Dynamic molecular and immune characterization for personalized treatment in locally advanced rectal cancer

Background: Locally advanced rectal cancer (LARC) shows highly variable responses to neoadjuvant radiotherapy with a minority of patients achieving complete tumor regression. Existing clinical tools cannot reliably predict responders using pre-treatment samples. Understanding the dynamic molecular and immune changes occurring during and after radiotherapy is initiated is therefore critical to guide personalized treatment strategies and improve outcomes. Aims/Objectives: This study employed a serial rectal tumor sampling protocol during neoadjuvant treatment. We aim to characterize longitudinal transcriptomic changes in LARC tumors during radiotherapy and to identify early molecular and microenvironmental features associated with treatment response. Methods/Results: Tumor biopsies were collected from 38 patients at baseline, week 2, week 6, and week 12 during short-course or long-course radiotherapy. Bulk RNA-seq was used to profile gene expression across time. PCA and differential expression analyses showed that most transcriptional reprogramming occurred between baseline and week 2, with minimal changes at later time points. Responders exhibited early downregulation of proliferation-related genes alongside sustained activation of immune pathways. Divergence from non-responders was most evident by week 2, highlighting a critical window for treatment response. Consensus Molecular Subtype (CMS) classification showed enrichment of CMS2 in non-responders and CMS4 in responders. Tissue composition further influenced transcriptional patterns, with stromal-rich samples in responders displaying stronger immune signatures. Based on Cell deconvolution tools, responders showed pronounced week-2 activation of myeloid and lymphoid populations, while non-responders exhibited delayed immune activation. Conclusions: This study shows that radiotherapy response in LARC is determined early, with week 2 emerging as a key point where responders and non-responders clearly diverge. Responders rapidly suppress proliferation and activate strong immune programs, while non-responders show delayed or minimal immune engagement. These early molecular and microenvironmental shifts highlight an important role for early immune activation in directing successful RT responses. These features, present early during therapy suggest that on-treatment predictive biomarker development should be pursued to support development of adaptive, personalized treatment strategies.