A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients with Glioblastoma Multiforme is Safe and Prolongs Both Overall and Progress Free Survival

Abstract

The study was a non-randomized controlled phase I-II trial to study were to ascertain the safety, feasibility and efficacy of immunotherapy with autologous IFN-γ transfected tumour cells in patients with glioblastoma multiforme. Autologous tumour cells harvested during surgery were cultured and transduced with the human IFN-γ gene. Irradiated cells were administered as intradermal immunizations every third week. Endpoints for safety were records of toxicity and adverse events, for feasibility the per cent of treated patients out of eligible patients and time to treatment and for clinical efficacy overall survival (OS) and progress free survival (PFS). Eight eligible patients, between 50 and 69 years, were immunized between 8 and 14 times after treatment with surgery and radiotherapy without adverse events or toxicity. Neurological status and quality of life were unchanged during immunotherapy. The immunized patients had a significantly (p < 0.05) longer median overall survival (488 days, 16.1 months than a matched control group of nine patients treated with only surgery and radiotherapy (271 days, 9.0 months). The prolongation of survival was also significant compared to all GBM treated at the same institution during the same period and published control groups within the same age cohort.

1. Introduction

The most aggressive primary brain tumour, glioblastoma multiforme (GBM) [1], is the most therapy-resistant human tumour. The mean survival time after diagnosis for patients with GBM had been approximately a year for more than 30 years when our study was performed, despite advances in surgery and radiotherapy. Consequently, very few patients survive the disease [2].

By the use of combined radiotherapy and chemotherapy with temozolomide, Stupp et al. demonstrated a small but significant increase in mean survival time from 12.1 to 14.3 months [3]. Unlike most other tumours, there is a considerable age-related impact on the survival of patients with GBM, where patients under the age of 50 years have more prolonged survival than those over the age of 50 [4, 5]. The mechanisms behind this are not precise, and both diverse biologies of the tumours in patients of different ages and senescence of the immune system have been proposed [6, 7]. The importance of immune reactivity against tumours has been highlighted by several reports, demonstrating a clear correlation between the numbers of tumour infiltrating lymphocytes and the prognosis of survival in patients with various neoplastic diseases [8, 9].

Glioblastomas induce profound immune suppression by several proposed mechanisms, such as releasing immunosuppressive substances, such as prostaglandin E2 (PGE2) and interleukin-10 (IL-10). It also releases growth factor-beta (TGF-α) [10, 11], which up-regulate apoptotic ligands such as programmed death receptor 1-ligand (PD1-L) [12] and induction of regulatory T cells [13].

Experimental intracranial tumour models report successful immunotherapy results [14, 15]. In addition, several investigators have reported promising preliminary results of clinical immunotherapy in patients with glioblastoma multiforme [16, 17, 18]. However, the results have been difficult to interpret due to heterogeneity of patients regarding age, the extent of resection and additional therapy.

We have previously reported successful immunotherapy against rodent brain tumours using autologous tumour cells secreting the cytokines IFN-γ, IL-7, nor expressing the adhesion molecule B7-1, where immunizations with IFN-γ secreting tumour cells were the most potent treatment [19, 20]. In our models, the proportion of CD8+ T-cells and NK cells of tumour-infiltrating leukocytes from immunized animals was larger than in tumours from control immunized animals [21].

Here we report the result of those experiments translated into a clinical trial of patients diagnosed with GBM aged 50–69 years. The study's goal was to ascertain whether immunization with transduced autologous tumour cells secreting IFN-γ; was feasible, safe for the patients and could show any evidence of clinical responses.

2. Material and methods

2.1 Study design

We designed this study as a phase I-II, non-randomized, therapeutic, exploratory, controlled study. Endpoints for feasibility were the number of treated patients out of eligible patients and the time from surgery to the start of immunizations. Endpoints for safety were records of toxicity and adverse reactions. Immune responses became monitored with immunohistochemistry of skin biopsies from the vaccination sites. Overall survival (OS) and progression-free survival (PFS) set the endpoints for clinical responses.

2.2 Patients

The study was performed with the permission of the Swedish Medical Products Agency and with the acceptance of the Local Ethical Board of the University of Lund. All patients gave their written consent to participate in the study. The patients were recruited from glioma cases referred to the Department of Neurosurgery at Lund University Hospital during 2000–2004. It is to be noted that temozolomide or other chemotherapeutic drugs were not included in the normal therapy in this age cohort at the time of the study. All patients were recruited before the inclusion of temozolomide in the regular treatment of glioma.

A. Inclusion criteria:

• Only patients from the Southern Swedish referral area (which includes 1.6 million people) were eligible for the protocol.

• PAD: Astrocytoma grade IV, (WHO) = Glioblastoma Multiforme.

• Age 50–69 years.

• >80% resection of tumour volume.

• Patient’s written consent.

• Karnofsky performance score ≥70 preoperatively.

• Radiotherapy (RT) only other treatment after surgery.

B. Exclusion criteria:

• Severe systemic disease.

• Autoimmune disease.

• Psychiatric illness.

• Deviation (major) from protocol.

C. Patient recruitment

2.3 Provisional and definite inclusion of patients

Patients with a confirmed diagnosis of GBM according to WHO-criteria [1] and whose first postoperative MRI revealed the resection to comprise 80% or more of the preoperative tumour volume were provisionally included in the study. The tentatively included patients whose tumour cells did not exhibit in vitro growth sufficient enough for transduction and immunization or where the cells could not be transfected appropriately constituted the control group. Table 1 show criteria for inclusion (A), exclusion (B) and patient recruitment (C).

Group	No.	<80% resection	Not GBM	Other reason for exclusion
Treated	8	0	0	0
Control	9	0	0	0
Excluded	11	5	5	1
All	28	5	5	1

Table 1.

Criteria of inclusion, exclusion, and recruitment of patients.

GBM, glioblastoma multiforme.

2.4 Preoperative investigations

Preoperatively the patients were examined with MRI, including diffusion- and perfusion sequences [22]. In addition, preoperatively and postoperatively, the patients were also evaluated by neurological (NIHSS) and quality of life (QOL) assessments (SF 36).

2.5 Surgical treatment

Temozolomide or other chemotherapeutic drugs were not included in the normal therapy in this age cohort at the time of the study. Tumour resection was performed using standard neurosurgical techniques, frequently applying neuro-navigation and ultrasonic aspiration. Viable tumour tissue was harvested for histopathological diagnosis and for culturing in vitro.

Based on clinical experience and judgment, repeated surgery was considered and performed as needed for diagnostic or palliative purposes throughout the study.

2.6 Postoperative treatment

All patients received irradiation treatment of the brain (58 Gy in 29 fractions) commencing within five weeks from surgery. Steroids were administered when symptoms occurred after tumour recurrence. No patients received steroids during the immunization period.

2.7 Postoperative investigations

Postoperative MRI as above was performed within 48 hours after surgery before immunization was started and at every second immunization. At the same time, patients were evaluated preoperatively with National Institutes of Health Stroke Scale (NIHSS ) and Short-Form Health Survey (SF-36).

2.8 Cell culture

Tumour tissue obtained at surgery was cultured in vitro and regularly karyotyped until the cells exhibited an abnormal karyotype. Then the cultivated cells were transduced using an adenoviral vector carrying the IFN-γ Human gene as well as the gene for the Green Fluorescent Protein and subsequently irradiated with 100 Gy to prevent a further growth in vivo [23, 24].

2.9 Immunization procedure

The patients included in the study received intradermal injections of their own irradiated and transfected cultured tumour cells at five sites in the upper arm at alternating sides every third week. The immunizations were repeated up to fourteen times or until the patient deteriorated and required steroids to alleviate symptoms. Figure 1 shows the immunization and monitoring procedures.

Figure 1.

Timeline of immunization and monitoring procedures.

2.10 Histopathological studies

Besides establishing the WHO diagnosis at the first surgery, further histopathological investigations were performed in some surgical specimens and in material obtained at autopsy in some patients [25].

3. Results

3.1 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells is safe

There could be a potential danger of evoking immune responses against normal CNS cells or inducing an inflammatory response that might spread to the regular brain after immunotherapy utilizing autologous tumour cells. However, we did not observe any major side effects or toxicity after immunizations.

The induction of autoimmune responses would most definitively have influenced the patients’ neurologic status. Neurological and cognitive grades were evaluated with the NIH Stroke Scale (NHSS) and no deterioration before tumour recurrence were recorded during immunizations in any patient (data not shown). Neither did postoperative MRI investigations show any inflammatory changes around the resection cavity nor in surrounding brain tissue [22].

Apart from achieving prolonged survival, a novel therapy also aims at maintaining or improving the quality of life (QOL) of the treated patients. Assessment of QOL by SF-36 did not reveal any deterioration during immunizations. However, there was a tendency to short-term memory deficits in some of the treated patients (data not shown). Nevertheless, overall the patients in the study experienced an increase in QOL during period of the treatment.

3.2 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells is feasible in 45% of the eligible patients

In total 28 patients were provisionally included in the study before surgery. After surgery, only 17 patients fulfilled all criteria of inclusion in the study (Table 1).

Eleven patients were excluded - due to another diagnosis than GBM, (5/11), insufficient tumour resection (5/11) or major psychiatric illness (1/11) (Table 1).

In nine of the 17 included patients, malignant cells were successfully cultured in vitro and became transduced as described above. One of these patients underwent six immunizations but was excluded from the study due to incomplete resection at review (Table 1). Thus finally, the treatment group in the study consisted of eight immunized patients with detailed information shown in Table 2. It was possible to vaccinate 8/17(45%) of eligible patients with GBM and 8/23(35%) of patients diagnosed with GBM.

No.	Age	Sex	Imm*	Tumour location	OS	PFS
Treated patients
1	52	M	10+4	Right occipital	800	433
2	53	F	10	Left frontal	639	286
3	59	F	10	Left temporal	582	353
4	63	F	8	Left frontal	313	239
5	64	F	10	Left frontal	758	666
6	66	M	8	Left frontal	366	239
7	68	F	10	Left frontal	354	161
8	68	M	8	Left frontal	394	253
Control patients
1	50	M	—	Right frontal	461	161
2	55	M	—	Right frontal	414	185
3	55	F	—	Left frontal	173	110
4	55	F	—	Left temporal	505	263
5	57	M	—	Left parietal	515	169
6	58	F	—	Right parietal	155	102
7	61	M	—	Right occipital	245	46
8	62	F	—	Left frontal	271	96
9	69	F	—	Left occipital	188	7

Table 2.

Individual patient data of treated and control groups.

F, female; M, male; Imm*, number of immunizations; OS, overall survival days; PFS, progress free survival days.

The control group consisted of the remaining nine patients (Table 2). In the treatment group, patients became vaccinated between 8 and 14 times. Additional immunizations were given depending on the availability of cells and patient status, although the protocol stipulated a minimum of four immunizations (Table 3). One patient received four additional immunizations after special approval from the Medical Products Agency. In conclusion, the immunization procedure was feasible in 45% of eligible patients.

	Treated	Matched control	Other Ctrl
Number of patients	8	9	90
Female	5	5	45
Female %	63	57	50
Male	3	4	45
Male %	37	43	50
Age (mean)	62	58	61
Age (STDV)	6	5	6
Age (range)	53–68	50–69	50–69
% resection (mean)	94	86	NA
% resection (range)	90–99	80–97	NA
Time to imm (mean)	154	—	NA
Time to imm (range)	97–253	—	NA
Secondary surgery (n)	7	8	NA
Secondary surgery (pat)	6	7	NA
Radiotherapy	Y	Y	Y
Chemotherapy	N	N	N
OS (mean) days	525 (17.4*)	325 (10.8*)	262 (9.0*)
OS (median) days	488 (16.3*)	271 (9.0*)	193 (6.4*)
OS (range) days	313–800	155–515	38#-962
PFS (mean) days	306 (10.1*)	151 (5.0*)	NA
PFS (median) days	267 (8.8*)	161 (5.3*)	NA
PFS (range) days	99–617	76–240	NA

Table 3.

Group data of treated and matched control of the study and as well as other controls.

*

Months

^#

Patients surviving less than 30 days postoperatively were excluded due to presumed surgical mortality.

Other ctrl, all patients with the diagnosis of GBM between 50 and 70 years of age treated during 2000–2003 (2 years) at our institution except the treated and matched control patients involved in the study; OS, overall survival; PFS, progress free survival

3.3 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells prolongs survival

The eight treated patients had a significantly prolonged overall median survival (488 days, 16.3 months) compared to the control group (288 days, 9.0 months) (Figure 2 and Table 3). There was also a significantly longer progress-free survival in the treated group (Table 3). No noteworthy differences between the groups appeared regarding age, gender, or repeated surgery (Table 3).

Figure 2.

Kaplan-Maier graph showing overall survival of immunized matched and non-matched control patients. The survival was analysed with the logrank test, the p value depicted refers to comparison between immunized and matched control patients.

Serial MR examinations showed no or stable contrast-enhancing areas in the responding patients and progressing towards contrast-enhancing areas in non-responding patients during immunizations (Figure 3). To rule out selection bias, we compared the matched control group with all patients treated at the same institution (all patients 50–69 years during 2000–2003 minus treated and matched control groups, n = 91). The data given in Table 3 show no significant differences between the survival times of the matched control group and the other control group, which indicate no apparent selection bias.

Figure 3.

Representative MRI (T1 with gadolinium) images from nonresponding and responding patients preoperatively, postoperatively and at the sixth immunization. The postoperative image of the non-responding patient shows a dense area, which constituted a haemorrhage also seen on non-gadolinium enhanced images (not shown).

There was also a clear indication that age was a prognostic factor apart from immunotherapy. Non-immunized patients aged 50–59 years survived 12.2 months, and immunized patients survived 22.2 months while non-immunized patients in the group aged 60–69 years survived 7.7 months, and vaccinated patients survived 14.3 months. Of the non-immunized patients, 0/9 survived >18 months, while 4/8 of the vaccinated patients survived >18 months and 2/8 >24 months. However, the study and control groups were too small to conduct more detailed statistics as COX regression analysis. In summary, the immunized group of patients had a prolonged overall survival (7.3 months compared to matched controls and 9.9 months compared to unmatched controls) that was not previously reported for patients with GBM over 50 years.

Figure 4 shows the survival results from nine vaccinated patients and 11 patients treated with surgery only, and subsequent radiotherapy presented at the World Federation of Neuro-Oncology and the European Neuro-Oncology Association in Edinburgh, Scotland. Post-diagnosis survival in nine glioma patients treated with vaccination was 14.3 months, which is significant (P < 0.02) longer compared to the 9.6 months of 11 patients normally treated with surgery only, and subsequent radiotherapy [26, 27].

Figure 4.

Post-diagnosis survival in nine glioma patients treated with vaccination and 11 patients normally treated with surgery and subsequent radiotherapy alone. Regression equations: Survival vaccinated (month) = 62(±18) – 0.75(±0.29)⋅Age(a) Survival normally treated (month) = 46(±12) – 0.64(±0.23)⋅Age(a) (dashed line).

4. Discussion

Based on our experimental results, we have treated eight patients with the diagnosis of GBM using immunizations with autologous tumour cells transfected with the human IFN-γ gene and compared them to 9 untreated but otherwise identically treated patients. Immunotherapy of malignant primary CNS tumours is no novelty, and the different therapeutic modalities attempted in general immunotherapy has also been utilized in trials of immunotherapy of these tumours with limited results [28, 29].

Promising results have been reported from several clinical trials based on immune therapy against high-grade gliomas:

• the Victory trial utilizing the EGFRvIII peptide conjugated with keyhole limpet hemocyanin (KLH) combined with autologous dendritic cells for immunization [30],

• the use of protein extracts from tumours in combination with autologous dendritic cells [16, 17, 31, 32],

• the use of cultured autologous GBM cells irradiated and infected with Newcastle Disease Virus before immunization [18] and

• the use of immunizations with tumour cells transfected with the sense for TGF-ß [33].

We chose to select the patients within a defined age cohort and within an outlined resection volume to rule out confounding factors. Although under discussion, recent reports have indicated that the extent of resection of high-grade gliomas is a prognostic factor and therefore, we excluded patients with a resection less than 80% of the preoperative volume [34]. Other reports of immunotherapy of high-grade gliomas have claimed a higher success rate of a culture of explanted tumour biopsies than we have found [18, 32, 33] Although several putative tumour markers for glioblastoma multiforme have been proposed [35], there are no ubiquitous ones that can be used for the identification of tumour cells in culture.

Unlike other investigators, who have used panels of associated tumour markers, we have utilized karyotyping to detect tumour cells in cultures to avoid contamination of non-tumour cells [24]. This procedure may have excluded tumour cells with a near-normal karyotype, but it has reduced the probability of including contaminating non-tumour cells in the vaccine. The prognosis for patients with malignant primary CNS tumours varies depending on grade and type of tumour, age, performance status at diagnosis, and expression pattern of different proteins and genes [34, 36, 37]. Even within the entity of GBM, the survival range is extensive, and a major impact of age and performance status at diagnosis has been demonstrated. This makes the interpretation of results from clinical studies difficult when patients of different ages and grades of tumour are included.

In some of the studies published on immunotherapy of patients with primary malignant brain tumours, younger patients and also patients with the diagnosis of anaplastic astrocytoma have been included. The latter group has a substantially longer expected survival than patients with GBM, and therefore, it is hard to evaluate the actual effect of immunotherapy in some of these studies [18, 33, 38] The reported mean survival rates for treated patients with GBM in these studies were 700, 462 and 931 days with mean ages 49, 50, and 44 years respectively. In the report by Steiner et al., the survivals of individual patients were stated and the median survival of patients 50–69 was 500 days (range 252–868). Although the current patient group is too small for statistical sub-analysis, both age and immunotherapy were strongly indicated as independent predictors of increased survival (data not shown).

Additional patients have received immunizations after adjuvant temozolomide and radiotherapy followed by 4–6 cycles of temozolomide, and one other patient, not included in this study, aged 57, who received this therapy had an overall survival of 24,5 months. This is a preliminary indication that immunizations might be feasible in this setting, and another case of concurrent immunotherapy and administration of temozolomide has been reported [39]. DTH reactions in the skin at the immunization sites were recorded in all patients, but there was no correlation with overall survival (data not shown).

Analysis of peripheral blood, before and during the vaccinations, has shown signs of immune activation. Recombinant antibody micro-array technology [40] has been used to perform differential plasma protein profiling of the non-immunized and immunized GMB patients and of age-matched healthy controls from this study [41]. We have previously reported that in one patient who was re-operated on during immunizations and in the patients re-operated on after the cessation of immunizations, a transient influx of T cells into the tumour tissue could be observed [25]. This indicates that the same pattern of a lymphocyte influx as observed in our experimental model indeed occurs after clinical immunotherapy. However, whether there is a specific pattern in responders compared to serial biopsies of tumour tissue can only study non-responders and controls immunotherapy.

As reported previously, there were no signs of inflammation or oedema in the tumour tissue or the surrounding brain as judged by magnetic resonance tomography (MRT) after immunotherapy [22] which has been reported after treatment of high-grade gliomas with oncolytic viruses. This could be explained by inappropriate methods to detect an inflammatory reaction or by the minimal tumour volumes during immunizations in most patients. An alternative explanation is that the current immunotherapy does not induce a recordable inflammatory reaction that can be demonstrated with MRT. Immunotherapy has a potential risk of inducing autoimmune reactions that could damage normal tissue. In the CNS, these reactions could be deleterious and possibly life-threatening due to cerebral inflammation and oedema induction.

We have not recorded any such adverse reactions during immunizations. This agrees with additional immunotherapy trials of CNS tumours and is somewhat surprising as strong immune responses are evoked against antigens that might be shared with normal CNS resident cells. The reasons for this are unknown but could depend on the immune privilege of the normal CNS or the absence of shared antigens. GBM is, with anecdotal exceptions, an incurable disease in adults. Therapies that aim to cure the disease will realistically first prolong survival with gradual improvements in treating other tumours. It is now generally accepted that treatments that aim to lengthen survival should also strive to maintain or improve the quality of life. The treated patients in this study did not experience a diminished QOL during the immunizations, but further studies will have to confirm this. Neither do we know whether maintained QOL was related to the direct nor indirect effects of the immunizations.

The treated group had a statistically increased overall survival compared to both a matched control group and another control group encompassing all patients with GBM over 50 years of age, treated in our institution during the same period. There was no difference in survival between the matched control group and the non-matched control group. Furthermore, when considering RTOG-RPA classes (both treated and control patients belong to class IV-V) the expected overall survival in these groups (8.9 and 11.1 months) matches that of the overall survival of both control groups [41, 42].

5. Conclusion

In conclusion, this is the first study to show a significant prolongation of survival after immunotherapy of patients with GBM in the age group over 50 years. Taken into account that age is a predictor for survival of patients with glioblastoma multiforme; treatment of younger patients might result in longer periods of survival with unchanged or improved quality of life.

Acknowledgments

Dedicated to Hans and Märit Rausing who during 1996–2010 generously supported the research project “BRIGTT” (Brain Immuno Gene Tumour Therapy) which was performed at the Rausing Laboratory during 1996–2010.

We thank professor Hans-Olov Sjögren for his fruitful advice and discussions. We also thank research nurses Anna Evaldsson, Anita Nilsson and Charlotte Orre for coordination of pre and post-operative examinations.

The Hedvig Foundation, the Hans and Märit Rausing Charitable Foundation, the Hans and Märit Rausing Charitable Trust UK, the Swedish Cancer Foundation, the Lund University Hospital Funds, the Lund University funds and the County Council (ALF and Region Skåne) funds supported this project.