Hiroaki Hiraga

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2 alpha.

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

Phase I vaccination trial of SYT-SSX junction peptide in patients with disseminated synovial sarcoma

BackgroundSynovial 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.MethodsA 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.ResultsA 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.ConclusionsOur 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.

Neuropathological and molecular studies of spinocerebellar ataxia type 6 (SCA6)

Abstract SCA6 is an autosomal dominant spinocerebellar ataxia (SCA) caused by a small CAG repeat expansion of the gene encoding an α-1a-voltage-dependent Ca channel gene subunit on chromosome 19p13. A Japanese woman with SCA6, with a 7-year history of progressive pure cerebellar ataxia, died of malignant lymphoma. Systematic neuropathological examination showed that neuronal degeneration was confined to the cerebellar Purkinje cells and, to a lesser degree, the granular cells, without any involvement of other central nervous system structures. Such pathological selectivity correlates with the localized expression of the responsible gene, and coincides with the neurological manifestation. These findings might contribute to establishing the phenotype of the SCA6 via comparison with other dominant ataxias.

Diagnosis of Synovial Sarcoma with the Reverse Transcriptase-polymerase Chain Reaction: Analyses of 84 Soft Tissue and Bone Tumors

The chimeric transcript SYT-SSX is generated as a result of reciprocal translocation t(X;18), which is the primary cytogenetic abnormality found in, and appears to be specific for, synovial sarcoma. We performed a reverse transcriptase-polymerase chain reaction (RT-PCR) for SYT-SSX transcripts in a series of 84 tumors (61 soft tissue tumors and 23 bone tumors), including a variety of histologic types, to assess its usefulness in molecular diagnosis. Ten synovial sarcomas, three tumors initially unclassified, and one malignant peripheral nerve sheath tumor contained the chimeric transcripts. A review of the original slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. Additionally, in situ hybridization with an SSX1 probe indicated that the chimeric transcripts exist not only in the cells of special components but also in cells showing a variety of histologic patterns. Therefore, RT-PCR can be considered a useful molecular biological technique that can provide objective evidence for diagnosis of synovial sarcoma. Northern blot analysis with an SSX1 probe also detected chimeric SYT-SSX transcripts in the synovial sarcoma cases. The additional smaller bands, however, were also detected in six peripheral primitive neuroectodermal tumors (pPNETs) and one embryonal rhabdomyosarcoma. In five of these pPNETs, other bands ranging in size from 2.0 to 2.2 kb were also found, and it seems possible that these bands might represent novel karyotypic aberrations and/or splicing variants of SSX.

Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma

To investigate the clinical significance of circulating angiogenic factors, especially in association with early relapse of osteosarcoma, we quantified pre-therapeutic levels of vascular endothelial growth factor, basic fibroblast growth factor and placenta growth factor in the sera of 16 patients with osteosarcoma using an enzyme-linked immunosorbent assay. After a 1-year follow-up, the serum level of angiogenic factors was analysed with respect to microvessel density of the biopsy specimen and clinical disease relapse. The serum vascular endothelial growth factor levels were positively correlated with the microvessel density with statistical significance (P=0.004; Spearman rank correlation) and also significantly higher in seven patients who developed pulmonary metastasis than the remaining nine patients without detectable disease relapse (P=0.0009; The Mann–Whitney U-test). In contrast, the serum levels of basic fibroblast growth factor or placenta growth factor failed to show significant correlation with the microvessel density or relapse of the disease. Although there was no significant correlation between serum vascular endothelial growth factor levels and the tumour volume, the serum vascular endothelial growth factor levels were significantly higher in patients with a vascular endothelial growth factor-positive tumour than those with a vascular endothelial growth factor-negative tumour. These findings suggest that the pre-therapeutic serum vascular endothelial growth factor level reflects the angiogenic property of primary tumour and may have a predictive value on early disease relapse of osteosarcoma.

Trabectedin monotherapy after standard chemotherapy versus best supportive care in patients with advanced, translocation-related sarcoma: a randomised, open-label, phase 2 study.

BACKGROUND Trabectedin binds to the minor groove of DNA and blocks DNA repair machinery. Preclinical data have shown that trabectedin also modulates the transcription of the oncogenic fusion proteins of translocation-related sarcomas. We aimed to assess the efficacy and safety of trabectedin as second-line therapy or later for patients with advanced translocation-related sarcoma. METHODS We did a multicentre randomised open-label study in Japan. Eligible patients had pathological diagnosis of translocation-related sarcoma, were aged 19 years or older, were unresponsive or intolerant to standard chemotherapy regimens, no more than four previous chemotherapy regimens, Eastern Cooperative Oncology Group performance status 0 or 1, adequate bone marrow reserve, renal and liver functions, and had measurable lesions. Patients were randomly assigned (1:1) by the minimisation method to receive either trabectedin (1·2 mg/m(2) given via a central venous line over 24 h on day 1 of a 21 day treatment cycle) or best supportive care, which was adjusted centrally by pathological subtype. Investigators, patients, and the sponsor were unmasked to the treatment assignment. Progression-free survival and objective responses were assessed by a masked central radiology imaging review. Efficacy was assessed by masked central radiology imaging review. The primary endpoint was progression-free survival for the full analysis set population. Follow-up is ongoing for the patients under study treatment. The study is registered with Japan Pharmaceutical Information Center, number JapicCTI-121850. FINDINGS Between July 11, 2012, and Jan 20, 2014, 76 patients were enrolled and allocated to receive either trabectedin (n=39) or best supportive care (n=37). After central review to confirm pathological subtypes, 73 patients (37 in the trabectedin group and 36 in the best supportive care group) were included in the primary efficacy analysis. Median progression-free survival of the trabectedin group was 5·6 months (95% CI 4·1-7·5) and the best supportive care group was 0·9 months (0·7-1·0). The hazard ratio (HR) for progression-free survival of trabectedin versus best supportive care was 0·07 (90% CI 0·03-0·14 and 95% CI 0·03-0·16) by a Cox proportional hazards model (p<0·0001). The most common drug-related adverse events for patients treated with trabectedin were nausea (32 [89%] of 36), decreased appetite (21 [58%]), decreased neutrophil count (30 [83%]), increased alanine aminotransferase (24 [67%]), and decreased white blood cell count (20 [56%]). INTERPRETATION Trabectedin significantly reduced the risk of disease progression and death in patients with advanced translocation-related sarcoma after standard chemotherapy such as doxorubicin, and should be considered as a new therapeutic treatment option for this patient population. FUNDING Taiho Pharmaceutical Co., Ltd.

Detection and Induction of CTLs Specific for SYT-SSX-Derived Peptides in HLA-A24+ Patients with Synovial Sarcoma

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.

Identification of Human Autologous Cytotoxic T-Lymphocyte-Defined Osteosarcoma Gene That Encodes a Transcriptional Regulator, Papillomavirus Binding Factor

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.

Alternative EWS-FLI1 fusion gene and MIC2 expression in peripheral and central primitive neuroectodermal tumors.

Primitive neuroectodermal tumors (PNET) occur either in the central nervous system (CNS; central PNET, cPNET) or in the peripheral sites (peripheral PNET, pPNET). Recent molecular approaches have been defining a new concept of PNET, that is, the pPNET including Ewings sarcoma (ES) which expresses MIC2 glycoprotein and shows the specific chimeric gene of EWS‐FLI1. The expression of MIC2 and the genetic rearrangement of EWS‐FLI1 are considered to be highly specific to the pPNET/ES. This study examined the expression of MIC2 and EWS‐FLI1 gene by means of immunohistochemistry and reverse transcriptase–polymerase chain reaction (RT‐PCR) on various small round cell tumors originating in the CNS or non‐CNS organs. All peripheral PNET tested expressed MIC2 and were positive for EWS‐FLI1 (11/11). In contrast, all cPNET and other blastic CNS tumors were negative for MIC2: medulloblastoma (0/3), cerebral PNET (0/2), spinal PNET (0/2), glioblastoma (0/2), retinoblastoma (0/3), and pineoblastoma (0/2). These MIC2‐negative tumors were also negative for the chimeric gene product of EWS‐FLI1. Interestingly, one PNET originating in the intracranial dura mater was positive for both MIC2 and EWS‐FLI1 fusion gene. The results indicate that cPNET lacks any genetic or protein markers, except for a meningeal PNET which falls into the same phenotypic spectrum of pPNET.

Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Polymerase chain reaction (PCR)‐based nucleotide sequence analysis was performed in 12 cases of Ewing sarcoma on the cDNA and/or genomic DNA breakpoint regions of a t(11;22)(q24;q12), which joins the EWS gene located on chromosome 22 with the FLI1 gene located on chromosome 11, in order to understand the molecular mechanism of this translocation. Reverse transcriptase‐PCR on total tumor cell RNA from the examined cases showed five types of EWS‐FLI1 chimeric product, resulting from various junctions between EWS exon 7 or 10 with FLI1 exon 5, 6, or 8. Sequencing of the genomic fusion junctions of EWS‐FLI1 in seven cases showing three types of the chimeric cDNA products revealed that most of the breakpoint junctions shared common nucleotide(s) from both genes, and that the breakpoints in EWS introns 7 and 10 clustered within 100 bp and 300 bp, respectively. All the junctions were found to be flanked by various oligomers, among which a consensus sequence, 5′‐AGAAAARDRR‐3′, was found near the breakpoints of both genes in four cases, suggesting that these oligomers may have a functional significance in the genesis of t(11;22). In addition to these oligomers, sequences highly homologous to Alu repeats and/or eukaryotic topoisomerase II cleavage sites were located near, or flanked, or even encompassed, the breakpoints in most of the cases examined. Thus, these sequences may also mediate DNA double‐strand breakage and rejoining to generate the t(11;22). Genomic sequence analysis of both EWS‐FLI1 and FLI1‐EWS chimeric genes in three of the seven cases demonstrated a deletion and duplication of both EWS and FLI1 sequences in two cases and no gain or loss in one case. The present findings suggest that multiple mechanisms may be operative for the break and rejoining of the fragments of chromosomes 11 and 22 in the genesis of t(11;22), and that some of these translocations are asymmetric at the molecular level. Genes Chromosomes Cancer 25:6–15, 1999.