Tomohide Tsukahara

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.

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.

Prognostic significance of HLA class I expression in osteosarcoma defined by anti‐pan HLA class I monoclonal antibody, EMR8‐5

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 β‐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. (Cancer Sci 2006; 97: 1374–1380)

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.

A novel CIC-FOXO4 gene fusion in undifferentiated small round cell sarcoma: a genetically distinct variant of Ewing-like sarcoma.

Differential diagnosis of small round cell sarcomas (SRCSs) grouped under the Ewing sarcoma family of tumors (ESFT) can be a challenging situation for pathologists. Recent studies have revealed that some groups of Ewing-like sarcoma show typical ESFT morphology but lack any EWSR1-ETS gene fusions. Here we identified a novel gene fusion, CIC-FOXO4, in a case of Ewing-like sarcoma with a t(X;19)(q13;q13.3) translocation. The patient was a 63-year-old man who had an asymptomatic, 30-mm, well-demarcated, intramuscular mass in his right posterior neck, and imaging findings suggested a diagnosis of high-grade sarcoma. He was treated with complete resection and subsequent radiotherapy and chemotherapy. He was alive without local recurrence or distant metastasis 6 months after the operation. Histologic examination revealed SRCS with abundant desmoplastic fibrous stroma suggesting a desmoplastic small round cell tumor. Immunohistochemical analysis showed weak to moderate and partial staining for MIC2 (CD99) and WT1, respectively. High-throughput transcriptome sequencing revealed a gene fusion, and the genomic rearrangement between the CIC and FOXO4 genes was identified by fluorescence in situ hybridization. Aside from the desmoplastic stroma, the CIC-FOXO4 fusion sarcoma showed morphologic and immunohistochemical similarity to ESFT and Ewing-like sarcomas, including the recently described CIC-DUX4 fusion sarcoma. Although clinicopathologic analysis with additional cases is necessary, we conclude that CIC-FOXO4 fusion sarcoma is a new type of Ewing-like sarcoma that has a specific genetic signature. These findings have important implications for the differential diagnosis of SRCS.

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.

Immunotherapeutic benefit of α-interferon (IFNα) in survivin2B-derived peptide vaccination for advanced pancreatic cancer patients

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA‐A24‐restricted antigenic peptide, survivin‐2B80–88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin‐2B80–88 plus incomplete Freunds adjuvant (IFA) and α‐interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme‐linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin‐2B80‐88 peptide‐specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor‐prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905. (Cancer Sci 2013; 104: 124–129)

Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients

We previously identified a human leukocyte antigen (HLA)‐A24‐restricted antigenic peptide, survivin‐2B80‐88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin‐2B80‐88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin‐2B80‐88 plus incomplete Freund’s adjuvant (IFA); and (ii) survivin‐2B80‐88 plus IFA and a type‐I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin‐2B80‐88 plus IFA was not significantly different from that with survivin‐2B80‐88 alone, treatment with the vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin‐2B80‐88 peptide‐specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme‐linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single‐cell clone separation by cell sorting of peptide‐specific CTL showed that each CTL clone was indeed not only peptide‐specific but also cytotoxic against human cancer cells in the context of the expression of both HLA‐A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin‐2B80‐88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers. (Cancer Sci 2011; 102: 1181–1187)

SYT-SSX breakpoint peptide vaccines in patients with synovial sarcoma: A study from the Japanese Musculoskeletal Oncology Group†

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 Freunds adjuvant and then administered subcutaneously six times at 14‐day intervals. In addition, interferon‐α 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.

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)