dataesr/scientific-paragraphs-categorization huggingface dataset:
DISCUSSION We show, in a large multicentre real-world database of MBC patients, that breast cancer molecular subtype strongly impacts the occurrence, kinetics and prognosis of CNS metastases. Only few data are available regarding prevalence and incidence rates of CNS metastases in breast cancer patients, as, to our knowledge, no nationwide reporting system for breast cancer patients diagnosed with CNS metastases exists. The data come from autopsy studies and population-based studies with various methodologies. Our analysis of the EMSE MBC database provides an estimate of the cumulated incidence rate of CNS metastases among MBC patients of 24. 6%. At the time of metastatic disease diagnosis, 7. 2% of patients were diagnosed with CNS metastases (12. 3% in patients with HER2+ tumours). In a population-based study from the "Surveillance, Epidemiology and End Results" (SEER) database, 614/7575 patients (8.
4%, 40. 6% and 25. 6% of patients with CNS metastases. This compares with 18. 8%, 25. 5% and 17. 7% of patients in the whole ESME database and with 16. 6%, 26. 4% and 19. 5% in a series of 11 011 unselected stage I-III breast cancer patients. 40 These proportions in patients with CNS metastases are consistent with previous data for HR-negative  19, 35, 36, 41, 42  and triple-negative tumours,  15, 21, 41, 42  but not for HER2-positive tumours. Indeed, previous reports have described higher proportions (34. 8-43. 1%) Fig. 2  CNSM-FS according to the tumour biology of HER2-positive tumours amongst patients with BM. 21, 41, 42  In our study, patients were included more recently, and HER2-targeted therapies might have been used more often in the early and metastatic phase of the disease. This could have led to a selection bias favouring HER2-negative tumours among patients with CNS metastases.
In our study, the median time interval between MBC diagnosis and the occurrence of CNS metastases was 10. 8 months amongst patients who developed CNS metastases, compared with 10-19 months in previous studies. 12, 21, 39 Because patients with CNS metastases occurring after the closing date of study analysis, lost to follow-up or dead without CNS metastases were censored at the closing date of analysis, this time interval is quite prolonged as compared with reported survivals of MBC patients (37. 2 months in the ESME MBC database  30  ), suggesting an increased risk of CNS metastases in long MBC survivors (consistently with clinical observation in daily practice). We confirmed a significant impact of tumour biology, HER2 positivity and HR negativity associated with a shorter CNSM-FS. 12, 21, 34 These patients at high risk of CNS metastases could potentially benefit from screening strategies, as CNS metastases can be asymptomatic, in order to improve the patients' outcomes by allowing localised BM treatments in a higher proportion of patients.
Further studies are warranted to better clarify these hypotheses. 12, 34, 46 Moreover, it must be noted that once the BM has occurred, a number of studies have demonstrated a clinical efficacy of systemic treatment including HER2-targeted therapies. 47, 48 he prognosis of patients with CNS metastases is poor. In our study, the median OS after the CNS metastasis diagnosis was 7. 9 months, lower than that reported in previous breast cancerrelated BM series (median OS around 14 months  19, 24  ), possibly linked to differences in the inclusion criteria (in the Sperduto's study, only patients referred for radiation therapy were included, whereas this database includes all patients with BM, whatever the treatment modalities). Another important difference is the fact that we included patients with both BM and LM.
49 e report that a low proportion of patients treated with surgery (2. 3%) or stereotactic radiation therapy (10. 5%) following CNS metastases should be investigated further in another study. Our study has some limitations. First, because the database was not specifically designed to study CNS metastases, it was not possible to distinguish intraparenchymal BM from LM. Second, because of unavailable data, some parameters could not be included in the multiparameter analysis of OS despite their demonstrated prognostic value. Indeed, for example, the number  Fig. 3  OS after CNS metastases diagnosis according to the tumour biology of BM was not described in the ESME MBC database, while it was shown to be prognostic in these patients. Also, it must be acknowledged that the proportion of patients diagnosed with CNS metastases depends on the duration of the patients' follow-up.
ACKNOWLEDGEMENTS We would like to thank  Dr. Hélène de Forges  ( Montpellier Cancer Institute ) for editing the paper. We thank the 18  French Comprehensive Cancer Centers  for providing the data and each ESME local coordinator for managing the project at the local level. Moreover, we thank the  ESME Scientific Committee  members for their ongoing support. ESME central coordinating staff:  Head of Research and Development: Claire Labreveux. Program director:  Mathieu Robain. Data Management team:  Coralie Courtinard ,  Emilie Nguyen ,  Olivier Payen ,  Irwin Piot ,  Dominique Schwob  and  Olivier Villacroux. Operational team:  Michaël Chevrot ,  Daniel Couch ,  Patricia D'Agostino ,  Pascale Danglot ,  Tahar Guesmia ,  Christine Hamonou ,  Gaëtane Simon  and  Julie Tort. Supporting clinical research associates:  Elodie Kupfer  and  Toihiri Said.
Most of the methodological developments of diatom DNA metabarcoding, presented in this article, were performed from this dataset. For river sites sampled in metropolitan France, they were selected to meet two objectives: (i) complete the DNA barcode reference database for some missing metropolitan important diatom species and (ii) compare ecological status assessments obtained using morphological and molecular approaches on a large scale. In collaboration with the regional environmental services in charge of WFD monitoring (DREAL), 461 sites were chosen to meet these two objectives (Figure  2 ). For the first purpose (completing the reference database), on the basis of previous morphological inventories, we selected sites known to contain a high abundance of taxa missing in the database (in particular rivers located in the Pyrenees, the Alps, the Massif Central and Brittany).
(2018). The pool of PCR products is then sent to a sequencing platform (e. g. GeT-Plage Toulouse (France), PGTB Bordeaux (France)) which carried out: (i) purification of PCR products; (ii) preparation of sequencing libraries by adding a sample-specific tag and sequencing adapters to the PCR products; (iii) preparation of the final "pool" corresponding to the equimolar mixture of the sample libraries; (iv) paired-end sequencing of the library pool performed with the Illumina Miseq technology  (Keck et al. 2018). The first processing steps of the sequence data ("demultiplexing" and "pair contigating") were performed by the sequencing platform which provides a "fastq" file. All bioinformatics steps were carried out with the Mothur programme  (Schloss et al. 2009 ). The second processing step is dedicated to cleaning sequence data by eliminating sequences of incorrect length, containing errors (e.
g. ambiguous bases "N", homopolymers), as well as chimeric sequences. Next, a "de-replication" step allows the grouping of similar sequences together in order to simplify the dataset and facilitate further processing. Only unique sequences represented by more than 2 reads, over the full dataset, are conserved. These de-replicated DNA sequences are then compared with the Diat. barcode reference base  (Rimet et al. 2019; previously called RSyst::diatom, Rimet et al. 2016 ) using a naïve Bayesian classification method  (Wang et al. 2007) , to assign a taxonomy to each sequence (confidence level > 85%). Only the sequences assigned to the phylum "Bacillariophyta" (diatoms) are kept for further analysis and grouped into OTUs (Operational Taxonomic Units), according to their genetic similarity (> 95% similarity threshold).
Calculation of water quality indices Although the BDI is the French diatom index used for WFD, the Indice de Polluosensibilité Spécifique (IPS)  (Cemagref 1982)  was chosen in this study because it includes a greater number of diatom species into its calculation and is frequently used in WFD intercalibration exercises  (Coste et al. 2009; Kelly et al. 2014). For each site, morphological and molecular IPS values were calculated  from morphological and molecular inventories using the Omnidia software (version 6. 0. 2s) (Lecointe et al. 1993). With regard to molecular inventories, it has been shown that the copy number of the rbcL gene is directly correlated to the biovolume of diatom species  (Vasselon et al. 2018). Compared to the morphological approach, the molecular approach, therefore, tends to overestimate species with a high biovolume.
Development and optimisation of DNA metabarcoding of diatoms The use of diatom DNA metabarcoding as a tool for assessing the ecological status of watercourses has been explored over the past 10 years (beginning in France with the PhD thesis of L. Kermarrec (2012) ). During this period, complementary studies have made it possible to remove certain methodological obstacles, such as: the validation of the suitability of the rbcL DNA barcode for diatoms identification  (Kermarrec et al. 2013 (Kermarrec et al. , 2014)) , the definition of good practices for the creation of a barcode reference database  (Zimmermann et al. 2014)  or the implementation of the first complete methodological framework from the biofilm sample to the assessment of the ecological status of the river site from where it was sampled  (Kermarrec 2012; Visco et al.
2015; Zimmermann et al. 2015). However, some methodological biases were still limiting the production of reliable molecular taxonomic inventories through DNA metabarcoding, particularly in terms of taxon quantification, thereby producing weak ecological status assessments. As a result, projects were carried out on the French rivers of Mayotte and of the France metropolitan territory in order to consolidate the DNA approach by optimising certain crucial parameters to increase bioassessment efficiency, namely: the coverage of the barcode reference database, the DNA extraction lab step and the quantification of diatom taxa from rbcL HTS data.
Development and completion of the barcode reference database "Diat. barcode" The ability of DNA metabarcoding to accurately identify diatom taxa in an environmental sample is directly related to the quality of the DNA barcode reference database used to assign a taxonomy to OTUs. This relies largely on its diversity coverage, with one quality criterion being its completion (ideal objective of hosting at least one reference DNA barcode per diatom species,  Weigand et al. 2019 ) and another quality criterion being the level of information provided for each barcode (DNA sequences, taxonomic and autecological information, related metadata etc. ). Although there are several international reference databases gathering genetic information obtained by the scientific community, such as the National Center for Biotechnology Information (NCBI)  (Federhen 2012)  or the Barcode Of Life Data system (BOLD)  (Ratnasingham and Hebert 2007) , the information they contain is generally very heterogeneous in terms of quality (e.
g. origin of DNA sequences, wet-lab protocols) and reliability (e. g. traceability of sequences, taxonomic identification). For this reason, an expert reference database has been developed for diatoms: R-Syst::diatom  (Rimet et al. 2016) , for which the traditional completion strategy is based on the isolation and culture of environmental cells, their taxonomic identification and the sequencing of the gene of interest from the DNA extracted from the monospecific culture. This diatom database incorporates information about the genes most frequently used as DNA barcodes for diatom metabarcoding: 18S (ribosomal RNA 18S) and rbcL. Data from algae culture collections (e. g. Thonon Culture Collection, http://www6. inra. fr/carrtel-collection_eng/), the NCBI database and scientific articles are used to annually update this database.
Before being integrated into the database, each new DNA sequence is submitted to several curation steps in order to keep only quality sequences with correct taxonomic identification  (Rimet et al. 2016). The data from strains that were isolated, identified and sequenced during the French projects have thus contributed to the development of this database, in particular with the addition of 112 rbcL sequences, corresponding to 29 Mayotte diatoms species, some of them tropical or endemic. Despite these efforts, the completeness of this database remains partial as stated by  Weigand et al. (2019)  in their large gap analysis. For example, of the 100 taxa most frequently identified in morphological inventories during the last monitoring campaigns (1992 to 2014) of French metropolitan rivers, 47 were still absent from the database in 2016 (version 6).
As these taxa are very abundant, their weight in the calculation of diatom indices is important and not detecting them is detrimental to the efficiency of molecular indices. To overcome the traditional completion strategy, which is time-consuming and has a moderate success rate, we recently proposed an alternative strategy to recover barcodes from environmental DNA sequences  (Rimet et al. 2018). Based on morphological inventories, we focused on low-diversity samples, characterised by diatom assemblages dominated by one taxon absent from the reference database. It was therefore highly likely that a large majority of the metabarcoding DNA sequence reads that were unidentified correspond to this missing taxon. Based on several validation criteria (morphological, genetic, phylogenetic), we associated this environmental DNA sequence with the taxonomy of the species observed in morphology and integrated it into the reference database  (Rimet et al.
2018). This operational approach allowed us to easily and quickly fill crucial gaps in the reference database by targeting abundant taxa, important for index calculation. Thus, from the French metropolitan river samples, we could identify 61 "environmental" rbcL barcodes corresponding to 21 species of diatoms, 18 of which were amongst those 100 most frequently identified species in France. Moreover, since 2018, this database has been enriched with collections from other countries (England, Russia) and is managed by a collective of International diatom experts  (Rimet et al. 2019 ), which gives it significant visibility  (Pawlowski et al. 2018). This international evolution has led to a change in the name of the reference database now called Diat. barcode, which is hosted and available in open-access on http://www6.
Potential of diatom DNA metabarcoding to characterise river communities Molecular inventories have identified 66 diatom species in Mayotte rivers  (Vasselon et al. 2017b ) and 288 species in those of metropolitan France  (Rivera et al. 2020). Morphological taxonomic inventories have identified 204 and 783 species of diatoms in these same rivers, respectively, which is about three times higher. The structures of diatom communities (richness and relative abundances of species), obtained with the two approaches, show a significant but partial correlation (Mantel test; Mayotte: r = 0. 42, Metropolitan France: r = 0. 63). There are several reasons for the discrepancies observed between morphological and molecular taxonomic inventories: Gaps in the reference database: despite the efforts made to complete the DNA barcode reference database, it remains incomplete, avoiding the full assignment of DNA read sequences to diatom species.
Unassigned sequences represent 12. 4% of the dataset in metropolitan France and 40. 7% in Mayotte. This percentage is higher in Mayotte due to the poor knowledge of diatom diversity in this tropical island, which includes endemic and tropical species that are not yet morphologically described  (Vasselon et al. 2017b ). Taxonomic differences: the presence of morphologically-related species and the constant evolution of diatom taxonomy  (Mann et al. 2016 ) make it difficult to obtain reliable taxonomic identification with microscopy (Figure  1b ). In addition, morphological inventories were produced by several taxonomic experts, which may have led to different identifications of the same morphological entity (species). This phenomenon tends to artificially increase the number of species detected through the morphological approach and thus create variability in ecological status assessments.
2017) , different kinds of false positives can be detected by both approaches (Figure  1c-e ). Although this artificially increases the discrepancy between methods and the number of species detected, these species generally have a low relative abundance and therefore a moderate impact on final index values. The taxonomic differences between molecular and morphological inventories mainly affect species present in low abundance. Overall, dominant species, when present in the reference database, are properly detected by both approaches. The application of CFs to molecular inventories has thus made it possible to obtain DNA sequence proportions closer to cell proportions obtained by microscopy. This correction was most effective on molecular inventories from Mayotte samples. This was probably due to the fact that they were strongly dominated by genera with high biovolume (Eunotia: 31.
9 points higher than the morphological IPS values  (Vasselon et al. 2018 ). This overestimation is partly explained by the lack of some poor quality indicator taxa in molecular inventories. Parts of them were not referenced in the database. Moreover, some of them could still not be identified in metabarcoding, despite the presence of a reference barcode in the database. This is particularly the case of Nitzschia inconspicua, which was a paraphyletic species whose taxonomy was poorly defined until recently  (Rovira et al. 2015). This taxonomic instability limited its identification in metabarcoding at the genus level. As this species was abundant in some samples (up to 33. 5% of the valves observed in microscopy) and is an indicator of rather degraded environments, we failed to correctly identify it, thereby raising the IPS scores.
These taxonomic problems were solved in the latest version (version 7) of the Diat. barcode database  (Rimet et al. 2019 ) and this species is now correctly identifiable. With regard to rivers in metropolitan France, the ecological status obtained using both approaches are congruent (Figure  6 ). Some 66. 2% of the samples belong to the same quality class, 31. 8% of the samples have a deviation of one class and only 2% of the samples have a deviation of two classes. In addition, the average difference between the morphological and corrected molecular IPS values is only 1. 5. These results are similar to what could be obtained by comparing morphological IPS scores produced by two different diatom experts, with differences of three points already observed for the same sample, corresponding to a difference of one quality class  (Kahlert et al.
For diatom metabarcoding, two CEN technical reports TR 17245  (CEN 2018a) and TR 17244, (CEN 2018b)  are already available for the field sampling and the management of reference databases, respectively. The recently launched EcoAlpsWater (INTERREG Alpine Space) project aims at pursuing these efforts at the scale of Alpine region by involving scientists and environmental agencies from 6 countries (France, Italy, Switzerland, Slovenia, Germany and Austria). Further work is required to propose European standards at each step of the workflow, to ensure proper sample preservation, DNA extraction and sequencing, data analysis etc. Transfer knowledge: it is crucial to set up training courses to enable the transfer of technical and theoretical knowledge to environment managers and stakeholders. The definition and transfer of good practices for implementing the molecular approach will be essential for their effective use in river monitoring and future implementation in environmental regulations.
Deploying the molecular approach in parallel with the morphological during the next WFD monitoring cycle on a large set of WFD river sites will require the definition of a common strategy for: (i) sample collection and storage, (ii) laboratory molecular biology steps (DNA extraction, PCR, sequencing), (iii) bioinformatics processing of DNA sequencing data and (iv) calculation of diatom indices and evaluation of water quality status. Considering the high throughput provided by the molecular approach, it is necessary to meet new needs, such as the sustainability of DNA barcode reference databases, the storage of samples and sequence data and the maintenance and improvement of the molecular approach. To provide a solution for these needs, it is necessary to bring together the different actors (e.
Acknowledgements We would like to thank the  French Agency for Biodiversity (AFB)  for funding the studies on the rivers of Mayotte ( 2013-2018 ) and metropolitan France (2016-2018), as well as  INRA , which directed this research. We would also like to thank all the actors who participated in this work through sampling and morphological analysis:  French DREALs (Aquitaine -D. Sagnet, Auvergne -F. Véry, Bourgogne -V. Peeters, Bretagne -G. Gicquiaud, Centre -S. Saadat and C. Karabaghli, Corse -I. Boulier, Franche-Comté -E. Parmentier, Ile-de-France -O. Cortial, Limousin -J. M. Vouters, Lorraine -D. Heudre, Midi-Pyrénées -E. Seigneur, Pays-de-la-Loire -D. Guillard, Rhône-Alpes -R. Chavaux, Normandie -F. Pétel, PACA -V. Vassal, Picardie -D. Fouré, Nord-Pas-de-Calais -N. Zydek), French Water Agencies (Artois-Picardy -C.
Data availability The data from the WFD river network on the French metropolitan territory are available at the following open-access repositories: -Sampling sites and floristic lists: https://data. inra. fr/dataset. xhtml?persistentId=doi%3A10. 15454%2FWNI6FQ -Raw sequencing data: https://data. inra. fr/dataset. xhtml?persistentId=doi%3A10. 15454%2F9EG5Z4 The data from the WFD river network on the French overseas department of Mayotte are available at the following open-access repositories: -Sampling sites and floristic lists: https://data. inra. fr/dataset. xhtml?persistentId=doi:10. 15454/6Z5IAH&version=1 -Raw sequencing data: https://zenodo. org/record/400160#. XbFuKdU6-Uk.
All assays were performed at least 3 times independently, several days or weeks apart. Gene Expression Analysis by Northern Blot, RNAseq, and RNA Decay. Total RNA extraction and Northen blot analysis were performed as previously described  (36). For RNAseq analysis, RNAs from cultures grown to OD 600 = 2 at 30 °C were treated with DNase I and purified. Following ribosomal RNA depletion, strand-specific cDNA libraries were sequenced (Illumina) and enriched transcripts were determined using DESeq2  (47). Half-lives of RocR and RocRp were determined by RNA decay experiments. Bacterial cultures were treated with 100 μg/mL of rifampicin to stop transcription. RocR and RocRp levels were detected by Northern blot analysis and quantitated by densitometry with ImageJ and fit to a first-order exponential decay.
The resulting PCR products, a mix of RocR (86 bp) and RocRp (85 bp), were digested with NruI, which cleaves only RocRp, giving 2 fragments of 42 and 43 bp. The restriction digests were quantified by capillary electrophoresis. Accession Numbers. Genome assemblies have been deposited in GenBank (Bioproject PRJNA528641). RNAseq reads have been deposited to the European Nucleotide Archive (PRJEB31835). Sequence data of all clinical isolates are available from the European Nucleotide Archive (PRJEB15241)  (48).