NKG2D ligand expression in human colorectal cancer reveals associations with prognosis and evidence for immunoediting

Abstract

Purpose: NKG2D (natural killer group 2, member D) binds to cellular ligands of the MIC and ULBP/RAET family. These ligands have restricted expression in normal tissue, but are frequently expressed on primary tumors. The role of NKG2D ligands is thought to be important in carcinogenesis but its prognostic effect has not been investigated in such a large cohort.

Experimental design: In our study, 462 primary colorectal tumors were screened for the expression of all MIC/ULBP/RAET proteins and NK cell infiltration. Tumor microarray technology was used for the purpose of this investigation.

Results: NKG2D ligands were expressed by the majority of colorectal tumors; however, the level of expression varied considerably. High expression of MIC (68 versus 56 months) or RAET1G (74 versus 62 months) showed improved patient survival. Tumors expressing high levels of MIC and RAET1G showed improved survival of 77 months over tumors that expressed high levels of one ligand or low levels of both. High-level expression of all ligands was frequent in tumor-node-metastasis stage I tumors, but became progressively less frequent in stages II, III, and IV tumors. Expression of MIC was correlated with NK cellular infiltration.

Conclusion: The observations presented are consistent with an immunoediting mechanism that selects tumor cells that have lost or reduced their expression of NKG2D ligands. The combination of MIC and tumor-node-metastasis stage was found to be the strongest predictor of survival, splitting patients into eight groups and suggesting prognostic value in clinical assessment. Of particular interest were stage I patients with low expression of MIC who had a similar survival to stage III patients, and may be candidates for adjuvant therapy.

Figures

Figure 1. Specificity of α-ULBP1, α-ULBP2, α-ULBP3 and α-RAET1E antibodies

(A) Western blot demonstrates that the antibodies to ULBP1, ULBP3 and RAET1E are specific to extracts from cells expressing the appropriate ligand. Cos7 non-transfected cells were included as a negative control. Anti-actin antibody positive control shows bands in each lane. (B) Flow cytometry confirmed the specificity of anti-ULBP2.

Figure 2. Photographs showing typical staining for MIC, RAET1E, RAET1G, ULBP1, ULBP2, and ULBP3

The photographs presented are from representative tissue micro array cores showing high (A) and low (B) expression at x100 magnification. Scale bar represents 0-500μm in 100μm increments.

Figure 3

Kaplan-Meier plots showing disease specific survival in statistically significant cases.

Figure 4. Tumor Expression of NKG2D Ligands Decreases as Tumor Grade Increases

The graphs above show the number of patients classified as low or high expression represented as a percentage of the total for each TNM stage where stages 0 and 1 were classified together as 1. Stages 2, 3, and 4 were left as individual groups.

Figure 5. 10 Year Survival Plot of Patient Prognostic Groups According to Their Prognostic Index Score

The graph shows cumulative survival for each of the prognostic groups as sub-divided according to our prognostic model generated from the Cox Regression analysis. The prognostic model was generated based on TNM category and MIC expression level. Each prognostic group is indicated in the legend with the corresponding prognostic index score.