Intra‑tumoral treatment with oxygen‑ozone in glioblastoma: A systematic literature search and results of a case series
- Authors:
- Published online on: September 5, 2018 https://doi.org/10.3892/ol.2018.9397
- Pages: 5813-5822
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Copyright: © Megele et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Despite significant advances in the basic understanding of tumor pathogenesis, improvement in surgical techniques and new adjunct treatment, the median overall survival rate of patients with glioblastoma (GB) has increased only by 3.3 months (from 11.3 to 14.6 months) until 2010 (1,2) with a long-term survival rate above three years of only 3 to 5% (3). During the past decade, the consistent implementation of surgical re-resection in recurrent GB has helped to improve this devastating figure resulting in up to 25 months of overall survival rate, and 11.9 months following the first resection (4). The main predictive factors of survival are tumor localization in eloquent areas of the brain and the functional performance and tumor volume of the patient.
In GB, a small cell population with stem cell-like properties is resistant to chemotherapy. These cells exhibit unique energy metabolic characteristics (5), including low mitochondrial respiration and a preference for hypoxia, to maintain their tumor-forming capacity (6). In vitro, hypoxia promotes the survival of these clonogenic cancer stem cells (7) and inhibits their differentiation even if the cultures are returned to normoxic conditions (8). Furthermore, approximately half of all human GB respond to hypoxia by inducing c-Met, with subsequent enhancement of tumor cell migration and invasion (9).
Treatment strategies utilizing reactive oxygen (O2) species induce cell death by autophagy and apoptosis in glioma cell cultures (10). Employing positron emission tomography (PET) or advanced magnetic resonance imaging (MRI) techniques such as MR spectroscopy enable the monitoring of local O2 levels (11). The intracellular in vivo oxymetry in rats implanted with the aggressive glioma cell line 9L demonstrates the preference for low tumor pO2 levels around 45 mmHg (12). Treatment with bischloroethylnitrosourea (BCNU) resulted in an immediate and significant increase in pO2 up to 140 mmHg (12). These anti-tumorigenic properties of oxygen are accompanied by its key role in regulating the immunogenicity of primary human glioma cells (13). Furthermore, the application of O2 plus ozone (O3) in two different human neuroblastoma cell lines (SK-N-SH and SK-N-DZ) induced cell growth inhibition at G2 phase and cell cycle perturbation in both cell lines (14).
Ozone is a gas that is produced endogenously by granulocytes. In the presence of an electron donor, reactive O2 species are produced, particularly hydrogen peroxide (15). Clinical O3 application has resulted in improved wound healing (16,17), and the improvement of radiation-induced proctitis (18) and cystis (19), as well as the improvement of osteonecrosis (20–22). The toxicity of inhaled O3 can be reduced by applying the gas for a calculated period and at lower doses (23–25) although certain genetic variance in the O3 response exist (26). However, O3 acts antineoplastically in the presence of carcinogens if administered by inhalation (27,28), intra-vesicular (29) or intra-peritoneal (30,31) instillation. Since O3 stimulates the release of immunoactive cytokines (32), these effects are more likely to be the result of an immune-mediated reaction rather than being a direct consequence of administration (33).
Studies have emphasized that O3 therapy can be considered a serious adjuvant therapy in oncological patients receiving radiochemotherapy (34). The present study reports data from the off-label application of O2-O3 into the tumor vicinity of GB following surgery in a limited series of patients. In addition, the results of a systematic literature search on O3 treatment following surgery for malignancies are presented.
Materials and methods
Between January 2012 and December 2013, patients diagnosed with GB at the Klinikum Amberg were offered an intra-tumoral treatment with O2-O3 extending the standard therapy as determined by the local tumor board. Following extensive information about their options and the possible side effects of the treatment, five patients provided their informed consent, and the ethical committee of the Bavarian National Medical Association approved the study, based on the Helsinki Declaration of 1964, revised 2013 (EK-Nr. 2013-125). Informed consent included publication of the case report and any accompanying images. Part of the illustrative case was presented as a poster on the Brain Tumor Meeting May 2013 in Berlin.
Treatment
Together with the re-resection in recurrent GB, a Rickham reservoir was implanted with the tip ending in the middle of the resection area. The exact catheter localization and the extent of tumor resection were confirmed by an early (<72 h post-operatively) contrast-enhanced MRI. Subsequently, five ml of O2-O3 were applied monthly through the reservoir at a concentration of 40 µg O3 per ml.
The standard adjuvant therapy following the initial surgery for GB consisted of radiotherapy with a local boost and chemotherapy according to Stupp et al (2); 75 mg/m2 body surface temozolomide followed by cycles with 150 mg/m2 body surface, five days per week for the irradiation period, followed by a three week off/one week on schedule. Following progression or recurrence and re-section, the patients were switched to PCV chemotherapy (procarbacine 60 mg/m2 day 8 to 21, CCNU 100 mg/m2 day and vincristine 1.4 mg/m2 day 8+29).
Histological assessment
Tumor biopsies at primary resection and recurrence(s) were evaluated by an experienced neuropathologist and classified according to the criteria of the WHO 2007 classification of tumors of the central nervous system. Prior to publication, the histological results were reviewed and adapted to the current WHO classification of 2016 (35). The standard work-up for diagnostics comprised routine histological stains, immunohistochemistry, including the p53 (BP53-12) and Ki-67 (Mib-1) antigens, and molecular diagnostics for O-6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and isocitrate dehydrogenase (IDH) 1/2 mutations. Additionally, immunohistochemistry for hypoxia-inducible factor (HIF)-1α and HIF-2α was performed for investigative purposes (Fig. 1). Immunohistochemistry on formalin-fixed and paraffin-embedded tissue samples was performed as described previously (36). In brief, 5-mm sections were cut and slides were deparaffinized and, following microwave antigen retrieval, stained using the EnVision+ Dual Link System-HRP (DAB+; K4065; Dako; Agilent Technologies GmbH, Waldbronn, Germany) according to the manufacturer's protocol. Anti-HIF-1α (NB100-123; Novus Biologicals, Ltd., Cambridge, UK) and anti HIF-2α ha (NB100-122; Novus Biologicals, Ltd.) primary antibodies were used at 1:50 in phosphate buffered saline supplemented with 1% bovine serum albumin. Pre-treatment for antigen retrieval was not applied.
Magnetic resonance tumor volumetry. Volumetric analysis was performed on a GE Advantage Workstation (GE Healthcare Life Sciences, Little Chalfont, UK; version 4.7, Operation System 2.0 on a HELiOS 6.6.1 subsystem) by a radiologist. Tumor size and volume were calculated by semi-automated contouring of tumor borders on each T1 weighted-slice (post gadolinium) in cm3 and maximum 2D diameter in mm, as outlined in Fig. 2. Slice thickness was 1 and 5 mm, dependent on the subsequent protocols of MRI examination sequences applied between January 2012 and July 2017. Enhancing areas were considered as tumor with the exception of obvious vessels, artifacts or postoperative resection defects.
Statistical analysis
Dichotomous or categorical parameters are presented in frequencies and percentages. Metric variables are provided as median and range. Statistical analyses were performed using SPSS version 21 (IBM Corp., Armonk, NY, USA).
Systematic literature search
A systematic literature search was performed for observational and clinical studies utilizing an O3 treatment for malignancies. Studies published between 01/01/1987 and 01/12/2017 were included, with no limitation regarding study type and sample unit (observational or experimental, animal or patient, in vitro or in vivo). The retrieval of studies was performed in PubMed using the combined filter and Medical Subject Headings (MeSH) term: [‘Ozone/surgery’ (Mesh) OR ‘Ozone/therapeutic use’ (Mesh) OR ‘Ozone/therapy’ (Mesh)) AND ‘Neoplasms’ (Mesh)] The records were screened based on title and abstract independently. Finally, the remaining records were evaluated by reading the full-text papers. All relevant characteristics (study type, disease, route of O3 application, unit of analysis, sample size and main findings) reported in the studies were extracted into an evidence table.
Results
The demographic and disease-related characteristics are summarized in Table I. The patient group comprised two women and three men with a median age of 48 years at diagnosis (range 31 to 68 years), the Karnofsky score (37) was 80% (range 50 to 90%).
Primary treatment/initial surgery
In two patients the diagnosis was established following a biopsy, in two patients following a partial resection. In one patient the initial resection was complete and at her request the O2-O3 therapy was started following diagnosis.
In four out of five patients molecular neuropathology was IDH wild-type GB; in the youngest patient the GB exhibited an IDH mutation corresponding to (secondary) GB, IDH-mutant. The MGMT promoter methylation status ranged between <3 and 25%. The median Ki67 proliferation index was 30% (range 20 to 40%), and the median nuclear score for p53 positivity was 20% (range 0 to 75%).
Of the five patients, four received a radiotherapy with a median of 49 Gy (range, 46 to 50 Gy) accompanied by a local boost of 11 Gy (range, 10 to 14 Gy). In all patients, the initial chemotherapy was started with temozolomide according to Stupp et al (2).
Surgery for first recurrence/progression: Further treatment
The patient who insisted on receiving the O2-O3 treatment following the initial diagnosis remains in remission. In the other four patients, the first recurrence occurred in a median time of five months (range, 4 to 9 months). Together with the re-resection in recurrent GB, a Rickham reservoir for O2-O3 application was implanted. Five milliliter of O2-O3 were applied monthly through the reservoir at a concentration of 40 µg O3 per ml. The chemotherapy was changed in three out of four patients to PCV; one patient refused further chemotherapy.
Overall survival and survival following initiation of ozone treatment
The patients received a median of 27 (range, 3 to 44) O2-O3 applications. The overall median survival rate was 40 months (range, 16 to 53 months). The median survival rate following the first recurrence subsequent to the initiation of the O2-O3 treatment was 34 months (range, 12 to 53 months). The progression-free survival rate was positively associated with a more extensive surgical re-resection (P=0.021), while the tumor volume was negatively correlated (P=0.027).
In one patient, a local infection occurred resulting in temporary removal of the reservoir. In another patient, a hemorrhage in the tumor vicinity around the catheter four months following the implantation necessitated the temporary removal of the reservoir. Based on a total of 115 O2-O3 applications in five patients, the complication rate per application was 1.7%.
Illustrative case
A 59-year-old male patient developed a seizure with a persisting hemiparesis. MRI demonstrated a contrast-enhancing lesion in the motor cortex (10.5 cm3; Fig. 3A). The histological examination confirmed a GB, IDH wild-type with a Ki-67 proliferation rate of 20% and no relevant MGMT promoter methylation (Fig. 1A). The patient was treated with radiochemotherapy (50 Gy with local boost of 10 Gy), temozolomide at 75 mg/m2 body surface for four weeks, followed by one three weeks off and one cycle at 150 mg/m2. The symptoms worsened and a tumor progression was evident at six months with a volume of 29.3 cm3 (Fig. 3B). At the time of tumor resection, a catheter connected to a reservoir was placed in the area of the former tumor cavity (Fig. 3C). Histology was performed and again demonstrated pleomorphic astrocytic tumor cells and fibroid changes of tumor vessels as a probable therapy-induced alteration (Fig. 1B). In addition, HIF-2α staining could be observed (Fig. 1E). O2-O3 treatment was employed monthly together with a second-line PCV chemotherapy. Then, 13 months following surgery, the catheter was removed due to a local infection accompanied by seizures. The tissue demonstrated reactive and resorptive changes with an accumulation of macrophages (Fig. 1C). Next, six weeks later, the catheter was re-implanted and the intra-tumoral O2-O3 treatment was resumed together with the PCV chemotherapy. The patient returned to work and was in a stable neurological condition for almost 40 months. However, paresis of the arm worsened and MRI demonstrated tumor progression. A second re-section was performed, and presented with the histology of a full-blown cell-rich tumor recurrence (Fig. 1D) but with lowered HIF-2α immunostaining (Fig. 1F) compared with the first recurrence and the situation prior to O2-O3 treatment. Comparing the two time points, no differences could be observed in HIF-1α staining. The patient died 40 months after the initial diagnosis, and 33 months following the initiation of O2-O3 treatment. Another illustrative case is depicted in Fig. 4. This patient is still alive, more than 4 years after the diagnosis GB.
Findings from the literature search
The systematic literature search revealed 79 scientific studies. Following the screening of titles and abstracts, 20 articles fulfilled the criteria for eligibility. In three cases, no English full-text articles were available. Finally, a total of seven clinical and ten basic research studies met the eligibility criteria applied to the studies. Data extracted from these studies are shown in Table II.
All cohorts/samples were independent and no overlap of included individuals could be identified. Overall, the sample sizes of the clinical studies detected by the literature search ranged from case reports to a prospective phase I–II study including one to 24 patients. The basic research studies adopted the guidelines for the care and use of animals and included 20 to more than 100 animals.
Discussion
The rationale to perform an individual off-label treatment in GB patients was three-fold: There was i) in vitro and in vivo evidence for a preference for hypoxia in GB, ii) in vitro and in vivo evidence for the treatment efficacy of hyperoxia generating reactive O2 species in GB, and iii) in vivo evidence for the treatment effect of O3 in stimulating the immune system in other types of cancer (31,33).
Tumor cells, particularly those with stem cell-like properties, exhibit a preference for hypoxia (6,12). Therefore the intra-tumoral application of O2 was aimed at establishing normoxia, thereby inhibiting proliferation and migration (9) in addition to promoting differentiation (5,8). Whether this target was accomplished, was not verified. Five ml of O2-O3 were applied intra-tumorally at a concentration of 40 µg ozone per ml once per month. While direct intracellular oxymetry can be utilized in basic research models, the measurement of tissue O2 levels in the clinical setting is restricted to advanced and not readily available techniques such as PET or MR spectroscopy (11).
HIFs are transcription factors that respond to decreases in available O2 in the cellular environment, or hypoxia. HIF-1 consists of an O2-sensitive α-subunit and a constitutively expressed β-subunit, and belongs to a family of transcription factors [PER/aryl hydrocarbon receptor nuclear translocator (ARNT)/single minded (SIM)] (38–40). HIF-1α facilitates, together with HIF-2α, O2 delivery and cellular adaptation to hypoxia by stimulating multiple biological processes, including erythropoesis, angiogenesis and anaerobic glucose metabolism (41).
Histologically, following the intra-tumoral O2-O3 application mainly reactive and resorptive changes were identified (Fig. 1C). Notably, while relevant HIF-1α staining was not present prior to or following the intra-tumoral O2-O3 application, the HIF-2α expression (particularly in tumor blood vessels) was higher under the hypoxic conditions prior to, compared with following, the treatment (Fig. 1E and F).
Treatment strategies utilizing reactive O2 species in glioma cell cultures have been demonstrated to induce cell death by autophagy and apoptosis (10) or at least cell growth inhibition (14). Histologically, the present illustrative case identified mainly reactive and resorptive changes following the intra-tumoral O2-O3 application for a long time period. Only single scattered pleomorphic astrocytic tumor cells could be detected (Fig. 1C).
O3, a gas that is produced endogenously by granulocytes, induces the generation of reactive O2 species, stimulates the release of immunoactive cytokines (12) and regulates immunogenicity of human glioma cells (13). The intra-peritoneal insufflation of O2-O3 resulted in a tumoricidal immune response in experimental head and neck squamous carcinoma (32) with subsequent complete tumor remission (31). Oxidative stress converts the immune response from a tumor permissive to a tumoricidal one, probably through the stimulation of systemic T cells, resident macrophages and dendritic cells (32).
Human natural killer cells as part of a systemic immune response are most likely at least in part, responsible for the absence of metastases in GB patients (42). The exposure of peripheral blood mononuclear cells to a single dose of 1 µg/ml O3 increased the numbers of CD3-CD16+/56+ natural killer cells in vitro (43). In human GB tissue of long-term (>36 months) survivors, the number of CD 8, CD 20, CD 25 and CD 95 positive lymphocytes was significantly increased compared with short-term (<1 year) survivors. Therefore, the O2-O3 application in GB may act as an immunotherapy through the enhancement of human natural killer cells. Histologically, in this context an accumulation of macrophages was identified that immunohistochemically stained for the CD68 (PGM1) antigen following the intra-tumoral O2-O3 application in our illustrative case (Fig. 1C).
The intra-peritoneal application of 1 ml O3 at 20 or 40 µg/ml into the peritoneal cavity of mice has been performed without side effects in a control group confirming thereby the safety of the selected concentration (30). Andreula et al (44) safely injected four ml O3 at 27 µg/ml into the lumbar disc and periganglionic in 600 patients with clinical signs of lumbar disk nerve root compression. The present study applied five ml O3 at 40 µg/ml into the tumor cavity and could not identify any direct cell damage or necrosis around the cavity histologically. Nevertheless, the safety of this application has to be confirmed by cell culture of normal CNS tissue and glioma cells in addition to in vivo studies.
The median overall survival rate in our series comprising of five patients was 40 months. Since in one patient the O2-O3 treatment was started following diagnosis, the median survival following recurrence was 30.5 months (range, 12 to 37 months) in the remaining four patients. These data outperform previously published data (3) and the data from a recent multicenter trial including 505 patients from 20 institutions undergoing re-resection in recurrent GB (4). In that multicenter study, the median overall survival rate was 25 months and 11.9 months following the first resection. Furthermore, one of the patients in the present study treated with local O2-O3 in addition to the standard treatment following initial surgery remains alive, 53 months following the diagnosis of GB (Fig. 4A-C). The patients in our series did not differ substantially with regard to known prognostic factors: The median age at diagnosis was 48 years (range, 31 to 68 years), the median Karnofsky score at presentation 80% (range, 50 to 90%), the median proliferation index 30% (range, 20 to 40%) and the median quantitative MGMT promoter methylation was 5% (range, 3 to 25%).
However, the authors are aware that a study focusing on long-term survivors following GB presented even better data (45). That study retrospectively identified 50 long-term GB survivors (>36 months). In this selected cohort, the median progression-free survival rate was 25.4 months (range, 2.3 to 97.8 months) compared with six months (range, 4 to 52 months) in our series, and the overall survival rate of long-term survivors was 55.9 months (range, 38.2 to 98.6 months) compared with 40 months (range, 16 to 52 months) in our series.
The authors are aware of several limitations of the present study. The exact dose and the timing of the application were based on estimates and patient comfort. Optimization of both should be performed either in a basic research setting utilizing tissue oxymetry or mapping brain tissue O2 saturation in the clinical setting with PET or MRI. Furthermore, this off-label case series should be transferred into a clinical trial.
Taken together, the overall survival rate of our patient series is longer than obtained in an unselected multicenter study. Taking a closer look at a long-term GB survivor cohort (44), both progression-free survival and overall survival rate are slightly shorter. These results and the existing evidence revealed by the systematic literature search highlighted that O3 therapy could be considered a viable adjuvant therapy in oncological patients receiving radiochemotherapy (34). The case series of the present study indicated the potential benefit and efficacy of intra-tumoral O2-O3 application following surgery for GB. Following this descriptive approach, further observational and experimental research is warranted to elucidate cellular and systemic effects, in addition to ensuring safety by applying inference statistical analyses based on an appropriate sample size.
Acknowledgements
Part of the illustrative case was presented as a poster on the Brain Tumor Meeting May 2013 in Berlin (abstract no. 36).
Funding
No funding was received.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
RM designed the study. MJR performed the histological examination and contributed to the drafting and revision of the manuscript. FDS performed the literature search, data analysis and interpretation and contributed to writing the manuscript. MF analyzed and interpreted the radiological patient data. AK revised the study design, structured the data aquisition, analysed and interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the ethical committee of the Bavarian National Medical Association (approval no. EK-Nr. 2013-125). All patients provided written informed consent.
Patient consent for publication
Following extensive information about their options and the possible side effects of the treatment, all patients provided their informed consent, including the publication of the case report and any accompanying images.
Competing interests
The authors declare that they have no competing interests.
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