研究者詳細 - 塚原　智英

論文 - 塚原　智英

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Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells

Tsutomu Tanaka, Koichi Okuya, Goro Kutomi, Akari Takaya, Toshimitsu Kajiwara, Takayuki Kanaseki, Tomohide Tsukahara, Yoshihiko Hirohashi, Toshihiko Torigoe, Koichi Hirata, Yoshiharu Okamoto, Noriyuki Sato, Yasuaki Tamura

CANCER SCIENCE ( WILEY-BLACKWELL ) 106 ( 1 ) 18 - 24 2015年01月 [査読有り]

The presentation of an exogenous antigen in a major histocompatibility complex class-I- restricted fashion to CD8(+) T cells is called cross-presentation. Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit efficient CTL responses by cross-presentation through an as-yet entirely unknown mechanism. Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We have shown that a tumor antigen peptide complexed with Hsp90 could be cross-presented by dendritic cells (DCs) through an endosomal pathway in a murine system. However, it has not been determined whether human DCs also cross-present an Hsp90-peptide complex and induce peptide-specific CTLs. In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway. Our data indicate that targeting of the antigen to a static early endosome by Hsp90 is essential for efficient cross-presentation.

A Novel CIC-FOXO4 Gene Fusion in Undifferentiated Small Round Cell Sarcoma A Genetically Distinct Variant of Ewing-like Sarcoma

Shintaro Sugita, Yasuhito Arai, Akiko Tonooka, Natsuko Hama, Yasushi Totoki, Tomoki Fujii, Tomoyuki Aoyama, Hiroko Asanuma, Tomohide Tsukahara, Mitsunori Kaya, Tatsuhiro Shibata, Tadashi Hasegawa

AMERICAN JOURNAL OF SURGICAL PATHOLOGY ( LIPPINCOTT WILLIAMS & WILKINS ) 38 ( 11 ) 1571 - 1576 2014年11月 [査読有り]

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.

Specific Targeting of a Naturally Presented Osteosarcoma Antigen, Papillomavirus Binding Factor Peptide, Using an Artificial Monoclonal Antibody

Tomohide Tsukahara, Makoto Emori, Kenji Murata, Takahisa Hirano, Norihiro Muroi, Masanori Kyono, Shingo Toji, Kazue Watanabe, Toshihiko Torigoe, Vitaly Kochin, Hiroko Asanuma, Hiroshi Matsumiya, Keiji Yamashita, Tetsuo Himi, Shingo Ichimiya, Takuro Wada, Toshihiko Yamashita, Tadashi Hasegawa, Noriyuki Sato

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC ) 289 ( 32 ) 22035 - 22047 2014年08月 [査読有り]

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in similar to 90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA.peptide complex. For detection and qualification of the HLA-A*02:01.PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201.PBF A2.2 complex using a naive scFv phage display library. As a result, scFv clone D12 with high affinity (K-D = 1.53 x 10(-9) M) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA.peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.

Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors

Shintaro Sugita, Tomoyuki Aoyama, Kei Kondo, Yoshiko Keira, Jiro Ogino, Katsuya Nakanishi, Mitsunori Kayo, Makoto Emori, Tomohide Tsukahara, Hisaya Nakajima, Masayuki Takagi, Tadashi Hasegawa

HUMAN PATHOLOGY ( W B SAUNDERS CO-ELSEVIER INC ) 45 ( 8 ) 1588 - 1596 2014年08月 [査読有り]

Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign flbrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant flbrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, MC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low:grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens. (C) 2014 Elsevier Inc. All rights reserved.

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells

Rena Morita, Satoshi Nishizawa, Toshihiko Torigoe, Akari Takahashi, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Alice Sokolovskaya, Vitaly Kochin, Toru Kondo, Satoshi Hashino, Masahiro Asaka, Isao Hara, Yoshihiko Hirohashi, Noriyuki Sato

Cancer Science ( Blackwell Publishing Ltd ) 105 ( 4 ) 389 - 395 2014年 [査読有り]

The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

The path to innovative drug development of cancer vaccine: From discovery of tumor antigens to clinical trials

Toshihiko Torigoe, Yoshihiko Hirohashi, Tomohide Tsukahara, Takayuki Kanaseki, Vitaly Kochin, Noriyuki Sato

Japanese Journal of Clinical Immunology ( 2 ) 37 ( 2 ) 90 - 95 2014年 [査読有り]

Tumor immunology has been advancing after the great discovery of tumor-specific antigen MAGE in 1991, and a number of tumor antigens have been reported to date. We have also found novel tumor antigens through various methodologies such as gene expression cloning, bioinformatics, reverse immunology, transcriptome analysis and peptidome analysis. Recently, we made a success of defining cancer stem cell-specific antigens. The fruits of our basic research have been applied to clinical trials of cancer vaccine. The long path and future perspectives of innovative immunotherapeutic drug development are described. © 2014 The Japan Society for Clinical Immunology.

High expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells in the novel epithelioid sarcoma cell line ESX and is related to poor prognosis of soft tissue sarcoma

Makoto Emori, Tomohide Tsukahara, Masaki Murase, Masanobu Kano, Kenji Murata, Akari Takahashi, Terufumi Kubo, Hiroko Asanuma, Kazuyo Yasuda, Vitaly Kochin, Mitsunori Kaya, Satoshi Nagoya, Jun Nishio, Hiroshi Iwasaki, Tomoko Sonoda, Tadashi Hasegawa, Toshihiko Torigoe, Takuro Wada, Toshihiko Yamashita, Noriyuki Sato

PLoS ONE ( 12 ) 8 ( 12 ) e84187 2013年12月 [査読有り]

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. © 2013 Emori et al.

Ectopically Expressed Variant Form of Sperm Mitochondria-Associated Cysteine-Rich Protein Augments Tumorigenicity of the Stem Cell Population of Lung Adenocarcinoma Cells

Akari Takahashi, Yoshihiko Hirohashi, Toshihiko Torigoe, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Vitaly Kochin, Hiroshi Saijo, Terufumi Kubo, Munehide Nakatsugawa, Hiroko Asanuma, Tadashi Hasegawa, Toru Kondo, Noriyuki Sato

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 8 ( 11 ) e69095 2013年11月 [査読有り]

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have self-renewal ability, differentiation ability and high tumor-initiating ability. CSCs/CICs are resistant to cancer therapies including chemotherapy and radiotherapy. Therefore, CSCs/CICs are thought to be responsible for cancer recurrence and distant metastasis after treatment. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we isolated CSCs/CICs as side population (SP) cells from lung carcinoma, colon carcinoma and breast carcinoma cells and analyzed the molecular mechanisms of CSCs/CICs. cDNA micro-array screening and RT-PCR analysis revealed that sperm mitochondria-associated cysteine-rich protein (SMCP) is ectopically expressed in SP cells. 5'-Rapid amplification of cDNA end (RACE) analysis revealed that the SMCP transcript in SP cells was a variant form (termed vt2) which is composed from only one exon. SMCP vt2 was detected in only cancer cells, whereas the wild-type (vt1) form of SMCP was expressed in the testis. SMCP was shown to have a role in tumor initiation by SMCP overexpression and SMCP knockdown using siRNAs in lung cancer cells. Taken together, the initiation results indicate that an ectopically expressed variant form of SMCP has a role in tumor initiation of CSCs/CICs and that the variant form of SMCP might be a novel CSC/CIC marker and a potential and promising target of CSC/CIC-targeting therapy.

Small proline-rich protein-1B is overexpressed in human oral squamous cell cancer stem-like cells and is related to their growth through activation of MAP kinase signal

Yoshitaka Michifuri, Yoshihiko Hirohashi, Toshihiko Torigoe, Akihiro Miyazaki, Jyunki Fujino, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Junichi Kobayashi, Takanori Sasaki, Akari Takahashi, Kenji Nakamori, Akira Yamaguchi, Hiroyoshi Hiratsuka, Noriyuki Sato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 439 ( 1 ) 96 - 102 2013年09月 [査読有り]

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1(br)) cells by the ALDEFLUOR assay. Small praline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1(br) cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1(br) cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4. (C) 2013 Elsevier Inc. All rights reserved.

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

Kameshima H, Tsuruma T, Kutomi G, Shima H, Iwayama Y, Kimura Y, Imamura M, Torigoe T, Takahashi A, Hirohashi Y, Tamura Y, Tsukahara T, Kanaseki T, Sato N, Hirata K

Cancer science ( 1 ) 104 ( 1 ) 124 - 129 2013年01月 [査読有り]

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-2B8088 (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-2B8088 plus incomplete Freund's adjuvant (IFA) and alpha-interferon (IFN alpha) 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 IFN alpha 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 IFN alpha 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: 124129)

Heat shock enhances the expression of cytotoxic granule proteins and augments the activities of tumor-associated antigen-specific cytotoxic T lymphocytes

Akari Takahashi, Toshihiko Torigoe, Yasuaki Tamura, Takayuki Kanaseki, Tomohide Tsukahara, Yasushi Sasaki, Hidekazu Kameshima, Tetsuhiro Tsuruma, Koichi Hirata, Takashi Tokino, Yoshihiko Hirohashi, Noriyuki Sato

CELL STRESS & CHAPERONES ( SPRINGER ) 17 ( 6 ) 757 - 763 2012年11月 [査読有り]

Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients' peripheral blood, and the CTL activities were investigated in several temperature conditions (37-41 A degrees C). Cytotoxicity and IFN-gamma secretion of CTL were greatest under 39 A degrees C condition, whereas they were minimum under 41 A degrees C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCR alpha beta by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 A degrees C. On the other hand, CTL cell death was induced under 41 A degrees C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 A degrees C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

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

Satoshi Kawaguchi, Tomohide Tsukahara, Kazunori Ida, Shigeharu Kimura, Masaki Murase, Masanobu Kano, Makoto Emori, Satoshi Nagoya, Mitsunori Kaya, Toshihiko Torigoe, Emiri Ueda, Akari Takahashi, Takeshi Ishii, Shin-ichiro Tatezaki, Junya Toguchida, Hiroyuki Tsuchiya, Toshihisa Osanai, Takashi Sugita, Hideshi Sugiura, Makoto Ieguchi, Koichiro Ihara, Ken-ichiro Hamada, Hiroshi Kakizaki, Takeshi Morii, Taketoshi Yasuda, Taisuke Tanizawa, Akira Ogose, Hiroo Yabe, Toshihiko Yamashita, Noriyuki Sato, Takuro Wada

CANCER SCIENCE ( WILEY-BLACKWELL ) 103 ( 9 ) 1625 - 1630 2012年09月 [査読有り]

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.

NK/T-cell lymphoma of bilateral adrenal glands in a patient with pyothorax

Tomohide Tsukahara, Akira Takasawa, Masaki Murata, Kazuyoshi Okumura, Masato Nakayama, Noriyuki Sato, Tadashi Hasegawa

DIAGNOSTIC PATHOLOGY ( BIOMED CENTRAL LTD ) 7 114 2012年08月 [査読有り]

Primary lymphoma of adrenal glands is rare, and non-B-cell lymphoma associated with pyothorax is also very rare. Here we report the first autopsy case of non-B-cell lymphoma in bilateral adrenal glands of a 79-year-old woman with pyothorax who had an aggressive clinical course. Immunohistochemically, tumor cells showed CD3+, CD45RO+, CD5-, CD7-, CD4-, CD8-, CD10-, CD20-, CD30-, CD79a-, CD138-, CD56-, granzyme B-, TIA-1+ and ALK-. In addition, tumor cells were strongly EBER1-positive by in situ hybridization. In genomic DNA of tumor cells, T-cell receptor rearrangements were not detected by southern blotting. We finally diagnosed this case as extranodal NK/T-cell lymphoma (nasal type).

ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells

Junichi Matsuzaki, Toshihiko Torigoe, Yoshihiko Hirohashi, Kenjiro Kamiguchi, Yasuaki Tamura, Tomohide Tsukahara, Terufumi Kubo, Akari Takahashi, Emiri Nakazawa, Eri Saka, Kazuyo Yasuda, Shuji Takahashi, Noriyuki Sato

CARCINOGENESIS ( OXFORD UNIV PRESS ) 33 ( 5 ) 996 - 1003 2012年05月 [査読有り]

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

[Peptide vaccination therapy for bone and soft tissue sarcoma].

Emori M, Tsukahara T, Kawaguchi S, Wada T

Nihon rinsho. Japanese journal of clinical medicine ( 9 ) 69 ( 9 ) 1670 - 1673 2011年09月 [査読有り]

Autologous CTL response against cancer stem-like cells/cancer-initiating cells of bone malignant fibrous histiocytoma

Masanobu Kano, Tomohide Tsukahara, Makoto Emori, Masaki Murase, Toshihiko Torigoe, Satoshi Kawaguchi, Takuro Wada, Toshihiko Yamashita, Noriyuki Sato

CANCER SCIENCE ( WILEY-BLACKWELL ) 102 ( 8 ) 1443 - 1447 2011年08月 [査読有り]

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. (Cancer Sci 2011; 102: 1443-1447)

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

Hidekazu Kameshima, Tetsuhiro Tsuruma, Toshihiko Torigoe, Akari Takahashi, Yoshihiko Hirohashi, Yasuaki Tamura, Tomohide Tsukahara, Shingo Ichimiya, Takayuki Kanaseki, Yuji Iwayama, Noriyuki Sato, Koichi Hirata

CANCER SCIENCE ( WILEY-BLACKWELL ) 102 ( 6 ) 1181 - 1187 2011年06月 [査読有り]

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), IFN alpha. 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 IFN alpha 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 IFN alpha 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).

Prognostic Significance of HLA Class I Expression in Ewing's Sarcoma Family of Tumors

Hiroki Yabe, Tomohide Tsukahara, Satoshi Kawaguchi, Takuro Wada, Toshihiko Torigoe, Noriyuki Sato, Chihiro Terai, Masaya Aoki, Shigemichi Hirose, Hideo Morioka, Hiroo Yabe

JOURNAL OF SURGICAL ONCOLOGY ( WILEY-BLACKWELL ) 103 ( 5 ) 380 - 385 2011年04月 [査読有り]

Background: Ewing's 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. Methods: Biopsy specimens from 28 ESFT patients were evaluated by immunohistochemistry with the anti-HLA class I monoclonal antibody (mAb) EMR8-5 and anti-CD8 mAb, respectively. Results: Expression of HLA class I was negative in 10 tumors and down-regulated in 22 tumors. The status of CD8+ T cell infiltration was closely associated with the expression levels of HLA class I. ESFT patients with down-regulated or negative expression of HLA class I showed significantly poorer survival than the rest of the patients. Conclusions: Our results suggested that CD8+T cell-mediated immune response restricted by HLA class I might play an important role in immune surveillance of ESFT, and we revealed for the first time that the status of HLA class I expression affects the survival of the patients with ESFT. J. Surg. Oncol. 2011; 103: 380-385. (C) 2010 Wiley-Liss, Inc.

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

Tomohide Tsukahara, Satoshi Kawaguchi, Toshihiko Torigoe, Akari Takahashi, Masaki Murase, Masanobu Kano, Takuro Wada, Mitsunori Kaya, Satoshi Nagoya, Toshihiko Yamashita, Noriyuki Sato

JOURNAL OF TRANSLATIONAL MEDICINE ( BIOMED CENTRAL LTD ) 7 44 2009年06月 [査読有り]

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 (51)Cr 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 2 x 10(-7) to 5 x 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. 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.

Molecular pathological approaches to human tumor immunology

Noriyuki Sato, Yoshihiko Hirohashi, Tomohide Tsukahara, Tomoki Kikuchi, Hiroeki Sahara, Kenjiro Kamiguchi, Shingo Ichimiya, Yasuaki Tamura, Toshihiko Torigoe

PATHOLOGY INTERNATIONAL ( WILEY-BLACKWELL PUBLISHING, INC ) 59 ( 4 ) 205 - 217 2009年04月 [査読有り]

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.

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

Tomohide Tsukahara, Shigeharu Kimura, Shingo Ichimiya, Toshihiko Torigoe, Satoshi Kawaguchi, Takuro Wada, Toshihiko Yamashita, Noriyuki Sato

CANCER SCIENCE ( WILEY-BLACKWELL PUBLISHING, INC ) 100 ( 1 ) 47 - 53 2009年01月 [査読有り]

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).

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

Tomohide Tsukahara, Satoshi Kawaguchi, Toshihiko Torigoe, Shigeharu Kimura, Masaki Murase, Shingo Ichimiya, Takuro Wada, Mitsunori Kaya, Satoshi Nagoya, Takeshi Ishii, Shin-ichiro Tatezaki, Toshihiko Yamashita, Noriyuki Sato

CANCER SCIENCE ( BLACKWELL PUBLISHING ) 99 ( 2 ) 368 - 375 2008年02月 [査読有り]

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.

Clonal T-Cell response against autologous pleomorphic malignant fibrous histiocytoma antigen presented by retrieved HLA-A*0206

Shigeharu Kimura, Yuriko Kozakai, Satoshi Kawaguchi, Tomohide Tsukahara, Kazunori Ida, Masaki Murase, Tadaki Matsumura, Mitunori Kaya, Toshihiko Torigoe, Takuro Wada, Noriyuki Sato, Toshihiko Yamashita

JOURNAL OF ORTHOPAEDIC RESEARCH ( JOHN WILEY & SONS INC ) 26 ( 2 ) 271 - 278 2008年02月 [査読有り]

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-restrieted 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. (c) 2007 Orthopaedic Research Society.

Overexpression of papillomavirus binding factor in Ewing's sarcoma family of tumors conferring poor prognosis

Hiroki Yabe, Tomohide Tsukahara, Satoshi Kawaguchi, Takuro Wada, Noriyuki Sato, Hideo Morioka, Hiroo Yabe

ONCOLOGY REPORTS ( PROFESSOR D A SPANDIDOS ) 19 ( 1 ) 129 - 134 2008年01月 [査読有り]

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 I I 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.

Identification of CLUAP1 as a human osteosarcoma tumor-associated antigen recognized by the humoral immune system

Hisamitsu Ishikura, Hiroaki Ikeda, Hiroyuki Abe, Takayuki Ohkuri, Hiroaki Hiraga, Kazuo Isu, Tomohide Tsukahara, Noriyuki Sato, Hidemitsu Kitamura, Norimasa Iwasaki, Naoki Takeda, Akio Minami, Takashi Nishimura

INTERNATIONAL JOURNAL OF ONCOLOGY ( PROFESSOR D A SPANDIDOS ) 30 ( 2 ) 461 - 467 2007年02月 [査読有り]

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.

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

Tomohide Tsukahara, Satoshi Kawaguchi, Toshihiko Torigoe, Hiroko Asanuma, Emiri Nakazawa, Kumiko Shimozawa, Yuki Nabeta, Shigeharu Kimura, Mitsunori Kaya, Satoshi Nagoya, Takuro Wada, Toshihiko Yamashita, Noriyuki Sato

CANCER SCIENCE ( BLACKWELL PUBLISHING ) 97 ( 12 ) 1374 - 1380 2006年12月 [査読有り]

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.

HLA-restricted specific tumor cytolysis by autologous T-lymphocytes infiltrating metastatic bone malignant fibrous histiocytoma of lymph node

T Tsukahara, S Kawaguchi, K Ida, S Kimura, Y Tamura, T Ikeda, T Torigoe, S Nagoya, T Wada, N Sato, TS Yamashita

JOURNAL OF ORTHOPAEDIC RESEARCH ( ELSEVIER SCI LTD ) 24 ( 1 ) 94 - 101 2006年01月 [査読有り]

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 I 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. (c) 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

A quest for therapeutic antigens in bone and soft tissue sarcoma

S Kawaguchi, T Wada, T Tsukahara, K Ida, T Torigoe, N Sato, T Yamashita

JOURNAL OF TRANSLATIONAL MEDICINE ( BIOMED CENTRAL LTD ) 3 31 2005年08月 [査読有り]

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.

Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family, Livin/ML-IAP in lung cancer

H Hariu, Y Hirohashi, T Torigoe, H Asanuma, M Hariu, Y Tamura, K Aketa, C Nabeta, K Nakanishi, K Kamiguchi, Y Mano, H Kitamura, J Kobayashi, T Tsukahara, N Shijubo, N Sato

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 11 ( 3 ) 1000 - 1009 2005年02月 [査読有り]

CD8(+) CTLs have an essential role in immune response against tumor. Although an increasing number of tumor-associated antigens that can be recognized by CTLs have been identified from human tumors, a limited number of tumor-associated antigens is known in lung cancer. In addition, because some of them are expressed in noncancerous tissues, there exist limitations in their application to tumor immunotherapy. Livin/ML-IAP is one of recently identified inhibitor of apoptosis protein (IAP) family, which is overexpressed in melanoma cells. In this report, we show that Livin/ML-IAP is aberrantly expressed in many lung cancer cell lines and primary lung cancer tissues, whereas it is not detectable in normal tissues, including lung by reverse transcription-PCR methods. To identify HLA-A24-restricted T-cell epitopes of Livin/ML-IAP, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. It was revealed that Livin7 peptide (amino acid sequence, KWFPSCQFLL) had the highest affinity to HLA-A24. By stimulating peripheral blood lymphocytes of HLA-A24-positive lung cancer patients with Livin7 peptide in vitro, the peptide-specific CTLs were successfully induced from four of five patients with Living ML-IAP-positive lung cancer but not from any of four patients without Livin/ML-IAP expression in their cancer tissues. Furthermore, the CTLs induced by Livin7 peptide showed cytotoxicity against Livin/ML-IAP(+) lung cancer cell lines in an HLA-A24-restricted manner. Our data suggest that Livin/ML-IAP may be an excellent target antigen in immunotherapy for lung cancer and Livin7 peptide may serve as a potent peptide vaccine for HLA-A*2402(+)/Livin(+) lung cancer patients.

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

S Kawaguchi, T Wada, K Ida, Y Sato, S Nagoya, T Tsukahara, S Kimura, H Sahara, H Ikeda, K Shimozawa, H Asanuma, T Torigoe, H Hiraga, T Ishii, S Tatezaki, N Sato, T Yamashita

JOURNAL OF TRANSLATIONAL MEDICINE ( BIOMED CENTRAL LTD ) 3 1 - 9 2005年01月 [査読有り]

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.

Identification of human autologous cytotoxic T-lymphocyte-defined osteosarcoma gene that encodes a transcriptional regulator, papillomavirus binding factor

T Tsukahara, Y Nabeta, S Kawaguchi, H Ikeda, Y Sato, K Shimozawa, K Ida, H Asanuma, Y Hirohashi, T Torigoe, H Hiraga, S Nagoya, T Wada, T Yamashita, N Sato

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 64 ( 15 ) 5442 - 5448 2004年08月 [査読有り]

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.

Crisscross CTL induction by SYT-SSX junction peptide and its HLA-A*2402 anchor substitute

K Ida, S Kawaguchi, Y Sato, T Tsukahara, Y Nabeta, H Sahara, H Ikeda, T Torigoe, S Ichimiya, K Kamiguchi, T Wada, S Nagoya, H Hiraga, A Kawai, T Ishii, N Araki, A Myoui, S Matsumoto, T Ozaki, H Yoshikawa, T Yamashita, N Sato

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 173 ( 2 ) 1436 - 1443 2004年07月 [査読有り]

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.

Simultaneous radial closing wedge and ulnar shortening osteotomies for distal radius malunion

T Wada, S Isogai, K Kanaya, T Tsukahara, T Yamashita

JOURNAL OF HAND SURGERY-AMERICAN VOLUME ( W B SAUNDERS CO ) 29A ( 2 ) 264 - 272 2004年03月 [査読有り]

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 (20degrees-22degrees) and shortening (3-7/mm); the other had the fracture with volar angulation (24degrees) 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, 8degrees-15degrees) 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. Copyright (C) 2004 by the American Society for Surgery of the Hand.

Recognition by cellular and humoral autologous immunity in a human osteosarcoma cell line

Yuki Nabeta, Satoshi Kawaguchi, Hiroeki Sahara, Hideyuki Ikeda, Yoshihiko Hirohashi, Takahiro Goroku, Yuriko Sato, Tomohide Tsukahara, Toshihiko Torigoe, Takuro Wada, Mitsunori Kaya, Hiroaki Hiraga, Kazuo Isu, Shinya Yamawaki, Seiichi Ishii, Toshihiko Yamashita, Noriyuki Sato

Journal of Orthopaedic Science ( Springer Japan ) 8 ( 4 ) 554 - 559 2003年 [査読有り]

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.

Improved generation of HLA class I/peptide tetramers

Y Sato, H Sahara, T Tsukahara, M Kondo, Y Hirohashi, Y Nabeta, S Kawaguchi, H Ikeda, T Torigoe, S Ichimiya, Y Tamura, T Wada, T Yamashita, M Goto, H Takasu, N Sato

JOURNAL OF IMMUNOLOGICAL METHODS ( ELSEVIER SCIENCE BV ) 271 ( 1-2 ) 177 - 184 2002年12月 [査読有り]

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. (C) 2002 Elsevier Science B.V. All rights reserved.

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

Y Sato, Y Nabeta, T Tsukahara, Y Hirohashi, R Syunsui, A Maeda, H Sahara, H Ikeda, T Torigoe, S Ichimiya, T Wada, T Yamashita, H Hiraga, A Kawai, T Ishii, N Araki, A Myoui, S Matsumoto, T Umeda, S Ishii, S Kawaguchi, N Sato

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 169 ( 3 ) 1611 - 1618 2002年08月 [査読有り]

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 (GYD QIMPKK), 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.

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