BACKGROUND: Several human cancers have been found to contain cancer stem-like cells (CSCs) having cancer-initiating ability. However, only a few reports have shown the existence of CSCs in bone and soft tissue sarcomas. In this study, we identified and characterised side population (SP) cells that showed drug-resistant features in human bone sarcoma cell lines. METHODS: In seven osteosarcoma cell lines (OS2000, KIKU, NY, Huo9, HOS, U2OS and Saos2) and in one bone malignant fibrous histiocytoma (MFH) cell line (MFH2003), the frequency of SP cells was analysed. Tumourigenicity of SP cells was assessed in vitro and in vivo. Gene profiles of SP cells and other populations (main population; MP) of cells were characterised using cDNA microarrays. RESULTS: SP cells were found in NY (0.31%) and MFH2003 (5.28%). SP cells of MFH2003 formed spherical colonies and re-populated into SP and MP cells. In an NOD/SCID mice xenograft model, 1 Â 10 3 sorted SP cell-induced tumourigenesis. cDNA microarray analysis showed that 23 genes were upregulated in SP cells. CONCLUSIONS: We showed that SP cells existed in bone sarcoma cell lines. SP cells of MFH2003 had cancer-initiating ability in vitro and in vivo. The gene profiles of SP cells could serve as candidate markers for CSCs in bone sarcomas.

In the present study, we evaluated the safety and effectiveness of SYT-SSX-derived peptide vaccines in patients with advanced synovial sarcoma. A 9-mer peptide spanning the SYT-SSX fusion region (B peptide) and its HLA-A*2402 anchor substitute (K9I) were synthesized. In Protocols A1 and A2, vaccines with peptide alone were administered subcutaneously six times at 14-day intervals. The B peptide was used in Protocol A1, whereas the K9I peptide was used in Protocol A2. In Protocols B1 and B2, the peptide was mixed with incomplete Freund's adjuvant and then administered subcutaneously six times at 14-day intervals. In addition, interferon-a was injected subcutaneously on the same day and again 3 days after the vaccination. The B peptide and K9I peptide were used in Protocols B1 and B2, respectively. In total, 21 patients (12 men, nine women; mean age 43.6 years) were enrolled in the present study. Each patient had multiple metastatic lesions of the lung. Thirteen patients completed the six-injection vaccination schedule. One patient developed intracerebral hemorrhage after the second vaccination. Delayed-type hypersensitivity skin tests were negative in all patients. Nine patients showed a greater than twofold increase in the frequency of CTLs in tetramer analysis. Recognized disease progression occurred in all but one of the nine patients in Protocols A1 and A2. In contrast, half the 12 patients had stable disease during the vaccination period in Protocols B1 and B2. Of note, one patient showed transient shrinkage of a metastatic lesion. The response of the patients to the B protocols is encouraging and warrants further investigation. (Cancer Sci 2012; 103: 1625-1630 S ynovial sarcoma is a malignant tumor of soft tissue characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion genes.(1,2) Reported 5-year survival rates of patients with synovial sarcoma range from 64% to 77%.(3-7) In contrast, most metastatic or relapsed diseases remain incurable, indicating a need for new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy.Antigen-specific peptide immunotherapy is one such option. (8)(9)(10)(11)(12) Previously, we demonstrated that SYT-SSX fusion gene-derived peptides (wild type and agretope modified) are recognized by circulating CD8 + T cells in HLA-A24 + patients with synovial sarcoma and elicit human leukocyte antigen (HLA)-restricted, tumor-specific cytotoxic responses. (13,14) Subsequent to these preclinical studies, we started a pilot clinical trial with a wild-type SYT-SSX-derived peptide vaccine. (15) In the present study, we evaluated immunologic and clinical outcomes of the vaccination trials using an agretope-modified SYT-SSX peptide and a combination of the peptide vaccine with adjuvant and interferon (IFN)-a.

Epithelioid sarcoma (ES) is a relatively rare, highly malignant soft tissue sarcoma. The mainstay of treatment is resection or amputation. Currently other therapeutic options available for this disease are limited. Therefore, a novel therapeutic option needs to be developed. In the present study, we established a new human ES cell line (ESX) and analyzed the characteristics of its cancer stem-like cells/cancer-initiating cells (CSCs/CICs) based on ALDH1 activity. We demonstrated that a subpopulation of ESX cells with high ALDH1 activity (ALDHhigh cells) correlated with enhanced clonogenic ability, sphere-formation ability, and invasiveness in vitro and showed higher tumorigenicity in vivo. Next, using gene expression profiling, we identified CD109, a GPI-anchored protein upregulated in the ALDHhigh cells. CD109 mRNA was highly expressed in various sarcoma cell lines, but weakly expressed in normal adult tissues. CD109-positive cells in ESX predominantly formed spheres in culture, whereas siCD109 reduced ALDH1 expression and inhibited the cell proliferation in vitro. Subsequently, we evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma. We found a strong correlation between CD109 protein expression and the prognosis (P = 0.009). In conclusion, CD109 might be a CSC/CIC marker in epithelioid sarcoma. Moreover, CD109 is a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas including ES.

Osteochondromas are the most common benign tumors of the bone. They occur in young adolescent patients and are frequently located in the metaphyses of the long bones; they do not grow after skeletal maturity. The incidence of osteochondroma in the spine is reported to be rare. Moreover, patients with spinal osteochondroma who develop symptoms of myelopathy are extremely rare. We report the case of an 8-year-old girl who experienced myelopathy due to spinal compression of the cervical osteochondroma. This case suggests that if a cartilage cap is observed on the spinal canal with magnetic resonance imaging (MRI), the tumor may extend to the spinal canal, resulting in neurologic dysfunction. Therefore, careful follow-up until bone maturity should be performed.

BackgroundTo 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.MethodsTen 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.ResultsPeptide 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 2 × 10-7 to 5 × 10-6. 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.ConclusionThese 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.

Malignant fibrous histiocytoma (MFH) of the bone is an aggressive tumor with high rates of local recurrence and metastasis. The development of novel therapeutic approaches is critical to improve the prognosis of patients with MFH. We reported previously that the side population (SP) cells of the MFH2003 bone MFH cell line have the characteristics of cancer stem-like cells (CSC)/cancer-initiating cells. In the present study, to establish immunotherapy targeting CSC, we analyzed cell surface immune molecules on SP cells of the MHF2003 cell line, as well as autologous CTL responses against these SP cells in the tumor microenvironment and peripheral circulating lymphocytes, using autologous tumor-infiltrating lymphocytes and autologous CTL clones derived from peripheral blood, respectively. We found that the SP cells expressed human leukocyte antigen (HLA) Class I molecules on the cell surface. The autologous tumor-infiltrating lymphocyte line TIL2003 recognized both the SP and main population cells of the MFH2003 cell line. Next, we induced the CTL clone Tc4C-6 by mixed lymphocyte tumor cell culture using autologous peripheral blood mononuclear cells and freshly isolated SP cells, followed by a limiting dilution procedure. The Tc4C-6 clone showed specific cytotoxicity against the SP cells. Moreover, the cytotoxicity against SP cells was blocked by the anti-HLA Class I antibody W6/32. In conclusion, the findings of the present study support the idea that CSC of bone MFH are recognized by autologous CTL in the tumor microenvironment and peripheral circulating lymphocytes. Thus, CTL-based immunotherapy could target CSC of bone sarcoma to help prevent tumor recurrence.

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