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. 2016 Sep 29;65(12):1499–1509. doi: 10.1007/s00262-016-1905-7

Phase I/II trial of combination of temozolomide chemotherapy and immunotherapy with fusions of dendritic and glioma cells in patients with glioblastoma

Yasuharu Akasaki 1,✉, Tetsuro Kikuchi 2, Sadamu Homma 2, Shigeo Koido 3, Toshifumi Ohkusa 3, Tetsunori Tasaki 4, Kazumi Hayashi 2, Hideo Komita 2, Nobuyuki Watanabe 1, Yuta Suzuki 1, Yohei Yamamoto 1, Ryosuke Mori 1, Takao Arai 1, Toshihide Tanaka 1, Tatsuhiro Joki 1, Takaaki Yanagisawa 1, Yuichi Murayama 1
PMCID: PMC11028634  PMID: 27688162

Abstract

Background

This trial was designed to evaluate the safety and clinical responses to a combination of temozolomide (TMZ) chemotherapy and immunotherapy with fusions of DCs and glioma cells in patients with glioblastoma (GBM).

Method

GBM patients were assigned to two groups: a group of recurrent GBMs after failing TMZ-chemotherapy against the initially diagnosed glioma (Group-R) or a group of newly diagnosed GBMs (Group-N). Autologous cultured glioma cells obtained from surgical specimens were fused with autologous DCs using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region. Toxicity, progression-free survival (PFS), and overall survival (OS) of this trial were evaluated. Expressions of WT-1, gp-100, and MAGE-A3, recognized as chemoresistance-associated peptides (CAP), were confirmed by immunohistochemistry of paraffin-embedded tumor samples. Patient’s PBMCs of pre- and post-vaccination were evaluated by tetramer and ELISPOT assays.

Results

FC-immunotherapy was well tolerated in all patients. Medians of PFS and OS of Group-R (n = 10) were 10.3 and 18.0 months, and those of Group-N (n = 22) were 18.3 and 30.5 months, respectively. Up-regulation and/or cytoplasmic accumulation of CAPs was observed in the recurrent tumors of Group-R patients compared with their initially excised tumors. Specific immune responses against CAPs were observed in the tetramer and ELISPOT assays.

Conclusions

The combination of TMZ-treatment leading to up-regulation and/or cytoplasmic accumulation of CAPs, with FC-immunotherapy as a means of producing specific immunity against CAPs, may safely induce anti-tumor effects in patients with GBM.

Keywords: Glioma, Dendritic cell, Chemoimmunotherapy, Temozolomide, Chemoresistance

Introduction

The current therapeutic standards against malignant glioma, the most common and lethal primary brain tumor in adults, include surgery followed by concomitant radiation and chemotherapy with temozolomide (TMZ). Despite the benefits of adjuvant TMZ-chemotherapy that had been attested in an international randomized trial for patients with glioblastoma multiforme (GBM), the highest malignancy grade of glioma, the median survival time of these patients is still less than 2 years [1]. A novel therapy to improve the prognosis is therefore eagerly awaited for them. In this context, a combination of cytotoxic chemotherapy and immunotherapy, so-called chemoimmunotherapy, has been investigated as a novel paradigm for different types of tumors [2], including malignant glioma [3].

In cancer immunotherapy, effective antigen presentation to T cell subsets can be considered a critical step for generation and maintenance of immune responses against tumor cells. [4]. Among antigen-presenting cells, DCs are the most potent in this specialized function [5]. After exposure to TAA, DCs process and express TAA-derived epitopes in combination with MHC class I and II molecules on their cell surface. They are recognized by both the naïve CD8+ T cells known as precursors of CTL and naïve CD4+ T cells as T helper type 1 (Th1) precursors, respectively [6].

We had shown earlier that immunotherapy against glioma with fusion cells (FC) of DCs and glioma cells induces safe, tumor-specific CTL response in a phase I clinical trial [7]. Therapeutic significance of recombinant IL-12 (rIL-12) supplementation in FC-immunotherapy has also been shown in an animal experiment [8] and a phase II clinical trial [9]. In our current study, we observed that stimulation with a synthetic analog of double-stranded RNA, poly I:C, and small interference RNA (siRNA) of IL-10 (IL-10-siRNA) induced high levels of IL-12p70 secretion from FCs and consequently elicited an efficient tumor-specific Th1 response [10].

Based on these results, we designed a clinical trial of chemoimmunotherapy against GBM using TMZ and IL-10-siRNA/poly I:C co-transfected FCs. In this trial, GBM patients received surgical tumor resection followed by adjuvant chemotherapy with TMZ and FC-immunotherapy. The safety, feasibility, mechanisms, and immunologic and clinical responses of this approach were evaluated.

Patients and methods

Patient selection

Patients were selected using the following inclusion criteria: histologically proven GBM according to the WHO criteria, preoperative Karnofsky performance status (KPS) ≥50 %, 75 years ≥ age ≥ 20 years, no past or current history of autoimmune disease or severe immune deficiency, and no use of glucocorticoid medication. From January 2007 to December 2014, a total of 32 patients, ranging in age from 30 to 74 years (average 54.6 years), were enrolled in this study. These patients were assigned to one of the following two groups: a group of recurrent GBM patients who underwent failing TMZ-chemotherapy against initially diagnosed glioma (Group-R) or a group of patients with newly diagnosed GBM (Group-N). The patient’s characteristics, categorized variables on the outline of FC-immunotherapy and toxicity profiles, and clinical outcome are summarized in Table 1. All patients gave written informed consent in accordance with the Declaration of Helsinki; study approval was obtained from the Institutional Review Board of Jikei University School of Medicine (Trial registration ID: 16-184-4412).

Table 1.

Case summary of Group-R and Group-N

Group R (n=10) N (n=22)
Age (years)
 Mean 50.1 56.7
 Range 35–65 30–74
Male/female (cases) 5/5 13/9
Initial diagnosis (cases)
 GBM 7 N.A.
 AA 2 N.A.
 AO 1 N.A.
KPS (cases)
 30–40 0 0
 50–60 1 5
 70–80 5 8
 90–100 4 9
Times of FC-vaccination (times/patient)
 Mean 4.2 4.3
 Range 3–8 3–9
FC-numbers/one-time inoculation (cases)
 <1 × 106 3 6
 1–2 × 106 5 12
 >2 × 106 2 4
Toxicity (cases)
 IR Grade 1 4 11
 LL Grade 1 5 4
 LL Grade 2 2 1
PFS (months)
 Median 11 17
 Range 7–29 8–48<
OS (months)
 Median 17 30
 Range 9–38 11–48<
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GBM glioblastoma, AA anaplastic astrocytoma, AO anaplastic oligodendroglioma, IR injection site reaction, LL leukopenia/lymphopenia

Generation of primary cultured glioma cells from surgical specimens

Single-cell suspensions of glioma cells were obtained by enzymatic digestion using a gentle MACS dissociator and brain tumor dissociation kit (Miltenyi Biotec, Auburn, CA), respectively. Briefly, each glioma specimen was collected from surgery and handled under sterile conditions. Necrotic tissue, clotted blood, and apparently normal tissues were removed, and the remaining specimen was minced into small pieces using surgical blades. The chopped tissue was dissociated into single-cell suspensions using a gentle MACS dissociator according to the manufacturer’s protocol. The resulting mixtures were re-suspended in DMEM F12 (Gibco, Gaithersburg, MD) containing 10 % heat-inactivated autologous serum and were cultured at 37 °C in 5 % CO2.

Generation of DC

DCs were generated from peripheral blood as described previously [10]. Briefly, PBMC were separated from patient’s peripheral blood by an apheresis collection method using a COBE Spectra (Terumo BCT, Lakewood, CO) or Ficoll–Hypaque density centrifugation. PBMCs were suspended in AIM-V medium (Gibco) and allowed to adhere to 100-mm petri dishes at 37 °C for 2 h. The non-adherent cells were removed. The adherent cells were subsequently cultured in AIM-V with 1 % heat-inactivated autologous serum, GM-CSF (20 ng/ml), and IL-4 (20 ng/ml) (PeproTech, Rocky Hill, NJ). TNF-α (20 ng/ml) (PeproTech) was added on day 5. Thereafter, the semi-adherent and non-adherent cells were harvested on day 7 and used as DCs for fusion.

Preparation of fusion cells

DCs, ranging in numbers from 2 × 106 to 1 × 107, were mixed with lethally irradiated (300 Gy, Hitachi MBR-1520R-3, dose rate:1.1 Gy/min) glioma cells, ranging in numbers from 1 × 106 to 2 × 106. The ratio of DCs and glioma cells ranged from 2:1 to 10:1 depending on the numbers of acquired DCs and glioma cells. Fusion was started by adding 500 μl of 50 % solution of polyethylene glycol (PEG; Sigma-Aldrich, St. Louis, MO), drop by drop, for 60 s, and then was stopped by stepwise addition of serum-free RPMI medium. After washing three times with phosphate-buffered saline (PBS; Cosmo Bio), the fusion cells (FC) were plated onto 100-mm petri dishes in the presence of GM-CSF, IL-4, and TNF-α in AIM-V with 1 % heat-inactivated autologous serum. Poly I:C (Sigma-Aldrich) and ON-TARGETplus SMARTpool siRNA of IL-10 (IL-10-siRNA, Dharmacon, Lafayette, CO) were transfected into the FCs using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) as described previously [10]. FCs were cultured with the RNA-Lipofectamine complexes for 24 h.

Fusion efficiency was assessed as follows. Glioma cells were dyed green with PKH-67 (Sigma-Aldrich) before cell fusion. After the procedure for making FCs, DCs were stained with mouse mAb of PE-conjugated anti-human CD11c (BioSource International) and analyzed using a flow cytometer (Miltenyi Biotec). Double-positive cells were determined to be FCs. The fusion efficiency was represented by the percentage of double-positive cells within all CD11c positive cells.

Therapeutic schedule

The therapeutic schedules after the surgery for Group-R and Group-N are shown in Fig. 1. Briefly, the patients in Group-N had surgery followed by concomitant radiation (2 Gy/day × 30 days) and TMZ-chemotherapy (75 mg/m2/day × 42 days). Maintenance chemotherapy with TMZ at 150–200 mg/m2/day for 5 days in each 28-day cycle was repeated for patients in both groups for the period when their general condition permitted. FCs suspended in 0.5 mL normal saline were inoculated intradermally in the cervical region. FC-inoculation was applied 2 weeks after the first maintenance TMZ-chemotherapy in both group and was repeated at least three times in each 28-day cycle. In the absence of progressive disease or major organ toxicity after the third one, FC-inoculation was repeated every 6–12 months. Patients were monitored for immediate and delayed toxicities and the inoculation sites were examined at 48 h. All toxicity was graded using the National Cancer Institute Common Toxicity Criteria. The response to the treatment was evaluated by clinical observations and MRI was performed every 2 months.

Fig. 1.

Fig. 1

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Therapeutic schedules after surgery for Group-R and Group-N. Concomitant radiation (arrow) and TMZ-chemotherapy (long open arrow) for 42 days were performed for the patients in Group-N. Maintenance TMZ-chemotherapy (short open arrow) in each 28-day cycle was repeated for the patients in both groups. FC-inoculation (arrow head) was applied 2 weeks after the first maintenance TMZ-chemotherapy in both group and was repeated at least three times in each 28-day cycle. After the third one, FC-inoculation was repeated every 6–12 months. # represents a start of therapy for Group-N. ## represents a start of therapy for Group-R

Tetramer assay

Tetramer assay was performed as described previously [11]. Briefly, PBMCs of HLA-A24 positive patients were incubated in 12-well plates (2 × 106 cells/well) in RPMI 1640 medium containing 10 % fetal calf serum (FCS), 55 μM mercaptoethanol, 0.1 mM MEM non-essential amino acids, and 1 mM sodium pyruvate. Cell lysate of autologous glioma cells (1–2 × 106/well), rIL-2 (10 U/ml, Shionogi & Co. Ltd, Osaka, Japan) and rIL-7 (10 ng/ml, PeproTech) were added to the culture. The cells were cultured for 6 days in these conditions, and then medium was changed to RPMI 1640 containing 10 %FCS and rIL-2 (10 U/ml). After 3-day incubation, the cells were stimulated overnight with the lysate of autologous glioma cells. The samples were analyzed by T-Select MHC Tetramer assay kit for WT-1, gp-100 and MAGE-A3 (Medical & Biological Laboratories Co. Ltd, Nagoya, Japan) according to the manufacturers’ instruction.

ELISPOT assay

PBMCs were incubated in 12-well plates (2 × 106 cells/well) in RPMI 1640 medium containing 10 % FCS, 55 μM mercaptoethanol, 0.1 mM MEM nonessential amino acids, and 1 mM sodium pyruvate. Irradiated autologous glioma cells (1 × 104 cells/well) and rIL-2 (10 U/ml) were added to the culture. Some PBMCs were cultured with only rIL-2 (10 U/ml) as a control. After 5-day incubation, CD4+ lymphocytes were isolated from the PBMC samples with a MiniMACS cell separation unit (Miltenyi Biotec) according to the manufacturer’s protocol. An ELISPOT assay was done by using a human INF-γ and IL-10 ELISPOT kit (MabTech, Cincinnati, OH) on PVDF-bottomed 96-well plate (MabTech). Samples (1 × 105 cells/well) were plated onto an IL-10 or INF-γ capture antibody-coated well and cultured for 48 h. All samples were run in triplicate. The spot-forming counts of controls were subtracted from the stimulated cells’ for evaluation of the tumor-specific CD4+ T cell response.

Light microscopic immunohistochemical analysis

Formalin-fixed, paraffin-embedded tissue sections were obtained from the tumor samples and were immunostained with ab of anti-WT-1 (C-19; Santa Cruz Biotechnology, Dallas, TX), gp-100 (ab27435; Abcam, Cambridge, UK), and MAGE-A3 (ab115332; Abcam) using an avidin–biotin immunoperoxidase technique. Percentages of nuclei-staining or cytoplasm-staining cells were individually calculated for patients in Group-R.

Statistical analysis

Overall survival (OS), the primary endpoint of this study, was defined as the time from the day of surgical tumor resection until the date of death due to any cause. For patients who were alive beyond October 1, 2015, times to death were censored on September 30, 2015. Progression-free survival (PFS), the secondary endpoint of this study, was defined as the time from the day of surgical tumor resection until the first documented progression in MRI or death due to any cause whichever is earlier. For patients who were alive without progression beyond October 1, 2015, times to progression were censored on September 30, 2015. The Kaplan–Meier method was used to estimate rates of OS and PFS as well as median, first and third quartiles. The standard errors of the estimated rates were calculated using the Greenwood formula. The 95 % confidence intervals (95 % CI) of median estimates on PFS and OS were computed by the nonparametric method of Brookmeyer and Crowley. The statistical analysis was performed using SAS 9.4 (SAS Institute Inc., Cary, NC).

Results

Characterization of FCs

To analyze fusion efficiency, glioma cells were stained with PKH-67 and were fused with autologous DCs in a ratio of DC: glioma = 2:1, 5:1, or 10:1. These cells were stained with PE-conjugated anti-CD11c and analyzed using a flow cytometer (Fig. 2a, c). The fusion efficiency was represented by the percentage of double-positive cells (upper right in Fig. 2a, c, i.e., FCs) within all CD11c-positive cells (sum of upper left and upper right in Fig. 2a, c, i.e., DCs). The fusion efficacies in ratios of DC: glioma = 2:1, 5:1, and 10:1 were 61.6 % (Fig. 2a), 16.0 % (Fig. 2b) and 7.2 % (Fig. 2c), respectively. Since DCs, ranging in numbers from 2 × 106 to 1 × 107, and glioma cells, ranging in numbers from 1 × 106 to 2 × 106, were used for a procedure of cell fusion, the numbers of acquired FCs were approximated to a range from 7.2 × 105 to 2.5 × 106. Based on these results, the total numbers of inoculated FCs in each patient were approximated to a range from 2.2 × 106 to 2.0 × 107.

Fig. 2.

Fig. 2

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Analysis of fusion efficiency by flow cytometer. Glioma cells were fused with autologous DCs using PEG in a ratio of DC: glioma = a 2:1, b 5:1 or c 10:1. Glioma cells and DCs were stained with PKH-67 (vertical axis) and CD 11c-PE (horizontal axis), respectively. Double-positive cells were determined to be fusion cells. The numbers show the percentage of cells

Background and clinical response of the patients

Ten patients were enrolled in Group-R. The pathological diagnoses of their initial tumors were seven cases of GBM, two cases of anaplastic astrocytoma (AA), and one case of anaplastic oligodendroglioma (AO). The rates of PFS and OS counted from the day of surgical treatment against the recurrent tumor were evaluated by using the Kaplan–Meier method (Fig. 3a, b). The medians, first quartiles, third quartiles, and lower limit on 95 % CI (95 % CIlow) of medians on PFS and OS were calculated. Briefly, medians of PFS and OS were 10.3 and 18.0 months, respectively. The results of the 95 % CIlow indicated that the medians of PFS and OS in Group-R can be expected to be more than 8.2 and 12.6 months, respectively.

Fig. 3.

Fig. 3

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Evaluation of clinical response and toxicity. (a–d) The Kaplan–Meier curves of a PFS and b OS on Group-R (n = 10), c PFS and d OS on Group-N (n = 22). Medians, first quartiles, third quartiles, and lower limit on 95 % CI (95 % CIlow) of medians were noted. e–h The Kaplan–Meier curves stratified for numbers of inoculated FCs of e PFS and f OS on Group-R, g PFS and h OS on Group-N. Medians of each stratum were noted. i A scatter plot showing the numbers of inoculated FCs on each adverse event. IR-G1 represents an event of injection site reaction grade 1. LL-G1 and LL-G2 represent events of leukopenia/lymphopenia grade 1 and grade 2, respectively

Twenty-two patients were enrolled in Group-N. The rates of PFS and OS counted from the day of surgical treatment against the tumor were evaluated by using the Kaplan–Meier method (Fig. 3c, d). The medians, first quartiles, third quartiles, and 95 % CIlow of medians were calculated. Briefly, medians of PFS and OS were 18.3 and 30.5 months, respectively. The results of 95 % CIlow indicated that the medians of PFS and OS in Group-N can be expected to be more than 13.7 and 25.3 months, respectively.

In order to determine whether numbers of inoculated FCs correlate with the clinical outcome of patients, the rates of PFS and OS were stratified for numbers of FCs in the one-time inoculations and were evaluated by using the Kaplan–Meier method for each group. As shown in Fig. 3e, h, there was not any specific correlation between clinical outcome and numbers of FCs in one-time inoculation within a range from 7.2 × 105 to 2.5 × 106.

Toxicity of FC-immunotherapy

FC-immunotherapy was well tolerated in all patients. We observed no serious adverse effects, clinical signs of autoimmune reaction, or substantial changes in the results of routine blood tests. In 15 cases, transient grade 1 toxicity of injection site reaction (IR), such as erythema and/or induration, was observed after the second or the third inoculation. Transient grades 1 and 2 leukopenia/lymphopenia (LL) occurred in nine and three patients, respectively. This toxicity was considered to be due to the TMZ-chemotherapy, because it improved when the TMZ-chemotherapy was delayed for a few weeks. There were no patients with discontinued treatment due to adverse effects. Furthermore, in order to determine whether the incidence of adverse events correlates with the numbers of inoculated FCs, FC-numbers of one-time inoculation on each adverse event were plotted in a scatter plot (Fig. 3i). In this evaluation, we could not find any specific correlation between the adverse events and numbers of inoculated FCs in each group.

Expressions of WT-1, gp-100, and MAGE-A3 in glioma cells

In order to determine whether glioma cells express WT-1, gp-100, or MAGE-A3, paraffin-embedded tumor sections were obtained from the samples of all patients in Group-N. The expression levels of them were confirmed by immunohistochemical stains. Although the tumor samples of all patients in Group-N expressed at least one or more of these TAAs (not shown), there was not any specific correlation between their expression levels and clinical outcome. Furthermore, in order to determine whether TMZ-treatment alters these TAA expressions, paraffin-embedded samples obtained from initially excised and recurrent tumors of patients in Group-R were analyzed by immunohistochemical examination. Although Group-R had ten patients, we obtained the data from three patients among them. The reasons of lacking in the results of other seven patients were as follows: (1) Five patients received their first surgery at other institutions, and (2) two patients had poor quality of samples at initial excision, unsuitable for immunohistochemical evaluation. In this analysis, the glioma cells in both initially excised and recurrent tumor samples stained positive with WT-1 (Fig. 4a, d), gp-100 (Fig. 4b, e), and MAGE-A3 (Fig. 4c, f). The stain levels of these antigens were up-regulated in recurrent tumor samples (Fig. 4d, e, f), compared with initially excised tumor (Fig. 4a–c). Interestingly, these TAAs were predominantly localized to the nuclei in initially excised tumors (arrows in Fig. 4a–c) and were strongly accumulated in the cytoplasm in recurrent lesions (blue arrow heads in Fig. 4d, e, f). Percentages of cytoplasm-staining (including both nuclei- and cytoplasm-staining) cells with WT-1 (Fig. 4g), gp-100 (Fig. 4h), and MAGE-A3 (Fig. 4i) in the recurrent tumors (rec.) were increased compared with those the initially excised (init.). These data indicate that TMZ-chemotherapy may induce up-regulation and/or cytoplasmic accumulation of WT-1, gp-100, and MAGE-A3 in glioma cells.

Fig. 4.

Fig. 4

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Immunohistochemical analyses on tumor samples of patients in Group-R. a–f Immunohistochemical features on a WT-1, b gp-100, and c MAGE-A3 stains of an initially excised tumor (init.), and d WT-1, e gp-100, and f MAGE-A3 stains of a recurrent tumor (rec.). Arrows were pointing nuclei-staining cells. Blue arrowheads were pointing cytoplasm-staining cells. Scale bar represents 50 μm. g–i Percentages of cytoplasm-staining (including both nuclei- and cytoplasm-staining) cells with g WT-1, h gp-100, and i MAGE-A3 in the initially excised tumor sections (init.) and recurrent tumor sections (rec.) of three patients in Group-R were show graphically

T cell immune response induced by FC-immunotherapy

We also determined whether FC-immunotherapy induces TAA-specific CTL response. To confirm this ability of FCs in vivo, PBMCs of HLA-A24-positive patients at prevaccination and 4 weeks after each FC-inoculation were subjected to tetramer assay of WT-1, gp-100, and MAGE-A3. Although Group-R and -N had five and ten of HLA-A24 positive patients respectively, we could obtain sufficient amounts of PBMCs for this assay from two patients each in Groups-R (Case 1 and 2) and Group-N (Case 3 and 4). As shown in Fig. 5a–c, antigen-specific CTL responses against WT-1 (Fig. 5a), gp-100 (Fig. 5b), and MAGE-A3 (Fig. 5c) were acquired in all of four patients after the first or second FC-inoculation. Since all of them had relatively prolonged OS (case 1:17.8 months, case 2:21.2 months, case 3: more than 36 months, case 4: more than 48 months), the CTL responses against these TAAs might be correlating with good clinical outcome.

Fig. 5.

Fig. 5

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T cell response assays using patients’ PBMCs on pre- and post-vaccine. Tetramer assay of a WT-1, b gp-100, and c MAGE-A3. Cases 1 and 2 were patients in Group-R. Cases 3 and 4 were patients in Group-N. The highest value of post-vaccination and the value of prevaccination in each patient were summarized by descriptive statistics, respectively. d, e ELISPOT assay of IFN-γ and IL-10. (d; Case 5) a patient in Group-R. (e; Case 6) a patient in Group-N’s patient

Following these observations, we sought to determine whether FC-immunotherapy induced tumor-specific CD4+ T cell immune response. To confirm this ability of FCs in vivo, PBMC samples from one patient each in Groups-R (case 5; Fig. 5d) and—N (case 6; Fig. 5e) at prevaccination and 4 weeks after each FC-inoculation were obtained. These PBMCs were co-cultured with irradiated autologous glioma cells and rIL-2 in order to stimulate tumor-specific response. Some PBMCs were stimulated with only rIL-2 as a control. After 5-day incubation, CD4+ lymphocytes isolated from the PBMCs in the samples were subjected to an ELISPOT assay for INF-γ and IL-10. In order to evaluate the tumor-specific response, the spot-forming counts of controls were subtracted from those of stimulated cells. As shown in Fig. 5d, e, FC-immunotherapy generated both phenotypes of IFN-γ and IL-10 secretory CD4+ T cell. In this response, IFN-γ secretory phenotype, i.e., Th1, predominated IL-10 secretory phenotype, or regulatory T cell, in both patients. These data indicate that FC-immunotherapy is capable to induce tumoricidal Th1 immune responses in glioma patients.

Discussion

It is widely accepted that defects in the process of endogenous antigen presentation underlie the depressed cellular immune response in patients with malignant glioma [12]. The strategies to eliciting efficient uptake of TAA by DCs are of special interest for developing effective immunotherapy against glioma. One such strategy for glioma immunotherapy is the fusion technique using DC and glioma cells [7–9]. Since FCs are designed to soak up the whole spectrum of TAAs contained by autologous glioma cells, the benefits of FC-immunotherapy include FCs’ capability to process and express several TAAs in combination with MHC molecules on their cell surface. Phuphanich et al. [13] demonstrated in their clinical trial of glioma immunotherapy that glioma cells of GBM patients were containing 3–6 types of TAAs. In fact, we confirmed that FC-immunotherapy induces antigen-specific CTL responses against at least three different types of glioma antigens: WT-1, gp-100, and MAGE-A3. Another benefit in FC-immunotherapy is that there is no limitation on patients` HLA typing for receiving immunotherapy. The induction of immune response against unidentified TAAs, such as mutated TAAs, may also be one of the benefits in FC-immunotherapy, although the existence of this additional advantage was not investigated in our study.

In a previous animal experiment and phase II clinical trial of FC-immunotherapy, we demonstrated that systemic administration of rIL-12 was required for induction of effective antitumor immunity [8, 9]. Production of heterodimeric IL-12p70 by human DC is a critical step for defining the stability of DC vaccines [14]. The IL-12p70 plays the role of a bridge between innate resistance and adaptive immunity as a potent inducer of Th1 responses [15]. However, the severe side effects associated with systemic administration of rIL-12 in clinical investigations and the very narrow therapeutic index of this cytokine markedly tempered enthusiasm for its application in cancer patients [16]. It has recently been reported that TAA-containing DCs with administration of poly I:C, synthesized as a Toll-like receptor (TLR)-3 agonist, secrete high levels of IL-12p70 and induce antigen-specific CTL and tumoricidal Th1 response in vitro [10] and in vivo [17]. These findings support the use of fusion technique with TLR stimulation in DC-based immunotherapy that promotes MHC-epitope complex presentation directing not only TAA-specific CTL, but also Th1 responses. Although the requirement of culturing autologous glioma cells is one of the disadvantages of FC-immunotherapy, this cumbersome technique may be indispensable for inducing effective immune responses against malignant gliomas.

Based on the data with 95 % CI, the median of PFS and OS in Group-R is expected to be more than 8.2 and 12.6 months, respectively. Since the patients in Group-R had acquired resistance to TMZ before enrolling this study, FC-immunotherapy seemed being capable of improving the chemotherapeutic effect of TMZ. Furthermore, the data with 95 % CI in Group-N indicates that the medians of PFS and OS of this group are expected to be more than 13.7 and 25.3 months, respectively. These data surpass in the medians of PFS (6.9 months) and OS (14.6 months) of the international randomized trial of adjuvant TMZ-chemotherapy [1]. Although a non-randomized study has only the ability to suggest clinical effects at a limited evidence level, we propose that FC-immunotherapy may have a capability to enhance the TMZ-based standard adjuvant therapy for patients with GBM. Regarding the relevance of immune response to the chemosensitivity on tumor cells, Liu et al. reported that tyrosinase-related protein 2 (TRP-2) up-regulated glioma cells to acquire TMZ-resistance, and down-regulation of TRP-2 by the specific CTL reaction restored therapeutic sensitivity [18]. Based on this report, we hypothesized that the augmented effect of TMZ in a combination with FC-immunotherapy may correlate with the biological function of TAAs. It is now recognized that up-regulation of several TAAs such as WT-1 [19], gp-100 [20], and MAGE-A3 [21] induces chemoresistance in tumor cells, indicating that these are recognized as chemoresistance-associated peptides (CAPs) within the glioma-associated antigen group. Based on this recognition, we evaluated the induction of antigen-specific CTL responses against these TAAs in the patients who underwent FC-immunotherapy. Given the already described ability of tumor cells to escape from chemotherapy-induced apoptosis by means of up-regulation of CAPs, the specific T cell immunity against CAPs may augment the therapeutic effect of cytotoxic chemotherapy.

On the other hand, the alteration of TAA-expression pattern between initial and recurrent gliomas observed in the tumor samples of Group-R patients seems to have a critical relevance to the synergistic effect in chemoimmunotherapy. Up-regulation and/or cytoplasmic accumulation of WT-1, gp-100, and MAGE-A3 was observed in the recurrent gliomas, suggesting that these alterations probably correlated with TMZ-treatment. Since adaptation of the proteasome–endoplasmic reticulum pathway for MHC–epitope expression enables recognition of endogenous TAAs by CTLs [22], accumulation of TAA in the cytoplasm may accelerate the expression process of the MHC–epitope complex. In this regard, Benteyn et al. demonstrated that WT-1 mRNA designed to enhance cytoplasmic expression improves the ability to stimulate WT1-specific T cell immunity [23]. Furthermore, Takahara et al. demonstrated that gemcitabine induces cytoplasmic accumulation of WT-1 protein in human pancreatic cancer cells and sensitizes the cells to WT-1-specific T cell immune response [24]. Thus, TMZ-treatment may induce cytoplasmic accumulation of TAAs, resulting in the augmentation of MHC–epitope complex expression on target cells, and then may achieve susceptibility of TAA-expressing glioma cells to the specific immune response in FC-immunotherapy.

Since TMZ has relatively low hematologic toxicity and thus seems to be preferable therapeutic agent in the combination with glioma immunotherapy, we propose that FC-immunotherapy should be combined with TMZ-chemotherapy in a standard adjuvant therapy for patients with malignant glioma as a means of producing TAA-specific immune responses that may augment the therapeutic effect of TMZ. Despite the confirmed ability of FCs to induce tumoricidal immune responses, most patients in this study had tumor progressions within 4 years on their clinical courses. In this regard, it was recently recognized that the blockade of immune checkpoints using inhibitors of PD1 or its ligand (PDL1) may enhance antitumor immunity with the potential to produce durable clinical responses [25]. In the study of glioma immunotherapy, therefore, researching the correlation between PD1/PDL1 interaction and clinical responses may be the next area of major interest.

Acknowledgments

The authors thank Dr. Masako Nishikawa for her advice on statistical analyses, Dr. Kostadin L. Karagiozov for editing the manuscript, and Ms. Yukiko Kobayashi and Ms. Akiko Kuhara for their technical assistance.

Abbreviations

AA

Anaplastic astrocytoma

AO

Anaplastic oligodendroglioma

CAP

Chemoresistance-associated peptide

CI

Confidence intervals

FC

Fusion cell

GBM

Glioblastoma multiforme

gp-100

Glycoprotein-100

IR

Injection site reaction

KPS

Karnofsky performance status

LL

Leukopenia/lymphopenia

MAGE

Melanoma-associated antigen gene

PFS

Progression-free survival

siRNA

Small interference RNA

TMZ

Temozolomide

TRP

Tyrosinase-related protein

WT-1

Wilms’ Tumor 1

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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