IMMUNOREACT 4: Peritumoral Microenvironment Associated with Anastomotic Leaks After Surgery for Rectal Cancer

<b>Background:</b> Anastomotic leaks (ALs) remain a critical complication after rectal cancer surgery. Emerging evidence suggests that local immune dysregulation may play a key role in anastomotic healing. We investigated the immune microenvironment of histologically normal, tumor-adjacent rectal mucosa-a tumor-conditioned field-as a potential substrate for AL predisposition. <b>Methods:</b> IMMUNOREACT 4 is a sub-analysis of the IMMUNOREACT project (clinicaltrials.gov NCT04915326 and NCT04915326), a multicenter translational study evaluating immune features of histologically normal, tumor-adjacent rectal mucosa of patients undergoing colorectal anastomosis. A prospective cohort (n = 121) was analyzed using flow cytometry, in addition to a retrospective cohort (n = 262) using immunohistochemistry. Immune markers of epithelial activation and lymphocyte subsets were compared between patients with and without postoperative ALs. Exploratory predictive models combining immune and clinical variables were developed and evaluated using discrimination, calibration and decision curve analyses. <b>Results:</b> At flow cytometry, the CK⁺HLAabc⁺ MFI (AUC 0.66, 95% CI 0.52-0.80), CD8⁺CD38⁺ cell rate (AUC 0.65, 95% CI 0.52-0.78) and CD3⁺CTLA4⁺ cell rate (AUC 0.65, 95% CI 0.51-0.80) showed moderate predictive potential for ALs. In immunohistochemistry, CD3⁺ (AUC 0.57, 95% CI 0.54-0.60), CD8⁺ (AUC 0.57, 95% CI 0.52-0.62), CD8β⁺ (AUC 0.59, 95% CI 0.53-0.65) and Tbet⁺ (AUC 0.60, 95% CI 0.56-0.64) showed some predictive ability for ALs. The model including CD8β⁺, the BMI, neutrophile/lymphocyte ratio and tumor location had an AUC of 0.67 (95% CI 0.62-0.72). <b>Conclusions:</b> Immune activation within histologically normal, tumor-adjacent rectal mucosa-characterized by epithelial HLA upregulation and cytotoxic or Th1 T cell infiltration-is associated with postoperative ALs. Although predictive accuracy is limited, these findings support the concept that a tumor-conditioned immune microenvironment may predispose patients to impaired anastomotic healing. Integration of mucosal immune profiling with clinical variables represents a promising exploratory approach that warrants further prospective validation.