dataesr/scientific-paragraphs-categorization huggingface dataset:
/github. com/MIAPPE/MIAPPE (accessed on 15 August 2021). Alternatively, we recommend downloading the data templates created for the Germinate platform, which are created in accordance with metadata standards, data templates available online: https://github. com/germinateplatform/germinate/tree/master/datatemplates (accessed on 15 August 2021). Templates specifically for quinoa are available at the Quinoa Germinate Database. Available online: http://germinate. quinoadb. org (accessed on 15 August 2021). A MIAPPE spreadsheet tem- plate is published online for guidance, MIAPPE available online: https:/.
the EMPULSE trial. The aim of this paper is to describe the rationale and design of this trial. EMPULSE is a multinational, multicentre, randomized, double-blind superiority trial to evaluate the effects of once daily oral empagliflozin 10 mg compared to placebo on clinical benefit, safety, and tolerability in patients hospitalized for AHF after initial stabilization. The trial is registered at ClinicalTrials. gov identifier NCT04157751, and is being conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice.
the EMPULSE trial. The aim of this paper is to describe the rationale and design of this trial. EMPULSE is a multinational, multicentre, randomized, double-blind superiority trial to evaluate the effects of once daily oral empagliflozin 10 mg compared to placebo on clinical benefit, safety, and tolerability in patients hospitalized for AHF after initial stabilization. The trial is registered at ClinicalTrials. gov identifier NCT04157751, and is being conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice Methods Trial structure and oversight. initiated guidelines. The institutional review board, ethics committee or relevant national competent authority of each participating centre has to approve the study, and all participants provide written informed consent prior to study entry.
Validation of the Social Force Model To validate and calibrate the parameters of the SFM, experimental data by Klüpfel  [28]  is used as reference case. Klüpfel carried out an evacuation exercise of a cinema with 101 students (see Fig. 1 ). While the commercials were running, the movie was stopped and the students were asked to leave the cinema. At the two exits (Route A and B) cameras were installed to record the evacuation of the cinema. The evaluation of the records provided the escape times of each individual. There are some constraints that differ from a real emergency situation pointed out by Klüpfel himself and should be kept in mind:   • Students are not a representative group for an average population. • The students were informed in advance about the procedure. • The students were asked to act carefully to avoid injuries.
Validation of the CFD solver The accuracy of LBM-Large Eddy Simulations using the HRR-BGK model with force terms, which is used in the present work, has been proven in a couple of publications, including complex geometries and pollutant dispersion. The HRR-BGK model with dynamic , which is used in this work, was presented by Jacob et al. [32]. A simple test case with a flow around a cylinder showed very good agreement with measurements and other well established numerical methods. Jacob and Sagaut  [34]  evaluated the method on a test case with a complex urban geometry aiming on pedestrian wind comfort assessment. Reference data is provided by the Architectural Institute of Japan (AIJ) open database and consists of field and wind tunnel measurements of wind velocities in the Shinjuku area in Tokyo, which contains small and large buildings.
Acknowledgments Many thanks to  Patrick Teyou  for his valuable work in the development of the Social Force Model described in Section 2. 1. Dr. Lucie Merlier  from  CETHIL  is warmly acknowledged for designing the three room geometries presented in Section 6. Centre de Calcul Intensif d'Aix-Marseille  is acknowledged for granting access to its high performance computing resources. This work was granted access to the HPC resources of CINES under the allocation 2021-A0092A07679 made by  GENCI.
BACKGROUND: Albumin-bilirubin (ALBI) grade is an objective measure of liver function for patients with hepatocellular carcinoma (HCC). The tyrosine kinase inhibitor cabozantinib is approved for patients with advanced HCC who have received prior sorafenib based on the phase 3 CELESTIAL trial (NCT01908426). Cabozantinib improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with previously treated HCC. METHODS: Patients were randomised 2:1 to receive cabozantinib 60 mg or placebo orally every day. Clinical outcomes in patients with ALBI grade 1 or 2 at baseline were evaluated in CELESTIAL. ALBI scores were retrospectively calculated based on baseline serum albumin and total bilirubin, with an ALBI grade of 1 defined as ≤ -2. 60 score and a grade of 2 as a score of > -2.
Future analyses are warranted to compare the relative prognostic value for Child-Pugh scores of A5 versus A6 with ALBI grades 1 versus 2 in prospective clinical trial populations with clinical annotation for presence of ascites and encephalopathy as well as ECOG score, subjective factors which may be missing from retrospective databases and which may not be captured consistently in clinical practice outside of clinical trials. These analyses could support use of ALBI grade as an objective stratification factor in future clinical trials. For clinical practice, the more accessible EZ ALBI score, which uses a more simplified formula than ALBI grade, could potentially be used, if prospective studies support this approach  [11].
Background. EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. Methods. To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 wholegenome samples from two datasets. Results. The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients.
Importance of the Study Glioblastoma is the most prevalent and aggressive form of malignant primary brain tumours, often characterized by EGFR mutations for which no effective treatments is available. We aimed to understand the role of its most common mutation, EGFRvIII (in-frame deletion of exons 2-7). By exploiting six combined datasets, we show the interplay between pan-EGFR and EGFRvIII levels, find an absence of positive selection towards EGFRvIII expression and demonstrate that EGFRwt and EGFRvIII largely activate similar pathways. However, significant and unique EGFRvIII mutationspecific associations were found with Cell Cycle (e. g. CDK4) and RTK/RAS/PI3K genes (e. g. MDM2) which provide new insights for tumour targeting. A preference in breakpoint location in intron-1 not only results in a distinct variant of EGFRvIII but also preserves an enhancer region, and so provides new insights into EGFR(vIII) gene regulation.
Protein levels of EGFRvIII vary widely across and spatially within glioblastoma tumours  [13] [14] [15]. Moreover, recent observations show changes in EGFRvIII levels during tumour evolution after initial resection  6, 16, 17  , including cases with complete loss of EGFRvIII over time. That such a common presumed driver mutation gets lost, or levels get reduced during tumour evolution is paradoxical and will complicate targeting it for clinical benefit. In this study, we aim to unravel EGFRvIII specific mechanisms related to glioblastoma tumorigenesis. We examined EGFRvIII expression, its genomic breakpoints and cooccurrence with other genetic changes using a large combined dataset.
Sequencing data Sequencing of the Intellance-2  18  (paired-end; 2x151bp total RNA + paired-end; 2x76bp TruSight Tumor 170 panel) and BELOB (single-end; 50bp)  19  data were described elsewhere. For G-SAM, RNA extraction was performed using the AllPrep DNA/RNA FFPE kit or the RNeasy FFPE kit (Qiagen, Venlo, The Netherlands). G-SAM samples were sequenced (150bp paired-end reads) on the Illumina NovaSeq at the GIGA-Genomics Core Facility University of Liège. Each of these datasets were non-poly(A) + -enriched and thus also include non-polyadenylated transcripts  20. Raw sequencing data is available (BELOB: EGAS00001004570, Intellance-2: EGAS00001005437; G-SAM: EGAS00001005436). TCGA-GBM (poly(A) + RNA and DNA mutations) and CPCT-02 and PCAWG DNA data were obtained from their public repositories.
Sequencing data processing For each RNA-seq sample, FASTQ files were cleaned using fastp (https://github. com/OpenGene/fastp), aligned to hg19 using STAR  21  and then de-duplicated with sambamba. For the Dr. Disco 20 pipeline, samples were first FASTQ-level de-duplicated level using HTStream deduper (https://github. com/ibest/HTStream). EGFRvIII and EGFRwt expression was estimated directly from BAM files using junction-reads (https://github. com/yhoogstrate/egfr-v3-determiner v0. 7. 4: --spliced-reads-only). Reads considered EGFRvIII spanned the splice junction of exons 1-8, and reads considered EGFRwt exons 1-2. Samples with <10 such reads were excluded, except for TCGA-GBM, where EGFRwt read counts for EGFRvIII negative samples were missing. Junction read counts of replicated samples were merged by summing the spliced read counts.
The EGFRvIII percentage was defined as the average percentage from different assays were present (Intellance-2). Gene level read-counts were obtained using featureCounts and Gencode v31. EGFRvIII counts from TCGA-GBM were taken from elsewhere  4. Junctioncounts involving non-canonical exons A, B and C were determined using egfr-v3-determiner with modified exon annotations. Genomic events were taken from processed WES data or public resources (Supplementary Methods).
Expression analysis Samples with an EGFRwt+EGFRvIII read count ≥10 were eligible for EGFRvIII status and percentage determination and for DE analysis. For DE analysis, only genes with on average ≥3 reads per sample were included. Only genes marked as "protein_coding" were included. DE analysis was performed using DESeq2 (Wald test)  22  , in which EGFRvIII was excluded in estimateSizeFactors to avoid redundant counts. The FDR adjusted p-value reflects the qvalue. For the tests with four datasets combined, the intersected protein-coding genes with on average ≥3 reads per sample, per dataset, were included. Normalised expression levels were estimated using DESeq2 followed by the VST transformation (blind=TRUE) to ensure homoscedasticity  22. A batch correction was performed for DE and for correlation analysis to correct per-dataset differences (DESeq2 for count data; limma::removeBatchEffect  23  for VST transformed data).
Volcano plots were generated with the EnhancedVolcano package (https://github. com/kevinblighe/EnhancedVolcano). Kaplan-Meier analysis was performed using R"s survival package. Survival analysis on EGFRvIII expression was performed with a Cox Proportional Hazard survival using R"s survival package on the normalised VST transformed expression values. Because the Depatux-M antibody binds EGFRvIII with high affinity  24  and the Intellance-2 trial reported a benefit from Depatux-M in EGFRvIII positive samples  18  , Depatux-M arms were excluded from survival analysis.
Breakpoint analyses Non-poly(A) + -enriched RNA-seq samples include relatively large proportions of intronic reads derived from actively transcribed pre-mRNA. This allows detection of genomic breakpoints when corresponding introns are sufficiently covered  19, 20. Settings for Chimeric alignment are given in Supplementary Methods. The 100-vertebrates-phastCons track was obtained from UCSC and smoothened by a running mean of 200bp fixed windows. H3K27ac Chip-Seq data was obtained from GSM3382305  25  , GSM3670052, GSM3670055 and GSM3670058  26. Actual genomic enhancer locations were not provided in the original manuscript  25. Their raw CRISPRi-assay data (GSM4141363 + GSM4141364) was used to reproduce their findings according to their described methodology (Supplementary Table  3 ).
A total of sixteen different constructs were made: those that initiated translation in exons-2 or 8 with i) either an inframe eGFP (located C-terminal to the transmembrane region  27  ) or eGFP co-expressed via an IRES sequence; ii) with and without the L858R activating mutation (to compare the activation state of the novel isoforms with a constitutively active isoform) and; iii) without/with a canonical Kozak sequence (to ensure optimal translation of the latter). Constructs were generated by in-fusion cloning into a piggyback vector. Constructs were stably transfected in HeLa cells, imaged using an Opera Phenix (Perkin Elmer, Hamburg, Germany) high content imager and analysed using Harmony software (Perkin Elmer, Hamburg, Germany).
Molecular differences of EGFRvIII expressing tumours To determine EGFRwt (spliced across exons 1-2) and EGFRvIII (spliced across exons 1-8) expression, we first developed egfr-v3-determiner (publicly available, see Methods). Out of the 839 available RNA-seq samples, we included samples with a combined EGFR junction read count (spliced across exons 1-2 and 1-8) of ≥10 into our combined study RNA dataset: n=741 from 622 patients; BELOB (n=69/92), Intellance-2 (n=224/239), G-SAM (n=285/345) complemented with all primary TCGA-GBM samples (n=163). In this combined dataset, 464/741 (62. 6%) samples had EGFR gene amplification or upregulation if copy-number data was absent. Using the transcript-specific junction-counts, we calculated the ratio EGFRvIII (. Of the EGFR amplified samples, 225/464 (48.
5%) were considered EGFRvIII expressing ( , consistent with observations in literature  9, 30. These ratios revealed a high dynamic from 1% to 95%, consistent in all datasets (Figure  1 ). Lower EGFRvIII expression ratios were slightly over represented (1%-10%; p=3. 2e -9 ; Wilcoxon test on the first derivative of the ordered percentages). The total EGFR expression levels are on average lower for samples with higher EGFRvIII percentages, implying that EGFRvIII is more potent in EGFR signalling (Figure  2 ,S1D). Several reports have indicated that EGFRvIII and EGFRwt activate different signal transduction pathways  9, 31. To assess if such differences are reflected in their transcriptomes, we performed differential gene expression (DE) analysis comparing the transcriptomes of EGFRvIII positive (using two cut-offs: ≥1.
0% or ≥10. 0%) with EGFRvIII negative (<1. 0%) but EGFR amplified tumours. Tests were performed for all four datasets separately, to correlate the logarithmic fold changes (LFC) of the genes between the datasets. Markedly higher LFC correlations were found across the datasets using ≥10% EGFRvIII as cut-off (Figure  S2 ), which suggests lower percentages (1%-10%) harbour a limited EGFRvIII response signal. We therefore proceeded with the combined dataset using only ≥10% EGFRvIII as cut-off (n=368) and found 213 genes significantly (q-value<0. 01, |LFC|>0. 5) differentially expressed (Figure  3A ) They showed enrichment in genes related to microtubule, cilium and axoneme related pathways (Figure  S3 ). The 187 significantly downregulated genes in ≥10% EGFRvIII included CDK4 and MDM2, genes that are frequently hyper-and co-amplified in glioblastoma.
EGFRvIII prognostic value There have been conflicting data on the association of EGFRvIII with prognosis  9. We interrogated the patient survival between EGFRvIII positive and negative patients in the BELOB, G-SAM and TCGA-GBM and Intellance-2 (control arm) datasets. Within patients with EGFR amplified tumours, there was no significant difference in overall survival between patients with EGFRvIII positive and negative tumours (n=327) in each dataset or combined.
EGFRvIII breakpoints preferentially retain intronic enhancer With a transcription rate of 1-6 kb/min  39  , transcription of the ~120 kb EGFR intron-1 can take up to two hours. The closer the breakpoint of the causal EGFRvIII deletion is to exon-1, the shorter its intron. Given the large size of intron-1, breakpoints at the beginning of the intron (early breakpoints) may provide an energetic and temporal benefit over breakpoints at the end of the intron (late breakpoints). We screened ≥1% EGFRvIII positive samples for their genomic EGFRvIII breakpoints based on the presence of pre-mRNA  19, 20. We found 44 breakpoints within our datasets (Supplementary Table  1 ; Figure  5 ). One sample harboured two unique EGFRvIII breakpoints. We complemented these breakpoints with those identified from CPCT-02 (8/41 patients)  28  and PCAWG (11/40 patients)  29  whole-genome sequencing datasets.
We refer to these as exons A, B and C. The EGFRvIII breakpoint preference region is located 3" of exon-B (Figure  5 ) and thus preserves this exon at the genomic level. All datasets examined revealed junction-reads that initiated in exon-B and were spliced to exon-2 (EGFRwt) or exon-8 (EGFRvIII) (Figure  6 ). However, the fraction of transcripts containing exon-B was low compared to those initiating in exon-1 (≤1. 05%; Figure  S8C ), indicating exon-B expression is driven by a weak promoter. Transcripts spliced from exons A or C to exon-2 were extremely rare. In samples with breakpoints retaining exon-B, a novel exon-B-exon-8 EGFR(vIII/B) variant is created (Figure  6 ). This variant was confirmed with RT-PCR in six out of ten tested tumour samples (Supplementary Table  2 ). We verified the presence of the exon-B→exon-8→exon-9 sequence in three samples (GenBank: MZ484953, MZ484954 and MZ484955).
Discussion EGFR is commonly amplified, mutated and activated in glioblastoma, resulting in increased cell invasion and proliferation  41. EGFRvIII is a specific tumour marker often present in glioblastoma, that has been intensively investigated  9, 18. Here, we report on this genomic mutation using a large glioblastoma EGFRvIII omics dataset. To maximize statistical power, analysis was performed across a combined cohort of four RNA datasets and two independent whole-genome sequencing datasets. Previous data on the prognostic value of EGFRvIII was conflicting, with some suggesting EGFRvIII is a negative  42, 43  or a positive  44  prognostic marker, where other studies also suggested it did not affect survival  45, 46. Here, we demonstrate that within patients with EGFR amplified glioblastoma, we observed no difference in survival between EGFRvIII positive and negative tumours.
Multiple mutations can co-exist in the same tumour, sometimes subclonal and with reported longitudinal differences, with a unique, different ligand dependency. An earlier study proposed defining samples with a read count of at least 1% or 10% EGFRvIII compared with total EGFR as EGFRvIII positive  4. We recommend similarly rather than using the presence of any EGFRvIII read, as mapping artefacts and index hopping/switching derived reads are common in multiplexed RNA-seq and because higher EGFRvIII percentages showed a stronger response signal. Determination of the subclonal breakpoints in pre-mRNA data was more complicated than in datasets where breakpoints were clonal  20. Breakpoints were found predominantly in samples with high fractions of EGFRvIII. The median EGFRvIII percentage in samples with detected breakpoints was 55%, whereas 29% in samples without.
Intriguingly, we find a minority of EGFR transcripts starting with a cryptic exon preferentially preserved in EGFRvIII expressing tumours. The first translation initiation site is located in exon-8, but the total exon-B read count is very low and, combined with a weak Kozak sequence, we did not consider this variant to be the main reason for a breakpoint preference. Recently, the promotor and functional enhancers specifically retained in extrachromosomal EGFR fragments in glioblastoma and neuroblastoma cells have been interrogated  25. These enhancers, including "E3", were discovered using 4C-seq, H3K27ac ChIP-seq and a CRISPRi knock-down proliferation dropout assay. The E3 enhancer also showed H3K27ac in an independent dataset  26. The preferential retention of intragenic enhancer E3 in EGFRvIII is in line with these observations.
As the E3 enhancer is also conserved across vertebrates, it likely results in higher EGFR transcription rates. Unfortunately, both absolute A c c e p t e d M a n u s c r i p t and relative EGFRvIII levels differ essentially between samples, which combined with a high level of EGFRwt heterogeneity makes it difficult to confirm this hypothesis. In summary, using the largest combined EGFRvIII omics dataset to date, we find that the expression profiles of EGFRvIII positive tumours differ only marginally from EGFRvIII negative tumours. The results suggest that EGFRvIII mainly performs a similar role as EGFRwt but with a stronger affinity to activate EGFR downstream pathways, possibly linked to persistent activity independent of ligand(s). Furthermore, genomic breakpoints in intron-1 retain an enhancer that likely increases the expression of EGFRvIII transcripts.
In this retrospective setting, no prognostic difference was found between EGFRvIII positive patients compared with those harbouring EGFRwt amplifications. However, associations between EGFRvIII and genes such as CDK4, MDM2 and PTPRZ1 suggest that the relation between EGFR and EGFRvIII is not fully understood and further research is needed, ideally to find therapies targeting both isoforms. (A) DE analysis between EGFR amplified samples with (≥10%) and without EGFRvIII (<1%), with batch correction for the four datasets (Intellance-2, G-SAM, BELOB and TCGA-GBM). 213/15. 617 protein coding genes were differentially expressed, including DLX1, DLX2, TSPAN31, TMPRSS7, PPBP and DPT. Classical subtype genes are marked black. Overall LFCs were more often negative while the majority of the classical subtype genes had a positive LFC.
Acknowledgements We thank the  GIGA facility of University of Liège  for RNA-sequencing. This publication and the underlying study have been made possible on the basis of data that  Hartwig Medical Foundation  and the  Center of Personalised Cancer Treatment (CPCT)  have made available. The authors thank the  European Organization for Research and Treatment of Cancer  for permission to use the data from  EORTC  studies  EORTC_1410 (Intellance-2 ) and EORTC_1542 (G-SAM) for this research. We thank  Martin E. van Royen  for contributing.
METHODS The design and methodology used in the EMPEROR-Reduced trial have been described in detail in previous publications. 7, 24 he trial was a randomized, double-blind, parallel-group, placebo-controlled, and event-driven study. Ethics approval was obtained at each study site, and all patients provided informed consent to participate in the study; the registration identifier at ClinicalTrials. gov is NCT03057977. Data will be made available on request in adherence with transparency conventions in medical research and through requests to the corresponding author. The executive committee of EMPEROR has developed a comprehensive analysis plan and numerous prespecified analyses, which will be presented in future scientific meetings and publications. At a later point in time, the full database will be made available in adherence with the transparency policy of the sponsor (available at https://trials.