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

A Prospective Observational Study of Tumour Microenvironment Remodelling During Neoadjuvant Therapy in Locally Advanced Rectal Cancer Using Single-Cell and Spatial Multi-omics

This prospective observational study characterises the tumour immune microenvironment and microbial ecology of locally advanced rectal cancer before and during neoadjuvant therapy. Tumour tissue and peripheral blood are profiled by whole-genome sequencing, single-cell RNA sequencing with paired T-cell and B-cell receptor repertoire sequencing, spatial transcriptomics of formalin-fixed paraffin-embedded sections, multiplex immunofluorescence, and blood-based proteomic, cytokine and bulk T-cell receptor analyses. The study examines how immune and stromal states change during treatment and how they relate to major pathological response.

Study Overview

• Status: Recruiting
• Conditions: Rectal Neoplasms

Detailed Description

Locally advanced rectal cancer is increasingly treated with total neoadjuvant therapy combining short-course radiotherapy, chemotherapy and programmed cell death protein 1 (PD-1) blockade, but the effect of treatment sequence on the tumour microenvironment is unresolved, and few patients have been profiled with matched pre- and on-treatment single-cell data. In this observational study, we collect tumour tissue and peripheral blood from patients with locally advanced rectal cancer at baseline, before any neoadjuvant treatment, and again after completion of short-course hypofractionated radiotherapy (5 Gy in 5 fractions) followed by one cycle of chemo-immunotherapy, together with archival formalin-fixed paraffin-embedded tissue where available. Additional formalin-fixed paraffin-embedded specimens from treatment-naive patients undergoing primary surgery, and from end-of-treatment biopsies, are used as controls for the spatial analyses. Fresh tumour tissue is analysed by whole-genome sequencing and by single-cell RNA sequencing with paired T-cell and B-cell receptor repertoire sequencing. Formalin-fixed paraffin-embedded tissue is analysed by spatial transcriptomics and multiplex immunofluorescence. Peripheral blood mononuclear cells are analysed by bulk T-cell receptor repertoire sequencing, and plasma or serum by proteomic and cytokine profiling where material permits. The principal clinical endpoint is major pathological response (mPR) at resection. We examine changes in immune and stromal populations between baseline and the on-treatment time point, features of the immune microenvironment associated with mPR, and, as exploratory analyses, the tumour microbial ecology associated with response together with differences between neoadjuvant treatment sequences. Analyses are descriptive and hypothesis-generating; no intervention, treatment assignment or change in clinical care is involved. The study was approved by the Ethics Committee of Fujian Medical University Union Hospital (2024KY297, approved 30 December 2024; and 2024KJCX060, approved 18 September 2024); archival specimens are used under a waiver of additional informed consent, and fresh biopsy or surgical tissue and blood are collected with written informed consent.

• Study Type: Observational
• Enrollment (Estimated): 100

Contacts and Locations

• Study Contact: Name: Wan-cai Que; Phone Number: 0086-0591-86218304; Email: xhyyjgb@163.com

Study Locations

• China
  • Fujian
    • Fuzhou, Fujian, China, 350001
      • Recruiting
      • Fujian Medical University Union Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

Patients with locally advanced rectal adenocarcinoma treated at Fujian Medical University Union Hospital, China, from whom tumour tissue and peripheral blood were collected before and during neoadjuvant therapy.

Description

Inclusion Criteria:

• Age 18 to 75 years
• Eastern Cooperative Oncology Group performance status 0-1
• Histologically confirmed rectal adenocarcinoma
• Locally advanced disease staged as cT3-T4N0 or cT2-T4N+ without distant metastasis (AJCC 8th edition)
• Lower border of the tumour within 10 cm of the anal verge
• Scheduled to receive neoadjuvant therapy followed by surgery, with tumour tissue and peripheral blood available before and during neoadjuvant therapy
• Written informed consent for fresh biopsy or surgical tissue, or a waiver granted by the institutional ethics committee for archival specimens

Exclusion Criteria:

• Insufficient tissue or nucleic acid quality for the planned profiling assays
• Incomplete clinical records precluding assessment of pathological or clinical response
• A history of other malignancy within 5 years other than adequately treated carcinoma in situ or basal cell carcinoma

Study Plan

How is the study designed?

Design Details

• Observational Models: COHORT
• Time Perspectives: PROSPECTIVE

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort
Achieved pathological or clinical complete response; 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.
Did not achieve pathological or clinical complete response; 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.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in immune and stromal cell composition in tumour tissue during neoadjuvant therapy	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.	Baseline (before treatment) and week 4 after treatment initiation
Major pathological response (mPR) and its association with immune microenvironment features	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.	Through study completion, an average of 4 years

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Transcriptional states of immune populations before and during treatment	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.	Baseline (before treatment) and week 4 after treatment initiation
Spatial organisation of tumour and immune compartments before and during treatment	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.	Baseline (before treatment) and week 4 after treatment initiation
Comparison of microenvironment remodelling between neoadjuvant treatment sequences	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.	Through study completion, an average of 4 years
Somatic genomic alterations associated with response to neoadjuvant therapy	Somatic mutations, copy-number alterations and mutational signatures determined by whole-genome sequencing of tumour tissue before and during neoadjuvant therapy.	Baseline (before treatment) and week 4 after treatment initiation
Clonal diversity of the peripheral blood T-cell receptor repertoire	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.	Baseline (before treatment) and week 4 after treatment initiation
Correlation of tumour microbial composition with major pathological response	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).	Baseline (before treatment) and week 4 after treatment initiation
Plasma protein and cytokine concentrations before and during neoadjuvant therapy	Concentrations of plasma or serum proteins and cytokines, expressed in pg/mL, measured by multiplex proteomic and cytokine assays.	Baseline (before treatment) and week 4 after treatment initiation

Collaborators and Investigators

• Sponsor: Fujian Medical University Union Hospital
• Investigators: Study Director: Jian Liu (jianliu@nankai.edu.cn), PhD, Nankai University

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2025
• Primary Completion (Estimated): December 31, 2028
• Study Completion (Estimated): December 31, 2028

Study Registration Dates

• First Submitted: October 27, 2026
• First Submitted That Met QC Criteria: November 2, 2026
• First Posted (Actual): November 6, 2026

Study Record Updates

• Last Update Posted (Actual): November 6, 2026
• Last Update Submitted That Met QC Criteria: November 2, 2026
• Last Verified: November 1, 2026

More Information

Terms related to this study

• Keywords: rectal cancer; neoadjuvant therapy; pathological complete response; single-cell RNA sequencing; spatial transcriptomics; tumour immune microenvironment
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Intestinal Diseases; Gastrointestinal Neoplasms; Digestive System Neoplasms; Digestive System Diseases; Gastrointestinal Diseases; Colorectal Neoplasms; Intestinal Neoplasms; Rectal Diseases; Rectal Neoplasms
• Other Study ID Numbers: FJMUUH-RECT-OMICS-2024-01

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

IPD Plan Description

Individual participant data, including clinical records and sample-level identifiers, will not be shared. De-identified single-cell, bulk RNA sequencing and spatial transcriptomic data will be deposited in the Genome Sequence Archive for Human (GSA-Human), National Genomics Data Center, China, and will be made available for research use through the repository according to its access policy after publication.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No