Takuro Wada

Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas

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 × 103 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.

Scythe/BAT3 regulates apoptotic cell death induced by papillomavirus binding factor in human osteosarcoma

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 2009; 100: 47–53)

The level of vascular endothelial growth factor as a predictor of a poor prognosis in osteosarcoma

We undertook a prospective study to evaluate the prognostic significance of the serum levels of vascular endothelial growth factor (VEGF) in predicting the survival of patients with osteosarcoma. The levels were measured by an enzyme-linked immunosorbent assay in 15 patients with osteosarcoma before commencing treatment. The patients were divided into two groups, with a high or a low serum VEGF level, and the incidence of metastases and overall survival rate were compared. No significant relationship was observed between the serum VEGF levels and gender, age, the size of the tumour or the response to pre-operative chemotherapy. Patients with a serum VEGF > 1000 pg/ml had significantly worse survival than those with a level < 1000 pg/ml (p = 0.002). The serum VEGF level may be useful in predicting the prognosis for survival in patients with osteosarcoma.

Prognostic impact and immunogenicity of a novel osteosarcoma antigen, papillomavirus binding factor, in patients with osteosarcoma

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 × 10−7 to 7 × 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. (Cancer Sci 2008; 99: 368–375)

Angiomatoid fibrous histiocytoma including cases with pleomorphic features analysed by fluorescence in situ hybridisation

Background Angiomatoid fibrous histiocytoma (AFH) is a rare soft-tissue tumour of uncertain differentiation and low metastatic potential. Cytogenetics and/or molecular genetics have revealed that most have a rearrangement of the EWSR1 gene, whereas a FUS gene rearrangement is present in a minority of cases. Although some cases of AFH display striking pleomorphism and mitotic activity, there are no known clinical, morphological or genetic factors that predict metastasis. The authors present clinicopathological features of AFH, including cases showing a pleomorphic histological appearance, and results of fluorescence in situ hybridisation analysis of EWSR1 and FUS rearrangements. Methods Tumour samples from 10 patients were subjected to clinicopathological and immunohistochemical analysis and dual-colour fluorescence in situ hybridisation for EWSR1 and FUS with split-signal probes. Results All cases showed clinical features (sites: extremities followed by trunk; age: adolescent to young adult), morphology (multinodular proliferation of spindle cells, lymphoid cuffs and pseudovascular spaces) and immunohistochemical results (more than half were positive for CD68, CD99, desmin and epithelial membrane antigen) typical of AFH. There were two local recurrences in each of two patients. Two patients developed distant metastases and died from the disease; tumours of these two patients showed focal proliferation of large pleomorphic cells with hyperchromatic nuclei and high proliferative activity (>10/10 high-power field and Ki-67 labelling index >10%). There were no clinical, histological or immunohistochemical differences between the nine cases with EWSR1 rearrangement and one case with FUS rearrangement. Conclusions Wide surgical excision and careful follow-up are necessary for patients with AFH in view of its risk of local recurrence and metastasis leading to a fatal outcome.

Osteosarcoma in the elderly over 60 years: A multicenter study by the Japanese musculoskeletal oncology group†

Few studies have described the characteristics and prognostic factors of osteosarcoma patients aged over 60 years.

HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor

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.

Advantage of FISH analysis using FKHR probes for an adjunct to diagnosis of rhabdomyosarcomas

Translocations can be detected using fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tissues. Recently, a commercially available FKHR (13q14) dual-color, break-apart rearrangement probe has been developed. However, the advantages of using this probe have not been reported. This study demonstrated the usefulness of this probe for the clinical diagnosis of rhabdomyosarcomas (RMS). We studied 33 RMS (19 embryonal rhabdomyosarcomas [ERMS], including three sclerosing-type RMS, and 14 alveloar rhabdomyosarcomas [ARMS]). Fluorescence signals were detected for 18 of the 19 (94.7%) ERMS and 13 of the 14 (92.8%) ARMS. A split-signal pattern was detected in 12 of 13 (92.3%) ARMS but was not detected in any of the ERMS, including the three sclerosing-type RMS. Amplification and polyploidy were present in both the ERMS and the ARMS. Our FISH study highlighted the excellent performance of the presently reported commercial break-apart probe for the detection of FKHR gene rearrangements in RMS. Because amplification and polyploidy were detected in both the ERMS and the ARMS, sufficient care should be taken when counting the nuclear signals. No rearrangements of the FKHR gene were found in any of the three sclerosing-type RMS when examined using a FISH assay, supporting the hypothesis that sclerosing RMS can be included as an ERMS.

Giant cell tumor of the sacrum treated with selective arterial embolization

Giant cell tumor of the sacrum is extremely difficult to manage. Standard treatments, including surgery and radiation, are associated with significant complications and recurrence rates. In this manuscript, we report an early clinical result of a case of giant cell tumor of the sacrum successfully managed with selective arterial embolization. A 56-year-old woman underwent selective embolization for management of giant cell tumor of the sacrum. Radiologically, massive shrinkage of the extraosseous mass and increased peripheral ossification were obvious. Clinically, rapid pain relief was achieved and gait disability recovered. At final follow-up 28xa0months after completion of treatment, she retained normal activity in daily life. We stress the effectiveness of selective arterial embolization as a less invasive and less complicated primary treatment of giant cell tumors of the sacrum.

Prognostic significance of HLA class I expression in Ewing's sarcoma family of tumors.

Ewings sarcoma family of tumors (ESFT) is one of the most malignant groups of tumors in young people. Human leukocyte antigen (HLA) class I displays endogenously processed peptides to CD8+ T lymphocytes and has a key role for host immune surveillance. In ESFT, the investigation concerning both HLA class I expression and T‐cell infiltration has yet to be reported.