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

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.

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.

Background
Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation t(X; 18), and its resultant SYT-SSX fusion gene [1]. This tumor affects mostly adolescents and young adults. The 5year survival rates of patients with this localized disease have ranged from 66% to 80% in the current literature [2][3][4][5]. However, the majority of metastatic or relapsed diseases still remain incurable despite intensive multimodality therapy. Therefore there is a need for additional new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy.
Vaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy [6,7]. This is based on the rationale that T cells recognize antigenic peptide in the context of MHC molecules on the tumor cell or antigen presenting cells through the T cell receptor, which elicits subsequent anti-tumor immune responses. Identification of antigenic peptides recognized T cells enabled us to apply vaccination trials to a variety of tumors, except bone and soft tissue sarcomas [8].
Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas [9,10]. The fusion regions of translocation products are specifically expressed by its corresponding tumors, thereby serving as targets of great potential for tumor specific therapies, including immunotherapy [11,12]. We [13,14] found that SYT-SSX fusion gene-derived peptides can be recognized by circulating CD8+ T cells in patients with synovial sarcoma and elicit HLA-restricted, tumor specific cytotoxic responses by in vitro stimulations. In this study, we conducted a phase I pilot trial of vaccination of a SYT-SSX-derived junction peptide in elected synovial sarcoma patients.

Peptide
A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies [13,14].

Eligibility
The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. 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. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004.

Vaccination schedule
Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg.

Delayed-type hypersensitivity (DTH) skin test
Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 µg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection.

Toxicity evaluation
Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC).

Clinical response evaluation
Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD.

Tetramer staining
HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/ R49.2, and HLA-A24/HIV) were previously constructed [13][14][15]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1 st , 3 rd , and 6 th vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8 + cells.

CTL induction
Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before [13,14]. The cytotoxic activity was evaluated by 6-h 51 Cr release assay [13]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A*2402 (T2-A*2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A*2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8 + T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the 51 Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific 51 Cr release: [(experimental 51 Cr release -spontaneous 51 Cr release) / (maximum 51 Cr release -spontaneous 51 Cr release)] × 100. Maximum 51 Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8 + T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.

Patient profiles
Six patients were enrolled in the study (Table 1). There were four men and two women with an average age of 34.7 years old (range 21-69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the

Clinical response
Recognized disease progression occurred in five out of six patients during the vaccination period (Table 1, Fig. 1). In contrast, one patient (case 5) showed no such rapid progression (Table 1, Fig. 2). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study.

Tetramer analysis and CTL induction
Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers ( Table 2), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig. 3). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients. Table 3 depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure  4 represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/ target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A*2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific CT scan image of the lung of case 3 patient

A B
immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine.

Discussion
The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (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) CT scan image of the lung of case 5 patient   Frequency of CTLs analyzed by HLA-A24/peptide tetramers in the case 4 patient. Frequencies of each analysis were described in Table 2. Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia [16], EWS-FLI1 in 12 patients with Ewing's sarcoma [17], and PAX3-FKHR in four patients Cytotoxicity of CTLs induced from the case 4 patient %Specific lysis with alveolar rhabdomyosarcoma [17]. In addition, Matsuzaki et al. [18] reported a case of a synovial sarcoma patient who were treated with autologous dendritic cells pulsed with a mixture of SYT-SSX junction peptides (8-16 mer). In these studies, tumor remission was noted only in one patient with Ewing's sarcoma where IL-2 was concomitantly administered. These findings together with the results of our present trial have indicated the limited therapeutic efficacy of natural junction peptides. In this regard, we discovered improved in vitro immunogenicity of the SYT-SSX junction peptide by the substitution of an HLA-A24 anchor residue (position 9) [14]. Clinical study of this anchor-substituted peptide is currently underway. Besides modification of the peptide itself, concurrent use of adjuvants and cytokines, and adoptive T cell or/and dendritic transfer should further improve the therapeutic efficacy [8,19]. Also, it is important to determine the appropriate timing of vaccination and proper endpoints of clinical studies.
To monitor the immunological responses, we used HLA/ peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table 2 and Fig. 4, reactivity of circulating T cells to HIV peptide tetramer remained under the background level throughout the vaccination period.
Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma [17]. Another limitation in the current study is lack of analysis on immunological significance of SYT-SSX variants (SYT-SSX1 and SYT-SSX2). This question should be addressed in a larger scale analysis.

Conclusions
This is the first clinical trial of SYT-SSX fusion genederived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.