Background Stage II colorectal cancer should undergo adjuvant therapy only in the presence of risk factors and vascular, lymphatic, and perineural invasion (VELIPI) are risk factors for recurrence after surgery. Immune surveillance mechanisms have been demonstrated to influence the natural history of colorectal cancers. The purpose of this study was to identify potential immune markers of VELIPI in the healthy mucosa to implement decision-making in patients with stage II rectal cancer.
Methods This study is a sub-analysis of data from the IMMUNOREACT project (clinicaltrials.gov NCT04915326 and NCT04917263) obtained from the healthy mucosa surrounding rectal cancer. A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8beta, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. A prospective analysis was performed with flow cytometry to determine the proportion of epithelial cells expressing CD80, CD86, CD40, HLA ABC, or HLA DR and of activated CD8+ T cells, CD4+ Th1 cells, and T reg. NanoString gene expression assay with the panCancer Immune profile was performed. Data on the mutational profile of rectal adenocarcinoma were extracted from the Cancer Genome Atlas (TCGA) deposited public database (https://gdc.cancer.gov/resourcestcga-users/tcga-code-tables/tcga-study-abbreviations). Nonparametric tests were used for comparison.
Results A total of 1164 patients with rectal cancer included in the IMMUNOREACT cohort were analyzed and 148 of them had stage II rectal cancer. In therapy-naïve patients, the CD8/CD3 ratio in peritumoral healthy rectal mucosa was increased in patients without VELIPI (p=0.021), and, a high CD8+ T-cell rate was associated with increased disease-free survival (p=0.045) while an increase in CD8beta+ T-cell rate was associated with decreased overall survival (p=0.0071). In the TCGA cohort, which included 44 patients, we observed a higher expression of CTLA-4 and PD-L1 in patients with VELIPI. Moreover, in patients who had neoadjuvant therapy, no significant differences in the immune microenvironment of healthy peritumoral tissue were observed but the expression of several genes associated with MAPK and metabolic stress pathways were significantly different in patients with VELIPI and those without it.
Conclusion Our study showed that, in stage II rectal cancer patients, cytotoxic T-cell activity may be crucial to predicting the presence of VELIPI and, consequently, the need for adjuvant therapy. In addition, transcriptomic analysis proved to be a valuable tool in developing predictive models for VELIPI by analyzing mRNA expression in the healthy peritumoral mucosa when neoadjuvant therapy may have blurred the most common markers.

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