mitox

Supportive scripts for the clinical trial, "A Pilot Study of the Effect of the Microbiome on Immune Checkpoint Inhibitor Response in Melanoma (NCT05102773, "OSU-19125)". The goal of the study is to predict which melanoma patients will respond to immunotherapy or experience immune-related adverse events using the gut microbiome and other biomarkers.

These analyses are reported in:

Dravillas C, Williams N, Husain M, Hoyd R, Hussein A, Meara A, et al. The Association of the Microbiome with Melanoma Tumor Response to Immune Checkpoint Inhibitor Treatment and Immune-Related Adverse Events (NCT05102773). medRxiv 2025. https://doi.org/10.1101/2025.01.30.25321413. PubMed

Code to support the protocol submission, including power calculations, sample size estimates, estimated accrual dates, etc., can be found at:

Spakowicz, D., and Muniak, M. (2019). spakowiczlab/mitox-protocol: IRB submission (Zenodo).

This work is supported by a Pelotonia (DS) and the Ohio State University's Translational Therapeutics Program (CV).

Manuscript directory

Analyses for the manuscript live in manuscript/. Figure notebooks read derived objects from manuscript/data/ and write images and table exports to manuscript/figures/.

Path	Contents
manuscript/scripts/processing/	HPC batch scripts and the notebook that builds de-identified analysis objects
manuscript/scripts/analysis/	Statistical analyses and the notebooks that assemble each manuscript figure and table
manuscript/data/	Small lookup tables and model-comparison metrics committed with the repo
manuscript/figures/	Saved figure panels (SVG, and PNG when both were exported) plus table CSV/HTML exports

Run notebooks from manuscript/scripts/analysis/ (or processing/) so relative paths of the form ../../data/ and ../../figures/ resolve.

Clinical and microbiome objects used by the figure notebooks (for example clinical.rds, counts-long.rds, ANCOM-BC2 and random-forest outputs) are produced by the processing and analysis scripts below. They are not all stored in git.

Processing scripts

File	Role
publication_objects.Rmd	Builds the de-identified R objects that later notebooks load
generate-metaphlan-batch.Rmd	Writes the MetaPhlAn batch job used to profile shotgun reads
merge_metaphlan-viruses.Rmd	Aggregates MetaPhlAn taxon and virus tables
humann-batch.Rmd	Writes the HUMAnN functional-profiling batch job

Analysis scripts that feed the figures

These notebooks compute the results that the figure scripts plot. They do not write the manuscript figure files themselves.

File	Role
ANCOM-BC2.Rmd	Baseline differential abundance for response and irAE
ANCOM-BC2-respondersig.Rmd	ANCOM-BC2 on the published Respondersig cohorts
ANCOM-BC2-results-overlap.Rmd	Overlap of response and irAE taxa
respondersig.Rmd, respondersig-irae.Rmd	Respondersig scores in this cohort
alphadiversity.Rmd, alpha-diversity.Rmd	Alpha-diversity summaries (Figure 2B / 3B)
longitudinal-modeling-response.Rmd, longitudinal-modeling-irAE.Rmd	Zero-inflated Gaussian mixed models (Figures 4 and 5)
supplemental-figureS1.Rmd	Longitudinal model comparison using data/model-metrics.csv; Supplemental Figure S1
core-microbiome.Rmd	Per-patient core microbiome score (Figure 5E)
random-forest.Rmd, rf-functions.R, corescore-predictive-modeling.Rmd, random-forest-validation.Rmd	Random-forest models and external validation (Figure 6 and Supplemental Figures S3–S7)
examine-timing.Rmd, window_size.Rmd	Definitions of baseline, irAE-adjacent, and end-of-study samples
labels_ANCOM-resist.Rmd, examine-viruses.Rmd	Taxonomy labels and virus-table checks

Figures and the files that create them

Filenames in manuscript/figures/ use the manuscript panel letter when that panel is a single exported plot. Multi-part schema panels and a few Illustrator composites are noted below.

Figure 1 — study schema and sampling timeline (figure1.Rmd)

Panel	Saved file	Description
1A	Fig1A-Baseline.svg	Timing of baseline stool samples (within 10 days of C1D1)
1A	Fig1A-irAE.svg	Timing of samples collected within 10 days of an irAE
1A	Fig1A-eos.svg	Timing of ~12-week / end-of-study samples
1C	timeline-MET-restricted-dates.svg	Per-patient event timeline for metastatic participants

Figure 1B (CONSORT-style flow) is assembled outside these scripts.

Figure 2 — baseline microbiome and response (figure2.Rmd)

Panel	Saved file	Description
2A	Fig2A-response-baseline-stackedbar.svg	Phylum stacked bars, baseline samples, faceted by response
2B	Fig2B-boxplots_diversity-response.svg	Alpha diversity by response
2C	Fig2C-response-baseline-volcano.svg	ANCOM-BC2 volcano plot, responders vs non-responders
2D	Fig2D-respondersig-plot.svg	Respondersig ordination (written by the notebook; add to figures/ after knitting)
2E	Fig2E-response-s0-baseline-boxplots.svg	Structural-zero taxa at baseline by response
2F	Fig2F-rsig-overlap-effect-size.svg	Effect sizes of taxa shared with Respondersig

Fig2E-labels.svg is a legend/label companion for 2E.

Figure 3 — baseline microbiome and irAE (figure3.Rmd)

Panel	Saved file	Description
3A	Fig3A-irAE-baseline-stackedbar.svg	Phylum stacked bars, baseline samples, faceted by irAE
3B	Fig3B_boxplots_diversity-irAE.svg	Alpha diversity by irAE
3C	Fig3C-irae-baseline-volcano.svg	ANCOM-BC2 volcano plot, irAE vs no irAE
3D	Fig3D-respondersig-plot.svg	Respondersig ordination colored by irAE
3E	Fig3E-irAE-s0-baseline-boxplots.svg	Structural-zero taxa at baseline by irAE
3F	Fig3F-pairwise-dist_boxplots.svg	Pairwise distances between irAE and no-irAE groups

Fig3E-labels.svg is a legend/label companion for 3E.

Figure 4 — longitudinal models of response (figure4.Rmd)

Panel	Saved file	Description
4A	Fig4A-ZIGMM-nonquad-volcano.svg	ZIGMM volcano for the linear response × time term
4B	Fig4B-ZIGMM-quad-volcano.svg	ZIGMM volcano for the quadratic time term
4C	Fig4C-lineplot.svg	Longitudinal abundance of Phocaeicola plebeius by response
4D	Fig4D-venn.svg	Overlap of ANCOM-BC2 and ZIGMM taxa

The notebook currently also writes PNG copies of 4A–4C.

Figure 5 — longitudinal models of irAE (figure5.Rmd)

Panel	Saved file	Description
5A	Fig5A-ZIGMM-nonquad-volcano-irae.svg	ZIGMM volcano for irAE-associated taxa
5B	Fig5C-lineplot.svg	Longitudinal abundance of Phocaeicola dorei, with irAE event times marked
5C	Fig5C-venn.svg	Overlap of ANCOM-BC2 and ZIGMM irAE taxa
5E	Fig5E-boxplot.svg	Core microbiome score by irAE

Figure 5D (schema) is assembled outside these scripts.

Figure 6 — prediction of response and irAE (figure6.Rmd)

Panel	Saved file	Description
6A	Fig6A-Forestplot-response.svg	Univariate clinical predictors of response
6B	Fig6B-Forestplot-irae.svg	Univariate clinical predictors of irAE
6C	Fig6C-AUCPR-boxplots-response.svg	Random-forest AUPRC by feature set, response
6D	Fig6D-AUCPR-boxplots-irae.svg	Random-forest AUPRC by feature set, irAE
6E	Fig6E-corescore-AUCPR-boxplots-irae.svg	AUPRC when the core score is included, irAE
—	Fig6I-combined.svg	External-validation ROC curves by published cohort
—	Fig6J-varImp-response.svg	Variable importance for response models
—	Fig6K-varImp-irae.svg	Variable importance for irAE models

Fig6I-valROC-by-study.svg is an alternate validation plot (not used in the main figure). Earlier ROC exports remain in figures/ as Fig6E-ROC-response.svg, Fig6F-ROC-irae.svg, Fig6G-valROC-response.svg, and Fig6H-valROC-irae.svg; those views now correspond to Supplemental Figures S3, S4, S6, and S7. Fig6A-Forestplot-irae.svg and Fig6B-Forestplot-response.svg are previous filename assignments (the current 6A/6B files above are the ones used in the manuscript). corescore-AUCPR-boxplots-response.svg and corescore-AUCPR-boxplots-irae.svg are exploratory core-score plots.

Supplemental figures

Figure	Script	Saved file	Description
S1	supplemental-figureS1.Rmd	supplemental-model-metrics.svg	Comparison of longitudinal model families (AIC/MSE); also writes data/model-metrics.csv
S2A	supplemental-figureS2-S7.Rmd	supplemental-figuresS2A.svg	Full univariate clinical forest plot, response
S2B	supplemental-figureS2-S7.Rmd	supplemental-figureS2B.svg	Full univariate clinical forest plot, irAE
S3	supplemental-figureS2-S7.Rmd	supplemental-figureS3.svg	ROC: microbiome vs clinical models, response
S4	supplemental-figureS2-S7.Rmd	supplemental-figureS4.svg	ROC: microbiome vs clinical models, irAE
S5	supplemental-figureS2-S7.Rmd	supplemental-figureS5.png	Variable importance for irAE models that include the core score
S6	supplemental-figureS2-S7.Rmd	supplemental-figureS6.png	External-validation ROC for response (all cohorts combined)
S7	supplemental-figureS2-S7.Rmd	supplemental-figureS7.png	External-validation ROC for irAE

S5–S7 are written as PNG only. window_size.Rmd writes diagnostic plots irae_windowsize.png and irae_windowsize_closeup.png that are not manuscript figures.

Tables

Table	Script	Saved file	Description
1	tables.Rmd	table1.html	Cohort characteristics stratified by response
2	tables.Rmd	table2.html	Cohort characteristics stratified by irAE
3	tables.Rmd	table3.html	irAE types and grades
S1	tables.Rmd	supplemental-table1.csv	irAE type/grade counts (CSV export of Table 3)
S2A/B	supplemental-tables.Rmd	supplemental-table2A.csv, supplemental-table2B.csv	ANCOM-BC2 response results and structural zeros
S3A/B	supplemental-tables.Rmd	supplemental-table3A.csv, supplemental-table3B.csv	ANCOM-BC2 Respondersig results and structural zeros
S4A/B	supplemental-tables.Rmd	supplemental-table4A.csv, supplemental-table4B.csv	ANCOM-BC2 irAE results and structural zeros
S5	supplemental-tables.Rmd	supplemental-table5.csv	ZIGMM coefficients for response
S6	supplemental-tables.Rmd	supplemental-table6.csv	ZIGMM coefficients for irAE
S7	supplemental-tables.Rmd	supplemental-table7.csv	Participant-level clinical variables
S8	supplemental-tables.Rmd	supplemental-table8.csv	Stool-sample metadata

Data files in manuscript/data/

File	Description
model-metrics.csv	Fit statistics comparing longitudinal model families; input to Supplemental Figure S1
CRISPR_VSG-to-species.csv	Virus sequence group to species lookup
CRISPR_VSG-to-SGBs.csv	Virus sequence group to SGB lookup