Efficient identification of a novel cancer/testis antigen for immunotherapy using three-step microarray analysis

Advanced technology in molecular biology has provided us powerful tools for the diagnosis and treatment for cancer. We herein adopted a new methodology to identify a novel cancer/testis (CT) antigen with high frequency of expression in colorectal cancer as follows: (a) combining laser microdissection and cDNA microarray was used to analyze the gene expression profile of colorectal cancer cells; (b) genes over-expressed in testis and underexpressed in normal colon epithelium were analyzed using cDNA microarray; and (c) the gene expression profile of colorectal cancer cells was compared with that of normal testis. Using this methodology, we selected 38 candidates for CT antigen. Among these genes, we identified a novel CT antigen, serine/threonine kinase 31 (STK31), which was previously reported as a gene expressed in spermatogonia. Reverse transcription-PCR analysis showed that STK31 gene expression levels in cancer samples were significantly higher (P < 0.0001) than those in normal samples. The STK31 gene was frequently expressed not only in colorectal cancer but also in gastric and esophageal cancer. Moreover, STK31 peptide was able to elicit specific CTLs and induced CTLs lysed either peptide-loading or endogenously STK31-expressing target cells. These results showed that the new methodology in this study facilitated identification of CT antigens and that STK31 may be a candidate for cancer immunotherapy against gastrointestinal cancer.