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

## TL;DR

This study shows that immune markers in normal-looking tissue near rectal tumors can predict the risk of anastomotic leaks after surgery.

## Contribution

The first evidence that immune profiling of tumor-adjacent mucosa can predict anastomotic leaks.

## Key findings

- CD3+, CD8+, CD8β+, and Tbet+ immune markers in tumor-adjacent mucosa are predictive of anastomotic leaks.
- Combining immune markers with clinical variables like BMI and tumor location achieved ~70% accuracy in predicting leaks.
- Immune activation in normal-appearing mucosa is linked to impaired anastomotic healing.

## Abstract

In this multicenter study of 363 patients, we provide the first evidence that immune profiling of histologically normal, tumor-adjacent mucosa may identify patients at risk for anastomotic leaks (ALs) after rectal surgery. Among the markers assessed, only CD3+, CD8+, CD8β+ and Tbet+ showed predictive value, suggesting that the mucosa intended for anatomosis may already be compromised by hypoxia, extensive cell death, autoimmune inflammation or infection. We then explored combinations of clinical and immunological markers to obtain predictive models of ALs. Models including CD8+ (or CD8β+), BMI, neutrophil-to-lymphocyte ratio and tumor location achieved ~70% accuracy. Although the predictive accuracy of our models remains relatively limited, combining immune microenvironmental data from the healthy mucosa with clinical variables may represent a promising approach to identifying patients at risk of ALs and deserves further testing in clinical practice.

Background: 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. Methods: 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. Results: 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). Conclusions: 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.