Osteosarcoma is the most frequent primary malignant bone tumor, excluding plasma cell myeloma. Although multimodal treatment includes wide local excision and adjuvant chemotherapy, the survival rates of patients with osteosarcoma have hovered at the 60–70% level. 1 Disease-free survival rates have not changed over the past 15 years despite alterations in the chemotherapeutic regimens. Therefore, the development of new treatments and the improvement of existing approaches are imperative.

The SSX gene family is composed of 5 members, including the SSX1 and SSX2 genes, which were initially identified as fusion partners of the SS18 (previously called SYT or SSXT) gene in human synovial sarcomas carrying a recurrent t (X; 18)(p11. 2; q11. 2) chromosomal translocation. 2–4 All 5 members of the SSX gene family exhibit close nucleotide homologies (ranging from 88–95%) and encode proteins of 188 amino acids (homologies ranging from 77–91%). 5 A recent study revealed that the conserved COOH-terminus of SSX could repress the transcription of a reporter gene. 6 To date, expression of the SSX genes has been found in adult testis and, at very low levels, in thyroid. However, recent investigations disclosed that all SSX genes, except SSX3, are ectopically expressed at varying frequencies (0–57%) in a number of malignancies. 7, 8 Additionally, it was shown that ectopically expressed SSX2 can elicit humoral immune responses in a subset of melanoma patients. 9 Combined, these data indicate that the SSX proteins constitute new members of the family of cancer/testis antigens, which are potential targets for the development of cancer immunotherapy. After informed consent was obtained, 17 tumor samples of osteosarcomas were collected during routine diagnostic and therapeutic procedures at the Osaka Medical Center for Cancer and Cardiovascular Diseases (Osaka, Japan). Histologic subtypes were as follows: conventional osteosarcoma (11 cases); telangiectatic osteosarcoma (1 case); high-grade surface osteosarcoma (1 case); periosteal osteosarcoma (1 case); parosteal osteosarcoma (1 case); secondary osteosarcoma (1 case); and extraskeletal osteosarcoma (1 case). Primary cultured human osteoblastoma was provided by the Osaka University Hospital (Osaka, Japan). The human osteoblast cell (NHOst) was purchased from Clonetics (San Diego, CA). Normal tissues including muscle, fat, synovium and testis were obtained from autopsies of unrelated individuals. Total RNA was prepared from frozen tissues or cells with the acid