IMMUNOREACT 9 metachronous rectal cancers have high HLA-ABC expression on healthy epithelium but a lower infiltration of CD3+ T cells than primary lesions

Abstract

Lynch syndrome is rarely associated with rectal cancer (RC) and thus, metachronous RC has been scarcely investigated. This study aimed to analyze the mucosal immune microenvironment in sporadic and metachronous RC. We analyzed the mucosal immune microenvironment in the 25 metachronous RCs present in the IMMUNOREACT 1 and 2 multicentre observational studies (624 patients). A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8b, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Single-cell suspensions were subjected to flow-cytometry to determine the proportion of epithelial cells (pan-cytokeratin) acting as antigen-presenting cells (expressing CD80, CD86, HLA-ABC) and the proportion of activated CD8 + T cells (CD8 + positive for CD28, CD38), inhibitory T cells (CD3 + CTLA-4+) of activated CD4 + T helper cells (CD4 + CD25+) and activated T regulatory cells (CD4 + CD25 + FoxP3+). No mismatch repair gene deficiencies were observed in the patients. The previous history of colorectal adenoma was significantly more frequent in metachronous RC. In healthy epithelial cells, HLA-ABC expression was significantly higher in patients with metachronous RC. In therapy-naïve metachronous RC patients, a significantly lower level of circulating lymphocytes and CD3 + T-cell infiltration in the healthy mucosa surrounding the RC was observed compared to patients with non-metachronous cancer. Our study supports the hypothesis that metachronous RC can occur in a cancerization field in patients with weak systemic and local immune systems. The peculiar site of RC makes the mismatch-repair genes deficiency in metachronous cancer onset less relevant.

Fig. 1

Metachronous RC and patients’ characteristics(IMMUNOREACT 9: whole cohort). (A) Representative immunohistochemical analysis of PMS2, MLH1, MSH2, and MSH6, (20x bar = 0.050 mm) in the cancer tissue. (B) Frequency of a previous history of colorectal adenoma (CRA) in metachronous and non-metachronous RC patients. (C) Illustration of primary cancer site in the metachronous cancers group. (D) Flow cytometric analysis of HLAabc + epithelial (cytokeratin+, CK+) cells within the cancerization field. Representative images of flow cytometric analysis of CK + HLAabc + cells are shown. ROC curve showing the accuracy of CK + HLAabc + mean fluorescence intensity (MFI) in predicting metachronous RC.

Fig. 2

Metachronous RC and differential gene expression in the cancerization field ((IMMUNOREACT 9: whole cohort). (A) Volcano plot of differential gene expression in the cancerization field of metachronous RC patients (n = 3) vs. non-metachronous RC patients (n = 30). The 20 statistically significant genes are labeled in the plot. (B) Differential expression of gene-based cell types abundance scores in the cancerization field of metachronous RC patients vs. non-metachronous RC patients. (C) Differential expression of gene signatures pathways scores in the cancerization field of metachronous RC patients vs. non-metachronous RC patients.

Fig. 3

Metachronous RC and immunological response (IMMUNOREACT 9: therapy naïve patients). (A) Association between metachronous RC and circulating lymphocytes. ROC curve showing the accuracy of circulating lymphocytes in predicting metachronous RC.RC (B) Association between metachronous RC and CD3 + T-cell infiltration in the RCcancerization field. Representative immunohistochemical analysis of CD3 is shown (20x bar = 0.050 mm). ROC curve showing the accuracy of CD3 + cells in predicting metachronous RC.

Fig. 4

Metachronous RC and immunological response (IMMUNOREACT 9: therapy naïve patients). Representative immunohistochemical analysis of CD4+, FoxP3+, Tbet+, CD8beta+, and CD8+, T cells within the cancerization field (20x bar = 0.050 mm).

Fig. 5

Metachronous RC and immunological response (IMMUNOREACT 9: therapy naïve patients). Representative immunohistochemical analysis of CD80, and PDL-1 + leukocytes within the cancerization field, (20x bar = 0.050 mm). Representative immunohistochemical analysis of PMS2, and MSH6 within the cancerization field, (20x bar = 0.050 mm).