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. 2018 Jul 3;67(9):1371–1380. doi: 10.1007/s00262-018-2197-x

Immunotherapy with cancer peptides in combination with intravesical bacillus Calmette–Guerin for patients with non-muscle invasive bladder cancer

Wataru Obara 1,✉, Isao Hara 2, Yoichiro Kato 1, Renpei Kato 1, Keiji Inoue 3, Fuminori Sato 4, Hiromitsu Mimata 4, Yusuke Nakamura 5, Tomoaki Fujioka 1
PMCID: PMC11028097  PMID: 29971464

Abstract

Purpose

A phase I study using two peptide vaccines derived from M phase phosphoprotein 1 (MPHOSPH1) and DEP domain containing 1 (DEPDC1) demonstrated promising results for the treatment of advanced bladder cancer. Therefore, we further tested the ability of these peptides to prevent recurrence after transurethral resection of the bladder tumor in patients with non-muscle invasive bladder cancer (NMIBC).

Materials and methods

127 patients were enrolled in a multicenter, non-randomized phase II clinical trial. The primary endpoint was recurrence-free survival (RFS) rate, and secondary endpoints were safety and immunological response. HLA-A24-restricted peptides were subcutaneously administered in addition to intravesical BCG therapy. The exploratory endpoint evaluated differences of RFS rate between HLA-A*2402-positive (A24(+)) and -negative (A24(−)) groups.

Results

A 2-year RFS rate in all patients was 74.0%. The RFS rate in the A24(+) group (n = 75) and in the A24(−) group (n = 52) were 76.0 and 71.2%, respectively. This vaccine therapy was well-tolerated and feasible. MPHOSPH1 and DEPDC1 peptide-specific cytotoxic T lymphocyte responses were observed in 75.8 and 77.5% of the A24(+) group, respectively. Patients having both peptide-specific CTL responses showed significantly better RFS than patients without CTL response (P = 0.014). In the A24(+) group, patients who had positive reaction at the injection sites (RAI) had significantly lower rates of recurrence than RAI-negative patients (P = 0.0019).

Conclusions

Cancer peptide vaccines in combination with intravesical BCG therapy demonstrated good immunogenicity and safety, and may provide benefit for preventing recurrence of NMIBC.

Keywords: Cancer peptide vaccine, Adjuvant therapy, Non-muscle invasive bladder cancer, M phase phosphoprotein 1, DEP domain containing 1

Introduction

Non-muscle invasive bladder cancer (NMIBC) accounts for approximately 70% of bladder cancer cases, with transurethral resection of the bladder tumor (TURBT) as the primary treatment of choice. Intravesical bacillus Calmette–Guérin (BCG) therapy is currently the global standard adjuvant therapy for NMIBC after TURBT as it significantly reduces the risk of disease recurrence or progression [1, 2]. Intravesical BCG therapy is a nonspecific immunotherapeutic method known to activate cellular immune responses; however, its immunological mechanism of action is not fully understood [3]. Despite receiving BCG therapy, 30–50% of patients fail to respond, and 15% show muscle-invasive disease progression [4, 5]. Moreover, BCG therapy is also known to cause local and systemic side effects, including drug-induced cystitis, hematuria, and fever, resulting in treatment cessation in nearly 30% of the affected patients, or delay or reduction in the number of instillations [6]. Hence, the development of a novel adjuvant therapy with a lesser risk for toxicity is eagerly awaited to improve the clinical outcomes of patients with NMIBC.

There is remarkable progress in cancer immunotherapy with the use of antibodies raised against CTLA-4, programmed death-1 (PD-1), or programmed death-ligand 1 (PD-L1) for advanced stages of several cancer types. PD-1/PD-L1 checkpoint inhibitors have dramatically changed the treatment strategy for metastatic urothelial cancer (UC). Two phase II studies using atezolizumab (anti-PD-L1) and nivolumab (anti-PD-1) in patients with metastatic UC who had progressed on platinum based chemotherapy showed a high response rate and longer survival effect [7, 8]. Pembrolizumab (anti-PD-1) is the first immunotherapeutic agent to demonstrate improved overall survival (OS) over chemotherapy in patients with advanced UC following progression after first-line platinum-based therapy [9]. Avelumab (anti-PD-L1) was also associated with durable responses and prolonged survival in patients with refractory metastatic UC [10]. Durvalumab (anti-PD-L1) demonstrated a manageable safety profile with evidence of remarkable clinical activity in PD-L1-positive patients with UC [11]. Based on these trials, the Food and Drug Administration (FDA) recently approved these five PD-1/PD-L1 checkpoint inhibitors for the treatment of advanced UC. Furthermore, active immunotherapy inducing CTLs with neoantigens, and T cell receptor (TCR)-engineered adaptive immunotherapy, have also demonstrated very promising effects [12, 13].

We performed a genome-wide expression profile analysis of bladder cancers a decade ago, and identified two typical cancer–testis antigens that showed oncogenic activity: the M phase phosphoprotein 1 (MPHOSPH1) and the DEP domain containing 1 (DEPDC1), which also play critical roles in bladder carcinogenesis [14, 15]. We also identified human leukocyte antigen (HLA)-A*2402-restricted epitope peptides derived from these proteins that successfully induced peptide-specific CTLs. The application of these MPHOSPH1- and DEPDC1-derived peptides as vaccine therapies confirmed their safety for usage and that they induced immunogenic reactions in patients with advanced bladder cancer [16, 17]. In the present study, we conducted a phase II clinical trial to examine the efficacy of a combination of these peptide vaccines and intravesical BCG therapy for the prevention of disease recurrence and immune responses in patients who had received TURBT for NMIBC.

Materials and methods

Study design

This exploratory phase II, open-label, non-randomized, multicenter cancer vaccine trial was conducted at 12 centers in Japan. All patients were administered the peptide vaccine regardless of HLA-A status, and the HLA-A genotypes were employed at the point of data analysis (Fig. 1a). The point of analysis point was decided by the efficacy and safety evaluation committee, including the external members. The primary endpoint was the difference between recurrence-free survival (RFS) in the HLA-A*2402-positive (A24(+)) and -negative (A24(−)) groups. The RFS was counted as days from the first vaccination to recurrence. The secondary endpoints were immunological response and adverse effects. Immunological monitoring was performed at the central laboratory using enzyme-linked immunospot (ELISPOT) assays with in vitro cultured lymphocytes that were derived from peripheral blood lymphocytes (PBLs). Toxicity assessments were performed in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. Immunological reactions at the injection sites (RAI) were evidenced by erythema and/or induration. We estimated that 70 patients would be required in the A24(+) group and 40 patients in the A24(−) group, based on the assumption of 2-year RFS rates of 65% in the A24(−) group and 80% in the A24(+) group, with overall alpha levels of 0.2 and beta levels of 0.5 in this exploratory setting. About 60% of the Japanese population have at least one HLA-A*2402 allele [18], and allowing for some study dropouts, we decided to enroll a total of 110 patients.

Fig. 1.

Fig. 1

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HLA key open method and Treatment schedule. a This study was a non-randomized trial. HLA-A genotype were checked at entry for all patients. An effect safety assessment chairperson managed the frequency of HLA-A genotype. HLA-A genotype were key open when effective cases were achieved. Effective cases were more 70 in A24 and more 40 in non-A24 genotypes. b Either one or both of MPHOSPH1 and DEPDC1 peptide vaccines (PV) were injected according to the antigen expression examined by immunohistochemistry (IHC). The intravesical BCG was given once a week for 8 weeks. The CTL analysis was performed at the pre, during and after-vaccination period

Patient eligibility

The eligibility criteria for participants were as follows: (1) pathologically confirmed urothelial carcinoma with intermediate or high risk, according to the European Organization for Research and Treatment of Cancer (EORTC) risk classification [19]; (2) an Eastern Cooperative Oncology Group performance status of 0 or 1; (3) age between 20 and 85 years; (4) adequate organ function; (5) no chemotherapy, radiotherapy, or other immunotherapy within 4 weeks before the vaccination; and (6) confirmed HLA class I expression, with MPHOSPH1 and/or DEPDC1 expression in tumor tissues detected by immunohistochemical analysis, as previously reported [16]. The exclusion criteria were as follows: (1) primary carcinoma in situ; (2) active infection or other active malignancy; (3) pregnancy or lactation; (4) concomitant treatment with steroids or immunosuppressant agents; and (5) unsuitability for the study determined by the principal investigator or the attending physician.

Treatment protocol

Figure 1 shows the HLA key open method and the treatment schedule employed in this study. All patients underwent an initial TURBT, and then a second TURBT after 4–6 weeks to confirm the absence of residual tumor tissue. Intravesical BCG of 40 mg (Tokyo 172 strain, purchased from Nihon BCG, Tokyo, Japan) was administered after the second TURBT. BCG was then administered once a week for 8 weeks. Based on the immunohistochemical analysis, one or both of the peptides were used for the vaccine treatment. MPHOSPH1 and/or DEPDC1derived peptide was administered depending on the expression of MPHOSPH1 and DEPDC1. One or both peptides (1 mg each) were emulsified in 1 ml incomplete Freund’s adjuvant, and injected into the axilla or inguinal regions. The vaccination was administered subcutaneously once a week for 8 weeks, and thereafter given once a month for 3 months (11 doses in total). For the immunological evaluation, PBLs were collected from the patients during the pre-vaccination period, after the 4th, 8th, 9th, 10th, and 11th injections, as well as 1 year and 1.5 and 2 years after the first vaccination, if possible. Clinical and laboratory assessments were performed at each visit. A recurrence check by cystoscopy and urine cytology was performed after the 8th vaccination, and then every 3 months. An assessment of adverse events was performed once a week during the weekly treatment periods, and then every 3 months after the vaccinations.

Peptides

Peptides derived from MPHOSPH1-278 (IYNEYIYDL) and DEPDC1-294 (EYYELFVNI) that showed a high binding affinity to an HLA-A24 molecule were synthesized as described previously [16]. Peptide purity (> 97%) was determined by analytical high-performance liquid chromatography and mass spectrometry analysis. The endotoxin levels and bioburdens of these peptides were tested and determined to be within levels that are in accordance with good manufacturing practice for vaccines (NeoMPS, Inc., San Diego).

Lymphocyte preparation for immunological monitoring

ELISPOT assay at the central laboratory was periodically standardized and validated in accordance with the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use guidelines. PBLs were obtained from the patients before, during, and after the vaccination period. Peripheral blood was taken by venipuncture, collected in ethylenediaminetetraacetic acid tubes, and transferred to the central laboratory at room temperature. Within 24 h of blood collection, PBLs were isolated using Ficoll-Paque Plus (GE Healthcare Bio-sciences, Piscataway, NJ, USA) density gradient solution and were then stored at − 80 °C in cell stock media (Juji field) containing serum at 5 × 106 cells/ml. After thawing, cell viability was confirmed to be > 90% using trypan-blue dye. For the in vitro culture, PBLs were thawed and 5 × 105 cells per well were incubated in medium containing 120 units/ml of recombinant interleukin-2; Novartis, with peptide stimulation (10 μg/ml) performed twice (day 1 and 8); human immunodeficiency virus (HIV)-specific peptide (ILKEPVHGV, 10 μg/ml) was used as a negative control, whereas cytomegalovirus (CMV)-specific peptide (RYLRDQQLL, 10 μg/ml) was used as a positive control. On day 16, the cultured lymphocytes were subjected to ELISPOT assay, after depletion of CD4 cells by magnetic beads (Invitrogen, Grand Island, NY, USA).

ELISPOT assay

ELISPOT assays were performed using the human interferon (IFN)-γ ELISPOT PLUS kit (Mabtech, Nacka Strand, Sweden). Ninety-six-well plates with nitrocellulose membranes (Millipore, Molshelm, France) were pre-coated with the primary anti-IFN-γ antibody (1-D1K) at 4 °C overnight. The plates were then pre-reacted with RPMI medium containing 10% fetal bovine serum (Invitrogen). For the A24(+) group, the TISI stimulator cells (2 × 104/well) pulsed with the vaccine peptide (10 μg/ml), HIV-specific peptide (ILKEPVHGV, 10 μg/ml) or CMV-specific peptide (RYLRDQQLL, 10 μg/ml) were incubated overnight in triplicate with responder cells (from 2 × 104/well to 2.5 × 103/well) at the indicated responder/stimulator ratios in a total volume of 200 μl/well. Stimulation with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml; Sigma-Aldrich, St. Louis MO, USA) and ionomycin (1 μg/ml, Sigma-Aldrich) was used as a positive control for T cell activity. For the A24(−) group, responder cells (2 × 104/well) and each peptide (10 μg/ml) or HIV-specific peptide (10 μg/ml) were cultured in a total volume of 200 μl/well without antigen-presenting cells (APC) overnight in triplicate, in combination with PMA + ionomycin, as a positive control. These cell mixtures were then treated with a biotinylated secondary anti-IFN-γ antibody (7-B6-1) and incubated for 2 h. The plates were subsequently incubated with horseradish peroxidase reagent, which catalyzed the conversion of 3,3′,5,5′-tetramethylbenzidine (Mabtech) to a colored product. The spots were quantified using an auto-analyzing system composed of ImmunoSPOT S4 and S5 Versa (Cellular Technology Ltd). A positive antigen-specific T-cell response was quantitatively defined using an evaluation tree algorithm [20]. In brief, the average of triplicate HIV peptide-pulsed stimulator well spots were subtracted from the average of triplicate immunized peptide-pulsed stimulator wells. The positivity of the antigen-specific T-cell response was classified into four grades (−, +, ++, and +++) depending on the amount of peptide-specific spots [17] and their reproducibility at different responder/stimulator ratios. Individuals in the A24(+) group were classified as showing a positive response when the algorithm indicated +, ++, or +++ after vaccination. Individuals in the A24(−) group were classified as cross-reactive when the algorithm indicated +++ after vaccination.

Statistical analysis

Chi square and Fisher’s exact test were used to compare patient characteristics. Cumulative (RFS) rates were estimated using the Kaplan–Meier method, and the significance of differences between the curves was tested using the log-rank test. All of the statistical analyses were performed using JMP version 10.0.0 (SAS Institute Inc., Cary, NC, USA). Statistical significance was defined by a value of P < 0.05.

Results

Patient characteristics

We evaluated the expression of MPHOSPH1, DEPDC1, and HLA class I on bladder cancer cells, which were obtained by TURBT. Of the 133 patients assessed prior to enrollment in the study, 127 patients (95.5%) showed expression of either MPHOSPH1 or DEPDC1. Moreover, the expression of both MPHOSPH1 and DEPDC1 was detected in 94 (74.0%) of these 127 patients. The expression of MPHOSPH1 only or DEPDC1 only was detected in 10 cases (7.9%) and 23 patients (18.1%), respectively. Cell membrane expression of HLA class I was confirmed in all 127 patients. Finally, 127 eligible patients were enrolled in the study. Table 1 shows the characteristics of these patients. The median age was 68 years, 108 patients (85.0%) were male, 70 patients (55.1%) had primary tumors, 76 patients (59.8%) had multiple tumors, 40 patients (31.5%) had tumors of larger than 3 cm, 94 patients (74.0%) had T1 tumors, and 31 patients (24.4%) had grade 3 tumors. The median EORTC recurrence score was 6. One hundred and four patients (81.9%) were classified as belonging to the intermediate risk group, and 23 patients (18.1%) belonged to the high-risk group. Regarding the HLA genotypes, 75 patients (59.1%) possessed at least one HLA-A*2402 allele, whereas 52 patients (40.9%) were negative for HLA-A*2402, as we expected [18]. These patient characteristics, with the exception of sex, did not differ significantly between the A24(+) and A24(−) groups.

Table 1.

Patients’ characteristics

Total (n = 127) % A24 (+)
n = 75		 % A24 (−)
n = 52		 % P value
Age (median) 68 68 68 0.67
Range 33–82 33–82 42–79
Sex
 Male 108 85.0 68 90.7 40 76.9 0.033
 Female 19 15.0 7 9.3 12 23.1
Primary recurrence rate, n (%)
 Primary 70 55.1 36 48.0 34 65.4 0.114
 Recurrent ≤ 1/year 24 18.9 15 20.0 9 17.3
 Recurrent > 1/year 33 26.0 24 32.0 9 17.3
Tumors, n (%)
 Single 51 40.2 27 36.0 24 46.2 0.504
 2–7 67 52.8 42 56.0 25 48.1
 ≥ 8 9 7.1 6 8.0 3 5.8
Tumor size, n (%) (cm)
 < 3 87 68.5 49 65.3 38 73.1 0.438
 ≥ 3 40 31.5 26 34.7 14 26.9
T category, n (%)
 Ta 33 26.0 21 28.0 12 23.1 0.681
 T1 94 74.0 54 72.0 40 76.9
WHO 1973 grade, n (%)
 G1 12 9.4 8 10.7 4 7.7 0.787
 G2 84 66.1 48 64.0 36 69.2
 G3 31 24.4 19 25.3 12 23.1
EORTC risk group, n (%)
 Intermediate 104 81.9 59 78.7 45 86.5 0.350
 High 23 18.1 16 21.3 7 13.5
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Clinical outcomes and immunological evaluation

A total of 33 patients (26.0%) showed recurrence, and the RFS rate was 74.0% at 2 years (Fig. 2a). The RFS rate in the A24(+) group was 76.0%, whereas that in the A24(−) group was 71.2% (P = 0.47) (Fig. 2b). The ELISPOT assay revealed strong specific T-cell responses to the MPHOSPH1 and DEPDC1 peptides. In the A24(+) group (n = 75), 58 patients (77.3%) received both MPHOSPH1 and DEPDC1 peptides, 4 patients (5.3%) received the MPHOSPH1 peptide only, and 13 (17.3%) received the DEPDC1 peptide only. Positive CTL responses to both the MPHOSPH1 and DEPDC1 peptide were observed in 43 (74.1%) of the 58 patients who received both vaccines. Positive responses to the MPHOSPH1 peptide were observed in all 4 cases who received this peptide only, and 12 of the 13 patients who received the DEPDC1 peptide only responded positively to this peptide. Therefore, the MPHOSPH1 peptide-specific CTL response was detected in 47 (75.8%) of the 62 patients treated with this peptide, while DEPDC1 peptide-specific CTL responses were detected in 55 (77.5%) of the 71 patients treated with this peptide. In these CTL-positive cases, about 30% showed a persistent and strong CTL response, even 1–2 years after the completion of the vaccine administration (Fig. 3). We investigated the outcome of persistent CTL response cases in detail. A total of 21 (33.4%) out of 62 patients receiving MPH peptide showed a persistent CTL response for 2 years, whereas 20 (95.2%) of these patients showed no recurrence. On the other hand, 25 (35.2%) of 71 patients receiving DEP showed a persistent CTL response for 2 years, whereas 23 patients (92.0%) of these patients showed no recurrence.

Fig. 2.

Fig. 2

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Kaplan–Meier curves for recurrence-free survival (RFS). a All patients treated with the peptide vaccine and intravesical BCG therapy. b RFS of HLA-A 24 positive and A24 negative patients

Fig. 3.

Fig. 3

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Time-dependent changes of the CTL response. a Frequency of MPHOSPH1 peptide-specific CTL response pre, during and after-vaccination period in the A24(+) group. b Frequency of DEPDC1 peptide-specific CTL response pre, during and after-vaccination period in the A24(+) group. Positivity of antigen-specific T-cell response was quantitatively defined according to evaluation tree algorithm as previously described [20]

When the patients who were treated with both MPHOPSH1 and DEPDC1 peptides were divided into three groups according to their CTL response positivity, RFS was found to be longer in patients who showed a positive reaction to at least one peptide (Fig. 4a). Notably, patients who showed CTL responses to both peptides had a significantly greater RFS than those with no CTL response (P = 0.0104) (Fig. 4b). Few patients showed CTL responses, nevertheless, the CTL induced by peptide vaccination might have contributed to the prevention of NMIBC recurrence.

Fig. 4.

Fig. 4

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RFS according to CTL and RAI responses against peptides in the A24(+) group. a RFS according to CTL responses against MPHOSPH1 and DEPDC1 peptides. b RFS by CTL responses group to both peptides and no CTL response group. Double positive means patients having CTL responses against both peptides. Single positive means patients having CTL responses against either peptide. Double negative means patients having no CTL response against both peptides. c RFS of RAI-positive and RAI-negative patients

Immunological evaluation of the A24(−) group

In vitro cultured T cells from 57 patients in the A24(−) group were used for the modified ELISPOT assay by applying the dump assay. This modified ELISPOT assay suggested that the MPHOSPH1-specific CTL response was induced in 5 (8.8%) patients with HLA-A genotypes of 3101/3303, 0201/2601, 1101/3303, 0201/3303, and 0207/3303. This observation implied the possibility of cross-reactivity between different HLA allele(s); in this case, HLA-A*3303 is the most likely candidate. It was also noted that these five patients showed no recurrence for at least 2 years after peptide administration.

Adverse reactions

The adverse events are listed in Table 2. Most of the patients experienced irritative voiding symptoms, including urinary tract pain and increased urinary frequency. Grades 1–2 hematuria and temperature elevation (38 °C or higher) were also observed. Grade 3 adverse events were observed in two patients: a myocardial infarction occurred in one patient, who had received an anticoagulant for atrial fibrillation, whereas the other patient had interstitial pneumonitis, which was confirmed to be caused by intravesical BCG by a lymphocyte blast transformation test. The efficacy and safety evaluation committee judged that these events were not related to the peptide vaccine treatment. There was no difference in the frequency of the adverse events between A24(+) and A24(−) groups. These results suggest that the combined treatment using intravesical BCG and peptide vaccine was well-tolerated and feasible.

Table 2.

Adverse events

A24(+)
n = 75		 A24(−)
n = 57
Grade 1 (n, %) Grade 2 (n, %) Grade 3 (n, %) Grade 1 (n, %) Grade 2 (n, %) Grade 3 (n, %)
Blood and lymphatic system disorders
 Anemia 1 1.3
Gastrointestinal disorders
 Diarrhea 2 2.7
 Ileus 1 1.3
General disorders and administration site conditions
 Fever 5 6.7 5 6.7 3 5.3 2 3.5
Investigations
 Aspartate aminotransferase increased 2 2.7 1 1.3 1 1.8
 Alanine aminotransferase increased 2 2.7 1 1.8
 Creatinine increased 1 1.3
 CPK increased 1 1.3
Cardiac disorders
 Myocardial infarction 1 1.3
Musculoskeletal and connective tissue disorders
 Arthralgia 3 4.0
Renal and urinary disorders
 Urinary frequency 8 10.7 3 4.0 3 5.3 1 1.8
 Hematuria 6 8.0 2 2.7 3 5.3 2 3.5
 Urinary tract pain 4 5.3 13 17.3 5 8.8 12 21.1
 Urinary incontinence 1 1.8
 Cystitis noninfective 3 4.0 1 1.8
 Urinary tract infection 7 9.3 1 1.8
Respiratory, thoracic and mediastinal disorders
 Pneumonitis 1 1.3
Skin and subcutaneous tissue disorders
 Rash acneiform 1 1.3
Immune system disorders
 Allergic reaction 1 1.8
General disorders and administration site conditions
 Malaise 1 1.8
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RAI, including redness and swelling, were observed in 57 patients (76.0%) in the A24(+) group during the vaccine administration and follow-up periods. The RFS rate in the RAI (+) group was significantly higher than that of the RAI(−) group (84.2 vs. 50.0%, respectively, at 2 years; P = 0.0019; Fig. 4c).

Discussion

In general, the following characteristics are desirable in target molecules for the development of cancer vaccines: (1) high immunogenicity; (2) specific expression in a large number of cancer cells; and (3) essential for cell survival [21, 22]. The MPHOSPH1 and DEPDC1 molecules used in this study are considered to be highly appropriate because they were overexpressed in bladder cancer tissues, they were expressed specifically by cancer cells and the testes (cancer-testis antigens), they were essential molecules for cancer cell survival, and more importantly, they showed extremely strong immunogenicity [14–16].

In this study, we compared the RFS rate in the HLA-A24(+) and A24(−) groups. Patients were assigned to these groups when the HLA genotypes were revealed at the end of this exploratory trial. This HLA-key open design provides natural randomization so that all patients can participate in the clinical trial. This design also facilitated the examination of safety for the non-adjustment patient and pharmacological activity. Kono et al. reported a phase II clinical trial of peptide vaccination for advanced esophageal cancer using the HLA-key open method. This study demonstrated the utility of the classification by HLA-A status [20]. In our study, the RFS rate for the entire study group was 74% at the 2-year time-point. This RFS rate was approximately equivalent to that reported for BCG maintenance therapy [23, 24]. Although BCG maintenance therapy was effective, local and systemic adverse events tend to reduce patient quality of life, increasing the rate of treatment interruption. The RFS rate in the A24(+) group was slightly greater than that of the A24(−) group although there was no significantly difference. We considered that all patients should had received both the MPHOSPH1 and DEPDC1 peptides regardless of the presence or absence of that expression.

The secondary endpoint was the immunological response as assessed by the ELISPOT assay. As a result, peptide-induced immune response correlated with better RFS in NMIBC patients. We considered that the combined BCG and peptide vaccine treatment might result in a clinical benefit. To our knowledge, this study is the first to present data indicating that therapeutic cancer peptide vaccines may prevent NMIBC recurrence following TURBT. In accordance with the recommendation of the iSBSTc SITC/FDA/NCI Workshop on Immunotherapy Biomarkers,[25–27] we performed the immunological analysis at different time-points. The MPHOSPH1 and DEPDC1 peptides both effectively induced peptide-specific CTL responses that were sustained throughout this 2-year study and these responses appeared to improve the RFS, as compared with A24(+) patients who did not show a CTL response. This result suggested that MPHOSPH1 and DEPDC1 peptides induced strong and sustained CTL responses, which may help to prevent tumor relapse for a long period of time. In the A24(−) group, the modified ELISPOT assay without APC suggested that there may be some cross-reactivity between other HLA allele and the peptide vaccine. Although the modified ELISPOT assay was not fully standardized because of a lack of APC and adequate positive controls, we speculate that this cross-reactivity in A24(−) patients might influence their clinical outcomes. We did not find a common HLA-A allele in the five patients who had positive ELISPOT assays to the MPHOSPH1 peptide. However, since four of them had HLA-A*3303, this allele is a strong candidate for cross-reactivity with this peptide. It is also possible that there is peptide cross-reactivity with other HLA class I, HLA-B, or -C molecules, and further studies will be required to clarify this.

The current study investigated adverse events to the combined intravesical BCG and peptide vaccine treatment. We observed no severe adverse events that were attributable to the peptide vaccine treatment. The most common adverse event was related to intravesical BCG and there was no difference in their frequency between the A24(+) and A24(−) groups. Therefore, this combined therapy was considered to be safe and well-tolerated. RAI was a vaccine-specific adverse event, but none of the patients showed a grade level greater than 4. Skin reactions to vaccinations have been suggested to correlate with clinical outcome [28]. This was consistent with our study, where the appearance of RAI in the A24(+) group correlated significantly with a better clinical outcome, suggesting that RAI might serve as a good surrogate marker for the prediction of clinical responses to this treatment. This could provide useful clinical information to inform the decision to continue or discontinue treatment.

In this study, the expression of MPHOSPH1, DEPDC1, and HLA-class I in the primary bladder tumor was required for patient inclusion. As a result, 95.5% of study participants expressed either MPHOSPH1 or DEPDC1. These proteins, therefore, serve as useful tumor antigens in bladder cancer. Expression of HLA-class I was observed in all study participants. However, Homma et al. observed downregulation of HLA-class I expression in about one-third of patients with muscle-invasive bladder cancer [29]. Hence, the progression of bladder cancer may be associated with a loss of HLA class I expression.

Our study had several limitations. First, although we performed the central pathological evaluation using a small amount of cancer tissue, any component of rare histological variant may have been included in the analysis. Other histological variants such as squamous cell carcinoma or adenocarcinoma might have an influence on the outcomes. Second, the follow-up period after the combined BCG and peptide vaccine therapy might not be long enough. Prolonged monitoring is required to determine the recurrence rates after combined therapy. Third, it was difficult to accurately measure the effects of peptide vaccines because almost all of the patients received BCG therapy. Finally, the number of peptide administrations and the durable effect of A24-restricted peptides were not evaluated, and these require further investigation. It will be necessary to conduct a prospective controlled trial in A24(+) patients using a three arm (peptide alone, BCG alone, combined peptide with BCG) study design to evaluate the efficacy of these peptide vaccines.

To our knowledge, this study is the first to demonstrate that the immune response induced by peptide vaccination could prevent NMIBC recurrence after TURBT with minimal adverse reactions, implying that treatments using cancer peptide vaccines have the potential to become viable treatment options for bladder cancer.

Acknowledgements

We thank Dr. Keiichi Tozawa and Dr. Kenjiro Kohri (Nagoya City University), Dr. Kazuya Mikami and Dr. Tsuneharu Miki (Kyoto Prefectural University of Medicine), Dr. Takashi Kasrashima and Dr. Taro Shuin (Kochi Medical School), Dr. Naoki Mitsuhata (Kure-Kyosai Hospital), Dr. Mitsuo Nishi (Saint Martin’s Hospital), Dr. Yoshihide Ogawa (Tokyo-West Tokushukai Hospital), Dr. Hisaaki Afuso (Nambu Tokushukai Hospital), Dr. Takahito Nasu (Tokuyama Central Hospital), and Dr. Ichiro Miura (Shonan Kamakura General Hospital) for case enrollment. We also thank Dr. Koji Yoshida and Dr. Takuya Tsunoda for providing all of the peptides. We thank Reiko Shinagawa for technical assistance.

Abbreviations

APC

Antigen-presenting cells

BCG

Bacillus Calmette–Guérin

CTL

Cytotoxic T lymphocyte

DEPDC1

DEP domain containing 1

ELISPOT

Enzyme-linked immunospot

EORTC

European Organization for Research and Treatment of Cancer

HLA

Human leukocyte antigen

MPHOSPH1

M phase phosphoprotein 1

NMIBC

Non-muscle invasive bladder cancer

PBLs

Peripheral blood lymphocytes

PD-1

Programmed death-1

PD-L1

Programmed death-ligand 1

RAIs

Reaction at the injection sites

RFS

Recurrence-free survival

TURBT

Trans-urethral resection of the bladder tumor

UC

Urothelial cancer

Author contributions

Clinical trial was designed by WO, YN and TF; patient registrations were provided by WO, YK, RK, KI, FS and HM; data analysis was done by IH; the manuscript was written by WO and YN and was reviewed by all co-authors.

Compliance with ethical standards

Conflict of interest

Y. Nakamura reports receiving a commercial research grant from and has ownership interest (including patents) in OncoTherapy Science, Inc. No potential conflicts of interest were disclosed by the other authors.

Ethical approval

This study was approved by the institutional review board at each hospital and was registered with Clinical Trials.gov as NCT 00633204. This study was carried out according to the Helsinki declaration on experimentation on human subjects.

Informed consent

Informed consent was obtained from all individual participants included in the study.

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