PharmD Marion Beaumont

Frédéric Dugay

MD Solène-Florence Kammerer-Jacquet

MD Sylvie Jaillard

PharmD Florian Cabillic

MD Romain Mathieu

MD Gregory Verhoest

MD Karim Bensalah

MD Nathalie Rioux-Leclercq

PharmD Marc-Antoine Belaud-Rotureau
email: marc-antoine.belaud-rotureau@univ-rennes1.fr

Dugay Frédéric

PhD Pharmd

Diagnosis of uncommon renal epithelial neoplasms performances of fluorescence in situ hybridization

Keywords: Renal epithelial neoplasms, Fluorescence in situ hybridization, Chromosomal abnormalities, Histological classification

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

INTRODUCTION

Renal cell carcinomas (RCC) represent more than 90% of adult renal malignancies [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF].

These tumors preferentially affect men, with a mean age at diagnosis of 64 years [START_REF] Hsieh | Renal cell carcinoma[END_REF].

According to the WHO classification, RCC are divided in several subtypes, characterized by morphological and histological features, protein expression patterns and genetics criteria [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF].

The conventional or clear cell renal cell carcinoma (CCRCC) is the most frequent subtype [3].

The cytoplasm of tumor cells appears optically empty after hematoxylin eosin safran (HES) staining and immunohistochemistry (IHC) assay frequently reveals positivity for CAIX [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF].

Moreover, an inactivation of the VHL gene (3p25.3) is found in at least 70% of the CCRCC [START_REF] Dagher | Wild-type VHL Clear Cell Renal Cell Carcinomas Are a Distinct Clinical and Histologic Entity: A 10-Year Follow-up[END_REF]. Papillary RCC (PRCC) encompassed two morphological types (type 1 and 2). Cylindrical eosinophilic cytoplasms with nuclei pseudostratifications are observed in type 2 while these features are absent in type 1 [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF]. Both types commonly express P504S (AMACR) and CK7 [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF]. Trisomies of chromosomes 7 and 17 are associated to PRCC [START_REF] Kovacs | Molecular cytogenetics of renal cell tumors[END_REF][START_REF] Pignot | Survival analysis of 130 patients with papillary renal cell carcinoma: prognostic utility of type 1 and type 2 subclassification[END_REF]. Chromophobe RCC (ChRCC) is characterized by large polygonal cells with transparent cytoplasm [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF]. The main differential diagnosis is oncocytoma, which is a benign tumor presenting round or polygonal cells with an eosinophilic cytoplasm and a round and regular nuclei [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF][START_REF] Wobker | Modern Pathologic Diagnosis of Renal Oncocytoma[END_REF][START_REF] Amin | Prognostic impact of histologic subtyping of adult renal epithelial neoplasms: an experience of 405 cases[END_REF]. However, the eosinophilic variant of ChRCC can also exhibit eosinophilic cells [START_REF] Wobker | Modern Pathologic Diagnosis of Renal Oncocytoma[END_REF]. CK7 immunostaining shows diffuse positivity in the majority of ChRCC cases whereas oncocytomas exhibit negative or focally positive patterns [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF][START_REF] Wobker | Modern Pathologic Diagnosis of Renal Oncocytoma[END_REF]. Nevertheless, a negative pattern can be observed in about 20% of the ChRCC [START_REF] Wu | Cytokeratins 7 and 20 immunoreactivity in chromophobe renal cell carcinomas and renal oncocytomas[END_REF]. Moreover, ChRCC present multiple chromosomal losses while rearrangement of the CCND1 gene is associated to oncocytomas [START_REF] Hsieh | Renal cell carcinoma[END_REF][START_REF] Kovacs | Molecular cytogenetics of renal cell tumors[END_REF][START_REF] Brunelli | Diagnostic Usefulness of Fluorescent Cytogenetics in Differentiating Chromophobe Renal Cell Carcinoma From Renal Oncocytoma: A Validation Study Combining Metaphase and Interphase Analyses[END_REF]. Translocation renal cell carcinoma (TRCC) affects preferentially children and young patients [START_REF] Argani | MiT family translocation renal cell carcinoma[END_REF]. TRCC encompasses TFE3 TRCC and TFEB TRCC, implying respectively rearrangements of TFE3 (Xp11. [START_REF] Belaud-Rotureau | Interphase fluorescence in situ hybridization is more sensitive than BIOMED-2 polymerase chain reaction protocol in detecting IGH-BCL2 rearrangement in both fixed and frozen lymph node with follicular lymphoma[END_REF] and TFEB (6p21.1) genes and overexpression of the corresponding fusion proteins [START_REF] Camparo | Renal translocation carcinomas: clinicopathologic, immunohistochemical, and gene expression profiling analysis of 31 cases with a review of the literature[END_REF]. The TFE3 TRCC can show clear cells or papillary architecture, challenging the differential diagnosis with CCRCC and PRCC, particularly in young patients [START_REF] Argani | MiT family translocation renal cell carcinoma[END_REF][START_REF] Argani | PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21)[END_REF]. The TFEB TRCC typically demonstrates biphasic morphology with epithelioid cells and small cells clustered around membrane basement material [START_REF] Argani | Molecular Confirmation of t(6;11)(p21;q12) Renal Cell Carcinoma in Archival Paraffin-embedded Material Using a Break-apart TFEB FISH Assay Expands its Clinicopathologic Spectrum[END_REF]. This subtype consistently expressed melanoma markers such as Melan A and HMB45 [START_REF] Argani | MiT family translocation renal cell carcinoma[END_REF]. Recently, amplifications of the TFEB gene have been described [START_REF] Argani | TFEB-amplified Renal Cell Carcinomas: An Aggressive Molecular Subset Demonstrating Variable Melanocytic Marker Expression and Morphologic Heterogeneity[END_REF]. Additionally tumors that do not resemble those of any well characterized RCC subtypes, or low/high grade unclassified oncocytic neoplasms, or RCC with pure sarcomatoid/rhabdoid morphology with no recognizable epithelial component corresponded to Unclassified RCC [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF].

Prognoses and treatments differ between RCC subtypes [START_REF] Hsieh | Renal cell carcinoma[END_REF][START_REF] Amin | Prognostic impact of histologic subtyping of adult renal epithelial neoplasms: an experience of 405 cases[END_REF], shedding the light on the importance of an accuracy classification of these tumors. In most cases, RCC can be easily classified according to histological criteria. Nevertheless, some samples can present a combination of features occurring in different subtypes or uncommon characteristics [START_REF] Hsieh | Renal cell carcinoma[END_REF]. Furthermore, each tumor occurring in young patient (≤ 40 years) should be screened for a TFE3 TRCC [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF]. Thus, uncommon RCC may require complementary analyses to support the diagnosis. Several immunohistochemical markers can lead to equivocal or contradictory results and distinct RCC subtypes can show overlapping histologic patterns [START_REF] Roh | The application of cytogenetics and fluorescence in situ hybridization to fine-needle aspiration in the diagnosis and subclassification of renal neoplasms[END_REF]. As each RCC subtypes exhibit distinct chromosomal abnormalities, cytogenetics analysis could also be helpful for tumor classification. Fluorescence in situ hybridization (FISH) is a powerful tool to detect balanced and unbalanced-chromosomal rearrangements in cancers [START_REF] Jehan | In-situ hybridization as a molecular tool in cancer diagnosis and treatment[END_REF]. FISH is the principal molecular assay used in clinical diagnosis of RCC [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF]. Several authors have assessed the suitability of FISH analysis in the RCC subtyping [START_REF] Barocas | Renal cell carcinoma sub-typing by histopathology and fluorescence in-situ hybridization on a needle-biopsy specimen[END_REF][START_REF] Receveur | Characterization of quantitative chromosomal abnormalities in renal cell carcinomas by interphase four-color fluorescence in situ hybridization[END_REF][START_REF] Sanjmyatav | Identification of tumor entities of renal cell carcinoma using interphase fluorescence in situ hybridization[END_REF], but these studies were carried out on small cohorts. Moreover, the impact of such assays for the classification of RCC with unusual histological characteristics and/or occurring in young people remains to be determined. In this aim, we evaluated the interest of FISH analysis in the histological classification of 359 uncommon renal epithelial neoplasms. Histopathological and immunohistochemical assays were performed on each tumor by 2 independent pathologists (NRL and SFKJ) on formalin-fixed paraffin-embedded (FFPE) tissue sections stained by hematoxylin eosin safran (HES). According to these analyses each case was classified into one of the following subgroups: 1) CCRCC or TFE3 TRCC, 2) PRCC or TFE3 TRCC, 3) Oncocytic renal tumors, corresponding to oncocytoma or eosinophilic variant of ChRCC, 4) TFEB TRCC and 5) Unclassified RCC.

MATERIAL AND METHODS

PATIENTS AND HISTOLOGICAL ANALYSIS

IMMUNOHISTOCHEMISTRY (IHC)

The protein expression patterns were assessed by IHC using the following antibodies: anti-CAIX (Abcam, Cambridge, UK), anti-CK7 (Dako, Agilent Technologies, USA), anti-P504S (Dako, Les Ulis, France), anti-TFE3 (Cell Marque Corporation, Rocklin, California, USA), anti-TFEB (Abcam). Briefly, the reactivity of antibodies was revealed with HRPlabeled polymer conjugated secondary antibodies using di-aminobenzidine (DAB) as chromogen (Sigma-Aldrich, Saint-Quentin-Fallavier, France). Antibody staining was observed using an Olympus BX51 microscope and images recorded with an Olympus DP70 camera. The tumor expression for each antibody were independently evaluated (NRL, SFKJ).

Negative control was performed by omitting the primary antibody.

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

FISH analysis was performed as previously described [START_REF] Dagher | Histologic prognostic factors associated with chromosomal imbalances in a contemporary series of 89 clear cell renal cell carcinomas[END_REF][START_REF] Belaud-Rotureau | Interphase fluorescence in situ hybridization is more sensitive than BIOMED-2 polymerase chain reaction protocol in detecting IGH-BCL2 rearrangement in both fixed and frozen lymph node with follicular lymphoma[END_REF]. Briefly, the 5 µm thick, paraffin-embedded sections fixed on slides were deparaffinized and pre-treated using pretreatment solution (Dako). Pepsin solution was added on preparations for 6 minutes (Sigma, 100 mg/l) and then dehydrated in ethanol 70°, 85°, and 100° (2 minutes each). Specimens and probes were codenatured (10 minutes at 75°C +/-2°C) and hybridized overnight at 37°C. for the detection of a chromosomal loss/gain or a gene rearrangement were respectively 30% and 15% as previously reported [START_REF] Jiang | 1p/19q codeletion and IDH1/2 mutation identified a subtype of anaplastic oligoastrocytomas with prognosis as favorable as anaplastic oligodendrogliomas[END_REF][START_REF] Sukov | TFE3 Rearrangements in Adult Renal Cell Carcinoma: Clinical and Pathologic Features With Outcome in a Large Series of Consecutively Treated Patients[END_REF].

The FISH hybridization patterns were classified as positive or negative as indicated in Table I. The FISH analysis was considering "contributive" when the results led to RCC subtype classification or "non-contributive" when the hybridization patterns did not provide any information for RCC subtyping. Cases with hybridization failure were also mentioned.

STATISTICAL ANALYSIS

Results are expressed as mean +/-standard error of the mean. Data were compared using the Chi2 test and the Fisher's Exact test. The Pvalue of <0.05 was considered statistically significant. 3. RESULTS

3.1.PATIENTS AND TUMORS

Patients and tumors characteristics are summarized in Table II and Figure 1. In this study, male patients were mostly represented (225/359; 63%). The mean age at diagnosis was 47,4 ± 0,9 years and 38% of patients were less than 40 years (137/359). Tumors were mainly originated from french medical centers of the CARARE network (324/359; 89%) and sent for a second opinion by an uropathologist expert in the Rennes University Hospital. RCC were classified in the following subgroups according to their histological and immunohistochemical features. The most important one was the CCRCC or TFE3 TRCC subgroup, which represented 173/359 cases (48%). In this group, all the tumors had solid, nested or papillary patterns and consisted of a majority of cells with clear cytoplasm. In some areas, tumor cells displayed granular eosinophilic cytoplasm with more prominent nucleoli, irregular nuclei and clumped chromatin. Two neoplasms contained psammoma bodies (Figure 1A). Unclassified RCC were observed in 127/359 tumors (35%). These cases represent a heterogeneous group of carcinomas that do not fit into any of the well-known histologic subtypes. They include also tumors with pure sarcomatoid histology with no recognized epithelial subtypes (Figure 1B and1C).

The distinction between oncocytoma and eosinophilic variant of ChRCC was required in 31/359 RCC (9%). In this group, all the tumors consisted of tubular or focal/diffuse solidsheet pattern. Tumor cells were cuboidal with oncocytic cytoplasm. The cell borders were usually indistinct to focally slightly distinct. Nuclei were round to irregular wrinkled appearance. Perinuclear haloes were rare but focally present (Figure 1D). Differential diagnosis between PRCC and TFE3 TRCC concerned 20/359 tumors (6%). These neoplasms demonstrated a papillary architecture with fibrovascular cores. Some tumors had more compact and solid patterns. Papillae were lined by columnar eosinophilic cells with more often prominent nucleoli, and a nuclear pseudostratification. In some areas but in all tumors, a clear cell component was present (Figure 1E).

Finally, histological features of TFEB TRCC were observed in only 8/359 cases (2%).

All these tumors had a solid pattern of growth with two types of tumor cells. The predominant cells were epithelioid with abundant clear or finely eosinophilic cytoplasm. The second population of tumor cells consisted of smaller cells with dense chromatin around nodules of hyaline basement membrane (Figure 1F). A majority of RCC (261/359; 73%) was successfully classified after FISH analysis.

3.2.FISH ANALYSIS

Conversely, these cytogenetics investigations did not contribute to the tumor classification in 69/359 of the cases (19%) and a hybridization failure was observed for 29/359 of the tumors (8%). The impact of the FISH was evaluated for each subgroup of tumors (Figures 3 and4).

The FISH results allowed the differential diagnosis between a CCRCC and a TFE3 TRCC in 156/173 of cases (90%; Figure 3A). A total of 133 CCRCC were diagnosed. A chromosome 3 loss, encompassing the VHL gene was observed in 94/133 tumors. This chromosomal loss was always heterozygous and corresponded most frequently to a deletion on the short arm of the chromosome 3 (1G, 2O signals in 70/94 CCRCC (74%)). For 24/94 cases a larger chromosomal imbalance, including the centromere of the chromosome 3 (1G, 1O) was observed. FISH analysis did not showed a VHL loss in 39/133 cases. However, these tumors were classified as CCRCC according to their histological features and a negative TFE3 FISH pattern. A TFE3 gene rearrangement was detected in 23 cases, corresponding to TFE3 TRCC. Among these tumors, TFE3 immunostaining was negative in one case. The hybridization failed in the 17/173 cases (10%).

The FISH analysis contributed to the differential diagnosis between PRCC and TFE3

TRCC in 19/20 RCC (95%; Figure 3B). A majority of these tumors (18/19) exhibited gains of chromosomes 7/17 or a normal hybridization pattern for the TFE3 gene and were classified as PRCC. The gain of the chromosome 17 was observed more frequently but not significantly in type 2 PRCC than in type 1 PRCC (respectively 4/8, 50%, and 8/10, 80%; p=0.32). Only one case (1/19) showed a TFE3 gene rearrangement but a negative TFE3 immunostaining and was classified as a TFE3 TRCC. A hybridization failure was observed in one case.

A TFEB gene rearrangement (6p21.1) was observed in all but one tumor exhibiting TFEB TRCC histological features (7/8; 88%; Figure 3C). Among these tumors, one case showed a complex rearrangement including a distal 6p21.1 deletion (no red signals) associated to a TFEB gene amplification (numerous green signals). This result was confirmed by array-CGH (data not shown). A TFEB gene rearrangement could not be retained for the last RCC, showing a splitted FISH signals in only 10% of nuclei, lower than the positive threshold (15%).

A differential diagnosis between oncocytoma and an eosinophilic variant of ChRCC was only achieved after FISH analysis in 9/31 cases (29%, Figure 4A). A rearrangement of the CCND1 gene was detected in 5/9 tumors classified as oncocytomas. Multiple chromosomal losses were observed 4/9 cases, corresponding to eosinophilic variant of ChRCC. Conversely, cytogenetics investigations did not contribute to the determination of the RCC subtype in 15/31 cases (48%) exhibited none of the previous chromosomal abnormalities and a hybridization failure was observed in 7/31 tumors (23%).

Unclassified RCC was a heterogeneous group included tumors with different cytogenetics profiles. The FISH analysis was contributive in 70/127 cases (55%; Figure 4B) corresponding to 39 CCRCC (deletion of the VHL gene), 24 PRCC (chromosome 7/17 gains), 5 TFE3 TRCC, 1 TFEB TRCC (rearrangement of the corresponding genes) and 1 RCC with a TFEB amplification without rearrangement of the gene (numerous fusions signals). The FISH assays were non contributive in 53/127 tumors (42%) and a hybridization failure was observed in the remaining cases (4/127; 3%).

DISCUSSION

RCC encompass several histological subtypes, differing by their clinical outcomes and treatments [START_REF] Hsieh | Renal cell carcinoma[END_REF][START_REF] Amin | Prognostic impact of histologic subtyping of adult renal epithelial neoplasms: an experience of 405 cases[END_REF]. The classification of renal tumors is mainly based on morphological and histological data. Nevertheless, RCC are characterized by a huge heterogeneity and several tumors require ancillary assays, as IHC or FISH analyses [START_REF] Tan | Renal Tumors: Diagnostic and Prognostic Biomarkers[END_REF][START_REF] Receveur | Characterization of quantitative chromosomal abnormalities in renal cell carcinomas by interphase four-color fluorescence in situ hybridization[END_REF][START_REF] Skinnider | An immunohistochemical approach to the differential diagnosis of renal tumors[END_REF]. To assess the impact of the latest one, we studied 359 RCC with uncommon characteristics. In routine diagnosis, a majority of RCC exhibits classical histological features and FISH assay is not necessary for their classification. However, numerous uncommon renal epithelial neoplasms are addressed to our department which is the French reference center for the diagnosis of renal tumors (CARARE network). This large and specific recruitment and our high-volume activity (more than 5000 analyses per year of all tumors), allowed the analysis of large series of tumors using rapid and automatized processes (deparaffinisation, sample preatreatment, nuclei pictures acquisition). As previously reported, this strategy is cost effective and suggests that testing cost is an insufficient reason to limit the use of FISH [START_REF] Parker | Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy in NSCLC: Implications for Cost Effectiveness of Testing and Treatment[END_REF]. To our knowledge, this study is the first and larger one showing 1) that FISH assay improves the histological classification of 73% of the RCC with uncommon characteristics and 2) that this strategy is highly efficient on targeted-RCC subgroups. A majority of the RCC studied herein (89%) were originated from various medical centers. Consecutively, variations in the pre-analytic parameters, such as time of fixation, may lead to a limitation of FISH analysis [START_REF] Khoury | Delay to formalin fixation effect on breast biomarkers[END_REF].

Interestingly, the hybridization failure rate was low (8%) and similar to previous studies, confirming that this testing is a robust ancillary assay [START_REF] Belaud-Rotureau | A comparative analysis of FISH, RT-PCR, PCR, and immunohistochemistry for the diagnosis of mantle cell lymphomas[END_REF]. FISH assay does not require DNA extraction and appears as a quick and easy method to assess chromosomal aberrations on RCC FFPE samples.

According to the literature, the mean age for RCC diagnosis is 64 years [START_REF] Hsieh | Renal cell carcinoma[END_REF]. Interestingly, it was only 47,4 years in this cohort. This difference could reflect the high rate of patients from the CARARE network, which includes especially all the RCC occurring in young people. As a TFE3 TRCC should be suspected for each tumor with a clear cell contingent occurring in a young patient, the distinction between a CCRCC and a TFE3 TRCC is frequently observed in our study. In this subgroup, FISH analysis was 100% contributive for the histological classification and supported the diagnosis of 133 CCRCC. A biallelic inactivation of the VHL gene is associated to CCRCC. Three different mechanisms are involved: chromosome 3 deletion encompassing 3p25-p26 region, mutations on the coding regions of the gene and/or methylation of its promotor [START_REF] Gossage | VHL, the story of a tumour suppressor gene[END_REF]. Two positive FISH patterns were observed herein: loss of the VHL gene and losses of both VHL gene and centromere of the chromosome 3. In the last case, FISH analysis does not allow the distinction between partial and complete chromosome 3 losses. Array-CGH analysis can overlap this limitation by an accurate determination of the extent of the deletion but no prognostic value was associated to these monosomies. In this series, some CCRCC cases (39/133) did not exhibit a loss of the VHL gene by FISH analysis. The classification of these tumors as CCRCC was based on their histological characteristics and the absence of TFE3 rearrangement. As previously reported, these CCRCC may present other VHL inactivating events (mutation and/or promoter methylation) or a wild-type VHL gene leading to a worse clinical outcome [START_REF] Dagher | Wild-type VHL Clear Cell Renal Cell Carcinomas Are a Distinct Clinical and Histologic Entity: A 10-Year Follow-up[END_REF]. The diagnosis of other clear cell renal tumors such as clear cell papillary RCC was excluded by morphological analysis and immunohistochemistry (CK7, CAIX, P504S) [START_REF] Moch | WHO Classification of Tumours of the Urinary System and Male Genital Organs[END_REF] and did not require complementary analyses. FISH testing also allowed the diagnosis of 23 TFE3 TRCC.

The TFE3 immunostaining was negative for one of these cases supporting, as previously described, that IHC and FISH analysis should be associated [START_REF] Rao | TFE3 Break-apart FISH Has a Higher Sensitivity for Xp11.2 Translocation-associated Renal Cell Carcinoma Compared With TFE3 or Cathepsin K Immunohistochemical Staining Alone: Expanding the Morphologic Spectrum[END_REF][START_REF] Just | Identification by FFPE RNA-Seq of a new recurrent inversion leading to RBM10-TFE3 fusion in renal cell carcinoma with subtle TFE3 break-apart FISH pattern[END_REF].

As for CCRCC, FISH analysis always contributed to the differential diagnosis between PRCC and TFE3 TRCC. Centromeric probes of chromosomes 7 and 17 were used to identify gains of the corresponding chromosomes observed in PRCC. PRCC is divided in type 1 and type 2, respectively associated to favorable and pejorative outcomes [START_REF] Hsieh | Renal cell carcinoma[END_REF][START_REF] Pignot | Survival analysis of 130 patients with papillary renal cell carcinoma: prognostic utility of type 1 and type 2 subclassification[END_REF]. The gain of chromosome 17 has been described more frequently in the type 1 and is correlated to an improvement of the survival [START_REF] Klatte | Cytogenetic and Molecular Tumor Profiling for Type 1 and Type 2 Papillary Renal Cell Carcinoma[END_REF]. Conversely, in our study, this aneuploidy was observed more frequently but not significantly in the type 2 (p=0.32). Characteristics of our tumors or recruitment of young patients may explain differences between our study and data of the literature. In this subgroup, one tumor exhibited a rearrangement of the TFE3 gene. As previously, the TFE3 immunostaining was negative, highlighting the interest of FISH assay.

The TFEB gene rearrangement is the molecular hallmark of TFEB TRCC [START_REF] Argani | MiT family translocation renal cell carcinoma[END_REF]. FISH analysis confirmed a rearrangement of this gene in almost all the cases with characteristic histological features. Interestingly, a complex 6p21.1 rearrangement associated to a TFEB gene amplification was detected by FISH in one RCC and confirmed by array-CGH. Another case (Unclassified-RCC) showed a TFEB amplification without gene rearrangement. The TFEB gene amplifications have been recently reported and individualized as a rare RCC subtype, more aggressive and occurring in older patients than rearrangements [START_REF] Argani | TFEB-amplified Renal Cell Carcinomas: An Aggressive Molecular Subset Demonstrating Variable Melanocytic Marker Expression and Morphologic Heterogeneity[END_REF][START_REF] Williamson | Renal Cell Carcinoma With Chromosome 6p Amplification Including the TFEB Gene: A Novel Mechanism of Tumor Pathogenesis?[END_REF]. Thus, FISH analysis is a particularly useful all-in-one assay for the detection of these different TFEB gene aberrations.

Oncocytoma is a benign tumor. However, some malignant ChRCC cases showed histological characteristics of oncocytoma (eosinophilic variant), shedding the light on the importance of the differential diagnosis [START_REF] Zheng | LMP2, a novel immunohistochemical marker to distinguish renal oncocytoma from the eosinophilic variant of chromophobe renal cell carcinoma[END_REF]. FISH analysis contributed to the histological classification in only 29% of the cases. These results are consistent with the literature: rearrangement of the CCDN1 gene is observed in 22% of the oncocytomas and multiple chromosomal imbalances in ChRCC can concern other chromosomes than those targeted by the selected FISH probes [START_REF] Hsieh | Renal cell carcinoma[END_REF][START_REF] Kovacs | Molecular cytogenetics of renal cell tumors[END_REF][START_REF] Sukov | CCND1 rearrangements and cyclin D1 overexpression in renal oncocytomas: frequency, clinicopathologic features, and utility in differentiation from chromophobe renal cell carcinoma[END_REF]. As Array-CGH is a very sensitive tool allowing a pangenomic analysis of chromosomal imbalances, the combination of these assays could be considered to improve the differential diagnosis between oncocytomas and ChRCC.

Unclassified RCC represented 35% of this cohort. Interestingly, FISH analysis led to the classification of 55% of these RCC showing more frequently CCRCC or PRCC cytogenetics patterns. Other analyses such as array-CGH and next generation sequencing could be useful for the diagnosis of the cases remaining unclassified.

This study shed the light on the interest of FISH analysis for the histological classification of uncommon renal epithelial neoplasms. FISH is a powerful tool to identify chromosomal aberrations associated to CCRCC, PRCC, and TRCC subtypes. Moreover, it allows the distinction between a rearrangement and an amplification of the TFEB gene. When FISH results are non-contributive, additional molecular assays could be considered to improve the diagnosis, especially for oncocytic-type and unclassified RCC.

FIGURE / TBALE LEGENDS

FISH

  probes were selected to detect the main chromosomal abnormalities associated to each RCC subtypes. Different probes were assayed according to the previous subgroups of tumors. The VHL and TFE3 genes status were assessed for the CCRCC or TFE3 TRCC cases using the Zyto-Light SPEC VHL/CEN3 Dual Color Probe (Zytovision, Clinisciences, Nanterre, France) and the Zyto-Light SPEC TFE3 Xp11 Dual Color Break Apart (Zytovision). Similarly, PRCC or TFE3 TRCC were distinguished using centromeric probes for chromosomes 7 and 17 (CEP 7 and CEP 17 probes; Abbott, Rungis, France) and the Zyto-Light SPEC TFE3 Xp11 Dual Color Break Apart (Zytovision). For tumors with TFEB TRCC histological features, the TFEB gene status was assessed using the TFEB Break apart Probe 6p21.1 (Empire Genomics, Buffalo, United States). Concerning ChRCC or oncocytomas cases, the ZytoLight SPEC CCND1 Break Apart/2q11/CEN 6 Quadruple Color Probes (Zytovision) and the ZytoLight SPEC VHL/1p12/CEN 7/17 Quadruple Color Probes (Zytovision) were assayed to highlight multiple chromosomal losses or a CCND1 gene rearrangement. Finally, Unclassified RCC were studied for VHL, TFEB, TFE3 and the number of chromosomes 7 and 17, according to the histological or clinical features. After hybridization, slides were washed according to the manufacturers' instructions, and the nuclei were counterstained by DAPI (Dako). Cells were viewed using a fluorescent Axioplan II microscope (Zeiss, Le Pecq, France) or the automatized microscope Bioview Encore (Bioview, Rehovot, Israel) with appropriate filters and 100 non-overlapped nuclei were analysed for each tumor by 2 independents observers (FD and FC). The positive thresholds

FISH

  analyses were performed for each subgroup of tumors as described in Patients and Methods. The results are indicated in Figure2. The TFE3 gene rearrangement was assessed in 279/359 cases (78%), following by the status of the VHL gene (246/359; 69%), gain(s) of the chromosomes 7 and/or 17 (103/359; 29%), the TFEB gene rearrangement (81/359; 23%), multiple losses or the CCND1 gene rearrangement (31/359; 9%). The more frequent abnormalities were a loss of the VHL gene (144/246; 59%), following by gains of the chromosomes 7 and/or 17 (52/103; 50%), multiple chromosomal losses or CCND1 rearrangement (9/31; 29%), and rearrangements of the TFEB genes (10/81; 12%) and TFE3 (29/279; 10%).

Figure 1 .

 1 Figure 1. Histological RCC features

Figure 2 .

 2 Figure 2. FISH analysis of 359 RCC

Figure 3 .

 3 Figure 3. FISH analysis: CCRCC, PRCC, and TRCC

Figure 4 .

 4 Figure 4. FISH analysis: oncocytic and unclassified RCC

  This study focused on 359 RCC addressed for FISH analysis in the Cytogenetics

	department of Rennes University Hospital from January 2014 to June 2017. Patients
	originated from the Rennes University Hospital or from others French medical centers. Most
	of them were included in the French CARARE network (Rare Renal Cancer in Adults) of the
	INCa (National Institute of Cancer, France) focused on RCC occurring in young people (≤ 40
	years) and/or every subtypes of RCC, excepted CCRCC and non-metastatic PRCC.

Supported by grants from the French National Institute of Cancer (INCa-CARARE) for the purchase of FISH probes. The institute had no implication in study design; in


Nicolas Gauthier

Cécile Courrèges
email: cecile.courreges@univ-pau.fr

Julien Demeaux

Cécile Tessier

Hervé Martinez

Probing the in-depth distribution of organic/inorganic molecular species within the SEI of LTO/NMC and LTO/LMO batteries: a complementary ToF-SIMS and XPS study

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Spinel Li4Ti5O12 (LTO) is an attractive candidate for negative electrode materials of Li-ion batteries because of its outstanding safety characteristics. In this paper, the influence of common high voltage cathodes, LiNi3/5Co1/5Mn1/5O2 (NMC) and LiMn2O4 (LMO), upon the electrochemical performances of LTO/LMO and LTO/NMC cells, in relation with the Solid

INTRODUCTION

Li-ion batteries are widely used nowadays for electric energy storage such as in electric vehicles which have increased the demand and the requirements in terms of voltage and safety [START_REF] Goodenough | Challenges for rechargeable Li batteries[END_REF][START_REF] Wu | Investigation on Li4Ti5O12 Batteries Developed for Hybrid Electric Vehicle[END_REF] . Their electrochemical performances rely on the intrinsic properties of the active materials and on the electrode/electrolyte interfaces. Thus, the formation of a stable surface layer (5-10 nm), called "Solid Electrolyte Interphase" (SEI) plays a major role in Li-ion batteries working. This SEI is composed of solvent and lithium salt degradation products and should ensure the passivation of the electrode surface while maintaining an efficient diffusion of lithium ions [START_REF] Peled | Advanced Model for Solid Electrolyte Interphase Electrodes in Liquid and Polymer Electrolytes[END_REF][START_REF] Peled | Review, SEI: Past, Present and Future[END_REF] . For high current densities, carbon-based anodes, are associated with the risk of lithium plating and dendrites formation, which presents a real safety hazard. In this context, other active materials, such as lithium titanate Li4Ti5O12 (LTO), have been considered and extensively studied. Indeed, LTO presents interesting characteristics with a high plateau intercalating potential of 1.55 V vs Li + /Li 0 , avoiding any dendrite plating [START_REF] Goodenough | The Li-Ion Rechargeable Battery: A Perspective[END_REF][START_REF] Chen | Li4Ti5O12 Anode: Structural Design from Material to Electrode and the Construction of Energy Storage Devices[END_REF] , and a negligible change of lattice parameter during Li + insertion/extraction, resulting in a very low capacity decrease upon cycling [START_REF] Ohzuku | Zero-strain insertion material of Li[Li1/3Ti5/3]O4 for rechargeable lithium cells[END_REF][START_REF] Zaghib | Electrochemical study of Li4Ti5O12 as negative electrode for Li-ion polymer rechargeable batteries[END_REF] . Nevertheless, different studies have shown the formation of a SEI (composed of LiF, fluorophosphates, alkylcarbonates, oxalates, ethylene oxides) and gassing occurring at the LTO electrode surface at different temperatures in half and full cells, which can affect cell electrochemical performances [START_REF] El Ouatani | The effect of vinylene carbonate additive on surface film formation on both electrodes in Li-Ion batteries[END_REF][START_REF] Dedryvère | Electrode/Electrolyte Interface Reactivity in High-Voltage Spinel LiMn1.6Ni0.4O4/Li4Ti5O12 Lithium-Ion Battery[END_REF][START_REF] Song | Is Li4Ti5O12 a solidelectrolyte-interphase-free electrode material in li-ion batteries? Reactivity between the Li4Ti5O12 electrode and electrolyte[END_REF][START_REF] Gieu | Temperature effects on Li4Ti5O12 electrode/electrolyte interfaces at the first cycle: A X-ray Photoelectron Spectroscopy and Scanning Auger Microscopy study[END_REF][START_REF] Gieu | Influence of Vinylene Carbonate additive on the Li4Ti5O12 electrode/electrolyte interface for lithium-ion batteries[END_REF][START_REF] Wu | Investigation on gas generation of Li4Ti5O12/LiNi1/3Co1/3Mn1/3O2 cells at elevated temperature[END_REF][START_REF] Wang | Quantitative investigation of the gassing behavior in cylindrical Li4Ti5O12 batteries[END_REF] . In addition, even if the high working potential of spinel LTO renders the system safer, it is at the cost of a loss of energy density.

Consequently, the LTO implementation requires the use of high voltage cathodes such as layered metal oxide LiNi3/5Co1/5Mn1/5O2 (NMC) electrode [START_REF] Chancelier | Characterization of LTO//NMC Batteries Containing Ionic Liquid or Carbonate Electrolytes after Cycling and overcharge[END_REF] and spinel oxide LiMn2O4 (LMO) electrode [START_REF] Chen | Li4Ti5O12 Anode: Structural Design from Material to Electrode and the Construction of Energy Storage Devices[END_REF][START_REF] Belharouak | Electrochemistry and safety of Li4Ti5O12 and graphite anodes paired with LiMn2O4 for hybrid electric vehicle Li-ion battery applications[END_REF] , which are commonly used until 4V. However, the manganese-based materials exhibit transition metal dissolution in the electrolyte during cycling and deposition on the negative electrode surface, identified as a potential issue for batteries cycle life [START_REF] Joshi | Effects of Dissolved Transition Metals on the Electrochemical Performance and SEI Growth in Lithium-Ion Batteries[END_REF][START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF][START_REF] Gilbert | Transition Metal Dissolution, Ion Migration, Electrocatalytic Reduction and Capacity Loss in Lithium-Ion Full Cells[END_REF][START_REF] Gieu | New insights in the characterization of the electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF] . In a previous study, we showed that LTO/NMC full cell cycled at 3.0V presents better electrochemical performances over the first hundred cycles compared to the LTO/LMO system cycled at 3.2V [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Indeed, the capacity becomes stable faster for LTO/NMC and remains higher (147±3 mAh.g -1 after 100 cycles) compared to LTO/LMO (140±3 mAh.g -1 after 100 cycles). These electrochemical results were correlated with the formation and the evolution of both LTO electrode/electrolyte interface layers cycled against NMC or LMO electrodes by X-ray Photoelectron Spectroscopy (XPS) and Scanning Auger Microscopy (SAM) measurements. XPS analysis quantitatively evidenced that the SEI formed on the LTO electrode cycled facing LMO is thicker than the one formed facing NMC electrode. Besides, the SEI formed on the LTO electrode surface vs LMO contains small amounts of manganese compounds (detected by XPS), homogeneously spread over the surface, as revealed by SAM.

Thanks to its high sensitivity and in-depth resolution (< 1 nm), ToF-SIMS can also be very useful to characterize the in-depth distribution of molecular SEI species, through sputterdepth-profiling experiments. For instance, the accumulation of manganese at the graphite electrode surface has already been observed by secondary ion mass spectrometry [START_REF] Vissers | Role of Manganese Deposition on Graphite in the Capacity Fading of Lithium Ion Batteries[END_REF] . Moreover, the surface of graphite and lithium electrodes have been analyzed by ToF-SIMS and the formation of a surface layer, where the organic products are mostly located in the outer part, has been observed [START_REF] Lu | Chemistry, Impedance, and Morphology Evolution in Solid Electrolyte Interphase Films during Formation in Lithium Ion Batteries[END_REF][START_REF] Busche | Dynamic formation of a solid-liquid electrolyte interphase and its consequences for hybrid-battery concepts[END_REF] .

The objective of the present work is to characterize the in-depth spatial distribution of SEI species at the LTO electrode surface over cycling by coupling ToF-SIMS (surface and depthprofile experiments) and XPS using two different sources (Al: 1486.6 eV and Ag: 284.3 eV with a depth of analysis of 5-10 nm and 10-20 nm respectively). The deposition of manganese species at the LTO electrode surface is particularly followed. Moreover, the interactions between the two electrodes during cycling are investigated, which can directly influence on the capacity and the stability of cells. Finally, the in-depth spatial distribution of inorganic and organic SEI molecular species upon cycling is reported for the first time to better understand the SEI impact at the LTO electrode surface on batteries electrochemical performances.

EXPERIMENTAL SECTION

Cell preparation and cycling protocol

The electrodes formulation requires first the preparation of an ink which is then coated on a current collector (in aluminum), calendered and then dried (100°C under vacuum for 12 h to remove residual water). To prepare this ink, 93 wt% of anhydrous active material (LTO, NMC or LMO) supplied by SAFT, 4 wt% of carbon black (CB) as additive with a specific surface of 65 m 2 .g -1 , (99.8% purity) Super C65 TIMCAL from Alfa Aesar, and 3 wt% of poly(vinylidene fluoride) (PVdF) used as polymeric binder, supplied by Solvay (Solef 5130), are mixed together using a T25 digital ULTRA-TURRAX homogenizer. Note that the electrodes are prepared so that they are limited by the LTO negative electrode active mass.

The liquid electrolyte is a mixture of ethylene carbonate (EC) and ethylmethyl carbonate (EMC) (3:7 in volume), containing LiPF6 as lithium salt (1 mol.L -1 ) from BASF (battery grade) with a water content <50 ppm. Coin cells are assembled in an argon-filled glove box using 0.25 ml of electrolyte and two polypropylene (Celgard) separators. Using a MPG2 battery cycler (Biologic SA, Claix, France), cells are cycled at 60°C (in a thermostatic oven) under galvanostatic mode at C/10 rate from open circuit voltage for the first cycle, and at 40°C under galvanostatic mode at C/2 rate (based on the LTO theoretical capacity, 175 mAh.g -1 ) for the next hundred cycles. Full-cells cycling experiments are controlled in order to cycle LTO electrode between 1,0 V and 2,0 V: NMC/LTO cells are then cycled between 1,9 V and 3,0 V and LMO/LTO cells between 2,0 V and 3,1 V. After cycling, coin cells are opened in an argon-filled glove box (<1 ppm H2O and O2) and all LTO, NMC and LMO electrodes are extracted and washed with 1.0 ml of DMC for 30 s in a beaker to remove residus of electrolyte. Note that the washing procedure (which can partially dissolve species deposited at the electrode surface) was strictly the same for all samples so that the results are comparable.

Surface Analyses

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). ToF-SIMS analysis are performed using a TRIFT V nanoToF II (Physical Electronics, Chanhassen (MN), US) equipped with a 30 kV Au-LMIG. The electrode samples are fixed with a metallic mask on the sample holder and transferred from the gloves box to the spectrometer using a transfer vessel to avoid any air exposure. For surface analysis, all mass spectra are acquired using the same experimental conditions which allow performing semi-quantitative analysis (comparing the ratio of peaks intensities): the LMIG gun is tuned to deliver Au + primary ions with a DC current of 12 nA over a 50x50 µm 2 raster size, the mass range is fixed between 0 and 800 um and the number of frames is set to 30 (dose: 5x10 [START_REF] Gieu | Influence of Vinylene Carbonate additive on the Li4Ti5O12 electrode/electrolyte interface for lithium-ion batteries[END_REF] ions/cm 2 ) for both positive and negative polarities. Depth profile experiments (series of 30 cycles "analysis/sputtering") are carried out using the previous conditions for surface analysis (Au + , 50x50µm 2 raster size) and a 5 kV Ar + gas gun for etching (with an etching time of 20 seconds for each cycle, a DC current of 300 nA and a sputtered area of 500x500 µm 2 ). Note that the lithium ( 6 Li + and 7 Li + ) peaks appear very intense and have to be blanked during the measurement so that to detect better the smaller peaks. Data processing is performed using ToF-DR software provided by Physical Electronics. All positive polarity mass spectra are calibrated using K + (m/z 39), Ti + (m/z 55) and TiO + (m/z 64) peaks and all negative polarity spectra are calibrated using F -(m/z 19) , O - (m/z 16) and P -(m/z 31) peaks.

X-ray Photoelectron Spectroscopy (XPS)

. XPS are carried out using an Escalab 250 Xi spectrometer (Thermo Fischer Scientific, Waltham (Massachusetts), US) equipped with two monochromatized sources, either Al Kα (hν =1486.6 eV) or Ag Lα (hν = 2984.3 eV).

Electrode samples are put on a sample holder using a metallic clamp and transferred through an argon-filled glove box with low H2O/O2 levels (< 5 ppm) directly connected to the spectrometer in order to prevent any moisture/air contamination. High resolved spectra are recorded using constant 20 eV and 200 eV pass energies for Al and Ag sources respectively.

Analyses are performed using a 0.15 eV step size and short time iterative scans to follow any sample degradation. Only "non degraded" spectra are taken in account and summed for data treatment with CasaXPS software. For spectral calibration, the CF2 component (at 290.7eV) from the PVdF binder in C 1s core peak is considered, as the hydrocarbon and carbon black binding energy (~ at 285eV) can be shifted due to the presence of SEI species containing C-C/C-H and due to the lithiation state of the electrode [12, 13, 22]. Peak fitting is processed with a non-linear Shirley-type background and the peak deconvolution is obtained with a weighted leastsquares calculation method using 30% Lorentzian and 70% Gaussian line shapes and full width at half maximum (fwhm) constraints.

RESULTS AND DISCUSSION

ToF-SIMS analysis

References and Pristine electrode

To make the interpretation of ToF-SIMS data easier, reference compounds from the electrolyte and the electrodes were first analyzed separately in order to establish a database with the main specific secondary ions for each species both in positive and negative polarities.

Only positive spectra are shown in this paper as they present the peaks of interest for our study. Therefore, relevant compounds like LTO-powder active material, PVdF binder, carbon additives, LiPF6 electrolyte salt and LiF were analyzed. Table 1 displays a list of the main peaks detected over a 0-100 m/z range for these compounds in the positive polarity. detected for all cycled electrodes whatever the state of charge, but in lower proportion compared to the other peaks, due to its dissolution during the washing of cycled electrodes.

At the end of the first charge (figure 2a and2c), the main positive secondary ion detected is Li2F + fluorine species for both LTO electrodes facing NMC and LMO, which is characteristic of the SEI formation from LiPF6 salt decomposition [START_REF] Daigle | Schiff Base as Additive for Preventing Gas Evolution in Li4Ti5O12-Based Lithium-Ion Battery[END_REF] . Organic species like carbonates or polymeric species m/z = 32 (CH4O + ) and m/z = 59 (C4H4Li + ) related to the EC/EMC solvent degradation, are also detected but in lower proportion. Moreover, specific secondary ions m/z = 48 (Ti + ), m/z = 64 (TiO + ) of the LTO active material are still observed.

Thus, ToF-SIMS evidences the covering process of the LTO electrodes by a thin (<5nm) and inhomogeneous (as the electrode compounds are still detected) surface layer since the first cycle, which is consistent with XPS results previously obtained in our group [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . At the end of the first discharge, SEI fragments (Li2F + and C4H4Li + ) are still visible on the mass spectra for the two systems but, the intensity of Ti + peak increases, which highlights the dissolution process of the surface layer formed upon charge, as already observed by XPS [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Note that this SEI dissolution phenomenon at the discharge state is partial; a passivation layer is still covering the surface of the negative electrode at the end of the first cycle, as similarly observed on graphite by Zhuo et al. using ToF-SIMS analysis [START_REF] Zhuo | Breathing and oscillating growth of Solid-electrolyte-interphase upon electrochemical cycling[END_REF] . Moreover, the ratio of the peaks intensities of Ti + /Li2F + and Ti + /C4H4Li + are respectively lower for the electrode cycled facing LMO (0.2 and 0.8) than facing NMC (0.4 and 1.6). This suggests that the SEI at the LTO electrode cycled facing LMO is thicker than the one cycled facing NMC, which is once again consistent with our previous XPS results [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . For the LTO/LMO system, manganese Mn + ion (fragment at m/z=55), is also detected in positive polarity at the surface of LTO electrodes since the first charge. This manganese comes from the LMO positive electrode dissolution and migrates through the electrolyte to the negative electrode during cycling. Due to the high sensitivity of ToF-SIMS, it is also possible to detect heavy metals (Ni + , Mn + and Co + ) in low intensity at the LTO electrode surface facing NMC, coming from the NMC electrode dissolution during cycling (figure 3). Note that the detection of elemental metals in the ToF-SIMS positive spectrum is mainly due to ionization process of SIMS, it does not mean they exist at the metallic state at the LTO electrode surface; these Mn + , Ni + and Co + secondary ions are likely to come from molecules containing these metals, like MnF2 for the LTO/LMO system, as suggested by XPS [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Indeed, this dissolution phenomenon of metals was already observed by XPS in our previous study for the LTO/LMO full cell but not for the LTO/NMC one. The number of counts for Mn + detected by ToF-SIMS at the LTO electrode surface cycled facing LMO is significant (around 2000 counts) whereas the one for LTO electrode cycled facing NMC is weaker (around 400 counts) so that it cannot be detected by XPS. These metals dissolution/migration processes are a proof of interactions between the two electrodes during cycling even for the LTO/NMC full cell. The presence of manganese in greater quantity over the LTO electrode surface vs LMO can destabilize the SEI and boost parasitic reactions at the electrode surface; which can entail the additional capacity loss observed for this system [START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF][START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Interestingly, ToF-SIMS also reveals (figure S1) the presence of Titanium (coming from the LTO electrode dissolution) in low intensity at the positive electrode surfaces after cycling which is not possible to detect by XPS. Even if the formation of layers at the surface of positive electrodes had only a limited impact on the electrochemical performance of the systems in these conditions, this is another proof of interactions between electrodes: from the positive electrode to the negative one but also from the negative electrode to the positive one.

After the hundredth charge (figure S2), the same main fragments are detected for both systems at the LTO electrode surfaces as after the first charge: m/z = 14 (Li2 + ) and 33 (Li2F + ) from LiPF6 salt decomposition; 32 (CH4O + ) and 59 (C4H4Li + ) from solvent degradation. At the end of the 100 th cycle, the intensity of the C4H4Li + peak is weaker compared to the 100 th charge whereas the number of counts detected for the Ti + peaks increase on both electrodes, which is in agreement with the SEI dissolution process during discharge already observed at the first cycle. Moreover, the ratios Ti + /Li2F + and Ti + /C4H4Li + are respectively lower for the LTO electrode cycled facing LMO (0.2 and 1.2) than for the one facing NMC (0.3 and 1.8), which is consistent with a thicker SEI layer for the LTO/LMO cell. Regarding the detection of Mn + , it is still detected at the LTO electrode surface facing LMO after 100 cycles and in similar quantity than after one cycle; Ni + , Co + and Mn + are also weakly detected on the LTO electrode surface facing NMC after 100 cycles. No difference in the amount of Mn-species on the LTO electrode (vs LMO) surface can be noticed on the ToF-SIMS spectra between charges and discharges and between the 1 st and the 100 th cycles. The concentration of Mnspecies seems therefore independent of the charging state and it seems that most of the dissolved Mn is deposited since the first charge.

In order to have more information about Manganese and SEI species spatial distribution at the LTO electrode surfaces and because of the high in-depth resolution of ToF-SIMS, depth-profiles experiments (series of "analysis/sputtering" cycles) were performed after the first and the hundredth cycles for the two systems. No sputtering experiment has been performed on the pristine electrode as the objective is to analyze the 3D distribution of SEI specific species deposited at the LTO electrode surface during cycling. Figure 4 presents the concentration depth profiles of Ti + (representing the electrode active material, in blue),

Li2F + (representing inorganic SEI species, in green), C4H4Li + (representing organic SEI species, in yellow) and Mn + (in red) secondary ions obtained in the positive polarity for the LTO electrode cycled vs LMO after the first cycle, over 300 seconds of etching. Organic species (C4H4Li + ) concentration decreases quickly with the etching whereas a distinct rise of the bulk active material species (Ti + ) concentration is observed. Concerning LiF related species (Li2F + ) and Manganese compounds, their concentration are gradually decreasing from the extreme surface to the bulk of the electrode but with a "lower" concentration gradient than C4H4Li + . Therefore, organic species are located mostly at the top surface of the LTO electrode while LiF and Manganese species are located both at the top surface and more deeply in the electrode surface layer, which is in agreement with a previous study by Nordh et al. [START_REF] Nordh | Depth profiling the solid electrolyte interphase on lithium titanate (Li4Ti5O12) using synchrotron-based photoelectron spectroscopy[END_REF] . Mn + ) is plotted over the analyzed area (50µm x 50µm = x and y dimensions) versus the etching time (the z axis, 800s). For a better visibility, the z axis is over-dimensioned compared to the other axes. Each species has a 3D spatial distribution over the analyzed volume with one specific color (Ti + in blue, Li2F + in green, C4H4Li + in yellow and Mn + in red). When a color is not appearing anymore on the image, it means the signal intensity of the secondary ion is too low in the mass spectrum to be plotted. The final 3D map corresponds to the overlay of all 3D spatial distributions of the considered species. Note that it was not possible to reconstruct the 3D spatial distribution of Mn + , Co + and Ni + secondary ions detected on the LTO electrode surface cycled facing NMC as their signal intensities in the mass spectra were too weak.

After the first cycle for the LTO/LMO system, organic species (in yellow) are After the hundredth cycle, organic species are still mostly concentrated at the top surface of the LTO electrode for both systems. Note that for LTO/LMO cells, organic species are spread more homogeneously all over the LTO electrode surface compared to the first cycle, which could be related to a surface structural rearrangement and a stabilization of the SEI after long cycling, meaning a better passivation of the LTO electrode in correlation with a stabilization of the capacity [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF][START_REF] Daigle | Schiff Base as Additive for Preventing Gas Evolution in Li4Ti5O12-Based Lithium-Ion Battery[END_REF][START_REF] Nordh | Depth profiling the solid electrolyte interphase on lithium titanate (Li4Ti5O12) using synchrotron-based photoelectron spectroscopy[END_REF] . 3D imaging also evidences that a greater amount of LiF inserts deeper within the SEI of the LTO electrode after hundred cycles than after the first cycle. Regarding the signal of 55 Mn + , its profile after 100 cycles is very similar to the one after the first cycle: it is gradually decreasing from the extreme surface layer to the inner of the SEI, which confirms that Manganese deposition mainly occurs at the first cycle. For LTO/NMC system, organic compounds are still mainly located at the extreme surface after hundred cycles, meaning a stable SEI correlated with a stable capacity, while LiF related species insert more deeply into the SEI of the LTO electrode after 100 cycles than after one cycle, like for LTO/LMO cells. However, more LiF related species are formed for LTO/LMO cells than for LTO/NMC ones after 100 cycles, which can be related to the longer time spent at a higher potential, degrading more LiPF6 salt and producing LiF in greater proportion, which is consistent with a ticker SEI for the LTO/LMO system.

Several hypothesis can be mentioned to explain why LiF is found in the inner part of the SEI for both systems: as suggested by Nordh et al [START_REF] Nordh | Depth profiling the solid electrolyte interphase on lithium titanate (Li4Ti5O12) using synchrotron-based photoelectron spectroscopy[END_REF] , it is the first one to be formed from LiPF6 salt degradation, but the presence of LiF both at the extreme surface and deeply in the SEI during all the cycling suggests that part of LiF may be formed elsewhere, particularly at the positive electrode surface; LiF related species then diffuse through the electrolyte to be finally deposited on the LTO electrode surface. Moreover, the smaller size of LiF related species compared to organic ones can also play a role in their spatial distribution into the SEI.

Consequently, these ToF-SIMS experiments highlight that salt degradation products (mostly LiF) and active materials dissolution species (Mn, Ni and Co) are also produced at the positive electrodes and then migrate to the negative electrodes surface where they are either reduced or simply deposited. These degradation phenomena seem to be accelerated at high voltage (here for LMO electrode) which is in agreement with the results of Madec et al. [START_REF] Madec | Exploring Interactions between Electrodes in Li[NixMnyCo1xy]O2/Graphite Cells through Electrode/Electrolyte Interfaces Analysis[END_REF] .

In order to complement and confirm the previous ToF-SIMS results, XPS analysis were performed using two different incident photons beams, either Al (1486.6 eV, depth of analysis of 5-10 nm) or Ag (2984.3 eV, depth of analysis of 10-20 nm), so that to obtain chemical information at the top of the electrode surface (with Al source) and deeper into the electrode (with Ag source). Note that with Al source, quantification is carried out using Scofield's relative sensitivity factors [START_REF] Scofield | Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV[END_REF] whereas with Ag source, the choice was made to calculate only the relative percentages (% reported in figure 6) which are sufficient to qualitatively compare the evolution of the different components in spectra. Atomic concentrations (at. %) of the different chemical environments identified by XPS with Al source at the surface of the cycled LTO electrodes are reported in Table S1. spectra at the LTO electrode surface than after the first cycle with Al source, but in greater quantities which proves the growth of the SEI thickness over cycling. With Ag source, the intensity of these components decreases which proves that organic species are still mainly located on top of the LTO electrode surface after the hundredth cycle like after the first one.

Concerning fluorine species, the ratio between CF2 and LiF peaks is independent of the number of cycles but it is greater when the depth of analysis is deeper. This result proves again that LiF diffuses into the SEI upon cycling, which is in agreement with ToF-SIMS results.

Finally, titanium, carbon, oxygen and fluorine species from LTO electrode active material are detected with Al source during all the cycling which proves that the thickness of the interfacial layer is lower than 10 nm for both systems as the XPS analysis depth is about 5-10 nm. Moreover, considering the 10-20 nm analysis depth of XPS with Ag source, this result allows estimating the ToF-SIMS sputtered depth (figures 4 and 5) between 20 nm and 30 nm, as Ti + active material (in blue) is detected in majority at the end of the etching.

CONCLUSIONS

LTO electrodes cycled facing either LMO or NMC were characterized by ToF-SIMS (surface analysis, depth-profile experiments and 3D image reconstruction) and XPS using 2 different sources (Al and Ag), which gives new insights on the in-depth distribution of SEI species over cycling and proves the consistency of the results obtained with these two techniques.

For both systems, it was found that organic SEI species are located exclusively at the top surface of the electrode whereas inorganic compounds like LiF are present both at the top of the surface layer and more deeply into it since the first cycle; this insertion of LiF within the SEI of the electrode is even more pronounced after 100 cycles. Moreover, Manganese is detected at the LTO electrode surface cycled vs LMO as well as deeply into the surface layer since the first cycle and its proportion does not seem to evolve with cycling; this proves the dissolution of the manganese at the LMO electrode, its migration through the electrolyte and its deposition and insertion into the SEI formed at LTO electrode during the first cycle.

Furthermore, the trace-sensitivity of ToF-SIMS also allows to detect small amounts of Ni, Mn and Co metals at the LTO electrode cycled facing NMC, as well as low amount of titanium at the LMO or NMC electrodes, which confirms the interactions between negative and positive electrodes during cycling.

The presence of manganese compounds and the formation of a thicker layer on the anode facing LMO than facing NMC, especially due to greater quantities of fluorinated species entering deeply in the SEI, can impede the diffusion of lithium and explain the decrease of the retention capacity for the LTO/LMO system. Overall, even if LiPF6 salt may be the first compound to degrade, our ToF-SIMS results highlight for the first time failure mechanisms which seem to be driven by oxidative parasitic reactions at the positive electrode and interactions with the negative one. To go further into this study, other types of salts like for instance LiClO4 could be tested [START_REF] Tarascon | New electrolyte compositions stable over the 0 to 5 V voltage range and compatible with the Li1+xMn2O4/carbon Li-ion cells[END_REF] .

Finally, this study proves that ToF-SIMS, thanks to its high sensitivity and high indepth resolution, is well adapted to characterize very thin surface layers commonly found at the electrolyte/electrode interfaces, whose properties greatly influence batteries electrochemical performances. Consequently, ToF-SIMS is a very promising analysis tool in the field of battery science, as a complement to XPS technique.

  Moreover, LTO pristine electrode was analyzed before cycled electrodes; the ToF-SIMS spectrum collected in the positive polarity in the 0-100 m/z range is presented in figure1. A large number of fragments is observed and assigned to organic fragments, for instance m/z = 26 (C2H2 + ), 27 (C2H3 + ) and 29 (C2H5 + ) from the carbon black (in blue) and m/z = 31 (CF + ), 51 (CHF2 + ), 69 (CF3 + ), 77 (C3H3F2 + ) and 95 (C2H2F3 + ) from the PVdF binder (in green). The secondary ions characteristic of the LTO active material are also detected: m/z = 14 (Li2 + ), 48 (Ti + ), 49 (TiH + ), and 64 (TiO + ). The ionization process causes the formation of species like TiF + (m/z = 67) or TiO3H + (m/z = 97) which come from the fragmentation of bigger molecules and/or re-arrangement of ions. Potassium (m/z=39), which has a high ionization yield, is also detected and attributed to surface contamination, coming from the chemical synthesis of the LTO powder.

Figure 1 .

 1 Figure 1. ToF-SIMS mass spectrum of the pristine LTO electrode over 0-100 m/z range in

Figure 2

 2 Figure 2 presents the mass spectra obtained over 0-100 m/z range of cycled LTO negative

Figure 2 .

 2 Figure 2. ToF-SIMS positive ion spectra over 0-100 m/z range for LTO electrodes cycled

Figure 3 .

 3 Figure 3. ToF-SIMS positive ion spectra of LTO electrodes cycled facing LMO a) and NMC b) after the first cycle over Mn + , Ni + , and Co + specific m/z ranges.

Figure 4 .Figure 5

 45 Figure 4. ToF-SIMS depth profiles of Ti + (blue), Li2F + (green), Mn + (red) and C4H4Li +

  located exclusively at the top surface of the electrode while LiF related species (in green) and Manganese (in red) are present both at the extreme surface and more deeply in the electrode surface layer. The same trend is observed for the LTO electrode cycled facing NMC, organic species (in yellow) are accumulated only at the top of the electrode surface, whereas LiF compounds diffuse more in the surface layer. Moreover, LiF species seem to insert deeper in the SEI of LTO electrode facing LMO than in the one of LTO electrode facing NMC after one cycle. Concerning organic species, their 3D spatial distribution seems more inhomogeneous over the LTO electrode surface cycled facing LMO than facing NMC, which can be explained by the presence of manganese inducing a destabilization of the SEI and a decrease of electrochemical performances of LTO/LMO cells. On the contrary, the SEI of LTO/NMC cells seems more protective (organic compounds are homogeneously spread over the surface) which is in good agreement with the coulombic efficiency evolution, indicating the passivation of the LTO electrodes (vs NMC) since the first cycle.

Figure 5 .Figure 6

 56 Figure 5. 3D images reconstruction from ToF-SIMS depth profile experiments showing the spatial distributions of Ti + , C4H4Li + , Li3F2 + and Mn + secondary ions over 50µm (x axis) by

Figure 6 .

 6 Figure 6. C 1s a), O 1s b) and F 1s c) XPS spectra of cycled LTO negative electrodes at the

Table 1 .

 1 Main secondary ions detected by ToF-SIMS for references powders (LTO, PVdF,

		LTO Powder	Reference PVdF	Reference CB	References LiPF6/LiF	Reference Li2CO3
	m/z	ID	m/z	ID	m/z	ID	m/z	ID	m/z	ID
	7	Li	12	C	13	CH	14	Li2
	27	C2H3	31	CF	14	CH2	19	F
	30	Li2O	41	H3F2	15	CH3			30	Li2O
	39	K	43	C3H3O	26	C2H2	33	Li2F	31	Li2OH
	48	Ti	51	CHF2	27	C2H3	59	Li3F2	37	Li3O
	64	TiO	69	CF3	29
	97	TiO3H	77	C3H3F2		C2H5	85	Li4F3	81	Li3CO3

CB, LiPF6/LiF, Li2CO3) over a 0-100 m/z range in the positive polarity (ID stands for identification).

  [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF][START_REF] Verma | A review of the features and analyses of the solid electrolyte interphase in Li-ion Batteries[END_REF] . All these results confirm the degradation process of the electrolyte LiPF6 salt and the solvent after the first cycle and the deposition of related species at the surface of the negative electrode. With Ag Lα radiation (second line), the SEI components are still visible on the O 1s spectrum but in lower relative intensity than with Al Kα radiation, which confirms that organic species are mainly located at the LTO electrode surface. This is accompanied by a decrease of C-C/C-H (from surface contamination and SEI species) component intensity at 285.0 eV on the C 1s spectrum (from 19.1% to 12.3%). However, regarding F 1s spectra, LiF is in greater proportion than CF2 component with Ag source (34% compared to 20% with Al source), which means LiF species are present deeper in the LTO electrode surface layer than the other SEI species, which is consistent with ToF-SIMS depth-

	profile results.

ACKNOWLEDGEMENTS

The work was supported by funding from The French National Research Agency via the project ANR-11-EQPX-0027 MARSS and SAFT company.

Competing financial interests

The authors declare that they have no competing financial interest.