Expression of SSX genes in human osteosarcomas (2002)

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 to 95%) and encode proteins of 188 amino acids (homologies ranging from 77 to 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 guanidinium-phenol-chloroform method. RT-PCR was performed on the RNA using the Amplitaq Gold kit (Perkin-Elmer, Weiterstadt, Germany). The thermocycling protocol and the specific primer sets for the respective SSX genes have been reported previously.7, 8 A primer set of SSX-Bcl and SSXL-rev was preliminarily used for detection of any SSX gene expression because this primer set can recognize and amplify the 3′ ends of all 5 characterized SSX genes (SSX1–SSX5). An aliquot of each PCR reaction was size-fractionated on a 2% agarose gel, visualized by ethidium bromide staining and evaluated for expected size. Using the SSX-Bcl/SSXL-rev primer set, we observed a specific band of 324 bp in 16 of 17 osteosarcomas tested. No PCR product could be found in the parosteal osteosarcoma sample (case 17), primary cultured osteoblastoma (data not shown), cultured osteoblast (NHOst, not shown) and normal tissues including muscle (not shown), fat (not shown) and synovium (Fig. 1). With the specific SSX primer sets we were able to show that SSX1 and SSX2 were most frequently expressed (13 cases, 76%), followed by SSX4 (10 cases, 59%) and SSX5 (8 cases, 47%). Since SSX3-derived transcripts have not been detected in any tumors previously reported,8 it should be noted that SSX3 expression was found in 4 of the osteosarcoma cases. These cases (1, 2, 3 and 4) show expression of all SSX family members (Fig. 1). Because the SSX genes show a very high degree of homology, we routinely sequenced all SSX amplification products using an automated capillary sequencer (Perkin-Elmer Applied System). In this way we could confirm that the sequences of all PCR fragments were identical to those reported for human SSX1 (Genbank accession no. X86174), SSX2 (X86175), SSX3 (U90840), SSX4 (U90841) and SSX5 (U90842), respectively. Detection of SSX gene expression in human osteosarcomas by RT-PCR. All primers used recognized sequences located in distinct exons, thereby preventing the amplification of putative contaminating DNA. RT-PCR with the primer sets of SSX-Bcl and SSXL-rev, SSX1-5′ and SSX1-3′, SSX2-5′ and SSX2-3′, SSX3-5′ and SSX3-3′, SSX4-5′ and SSX4-3′ and SSX5-5′ and SSX5-3′ yielded expected bands of 324, 422, 435, 378, 415 and 314 bp, respectively. Integrity of the obtained cDNA was tested by amplification of GAPDH transcripts (520 bp) in a 30-cycle standard PCR reactions. Strong expression of all SSX family genes (SSX1–SSX5) was seen in normal testis. SYT-SSX fusion gene product was not observed in all 17 samples of osteosarcoma (data not shown). Lane M, size marker; lanes 1–17, osteosarcoma; lane N, negative control (no RNA). In addition, we used immunohistochemistry to investigate SSX expression at the protein level in normal testicular tissue and the 17 osteosarcoma samples. The anti-SSX monoclonal antibody used in the present study (E3AS; kindly provided by Dr. Ad Geurts van Kessel) can detect SSX2, -3 and -4 but not SSX1.7 As shown before, SSX expression in the testis was found to occur in the nuclei of spermatogenic cells, mainly spermatogonia close to the basement membrane of the seminiferous tubule (Fig. 2a). Using this antibody, we observed nuclear staining of SSX in 7 of 17 osteosacoma samples (cases 1, 2, 3, 5, 6, 7 and 15; Fig. 2). The expression pattern among 7 positive samples was heterogeneous and in 2 samples very focal. In these experiments, we found some lack of correspondence between the results of the RT-PCRs and the immunohistochemistry. This was considered to be due to a bias in sampling different areas of the tumor with disparate SSX expression and/or the limitation of the E3AS MAb to recognize all SSX proteins. The same discrepancy has been noted in earlier studies using this antibody.7 Immunohistochemical staining of SSX in normal testicular tissue (a) and a representative sample of human osteosarcoma (b) using the anti-SSX E3AS MAb. (a) Seminiferous tubule exhibited intratubular staining of early spermatogenic cells, mainly spermatogonia. Neither Sertoli cells, interstitial cells nor maturated spermatogenic cells were stained by the E3AS MAb. (b) Osteosarcoma cells revealed heterogeneous nuclear staining of SSX in a representative case (case 6). We demonstrated by RT-PCR that all tested osteosarcomas (excluding 1 specimen of parosteal osteosarcoma) expressed at least 1 of the 5 SSX genes. In a parallel study, we also found SSX expression in 10 of 12 malignant fibrous histiocytomas, the most common soft tissue sarcoma (data not shown). Although ectopic SSX expression is shared by a broad spectrum of human cancers, the percentage of positive tumors varies and has been shown to reach 60% at most.8 In the current study, we found a much higher percentage of positive tumors (up to 76% for SSX1 and SSX2) in this (still limited) set of osteosarcomas. Considering these data together, we think that patients with musculoskeletal tumors, especially osteosarcoma, could be potential candidates for future immunotherapy and/or gene therapy targeted to the SSX genes and their gene products. In addition, the observed high incidence of SSX gene expression may be clinically useful in the diagnosis of musculoskeletal tumors, including estimation of surgical margins and detection of lymphatic or hematologic spread, since no distinctive molecular marker has been available to date in musculoskeletal tumors such as osteosarcomas and malignant fibrous histiocytomas. Recently, SYT-SSX1-expressing rat fibroblasts were reported to exhibit enhanced growth rates, anchorage-independent growth in soft agar and tumor-forming potential in nude mice, confirming that SYT-SSX gene fusion is the key event in the development of synovial sarcoma.10 In addition, SSX1-expressing fibroblasts were also shown to exhibit slightly enhanced growth rates as well as and tumor-forming potential in 1 of 4 clones in nude mice.10 Therefore, SSX genes and their products might be important in the pathogenesis of musculoskeletal tumors. Although previous cDNA cloning studies have shown that the SSX3 gene is one of the most abundantly expressed SSX genes in testis,5 it has neither been implicated in t(X;18)-positive synovial sarcomas, nor has it been found to be ectopically expressed in various tumors. In the present study we have shown, for the first time, that SSX3 is expressed in 4 osteosarcoma cases, together with all 4 of the other SSX genes. Very recently, we also found SSX3 expression in the osteosarcoma cell line Saos-2 (data not shown). Further investigation will be necessary to elucidate the specific function of SSX3 in the development and progression of human osteosarcomas. Yours sincerely, Norifumi NAKA*, Nobuhito ARAKI, Hirofumi NAKANISHI, Kazuyuki ITOH, Masayuki MANO, Shingo ISHIGURO, Diederick R. H. DE BRUIJN, Akira MYOUI, Takafumi UEDA AND HIDEKI YOSHIKAWA