The feasibility and clinical effects of dendritic cell-based immunotherapy targeting synthesized peptides for recurrent ovarian cancer

This retrospective study included patients who initially received chemotherapy for ROC followed by DC-based immunotherapy targeting synthesized peptides at the Seren Clinics in Nagoya and Tokyo between March 2007 and August 2013. The inclusion criteria were as follows: (1) a clinical diagnosis of inoperable ROC; (2) an expected prognosis of more than 3 months; (3) white blood cell count of 2,000 cells/μL or higher; (4) hemoglobin level of 7.0 g/dL or higher; (5) platelet count of 70,000 counts/μL or higher, and (6) no serious vital organ dysfunction. All participants provided signed informed consent for use of their data for this study. This study was approved by the institutional review board of Isoukai (approval number 25–2) and was conducted in accordance with the Declaration of Helsinki.

To determine the type of peptides for administration, we first evaluated each patient for human leukocyte antigen (HLA) expression. In cases of available patient tissue samples, WT1 and/or MUC1 expression was assessed via IHC staining. Serum CA125 levels were also evaluated to determine the peptide to be administered. Based on these results, DCs were prepared and pulsed with 1–3 synthesized peptides as previously described [18, 19]. Briefly, peripheral blood mononuclear cells (PBMCs) were prepared from leukapheresis products by Ficoll-Hypaque gradient density centrifugation. PBMCs were then plated on tissue culture vessels and continuously cultured for 5 days in medium containing granulocyte-monocyte colony-stimulating factor (GM-CSF; 50 ng/mL) and interleukin (IL) 4 (25 ng/mL) to generate immature DCs. To induce further differentiation, the immature DCs were stimulated with OK-432 (Chugai Pharmaceutical Co, Ltd, Tokyo, Japan) and prostaglandin-E2 (50 ng/mL; Daiichi Fine Chemical Co, Ltd, Toyama, Japan) for 24 hours. On day 7 in culture, the DCs were pulsed with MHC class I-restricted WT1 peptide antigens according to the HLA-A pattern (CYTWNQMNL [mutant WT1 peptide; Neo-MPS, San Diego, CA] for HLA-A*2402 or RMFPNAPYL [WT1 peptide; Neo-MPS] for HLA-A*0201/0206), MUC1 long peptide (30-mer [TRPAPGSTAPPAHGVTSAPDTRPAP-GSTAP] at 20 mg/mL; Greiner Japan, Tokyo, Japan) for any HLA-A type, and/or CA125 protein (500 U/mL) for any HLA-A type. Subsequently, the DCs were characterized by flow cytometry to ensure that they achieved the typical phenotype of mature DCs (CD14^-/low/HLA-DR⁺/HLA-ABC⁺/CD80⁺/CD83⁺/CD86⁺/CD40⁺/CCR7⁺). They were then cryopreserved until the day of administration.

The DC suspension was adjusted to a total volume of 1.0 mL using saline. All patients were intradermally injected 5–7 times with DCs (approximately 10⁷ cells/injection) in close proximity to the axial and/or inguinal lymph nodes. Injections were repeated every 14–21 days. OK-432, a streptococcal immunological adjuvant, was administered simultaneously with the DC vaccine to patients without serious allergies to penicillin or other drugs. Tolerable doses of OK-432 ranged from 0.5 to 5 KE, and the appropriate doses were determined according to the incidence of fever after administration.

Immunological function was found to be associated with survival on DC vaccine administration. For the functional analysis of cancer immunotherapy, CD4⁺ T cells, CD8⁺ T cells, and natural killer (NK) cells were obtained from blood samples before and after DC vaccine administration. Cells with a CD16⁺/CD56⁺ phenotype as determined by flow cytometry were considered to be NK cells.

WT1-specific cytotoxic T lymphocytes (CTLs) were also evaluated for adequate induction. The frequency of WT1-specific CTLs in each patient was determined with either WT1-HLA-A*2402 or 0201 tetramers by flow cytometry analysis as previously described [18]. Briefly, T cells were incubated with Clear Back (MBL, Nagoya, Japan) prior to tetramer staining to block the Fc receptors. They were then stained with phycoerythrin-labeled HLA-A*2402 WT1 mutated (CYTWNQMNL) and HLA-A*0201 WT1 wild-type (RMFPNAPYL) tetramers (MBL), followed by fluorescein isothiocyanate-labeled anti-human CD3, CD4, and CD8. The staining was performed at 4°C for 30 minutes, and the cells were washed twice before flow cytometry analysis.

All adverse events (AEs) were graded and documented according to the Common Terminology Criteria for Adverse Events, version 4.0. Clinical assessments were performed at baseline, 3 months, and 6 months via computed tomography (CT) or magnetic resonance imaging (MRI). Response was expressed as the proportion of patients with a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), as well as disease control rate (DCR) or objective response rate (ORR), as assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. We assessed transient erythema in the patients’ forearm skin within 24–48 h after vaccination. Fever after vaccination was also assessed and defined as a body temperature of ≥38°C at 48 hours after vaccination. In addition, we used the neutrophil-to-lymphocyte ratio (NLR) as a simple index of systemic inflammation [20].

The Kaplan-Meier probability estimates of overall survival (OS) were calculated, and statistical differences between the treatment arms were determined using the log-rank test. A multivariate analysis was performed using the Cox regression method to estimate the hazard ratios (HRs) with a 95% confidence interval (CI). Categorical data were compared using the Fisher exact probability test or the chi-square test. Differences were considered statistically significant when P values were <0.05.

A total of 71 patients who initially received chemotherapy for ROC followed by DC-based immunotherapy were included in our study. Of these, 8 patients who received less than 5 rounds of the DC vaccine and 5 who were unavailable for follow-up examinations were excluded. Two patients who received the DC vaccine pulsed with peptides eluted from autologous OCs were also excluded. Thus, the final analysis included 56 eligible patients whose clinicopathological characteristics are summarized in Table 1. The patients’ age ranged from 23 to 70 years (median, 55.0 years). Histological diagnoses included serous cystadenocarcinoma (n = 37), endometrioid adenocarcinoma (n = 6), clear cell adenocarcinoma (n = 5), others (n = 4, Additional file 1: Table S2), and unknown (n = 4). Forty-six patients received DCs pulsed with WT1 (WT1 only, n = 7; WT1 + MUC1, n = 31; WT1 + MUC1 + CA125, n = 3; and WT1 + CA125, n = 5), whereas the others received DCs pulsed with MUC1 and/or CA125. All patients in this study had initially received chemotherapy for ROC. Of these, 27 (48%) received platinum-based chemotherapy while the others (52%) received non-platinum-based chemotherapy or received no chemotherapy during DC vaccination.

The AEs were tolerable in all patients. No serious acute allergic reaction such as anaphylaxis was observed. The most common AEs were injection site reaction (68%) and fever (32%). Other common AEs such as arthralgia and elevated liver enzyme levels were not observed. No grade 3–4 toxicity or evidence of autoimmune sequelae was documented.

Fever and erythema after vaccination and frequencies of CD4⁺ T cells, CD8⁺ T cells, and NK cells were examined to assess immunological function. However, no remarkable changes were observed in CD4+ T cell, CD8⁺ T cell, and NK cell frequencies after vaccination (Figure 1A–C). In addition, none of these factors affected the median survival time (MST). Forty-six of the 56 patients received DCs pulsed with WT1 peptide, 17 of whom were evaluable for WT1-specific CTLs. The frequency of WT1-specific CTLs increased in 12 of these examined patients (70.6%; P = 0.04; Figure 2 and Additional file 1: Table S2). However, no significant differences in clinical outcomes were observed between patients with WT1-specific CTL increase and those without such an increase.

Clinical responses were evaluated in 56 patients. The MST from diagnosis was 30.4 months and that from the first vaccination was 14.5 months (Figure 3, left). The 1- and 2-year survival rates from diagnosis were 87% and 65%, respectively. Therapeutic efficacy was evaluated according to RECIST in all 56 patients at 3 months after the first vaccination, and none of the patients showed CR. However, 2 patients (3.6%) achieved PR, 14 (25%) had SD, 32 (57%) had PD, and 8 (14%) were not evaluated. The DCR and ORR were 29% and 3.6%, respectively (Table 2). At the time of the final analysis, 35 patients (63%) had died of cancer. Multivariate analysis revealed that an albumin level of ≥4.0 g/dL and a lactate dehydrogenase (LDH) level of <200 IU/L before vaccination were significantly associated with the MST from the first vaccination (Table 3). The albumin level before vaccination was a significant factor for MST prolongation, as determined by the log-rank test (P = 0.017; HR = 0.46; 95% CI, 0.18–0.85) and multivariate analysis. The MST from the first vaccination in patients with albumin levels of ≥4.0 and <4.0 g/dL were 19.9 and 11.6 months, respectively (Figure 3, Right). The DCR and ORR were 36% and 5.5%, respectively, in the 36 (64%) patients with albumin levels of ≥4.0 g/dL. In contrast, these rates were 15% and 0%, respectively, in patients with albumin levels of <4.0 g/dL (Table 2).

We used an NLR cutoff of 4 to evaluate the MST from the first DC vaccine and found that the NLR was significantly correlated with the MST by using the log-rank test (P = 0.02; HR = 2.16; 95% CI, 1.15–5.97). The MST of patients with an NLR of <4 was significantly longer than that of patients with an NLR of ≥4 (19.9 vs. 9.5 months, Additional file 2). The DCR was 35% in patients with an NLR of <4 compared to 13% in those with an NLR of ≥4 (Additional file 1: Table S1). Neither the use of OK-432 nor the amount of OK-432 administered was significantly associated with survival, indicating that OK-432 itself did not affect the survival of patients with ROC (log-rank test, P = 0.854, 14.5 vs. 19.9 months; Wilcoxon, P = 0.498).