The Induction of Immunogenic Cell Death (ICD) During Maintenance Chemotherapy and Subsequent Multimodal Immunotherapy for Glioblastoma (GBM)

Research Article

Austin Oncol Case Rep. 2018; 3(1): 1010.

The Induction of Immunogenic Cell Death (ICD) During Maintenance Chemotherapy and Subsequent Multimodal Immunotherapy for Glioblastoma (GBM)

Van Gool SW¹*, Makalowski J¹, Feyen O², Prix L³, Schirrmacher V¹ and Stuecker W¹

¹Immuno-Oncological Center Cologne, Hohenstaufenring 30-32, 50674 Cologne, Germany

²Zyagnum, Reißstrasse 1, 64319 Pfungstadt, Germany

³Biofocus, Berghäuser Strasse 295, 45659 Recklinghausen, Germany

*Corresponding author: Van Gool SW, Translational Oncology, IOZK, Hohenstaufenring 30-32, 50674 Köln, Germany

Received: June 01, 2018; Accepted: July 07, 2018; Published: July 18, 2018


The prognosis of Glioblastoma multiforme remains poor. Immunotherapy improved survival in a small fraction of patients. We studied the efficiency of multimodal immunotherapy as part of first line treatment for patients with GBM. Immunogenic Cell Death (ICD) was induced with Newcastle Disease Virus (NDV) and Modulated Electrohyperthermia (mEHT), and Dendritic Cell (DC) vaccinations loaded with autologous tumor proteins were performed. In a retrospective analysis of 60 adults, we detected 15 adults in whom NDV/mEHT were added at days 8/9/10 during Temozolomide Maintenance (TMZm) cycles, multimodal immunotherapy with NDV/mEHT/DC vaccinations were administered after TMZm, and further 3-day NDV/mEHT maintenance immunotherapy treatments were given thereafter. Median age was 60 years. Median Karnofsky was 90. There was no added toxicity due to immunotherapy. Median progression-free survival was 13 months (m). With a median follow up of 17m (ranging 4-30m), median overall survival was not reached, and estimated overall survival at 30m was 58% (95%CI: +27, -42). The detection of Apo10 protein epitope (Apo10) and Transketolase-like 1 (TKTL1) in monocytes, the mRNA expression level for PDL1 on circulating tumor cells, and the Th1/Th2 balance in CD4+ T cells showed a dynamic interaction between tumor cells and immune reactivity. The data suggest that the additional induction of ICD via NDV/ mEHT during TMZm is beneficial in improving overall survival. While TMZm only targets dividing tumor cells, ICD targets dividing and non-dividing tumor cells. DC vaccination induces an antitumoral and anti-viral immune response which is maintained by the 3-day NDV/mEHT maintenance immunotherapy treatments.

Keywords: Newcastle disease virus; Modulated electrohyperthermia; Glioblastoma; Immunogenic cell death; chemotherapy


Apo10: Apo 10 protein epitope; DC: Dendritic Cells; EDIM: Epitope Detection in Monocytes; E:T: Effector:Target;GBM: Glioblastoma Multiforme; ICD: Immunogenic Cell Death; IOZK: Immun-Onkologisches Zentrum Köln; mEHT: modulated Electrohyperthermia; NDV: Newcastle Disease Virus; NK: Natural Killer; OS: Overall Survival; PFS: Progression-Free Survival; POH: Perillyl Alcohol; TEVs: Serum-Derived Tumor Extracellular Vesicles; Th: T helper; TKTL1: Transketolase-Like 1; TMZm: Temozolomide maintenance


Diffuse astrocytic tumors are brain tumors occurring in adults and children [1]. The grade IV tumor, called Glioblastoma Multiforme (GBM) is the most frequent brain tumor in adults with an incidence of 3 to 4 per 100000 adults per year [2]. In spite of standard multimodal treatment, consisting of neurosurgery, radiotherapy and chemotherapy, the prognosis is poor with a median Overall Survival (OS) of only 15 months [3]. At time of relapse the Progression-Free Survival (PFS) is 6 months, and the median PFS and OS have not improved over the last decade [4]. In spite of being an orphan disease, GBMs cause the highest number of years of life lost due to cancer [5,6].

Amongst other innovative approaches like anti-angiogenesis and targeted therapy, immunotherapy has been developed as an innovative approach to control GBM [7]. Active specific immunotherapy is based on the injection of autologous mature dendritic cells loaded with tumor antigens derived from different sources. Numerous clinical studies and reviews have been published on the role of immunotherapy for patients with GBM [8,9]. All point to feasibility of the technology without major side effects. Recently a large phase III clinical trial integrating DC vaccination during first line treatment, or in cross-over at time of disease progression, demonstrated improved long-term overall survival [10]. Moreover, meta-analyses pointed out the significant effect of active specific immunotherapy on OS compared to intra-institutional historical control patients [11,12].

Immunotherapy based on immunomodulation with checkpoint blockers like anti-CTLA-4 or anti-PD1 monoclonal antibodies is focus of current clinical research to treat GBM, but did not lead to a break-through like in other tumors [13-15], except for hypermutant GBM [16], presumably because of lack of activated antitumoral immune cells. More recent immunotherapeutic approaches consist of combinations of several treatment modalities of which the antitumoral activities ultimately merges at the effector arm of the immune system.

In this regard, the combination of oncolytic virus therapy and immunotherapy is a promising strategy [17,18]. Virally infected tumor cells can be recognized by NK cells, macrophages, neutrophils and virus-specific T cells. Furthermore, ICD-induced dying tumor cells can lead to an efflux of tumor antigens and damage-associated molecular pattern molecules, which can be taken up by immature dendritic cells for presentation to the T cells in the draining lymph nodes. Similar to virus-mediated ICD of tumor cells, moderate hyperthermia can contribute as immunogenic treatment modality to strengthen antitumoral immune reactivity [19]. The need for rational combinations of immunotherapeutic modalities that work at multiple levels in the cancer immunity cycle in CNS malignancies has recently been reviewed [20]. The combination of Newcastle Disease Virus (NDV), Modulated Electrohyperthermia (mEHT) and DC vaccination has been published as an innovative immunotherapy concept [21].

A further challenge is the integration of the multimodal immunotherapy in the standard antitumoral treatment strategies like surgery, radiochemotherapy and Maintenance Temozolomide (TMZm) chemotherapy. Observations in small cohorts of GBM patients treated with multimodal immunotherapy integrated in the standard Stupp-based treatment might be of help for the scientific community to design proper clinical trials in future.

Patients and Methods


A retrospective analysis of 133 treated GBM patients was performed at the Immun-Onkologisches Zentrum Köln (IOZK). All patients were treated on an individualized basis outside clinical trial, upon patient request and after extensive explanation of the treatment and signed informed consent. Seventeen patients were IDH mutated or had prior low grade glioma medical history. One patient was classified as Diffuse Midline Glioma. 115 patients were left and classified as primary GBM, 63 of them being treated with multimodal immunotherapy together with standard therapy at primary diagnosis. Fifteen adults were detected in whom three days of NDV/mEHT were associated to TMZm courses. At first contact and during therapy, patient’s blood was investigated for immunologic parameters including PanTum detect Epitope Detection in Monocytes (EDIM) tests [22,23]via Biovis,


During the 28-day TMZm cycles, mEHT sessions and NDV injections were scheduled at days 8, 9 and 10. The mEHT was administered with the Oncothermia EHY‑2000 device (Oncotherm GmbH, Troisdorf, Germany) for 50 min at increasing intensity from 40 to 80 Watt. During mEHT, 250 ml NaCl 0.9% infusion supplemented with 7.5g Vitamin C, 40 mg MgCl2, 45 mg CaCl2, 15 mg KCl, 10 ml Magnesiocard containing 737.6 mg Magnesiumaspartahydrochlorid 3H2O with 72.9 mg Mg (Verla-Pharm Arzneimittel GmbH & Co. KG, Tutzing, Germany), and 5 ml Nervoregin comp. H containing 0.1 ml Agaricus (HAB 34) Dil. D 6 (HAB, V. 3a), 0.35 ml Asa foetida Dil. D 5, 2.0 ml Strychnos ignatii Dil. D 6, 0.1 ml Valeriana officinalis Dil. D 3 and 0.65 ml Zincum isovalerianicum Dil. D 8 (Pflüger, Rheda, Germany) was administered, followed by 100 ml NaCl 0.9% infusion, and finally 100 ml NaCl 0.9% infusion with 20 ml Selenase T containing Natriumselenit-Pentahydrat 50 μg/ml Selen (Biosyn GmbH, Fellbach, Germany). At the end of the mEHT session, mesogenic oncolytic MTH-68 strain Newcastle Disease Virus (NDV) was injected at a dose of 10 x 107 infectious particles. Short infusions of NDV with in 100 ml NaCl 0.9% over 20 minutes were switched towards bolus injections of NDV with in 2 ml NaCl 0.9% since September 2017.

After finishing the TMZm cycles, full vaccination cycles were administered with three weeks interval. Each full vaccination cycle consisted of NDV and mEHT administrations at days 1 to 5 and at day 8. An intradermal injection of autologous mature Dendritic Cells (DCs) loaded with autologous tumor antigens was administrated at day 8. Immature DCs were differentiated ex vivo out of adherent peripheral blood monocytes in the presence of 800 U/ml IL-4 and 1000 U/ml GM-CSF. DCs were loaded at day 5 with autologous tumor antigens, obtained via tumor lysate [24,25]or obtained from serum after induction of tumor-derived antigenic extracellular microvesicles [26,27], induced via ICD by mEHT and NDV [19]. DC maturation was induced with NDV (105 infectious particles per 106 DCs) and the cytokine cocktail 1000 U/ml IL-6, 1100 U/ml TNF-a and 1900 U/ml IL-1b. GMP-approved culture medium and cytokines were purchased from Cellgenix (Freiburg, Germany). The vaccine product is an approved medicinal product by the German authorities (DE-NW-04-MIA-2015-0033).

After the vaccination cycles, further maintenance immunotherapy was provided consisting of 3 days NDV/mEHT at intervals of about 6 weeks.

In some patients, immunomodulatory strategies were added. The anti-PD1 mAb Pembrolizumab (Keytruda®, MSD) was infused at 2 mg/kg each 3 weeks according to the instructions of the manufacturer. ATRA (all-trans-retinoic-acid) was used with the aim to deplete myeloid-derived suppressor cells, and was administered for three days at 150 mg/m²/day in three doses with at least 6 hours interval, as published [28].

Progression-Free Survival (PFS) was defined when treatment switch was needed. In case of doubt for pseudoprogression, PFS was eventually retro-actively defined after having the results of the subsequent MRI. All patients were followed further to define the overall survival.


Circulating Tumor Cell (CTC) analysis was performed via Biofocus ( Heparinized blood samples of patients were processed as described in detail previously [29]. In brief, CTCs from 30 ml blood were enriched by filtration cytometry [30] using 20 μm polyester filter meshes (Reichelt Chemietechnik, Heidelberg, Germany). RNA was extracted from cells retained on filter meshes with Trizol reagent. For proof of CTCs in these cell preparations, qRT-PCR for relative mRNA expression of a set of four genes (telomerase, ERBB2, c-KIT, EGFR) was performed. Assays were purchased (telomerase: Hs00972649, Applied Biosystems) or designed in-house [29]taking care that fluorescence probes are spanning exon-boundaries. Expression values in the enriched CTC preparation was normalized to the house-keeping gene GAPDH and compared to GAPDH-normalized expression values in mononuclear cells of the patients. Relative expression ratios of >2.0 (telomerase, c-KIT, ERBB2) or >1.0 (EGFR) in enriched CTC preparations were considered overexpressed and CTC-positive. In CTC-positive samples, relative mRNA expression of PD-L1 was subsequently determined by qRT-PCR in a similar manner.

PanTum detect tests were performed at presentation and during treatment. The tests were originally designed as a biologic biopsy (as a special form of liquid biopsy) test exploiting the innate immune system and its interaction with cancer, for early detection of cancerrelated biomarkers like the DNase/Apo10 protein epitope as maker of tumor cells with abnormal apoptosis and proliferation, and the Transketolase-like 1 (TKTL1) epitope as a biomarker for anaerobic glucose metabolism (Warburg effect). More in detail, both biomarkers have been detected intracellularly in monocytes, allowing a sensitive and specific noninvasive detection of cancer patients by blood samples (“biologic biopsy” as a special form of liquid biopsy). This blood test is based on the EDIM technology, which utilizes the fact that activated monocytes phagocytize and present tumor-related material even in the presence of low tumor mass. Those activated monocytes, which contain intracellular tumor epitopes, can be detected by CD14 and CD16 specific antibodies using flow cytometry [22,23,31,32].


Status at start of immunotherapy

GMB patients came to the IOZK with the request to add-on immunotherapeutic strategies during maintenance chemotherapy after neurosurgery and radiochemotherapy, in median 3.38 months after operation (range 1.11-8.72m). Patient characteristics are described in (Table 1). All patients had a Karnofsky score above 60 at presentation. MGMT was methylated in seven of the fifteen patients, four patients had an MGMT not-methylated tumor, while the MGMT status was not defined in another four patients. Four patients were already in the maintenance chemotherapy phase of their treatment, taking TMZm cycles.