Restriction of GAGE protein expression to subpopulations of cancer cells is independent of genotype and may limit the use of GAGE proteins as targets for cancer immunotherapy.

The GAGE cancer testis antigen gene family encodes products that can be recognized by autologous T cells, and GAGE proteins have been suggested as potential targets for cancer immunotherapy. Analysis of GAGE expression in tumours has primarily been performed at the level of gene transcription, whereas little is known about GAGE expression at the protein level. To evaluate the potential of GAGE proteins as targets for cancer-specific immunotherapy, we studied the expression of these proteins in normal and malignant cells/tissues using a novel panel of monoclonal antibodies. Immunohistochemical analysis of more than 250 cancer specimens demonstrated that GAGE proteins were frequently expressed in numerous cancer types and correlated with the expression of the cancer testis antigens MAGE-A1 and NY-ESO-1. Significant intercellular and subcellular differences in GAGE protein levels were observed, and most GAGE-positive tumours also contained cancer cells lacking GAGE expression. Studies of genetically homogenous cell lines with similar intercellular heterogeneous GAGE expression showed that GAGE expression was not associated with a specific genotype, but defined a phenotypically distinct population of cells. Surprisingly, in normal tissues we found that GAGE proteins were not restricted to testis, but were also present in a subset of oocytes of resting primordial follicles and in maturing oocytes. This is the first time that a cancer testis antigen has been reported in postfoetal oocytes. The lack of GAGE expression in a subset of cancer cells within GAGE-positive tumours has decisive implications for the development of GAGE-targeted cancer therapy.