Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer

Abstract

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8(+) T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.

Fig. 1

Analysis of CT gene expression by semi-quantitative RT-PCR. Expression of MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1, SSX2 and the housekeeping gene β-actin in normal and tumor samples of LAU852 and LAU911 as well as tumor and metastatic samples of LAU846 was analyzed by RT-PCR. For each tissue sample, PCR was performed on serial fivefold dilutions of the cDNA. The cDNA prepared from human testis and the melanoma cell line SK-MEL-37 were used as positive controls. Arrows indicate the specific product and stars genomic contamination

Fig. 2

Detection of CT antigen expression in CRC by immunohistochemistry. Sections of the primary CRC tumors isolated from the CT antigen-positive patients LAU846, LAU852 and LAU911 were stained with mAb anti-MAGE (57B and 6C1) and anti-NY-ESO-1 (D8.38). As positive control, sections from CT antigen-positive testicular seminoma were stained with the same set of antibodies. Hematoxylin/eosin staining of the individual sections is shown at lower magnification. Filled arrows indicate an example of positive stained tumor cell. Empty arrows indicate an example of negative stained tumor cell. The region shown at higher magnification after mAb staining is boxed

Fig. 3

T cell responses in CT antigen-positive CRC patients. a Detection of CT antigen-specific T cells in HLA-A2 patients by A2 tetramers. PBMC from patients LAU919 (MAGE-A3⁻ MAGE-A10⁻ NY-ESO-1⁻), LAU911 (MAGE-A3⁺ MAGE-A10⁺ NY-ESO-1⁺), LAU942 and 987 (MAGE-A3⁺) were stimulated individually in vitro with peptides MAGE-A3_112–120, MAGE-A10_254–262 and NY-ESO-1_157–165 (LAU919 and LAU911) or only MAGE-A3_112–120 (LAU942 and LAU987). At day 10, peptide-specific CD8⁺ T cells were detected by flow cytometry with A2 tetramers incorporating the corresponding peptides. Dead cells were excluded by PI staining. b IFN-γ ELISPOT assay of in vitro stimulated PBMC of patient LAU846. PBMC were stimulated once in vitro with peptides NY-ESO-1-ORF2_46–54 and MAGE-A3_167–176 and analyzed at day 10. Autologous cells were used as antigen-presenting cells in the presence of peptides. Quantification of spot-forming T cells was done after 16 h of incubation