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AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study
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doi:10.1016/j.ymthe.2004.08.002
AdvHSV-tk Gene Therapy with Intravenous Ganciclovir Improves Survival in Human Malignant Glioma: A Randomised, Controlled Study Arto Immonen,1,* Matti Vapalahti,1,2,* Kristiina Tyynel7,3 Heleena Hurskainen,1 Anu Sandmair,1 Ritva Vanninen,4 Gillian Langford,5 Neil Murray,5 and Seppo Yl7-Herttuala2,6,7,y 1
Department of Neurosurgery, 3Department of Oncology, 4Department of Radiology, 6Department of Molecular Medicine, A.I. Virtanen Institute, and 7Department of Medicine, University of Kuopio, FIN-70211 Kuopio, Finland 2 Gene Therapy Unit, Kuopio University Hospital, FIN-70211 Kuopio, Finland 5 Ark Therapeutics Ltd., London, UK
*These authors contributed equally to this article. y
To whom correspondence and reprint request should be addressesed at the A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Fax: +358 17 163751. E-mail: [email protected].
Available online 8 September 2004
Malignant glioma is a devastating brain tumour with no effective treatment. This randomised, controlled study involved 36 patients with operable primary or recurrent malignant glioma. Seventeen patients were randomised to receive AdvHSV-tk gene therapy (3 1010 pfu) by local injection into the wound bed after tumour resection, followed by intravenous ganciclovir (GCV), 5 mg/kg twice daily for 14 days. The control group of 19 patients received standard care consisting of radical excision followed by radiotherapy in those patients with primary tumours. The primary endpoint was survival as defined by death or surgery for recurrence. Secondary end-points were allcause mortality and tumour progression as determined by MRI. Overall safety and quality of life were also assessed. Findings were also compared with historical controls (n = 36) from the same unit over 2 years preceding the study. AdvHSV-tk treatment produced a clinically and statistically significant increase in mean survival from 39.0 F 19.7 (SD) to 70.6 F 52.9 weeks ( P = 0.0095, log rank regression vs. randomized controls). The median survival time increased from 37.7 to 62.4 weeks. Six patients had increased anti-adenovirus antibody titers, without adverse effects. The treatment was well tolerated. It is concluded that AdvHSV-tk gene therapy and GCV is a potential new treatment for operable primary or recurrent high-grade glioma. Key Words: adenovirus, malignant glioma, gene therapy, thymidine kinase, survival
INTRODUCTION Malignant glioma is a devastating and fatal disease. The estimated prevalence of brain and central nervous system tumours in the EU and USA is 1.3 per 10,000 persons [1]. Approximately 60% are gliomas and of these some 40 to 50% are glioblastoma multiforme, a particularly aggressive and rapidly fatal form of the disease. Despite surgical and radiotherapeutic advances there is currently no effective treatment and the prognosis for these patients remains poor. Adenovirus-mediated gene therapy may be suitable for the treatment of malignant glioma, which are good targets for gene therapy as they are generally well localised and rarely metastasise out-
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side the central nervous system [2–5]. Therefore, the necessary vectors can be delivered at high concentrations directly to the site of action, thus minimising systemic toxicity. The Thymidine Kinase enzyme (TK) produced by the Herpes simplex virus (HSV) is harmless to man since it lacks a substrate in the human body. However, in cells that express TK, the TK can metabolise intravenously administered ganciclovir (GCV) to produce a cytotoxic nucleotide, which is selective for dividing cells. The nucleotide is incorporated into the DNA of dividing cells during replication where it halts DNA synthesis and results in cell death mainly by apoptosis [2,6]. This is of particular significance in the brain where the normal
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neurons surrounding the tumour are non-proliferative and therefore not susceptible to toxic metabolites. The treatment effect is further strengthened by a bbystander effectQ [2,5]. The hypothesis was that transduction of the AdvHSVtk into the brain, followed by GCV administration, is a potential new treatment for malignant glioma. This randomised, controlled study was designed to assess the efficacy and safety of AdvHSV-tk and intravenous GCV for the treatment of operable primary and recurrent malignant gliomas.
RESULTS Demography We randomized 36 patients; 17 received AdvHSV-tk gene therapy and 19 received control treatment. All patients received the assigned treatment. Patient demographics are shown in Table 1 along with those of the additional cohort of historical controls. Efficacy The mean survival of patients on AdvHSV-tk was 81% longer than for the control group (70.6 F 52.9 [SD] vs. 39.0 F 19.7 weeks, Fig. 1). The median survival in the AdvHSV-tk group was 65% longer than the control group (62.4 weeks vs. 37.7 weeks). The difference in survival was statistically significant ( P = 0.0095), using Kaplan-Meier survival plots and log rank regression analysis. The data were normally distributed indicating a consistent effect across the study population. Statistical significance was confirmed using Cox regression analysis to test the influence of the prognostic factors age, tumour type, histology and Karnofsky score and it was determined that these parameters did not affect the overall outcome.
TABLE 1: Patient demographic characteristics AdvHSV-tk
Randomised controls
Historical controls
17
19
36
12 5
12 7
25 11
Mean Age (Years) and range
51.9 (39–68)
56.5 (35–75)
59.5 (35–75)
TumourType Primary Recurrent Number of GBMs Number of AAs Karnofsky Score (Mean)
12 5 13 4 85.2
12 7 18 1 84.2
36 0 34 2 85.0
Number Gender Male Female
GBM: glioblastoma multiforme; AA: anaplastic astrocytoma.
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Additional post hoc subgroup analyses of the primary endpoint showed an increased median survival in patients with all primary tumours (66.4 weeks vs. 40.3 weeks) and in those with glioblastoma multiforme only (55.3 weeks vs. 37.0 weeks). These differences were statistically significant using Kaplan-Meier survival plots and log rank regression ( P = 0.0322 and P = 0.0214, respectively). The difference between the treated patients with newly diagnosed primary glioblastoma multiforme vs. randomized controls (54.4 weeks vs. 42.8 weeks) also tended to be significant. The improvement in survival in the AdvHSVtk group was maintained at 12-month follow-up ( P = 0.04). All cause mortality was improved with AdvHSV-tk treatment, with an improvement in median survival from 45.0 weeks to 62.4 weeks ( P = 0.0256). Patients treated with AdvHSV-tk also showed a significantly longer mean survival than those in a historical control group: 70.6 F 52.9 weeks vs. 36.0 F 29.9 weeks (Fig. 2). The same was true for median survival (62.4 weeks vs. 30.9 weeks, respectively). The difference between the AdvHSV-tk group and the historical controls was also significant by log rank regression ( P b 0.0017). Furthermore, mean survival for the randomised controls was not significantly different from that of the historical controls (39.0 F 19.7 weeks vs 36.0 F 29.9 weeks). As is evident from Figs. 1 and 2 early survival in the randomized controls seems to be better than in the historical controls which is probably due to the introduction of neuronavigation to aid surgeons to remove as much of the tumour as possible. In relation to tumour progression as determined by MRI, at eight weeks post-operatively progression was observed in 6 out of 15 patients (40%) in the AdvHSV-tk group and 8 out of 16 patients (50%) in the control group. We observed no discernable trends in either an improvement or deterioration in quality of life using standardised neuropsychological tests including Wechsler’s memory scale, quickness and attention, flexibility of behaviour, psychomotor quickness and a symptom and mental agility questionnaire. Furthermore, there was no evidence that patients who survived longer required increasing use of concomitant medication. Safety AdvHSV-tk treatment was well tolerated and no significant safety issues emerged. Three patients in the active group developed transient reversible elevations of liver enzymes after 5 to 7 days of GCV treatment (AST from b36 IU/l at screening to 41-45 IU/l; ALT from b47 IU/l at screening to 101-212 IU/l), but in no case was bilirubin elevated. Aside from these changes no major alterations in routine laboratory parameters occurred (Table 2). Two patients in the AdvHSV-tk group developed localised post-operative intracerebral oedema during GCV treatment, 1 with an associated hemiparesis; both required further surgery but recovered without sequelae. Adenovirus was detected
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ments were well tolerated, and that the mean survival for the AdvHSV-tk group was approximately double (15.0 months, n = 7) that for patients treated with 109 retroviral packaging cells (7.4 months, n = 8) or control patients treated with a lacZ vector injected into the tumour 4-5 days before resection (8.3 months, n = 7). The difference in survival between AdvHSV-tk and retroviral HSV-tk treatment was statistically significant ( P b 0.012) [7]. The protocol-specified primary endpoint used in the current study was survival from the date of operation as defined by death or surgery for recurrence. This latter endpoint could theoretically cause a bias if clinicians know which patients are treated. However, this is unlikely since in the current study only three patients underwent reoperation which was performed to rescue the patients from immediate death thus avoiding potential bias caused by artificial prolongation of the survival time due to reoperation. The HSV-tk approach has been previously tested in retrovirus-based phase I/II studies [4,15,16] and in a large multicenter phase III trial [17]. All these studies used retrovirus-packaging cells producing HSV-tk retroviruses. However, this large Phase III trial failed to show any significant therapeutic effect probably due to low trans-
FIG. 1. Survival of Adv.HSV-tk treated patients and randomised controls. (A) All patients. (B) Patients with glioblastoma multiforme. Kaplan–Meier plots with log rank regression analysis.
by PCR in the plasma of 2 patients in the AdvHSV-tk group 3 days after gene therapy, but not thereafter. Antiadenovirus antibodies increased more than 4-fold in 6 patients in the AdvHSV-tk group, without adverse outcome (Table 2).
DISCUSSION The efficacy of AdvHSV-tk has been assessed in several experimental studies [2,9–13]. However this is the first randomised, controlled study to demonstrate increased survival in patients with operable primary or recurrent high-grade glioma. Our group has performed two previous clinical studies supporting the safety and efficacy of AdvHSV-tk. The first was a dose-ranging study from 1 108 pfu which examined the efficacy of different vectors in the transduction of a marker gene (lacZ) into human malignant gliomas. This study established 3 1010pfu as the dose of adenovirus for subsequent clinical trials [14]. The second study [7] compared the safety and efficacy of the AdvHSV-tk and HSV-tk retrovirus-packaging cells for the treatment of patients with operable primary or recurrent malignant glioma. The results showed that both treat-
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FIG. 2. Survival of AdvHSV-tk-treated patients and historical controls. (A) All patients. (B) Patients with glioblastoma multiforme. Kaplan–Meier plots with log rank regression analysis.
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7.3 (3.1) 87.8 (18.2) — 7.3 (3.3) 78.1 (11.9) — 7.5 (3.5) 73.5 (12.0) 8.2 (2.9) 7.9 (2.6) 77.4 (13.1) 44.4 (45.3)* 10.0 (4.2) 73.7 (11.9) —
P b 0.05.
*
All values are mean (SD).
11.4 (11.8) 86.3 (12.6) 18.4 (25.5) 7.5 (4.8) 87.7 (11.5) 11.8 (7.8)
10.3 (4.0) 73.9 (11.0) —
217.5 (60.0) 186.5 (70.4) 255.5 (16.3) 245.5 (177.1) 148.0 (14.1) 173.3 (43.2) 182.3 (71.2)
159.0 (63.7)
22.6 (15.1) 27.3 (18.7) 27.0 (19.8) 19.7 (10.6) 41.0 (2.8) 28.6 (30.2) 22.2 (12.4)
27.0 (18.5)
38.8 (26.9) 54.5 (51.4) 30.0 (31.0) 53.7 (52.4) 84.5 (55.9) 65.0 (42.3) 59.2 (79.1)
121.2 (14.4) 315.9 (90.3) 6.6 (2.2) 30.4 (41.9) (14.8) (76.6) (5.8) (8.4) 125.4 252.0 16.6 11.8 142.6 283.5 9.6 5.9
Haemoglobin (g/liter) Platelet count (10/liter) Leukocytes (10/liter) C-reactive protein (mg/liter) Alanine aminotransferase (IU/liter) Aspartate aminotransferase (IU/liter) Alkaline phosphatase (IU/liter) Total bilirubin (Amol/liter) Creatinine (Amol/liter) Anti-adenovirus antibodies (IU)
(10.2) (65.8) (4.5) (3.2)
Day 19
AdvHSV-tk
Randomised controls 128.3 (11.4) 219.4 (92.1) 15.7 (4.8) 13.3 (8.8)
Day 1
AdvHSV-tk
Randomised controls 142.9 (11.3) 246.7 (106.6) 9.5 (2.9) 12.3 (16.0)
Screening
AdvHSV-tk Laboratory parameter
TABLE 2: Haematology and clinical chemistry results
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45.9 (35.6)
(10.4) (81.3) (2.8) (38.1) 136.5 283.5 7.5 17.7
AdvHSV-tk
Randomised controls 139.5 (12.2) 253 (97.3) 7.4 (2.4) 25.1 (31.1)
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Randomised controls 124.0 (14.7) 220.5 (12.0) 7.3 (1.2) 89.5 (24.7)
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duction efficiency and low penetration of the brain tissue by the retroviruses and retrovirus producing cells [17]. We obtained similar negative results in our previous trial [7] using HSV-tk retrovirus packaging cells. We attribute the better outcome obtained in this trial with AdvHSV-tk gene therapy to improved gene transfer efficiency and stronger transgene expression in the target tissue [18]. We reasoned that the main problem in the treatment of malignant glioma which is in an anatomically accessible location and can be removed by surgery is not so much the removal of the tumour per se but the few remaining malignant cells scattered in the normal brain tissue outside the primary tumour in the walls of the resection cavity. The current treatment approach differs from the previous ones in that it specifically tries to hit these few glioma cells within the normal brain tissue. Also, making healthy brain cells as the main producers of HSV-tk is probably more efficient than what can be achieved by direct transduction of the vector into tumour tissue which is frequently necrotic and may not lead to efficient expression of the transgene. Indeed, we have previously demonstrated that adenoviruses, when used at the current dose (3 1010 pfu), can lead to approximately 10% transduction efficiency in human malignant glioma in vivo [14]. Furthermore, the use of 30-70 small injections covering as much of the surface area of the resected tumour cavity as possible probably leads to a more evenly distributed treatment effect than what has been achieved previously. Even though mostly normal neurons were transduced, the therapeutic effect of the AdvHSV-tk then spreads into the neighbouring cell, including the remaining scattered glioma cells, via the so-called bystander effect. This is the means by which cytotoxic nucleotide analogues can spread from transduced cells to neighbouring nontransduced cells [6,19]. Our earlier studies demonstrated that provided N10% of the target tumour cells contain HSV-tk gene, most of the tumour can be destroyed by GCV [10]. Current in vivo transduction efficiency with adenoviruses approach this level although potential improvements in efficacy may still be achieved by enhancing the delivery of viruses around the tumour cavity and by increasing the percentage of target cells expressing the transgene. In addition, new formulations of GCV with improved bioavailability and penetration into the brain tissue may lead to improved efficacy. In the current study AdvHSV-tk and GCV therapy was well tolerated. Apart from an increase in anti-adenovirus antibodies in 6 patients no major alterations were seen in routine laboratory parameters apart from the mild elevations in liver enzymes. Localised post-operative intracerebral oedema occurred during GCV treatment in 2 patients both of whom required surgical intervention. We believe that these events result from the activity of AdvHSV-tk/GCV therapy on tumour tissue that remained in the brain after surgery. Adenovirus was detected by
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PCR in the plasma of 2 AdvHSV-tk treated patients 3 days after the gene transfer but not thereafter. These results indicate that some systemic release of the adenovirus can occur during surgery, presumably via blood vessels exposed during the operation. However, the adenovirus DNA was eliminated from the circulation and the presence of DNA fragments detectable by PCR does not necessarily indicate the presence of functional transgene in the plasma. Adenovirus mediated HSV-tk gene therapy has been previously used for safety and dose-escalation phase I studies by four other groups [20–23]. Trask et al. [21] reported that a dose of 1 1010 pfu was well tolerated but that a dose of 1 1011 pfu caused central nervous system toxicity. However, this latter dose was found to be welltolerated in the study of Eck et al. [20]. Smitt et al. [22] also reported the lower dose (1 1010pfu) to be well tolerated. Nanda et al. [23] made similar observations using doses up to 4.6 1011 vps. However direct comparison of studies and pfu values should be approached with caution [24]. We conclude that gene therapy with AdvHSV-tk and GCV may be an effective adjuvant treatment for patients with operable primary or recurrent high-grade glioma.
MATERIALS AND METHODS HSV-tk adenovirus. AdvHSV-tk was produced in a HEK293 cell line with the ability to stably express E1 proteins (ECACC, European Collection of Cell Cultures, UK). The first generation serotype 5 adenovirus was used for the gene transfer. The technique involved deletion of the E1 region of the virus (rendering the virus unable to replicate or spread infection) and partial deletion of the E3 region. The HSV-tk was then cloned into the E1region and the structure of the AdvHSV-tk was verified by sequencing [7]. The crude viral lysate prepared in HEK293 cells was used as a crude master virus seed stock to produce clinical batches of AdvHSV-tk (EG009, Cereprok, Ark Therapeutics Ltd, London, UK) [7]. The virus underwent purification through two CsCl gradient centrifugations and was dialysed against 5 mM Hepes, pH 7.8, containing 20% (v/v) glycerol. The final product was sterilized by filtration and stored at –708C in sterile Herpes buffer containing 20% glycerol. Before use, clinical lots of AdvHSV-tk were analysed for quality [7]. Prior to administration, the virus was diluted in physiological saline to a volume of 10 ml and a total dose of 3 1010 plaque forming units (pfu). Patient population. All patients with operable primary or recurrent highgrade glioma referred to the Department of Neurosurgery at Kuopio University Hospital, Kuopio, Finland between May 1998 and January 2002 were considered for the study. Those patients 18 and 75 years of age, with Karnofsky score z70, capable of giving informed consent, were eligible for inclusion. Patients with significant co-existing diseases (including renal or hepatic disease), multifocal or intraventricular tumour and infiltration of the corpus callosum or allergy to GCV were excluded. A total of 36 patients entered the study (Table 1). No patient received AdvHSV-tk treatment more than once and no patient was previously included in any other types of treatment or control cohorts. An additional comparator cohort comprised 36 matched historical control patients derived from the same neurosurgery unit over a 2-year period preceding the study. However, it should be emphasized that the main statistical analysis was done in comparison to the randomised control group (see below).
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The University of Kuopio Ethics Committee and the Finnish National Agency for Medicinal Products approved the study protocol. Although originally designed to include 60 patients, results of an interim analysis of data from the first 36 patients demonstrated a significant improvement in survival for patients receiving AdvHSV-tk treatment. Further recruitment was therefore suspended. Treatment regimen. After diagnosis by clinical assessment and MRI, patients were randomised to receive either AdvHSV-tk or control treatment, having first given written informed consent. Randomization was made prior to beginning of the trial and was based on random digits, whereafter the treatment allotments were placed in sealed envelopes. These pre-made sealed envelopes were opened only after decision to operate had been made and informed consent obtained. Randomisation was stratified for tumour type (primary or recurrent malignant glioma) since the prognosis of the latter group is considerably poorer. Stratification was made to ensure that equal numbers of primary and recurrent malignant glioma patients were assigned to each treatment. All patients underwent craniotomy for radical resection of as much of the identified tumour tissue as possible using routine neuronavigation. Those patients randomised to the active group received 10 ml of 3 1010 pfu AdvHSV-tk injected in aliquots of 0.1 to 0.3 ml to a maximum depth of 10 mm. Administration was directly into the healthy tissue of the wound bed after tumour resection. Depending on the size of the tumour cavity, between 30 and 70 injections were given. This was followed by intravenous GCV at a dose of 5 mg/kg twice daily beginning 5 days after gene therapy, and continuing for 14 days [7]. Patients randomised to the control group underwent surgical resection but did not receive gene therapy or GCV. All patients received steroids and antiepileptics and those with primary tumours also received postoperative radiotherapy (max. 60 Gy). Radiotherapy commenced only after completion of the GCV therapy, between three and six weeks after the surgery. The protocol-specified primary endpoint was survival from the date of operation as defined by death or surgery for recurrence. The secondary endpoints were all cause mortality, tumour progression as determined by MRI and quality of life. MRI was performed at baseline, post-surgery and at 2-monthly intervals thereafter. The scans were interpreted in a blinded manner by at least two independent neuroradiologists. The immediate post-operative scan constituted the baseline to which all subsequent scans were compared. Quality of life was assessed using standardised neuropsychological testing [7]. Routine blood and urine clinical chemistry was performed in the ISO9002-accredited laboratory of Kuopio University Hospital. Samples were analysed before gene therapy, at initial hospitalisation, third postoperative day and then weekly during hospitalisation up to day 19, except for leukocyte differential count, which was measured every second day during GCV administration. Additional samples were taken at 2-monthly intervals. Anti-adenovirus antibodies were measured before and 2 weeks after AdvHSV-tk gene transfer [8]. Polymerase chain reactions (PCR) were performed (by Lark Technology Inc.) from plasma and serum samples taken before AdvHSV-tk gene transfer and at 1, 2, 4 and 7 days thereafter. Real-time methods (ABI Prism 7700) were used to detect adenovirus packaging sequences and HSV-TK sequences. The primers spanned the CMV promoter region and the HSV-tk transgene. Primers: forward primer: ACC GGA AGC TTG GTA CCG A, reverse primer: CCG AAG AGG TGC GGG AG, probe: 6FAM – TTC ACG CCA CCA AGA TCC GAG C – TAMRA. PCR conditions in 50 Al volume were as follows: 508C for 2 min, 958C for 10 min, followed by 45 cycles of 958C for 15 s and 608C for 1 min. The sensitivity of the assay was 16 copies of ppcDNA HSV-tk plasmid. Histological diagnosis was initially performed from frozen perioperative sections and later confirmed from paraffin sections by two independent neuropathologists from Kuopio University Hospital (ISO9002). All histological slides were re-evaluated in a blinded manner to confirm the original diagnosis and no changes in diagnoses resulted. Statistics. The primary endpoint was evaluated using the Kaplan-Meier survival plot with log rank regression. Covariate analysis was performed
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using a Cox regression model. ANOVA and modified t-tests were used for the clinical chemistry evaluation.
ACKNOWLEDGMENTS Authors want to thank Kuopio University Hospital (EVO grant no 5654112) and Ark Therapeutics Ltd for support. We would also like to thank Dr. Leo Palj7rvi, MD (Department of Pathology, University of Kuopio and Kuopio University Hospital) for histological analysis of the resected tumours, Henna Ahtinen, MSc (MedFiles Ltd) for statistics, Alan Boyd, MD (Ark Therapeutics Ltd) for valuable discussions, Riitta Kauppinen (Kuopio University Hospital) for help in collecting hospital records and Marja Poikolainen (A.I.Virtanen Institute, University of Kuopio) for preparing the manuscript. RECEIVED FOR PUBLICATION APRIL 30, 2004; ACCEPTED AUGUST 5, 2004.
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MOLECULAR THERAPY Vol. 10, No. 5, November 2004 Copyright C The American Society of Gene Therapy
doi:10.1016/j.ymthe.2004.08.002
AdvHSV-tk Gene Therapy with Intravenous Ganciclovir Improves Survival in Human Malignant Glioma: A Randomised, Controlled Study Arto Immonen,1,* Matti Vapalahti,1,2,* Kristiina Tyynel7,3 Heleena Hurskainen,1 Anu Sandmair,1 Ritva Vanninen,4 Gillian Langford,5 Neil Murray,5 and Seppo Yl7-Herttuala2,6,7,y 1
Department of Neurosurgery, 3Department of Oncology, 4Department of Radiology, 6Department of Molecular Medicine, A.I. Virtanen Institute, and 7Department of Medicine, University of Kuopio, FIN-70211 Kuopio, Finland 2 Gene Therapy Unit, Kuopio University Hospital, FIN-70211 Kuopio, Finland 5 Ark Therapeutics Ltd., London, UK
*These authors contributed equally to this article. y
To whom correspondence and reprint request should be addressesed at the A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Fax: +358 17 163751. E-mail: [email protected].
Available online 8 September 2004
Malignant glioma is a devastating brain tumour with no effective treatment. This randomised, controlled study involved 36 patients with operable primary or recurrent malignant glioma. Seventeen patients were randomised to receive AdvHSV-tk gene therapy (3 1010 pfu) by local injection into the wound bed after tumour resection, followed by intravenous ganciclovir (GCV), 5 mg/kg twice daily for 14 days. The control group of 19 patients received standard care consisting of radical excision followed by radiotherapy in those patients with primary tumours. The primary endpoint was survival as defined by death or surgery for recurrence. Secondary end-points were allcause mortality and tumour progression as determined by MRI. Overall safety and quality of life were also assessed. Findings were also compared with historical controls (n = 36) from the same unit over 2 years preceding the study. AdvHSV-tk treatment produced a clinically and statistically significant increase in mean survival from 39.0 F 19.7 (SD) to 70.6 F 52.9 weeks ( P = 0.0095, log rank regression vs. randomized controls). The median survival time increased from 37.7 to 62.4 weeks. Six patients had increased anti-adenovirus antibody titers, without adverse effects. The treatment was well tolerated. It is concluded that AdvHSV-tk gene therapy and GCV is a potential new treatment for operable primary or recurrent high-grade glioma. Key Words: adenovirus, malignant glioma, gene therapy, thymidine kinase, survival
INTRODUCTION Malignant glioma is a devastating and fatal disease. The estimated prevalence of brain and central nervous system tumours in the EU and USA is 1.3 per 10,000 persons [1]. Approximately 60% are gliomas and of these some 40 to 50% are glioblastoma multiforme, a particularly aggressive and rapidly fatal form of the disease. Despite surgical and radiotherapeutic advances there is currently no effective treatment and the prognosis for these patients remains poor. Adenovirus-mediated gene therapy may be suitable for the treatment of malignant glioma, which are good targets for gene therapy as they are generally well localised and rarely metastasise out-
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side the central nervous system [2–5]. Therefore, the necessary vectors can be delivered at high concentrations directly to the site of action, thus minimising systemic toxicity. The Thymidine Kinase enzyme (TK) produced by the Herpes simplex virus (HSV) is harmless to man since it lacks a substrate in the human body. However, in cells that express TK, the TK can metabolise intravenously administered ganciclovir (GCV) to produce a cytotoxic nucleotide, which is selective for dividing cells. The nucleotide is incorporated into the DNA of dividing cells during replication where it halts DNA synthesis and results in cell death mainly by apoptosis [2,6]. This is of particular significance in the brain where the normal
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neurons surrounding the tumour are non-proliferative and therefore not susceptible to toxic metabolites. The treatment effect is further strengthened by a bbystander effectQ [2,5]. The hypothesis was that transduction of the AdvHSVtk into the brain, followed by GCV administration, is a potential new treatment for malignant glioma. This randomised, controlled study was designed to assess the efficacy and safety of AdvHSV-tk and intravenous GCV for the treatment of operable primary and recurrent malignant gliomas.
RESULTS Demography We randomized 36 patients; 17 received AdvHSV-tk gene therapy and 19 received control treatment. All patients received the assigned treatment. Patient demographics are shown in Table 1 along with those of the additional cohort of historical controls. Efficacy The mean survival of patients on AdvHSV-tk was 81% longer than for the control group (70.6 F 52.9 [SD] vs. 39.0 F 19.7 weeks, Fig. 1). The median survival in the AdvHSV-tk group was 65% longer than the control group (62.4 weeks vs. 37.7 weeks). The difference in survival was statistically significant ( P = 0.0095), using Kaplan-Meier survival plots and log rank regression analysis. The data were normally distributed indicating a consistent effect across the study population. Statistical significance was confirmed using Cox regression analysis to test the influence of the prognostic factors age, tumour type, histology and Karnofsky score and it was determined that these parameters did not affect the overall outcome.
TABLE 1: Patient demographic characteristics AdvHSV-tk
Randomised controls
Historical controls
17
19
36
12 5
12 7
25 11
Mean Age (Years) and range
51.9 (39–68)
56.5 (35–75)
59.5 (35–75)
TumourType Primary Recurrent Number of GBMs Number of AAs Karnofsky Score (Mean)
12 5 13 4 85.2
12 7 18 1 84.2
36 0 34 2 85.0
Number Gender Male Female
GBM: glioblastoma multiforme; AA: anaplastic astrocytoma.
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Additional post hoc subgroup analyses of the primary endpoint showed an increased median survival in patients with all primary tumours (66.4 weeks vs. 40.3 weeks) and in those with glioblastoma multiforme only (55.3 weeks vs. 37.0 weeks). These differences were statistically significant using Kaplan-Meier survival plots and log rank regression ( P = 0.0322 and P = 0.0214, respectively). The difference between the treated patients with newly diagnosed primary glioblastoma multiforme vs. randomized controls (54.4 weeks vs. 42.8 weeks) also tended to be significant. The improvement in survival in the AdvHSVtk group was maintained at 12-month follow-up ( P = 0.04). All cause mortality was improved with AdvHSV-tk treatment, with an improvement in median survival from 45.0 weeks to 62.4 weeks ( P = 0.0256). Patients treated with AdvHSV-tk also showed a significantly longer mean survival than those in a historical control group: 70.6 F 52.9 weeks vs. 36.0 F 29.9 weeks (Fig. 2). The same was true for median survival (62.4 weeks vs. 30.9 weeks, respectively). The difference between the AdvHSV-tk group and the historical controls was also significant by log rank regression ( P b 0.0017). Furthermore, mean survival for the randomised controls was not significantly different from that of the historical controls (39.0 F 19.7 weeks vs 36.0 F 29.9 weeks). As is evident from Figs. 1 and 2 early survival in the randomized controls seems to be better than in the historical controls which is probably due to the introduction of neuronavigation to aid surgeons to remove as much of the tumour as possible. In relation to tumour progression as determined by MRI, at eight weeks post-operatively progression was observed in 6 out of 15 patients (40%) in the AdvHSV-tk group and 8 out of 16 patients (50%) in the control group. We observed no discernable trends in either an improvement or deterioration in quality of life using standardised neuropsychological tests including Wechsler’s memory scale, quickness and attention, flexibility of behaviour, psychomotor quickness and a symptom and mental agility questionnaire. Furthermore, there was no evidence that patients who survived longer required increasing use of concomitant medication. Safety AdvHSV-tk treatment was well tolerated and no significant safety issues emerged. Three patients in the active group developed transient reversible elevations of liver enzymes after 5 to 7 days of GCV treatment (AST from b36 IU/l at screening to 41-45 IU/l; ALT from b47 IU/l at screening to 101-212 IU/l), but in no case was bilirubin elevated. Aside from these changes no major alterations in routine laboratory parameters occurred (Table 2). Two patients in the AdvHSV-tk group developed localised post-operative intracerebral oedema during GCV treatment, 1 with an associated hemiparesis; both required further surgery but recovered without sequelae. Adenovirus was detected
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ments were well tolerated, and that the mean survival for the AdvHSV-tk group was approximately double (15.0 months, n = 7) that for patients treated with 109 retroviral packaging cells (7.4 months, n = 8) or control patients treated with a lacZ vector injected into the tumour 4-5 days before resection (8.3 months, n = 7). The difference in survival between AdvHSV-tk and retroviral HSV-tk treatment was statistically significant ( P b 0.012) [7]. The protocol-specified primary endpoint used in the current study was survival from the date of operation as defined by death or surgery for recurrence. This latter endpoint could theoretically cause a bias if clinicians know which patients are treated. However, this is unlikely since in the current study only three patients underwent reoperation which was performed to rescue the patients from immediate death thus avoiding potential bias caused by artificial prolongation of the survival time due to reoperation. The HSV-tk approach has been previously tested in retrovirus-based phase I/II studies [4,15,16] and in a large multicenter phase III trial [17]. All these studies used retrovirus-packaging cells producing HSV-tk retroviruses. However, this large Phase III trial failed to show any significant therapeutic effect probably due to low trans-
FIG. 1. Survival of Adv.HSV-tk treated patients and randomised controls. (A) All patients. (B) Patients with glioblastoma multiforme. Kaplan–Meier plots with log rank regression analysis.
by PCR in the plasma of 2 patients in the AdvHSV-tk group 3 days after gene therapy, but not thereafter. Antiadenovirus antibodies increased more than 4-fold in 6 patients in the AdvHSV-tk group, without adverse outcome (Table 2).
DISCUSSION The efficacy of AdvHSV-tk has been assessed in several experimental studies [2,9–13]. However this is the first randomised, controlled study to demonstrate increased survival in patients with operable primary or recurrent high-grade glioma. Our group has performed two previous clinical studies supporting the safety and efficacy of AdvHSV-tk. The first was a dose-ranging study from 1 108 pfu which examined the efficacy of different vectors in the transduction of a marker gene (lacZ) into human malignant gliomas. This study established 3 1010pfu as the dose of adenovirus for subsequent clinical trials [14]. The second study [7] compared the safety and efficacy of the AdvHSV-tk and HSV-tk retrovirus-packaging cells for the treatment of patients with operable primary or recurrent malignant glioma. The results showed that both treat-
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FIG. 2. Survival of AdvHSV-tk-treated patients and historical controls. (A) All patients. (B) Patients with glioblastoma multiforme. Kaplan–Meier plots with log rank regression analysis.
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7.3 (3.1) 87.8 (18.2) — 7.3 (3.3) 78.1 (11.9) — 7.5 (3.5) 73.5 (12.0) 8.2 (2.9) 7.9 (2.6) 77.4 (13.1) 44.4 (45.3)* 10.0 (4.2) 73.7 (11.9) —
P b 0.05.
*
All values are mean (SD).
11.4 (11.8) 86.3 (12.6) 18.4 (25.5) 7.5 (4.8) 87.7 (11.5) 11.8 (7.8)
10.3 (4.0) 73.9 (11.0) —
217.5 (60.0) 186.5 (70.4) 255.5 (16.3) 245.5 (177.1) 148.0 (14.1) 173.3 (43.2) 182.3 (71.2)
159.0 (63.7)
22.6 (15.1) 27.3 (18.7) 27.0 (19.8) 19.7 (10.6) 41.0 (2.8) 28.6 (30.2) 22.2 (12.4)
27.0 (18.5)
38.8 (26.9) 54.5 (51.4) 30.0 (31.0) 53.7 (52.4) 84.5 (55.9) 65.0 (42.3) 59.2 (79.1)
121.2 (14.4) 315.9 (90.3) 6.6 (2.2) 30.4 (41.9) (14.8) (76.6) (5.8) (8.4) 125.4 252.0 16.6 11.8 142.6 283.5 9.6 5.9
Haemoglobin (g/liter) Platelet count (10/liter) Leukocytes (10/liter) C-reactive protein (mg/liter) Alanine aminotransferase (IU/liter) Aspartate aminotransferase (IU/liter) Alkaline phosphatase (IU/liter) Total bilirubin (Amol/liter) Creatinine (Amol/liter) Anti-adenovirus antibodies (IU)
(10.2) (65.8) (4.5) (3.2)
Day 19
AdvHSV-tk
Randomised controls 128.3 (11.4) 219.4 (92.1) 15.7 (4.8) 13.3 (8.8)
Day 1
AdvHSV-tk
Randomised controls 142.9 (11.3) 246.7 (106.6) 9.5 (2.9) 12.3 (16.0)
Screening
AdvHSV-tk Laboratory parameter
TABLE 2: Haematology and clinical chemistry results
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45.9 (35.6)
(10.4) (81.3) (2.8) (38.1) 136.5 283.5 7.5 17.7
AdvHSV-tk
Randomised controls 139.5 (12.2) 253 (97.3) 7.4 (2.4) 25.1 (31.1)
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Randomised controls 124.0 (14.7) 220.5 (12.0) 7.3 (1.2) 89.5 (24.7)
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duction efficiency and low penetration of the brain tissue by the retroviruses and retrovirus producing cells [17]. We obtained similar negative results in our previous trial [7] using HSV-tk retrovirus packaging cells. We attribute the better outcome obtained in this trial with AdvHSV-tk gene therapy to improved gene transfer efficiency and stronger transgene expression in the target tissue [18]. We reasoned that the main problem in the treatment of malignant glioma which is in an anatomically accessible location and can be removed by surgery is not so much the removal of the tumour per se but the few remaining malignant cells scattered in the normal brain tissue outside the primary tumour in the walls of the resection cavity. The current treatment approach differs from the previous ones in that it specifically tries to hit these few glioma cells within the normal brain tissue. Also, making healthy brain cells as the main producers of HSV-tk is probably more efficient than what can be achieved by direct transduction of the vector into tumour tissue which is frequently necrotic and may not lead to efficient expression of the transgene. Indeed, we have previously demonstrated that adenoviruses, when used at the current dose (3 1010 pfu), can lead to approximately 10% transduction efficiency in human malignant glioma in vivo [14]. Furthermore, the use of 30-70 small injections covering as much of the surface area of the resected tumour cavity as possible probably leads to a more evenly distributed treatment effect than what has been achieved previously. Even though mostly normal neurons were transduced, the therapeutic effect of the AdvHSV-tk then spreads into the neighbouring cell, including the remaining scattered glioma cells, via the so-called bystander effect. This is the means by which cytotoxic nucleotide analogues can spread from transduced cells to neighbouring nontransduced cells [6,19]. Our earlier studies demonstrated that provided N10% of the target tumour cells contain HSV-tk gene, most of the tumour can be destroyed by GCV [10]. Current in vivo transduction efficiency with adenoviruses approach this level although potential improvements in efficacy may still be achieved by enhancing the delivery of viruses around the tumour cavity and by increasing the percentage of target cells expressing the transgene. In addition, new formulations of GCV with improved bioavailability and penetration into the brain tissue may lead to improved efficacy. In the current study AdvHSV-tk and GCV therapy was well tolerated. Apart from an increase in anti-adenovirus antibodies in 6 patients no major alterations were seen in routine laboratory parameters apart from the mild elevations in liver enzymes. Localised post-operative intracerebral oedema occurred during GCV treatment in 2 patients both of whom required surgical intervention. We believe that these events result from the activity of AdvHSV-tk/GCV therapy on tumour tissue that remained in the brain after surgery. Adenovirus was detected by
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PCR in the plasma of 2 AdvHSV-tk treated patients 3 days after the gene transfer but not thereafter. These results indicate that some systemic release of the adenovirus can occur during surgery, presumably via blood vessels exposed during the operation. However, the adenovirus DNA was eliminated from the circulation and the presence of DNA fragments detectable by PCR does not necessarily indicate the presence of functional transgene in the plasma. Adenovirus mediated HSV-tk gene therapy has been previously used for safety and dose-escalation phase I studies by four other groups [20–23]. Trask et al. [21] reported that a dose of 1 1010 pfu was well tolerated but that a dose of 1 1011 pfu caused central nervous system toxicity. However, this latter dose was found to be welltolerated in the study of Eck et al. [20]. Smitt et al. [22] also reported the lower dose (1 1010pfu) to be well tolerated. Nanda et al. [23] made similar observations using doses up to 4.6 1011 vps. However direct comparison of studies and pfu values should be approached with caution [24]. We conclude that gene therapy with AdvHSV-tk and GCV may be an effective adjuvant treatment for patients with operable primary or recurrent high-grade glioma.
MATERIALS AND METHODS HSV-tk adenovirus. AdvHSV-tk was produced in a HEK293 cell line with the ability to stably express E1 proteins (ECACC, European Collection of Cell Cultures, UK). The first generation serotype 5 adenovirus was used for the gene transfer. The technique involved deletion of the E1 region of the virus (rendering the virus unable to replicate or spread infection) and partial deletion of the E3 region. The HSV-tk was then cloned into the E1region and the structure of the AdvHSV-tk was verified by sequencing [7]. The crude viral lysate prepared in HEK293 cells was used as a crude master virus seed stock to produce clinical batches of AdvHSV-tk (EG009, Cereprok, Ark Therapeutics Ltd, London, UK) [7]. The virus underwent purification through two CsCl gradient centrifugations and was dialysed against 5 mM Hepes, pH 7.8, containing 20% (v/v) glycerol. The final product was sterilized by filtration and stored at –708C in sterile Herpes buffer containing 20% glycerol. Before use, clinical lots of AdvHSV-tk were analysed for quality [7]. Prior to administration, the virus was diluted in physiological saline to a volume of 10 ml and a total dose of 3 1010 plaque forming units (pfu). Patient population. All patients with operable primary or recurrent highgrade glioma referred to the Department of Neurosurgery at Kuopio University Hospital, Kuopio, Finland between May 1998 and January 2002 were considered for the study. Those patients 18 and 75 years of age, with Karnofsky score z70, capable of giving informed consent, were eligible for inclusion. Patients with significant co-existing diseases (including renal or hepatic disease), multifocal or intraventricular tumour and infiltration of the corpus callosum or allergy to GCV were excluded. A total of 36 patients entered the study (Table 1). No patient received AdvHSV-tk treatment more than once and no patient was previously included in any other types of treatment or control cohorts. An additional comparator cohort comprised 36 matched historical control patients derived from the same neurosurgery unit over a 2-year period preceding the study. However, it should be emphasized that the main statistical analysis was done in comparison to the randomised control group (see below).
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The University of Kuopio Ethics Committee and the Finnish National Agency for Medicinal Products approved the study protocol. Although originally designed to include 60 patients, results of an interim analysis of data from the first 36 patients demonstrated a significant improvement in survival for patients receiving AdvHSV-tk treatment. Further recruitment was therefore suspended. Treatment regimen. After diagnosis by clinical assessment and MRI, patients were randomised to receive either AdvHSV-tk or control treatment, having first given written informed consent. Randomization was made prior to beginning of the trial and was based on random digits, whereafter the treatment allotments were placed in sealed envelopes. These pre-made sealed envelopes were opened only after decision to operate had been made and informed consent obtained. Randomisation was stratified for tumour type (primary or recurrent malignant glioma) since the prognosis of the latter group is considerably poorer. Stratification was made to ensure that equal numbers of primary and recurrent malignant glioma patients were assigned to each treatment. All patients underwent craniotomy for radical resection of as much of the identified tumour tissue as possible using routine neuronavigation. Those patients randomised to the active group received 10 ml of 3 1010 pfu AdvHSV-tk injected in aliquots of 0.1 to 0.3 ml to a maximum depth of 10 mm. Administration was directly into the healthy tissue of the wound bed after tumour resection. Depending on the size of the tumour cavity, between 30 and 70 injections were given. This was followed by intravenous GCV at a dose of 5 mg/kg twice daily beginning 5 days after gene therapy, and continuing for 14 days [7]. Patients randomised to the control group underwent surgical resection but did not receive gene therapy or GCV. All patients received steroids and antiepileptics and those with primary tumours also received postoperative radiotherapy (max. 60 Gy). Radiotherapy commenced only after completion of the GCV therapy, between three and six weeks after the surgery. The protocol-specified primary endpoint was survival from the date of operation as defined by death or surgery for recurrence. The secondary endpoints were all cause mortality, tumour progression as determined by MRI and quality of life. MRI was performed at baseline, post-surgery and at 2-monthly intervals thereafter. The scans were interpreted in a blinded manner by at least two independent neuroradiologists. The immediate post-operative scan constituted the baseline to which all subsequent scans were compared. Quality of life was assessed using standardised neuropsychological testing [7]. Routine blood and urine clinical chemistry was performed in the ISO9002-accredited laboratory of Kuopio University Hospital. Samples were analysed before gene therapy, at initial hospitalisation, third postoperative day and then weekly during hospitalisation up to day 19, except for leukocyte differential count, which was measured every second day during GCV administration. Additional samples were taken at 2-monthly intervals. Anti-adenovirus antibodies were measured before and 2 weeks after AdvHSV-tk gene transfer [8]. Polymerase chain reactions (PCR) were performed (by Lark Technology Inc.) from plasma and serum samples taken before AdvHSV-tk gene transfer and at 1, 2, 4 and 7 days thereafter. Real-time methods (ABI Prism 7700) were used to detect adenovirus packaging sequences and HSV-TK sequences. The primers spanned the CMV promoter region and the HSV-tk transgene. Primers: forward primer: ACC GGA AGC TTG GTA CCG A, reverse primer: CCG AAG AGG TGC GGG AG, probe: 6FAM – TTC ACG CCA CCA AGA TCC GAG C – TAMRA. PCR conditions in 50 Al volume were as follows: 508C for 2 min, 958C for 10 min, followed by 45 cycles of 958C for 15 s and 608C for 1 min. The sensitivity of the assay was 16 copies of ppcDNA HSV-tk plasmid. Histological diagnosis was initially performed from frozen perioperative sections and later confirmed from paraffin sections by two independent neuropathologists from Kuopio University Hospital (ISO9002). All histological slides were re-evaluated in a blinded manner to confirm the original diagnosis and no changes in diagnoses resulted. Statistics. The primary endpoint was evaluated using the Kaplan-Meier survival plot with log rank regression. Covariate analysis was performed
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using a Cox regression model. ANOVA and modified t-tests were used for the clinical chemistry evaluation.
ACKNOWLEDGMENTS Authors want to thank Kuopio University Hospital (EVO grant no 5654112) and Ark Therapeutics Ltd for support. We would also like to thank Dr. Leo Palj7rvi, MD (Department of Pathology, University of Kuopio and Kuopio University Hospital) for histological analysis of the resected tumours, Henna Ahtinen, MSc (MedFiles Ltd) for statistics, Alan Boyd, MD (Ark Therapeutics Ltd) for valuable discussions, Riitta Kauppinen (Kuopio University Hospital) for help in collecting hospital records and Marja Poikolainen (A.I.Virtanen Institute, University of Kuopio) for preparing the manuscript. RECEIVED FOR PUBLICATION APRIL 30, 2004; ACCEPTED AUGUST 5, 2004.
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