemistry (scale bar: 400 µm). c, e Higher magnifications of the rectangles c and e showing a peripheral stellate deposit (c) and a central neuritic deposit (e). Scale bars: 20 µm. d, f Higher magnifications of the rectangles d and f showing axonal threads at the periphery of the mamillary body, where the PoF ends (d) and neurofibrillary tangles (f). Scale bars: 20 µm.

Fig. 4

 4 Fig. 4 Density maps of Aβ extracellular deposits and hp-Tau intraneuronal accumulation in the mamillary body. The maps were obtained from five post-mortem cases (listed in columns), corresponding to increased severities of Aβ and Tau pathologies in the mamillary body. Rows 1 and 3: maps in which the position of Aβ 6F3D positive extracellular deposits (row 1) or hp-Tau AT8 positive pre-NFTs, NFTs and ghost NFTs (row 3) is indicated by a dot. Black: medial nucleus (M) and grey: lateral nucleus (L) of the mamillary body. Rows 2 and 4:

Fig. 5

 5 Fig. 5 Homogenates of the pillar of the fornix sampled from Alzheimer brains can act as seeds and induce Tau aggregation in vitro, in biosensor cells sensitive to Tau seeding. a-c Homogenates of the pillar of the fornix (10% in PBS, 3000g, 5 min) were obtained from frozen unfixed samples derived from a control case (a, case 49, Braak 0), a case of intermediate severity (b, case 50, Braak IV) and a severe Alzheimer case (c, case 53, Braak VI). HEK cells developed Tau P301S-CFP positive inclusions (arrowheads) when treated with homogenates from cases 50 and 53 containing Tau pathology, but not when treated with the homogenate from the control case (case 49). Representative confocal images with maximal CFP and DAPI intensity projections from a stack of 59 sections (scale bar: 20 µm). d Mean percentage of HEK cells containing intracellular Tau P301S-CFP inclusions in each group, obtained after three repeated independent experiments (Student test, performed after arcsine square root transformation -applied to proportions in order to meet more closely a Gaussian distribution, n. s. : non-significant, * p < 0.05). Error bars: Standard error of the mean.

Fig. 6

 6 Fig. 6 Non-fibrillary hyperphosphorylated-Tau is found in the axons of the pillar of the fornix. a Tau pathology observed in the subiculum, pillar of the fornix and mamillary body (in rows) after immunohistochemistry (IHC) against phosphorylation (AT8, TG3, PHF1), conformation (Alz50) or truncation (C3) dependent epitopes and Gallyas silver technique staining fibrillary Tau, shown in columns (scale bar: 20 µm). b-d Mean numerical density (semi-quantitative score) of Tau alterations assessed in a subset of 19 cases at various Braak stages after AT8, TG3, PHF1, C3, Alz50 IHC or Gallyas silver stain in the subiculum (b), pillar of the fornix (c), mamillary body (d). Friedman and Dunn post-test in comparison with the values obtained after AT8 IHC, considered as the reference group (AT8): * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars: Standard error of the mean.

Fig. 7

 7 Fig. 7 Labeling profile of Tau axonal pathology in the pillar of the fornix. A subset of 19 cases at various Braak stages is listed according to the absence (-, light grey) or presence (+, dark grey) of Tau pathology in the pillar of the fornix, detected with AT8, TG3, PHF1, Alz50, C3 immunochemistry and Gallyas silver impregnation. Alz50 and C3 immunostained mainly small dots. The presence of rare dot-like immunoreactive material is indicated by "+/-". The control group refers to Braak stages 0, I or II, the intermediate group, to Braak stages III or IV, and the severe group to Braak stages V or VI.

Fig. 8

 8 Fig. 8 Intra-axonal aggregates of hyperphosphorylated-Tau, devoid of PHF, in the pillar of the fornix. a-b Diaminobenzidine electron-dense precipitates observed at electron microscopy after AT8 anti-hp-Tau immunohistochemistry in the subiculum (a) or in the pillar of the fornix (b) of an Alzheimer case (Braak VI, Thal 5; scale bar: 500 nm). a AT8 labelled PHFs located in the perikaryon of a neuron in the subiculum (arrowheads). n: nucleus. b hp-Tau AT8 positive staining (arrows) in an axon of the pillar of the fornix, devoid of any PHF. AT8 immunoreactivity was located in the axoplasm (solid line) and absent in the myelin sheath (dashed line). We could not identify cytoskeleton components or organelles associated with hp-Tau immunoreactivity.

Fig. 9

 9 Fig.[START_REF] Calafate | Synaptic contacts enhance cell-to-cell tau pathology propagation[END_REF] No relationship could be observed between Tau and Aβ pathology distribution. Cases listed according to the absence (-, light grey) or presence (+, dark grey) of Tau or Aβ pathology in the subiculum (Sub-Tau or Sub-Aβ), pillar of the fornix (PoF-Tau or PoF-Aβ) and mamillary body (MB-Tau or MB-Aβ). NB: Six mamillary bodies could not be sampled; the six corresponding cases were excluded from the analysis of the hierarchical distribution (in grey, end of the table).

Fig. 10 a

 10 Fig. 10 Hyperphosphorylated-Tau positive axonal threads are in contact with Aß deposits in the mamillary body. a Coronal section of the mamillary body after microcryodissection. Preserved aspect of the microcryodissected section at low magnification. Anti-MBP/NF immunohistochemistry (myelin basic protein -MBPin brown, neurofilament -NFin red). PoF: end of the afferent pillar of the fornix. MT: origin of the efferent mamillothalamic tract (scale bar: 400 µm). b Isolated axons of the pillar of the fornix filled with hp-Tau AT8 positive material (arrowheads), observed in the mamillary body after microcryodissection. Anti-hp-Tau AT8 immunohistochemistry (scale bar: 8 µm). c Microcryodissected Aß focal deposit in contact with a hp-Tau AT8 positive axonal thread (arrowhead) in the mamillary body. Anti-Aβ 6F3D/hp-Tau AT8 immunohistochemistry (Aβ in brown, Tau in red, scale bar: 8 µm). ac: amyloid core.

Table 1 : Cases Neuropathological analysis (n = 47)

 1

	Case	Braak (Tau)	Thal	Braak (α-Synuclein)	Age Gender	Diagnosis
	01	0	0	0	51	M	Coronary atheroma and fungal pneumopathy.
	02	0	1	0	66	M	Acute myeloblastic leukaemia.
	03	I	0	0	65	M	Brain donation. Some ischemic neurons.
	04	I	1	0	74	F	Ischemic lesions.
	05	II	3	0	83	M	Large T-cell lymphoma.
	06	II	0	0	62	M	Ischemic cardiopathy, infarcts in contralateral hemisphere.
	07	II	1	0	81	M	Brain donation. Control.
	08	II	1	5	67	M	Parkinson disease. Cortical Lewy-bodies.
	09	III	2	0	85	M	Perirenal angiosarcoma with pulmonary metastases.
	10	III	4	0	73	F	Peritonitis and acute bronchopneumopathy.
	11	III	0	0	75	M	Cardio-respiratory arrest after parotidectomy. Some dilations of the perivascular space in the basal ganglia.
	12	III	4	5	84	M	Diffuse Lewy-body disease. High probability*.
	13	III	0	0	71	M	Peritonitis.
	14	III	0	0	54	F	Cirrhosis, and pulmonary infarction.
	15	III	0	0	92	F	Brain donation. Control.
	16	III	0	5	60	F	Parkinson disease. Cortical Lewy-bodies.
	17	III	3	6	84	M	Diffuse Lewy-body disease. High probability*.
	18	III	0	0	66	M	Cirrhosis.
	19	III	0	0	70	F	Brain donation. Minimal neuropathological changes. Type 2 CAA (meninges).
	20	III	0	0	53	M	Sudden death.
	21	IV	2	0	86	M	Brain donation. Myocardial infarct. Type 1 CAA.
	22	IV	3	0	85	F	Lacune of the pons. Sclerohyalinosis.
	23	IV	3	0	71	M	Acute pancreatitis and cirrhosis.
	24	IV	4	0	74	M	Metastatic adenocarcinoma.
	25	IV	1	6	85	M	Parkinson disease. Cortical Lewy-bodies.
	26	IV	3	6	81	M	Parkinson disease. Cortical Lewy bodies, argyrophilic grain disease, lacune of the pallidum, old occipital infarct.
	27	IV	3	6	84	M	Diffuse Lewy-body disease, High probability*. CAA (type 2).
	28	IV	0	0	90	F	Non-demented individual with tau AD-related lesions.
	29	V	4	0	76	M	AD.
	30	V	5	6	81	M	AD, diffuse Lewy-body disease. Low probability*.
	31	V	4	6	76	F	AD, diffuse Lewy-body disease. Intermediate probability*.
	32	V	5	0	92	M	AD, Ammon's horn sclerosis and TDP-43 inclusions.
	33	V	4	6	87	M	AD, diffuse Lewy-body disease. Intermediate probability*.
	34	V	4	0	81	F	AD.
	35	V	4	Amygdala only	96	F	AD, amygdala Lewy bodies.
	36	V	5	6	80	M	AD, diffuse Lewy-body disease. Intermediate probability*.
	37	V	5	6	74	F	AD, diffuse Lewy-body disease. Intermediate probability*.
	38	VI	5	0	78	F	AD.
	39	VI	3	0	86	M	AD.
	40	VI	2	0	79	M	AD.
	41	VI	5	Amygdala only	73	F	AD, amygdala Lewy bodies.
	42	VI	5	0	68	F	Posterior cortical atrophy. AD.
	43	VI	5	0	79	M	AD.
	44	VI	5	0	60	M	AD.
	45	VI	5	0	66	M	AD.
	46	VI	4	0	79	M	AD.
	47	VI	3	0	65	M	AD.

table 1

 1

	Additional analyses (n = 6)
	Case	Braak (Tau)	Thal	Braak (α-Synuclein)	Age Gender	Diagnosis	Technique(s)
	48	0	0	0	62	F	Multi-organ dysfunction syndrome.	Electron microscopy.
	49	0	0	0	39	M	Brainstem glioma (samples were taken from uninvolved pillars of the fornix).	Cell culture.
	50	IV	5	0	91	M	AD.	Cell culture, electron microscopy.
	51	VI	5	Amygdala only	61	F	AD, amygdala Lewy bodies.	Microcryodissection.
	52	VI	5	0	81	F	AD.	Clarity, electron microscopy.
	53	VI	5	0	76	M	AD.	Cell culture.

Relationship between Tau and Aβ pathology No constant hierarchical order between Tau and Aβ pathology

We had evidence of a propagation of hp-Tau pathology in the subiculo-fornico-mamillary system through axons. This evidence was lacking for Aβ. We wondered if we could find a hierarchical relationship of Tau and Aβ pathology among the cases. We could not find, however, a constant hierarchical order linking directly axonal threads or NFTs on the one hand, and Aβ deposits on the other (Fig. 9). Yet, cross-correlation analysis of the densities of Tau and Aβ pathologies in the subiculo-fornico-mamillary circuit showed that the strongest correlations involved the numerical density of NFTs in the subiculum (or axonal threads in the PoF) and the volume of Aβ deposition in the mamillary body (supplementary Fig. 3, online resource, for detailed statistical analysis):

-NFTs in the subiculum vs. Aβ deposits in the mamillary body (Spearman test, ρS = 0.721, p < 0.0001) -Axonal threads in the PoF vs. Aβ deposits in the mamillary body (Spearman test, ρS = 0.718, p < 0.0001).

Ten of the 16 cases without Aβ deposit in the mamillary body had axonal threads in the PoF, while only one of the twenty-five cases with Aβ deposits in the mamillary body had no axonal threads in the PoF (case 05; Fig. 9). In that case, a few threads were seen in the mamillary body, in an area encompassing the border with the hypothalamus.

Anatomical relationships

Microcryodissection could dissociate in situ axons originating from the PoF and entering the mamillary body (Fig. 10a). Some of those dissociated axonal endings were filled with Tau AT8 positive material (Fig. 10b).

Microcryodissected Aß focal deposits were observed in contact with Tau AT8 positive axonal threads in the area of the mamillary body where fibers diverge from the bundle of the PoF (Fig. 10c).

CLARITY gave a 3-D view of the various types of Aβ deposits observed in the mamillary body. They were either diffuse (Video 1a, online resource) or cored and sometimes neuritic (Video 1b, online resource). Tau positive neurites were seen entangled into Aβ diffuse deposits but repelled from the amyloid core. They were sometimes in contact with Aβ immunoreactive material. Aβ deposits were observed in the lateral and peripheral parts of the mamillary body, in the PoF terminal fields (Video 2, online resource).

Discussion

We have shown that pathological Tau found in the axons of the PoF, in the human subiculo-fornicomamillary system, has a seeding capacitya result that supports previous observations in cell and animal models, in which Tau pathology is transmitted from cell to cell through neuronal connections [START_REF] Calafate | Synaptic contacts enhance cell-to-cell tau pathology propagation[END_REF][START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF][START_REF] Guo | Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice[END_REF][START_REF] Iba | Tau pathology spread in PS19 tau transgenic mice following locus coeruleus (LC) injections of synthetic tau fibrils is determined by the LC's afferent and efferent connections[END_REF][START_REF] Takeda | Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer's disease brain[END_REF][START_REF] Tardivel | Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies[END_REF][START_REF] Wang | The release and trans-synaptic transmission of Tau via exosomes[END_REF]. The study of Tau pathology in the subiculum, in the PoF, one of its major efferent tracts, and in the mamillary body, its main projection area, suggested that Tau pathology progressed in an anterograde direction.

Tau aggregates in the cell body of the subicular neurons were immunolabelled by antibodies against hp-Tau (AT8, TG3 and PHF1), misfolded Tau (Alz50) and N-truncated Tau (C3), and silver stained with Gallyas method, which is thought to stain only fibrillary Tau structures. The PHF nature of the aggregates was confirmed by electron microscopy. The PoF was cut longitudinally to facilitate the analysis of the fiber tract. The axons of

Legends of the table 1 and figures

Table 1 Cases. The cases are sorted according to Braak neurofibrillary stages [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF]. "Braak (Tau)" stands for Braak & Braak neurofibrillary stages [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF]; "Thal" refers to Thal phases [START_REF] Thal | Phases of Aβ-deposition in the human brain and its relevance for the development of AD[END_REF] and "Braak (α-Synuclein)" indicates Braak et al. staging of synucleinopathy, related to sporadic Parkinson's disease [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]. The hippocampus and the pillar of the fornix were sampled in all 47 cases. The hippocampus was studied in slices cut perpendicularly to its long axis. The fornix was cut parallel to its main axis. The mamillary body was studied in 41 cases (in 6 cases, the mamillary body was not available or too altered to be analyzed). Six additional cases were sampled for microcryodissection and CLARITY techniques (two formalin-fixed mamillary bodies), electron microscopy analysis (three formalin-fixed transversal sections of hippocampus and three formalin-fixed longitudinal sections of the pillar of the fornix) and cell culture (three frozen pillars of the fornix).

AD: Alzheimer's disease; CAA: Cerebral Amyloid Angiopathy; PD: Parkinson's disease. *The probability refers to the "assessment of the likelihood that the pathologic findings are associated with a typical, dementia with Lewy bodies, clinical syndrome", from McKeith et al. [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies[END_REF].


Luc Person

Brigitte Lacour

Laure Faure

Sandra Guissou

Claire Poulalhon

Daniel Orbach

Stéphanie Goujon

Claire Berger

Jacqueline Clavel

MD Emmanuel Desandes
email: emmanuel.desandes@univ-lorraine.fr

Childhood head and neck cancer in France: incidence, survival and trends from 2000 to 2015

Keywords: Childhood, Head and neck cancer, Epidemiology, Incidence, Survival

Introduction: Childhood head and neck cancers (HNC) are rare and represent a complex group of anatomical topographies. The aim of this study is to describe the distribution, the incidence and survival rates of children with malignant HNC in France.

Methods: A population-based study was conducted between 2000 and 2015 in children less than 15 years with a diagnosis of HNC using the French National Registry of Childhood Cancers database (RNCE). Age-standardized incidence rates (ASR) and survival analysis were performed.

Results:

The 1623 included HNC represented 5.6% of all cancers included in the RNCE. The thyroid was the leading tumor site category (26.6%), followed by head and neck soft tissue location (15.4%) and the nasopharynx (10.8%). The most common cancers were thyroid gland carcinomas (26.1%), rhabdomyosarcomas (23.9%) and Burkitt Lymphomas (8.6%).

Nasopharynx cancers and soft-tissue sarcomas were statistically more frequent in boys, while thyroid carcinomas were significantly more frequent in girls. The annual ASR was 8.6 new cancer cases per million children. For all HNC combined, the 5-year overall survival (OS) was 87.7% [95%CI: 85.9-89.2]. There was no statistically significant variation in 5-year OS between 2000-2007 and 2008-2015. Conclusions: Epidemiological data on HNC distribution, incidence and survival contributes to better understand these tumors by quantifying their impact on the French population and assessing their burden. Regarding the exclusion of topographies and some histological origins performed by some authors, this report proposes new recommendations to study HNC in a pediatric population.

Introduction

Head and neck cancers (HNC) are rare among children under 15 years of age but some studies show an increasing incidence of pediatric HNC compared to all childhood cancer [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF][START_REF] Gosepath | Incidence of childhood cancer of the head and neck in Germany[END_REF]. HNC account for about 12-15% of all reported pediatric cancers [START_REF] Albright | Pediatric head and neck malignancies: US incidence and trends over 2 decades[END_REF][START_REF] Lilja-Fischer | Pediatric malignancies presenting in the head and neck[END_REF][START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF], because there are differing opinions among authors and clinicians as to what exactly constitutes HNC regarding both reported sites and histologies [START_REF] Albright | Pediatric head and neck malignancies: US incidence and trends over 2 decades[END_REF][START_REF] Lilja-Fischer | Pediatric malignancies presenting in the head and neck[END_REF][START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF][START_REF] Gosepath | Incidence of childhood cancer of the head and neck in Germany[END_REF]. Indeed, HNC represent a complex group of anatomical topographies which can be affected by a multitude of malignant tumors, the most frequent of which are lymphomas, rhabdomyosarcomas (RMS), thyroid carcinomas and nasopharyngeal carcinomas [START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF][START_REF] Lemelle | NUT carcinoma in children and adults: A multicenter retrospective study[END_REF][START_REF] Orbach | Nonparameningeal head and neck rhabdomyosarcoma in children and adolescents: Lessons from the consecutive International Society of Pediatric Oncology Malignant Mesenchymal Tumor studies[END_REF].

Overall, the 5-year survival in children with HNC remains high (around 80-90%) [START_REF] Gatta | Childhood cancer survival trends in Europe: a EUROCARE Working Group study[END_REF], but analyzes by topographic and/or histological subgroup reveal some disparities: children with malignant thyroid or salivary gland tumors have excellent survival, while those with high-risk squamous cell carcinomas or neuroblastomas have poor survival rate [START_REF] Das | Non-hematological tumors of head and neck region in the pediatric age group in a tertiary care cancer center[END_REF][START_REF] Rebours | Pediatric French Rare Tumor Group, Pediatric salivary gland carcinomas: Diagnostic and therapeutic management[END_REF].

The aim of the study is to describe the main characteristics of HNC, their distribution by topography and histology, and to provide national estimates of the incidence and survival rates of children with malignant HNC in the French population over the 2000-2015 period. Then incidence and survival trends over the study period are analyzed.

Material & methods

Population

Study data was obtained from the French National population-based Registry of Childhood Cancers (RNCE), including about 1800 cases per year [START_REF] Lacour | Incidence of childhood cancer in France: National Children Cancer Registries, 2000-2004[END_REF]. Children from 0 to 14 years of age living in mainland France with HNC diagnosed between January 1, 2000 and December 31, 2015 were identified. The diagnoses were coded using third edition of the International Classification of Disease for Oncology (ICDO-3) [START_REF] Frizt | International Classification of Diseases for Oncology[END_REF] and grouped according the third version of the International Classification of Childhood Cancer (ICCC-3) [START_REF] Steliarova-Foucher | International Classification of Childhood Cancer[END_REF]. The HNC were selected according to their specific ICDO-3 topographic codes divided in 13 groups regarding to tumor location (Table 1) [START_REF] Frizt | International Classification of Diseases for Oncology[END_REF]. Staging at diagnosis was defined as metastatic (M+) or non-metastatic (M0) for solid tumors and using Ann Arbor or St Jude/Murphy systems for lymphomas.

Only primary head and neck malignant tumors were included. The selected sites are listed in Table 1, so all malignant tumors with any of these topographic ICDO-3 codes were included.

Nodal or extranodal lymphomas were included only if they were located in the head and neck area (Waldeyer's ring, tonsils, hypopharynx, nasopharynx, …). Retinoblastoma, central nervous system (CNS) tumors, and post-transplant lymphoproliferative diseases or Langerhans cell histiocytosis were excluded.

The RNCE provided information on child and cancer characteristics, vital status and last contact date. The methods used to record cases and ensure case completeness have been described previously [START_REF] Lacour | Incidence of childhood cancer in France: National Children Cancer Registries, 2000-2004[END_REF].

Data analysis

All data were retrieved, tabulated by tumor type, and categorized using the ICCC-3 classification [START_REF] Steliarova-Foucher | International Classification of Childhood Cancer[END_REF]. The male/female (M/F) ratio was the ratio of number of cases in males to that in females.

Age-standardized incidence rates (ASR) were calculated for the 16-year period, 2000-2015, based on the annual age-and sex-specific population estimates provided by the INSEE. ASR were estimated by the direct method using the world population age distribution. Methods based on Poisson distribution were used to specify the 95% confidence interval (95%CI) for the ASR [START_REF] Dobson | Confidence intervals for weighted sums of Poisson parameters[END_REF]. The conventional average annual percent change (AAPC) was considered to test for a time trend in ASR. AAPC was derived from the slope parameter estimate of a linear regression model considering the natural logarithm of the ASR as a function of the calendar year [START_REF] Howlader | SEER Cancer Statistics Review[END_REF].

Survival analysis was performed on a dataset containing all cases diagnosed between January 1, 2000 and December 31, 2015 and followed up until August 1, 2018. Median length of followup was calculated using the inverse Kaplan-Meier method [START_REF] Shuster | Median follow-up in clinical trials[END_REF]. The overall survival (OS) was estimated using the Kaplan-Meier method [START_REF] Kaplan | Nonparametric Estimation from Incomplete Observations[END_REF]. The end point of interest was death from any cause, with the date of diagnosis acting as the time origin. Survival curves between groups were compared using the log-rank test, and the trend in survival over time periods was estimated using the log-rank test for trend [START_REF] Mantel | Evaluation of survival data and two new rank order statistics arising in its consideration[END_REF][START_REF] Peto | Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. analysis and examples[END_REF]. For survival analysis conducted by ICCC-3 groups and subgroups [START_REF] Steliarova-Foucher | International Classification of Childhood Cancer[END_REF], and tumor stage, multiple HNC cases were counted once, considering the first HNC diagnosed. Multivariate statistical analyses were performed to examine the relationships between duration of survival and following variables: age groups (<1year, 1-4 years, 5-9 years, 10-14 years), periods of diagnosis (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015), metastatic disease (yes/no), and gender (boys, girls). The risks of death were estimated by hazard ratios (HR) and their 95% confidence intervals (95% CI) using Cox proportional hazards regression models [START_REF] Cox | Analysis of Survival Data[END_REF].

Statistical tests were two-sided with a significance level of 0.05. All statistical analyses were performed using the SAS version 9.4 (SAS Institute Inc., Cary, North Carolina, USA, 2013).

Results

Description of cases

Over the 2000-2015 period, 1623 cases of HNC were identified, i.e. 5.6% of all cancers included in the RNCE (28 998 malignancies). Four children were reported with multiple HNC (Table 2). Two of them had diagnosis of multiple cutaneous cancers in a context of xeroderma pigmentosum and two children had a post-radiation osteosarcoma after their first cancers. Table 3 describes the distribution of HNC by main histologic groups and anatomic sites. The thyroid was the leading tumor site category (26.6%), followed by head and neck soft tissue location (15.4%) and the nasopharynx (10.8%). The most common histologic groups were carcinomas (37.4%), especially affecting thyroid gland (26.1%), then rhabdomyosarcomas (23.9%), and Burkitt Lymphomas (8.6%), especially involving nasopharynx (6.8%). More boys were reported to have nasopharynx cancer (especially Burkitt lymphoma and undifferentiated carcinoma of nasopharyngeal type (UCNT)) or soft-tissue sarcomas as compared to girls with M/F ratios of 4.7 (p<0.01), 2.7 (p=0.01), and 1.4 (p=0.01), respectively. Conversely, thyroid carcinomas were significantly more frequent in girls (M/F ratio of 0.5, p<0.01). The mean age at diagnosis was 8.1 years (Standard deviation: 4.5). Neuroblastoma affected the youngest children (mean age: 1.5 years) and UCNT was found among the oldest (mean age: 12.4 years).

Incidence

The annual ASR over the 2000-2015 period was estimated to be 8.6 new HNC cases per million children [95%CI:7.8-9.5] (Table 4). Overall, ASRs varied from less than 1 per million for neuroblastoma and malignant bone tumors, to more than 3 per million for carcinoma and melanoma. HNC incidence was stable over time with an AAPC of 0.1% [95%CI: -1.1 % to 1.2 %, p-value = 0.94].

Survival

The median follow-up time in the cohort was 106 months (Interquartile Range: 55 -160 months), with 2.9% of cases lost to follow-up. 87.7% [95%CI: 85.9-89.2] at 1 year, 3 years and 5 years after diagnosis, respectively. The 5year OS>85% was observed for lymphomas, neuroblastomas, germ cells tumors (GCT), carcinomas and melanomas, whereas 5-year OS was around 65-75% for osteosarcomas and rhabdomyosarcomas. The 5-year OS was close to 29% for malignant rhabdoid tumors. There was no statistically significant variation in 5-year OS between 2000-2007 and 2008-2015 for all HNC combined and by diagnostic groups (Table 6).

Stage at diagnosis

Lymphomas

A total of 167 cases of head and neck lymphomas (66.8%) were stage I-II. The majority of Hodgkin lymphoma and mature-B cell lymphoma were stage I-II (90% and 74.2%, respectively), while high proportions of stage III-IV were observed for Burkitt lymphoma (38.5%) and precursor-cell lymphoma (32.3%). For all lymphomas, 5-year OS rates were 96.2% [CI95%:91.8-98.3] for stage I-II and 92.5% [CI95%:84.0-96.6] for stage III-IV (p =0.12) (Table 8A). There were no significant differences in survival by stage for all subgroup lymphomas.

Solid tumors

Metastatic diseases were observed for 137 of head and neck solid tumors (10.0%), with highest proportions for rhabdomyosarcomas and neuroblastomas (15.6% and 18.5%, respectively. Table 7B). For all solid tumors, 5-year OS rates were 88.7% [CI95%:86.7-90.4] for nonmetastatic diseases and 63.0% [CI95%:54.2-70.5] for metastatic stages (p < 0.01) (Table 8B).

There were statistically significant differences in survival by stage for all reported sub-groups of head and neck solid tumors. For metastatic diseases, the 5-year OS varied from 57.7% [CI95%: 45.3-68.2] for children with rhabdomyosarcoma to 91.5% [CI95%: 69.6-97.8] for those with thyroid carcinoma.

3.5.Multivariate analysis

For all HNC combined, children aged 1-4 years and with metastatic disease had a significant risk of death compared to those of another age group and with non-metastatic disease with hazard ratios of 2.2 (CI95%: 1.6-2.9) and 2.7 (CI95%: 2.0-3.6), respectively. In metastatic HNC, the proportion of children aged 1-4 years with soft tissue sarcoma, for which the lowest 5-year OS was observed (Table 8B), was higher compared to the other age group (<1 year: 3.8%, 1-4 years: 42.5%, 5-9 year: 31.2%, 10-14 year: 22.5%).

Discussion

This study describes the incidence and the overall survival of children with head and neck cancers in France during the 2000-2015 period, based on national population-based cancer registry data.

The study data displayed that 5.6% of all children with cancer in France were affected by HNC.

The HNC incidence was stable over the considered period, with an annual ASR of 8.6 cases per million children. The predominant malignant entities contributing to childhood HNC were thyroid carcinomas, rhabdomyosarcomas and Burkitt lymphomas. The leading tumor sites were thyroid gland, soft-tissue and nasopharynx. Highest proportions of girls were found for GCT and thyroid carcinoma. Differences in age groups were observed with GCT and neuroblastoma especially in youngest children and carcinomas among the oldest. A high proportion of children with cancer in this location survived with a 5-year OS of 87.7% [CI95%: 85.9-89.2].

The main strength of this study resides in the national registry-based, prospective cohort design.

The national coverage and the long study period enabled to estimate precisely incidence and survival rates, and trends over time, based on a large number of head and neck cancer cases.

The comparisons with other study results are however limited by the fact that no consensual definition of « head and neck cancer » exists to date. Previous studies had different inclusion criteria, especially in terms of histological categories and tumor sites [START_REF] Albright | Pediatric head and neck malignancies: US incidence and trends over 2 decades[END_REF][START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF][START_REF] Gosepath | Incidence of childhood cancer of the head and neck in Germany[END_REF][START_REF] Grønhøj | Incidence of head and neck cancer in children: A Danish nationwide study from 1978 to 2014[END_REF]. In accordance to global literature delineating common pediatric head and neck malignancies as lymphomas and all solid tumors [START_REF] Chadha | Pediatric head and neck malignancies[END_REF], the present study proposes a consensus to adopt standard recommendations in selection of head and neck cases, particularly in the exclusion of retinoblastoma (ICCC-3 group V) and CNS tumors (ICCC-3 group III) [START_REF] Steliarova-Foucher | International Classification of Childhood Cancer[END_REF], and in the inclusion of the lymphomas only if they involved head and neck area. Population-based registries are useful for determining cancer epidemiology, but the lack of some clinical data (granularities of histological subtypes, details on treatments, responses to each line of treatment) may make interpretation of their results difficult. In the future, the French Childhood Cancer Observation Platform (CCOP), a nationwide infrastructure based on the National population-based Registry of Childhood Cancers (RNCE) and medico-administrative databases will enable in-depth survival analyses by providing complementary information on specimens stored in the hospital biobanks and data on treatments, including doses of chemotherapy and radiotherapy [23].

Our proportions of head and neck Hodgkin lymphomas (HL) and Non-Hodgkin lymphomas (NHL) were lower than those observed in the United-States (US) [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF][START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF]: 1.2% versus 13.8% and 5.5% versus 10.0%, respectively. But neck and lymph nodes (not included in the present study) were the main anatomic topography in two American studies: 41% [START_REF] Albright | Pediatric head and neck malignancies: US incidence and trends over 2 decades[END_REF] and 61% [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF]. Conversely and surprisingly, our proportion of head and neck RMS was higher than those observed in the US [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF][START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF]: 23.9% versus 7.3%, respectively. Compared to 2 population-based registries in European countries (Germany and Denmark) [START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF], most proportions were similar, except for head and neck NHL in Germany (20.8% vs 5.5% in our study) [START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF][START_REF] Gosepath | Incidence of childhood cancer of the head and neck in Germany[END_REF]. The proportion of lymphomas may be under or overestimated, since studies excluded this malignancy totally or partially, because they were nonspecific HNC [START_REF] Arboleda | Global frequency and distribution of head and neck cancer in pediatrics, a systematic review[END_REF].

Incidence rates vary from 5.0 per million children in Denmark for 1978-2014 [START_REF] Grønhøj | Incidence of head and neck cancer in children: A Danish nationwide study from 1978 to 2014[END_REF], to 16.0 in the US for 2007-2009 [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF], depending on the inclusion and exclusion criteria. No time variation in HNC incidence was observed in the present study. In Denmark, Grønhøj et al [START_REF] Grønhøj | Incidence of head and neck cancer in children: A Danish nationwide study from 1978 to 2014[END_REF] reported a significant annual increase of 2.2% (95%CI, 0.8-3.7%) during 1978-2014 among children with HNC, explained by a rise mainly of sarcomas, where rhabdomyosarcomas accounted for 61.6%. A similar trend of increasing rates of pediatric HNC has been observed in the US between 1973 and 2010 [START_REF] Schwartz | Pediatric head and neck malignancies: incidence and trends, 1973-2010[END_REF], which is proportional to the overall cancer increase in children.

Sex and age variations in HNC are consistent compared to all childhood primary cancers in France [START_REF] Poulalhon | Factors associated with 5-and 10-year survival among a recent cohort of childhood cancer survivors (France, 2000-2015)[END_REF]: a female predominance is particularly seen for thyroid carcinomas and extracranial and extragonadal GCT, highest proportion of young children are found for neuroblastoma and extracranial and extragonadal GCT, and carcinomas occur most often in older children.

Considering the 5-year OS rates, our results were comparable to those reported in France during the 2000-2015 period for all childhood primary cancers [START_REF] Poulalhon | Factors associated with 5-and 10-year survival among a recent cohort of childhood cancer survivors (France, 2000-2015)[END_REF]. However, marked differences between head and neck location and all sites for neuroblastomas (5-year OS: 91.0% 5)).

The best survival observed in head and neck neuroblastomas can be explained by two factors: the slightly higher proportion of children aged less than one year (44.4% versus 38.5% for all neuroblastomas) [START_REF] Poulalhon | Factors associated with 5-and 10-year survival among a recent cohort of childhood cancer survivors (France, 2000-2015)[END_REF] and the lower proportion of metastatic stage (18.5%) while the metastatic stages in all neuroblastomas represent 39% in a recent European study [START_REF] Gatta | JARC Pilot Study Toronto Guidelines Working Group, Staging childhood cancers in Europe: Application of the Toronto stage principles for neuroblastoma and Wilms tumour. The JARC pilot study[END_REF]. Our results are in line with those reported by an American study which showed a higher disease-specific death risk for adrenal locations than for head and neck location (adjusted hazard ratio= 2.9; 95%CI, 1.5-5.3) [START_REF] Kaufmann | Factors Associated With the Improved Survival of Head and Neck Neuroblastomas Compared to Other Body Sites[END_REF]. Compared to non-head and neck sites, we reported higher 5-year OS for children with head and neck Ewing sarcoma. These survival disparities may be due to differences in tumor size or metastatic rate. Ellis et al [START_REF] Ellis | Ewing Sarcoma of the Head and Neck: Analysis of 183 Cases With Comparison to Non-Head and Neck Sites[END_REF] have reported that head and neck Ewing sarcomas were more commonly less than 5cm (61% vs 24%) and presented more commonly with local/regional disease (82% vs 66%) with impact on prognosis.

Although there are few studies analyzing survival in children with HNC, our 5-year survival rates were similar to those observed in Europe for thyroid cancer [START_REF] Gatta | Childhood cancer survival trends in Europe: a EUROCARE Working Group study[END_REF], in the US for head and neck neuroblastoma [START_REF] Kaufmann | Factors Associated With the Improved Survival of Head and Neck Neuroblastomas Compared to Other Body Sites[END_REF] and for head and neck melanoma [START_REF] Richards | Survival and Surgical Outcomes for Pediatric Head and Neck Melanoma[END_REF].

In our study, the overall survival among children with HNC incidence was stable over time.

Probably the number of our cohort prevents to depict some small outcome variations, as in pediatric nasopharyngeal carcinoma [START_REF] Jouin | Adapted strategy to tumor response in childhood nasopharyngeal carcinoma: the French experience, Strahlenther[END_REF]. It is already known that some tumors with intermediate survivals has not outcome improvement over time as osteosarcoma [START_REF] Gaspar | Recent advances in understanding osteosarcoma and emerging therapies[END_REF]. Some other studies have reported a stable outcome in large other series as RMS in favorable [START_REF] Glosli | EpSSG members, Non-parameningeal head and neck rhabdomyosarcoma in children, adolescents, and young adults: Experience of the European paediatric Soft tissue sarcoma Study Group (EpSSG) -RMS2005 study[END_REF] or unfavorable head and neck sites [START_REF] Darwish | Pediatric head and neck rhabdomyosarcoma: An analysis of treatment and survival in the United States (1975-2016)[END_REF].

Conclusion

Childhood HNC are of varied natures, have a low incidence, are often poorly described in terms of natural evolution and optimal treatment, and frequently involve functionally or vitally important anatomic structures [33]. Epidemiological data on HNC distribution, incidence and survival reported in this report contributes to our understanding of these tumors by quantifying their impact on the French population and assessing their burden. Regarding to the exclusion of topographies and some histological origins performed by some authors, we recommend that HNC studies should include all locations that are part of this region, except for central nervous system tumors, retinoblastomas, and all nodal or extranodal lymphomas without involvement of head and neck area. Therefore, this report could become the starting point for future HNC epidemiological studies. Table 1. Topographic codes used to identify Head and Neck Cancer cases [START_REF] Frizt | International Classification of Diseases for Oncology[END_REF].

Abbreviations: ICD-O-3, International Classification of Diseases for Oncology, Third Edition.

* Logrank test

Table 1

Topographic codes used to identify head and neck cancer cases [START_REF] Frizt | International Classification of Diseases for Oncology[END_REF].

Topography

ICD-O-3 site code [START_REF] Frizt | International Classification of Diseases for Oncology[END_REF] Orbit and adnexa (lacrimal gland) C69.5, C69.6

Middle ear

Table 2

Characteristics of children with multiple head and neck primary cancers (France, 2000(France, -2015)).

Children Gender Genetic abnormalities Age at diagnosis

Diagnoses

  Population data were provided by the French Institute of Statistics and Economics Studies (INSEE) for each year until 2015. The number of children under the age of 15 years varies overall from 11,138,557 in 2000 to 11,780,105 in 2015.

[

  CI95%:82.0-95.6] versus 76.1% [CI95% 74.3-77.9], respectively) and bone Ewing tumor (5year OS: 85.0% [CI95%:67.6-93.5] versus 75.1% [CI95% 71.3-78.4], respectively (Table

Table 8 .

 8 Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition

4 ,

 4 Abbreviations: ICD-O-3, International Classification of Diseases for Oncology, Third Edition.

Follow

  basal cell carcinoma Abbreviations: H&N, Head and Neck.

ICCC- 3 7 ]

 37 Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition; ASR, Age-Standardized rate; 95%CI, 95% confidence interval.

Table 5

 5

	describes the 1-, and 5-year overall

Table 2 .

 2 Characteristics of children with multiple head and neck primary cancers(France, 2000(France, -2015)).

	Abbreviations: H&N, Head and Neck.

Table 3 .

 3 Childhood head and neck cancer characteristics(France, 2000(France, -2015)).

	Abbreviations: M/F ratio, ratio of male over female cases; SD, Standard deviation; UCNT:
	Undifferentiated carcinoma of nasopharyngeal type; NRSTS: Non-rhabdomyosarcoma soft-
	tissue sarcomas

Table 4 .

 4 Annual standardized incidence rates of head and neck cancers per million children by ICCC-3 diagnostic groups and subgroups in France (2000-2015) by gender.

	Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition; ASR,
	Age-Standardized rate; 95%CI, 95% confidence interval.

Table 5 .

 5 One-year and 5-year overall survival (OS) among children with head and neck cancers by ICCC-3 diagnostic groups and subgroups: France, 2000 to 2015 and comparison to all childhood cancer.

	Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition; OS,
	Overall Survival; 95%CI, 95% confidence interval; pPNET, peripheral primitive
	neuroectodermal tumor.

Table 6 .

 6 Five-year overall survival (OS) for French children diagnosed with head and neck cancers, by period of diagnosis(2000-2007, and 2008-2015).

	Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition; OS,
	Overall Survival; 95%CI, 95% confidence interval; pPNET, peripheral primitive
	neuroectodermal tumor
	* Logrank test

Table 7 .

 7 Stage of childhood head and neck cancer by ICCC-3 diagnostic groups and subgroups:

	France, 2000 to 2015.

Table 3

 3 Childhood head and neck cancer characteristics(France, 2000(France, -2015)).

	Anatomic site	Morphology	n	%	M/F ratio	Age (Year)
						Mean (SD)
	Head and neck		541	33.3	1.2	6.3 (4.6)
	Bone		114	7.0	1.4	8.4 (3.9)
		Ewing sarcoma	36	2.2	1.6	7.4 (3.8)
		Chordoma	25	1.5	1.1	10.2 (2.8)
		Osteosarcoma	20	1.2	1.2	10.6 (3.2)
		Other malignancies	33	2.0	1.8	6.8 (4.2)
	Skin		103	6.3	1.2	8.5 (4.1)
		Melanoma	46	2.8	1.7	8.5 (4.6)
		Basal cell carcinoma	25	1.5	0.7	9.6 (2.9)
		Other malignancies	32	2.0	1.1	7.6 (3.9)
	Peripheral nerve		74	4.6	0.9	2.2 (3.3)
		Neuroblastoma	69	4.3	1.0	1.5 (2.2)
		Other malignancies	5	0.3	0.0	11.0 (2.8)
	Soft tissue		250

Table 3 (

 3 Continued)

	Localization	Morphology	n	%	M/F ratio	Age (Year)
						Mean (SD)
	Salivary glands (parotid gland and other major glands)	102	6.3	0.7 10.2 (3.3)
		Acinar cell carcinoma	22	1.4	0.2 11.9 (1.6)
		Rhabdomyosarcoma	17	1.0	1.8	6.8 (3.5)
		Adenocarcinoma	9	0.6	0.3 11.8 (2.0)
		Other malignancies	20	1.2	2.8	9.6 (4.0)
		Mucoepidermoid carcinoma	34	2.1	0.5 10.7 (2.6)
	Accessory sinuses (paranasal sinuses)	57	3.5	2.0	7.0 (4.0)
		Rhabdomyosarcoma	37	2.3	2.4	7.0 (4.1)
		Burkitt lymphomas	9	0.6	8.0	6.6 (3.0)
		Other malignancies	11	0.7	0.6	7.1 (4.7)
	Lips and oral cavity		51	3.1	1.0	8.0 (5.0)
		Rhabdomyosarcoma	18	1.1	1.0	5.4 (5.0)
		Mucoepidermoid carcinoma	10	0.6	1.5 12.0 (2.2)
		Other malignancies	23	1.4	0.8	8.2 (4.8)
	Nasal cavity and Middle ear		47	2.9	1.8	7.6 (4.4)
		Rhabdomyosarcoma	27	1.7	2.0	6.1 (3.9)
		Esthesioneuroblastoma	8	0.5	0.3	8.8 (5.5)
		Other malignancies	12	0.7	5.0 10.3 (3.2)
	Larynx and Trachea		5	0.3	1.5 10.6 (4.5)
		Rhabdomyosarcoma	2	0.1	1.0	8.0 (7.1)
		Other malignancies	3	0.2	2.0 12.3 (2.1)
	Total		1623	100.0	1.1	8.1 (4.5)
	Abbreviations: M/F ratio, ratio of male over female cases; SD, Standard deviation; UCNT: Undifferentiated
	carcinoma of nasopharyngeal type; NRSTS: Non-rhabdomyosarcoma soft-tissue sarcomas

Table 4

 4 Annual standardized incidence rates of head and neck cancers per million children by ICCC-3 diagnostic groups and subgroups in France(2000- 2015) by gender.

Table 5

 5 One-year and 5-year overall survival (OS) among children with head and neck cancers by ICCC-3 diagnostic groups and subgroups: France, 2000 to 2015 and comparison to all childhood cancer.

	ICCC-3 diagnostic groups and subgroups	N	1-year OS	5-year OS	5-year OS for all
					sites [24]
			% [95%CI]	% [95%CI]	% [95%CI]
	II. Lymphoma

Table 7

 7 Stage of childhood head and neck cancer by ICCC-3 diagnostic groups and subgroups: France, 2000 to 2015. (A) Lymphomas. (B) Solid tumors.

	A
	ICCC-3 diagnostic groups and subgroups	Missing	Stage I	Stage II	Stage III	Stage IV
		N (%)	N (%)	N (%)	N (%)	N (%)
	II. Lymphoma	1 (0.4) 71 (28.4)	96 (38.4)	40 (16.0)	42 (16.8)
	IIa. Hodgkin lymphoma	0 (0.0)	6 (30.0)	12 (60.0)	1 (5.0)	1 (5.0)
	IIb. Non-Hodgkin lymphoma	0 (0.0) 27 (30.3)	36 (40.4)	16 (18.0)	10 (11.2)
	IIb1. Precursor-cell lymphoma	0 (0.0)	6 (17.6)	17 (50.0)	6 (17.6)	5 (14.7)
	IIb2. Mature B-cell lymphoma except Burkitt	1 (2.6) 17 (43.6)	12 (30.8)	7 (17.9)	3 (7.7)
	IIc. Burkitt lymphoma	0 (0.0) 38 (27.1)	48 (34.3)	23 (16.4)	31 (22.1)
	Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition
	B
	ICCC-3 diagnostic groups and subgroups		Stage M0	Stage M+
			N (%)	N (%)
	IV. Neuroblastoma		66 (81.5)	15 (18.5)
	VIII. Malignant bone tumors		88 (93.6)	6 (6.4)
	VIIIa. Osteosarcoma		18 (90.0)	2 (10.0)
	VIIIc. Ewing tumor and related sarcomas of bone		32 (88.9)	4 (11.1)
	VIIId. Other malignant bone tumors		30 (100.0)	0 (0.0)
	VIIId2. Chordoma		25 (100.0)	0 (0.0)
	IX. Soft tissue and other extraosseous sarcomas		432 (84.4)	80 (15.6)
	IXa. Rhabdomyosarcoma		316 (81.4)	72 (18.6)
	IXd. Other specified soft tissue sarcomas		79 (91.9)	7 (8.1)
	IXd2. pPNET of soft tissue		24 (92.3)	2 (7.7)
	IXd3. Extrarenal rhabdoid tumor		20 (83.3)	4 (16.6)
	X. Head and Neck Germ-cell tumors		26 (96.3)	1 (3.7)
	XI. Carcinomas and melanomas		621 (94.7)	35 (5.3)
	XIb. Thyroid carcinomas		396 (93.6)	27 (6.4)
	XIc. Nasopharyngeal carcinomas		52 (89.7)	6 (10.3)
	XId. Malignant melanomas		46 (95.8)	2 (4.2)
	XIe. Skin carcinomas		33 (100.0)	0 (0.0)
	XIf. Other and unspecified		94 (100.0)	0 (0.0)
	XIf1. Carcinomas of salivary glands		66 (100.0)	0 (0.0)
	XII

. Other and unspecified malignant neoplasms 3 (100.0) 0 (0.0) Total 1236 (90.0) 137 (10.0)

  Abbreviations: ICCC-3, International Classification of Childhood Cancer, Third Edition; pPNET, peripheral primitive neuroectodermal tumor; M0, absence of distant metastasis; M+ presence of metastasis.

Table 8

 8 Five-year overall survival (OS) by stage for childhood head and neck cancer: France, 2000 to 2015. (A) Lymphomas. (B) Solid tumors. ICCC-3, International Classification of Childhood Cancer, Third Edition; 95%CI, 95% confidence interval. ICCC-3, International Classification of Childhood Cancer, Third Edition; pPNET, peripheral primitive neuroectodermal tumor; 95%CI, 95% confidence interval; M0, absence of distant metastasis; M+ presence of metastasis.

	A
	ICCC-3 diagnostic groups and subgroups	Stage I-II	Stage III-IV	P-value*
		5-year OS	5-year OS
		% [95%CI]	% [95%CI]
	II. Lymphoma	96.2 [91.8-98.3]	92.5 [84.0-96.6] 0.12
	IIa. Hodgkin lymphoma	92.9 [59.1-99.0]	100.0 [-]	0.79
	IIb. Non-Hodgkin lymphoma	95.0 [85.2-98.4]	88.5] [68.4-96.1	0.40
	IIb1. Precursor-cell lymphoma	100.0 [-]	90.9 [50.8-98.7]	0.15
	IIb2. Mature B-cell lymphoma (except Burkitt)	96.6 [77.9-99.5]	90.0 [47.3-98.5]	0.71
	IIc. Burkitt lymphoma	97.6 [90.9-99.4]	94.3 [83.4-98.1]	0.12
	Abbreviations: * Logrank test
	B
	ICCC-3 diagnostic groups and subgroups	Stage M0	Stage M+		P-value*
		5-year OS	5-year OS
		% [95%CI]	% [95%CI]
	IV. Neuroblastoma	98.5 [89.7-99.8]	60.0 [31.8-79.7]	<0.01
	VIII. Malignant bone tumors	80.6 [70.3-87.7]	66.7 [19.5-90.4]	0.25
	IX. Soft tissue and other extraosseous sarcomas	76.3 [71.9-80.1]	53.1 [41.5-63.4]	<0.01
	IXa. Rhabdomyosarcoma	77.8 [72.7-82.1]	57.7 [45.3-68.2]	<0.01
	X. Head and Neck Germ-cell tumors	100.0 [-]	100.0 [-]		-
	XI. Carcinomas and melanomas	97.5 [95.9-98.5]	85.3 [68.1-93.6]	<0.01
	XIb. Thyroid carcinomas	98.9 [97.2-99.6]	91.5 [69.6-97.8]	0.02
	XII. Other and unspecified malignant neoplasms	66.7 [5.4-94.5]	-[-]		-
	Total	88.7 [86.7-90.4]	63.0 [54.2-70.5]	<0.01
	Abbreviations:

* Logrank test

Acknowledgements

This work was supported by the "French National Cancer Institute", and the "French Public Health Agency". The authors are grateful to the numerous investigators who have meticulously contributed to the active search for information in clinical departments. The authors would also like to express their gratitude to the heads of the Medical Information Departments of all the University Hospitals and Comprehensive Cancer Centers.

Authors' contributions

All named authors have participated in the study to a sufficient extend to be named as authors.

All authors were involved in the study design and analysis plan. All authors critically reviewed the study results, commented on the drafts, and approved the final manuscript.

Declaration of Competing Interest

None declared.

Ethical Approval

This study was performed at the French National Registry of Childhood Cancer, CRESS Team