Cytotoxic immune activity reflects a coordinated immune microenvironment and is associated with response to total neoadjuvant therapy in rectal cancer.

BACKGROUND: Total neoadjuvant therapy (TNT) has emerged as a promising treatment strategy for locally advanced rectal cancer; however, robust biomarkers predicting treatment response remain lacking. We aimed to identify pretreatment transcriptomic determinants of TNT response, focusing on cytotoxic immune activity and its broader immune context. METHODS: We analyzed two independent cohorts of patients with rectal cancer treated with TNT: a laser-capture microdissection (LCM) cohort (n = 154) and a bulk tumor cohort (n = 80). Differential gene expression, gene set enrichment, and immune deconvolution analyses were performed using pretreatment RNA sequencing data. Associations between cytotoxic activity and treatment response were evaluated using multivariable models. RESULTS: Only a limited number of genes were reproducibly associated with response across cohorts. Pathway-level analyses revealed enrichment of immune-related signatures in complete responders in the bulk cohort, whereas no significant enrichment was observed in the LCM cohort. Cytotoxic scores showed a consistent directional association with complete response in both cohorts, although modestly, and did not reach statistical significance in the LCM cohort. Cytotoxic activity was strongly correlated with multiple immune cell populations and signaling pathways, including effector and regulatory components, indicating a coordinated immune microenvironment. Multivariable analyses demonstrated that cytotoxic activity was independently associated with treatment response in the bulk cohort and recurrence-free survival in the LCM cohort. CONCLUSIONS: Cytotoxic immune activity represents a biologically relevant but modest determinant of response to TNT in rectal cancer. These findings suggest that treatment sensitivity is shaped by a coordinated immune microenvironment rather than cytotoxic activity alone.