Abstract: Papillomavirus binding factor (PBF) was first identified as a transcription factor regulating the promoter activity of human papillomavirus. We previously demonstrated that PBF is an osteosarcoma-associated antigen and 92% of osteosarcoma tissues express PBF in the nucleus. Moreover, PBF-positive osteosarcoma has a significantly poorer prognosis than that with negative expression of PBF. In the present study, we assessed the biological role of PBF in cell survival. Overexpression of PBF induced cell death-mediated lactate dehydrase (LDH) release from 293EBNA cells. Cleaved poly(ADP-ribose) polymerase and active caspase-3 were also detected. However, PBF-induced apoptosis did not affect caspase-9 activity. Next, to identify the apoptosis regulator of PBF, we screened a cDNA library constructed from mRNA of the osteosarcoma cell line OS2000 using a yeast two-hybrid system and isolated Scythe/BAT3. Scythe/BAT3 mRNA was detected in 56% of osteosarcoma tissues and ubiquitously in various normal tissues. Although Scythe/BAT3 was localized to the cytoplasm in normal tissue, it was localized to the nucleus in osteosarcoma tissue. PBF and Scythe/BAT3 also colocalized to the cytoplasm in 293T cells and the nucleus in OS2000. Furthermore, overexpression of Scythe/BAT3 suppressed cell death events that resulted from overexpression of PBF in OS2000, but not in 293EBNA cells. Thus, our results support the ideas that: (i) PBF could induce apoptotic cell death via a caspase-9-independent pathway; (ii) the apoptosis regulator Scythe/BAT3 is a PBF-associated molecule acting as a nucleus-cytoplasm shuttling protein; and (iii) colocalization of PBF and Scythe/BAT3 in the nucleus might be an important factor for survival of osteosarcoma cells. (Cancer Sci 2008).

Abstract: Research on human tumor immunology has greatly advanced in the past two decades. Many immunogenic tumor antigens have been identified, and some of these antigens entered in clinical trials. Consequently, it has been shown that these antigens can inhibit tumor growth in patients to some extent, indicating that they act as potent immunogenic therapeutic vaccines in cancer patients with malignancies originating from various tissues. These patients had antigen-specific cytotoxic T-lymphocyte (CTL) responses when assessed on tetramer, enzyme-linked immunospot (ELISPOT), T-cell clonotype and CTL induction efficiency. Thus, it has become clear that human tumor vaccines can evoke clinical and immunological anti-tumor responses in patients. The tumor regression effects of tumor vaccines, however, are generally low, and it is obvious that current vaccination protocols are generally too weak to provide substantial and satisfactory clinical benefits. This means that other drastic and more potent clinical and immunological protocols are required in cancer immunotherapy. To find such efficient protocols the basic immunological and biological properties of cancers must be investigated. In the present review the identification of human tumor antigens recognized on CTL and the clinical trials are introduced. Next, the most recent analysis of human cancer-initiating cell (cancer stem cell)-associated antigens is described. These antigens might be able to act as 'universal, general and fundamental' tumor antigens. Also present is the authors' recent study for increasing cross-presentation efficiency in dendritic cells and subsequent enhancement of human leukocyte antigen (HLA)-class I-restricted peptide antigenicity by using HSP90 and ORP150 molecular chaperones that act as endogenous Toll-like receptor ligands. In addition to the aforementioned manipulation of the positive loop of tumor immunity, it is necessary to regulate and intervene in the negative loop. In particular, the potential of the expression of HLA class I molecule regulation by epigenetic mechanisms will be discussed. Finally, the type of basic and clinical tumor immunology research highly required currently, and in the very near future, are described.

Abstract: ABSTRACT: BACKGROUND: To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2. METHODS: Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201+ patients with osteosarcoma using limiting dilution (LD)/mixed lymphocyte peptide culture (MLPC) followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by 51Cr release assay. RESULTS: Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2x10-7 to 5x10-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2. CONCLUSION: These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.

Abstract: Ewing's sarcoma family of tumors (ESFT) is comprised of highly malignant bone and soft tissue tumors in children and young adults. Despite intensive treatments for patients with ESFT, disease which presents with metastatic spread or relapses after primary treatment remains incurable in the majority of cases, indicating the importance of efforts to develop new treatment modalities, including immunotherapy. The present study was designed to examine the expression profile of papillomavirus binding factor (PBF), which we previously defined as an osteosarcoma-associated antigen, and its prognostic significance for patients with ESFT. Biopsy specimens from 20 ESFT were stained with an anti-PBF antibody. Survival was estimated using Kaplan-Meier plots and the prognostic significance of several variables, including the expression status of PBF, on disease-free and overall survival was determined by univariate analysis using the log-rank test. Of 20 specimens, 18 (90%) reacted positively to the anti-PBF antibody. Fifteen specimens (75%) were graded as PBF overexpression. Of the 11 variables analyzed, stage III disease, inadequate surgical margins and PBF overexpression were significantly associated with decreased disease-free and overall survival. None of the other variables, including age, gender, origin of tumor, tumor site or levels of LDH, ALP, CRP and ESR, showed any significant association. These findings indicate that the overexpression of PBF is a factor indicative of poor prognosis in ESFT. PBF may also serve as a putative target antigen in immunotherapy for patients with ESFT that have a poor prognosis and PBF overexpression.

Abstract: To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a cytotoxic T lymphocytes (CTL)-defined osteosarcoma antigen in the context of human leukocyte antigen (HLA)-B55. In the present study, we analyzed the distribution profile of PBF in 83 biopsy specimens of osteosarcomas and also the prognostic impact of PBF expression in 78 patients with osteosarcoma who had completed the standard treatment protocols. Next, we determined the antigenic peptides from PBF that react with peripheral T lymphocytes of HLA-A24(+) patients with osteosarcoma. Immunohistochemical analysis revealed that 92% of biopsy specimens of osteosarcoma expressed PBF. PBF-positive osteosarcoma conferred significantly poorer prognosis than those with negative expression of PBF (P = 0.025). In accordance with the Bioinformatics and Molecular Analysis Section score, we synthesized 10 peptides from the PBF sequence. Subsequent screening with an HLA class I stabilization assay revealed that peptide PBF A24.2 had the highest affinity to HLA-A24. CD8(+) T cells reacting with a PBF A24.2 peptide were detected in eight of nine HLA-A24-positive patients with osteosarcoma at the frequency from 5 x 10(-7) to 7 x 10(-6) using limiting dilution/mixed lymphocyte peptide culture followed by tetramer-based frequency analysis. PBF A24.2 peptide induced CTL lines from an HLA-A24-positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA-A24. These findings suggested prognostic significance and immunodominancy of PBF in patients with osteosarcoma. PBF is the candidate target for immunotherapy in patients with osteosarcoma.

Abstract: Towards the goal of identifying tumor-rejection antigens on eradicated tumors in bone and soft tissue sarcomas, we evaluated the immune response against antigens presented by lost HLA class I molecules. Tumor specimens and peripheral blood samples were obtained from a 70-year-old woman with pleomorphic malignant fibrous histiocytoma. Over 1-year culture, a tumor cell line (MFH2004) was established. A B-cell line infected with Epstein-Barr virus (B2004-EBV) was developed from the blood samples. HLA genotypes of B2004-EBVcells were A*0206/2402, B*4006/4601, and C*0102/0801, whereas MFH2004 cells were defective for A*0206, B*4006, and C*0102. Loss of HLA-A2 expression was also proved immunohistochemically in the primary tumor tissues. Lost HLA-A2 in MFH2004 cells was retrieved by transfection of HLA-A*0206 cDNA to develop MFH2004-A2. Attempts to induce CTLs by mixed culture with autologous T cells and MFH2004 cells resulted in failure. In contrast, those with MFH2004-A2 induced CTL clones CTL2004-c6 and CTL2004-c17. These CTL clones specifically killed MFH2004-A2 but not MFH2004 or B2004-EBV in an HLA-A2-restricted manner. These findings suggest that CTL2004-c6 and CTL2004-c17 recognize autologous tumor-rejection antigens presented by HLA-A*0206, which may have been expressed by tumor cells that had been eradicated by the host's immunosurveillance system.

Abstract: Recent immunotherapy depends largely on understanding of the molecular interactions between T cell receptors (TCR) on cytotoxic T lymphocytes (CTL) and peptide/MHC class I complexes on tumor cells. Many tumor antigens identified by cDNA library expression cloning methods, especially from malignant melanoma, have greatly contributed to clarifying such mechanisms and led to peptide vaccination trials, mainly for patients with melanoma. Although the objective tumor regression rate mediated by peptide vaccination is still low compared to adoptive cell transfer therapy, antigenic peptide vaccination can cause a constant objective response generally evaluated as stable disease or decreased serum levels of tumor markers. In addition, recent trials in the adjuvant setting showed some suppressive effects against recurrence. Therefore, peptide vaccination still has potential for clinical benefits in patients with various cancers. For further improvement of peptide vaccination, we considered that (i) novel antigenic peptides, (ii) effective adjuvants, (iii) more sensitive immunological monitoring and (iv) drugs up-regulating HLA class I molecules might be important.

Abstract: Since the prognosis of human osteosarcoma in advanced stage remains poor, the development of new and effective therapies including immunotherapy is required. To identify tumor-associated antigens of osteosarcoma applicable to the immunotherapy of this malignancy, we employed the serological analysis of recombinant cDNA expression library (SEREX) technique that defines tumor antigens recognized by the humoral immune system. Screening a cDNA library derived from an osteosarcoma cell line MG63 with sera from osteosarcoma patients identified 43 positive clones, representing 14 distinct antigens. Among them, CLUAP1 (clusterin-associated protein 1) was highly expressed in osteosarcoma tissue samples and cell lines. Overexpression of CLUAP1 was observed in other malignancies including ovarian, colon, and lung cancers. Our results suggest that CLUAP1 may be useful as a prognostic/diagnostic marker and/or for a target of immunotherapy of osteosarcoma.

Abstract: With the goal of establishing efficacious peptide-based immunotherapy for patients with bone and soft tissue sarcomas, we previously identified the cytotoxic T lymphocyte-defined osteosarcoma antigenic gene Papillomavirus binding factor. The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas. Seventy-four formalin-fixed paraffin-embedded specimens of various bone and soft tissue sarcomas, including 33 osteosarcomas, were stained with the anti-HLA class I monoclonal antibody EMR8-5, which we recently generated. The expression of HLA class I was lost or downregulated in 46 of these specimens (62%). With respect to osteosarcoma, loss or downregulation of HLA class I expression was seen in 13 (52%) of 25 primary tumors and seven (88%) of eight metastatic tumors. In six of 11 HLA class I-negative osteosarcoma specimens, the expression of beta-2 microglobulin was also lost. Subsequently the prognostic significance of HLA class I expression was analyzed in 21 patients with osteosarcoma who had completed multidrug neoadjuvant chemotherapy and undergone adequate surgery. Patients with osteosarcoma highly expressing HLA class I showed significantly better overall and event-free survival than those with HLA class I-negative osteosarcoma. In contrast, such prognostic significance of HLA class I expression was not found in 15 patients with malignant fibrous histiocytoma of soft tissue. These findings suggest that the class I-restricted cytotoxic T lymphocyte pathway plays a major role in immune surveillance of patients with osteosarcoma.

Abstract: Malignant fibrous histiocytoma (MFH) of the bone is a high-grade sarcoma characterized histologically by the composition of fibroblasts and pleomorphic cells with a prominent storiform pattern. Despite institution of multi-modality treatments, the prognosis for patients with this tumor remains unsatisfactory. In the present study, towards the goal of developing active immunotherapy for those affected, we established four cell lines from a 53-year-old woman who suffered from MFH of the humerus with lymph node metastases. MFH2003 and MFH2003-B7.1 were the primary lesion-derived cell line and its B7.1-transfectant, respectively. B2003-EBV and TIL2003 were a B cell line established from peripheral blood lymphocytes and a T cell line established from tumor-invaded lymph nodes, respectively. MFH2003 cells could be maintained over a period of 1 year in vitro, and could be xenotransplanted into nude mice. The phenotype of cells analyzed by immunostaining was similar to the original tumor. TIL2003 cells were all CD8+ and specifically recognized MFH2003 cells and MFH2003-B7.1 cells, but not B2003-EBV cells. An anti-HLA-class I monoclonal antibody completely blocked the anti-MFH2003 response of TILs2003. These findings indicate the existence of an anti-MFH specific immune response in the microenvironment of metastatic lymph nodes. The present autologous cell lines provide the basis for identification of novel tumor-associated antigens and may be helpful in the establishment of immunotherapy for patients with MFH of the bone.

Abstract: Over the past three decades, there have been remarkable advances in the treatment of bone and soft tissue sarcomas. These include the introduction of adjuvant chemotherapy, establishment of guidelines for adequate surgical margins, and the development of post-excision reconstruction. There have also been advances in the field of immunotherapy against bone and soft tissue sarcomas, which, unfortunately, have received less attention. However, lack of progress in chemotherapy-based treatments for bone and soft tissue sarcomas has reignited interest in immunotherapeutic approaches. Here we summarize current progress in the immunotherapy of bone and soft tissue sarcomas including the strategies utilized to identify tumor-associated antigens, and the design of clinical trials.

Abstract: BACKGROUND: Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma. METHODS: A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction. RESULTS: A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. CONCLUSIONS: Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.

Abstract: The identification of tumor antigens recognized by cytotoxic T-lymphocytes and subsequent clinical trials using peptide immunization have shown tumor regression in advanced malignant melanoma patients. However, efficacious therapies for epithelial cancers, and certainly bone and soft tissue sarcomas, seemed to have been significantly delayed. For the development of a peptide-based immunotherapy for epithelial cancer and sarcomas, we have identified novel antigens recognized by CTLs using autologous pairs of tumor cell line-CTLs, or reversed immunological approach. Recently we started the phase I clinical trials of peptide-based immunotherapy. In this review, we described the recent study to establish a peptide-based immunotherapy for epithelial cancers, bone and soft-tissue sarcomas.

Abstract: To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24(+) synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24(+) synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24(+) synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.

Abstract: PURPOSE: Closing wedge osteotomies are an attractive treatment option for distal radius malunion in patients with osteopenia; however, they require an ulnar head resection to accommodate closure of corrective osteotomy and to address the issue of ulnocarpal abutment. The literature contains little information on concomitant ulnar shortening osteotomy despite a physiologic solution. We report the functional and radiographic outcomes of 5 patients treated for symptomatic distal radius malunion with simultaneous radial closing wedge and ulnar shortening osteotomies. METHODS: All 5 patients were women aged 52 to 69 years (average, 61 years). Four patients had extra-articular radius fractures with dorsal angulation (20-22 degrees ) and shortening (3-7/mm); the other had the fracture with volar angulation (24 degrees ) and shortening (11 mm). Through a volar approach an appropriate amount of bone wedge was removed from the distal radius. A small volar T-plate was used to secure the osteotomized bone fragment. Six to 11 mm of ulnar shortening osteotomy was performed by using transverse osteotomy and compression plating technique with an AO compression device. RESULTS: In all 5 wrists healing of radial and ulnar osteotomies occurred less than 3 months after surgery. There were no postsurgical complications. Postsurgical radiographs showed that the volar tilt angle of the radius was reduced to normal range (range, 8-15 degrees ) in all wrists. The ulnar variance was 0 mm in 4 wrists and 2 mm in 1 wrist. There were significant improvements in pain, function, and range of motion at an average follow-up evaluation of 17 months. The average grip strength as a percentage of the opposite side improved from 30% before to 73% after surgery. CONCLUSIONS: This study showed that closing wedge osteotomy of the radius concomitant with ulnar shortening osteotomy is technically and functionally adequate. Our procedure is indicated for patients with osteopenia for whom opening wedge osteotomy of the radius is inadequate.

Abstract: The prognosis for patients with osteosarcoma who do not respond to current chemotherapy protocols still remains poor. Toward the goal of establishing efficacious peptide-based immunotherapy for those patients, we previously developed an autologous pair of CTLs and an osteosarcoma cell line. In the current study, we screened the cDNA library of this osteosarcoma cell line using an autologous CTL clone and identified cDNA encoding an antigen. The isolated cDNA was identical to papillomavirus binding factor (PBF), which was recently reported as a DNA binding transcription factor cooperating with RUNX1. Reverse transcription-PCR analysis revealed that PBF was expressed in 16 of 19 cases of bone and soft-tissue sarcoma cell lines (5 of 6 of osteosarcoma lines) and 57 of 76 sarcoma tissue samples (11 of 14 of osteosarcoma tissues). Also, PBF was expressed in 10 of 13 epithelial cancer cell lines and 20 of 34 of cancer tissues. In contrast, PBF was detected in some normal organs including ovary, pancreas, spleen, and liver by reverse transcription-PCR but was restricted in the cytoplasm by immunostaining and undetectable by Western blotting. Furthermore, a 12-mer peptide, CTACRWKKACQR, located at the COOH terminus of PBF, was found to be a minimum requirement for recognition by the CTL clone in the context of the HLA-B*5502 molecule. These findings suggest that PBF is a shared tumor-associated antigen, which may serve as a source of peptides applicable to peptide-based immunotherapy for osteosarcoma and other malignant tumors.

Abstract: Because of the difficulty of developing pairs of osteosarcoma cell lines and cytotoxic T lymphocytes (CTLs), no osteosarcoma tumor antigens that are useful for antiosteosarcoma immunotherapy have yet been identified. In parallel with continuous attempts to develop such pairs from osteosarcoma, we employed serological identification using a recombinant expression cloning (SEREX) method to identify B cell-defined antigens. Consequently, a human osteosarcoma cell line, OS2000, was established from a primary osteosarcoma of a patient cured of hereditary retinoblastoma. Repetitious in vitro stimulations by OS2000 cells to the autologous peripheral T cells induced cytotoxic activity in the autologous osteosarcoma cells but not in the nontumor cells. The cytotoxicity was inhibited by anti-HLA class I monoclonal antibody. SEREX analysis revealed that autologous humoral immunity reacted to two proteins expressed in OS2000. One was the self HLA-Cw*0102 molecule, and the other was wild-type smooth muscle myosin light chain (SMMLC). However, no antigenicity of these proteins was seen versus the sera of the other patients. In conclusion, our results demonstrated the presence of host cellular and humoral immune responses to autologous osteosarcoma cells. This offered the opportunity to identify osteosarcoma antigens recognized by autologous immunity.

Abstract: As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL.

Abstract: To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.