High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability

Abstract

Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI- cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI- CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 +/- 6.2 versus 4.6 +/- 4.1, P < 0.001), CD8 (13 +/- 6.4 versus 3.7 +/- 3.8, P < 0.001), and TIA-1 (11.2 +/- 6.5 versus 1.9 +/- 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI- tumors, as revealed by the expression of granzyme B (5.3 +/- 4.5 versus 0.6 +/- 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 +/- 2.1 versus 2.6 +/- 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients.

Figure 1.

Characterization of intraepithelial lymphocytes in representative cases of MSI⁻ (a) and MSI⁺ (b to g) CRCs. Activated CTLs infiltrating within neoplastic epithelial structures are present in significantly higher numbers in MSI⁺ CRCs. CD8 (a and b), TIA-1 (c), perforin (d), and GrB (e). Immunoperoxidase with hematoxylin counterstain. Original magnification, ×400. f: Polarized GrB immunolocalization in IELs infiltrating MSI⁺ CRCs. Original magnification, ×1000. g: Co-localization of CD8 (red) and GrB (brown) in IELs in MSI⁺ CRCs. Double immunolabeling with Fast Red and DAB as chromogens; original magnification, ×400. h: Apoptotic cell bodies (TUNEL⁺, brown) in close contact with CD8⁺ (red) intraepithelial lymphocytes in MSI⁺ CRCs. TUNEL developed with DAB, followed by immunostaining with anti-CD8 antibody developed with Fast Red; original magnification, ×400. Inset: Cytokeratin/TUNEL double labeling demonstrating that the large majority of TUNEL⁺ cells were neoplastic epithelial cells. TUNEL developed with DAB, followed by immunostaining with anti-cytokeratin antibodies developed with Fast Red; original magnification, ×400.