Pre- and On-treatment Multi-omics Profiling of Locally Advanced Rectal Cancer

Patients whose resection specimen showed a pathological complete response (ypT0N0) or who achieved a sustained clinical complete response, from whom pre-treatment and on-treatment tissue and blood were profiled.

Patients with residual viable tumour at resection or without a sustained clinical complete response, from whom pre-treatment and on-treatment tissue and blood were profiled.

Cell-type composition and transcriptional states of T, natural killer (NK), B and myeloid populations and of stromal subsets before treatment and during neoadjuvant therapy, determined by single-cell RNA sequencing.

Proportion of patients achieving major pathological response, defined as 10% or less residual viable tumour at resection, and the association of pre-treatment and on-treatment immune microenvironment features with mPR.

Exhaustion, activation and memory programmes of T and NK cells and B-cell states, determined by single-cell RNA sequencing with paired T-cell and B-cell receptor repertoire sequencing.

Spatial distribution of tumour, immune and stromal compartments determined by spatial transcriptomics of formalin-fixed paraffin-embedded sections and multiplex immunofluorescence, compared with specimens from treatment-naive patients undergoing primary surgery and from end-of-treatment biopsies as controls.

Between-group comparison of the change in tumour microenvironment composition between baseline and week 4, expressed as the percentage of cluster of differentiation 8 (CD8)-positive T cells, NK cells, B cells and plasma cells among all cells measured by single-cell RNA sequencing, in patients receiving radiotherapy before versus after chemo-immunotherapy.

Somatic mutations, copy-number alterations and mutational signatures determined by whole-genome sequencing of tumour tissue before and during neoadjuvant therapy.

Diversity and distribution of T-cell receptor clonotypes in peripheral blood mononuclear cells, expressed as the Shannon diversity index and the proportion of expanded clonotypes, measured by bulk T-cell receptor repertoire sequencing.

Correlation between the relative abundance of tumour-associated bacterial taxa, expressed as the percentage of sequencing reads assigned to each taxon measured by small-subunit ribosomal RNA gene sequencing or shotgun metagenomic sequencing of tumour tissue, and major pathological response, expressed as the percentage of patients with 10% or less residual viable tumour at resection (exploratory).

Concentrations of plasma or serum proteins and cytokines, expressed in pg/mL, measured by multiplex proteomic and cytokine assays.