Transcriptomic Profiling of Normal Rectal Mucosa to Identify Biomarkers of Sustained Complete Response After Neoadjuvant Therapy in Rectal Cancer: An Exploratory Study

Background and Aim: Pathological complete response (pCR) after neoadjuvant therapy in locally advanced rectal cancer (LARC) is associated with improved outcomes, yet disease recurrence still occurs in a subset of patients. While tumor tissue has been extensively characterized, the biological features of histologically normal rectal mucosa adjacent to the tumor remain poorly understood. Increasing evidence suggests that non-neoplastic mucosa may reflect host immune status, therapy-induced remodeling, and field cancerization effects. This study aimed to characterize the transcriptomic and immune landscape of tumor-adjacent normal rectal mucosa in LARC patients, according to pathological response and disease recurrence. Methods: This retrospective analysis included 82 patients from the prospective IMMUNOREACT 2 cohort treated with neoadjuvant therapy followed by surgery. Histologically normal, tumor-adjacent rectal mucosa was analyzed using the NanoString nCounter PanCancer IO 360™ Panel. Differential gene expression, pathway activity, and immune cell composition were assessed using nSolver Advanced Analysis. Patients were stratified by pathological response (pCR vs non-pCR) and by recurrence status during follow-up. Results: Normal mucosa from patients achieving pCR exhibited a global downregulation of immune- and inflammation-related genes, particularly involving innate immune signaling, myeloid-associated pathways, cytokine and chemokine signaling, and antigen presentation. Pathway analyses indicated an immunologically quiescent mucosal profile, consistent with resolution of therapy-induced inflammation. In contrast, patients who developed recurrence showed upregulation of stromal, angiogenic, extracellular matrix, and Wnt-related pathways, alongside suppression of immune signaling, including interferon responses and antigen presentation. Immune cell profiling suggested functional impairment or polarization rather than simple depletion of immune populations, consistent with a permissive field effect. Conclusion: Histologically normal, tumor-adjacent rectal mucosa displays distinct transcriptional states associated with pathological response and disease recurrence in LARC. These findings support the biological relevance of the surrounding non-neoplastic tissue as a surrogate of host–tumor interactions and highlight its potential value for future integrative biomarker strategies aimed at improving patient stratification and long-term disease control.