• Rat prevention study
  • Preface
  • 1 Prerequisites
    • 1.1 Packages and Software
    • 1.2 External software
    • 1.3 Annotations
      • 1.3.1 Genomic properties
      • 1.3.2 Gene name homologs between organisms
      • 1.3.3 Gene signatures and data-bases
  • 2 Cohort characteristics
    • 2.1 Size information
    • 2.2 Calculating growth rates
    • 2.3 KM curves for survival
    • 2.4 FACS data (DN/CD45/EpCAM)
    • 2.5 FACS data
  • 3 Cohort Summary Table
    • 3.1 Total number of samples
    • 3.2 Compare the characterisation vs progression cohort
    • 3.3 Summary of the RNA data
  • 4 Whole-slide imaging
    • 4.1 Associate the frequencies with other data types
    • 4.2 Cellular composition
    • 4.3 Associate composition with other covariates
    • 4.4 Estimate tumor size
    • 4.5 Correlations between different subpopulations
    • 4.6 Associations between CD8 counts with other clinical variables
  • 5 Spatial statistics
    • 5.1 knn-Distances:
      • 5.1.1 Comparison to manual classification
      • 5.1.2 Associations with outcome to treatment
      • 5.1.3 Growth
    • 5.2 The interacting fraction
      • 5.2.1 Comparison to manual & select optimal r
      • 5.2.2 Growth
      • 5.2.3 Treatment
    • 5.3 M-H distances
      • 5.3.1 Comparison to Manual Scoring
      • 5.3.2 Growth
      • 5.3.3 Treatment
    • 5.4 Comparison between metrics
    • 5.5 Distances to “unclassified cells”
  • 6 Expression data
    • 6.1 Running alignment
    • 6.2 RNA Initial QC
    • 6.3 Normalisation
      • 6.3.1 preliminary visualisation (to remove outliers)
    • 6.4 Processing files for external software
  • 7 RNA data: preliminary plots
    • 7.1 PCA plots
    • 7.2 Expression patterns by cell type
  • 8 DESeq analysis: Progression/Immunotherapy cohort
    • 8.1 DN vs Ep
    • 8.2 No. samples in comparisons
    • 8.3 Set-up cell-type specific the comparisons
    • 8.4 PCA plots
      • 8.4.1 EpCAM
      • 8.4.2 CD45
      • 8.4.3 DN
    • 8.5 Pearson correlation plots of samples
  • 9 Collating results and running GSEA
  • 10 ER/Pgr Subtyping
    • 10.1 Gene List
    • 10.2 Progression cohort
    • 10.3 Charcterisation cohort
    • 10.4 DN samples
    • 10.5 Comparison with staining
    • 10.6 Summary of expression markers for each subtype/cell fraction
  • 11 Expression in Specific pathways
  • 12 DESeq analysis: Immunotherapy/Growth comparisons
    • 12.1 Summary of comparisons
    • 12.2 DESeq: immunotherapy
    • 12.3 Growing vs stable emphasis
      • 12.3.1 DN fraction
      • 12.3.2 CD fraction
      • 12.3.3 Ep Fraction
  • 13 Summary of GSEA runs
    • 13.1 Stable vs growing: all samples
      • 13.1.1 barplots of enriched pathways
    • 13.2 Comparisons based on treatment
    • 13.3 Pathways of Interest 2
  • 14 Epcam+ Inflammatory vs non-inflammatory samples
    • 14.1 Identification of inflammatory samples
    • 14.2 DEG: inflammatory vs non-inflammatory
    • 14.3 Finding 3 signatures for 3 branches
    • 14.4 Analyse the non-inflammatory samples
      • 14.4.1 GSEA
    • 14.5 Luminal-only non-inflammtory samples samples
  • 15 TCGA: Associate Epcam+ inflammatory with survival
    • 15.1 Associating CD74 with phenotype and outcome
    • 15.2 Associating ADMATS10 with phenotype and outcome
      • 15.2.1 All samples
      • 15.2.2 Redone using only ER cases
    • 15.3 Signature: Lum cases non-inflammatory: growing vs stable
    • 15.4 Comparison to oncotype and mammaprint
      • 15.4.1 Overall survival
      • 15.4.2 DFS survival
      • 15.4.3 TCGA: LumA high vs low signatures
  • 16 Read in ER i/o data
    • 16.1 Signature: Lum cases non-inflammatory: growing vs stable
    • 16.2 Do associative study with other features?
    • 16.3 Use raw counts and perform a batch correction if necessary
  • 17 DESeq analysis: Characterisation cohort (big vs small)
    • 17.1 CD45 samples
      • 17.1.1 PCA plot
      • 17.1.2 Differential Gene Expression
      • 17.1.3 GSEA
    • 17.2 Epithelial samples
      • 17.2.1 PCA plot
      • 17.2.2 GSEA
      • 17.2.3 Check expression of checkpoint proteins
  • 18 Signature analysis
    • 18.1 MHC signature analysis
      • 18.1.1 MHC-I
      • 18.1.2 MHC-II
      • 18.1.3 MHC presentation proteins
      • 18.1.4 inflammation related genes: IL6-JAK-STAT and TNF/NFKB
  • 19 Immune estimation
    • 19.1 Overview of the cell types
    • 19.2 characterisation cohort: assoc with size
    • 19.3 Comparison with FACS data
    • 19.4 Progression cohort
    • 19.5 Clinical associations
      • 19.5.1 Associate with Treatment
      • 19.5.2 Association with Growth
    • 19.6 Summary of the outcome
  • 20 BCR clonotype analysis
    • 20.1 Processing summary
    • 20.2 Summary Stats
    • 20.3 Diversity metrics
    • 20.4 Compare the characterisation cohort
    • 20.5 Associate with clinicopathological data (progression)
    • 20.6 Associate with signature scores
  • 21 TCR clonotype analysis
    • 21.0.1 Progression cohort
  • 22 Whole Genome Sequencing Mutations
    • 22.1 Data
      • 22.1.1 Extract mutation signatures
    • 22.2 Annotate the data with human common variants
    • 22.3 Plots
      • 22.3.1 Quick overview
      • 22.3.2 Coding variants & TMB
    • 22.4 Comparison with rat mutational datasets
      • 22.4.1 Sites which are commonly mutated?
    • 22.5 Overview of the mutations
      • 22.5.1 All mutations
    • 22.6 Metacore analysis of commonly mutated pathways
  • 23 Mutations in RNA
    • 23.1 Haplotype caller
    • 23.2 Load files
    • 23.3 Identifying polymorphisms: compare frequencies in CD45 and Ep data
    • 23.4 Find coding mutations which are specific to ep cells
    • 23.5 Filtering WGS data based on CD45 data
  • 24 Mutations in progression cohort
    • 24.1 Mutational burden
    • 24.2 Summary of common mutations:
    • 24.3 Look at the common mutations (cosmic ones)
  • 25 Trichrome staining
    • 25.1 Associations with cellular fraction (wsi)
    • 25.2 Associations with CD8 content
    • 25.3 Associations with growth and treatment
    • 25.4 Association with hyperinflammatory status
  • 26 CNV calling
    • 26.1 Sample data
    • 26.2 Data Summary
      • 26.2.1 Frequency of gains and losses across the genome
      • 26.2.2 Loci which have a hit in at least 30% of samples
      • 26.2.3 Annotated plot of genome and locations of genes
    • 26.3 Samples with CNVs in breast-related genes
    • 26.4 Summary of the sequencing depth
  • 27 Writing files to file
  • Appendix
  • A List of Figures
    • Main Figures
    • Figure 1: NMU-induced mammary tumors
    • Figure 2: Immune system of NMU-rat
    • Figure 3: Immunotherapy applied to NMU rats
    • Figure 4: Characterizing CD45 cells in tumors
    • Figure 5 : Epithelial cells in growing vs stable rats
  • Extended Figures
    • Extended data 1
    • Extended data 2
    • Extended data 3
    • Extended data 4
    • Extended data 5
  • Published with bookdown

NMU-induced rat tumor models for I/O

Extended Figures

Extended data 1

  • Ext Fig 1B : Number of tumors per rat in each cohort
  • Ext Fig 1C: growth of NMU treated tumors over time
  • Ext Fig 1F: PCA of growing vs stable epithelial cells
  • Ext Fig 1G: correlation plot epithelial samples
  • Ext Fig 1H-I: assoc of mutations with cohort
  • Ext Fig 1J: Alexandrov mutational signatures
  • Ext Fig 1K: most frequent mutations
  • Ext Fig 1L: CNV calls

Extended data 2

  • Ext2D PCA of CD45 cells
  • Ext2E clonotype assoc with size
  • Ext2F expression of checkpoint proteins in epithelial cells

Extended data 3

  • Ext Fig 3C : FACS associated with outcome
  • Ext Fig 3D : PCA plot all cell types
  • Ext Fig 3E : Cell type specific genes
  • Ext Fig 3H: DN assoc treatment GSEA
  • Ext Fig 3I : HR clustering output progression

Extended data 4

  • Ext Fig 4B association with facs
  • Ext Fig 4C-D WSI-normalised-all-samples
  • Ext Fig 4E Knn with treatment
  • Ext Fig 4F Interacting fraction with treatment
  • Ext Fig 4H MH with treatment

Extended data 5

  • Ext Fig 5A: Expression of inflammatory genes across samples
  • Ext Fig 5B growth profiles of inflammatory tumors
  • Ext Fig 5C: Differential gene exp inflammatory vs non-inflammatory
  • Ext Fig 5D: association signature with WSI
  • Ext Fig 5E: Collagen content in growing vs stable
  • Ext Fig 5F: Forest plot TCGA luminal ADAMTS10
  • Ext Fig 5G ADAMST10 expression associated with clinical variables
  • Ext Fig 5I-J: Associating growing signature with clinical variables
  • Ext Fig 5K: TCGA DEG for LumA samples