Hiroko Asanuma

Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL) recognized by CD8+ cytotoxic T lymphocytes (CTLs). We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2), general malaise (grade 1), and fever (grade 1). No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9) decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

Survivin Expression Is Regulated by Coexpression of Human Epidermal Growth Factor Receptor 2 and Epidermal Growth Factor Receptor via Phosphatidylinositol 3-Kinase/AKT Signaling Pathway in Breast Cancer Cells

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.

A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8+ CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24+ cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24+ cancer patients.

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.

Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

BackgroundWe previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to c linically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.MethodsWe set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1–1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks.ResultsIn the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-γ responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols.ConclusionThis phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity

Recently, the SOX2 gene has been reported to be amplified in human lung squamous cell carcinomas. However, its roles in human lung adenocarcinomas are still elusive. In this study, we analyzed the functions of SOX2 in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) derived from human lung adenocarcinoma. Human lung CSCs/CICs were isolated as higher tumorigenic side population (SP) cells using Hoechst 33342 dye from several lung cancer cell lines. Four of nine lung cancer cell lines were positive for SP cells (LHK2, 1-87, A549, Lc817). The ratios of SP cells ranged from 0.4% for Lc817 to 2.8% for LHK2. To analyze the molecular aspects of SP cells, we performed microarray screening and RT-PCR analysis, and isolated SOX2 as one of a SP cell-specific gene. SOX2 was expressed predominantly in LHK2 and 1-87 SP cells, and was also expressed in several other cancer cell lines. The expression of SOX2 protein in primary human lung cancer tissues were also confirmed by immunohistochemical staining, and SOX2 was detected in more than 80% of primary lung cancer tissues. To address SOX2 molecular functions, we established a SOX2-overexpressed LHK2 and A549 cell line (LHK2-SOX2 and A549-SOX2). LHK2-SOX2 cells showed higher rates of SP cells and higher expression of POU5F1 compared with control cells. LHK2-SOX2 and A549-SOX2 cells showed relatively higher tumorigenicity than control cells. On the other hand, SOX2 mRNA knockdown of LHK2 SP cells by gene-specific siRNA completely abrogated tumorigenicity in vivo. These observations indicate that SOX2 has a role in maintenance of stemness and tumorigenicity of human lung adenocarcinoma CSCs/CICs and is a potential target for treatment.

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)

HSP DNAJB8 Controls Tumor-Initiating Ability in Renal Cancer Stem-like Cells

Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC.

Cytotoxic T Lymphocytes Efficiently Recognize Human Colon Cancer Stem-Like Cells

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs.

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.