/Shiny_bc_paper

A repository that contains all the code for the interactive Shiny app of the models developed in our work on predicting response to neoadjuvant treatment.

Primary LanguageR

Shiny app for my PhD's breast cancer project

This repository that contains all the code for the interactive Shiny app of our paper on predicting response to neoadjuvant treatment. Due to browser host limits we suggest installing the repo locally and running the app through RStudio; see Installation.md.

Contents

Overview

This work focused on combining patient gene expression data from chemotherapy and endocrine treatment studies in an effort to analyze them in an integrative manner for the discovery of predictors of response to treatment.

  • Data from different studies were preprocessed and cleaned separately.
  • The expression values for each gene in each study were standardized.
  • The standardized gene expression matrices were merged using common genes as anchors into a comprehensive expression matrix.
  • This matrix was used as input for differential gene expression analysis (more details below) and the identification of markers that distinguish between responders and non-responders.
  • The identified markers were then used as coordinates for the identification of response subtypes using Monte Carlo Consensus Clustering (see John et al.).
  • Additionally, the markers were combined with phenotypic/clinical variables of interest in order to train response prediction models.

You can read more about work in our preprint.

Code organization 💻

  • global.R: A script that loads all required libraries and sets up the environment for the shiny app to run.
  • server.R: A script that sources all server scripts that define the functionality of the app.
  • ui.R: A script that defines the elements of the app's user interface.
  • App.R: A small script that sources the prep code (global.R), the full UI code (ui.R) and the full server code (server.R), and then runs the app using runApp().
  • test_files_generation.R: A script that generates appropriate and non-appropriate files for the sixth tab of the app (new predictions based on input). The files are used for debugging and error-handling.
  • 📂 current_scripts: modularized code for all six different tabs of the app (six folders, with sub-scripts of server code)
  • 📂 old_scripts: scripts from older versions
  • 📂 input_data: files that are sourced in the global.R script to build the environment of the app.
  • 📂 file_checks: files that are used for testing of the tab that produces new predictions for uploaded inputs
  • 📂 www: directory of additional .css files
  • 📂 www/GIFs: .gif files that are used for tutorials in each of the different tabs of the app.
  • 📂 rsconnect...: rsconnect deployment folder for shinyApps.io.

App features 💡

Exploratory plot generation 📊

The app offers functionality for generating exploratory plots: histograms and bar charts. In both cases, all data from the project are included by default.

  • ☑️ The user can use the checkbox input to include/exclude studies.
  • 🖌️ Click Draw! to generate plot.
  • ℹ️ Click Info for useful tips and instructions.
  • 🔄 Click Reset to default parameters to bring all options back to default.
  • ⬇️ The plots generated can be downloaded by hovering to the plot tools on the top right.

Histograms: the user can select a variable of interest and choose the type of the histogram (classic/histogram of counts, probability, percentage).

  • 🌈 The user can also choose the bin fill and bin color, as well as
  • #️⃣ the number of bins (for continuous variables only).
Short demo

Short demo

Bar charts: the user can select up to two variables for the bar charts. If a second variable is selected, then the user can also select the type of bar chart (grouped or stacked).

  • 🌈 Options for bin fill and outline color are also provided.
Short demo

Short demo

Sunburst plots 🌞

Dynamic and interactive plots to illustrate the distribution of up to four categorical variables in our data. Colors are preselected. Choose a root variable and add up to three more variables to produce an interactive sunburst plot.

  • 🖌️ Click Draw! to generate plot.
  • ℹ️ Click Info for useful tips and instructions.
  • 🔄 Click Reset to default parameters to bring all options back to default.
  • 🎚️ Choose color opacity using the slider.

Analytical sunbursts

These plots have access to all the data in our study (training, validation, test, external validation). You can use these plots to examine the nested distributions of subtypes, risk scores, response to treatment and more, within the different studies.

Consensus sunbursts

These plots can be used to illustrate the distribution of different variables within the clusters (Neoadjuvant treatment - NAT response subtypes) we derived.

  • 🟦 Cluster 1: NAT-responsive subtype; associated with more favorable profiles.
  • 🟧 Cluster 2: NAT-neutral subtype; associated with higher risk scores and subtypes with poor prognosis.
Short demo

Short demo

Volcano plot 🌋

Use the results from our differential expression analysis, to create customized volcano plots.

❓ What is a volcano plot? A volcano plot is typically used to illustrate results of differential expression analysis. It is a scatter plot, where each point represents a gene.

The $y$-axis contains the negative base-10 logarithm of the gene's differential expression $p$-value ($-\log_{10}p$), i.e. the higher a point is in the plot, the lower its $p$-value.

The $x$-axis shows differential expression (usually log2FoldChange). It's usually centered at 0; points right from 0 represent up-regulated genes; points left from 0, represent down-regulated genes.

Note on x-axis: In our case, differential expression analysis was performed on standardized data. Instead of $\log_{}FoldChange$, differential expression here is defined as the numerical difference between the model coefficient for responders and the model coefficient for non-responders. The (full) model for each gene used in our case is:

$$ \begin{align*} \hat{y_{g}} &= a_1 \cdot 1_{{Response = responder}} + a_2 \cdot 1_{{Response = non-responder}} + \\ &\quad b_1 \cdot 1_{{pam50 = Luminal A}} + b_2 \cdot 1_{{pam50 = Luminal B}} + b_3 \cdot 1_{{pam50 = HER2+}} + b_4 \cdot 1_{{pam50 = Normal-like}} + \\ &\quad c_1 \cdot 1_{{timepoint = T2}} + \\ &\quad d_1 \cdot 1_{{study = study_2}} + \dots + d_9 \cdot 1_{{study = study_{10}}} \end{align*} $$

and the differential expression value for each gene is:

$$DE = a_1 - a_2$$

The user can pick:

  • 🗒️ which type of $p$-value to plot (adjusted/unadjusted; we suggest the adjusted $p$-value)
  • 🛑 a differential expression threshold (DET) which will draw two vertical dashed lines at the positive and negative coordinates of the selected value on the $x$-axis
  • 🛑 a $p$-value threshold (PVT) lower than which a gene's differential expression is considered statistically significant (draws a single horizontal line)
  • 🌈 colors for:
    • ❌ a) non-significant genes,
    • 🟠 b) significant genes that don't pass the DET,
    • 🔵 c) down-regulated genes (pass both DET - left side - and PVT) and
    • 🔴 d) up-regulated genes (pass both DET - right side - and PVT)
  • 🎚️ opacity for the color of the points
Short demo

Short demo

Customized Differential Gene Expression Analysis 📈

Use the training and validation samples as input to perform customized DGEA. There are three tabs available:

  • 🗒️ Data Selection: select the studies you want to keep for your analysis
  • 🧮 Filtering and Adjustments:
    • Filter for clinical, demographic and risk score variable of interest
    • Select covariates for the linear models that will be fit for each gene
    • Pick the contrast variable of interest (the variable whose levels you want to compare)
    • Select the levels of interest (e.g. responder and non-responder). The kdifferential expression results represent the numerical difference between the model coefficients for level 1 and level 2: $$DE = coef_{level_{1}} - coef_{level_{2}}$$
  • 🌋 Plot Settings: choose colors and text for the volcano plot that will be produced (see the Volcano plot section for more details)

🔍 Click Analyse to perform the analysis and produce the table of results and the volcano plot. Both are downloadable.

🔄 Click Default settings to set everything back to default (applies to all tabs).

ℹ️ Click Info for useful tips and instructions

Short demo

Short demo

Machine Learning 🔮

A tab that allows the user to compare the performance of up to 3 pre-trained models on the full data and subets selected interactively.

There are two boxes for each model:

🧰 Box 1

Select a model from the following categories:

📉 Logistic regression 🌳 Decision Trees 🤖 Support Vector Machines
Backward Logistic Regression C5.0 (optimized with Cohen's kappa) Linear kernel
Regularised Logistic Regression C5.0 (optimized with ROC) L2-regularised linear kernel
Random Forest (optimized with Cohen's kappa) Radial Basis Function kernel
Random Forest (optimized with Cohen's ROC)
Boosting
Bagging (x100)

and a subset of studies of interest (or keep data from all studies - default option).

🧰 Box 2

  • 🧮 Filter the subset for clinical, demographic and risk score variables of interest
  • 📛 Pick a name for your model (e.g. C5.0 model or C5.0 model for ER- subset, etc.)
  • ➕ Click Add comparison if you want to add another model for comparison:
    • An additional set of boxes will appear
    • Choose model and studies of interest
    • Filter subset if desirable
    • Name the model and click Apply to enable the comparison with the first model
    • If you want a third model, click Add comparison and follow the same steps. Don't forget to click Apply in the end!
  • ❌ Click Remove if you want to remove a comparison you added
  • 🔮 Click Predict! (after applying additional comparisons - if any) to generate predictions of response to treatment and produce a ROC curve
  • 🔄 Click Default filters to set all filters back to default in Box 2
  • ℹ️ Click Info to see useful tips and instructions

Output

  • ROC curve(s) for the selected model(s) with info on the record AUC values. Downloadable.
  • Table of error metrics for each model used.
Short demo

Short demo

New prediction 🧪

In this tab, users can upload their own data (assuming appropriate formatting at least for gene names) and generate predictions about the probability of response to a selected treatment for a unique patient or a full dataset. In the case of a full dataset with a response column available, ROC curves can also be produced if the user desires so.

General instructions

The app will display helpful warning messages if input is not appropriately formatted but here are some quick tips on preparing the input:

Show good practice tips
  • The file must have one of the following extensions: .csv, .tsv, .xlsx, .txt.
  • The model can make a prediction provided all 166 genes identified in the project are present (you can find the list here: sheet 2, first 166 genes).
  • The order of the columns does not matter, but gene names must be given in NCBI Entrez identifiers or in the format "X_Entrez" (e.g. X_3489).
  • Gene columns must contain numeric values. All other columns must contain either 0 or 1 (binary values).
  • When uploading a unique sample without phenotypic annotation, the top right panel will be enabled and allow the user to annotate the sample, with respect to phenotypic variables (treatment, pam50 subtype, scmod1 subtype, iC10 subtype, rorS risk, Mammaprint risk and timepoint), themselves.
  • When uploading a unique sample with phenotypic annotation, the user must first choose whether an 'Endo' column is presented in the data (name: Endo, values: 0 for chemotherapy, 1 for endocrine treatment). Then, they can upload the sample data and choose whether the rest of the phenotypic variables are preset (choose the 'Preset' option for all of them) or select them using the drop-down menus for each variable. If 'Preset' is selected, then variables must have the correct names shown here:
    Variable Dummy column(s) Example (meaning)
    Treatment Endo 0 (Chemotherapy)
    pam50 LumA, LumB, HER2, Normal 0, 0, 0, 0 (Basal-like)
    scmod1 ER_hp, ER_lp, HER2_scmod1 0, 0, 0 (ER-/HER2-)
    timepoint T2 0 (pre-treatment, T1)
    rorS rorS_risk_interm, rorS_risk_high 0, 0 (low rorS risk)
    Mammaprint Mammaprint_risk_yes 0 (No Mammaprint risk)
    iC10 IC2, IC3, IC4, IC5, IC6, IC7, IC8, IC9, IC10 0, 0, 0, 0, 0, 0, 0, 0, 0 (IC1)
    The user can also set variables to 'Random' values.
  • When uploading a dataset, the user will again have to specify whether an 'Endo' column is present. If not, then the user can apply a prticular treatment to the full dataset. The top right panel will be disabled and the bottom panel will be enabled. If a response column is present, the user can select whether to produce a ROC curve or not.

❔ Random sample

When this option is chosen, the app will generate a sample with random numerical values for all genes. The user can then add phenotypic annotation ('Preset' not allowed), choose a treatment and then get the predicted probability of response to the selected treatment by clicking Predict! 🔮.

🧬 Unique sample (genes only)

When this option is chosen, the user must upload a file with at least 166 columns which are appropriately named (check out the good practice tips for more details). Subsqequently, the user must select phenotypic annotation for the sample based on either their knowledge of the sample's characteristics, additional laboratory results they have or genefu annotation results. After all variables have been set, the user can proceed with the prediction by clicking Predict! 🔮.

Short demo

Short demo

✅ Unique sample (pre-annotated)

When this option is chosen, the user must upload a file with the 166 gene columns and the required phenotypic annotation columns (check out the good practice tips for more details). The user is also required to specify whether a treatment column 'Endo' already exists or not. Phenotypic annotation for a unique sample can be modified using the drop-down menus for each variable prior to making a prediction. If no modification is required, all drop-down menus must be set to 'Preset'. Click Predict! to get an estimate of the probability of response to the selected treatment 🔮.

Short demo

Short demo

🗃️ Pre-annotated dataset

When this option is chosen, the user must upload a file with the 166 gene columns and the required phenotypic annotation columns (check out the good practice tips for more details). The file should contain more than one rows. The user must specify again whether an 'Endo' column is present. Otherwise, they have to select a treatment for the full set of samples. The radio buttons in the bottom panel can be used to filter the data for subsets of interest (by default, all levels are included for each variable). If a binary column named 'Response' is available in the data, the user can produce ROC curves along with predicted probabilities. Click Predict! to get the (optional) ROC curve and download it along with the predicted probabilities of response for each sample. An additional table with information on the error metrics of the model is also generated.

Short demo

Short demo

As always:

  • 🔄 Click Reset parameters to restore everything to default and upload a new file
  • ℹ️ Click Info for useful tips and instructions