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

Abstract

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‐α was injected subcutaneously on the same day and again 3 days after the vaccination. The B peptide and K9I peptide were used in Protocols B1 and B2, respectively. In total, 21 patients (12 men, nine women; mean age 43.6 years) were enrolled in the present study. Each patient had multiple metastatic lesions of the lung. Thirteen patients completed the six‐injection vaccination schedule. One patient developed intracerebral hemorrhage after the second vaccination. Delayed‐type hypersensitivity skin tests were negative in all patients. Nine patients showed a greater than twofold increase in the frequency of CTLs in tetramer analysis. Recognized disease progression occurred in all but one of the nine patients in Protocols A1 and A2. In contrast, half the 12 patients had stable disease during the vaccination period in Protocols B1 and B2. Of note, one patient showed transient shrinkage of a metastatic lesion. The response of the patients to the B protocols is encouraging and warrants further investigation.

Synovial sarcoma is a malignant tumor of soft tissue characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT‐SSX fusion genes.1, 2 Reported 5‐year survival rates of patients with synovial sarcoma range from 64% to 77%.3, 4, 5, 6, 7 In contrast, most metastatic or relapsed diseases remain incurable, indicating a need for new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy.

Antigen‐specific peptide immunotherapy is one such option.8, 9, 10, 11, 12 Previously, we demonstrated that SYT‐SSX fusion gene‐derived peptides (wild type and agretope modified) are recognized by circulating CD8⁺ T cells in HLA‐A24⁺ patients with synovial sarcoma and elicit human leukocyte antigen (HLA)‐restricted, tumor‐specific cytotoxic responses.13, 14 Subsequent to these preclinical studies, we started a pilot clinical trial with a wild‐type SYT‐SSX‐derived peptide vaccine.15 In the present study, we evaluated immunologic and clinical outcomes of the vaccination trials using an agretope‐modified SYT‐SSX peptide and a combination of the peptide vaccine with adjuvant and interferon (IFN)‐α.

Materials and Methods

Eligibility

The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Sapporo, Japan. Eligible patients were those who: (i) had histologically and genetically confirmed unresectable synovial sarcoma (SYT‐SSX1 or SYT‐SSX2 positive); (ii) were HLA‐A*2402 positive; (iii) were between 20 and 70 years of age; (iv) had Eastern Cooperative Oncology Group (ECOG) performance status between 0 and 3; and (v) provided informed consent. Exclusion criteria included: (i) prior chemotherapy, steroid therapy, or other immunotherapy within the previous 4 weeks; (ii) the presence of other cancers that may influence prognosis; (iii) immunodeficiency or a history of splenectomy; (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure; (v) ongoing breast‐feeding; and (vi) unsuitability for the trial based on the clinical judgment of the doctors involved.

Peptide

A 9‐mer peptide (Peptide B: GYDQIMPKK) spanning the SYT‐SSX fusion region and its HLA‐A*2402 anchor substitute (Peptide K9I: GYDQIMPKI), in which lysine at position 9 was substituted to isoleucine, were synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA, USA). The identity of the peptide was confirmed by mass spectral analysis, and it was shown to have >98% purity when assessed by HPLC. The peptides were delivered to us as sterile, freeze‐dried white powders. They were dissolved in 1.0 mL physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) and were stored at −80°C until just before use. The affinity of the peptides to HLA‐A24 molecules and their antigenicity has been determined in previous studies.13, 14

Vaccination schedule

Four protocols were used (Fig. 1). In Protocols A1 and A2, vaccines with the peptide alone (0.1 or 1 mg) were administered subcutaneously into the upper arm six times at 14‐day intervals. The SYT‐SSX B peptide (0.1 or 1 mg) was used in Protocol A1, as reported previously,15 whereas the K9I peptide (1 mg) was used in Protocol A2. In Protocols B1 and B2, 1 mL peptide was mixed with 1 mL incomplete Freund's adjuvant (IFA, Montanide ISA 51; Seppic Inc., Fairfield, NJ, USA). The mixture was then administered subcutaneously into the upper arm six times at 14‐day intervals. In addition, 3 × 10⁶ U IFN‐α (Sumiferon; Sumitomo Pharmaceuticals, Osaka, Japan) was injected subcutaneously into the upper arm on the same day together with the vaccination and again 3 days after vaccination. The B peptide (1 mg) was used in Protocol B1, whereas the K9I peptide (1 mg) was used in Protocol B2.

Figure 1.

Vaccination protocols. In Protocols A, vaccines with K9I peptide were administered subcutaneously six times at 14‐day intervals. In Protocols B, a mixture of SYT‐SSX B peptide and incomplete Freund's adjuvant (IFA) was administered subcutaneously six times at 14‐day intervals. In addition, interferon (IFN)‐α was injected on the same day as the vaccination and 3 days after the vaccination. The B peptide was used in Protocols A1 and B1, whereas the K9I peptide was used in Protocols A2 and B2.

Delayed‐type hypersensitivity skin test

A delayed‐type hypersensitivity (DTH) skin test was performed at the time of each vaccination. The peptide (10 μg) solution in physiological saline (0.1 mL) and the physiological saline itself (0.1 mL) were separately injected intradermally into the forearm. A positive reaction was defined as the presence of erythema (diameter >4 mm) 48 h after 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 Terminology Criteria for Adverse Events v3.0 (CTCAE; http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf).

Tetramer‐based frequency analysis

The frequency of peptide‐specific CTLs was determined by tetramer‐based analysis. The HLA‐A24/peptide tetramers (HLA‐A24/K9I, HLA‐A24/B and HLA‐A24/HIV) were constructed as described previously.13, 14, 16 The PBMCs were obtained prior to vaccination and then again 1 week after the first, third, and sixth vaccinations.

In Protocols A1, A2, and B2, cells were stained with phycoerythrin (PE)–Cy5‐conjugated anti‐CD8 antibody (eBioscience, San Diego, CA, USA), PE‐conjugated HLA‐A24/B tetramer or HLA‐A24/K9I tetramer, and FITC‐conjugated HLA‐A24/HIV tetramer. The CD8⁺ living cells were gated and cells labeled with the HLA‐A24/B (or K9I) tetramer, but not the HLA‐A24/HIV tetramer, were referred to as tetramer‐positive cells (Fig. S1). Analysis of stained PBMCs was performed using a FACS Caliber (Becton Dickinson, San Jose, CA, USA) and CellQuest software (Becton Dickinson). The frequency of CTLs was calculated as the number of tetramer‐positive cells/number of CD8⁺ cells.15

In Protocol B1, cells were first stimulated by limited dilution/mixed lymphocyte peptide culture (LD/MLPC) in 96‐well plates, as described previously.16, 17 Cells were stained with PE–Cy5‐conjugated anti‐CD8 antibody, PE‐conjugated HLA‐A24/K9I tetramer, and FITC‐conjugated HLA‐A24/HIV tetramer. Cells were analyzed by flow cytometry using FACS Caliber and CellQuest. The CD8⁺ living cells were gated and cells labeled with the HLA‐A24/K9I tetramer, but not the HLA‐A24/HIV tetramer, were referred to as tetramer‐positive cells. Wells containing tetramer‐positive cells were referred to as tetramer‐positive wells. The frequency of CTLs was calculated as follows (see Fig. S2): (number of tetramer‐positive wells)/(total number of CD8⁺ cells seeded).16, 17

Evaluation of the clinical response

Physical and hematological examinations were performed before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as the complete disappearance of all measurable disease. A partial response (PR) was defined as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. Progressive disease (PD) was defined as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study or by the appearance of new lesions. Stable disease (SD) was defined as the absence of matched criteria for CR, PR, or PD.

Results

In all, 21 patients were enrolled in the present study (Table 1). The initial six patients (Protocol A1) reported previously15 were included for reference. There were 12 men and nine women in the study, with a mean age of 43.6 years (range 21–69 years). Each patient had multiple metastatic lesions of the lung.

Table 1.

Profiles of participants and clinical responses

	Age (years)	Gender	Location of the metastatic tumor	No. vaccination	Adverse events	Evaluation of CT images	Follow up (months)	Status
Protocol A1
Patient 1	69	M	Bil. lungs	1	—	PD	1	DOD
Patient 2	32	M	Bil. lungs	3	—	PD	2	DOD
Patient 3	21	F	Bil. lungs	6	—	PD	5	DOD
Patient 4	21	M	Bil. lungs	6	—	PD	6	DOD
Patient 5	39	F	Bil. lungs	6	Fever	SD	12	DOD
Patient 6	26	M	Bil. lungs	4	—	PD	6	DOD
Protocol A2
Patient 7	30	M	Bil. lungs, RP	6	Fever	PD	8	DOD
Patient 8	63	F	Bil. lungs	6	—	PD	8	DOD
Patient 9	28	M	Bil. lungs	2	—	PD	1	DOD
Protocol B1
Patient 10	63	F	Bil. lungs	6	Fever	SD	10	DOD
Patient 11	28	F	Bil. lungs	6	Fever	SD	57	AWD
Patient 12	24	M	Bil. lungs	6	Fever	PD	14	DOD
Patient 13	60	M	Bil. lungs	6	Fever	SD	48	AWD
Patient 14	42	F	Bil. lungs	6	Fever	PD	7	DOD
Patient 15	36	M	Bil. lungs	4	Fever	PD	1	DOD
Protocol B2
Patient 16	52	F	Bil. lungs	2	Fever, ICH	PD	3	DOD
Patient 17	66	M	Bil. lungs	6	Fever	SD	27	DOD
Patient 18	61	F	Unilat. lung	6	Fever	SD	16	AWD
Patient 19	57	M	Bil. lungs	2	Fever	PD	1	DOD
Patient 20	64	F	Bil. lungs	2	Fever	PD	1	DOD
Patient 21	34	M	Unilat. lung	6	Fever	SD	6	AWD

AWD, alive with disease; Bil., bilateral; DOD, death of the disease; ICH, intracerebral hemorrhage; NA, not available; PD, progressive disease; RP, retroperitoneual space; SD, stable disease; Unilat., unilateral.

A six‐injection vaccination schedule was completed in 13 patients. Seven patients discontinued the vaccination regimen because of rapid disease progression. One patient (Patient 16) developed intracerebral hemorrhage after the second vaccination and discontinued thereafter. This patient had been on anticoagulation therapy (warfarin, cirostazol, and limaprost) for more than 2 years following vascular reconstruction surgery. The international normalized ratio (INR) and platelet count at the time of the second vaccination in this patient were 2.01 and 197 000/μL, respectively. The patient had no history of hypertension or diabetes and blood pressure was 109/65 mmHg at the time of vaccination. The intracranial hemorrhage was treated conservatively. One patient each in Protocols A1 and A2 and all six patients in Protocols B1 and B2 experienced fever after vaccination. One patient (Patient 11) had erythema on the vaccine injection site (Fig. 2).

Figure 2.

Vaccination site in Patient 11. The patient received a vaccination in the right upper arm and erythema developed at the vaccination site.

The DTH skin test was negative in all patients. Tetramer‐based frequency analysis was performed in 19 patients. As indicated in Table 2 and shown in Figure 3, three patients (Patients 2, 4, and 6) in Protocol A1, one (Patient 7) in Protocol A2 and three (Patients 16, 17, and 18) in Protocol B2 exhibited a greater than twofold increase in the frequency of CTLs.

Table 2.

HLA‐A24/peptide tetramer analysis

	Before‐vaccination	After 1st vaccination	After 3rd vaccination	After 6th vaccination
Protocol A1
Patient 1	NA	NA	NA	NA
Patient 2	2	2	305	NA
Patient 3	42	49	52	62
Patient 4	6	41	36	47
Patient 5	50	52	9	3
Patient 6	2	15	8	NA
Protocol A2
Patient 7	4	3	14	9
Patient 8	3	1	2	6
Patient 9	0	0	NA	NA
Protocol B1
Patient 10	0	0	0.01	0
Patient 11	0.12	0.04	0.09	0.06
Patient 12	0.07	0.01	0.06	0.02
Patient 13	0.13	0.13	0.13	0.13
Patient 14	0.21	NA	NA	0.22
Patient 15	0.14	0.14	0.17	NA
Protocol B2
Patient 16	0	27	NA	NA
Patient 17	8	1	25	34
Patient 18	7	24	13	39
Patient 19	NA	NA	NA	NA
Patient 20	16	25	NA	NA
Patient 21	24	22	18	11

For Protocols A1, A2, and B2, the data show the number of tetramer‐positive CD8⁺ cells in a population of 10 000 CD8⁺ cells. For Protocol B1, the data show the number of tetramer‐positive wells in a population of 10 000 CD8⁺ cells. NA, not available.

Figure 3.

Frequency of CTLs analyzed by HLA‐A24/peptide tetramers in Patient 17.

Recognized disease progression occurred in all but one (Patient 5) of the nine patients in Protocols A (A1 and A2) during the vaccination period (Table 1). In contrast, disease progression was noted in only half of the 12 patients in Protocols B (B1 and B2; Fig. 4). The remaining six patients had stable disease during the vaccination period. Of these, one patient (Patient 17) exhibited transient shrinkage of a metastatic lesion (Fig. 5).

Figure 4.

Computed tomography scans of the lung in Patient 12 before vaccination on the day of the first vaccination and 4 and 6 weeks after the first vaccination. Growth of metastatic tumors was seen during the vaccination period.

Figure 5.

Computed tomography scans of the lung in Patient 17 before vaccination on the day of the first vaccination and 4 and 6 weeks after the first vaccination. A decrease in the size of a metastatic tumor was seen during the first 6 weeks of the vaccination period.

Discussion

In the present study, we evaluated the safety and effectiveness of SYT‐SSX‐derived peptide vaccines in 21 patients with advanced synovial sarcoma using four different protocols. Vaccines were administered safely in 20 patients. However, one patient in Protocol B2 (Patient 16; K9I peptide, IFA and IFN‐α) developed intracerebral hemorrhage during the vaccination period. To our knowledge, such adverse events have not been reported previously in the literature of anticancer peptide vaccination trials. In contrast, there have been a few reported cases of intracerebral hemorrhage associated with IFN therapy.18, 19, 20 These patients were either long‐term users of IFN18, 19 or had comorbidities of hypertension or diabetes.20 Interferon is known to cause thrombocytopenia.21, 22 Nevertheless, Patient 16 was not a long‐term user of IFN and did not have any of those complications or thrombocytopenia. However, the patient had been on anticoagulants for more than 2 years. Intracranial hemorrhage is a known complication of anticoagulant therapy, with an estimated annual incidence in the US of nearly 3000.23 In addition, approximately half the cases of anticoagulant‐associated intracranial hemorrhage occurred within or below the INR therapeutic range (2.0–3.0).24 It is therefore more likely that the intracranial hemorrhage in Patient 16 was associated with anticoagulants rather than the peptide vaccine or IFN.

With regard to the efficacy of the protocols, the tumors showed dormancy during the vaccination period in six of 12 patients (50%) in Protocols B1 and B2, including one patient exhibiting transient shrinkage of a metastatic lesion. In contrast, only one (11%) of the nine patients who received the peptide vaccine by itself showed such tumor dormancy. In addition, a greater number of patients completed the six‐injection vaccination regimen in Protocol B (80%) than in the protocols with the peptide itself. These findings indicate that the adjuvant activity of IFA and IFN‐α enhance the antitumor effects of the peptide vaccine. Interferon‐α is a cytokine with various biological activities. In a murine model with antimelanoma peptide vaccination, administration of IFN‐α enhanced antigen presentation and promoted the effector function of peptide‐specific CTLs.25 This was attributed to the induction by IFN‐α of dendritic cell maturation and expression of HLA molecules on tumor cells. Such adjuvant activities of IFN‐α have also been seen in clinical vaccination trials.26, 27, 28 In contrast, IFN‐α itself has direct antitumor properties.29 Brodowicz et al.30 reported inhibitory effects of IFN‐α on the proliferation of a synovial sarcoma cell line in vitro. However, no clinical studies have addressed direct the antitumor effects of IFN‐α on synovial sarcoma. Unless we evaluate a protocol with IFN‐α by itself or a protocol with only IFA and IFN‐α, it remains unclear whether the clinical responses seen in patients in Protocol B are due to the effects of IFA and IFN‐α or to the synergistic effects of the peptide vaccine, IFA, and IFN‐α.

The K9I peptide is an agretope‐modified peptide in which an HLA‐A24 anchor residue of the B peptide (lysine at position 9) is substituted to isoleucine.14 In a previous study,14 this substitution enhanced the affinity for HLA‐A24 molecules and improved the capacity of the peptide to induce synovial sarcoma‐specific CTLs in vitro. In the present study, a greater than twofold increase in the frequency of CTLs was seen in three patients (25%) in Protocols A1 and B1 (B peptide) and in four patients (44%) in Protocols A2 and B2 (K9I peptide). Although the percentage of patients exhibiting tumor dormancy was 33% in both the B peptide and K9I peptide groups, tumor shrinkage was observed only in a patient treated with the K9I peptide (Protocol B2). These findings may reflect higher immunogenic properties of the K9I peptide than the B peptide. Indeed, an agretope‐modified peptide has been used in a mixture with wild‐type peptides in bcr‐abl fusion gene peptide vaccines for CML.31

Analysis of peripheral blood lymphocytes using HLA‐A24/peptide tetramers revealed a greater than twofold increase in the peptide‐specific CTL frequency in seven patients. However, the immune responses had no relevance to the clinical responses. One possible explanation for this is that analysis of peripheral lymphocytes does not properly reflect the immunological environment at the tumor site. It remains unknown how many vaccine‐specific CTLs were recruited into the tumor site. In addition, the cytotoxic function of CTLs may be suppressed in the tumor site by certain mechanisms, such as downregulation of Class I molecules and immunosuppressive effects of regulatory T cells. A combination of tetramer analysis with other monitoring assays, such as enzyme‐linked immunospot (ELISPOT), may provide more precise information about the immunological status of patients.

Vaccination trials of fusion gene‐derived peptides have been reported with EWS‐FLI1 in Ewing's sarcoma,32 PAX3‐FKHR in alveolar rhabdomyosarcoma,32 and BCR‐ABL in CML.31, 33, 34, 35, 36 Tumor regression was seen more frequently in studies with CML than in those with sarcomas. One reason for such differences in outcome is the additional use of Class II peptide vaccines in the CML studies.10, 11, 36, 37 Another possible explanation is the introduction of a highly effective therapy for CML, such as the administration of imatinib, which enables a reduction of the tumor mass prior to initiation of peptide vaccination.

Apart from direct vaccinations of peptides into patients, SYT‐SSX‐derived peptides have been used to stimulate dendritic cells in adoptive immunotherapy for patients with synovial sarcoma.38, 39 More recently, the T cell receptor was engineered to recognize an NY‐ESO‐1‐derived peptide and was transduced into autologous T cells. Adoptive immunotherapy using these genetically engineered T cells conferred objective clinical responses (PR) in four of six patients with synovial sarcoma.40 In contrast with adoptive immunotherapy, peptide vaccination would suit a setting with small tumor burden or an adjuvant setting. In this regard, an HER2‐derived peptide vaccine has been used to prevent recurrence from breast cancer in clinical trials.41, 42

In conclusion, the present study is the first clinical trial of SYT‐SSX breakpoint peptide vaccines combined with IFA and IFN‐α. The response of patients to Protocols B is encouraging and warrants further investigation, ideally in an adjuvant setting.

Disclosure Statement

The authors declare no conflicts of interest.

Supporting information

Fig. S1. Data acquisition and sequential gating (Protocols A1, A2 and B2).

Fig. S2. Limited dilution/mixed lymphocyte peptide culture (Protocol B1).

 

(Cancer Sci, 2012; 103: 1625–1630)