SQU Med J, February 2011, Vol. 11, Iss. 1, pp. 5-28, Epub. 12th Feb 11
Invited Article
Submitted 4th Aug 10
Revision ReQ. 5th Oct 10, Revision recd. 25th Oct 10
Accepted 8th Nov 10

Glia-derived neoplasms of the brain (gliomas) of the histological grade 3 (anaplastic astrocytoma, 
anaplastic oligoastrocytoma, and anaplastic 
oligodendroglioma) and grade 4 (glioblastoma 
multiforme, [GBM]) according to the morphology-
based classification of the World Health 

Organization (WHO),1 represent different stages 
of the same fatal neoplastic disease of the central 
nervous system (CNS). WHO grade 3 and 4 gliomas 
are also known as malignant gliomas and are the 
most frequent intrinsic brain tumours in adults.2 
According to the US central brain tumour registry, 
the annual rate of primary tumours of the CNS is 

Department of Neurosurgery, Klinikum Augsburg, D-86156 Augsburg, Germany. 
*Corresponding Author email: nikolai.rainov@klinikum-augsburg.de

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
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aBStract: Advances in medical and surgical treatments in the last two to three decades have resulted in 
quantum leaps in the overall survival of patients with many types of non-central nervous system (CNS) malignant 
disease, while survival of patients with malignant gliomas (WHO grades 3 and 4) has only moderately improved. 
Surgical resection, external fractionated radiotherapy and oral chemotherapy, during and after irradiation, 
remain the pillars of malignant glioma therapy and have shown significant benefits. However, numerous clinical 
trials with adjuvant agents, most of them administered systemically and causing serious complications and side 
effects, have not achieved a noteworthy extension of survival, or only with considerable deterioration in patients’ 
quality of life. Significant attention was focussed in the last decades on the cell biology and molecular genetics of 
gliomas. Improved understanding of the fundamental features of tumour cells has resulted in the introduction and 
increasing clinical use of local therapies, which employ spatially defined delivery methods and tumour-selective 
agents specifically designed to be used in the environment of a glioma-invaded brain. This review summarises the 
key findings of some of the most recent and important clinical studies of locally administered novel treatments for 
malignant glioma. Several such therapies have shown considerable anti-tumour activity and a favourable profile 
of local and systemic side effects. These include biodegradable polymers for interstitial chemotherapy, targeted 
toxins administered by convection enhanced delivery, and intra- and peritumourally injected genetically modified 
viruses conferring glioma-selective toxicity. Areas of possible improvement of these therapies and essential future 
developments are also outlined.

Keywords: Astrocytoma; Convection-enhanced delivery; Glioma; Immunotoxin; Virus

REVIEW

Clinical Development of Experimental 
Therapies for Malignant Glioma

*Nikolai G Rainov and Volkmar Heidecke 



Clinical Development of Experimental Therapies for Malignant Glioma

6 | SQU Medical Journal, February 2011, Volume 11, Issue 1

14.1 per 100,000 persons, of which more than 36% 
are malignant gliomas.3,4 

Malignant gliomas typically arise in the 
lobar white matter or in the deep grey matter 
of the brain and are characterised by diffusely 
infiltrating growth.2,5 These gliomas consist of 
highly proliferative and exceptionally migratory 
tumour cells with a variety of acquired genetic 
alterations which make them extremely resistant 
to antiproliferative therapies.6,7,8 There is no clear 
demarcation of tumour from normal surrounding 
brain tissue to allow the surgeon to completely 
remove a glioma. Neuroradiological imaging 
such as computed tomography (CT) or magnetic 
resonance imaging (MRI) can only visualise areas 
with a high percentage of tumour cells and strong 
neoangiogenesis, but not those with a low density 
of invading glioma cells and neoplastic capillaries.9 
Gliomas expand both by mitotic activity of tumour 
cells and by migration of these cells away from 
the initial tumour mass and into the surrounding 
brain.10 It is now generally accepted that widespread 
migration of glioma cells is a very early feature 
of these tumours, and by the time of clinical 
manifestation and histological diagnosis cells have 
spread throughout the CNS and are located beyond 
the extent of any feasible surgical resection.11,12 
There is evidence to suggest that the density of 
infiltrating malignant cells decreases according 
to the distance from the initial tumour mass, and 
there is a tendency of tumour cells to migrate along 
defined anatomical structures such as white matter 
tracts.5,13 Supporting the notion that malignancy 
declines with distance from the main tumour, the 
fact is well known that most malignant gliomas 
will recur within a few centimetres of the walls of 
the resection cavity or the visible tumour mass, 
respectively.6,10,14 Simple diffusion of cells could 
potentially explain this observation. On the other 
hand, recent insights in glioma cell biology suggest 
that more malignant and actively proliferating cells 
might be less migratory and less invasive, while less 
malignant cells may tend to invade more remote 
areas of the brain which surround the tumour 
mass.10,15

Survival of patients with malignant glioma has 
remained essentially the same in the last three 
decades, despite a quantum leap in medical and 
surgical technology and major advancements in 
understanding the natural history and biology of 

the disease, and the molecular genetics of glioma 
cells.11,16 Median survival time for malignant 
gliomas varies according to different studies, but is 
generally agreed to be 18–20 months for anaplastic 
astrocytomas and 8–14 months for GBM.2,3,11 There 
are no curative treatments for malignant glioma at 
present and no treatment able to prevent tumour 
progression or recurrence. Even the most radical 
surgical resection is unable to cure a malignant 
glioma, although it may significantly prolong 
survival and improve quality of life.6,12,14,17

The current standard therapy of newly 
diagnosed malignant gliomas consists of surgery, 
preferably a gross total resection of contrast-
enhancing areas, subsequent fractionated external 
radiotherapy up to a total dose of 60 Gy, and 
concomitant and adjuvant chemotherapy in WHO 
grade 4 tumours, with various combinations 
of these three approaches.6,8,16,18 The use of 
chemotherapy, in addition to surgery and radiation, 
was considered somewhat controversial until 
recent studies demonstrated significantly improved 
outcome for patients with previously untreated 
GBM receiving radiotherapy plus concomitant and 
adjuvant chemotherapy after surgery.18 However, 
in all clinical studies, significant improvements in 
overall survival are demonstrated mostly in selected 
subpopulations of patients with somewhat better 
prognostic factors, such as a higher Karnofsky 
Performance Status (KPS), younger age, specific 
molecular markers, complete surgical resection of 
tumour, and others.4,11,12,16-18 

Perhaps the most prominent feature, which 
renders malignant glioma a preferred target for 
tumour selective drugs and local treatments, is 
the unique combination of high mitotic activity of 
tumour cells against the background of the mostly 
post-mitotic environment of the adult brain.9,10 In 
addition, malignant gliomas do not metastasise 
systemically, but are extremely aggressive in their 
local environment within the closed compartment 
of the CNS. Multidrug and radiation resistance 
genes are upregulated in primary and recurrent 
glioma and allow tumour cells to escape the toxicity 
of the standard treatment modalities and to continue 
growing even during radio- or chemotherapy.6,10,16

Distinct biological features common in 
malignant gliomas, but unusual in normal brain 
tissue, have been the subject of many research 
studies. Differential expression of specific proteins 



Nikolai G Rainov and Volkmar Heidecke

review | 7

and clinically relevant approaches to local 
intracerebral therapy of malignant gliomas. All 
of these are currently employed in an adjuvant 
setting, in addition to the above mentioned and 
well established standard modalities of surgery 
and fractionated external radiotherapy with 
chemotherapy, and not as their replacements. We 
will focus mostly on therapeutic approaches that 
have demonstrated safety, feasibility, and potential 
anti-tumour efficacy in previous and ongoing 
clinical studies. 

i n t e r s t i t i a l c h e m o t h e r a p y w i t h 
b i o d e g r a d a b l e p o ly m e r wa f e r s 
(g l i a d e l )
Sustained and controlled local delivery of 
therapeutic agents to a brain glioma is one of the 
recent strategies for increasing toxicity to tumour 
cells while reducing systemic side effects. Gliadel  

(MCI Pharma Inc., Bloomington, MN, USA) wafers 
were developed as a means for controlled release 
of a chemotherapeutic agent from biodegradable 
polymer wafers to the surroundings of surgically 
resected malignant glioma.21,22 Their efficacy was 
investigated in several preclinical and clinical 
studies.23-26  Gliadel wafers are currently approved 
for use in patients with primary and recurrent 
glioblastoma as an adjunct to standard treatment 
modalities.

Clinical studies investigating local intracerebral 

in malignant glioma versus the normal brain and 
aberrant activation of signal transduction cascades 
in tumours seems to provide an opportunity for 
selective molecular targeting of tumour cells despite 
the molecularly targeted agents being given mostly 
systemically, thus associated with less toxicity to 
the patient and with a higher rate of tumour cell 
killing.6,8,10,16 All these features make gliomas a 
model disease for the design and testing of novel 
treatments with tumour-selective toxicity and a 
local rather than systemic mode of administration. 

Several novel approaches have been introduced 
into clinical practice in a continuous effort to 
improve the outcome of malignant glioma. In 
general, such approaches can be categorised as 
systemic or local, the latter being intratumoural, 
peritumoural, intracerebral, or combined.19,20 Novel 
systemic approaches demonstrating potential in 
early clinical studies are chemotherapy drugs with 
molecular selectivity mechanisms (such as receptor 
tyrosine kinase inhibitors), and active (e.g. dendritic 
cells) or passive (e.g. antibodies) immunotherapies. 
Novel local intracranial approaches include 
intracerebral delivery of standard chemotherapy 
drugs (interstitial chemotherapy) by slow 
release polymers, recombinant targeted toxins 
administered by convection enhanced delivery 
(CED), or genetically modified viruses with 
molecular selectivity for glioma cells.20,21

This review will summarise the most recent 

Figure 1: Handling and implantation of Gliadel® wafers during brain tumour surgery. A: A single wafer is removed from 
the sterile aluminium foil protective packaging. B: All 8 Gliadel® wafers belonging to a complete pack are removed from 
the packaging and prepared for intracerebral implantation. C: Gliadel® wafers lining the wall of a glioblastoma (GBM) 
resection cavity. Note that the relatively small tumour resection cavity can only accommodate 5 wafers. Slight overlapping 
of wafers is acceptable. D: Implanted wafers are covered with oxidised cellulose (Surgicel®, Ethicon, Hamburg, Germany) 
to prevent dislocation and intracavitary movement. 



Clinical Development of Experimental Therapies for Malignant Glioma

8 | SQU Medical Journal, February 2011, Volume 11, Issue 1

delivery of the well-known and frequently used 
chemotherapy drug, 1,3-bis-(2-chloroethyl)-1-
nitrosourea (BCNU or carmustine) began in the 
early 1990s.23 BCNU was initially chosen due to 
proven efficacy against glioma cell lines. For local 
delivery of BCNU, biodegradable polymer wafers 
were used so as to deliver the drug to the tumour 
resection cavity in a controlled-release fashion over 
time, which should circumvent the short in vivo half-
life of the drug, increase the local concentration of 
BCNU reaching glioma-invaded areas of normal 
brain, and avoid the concomitant toxicities of 
systemic drug application.21,22 

Polymer (prolifeprosan 20) wafers containing 
3.85% (w/w) BCNU, which are also known by the 
brand name Gliadel, are currently the only type of 
interstitial chemotherapy wafers licensed by U.S. 
and European agencies for treatment of de novo and 
recurrent malignant gliomas [Figure 1, A and B]. 
Wafers are placed directly into the resection cavity of 
a malignant glioma at the end of surgical resection of 
the tumour [Figure 1, B and C].22 This may result in 
local BCNU levels approximately 100-fold of those 
obtained with systemic delivery.27 Gliadel wafers 
have been shown to release BCNU in vivo over a 
period of approximately 5 days. When in continuous 
contact with interstitial fluid, wafers have been 
shown to degrade completely over a period of 6–8 
weeks. Polymer degradation products are excreted as 
expired CO2 or through the urine. BCNU degradation 
products are also excreted primarily through the 
urine.21,27

No direct pharmacokinetic measurements have 
been made in humans after the implantation of 
Gliadel wafers; however, drug distribution and 
clearance have been extensively studied in rodents 
and primates. In addition, degradation of the 
polymer matrix, the release kinetics of BCNU, and 
drug and polymer metabolism have been studied 
both in vitro and in vivo.27 Pharmacokinetic studies 
have demonstrated the capability of Gliadel wafers 
to deliver high (mM) concentrations of BCNU 
within a few millimetres of the polymer implant 
and with a limited penetration distance. The limited 
interstitial spread of the drug is presumably due to 
the high transcapillary permeability of this lipophilic 
molecule.22 However, convective flow in the existing 
peritumoural oedema may augment diffusion.27,28

In 1991, Brem et al. established a significant 
survival advantage of Gliadel wafers over placebo 

in patients with recurrent malignant glioma.23 
They conducted a randomised, placebo-controlled, 
prospective phase 3 clinical study to evaluate the 
effectiveness of Gliadel wafers. Their multicentre 
study enrolled 222 patients with recurrent malignant 
brain tumours requiring re-operation. Cases were 
randomly assigned to surgically implanted wafers 
with or without BCNU. Median survival of the 110 
patients who received Gliadel wafers was 31 weeks 
compared with 23 weeks for the 112 patients who 
received placebo wafers (P = 0.006). Among GBM 
patients, 6-month survival in the treated group was 
50% greater than in the placebo group. There were 
no clinically important adverse reactions related 
to the carmustine polymer, either in the brain or 
systemically.23,24 

A phase 1 trial was carried out in patients with 
newly diagnosed GBM.28 Twenty-two mostly elderly 
patients (mean age 60 years) with newly diagnosed 
malignant glioma (21 with GBM) were treated 
with Gliadel wafers. Postoperatively, all patients 
received standard radiation therapy, but no additional 
chemotherapy in the first 6 months. The neurotoxicity 
of this regimen was equivalent to that occurring in 
other series of patients undergoing craniotomy and 
radiation. No significant bone marrow suppression 
occurred and there were no wound infections. 
Median survival of the whole group was 42 weeks, 
8 patients survived 1 year, and 4 patients survived 
more than 18 months. The authors concluded that 
interstitial chemotherapy with Gliadel wafers with 
subsequent radiation appears to be safe as an initial 
therapy for malignant glioma.28 

In 2004, Kleinberg et al. described, in a 
retrospective review, the clinical course, toxicity, and 
pathologic findings after Gliadel wafer implantation 
for newly diagnosed malignant glioma.29 Forty-
five consecutive patients, most of them with GBM, 
received Gliadel wafer implants followed by 
radiotherapy and were available for follow-up. 
Postoperative infection, or the need for reoperation 
within 30 days was uncommon after Gliadel wafer 
placement. Full-dose external radiotherapy was 
well tolerated after Gliadel implantation; however, 
13 patients developed neurological symptoms 
during radiotherapy, but responded to medication 
(dexamethasone and/or anticonvulsants). Fifteen of 
these 45 patients underwent re-operation or biopsy 
for a new local contrast-enhancing lesion. In 5 of 
these, histological analysis revealed necrosis or 



Nikolai G Rainov and Volkmar Heidecke

review | 9

patients (P = 0.017), with a hazard ratio of 0.73 (P 
= 0.018), representing a significant risk reduction 
of 27%. This survival advantage was maintained 
at 1, 2, and 3 years and was statistically significant 
(P = 0.01) at 3 years. Two of the 207 GBM patients 
remained alive at the end of the follow-up period, 
both in the Gliadel group. The authors concluded 
that malignant glioma patients treated with Gliadel 
at the time of initial surgery in combination with 
radiation therapy demonstrated a continuous and 
significant survival advantage at 2 and 3 years 
follow-up compared with placebo patients.26

Additional studies were aimed at increasing the 
clinical effects of Gliadel treatment using different 
strategies. Olivi et al. treated 44 adult patients with 
surgery and implantation of BCNU wafers.31 Six 
patients per dose level were studied using wafers 
with 6.5%, 10%, 14.5%, 20%, and 28% BCNU 
(weight/weight). Toxicity was assessed one month 
after wafer implantation. No dose-limiting toxicity 
was identified at the 6.5%, 10%, or 14.5% dose 
levels, although delayed wound healing, seizures, 
and brain oedema were noted. At the 20% dose, 
these side effects seemed more prominent, and 6 
additional patients were treated at this dose and 
tolerated treatment well. Three of 4 patients receiving 
28% BCNU wafers developed severe brain oedema 
and seizures. Nine additional patients received 20% 
wafers, confirming this as the maximum tolerated  
dose (MTD). Maximum BCNU plasma concen- 
trations with the 20% wafers were 27 ng/ml. Overall 
median survival of the patients was 12 months. 
The authors concluded that 20% BCNU wafers 
are relatively well tolerated and result in minimal 
systemic BCNU exposure.31 Additional studies are 
however needed to establish the efficacy of high-dose 
BCNU wafers and a possible significant extension 
of survival due to the increased concentration of 
BCNU.

Beginning in 2004, concomitant temozolomide 
(TMZ) during fractionated brain irradiation (Stupp 
protocol) became the standard of care at most 
neurooncology centres in Europe and the USA.18 
Patients with newly diagnosed GBM and Gliadel 
implantation received therefore concomitant TMZ 
and irradiation radiochemotherapy); however, it 
remained unstudied whether combining Gliadel 
and radiochemotherapy is safe or further improves 
survival in such patients. In 2010, McGirt et al. 
reviewed retrospectively the initial experience with 

post-treatment scars without active tumour. Median 
survival was 12.8 months for all GBM patients.29

To corroborate the evidence from early clinical 
trials, a randomised controlled trial planned to 
include 100 cases was carried out in patients 
undergoing surgery for newly diagnosed gliomas, 
but was terminated prematurely after enrolment of 
32 patients because the Gliadel implants became 
unavailable.30 The median overall survival time was 
58.1 weeks for the active treatment group versus 39.9 
weeks for the placebo group (P = 0.012). At the end 
of the study, 6 patients were still alive, 5 of whom 
belonged to the active treatment group. Results were, 
however, not corrected for the early termination and 
thus a possible bias may have been introduced in the 
statistical evaluation.30 

Westphal et al. reported the results of a further 
multicentre phase 3 study investigating Gliadel 
wafer treatment in 240 patients with newly 
diagnosed GBM.25 Patients were randomised to 
receive either BCNU or placebo wafers at the time 
of primary surgical resection. All patients were given 
identical standard treatment consisting of surgical 
resection followed by fractionated external radiation. 
Exclusion criteria were recurrent tumour, prior brain 
radiotherapy, and multifocal disease. The primary 
clinical endpoint was overall survival. Median 
survival in the intent-to-treat (ITT) population was 
13.9 months for the Gliadel-treated group, and 11.6 
months for the placebo-treated group, with a 29% 
reduction in the risk of death in the treatment group. 
Time to decline in KPS and clinical parameters (e.g. 
vital signs, level of consciousness, neurological 
status), were also significantly improved in the 
treatment group compared to the placebo group. The 
adverse events (AE) were comparable for the two 
groups, except for cerebrospinal fluid (CSF) leak 
(5% in the Gliadel group versus 0.8% in the placebo 
group) and intracranial hypertension (9.1% in the 
Gliadel group versus 1.7% in the placebo group). 
This study is the largest clinical investigation of 
Gliadel activity to date.25 

In a later study, the authors reported the long-
term follow-up results of the above patient group 
regarding survival benefits at 2 and 3 years after 
Gliadel implantation.26 Of the 59 patients available 
for long-term follow-up, 11 were alive at 56 months; 
9 having received Gliadel and 2 placebo wafers. The 
median survival of patients treated with Gliadel was 
13.8 months versus 11.6 months in placebo-treated 



Clinical Development of Experimental Therapies for Malignant Glioma

10 | SQU Medical Journal, February 2011, Volume 11, Issue 1

application. Most importantly, there is only a 
modest short-term clinical benefit in most patients, 
while long-term survival is prolonged in a small 
subset of malignant glioma patients only. The side 
effects of Gliadel are not negligible, and the high 
cost of treatment is an important issue, at least in 
most European Union (EU) countries. In the future, 
local release wafers will need to be optimised for 
increased anti-tumour activity, either by increasing 
the concentration and improving the release 
kinetics of well-known agents such as BCNU, or 
by employing novel agents with a more potent 
anti-tumour effect, e.g. mitoxantrone or IL-2.34 
Combinations of local release wafers with standard 
adjuvant therapies such as radiochemotherapy or 
single-drug oral chemotherapy (e.g. with TMZ) may 
become a standard of care if more reliable clinical 
evidence is presented for increased survival and 
unchanged morbidity with these combinations.33

Targeted Toxins 
(Immunotoxins)
Targeted toxins represent a new class of anti-
cancer agents providing high specificity for 
tumour cells selectively overexpressing some 
surface proteins.35,36 Currently used toxins are 
recombinant polypeptide molecules consisting 

Gliadel and radiochemotherapy in GBM patients.32 
Thirty-three patients were treated with the combined 
modalities and the median survival in the group was 
20.7 months, with a 2-year survival rate of 36%. Six-
month morbidity included surgical site infection in 
1 case (3%), perioperative seizures in 2 cases (6%), 
deep-vein thrombus in 1 (3%), pulmonary embolism 
in 3 (9%), and cerebral oedema requiring intravenous 
dexamethasone administration in 1 case (3%). 
Myelosuppression required premature termination 
of TMZ in 7 patients (21%): (thrombocytopenia in 5, 
neutropenia in 2 cases). In patients < 70 years of age, 
Gliadel and radiochemotherapy were independently 
associated with improved median survival (21.3 
versus 12.4 months, P = 0.005). Moreover, the 
combined modalities were not associated with an 
increase in postoperative morbidity in comparison 
with Gliadel or radiation only. The authors 
concluded that radiochemotherapy can be safely 
administered to patients receiving Gliadel wafers 
after resection of newly diagnosed GBM.32,33

Conclusions and Future 
Developments - Gliadel ® 
Therapy
Despite the convenience and relative simplicity of 
use of intraoperatively placed Gliadel wafers, there 
are some factors limiting their widespread clinical 

Figure 2: Schematic and 3D-ribbon representations of the structure of the recombinant fusion toxin IL13-PE38 (IL13-
PE38QQR). A derivative of the native Pseudomonas exotoxin A (PE), PE38QQR,  in which domain Ia (amino acids 1 
to 252) has been completely deleted and domain Ib (amino acids 365 to 380) has been partially deleted; lysine residues 
at positions 590 and 606 have been replaced by glutamine, and at position 613 by arginine, and it has been fused to the 
carboxy terminus of the human IL-13 to produce the fusion toxin IL13-PE38QQR (IL13-PE38).  



Nikolai G Rainov and Volkmar Heidecke

review | 11

of these toxins reaching phase 3 trials.43  

il4-pe (nbi-3001)
This chimeric recombinant fusion protein (also 
known as NBI-3001, Neurocrine Inc., S. Diego, CA) 
is composed of circularly permuted interleukin-4 
(IL-4) and a truncated form of P. aeruginosa 
exotoxin (PE) A.44 PE is a 66 kD protein with three 
domains: Ia/Ib, II, and III. When domain Ia is 
removed, the resulting molecule (termed PE-40) 
retains its translocation function and elongation 
factor 2 (EF-2) inhibition properties, but is unable 
to bind to and kill human cells.35 Domain II is 
the site of proteolytic cleavage and is responsible 
for catalysing translocation of the toxin into the 
cytosol. Domain III, located at the C-terminus, 
possesses ADP-ribosylation activity which in turn 
leads to inactivation of EF-2 and to cell death. In the 
genetically engineered PE molecule PE38KDEL, 
amino acids (AA) 253–364 were linked to AA 
381–608, which in turn were fused to KDEL (an 
endoplasmic reticulum retaining sequence) at 
position 609–612. To improve the binding of IL-4 
toxin to the IL-4 receptor (IL4-R), a circularly 
permuted form of IL-4 was fused to the toxin. This 
new agent was termed IL-4(38-37)-PE38KDEL, 
or IL4-PE.45,46 IL4-PE was found to have a 16-fold 
higher affinity for binding to GBM cell lines than 
the native PE toxin, and was 3–30-fold more toxic 

of a tumour-selective ligand coupled to a highly 
potent peptide toxin, which is truncated to abolish 
native toxicity.37 The most frequently used and 
best researched ligands bind to tumour-associated 
molecules with receptor signalling properties, 
such as epidermal growth factor receptor (EGFR), 
transferrin receptor (TfR), interleukin-13 receptor 
(IL-13R), or interleukin-4 receptor (IL-4R).38 The 
toxin part of the molecule in all clinically used 
toxins is a polypeptide derived from bacteria 
(Corynebacterium diphtheriae, Pseudomonas 
aeruginosa), which has been modified by deleting 
the native targeting and internalisation chains of 
the polypeptide and replacing them with one of the 
above ligands [Figure 2].39,40 

The mechanism of action of targeted toxins may 
have important advantages over that of radiation and 
classic chemotherapeutic agents. Toxins are effective 
against radiation-resistant hypoxic tumour cells and 
far more potent than any chemotherapy drug. One 
single molecule of toxin is sufficient to cause tumour 
cell death independent of any malignancy-associated 
genetic alterations and/or mutations  [Figure 3].37 
Multidrug resistance and apoptosis resistance are 
therefore not an issue with toxins; after receptor 
binding and internalisation, no tumour cell is able to 
survive the toxin part of the molecule.38,41,42 A few 
targeted toxins have advanced from the laboratory to 
the stage of phase I and II clinical studies, with two 

Figure 3: Schematic representation of the mode of action of the recombinant fusion toxin IL13-PE38 (IL13-PE38QQR). 
IL-13 receptor (IL-13R-ß2 subunit) is a tumour-specific protein in human malignant glioma cells. Immune cells, 
endothelial cells and normal glia and neurons express none or very low amounts of IL-13R. IL13-PE38 is highly selective 
and potent in killing human glioblastoma multiforme cells by binding to IL-13R, but does not kill normal cells, which are 
IL-13R-negative.



Clinical Development of Experimental Therapies for Malignant Glioma

12 | SQU Medical Journal, February 2011, Volume 11, Issue 1

A multicentre, randomised, open-label phase 
2 study with IL4-PE was carried out in patients 
with recurrent GBM to investigate continuous 
intratumoural infusion of the toxin followed by 
surgical resection of the tumour. The study was 
designed to evaluate the efficacy of IL4-PE, with 
a secondary objective to evaluate the safety and 
tolerability of the toxin.52 In the toxin group, patients 
received an intratumoural infusion of toxin at 
total doses of up to 90 µg and underwent surgical 
resection of the tumour between 2 and 7 days after 
the end of toxin infusion. Patients in the control 
group underwent tumour resection without prior 
toxin treatment. A total of 30 adult patients with 
unilateral, unifocal tumours with a volume <100 
ml and a KPS ≤60 were enrolled. Recruitment was 
completed in 2003, but no final published results of 
the study are available yet.52 There are currently no 
phase 3 protocols using IL4-PE.

tp-38
TP-38 (Teva Pharmaceuticals, formerly IVAX Inc., 
Miami, FL, USA) is a recombinant chimeric protein 
composed of transforming growth factor α (TGF-α), 
an epidermal growth factor receptor (EGFR) ligand, 
and the genetically engineered form of PE described 
above (see IL4-PE). 

The 170-180 kD transmembrane glycoprotein, 
epidermal growth factor (EGF) receptor, is one of 
four members of the erbB family of receptor tyrosine 
kinases, which consist of an extracellular domain 
that can bind ligands, a transmembrane domain and 
an intracellular tyrosine kinase domain.53 Binding of 
a ligand (EGF or TGF-α) to EGFR causes receptor 
dimerisation leading to tyrosine kinase activation.54 

The resultant receptor autophosphorylation initiates 
signal-transduction cascades involved in cell 
proliferation and survival.53 After ligand binding, 
the whole receptor-ligand complex undergoes 
endocytosis and is translocated to the lysosomes, 
where the ligand dissociates from the receptor.55  

Human malignant gliomas and many other 
malignant tumours that metastasise to the brain 
express EGFR, and this is commonly associated with 
amplification and/or mutation of the EGFR gene 
during neoplastic transformation.54 Amplification 
and high expression of EGFR in gliomas may drive 
tumour growth and proliferation to a significant 
degree.55 By contrast, EGFR is expressed at very low 
levels, or is undetectable on normal human glial cells 

to GBM cells.42,47

IL-4 is a pleiotropic cytokine composed of 4 
α-helices which is primarily produced by Th2-type 
T lymphocytes. Whereas normal CNS cells such as 
endothelial cells, microglia, or astrocytes express 
low levels of IL4-R, malignant glioma cells express 
high levels of this receptor on their surface.44,48 In one 
series, 16 of 21 surgical samples were determined to 
express high numbers of IL4-R.36 In another series, 
all of the 25 tested surgical specimens of high-grade 
gliomas expressed IL4-R. In contrast, no detectable 
IL-4R was expressed in a normal brain as determined 
by immunohistochemistry.49

Cell culture studies and animal models have 
confirmed that IL4-PE is highly cytotoxic to glioma 
cells.50 IL4-PE has a 16-fold higher affinity for 
binding to GBM cell lines than the native PE toxin 
and is 3–30-fold more toxic to GBM cells.47,48 
Whereas normal CNS cells such as endothelial 
cells, microglia, or astrocytes express low levels 
of IL-4R, malignant glioma cells do express high 
levels of this receptor on their surface.42 A recent 
study demonstrated that 15 of 18 GBM and 12 other 
brain tumour samples were moderately to intensely 
positive for IL-4R. In contrast, no detectable IL-4R 
was expressed in a normal brain as determined by 
immunohistochemistry.44

Weber et al. carried out an open-label, dose-
escalation trial of intratumoural administration of 
IL4-PE in patients with recurrent malignant glioma.51 
A total of 31 adult patients were enrolled, 25 with 
GBM and 6 with anaplastic astrocytoma. Patients 
were assigned to one of 4 groups receiving different 
doses of toxin in different volumes of infusate. The 
toxin was administered by CED via an external 
pump connected to stereotactically placed catheters. 
The overall median survival for the whole group of 
WHO grade 3 and 4 tumours was 8.2 months, with 
a median survival of 5.8 months for GBM patients. 
Six months survival was 52% and 48%, respectively. 
Drug-related grade 3 or 4 CNS toxicity was seen in 
all groups in a total of 39% of all patients, and in 
22% of patients at the MTD of 6 µg/ml in 40 ml. 
Treatment-related AE were limited to the CNS. 
No deaths were attributable to treatment. No drug-
related systemic toxicity was apparent in any patient. 
Gadolinium-enhanced MRI of the brain in some of 
the treated patients showed areas of decreased signal 
intensity within the tumour consistent with toxin-
mediated tumour necrosis.51



Nikolai G Rainov and Volkmar Heidecke

review | 13

infusion rate was 200 µl/h per catheter. Each catheter 
delivered 13.4 ml over 67 h. The total volume 
infused was approximately 40 ml, and the total 
dose of TP-38 infused was 2 μg (50 ng/ml) or 4 μg 
(100 ng/ml). One patient had a complete response 
48 weeks after infusion, and another showed a 
partial response stable over 60 weeks. Twenty-four 
patients remained stable. Post-infusion MRI scans 
in most patients showed unspecific treatment-
related changes such as halo contrast enhancement 
around the infusion sites, which made assessment 
of response rather difficult. These changes usually 
resolved by 20 weeks post-treatment. There were 
no grade 3 and 4 toxicities related to TP-38, and all 
adverse effects of the treatment were reversible.58 A 
phase 3 clinical protocol employing TP-38 seems 
to be in development, but no further details are 
currently known.

il13-pe38
IL13-PE38 (IL13-PE38QQR, also known as 
cintredekin besudotox, (NeoPharm Inc., Lake 
Forest, IL, USA) is a recombinant chimeric toxin 
consisting of human IL-13 fused to the mutated 
form of PE mentioned above [Figure 2]. The 
T-helper cell 2-derived immunoregulatory cytokine 
interleukin-13 (IL-13) inhibits the production of 
inflammatory cytokines in monocytes. The cell 
surface receptor for IL-13 (IL-13R) is expressed on 
many human cancer cells, including glioblastoma, 
AIDS-associated Kaposi’s sarcoma, ovarian 
carcinoma,  renal cell carcinoma, but also fibroblast 
cell lines.59 The IL-13R structure varies according to 
cell type, existing in three different forms known as 
type I, type II and type III.60 

Subunit structure studies of the IL-13R complex 
in primary brain tumour cells established the IL-
13Rα2 subunit as a tumour-specific protein.59 
Immune cells, endothelial cells and normal glia and 
neurons generally express none or very low amounts 
of IL-13R.39 

IL13-PE38 was found to be highly selective and 
potent in killing human glioma cells in culture. The 
activity of IL13-PE38 is mediated via IL-13R, as its 
cytoxicity is blocked by exposing cell lines to a 10– 
to 100-fold excess of human IL-13.61 Furthermore, 
IL-13R-negative cell lines or cell lines expressing 
low numbers of IL-13R, including human bone 
marrow-derived cells and PHA-activated T-cells, are 
not susceptible to IL13-PE38 mediated cytotoxicity 

and neurons thus suggesting a potential therapeutic 
window. The ratio of EGFR expression in glioma 
versus normal control brain specimens has been 
shown to be as high as 300-fold.56

Animal studies with TP-38 have been carried 
out in rodent and primate models. In tumour bearing 
mice, a total dose of 0.1 µg TP-38 at a concentration 
of 5 µg/ml was found to be safe and efficacious in 
prolonging survival. The MTD of intracerebral 
TP-38 in normal rats was 0.666 µg at a toxin 
concentration of 33.3 µg/ml. In non-human primates, 
it was demonstrated that an intracerebral bolus dose 
of 2 µg TP-38 at a concentration of 10 µg/ml is safe.50

Sampson et al. investigated TP-38 in a phase 1 
clinical trial.57 The primary objective of the study 
was to define MTD and dose limiting toxicity of 
TP-38 delivered by CED in patients with recurrent 
malignant glioma. A secondary objective was to 
detect the efficacy of the toxin. Twenty patients 
were enrolled in the study and doses were escalated 
from 25 to 100 ng/ml. TP-38 was infused by two 
stereotactically placed catheters at a flow rate of 0.4 
ml/h for each catheter. A total volume of 40 ml was 
infused. TP-38 was tolerated well, and an MTD was 
not established. Non-specific toxicity was not found 
at any of the dose levels. The toxicity encountered 
was solely neurologic and mostly related either to 
infusion volume, recurrent tumour, or stereotactic 
catheter placement, but not directly to TP-38. Fifteen 
of the patients in this study died from progressive 
disease. When the study closed, 4 further patients 
had not had a recurrence of tumour and were 55, 56, 
69, and 116 weeks from the time of TP-38 therapy. 
Overall median survival after TP-38 for all patients 
was 23 weeks, whereas for those without radiographic 
evidence of residual disease at the time of therapy, 
the median survival was 31.9 weeks. Overall, 3 of 15 
patients with residual disease at the time of therapy 
have demonstrated radiographic responses.57

A multicentre randomised open-label phase 2 
study was conducted  in adult patients with recurrent 
GBM.58 Patients were randomised to two dose levels 
of TP-38, 50 ng/ml or 100 ng/ml, and toxin was 
administered by CED via stereotactically placed 
catheters. Patients did not have to undergo tumour 
resection prior to treatment. The end points of the 
study were time to progression, progression-free 
survival, and overall survival. Three catheters were 
stereotactically placed in investigator-determined 
locations within the enhancing tumour area. The 



Clinical Development of Experimental Therapies for Malignant Glioma

14 | SQU Medical Journal, February 2011, Volume 11, Issue 1

were placed into the brain adjacent to the tumour 
resection cavity and on days 10 to 14, IL13-PE38 was 
given at 750 µl/h for 96 h at a fixed dose of 0.25 µg/ml 
(total dose 18 µg). In stage 2, intratumoural infusion 
was omitted and patients underwent resection 
of the tumour followed by a 96-h peritumoural 
infusion of IL13-PE38 at 0.5 µg/ml and 1.0 µg/ml. 
In stage 3, the duration of peritumoural infusion 
was increased from 5 to 7 days at a fixed dose of 
0.5 µg/ml. Tumour necrosis up to 2.5 cm from the 
catheter tip was demonstrated radiologically in at 
least 5 patients with preoperative intratumoural 
toxin infusion. Tumour specimens from at least 
two patients after intratumoural injection of 0.5 
µg/ml IL13-PE38 toxin revealed regional necrosis 
in an ovoid zone extending 1 to 2 cm from the 
catheter tip. Peritumoural post-resection MTD was 
defined as 0.5 µg/ml. Similar AEs occurred across 
all cohorts, most being neurological. The most 
frequent AE were headache (53%), hemiparesis 
(27%), sensory disturbance (20%), fatigue (17%), 
aphasia (13%), facial paresis (13%), abnormal gait 
(13%), convulsions (10%), hypaesthesia (10%) and 
lymphopenia (10%). Prolonged individual patient 
survival has been observed after peritumoural 
therapy at concentrations of 0.25 µg/ml (total dose 
18 µg) and above.39,66,67

A further multicentre phase 1/2 study 
investigated IL13-PE38 in adult recurrent 
supratentorial malignant glioma with confirmed 
radiographic evidence of recurrent or progressive 
tumour. The primary objective of the phase 1 portion 
of the study was to determine the optimum duration 
of infusion and drug concentration of IL13-PE38 
delivered by CED via 1 or 2 intratumoural catheters 
prior to surgical resection of the tumour.64,67 The 
phase 2 objective was to determine the proportion 
of patients surviving at 6 months. In phase 1, 
cohorts of 3-6 patients received a fixed drug 
concentration (0.5 µg/ml) with escalating duration 
of infusion (4–7 days) to determine MTD based on 
duration. After determining maximum duration, 
the drug concentration was escalated from 1.0 to 
4.0 µg/ml in cohorts of 3–6 patients to determine 
a MTD based on concentration. Tumour necrosis 
was assessed 6 to 13 days after the end of infusion, 
at the time of tumour resection. There were no 
severe drug-related AE in the 10 treated patients. 
Five patients experienced AE grade 2–3. The most 
frequent AE were headache (70%), hemiparesis 

[Figure 3].62

The significant antitumour activity of IL13-
PE38 was observed in animal models of human 
glioma, with intratumoural injection proving more 
efficacious than intravenous (i.v.) or intraperitoneal 
(i.p.) administration. In nude mice with intracerebral 
glioma, the MTD of IL13-PE38 was 4 µg when 
delivered by bolus injection, and 10 µg when 
delivered by CED.39 

Several phase 1 and 2 studies have been 
initiated to investigate IL13-PE38 as an antitumour 
agent for the treatment of patients with recurrent 
malignant glioma. In a phase 1/2 study, IL13-PE38 
was infused intratumourally in adult patients with 
recurrent malignant glioma (including anaplastic 
oligoastrocytoma) to determine the MTD of the 
drug.63 Patients received two IL13-PE38 infusions 
at 8-week intervals via two stereotactically placed 
intratumoural catheters at a constant infusion rate. 
One aim of the study was to determine MTD and 
toxicity of IL13-PE38 administered by CED via 
intratumoural catheters at 200 µl/h/catheter for 
96 h (total 38.4 ml), in two courses 8 weeks apart. 
Escalation was planned through 9 levels ranging 
from 0.125 to 12.0 µg/ml (total dose 4.8 to 460.8 
µg) in cohorts of 3 patients per level. Cohorts 
of 3 patients received 0.5, 1.0, 2.0 and 4.0 µg/ml. 
Concentrations of up to 2.0 µg/ml were safe and 
well tolerated. The AE reported across all dose 
ranges were mild and mainly neurological. The 
most frequent drug-related AE were headache 
(24%), hemiparesis (24%), brain oedema (14%), 
aphasia (10%), and ataxia (10%). In the phase 1 
portion of the study, two histopathological and 
two radiographic responses were observed, with 
progression-free survival ranging from 3 to 88 
weeks and overall survival of 147 weeks. No results 
have been published yet for the phase 2 portion of 
this study.63

Another phase 1 study investigated IL13-PE38 
in adult supratentorial malignant glioma. In this 
four-stage study, the primary objectives were to 
determine effective dose of IL13-PE38 either prior 
to or post-resection of the tumour.64,65 In stage one, 
after biopsy and intratumoural catheter placement 
on day 1, IL13-PE38 was administered for 48 h at 
400 µl/h on days 2 to 4 at escalating doses ranging 
from 0.25 to 2 µg/ml. The tumour was resected on 
day 8 and tissue was evaluated for necrosis adjacent 
to the catheter. Following resection, 2 or 3 catheters 



Nikolai G Rainov and Volkmar Heidecke

review | 15

pulmonary embolism was higher in the toxin arm (8% 
versus 1%, P = 0.014). This was the first randomised 
phase 3 evaluation of an agent administered via 
CED and the first with an active control group in 
GBM patients. There was no survival difference 
between IL13-PE38 toxin administered via CED and 
Gliadel wafers placed at the time of surgery. Drug 
distribution was not assessed, but may be crucial for 
evaluating future CED-based therapeutics.43,68

TransMID-107  
(Tf-CRM107)
TransMIDTM (known as TransMID-107, Tf-
CRM107, or KSB-311; KS Biomedix Holdings 
plc, UK, now Xenova Group plc, UK, and Celtic 
Pharmaceutical Holdings PL, Bermuda) is a 
thioether conjugate of human transferrin (Tf ) with 
a truncated natural mutant form of diphtheria toxin 
(DT) known as CRM-107, which lacks receptor 
binding.69

Native DT is produced by the bacterium C. 
diphtheriae and is composed of two disulphide-
linked subunits. The A subunit catalyses ADP-
ribosylation of EF-2, thereby stopping protein 
synthesis and killing the cell. The B subunit has 
two functions: binding to cell-surface receptors and 
translocation of the A subunit into the cytosol.70 In 
the natural mutant form of DT, designated CRM-
107, two point mutations (phenylalanine at positions 
390 and 525 of the DT sequence) reduce the binding 
8,000-fold and its toxicity 10,000-fold, compared to 
native DT.71 However, conjugation of CRM-107 by 
thioether linkage to various new binding moieties was 
able to reconstitute the full toxicity of the mutant.72 
By linking CRM-107 with Tf, a receptor-ligand 
complex was created that is rapidly internalised to 
an environment that facilitates toxin translocation.70

The Tf receptor (TfR) is a transmembrane 
glycoprotein that mediates cellular uptake of 
iron. TfR are overexpressed on rapidly dividing 
cells, such as hematopoietic and neoplastic cells, 
including glioma cells.72 TfR in normal brains are 
sparse and their expression is largely restricted 
to the luminal surface of brain capillaries.73 It is 
this relative difference in the density of TfR that 
TransMIDTM uses to generate differential toxicity 
to highly TfR expressing neoplastic cells, while 
sparing low expressing normal brain cells.74 Low-
density background expression of the respective 

(40%), seizures (40%), oedema (40%) and aphasia 
(30%). Progression-free survival ranged from 6 to 30 
weeks and overall survival from 10 to 30 weeks.39,64

In all of the above studies, intratumoural 
infusion of IL13-PE38, with or without tumour 
resection in patients with recurrent or progressing 
malignant glioma, seemed to be well tolerated and 
did not result in any grade 3 and 4 AE, or in any 
AE in peripheral organs such as the liver, kidney 
and lungs. Neurological AE during and after toxin 
infusion were, however, encountered in a significant 
proportion of the treated patients. These included 
brain oedema, meningitis, seizures, headache, and 
symptoms of increased intracranial pressure. All 
these AE were temporary and mostly controllable 
by administration of steroids. In general, IL13-PE38 
doses (0.5–2 µg/ml) showing biological activity (e.g. 
necrosis on MRI scans) in tumours were below 
the threshold of widespread neurotoxicity (4–12 
µg/ml). Selecting of patients without incipient 
mass effect in the brain (due to tumour size and/
or peritumoural oedema) seemed a particularly 
important factor for avoiding serious unwanted 
side effects. Some prolonged survival was observed 
in this selected population of patients.39,66,67

A multicentre phase 3 randomised, open label, 
active control, parallel assignment efficacy study 
(PRECISE) was carried out in order to determine 
whether overall survival duration, safety, and quality 
of life were improved for patients treated with IL13-
PE38 compared to patients treated with Gliadel 
wafers following surgical tumour removal in the 
treatment of first recurrence of GBM after initial 
surgery and external beam radiation therapy.43,68 
Patients were randomised 2:1 to receive IL13-PE38 
or Gliadel. The IL13-PE38 toxin (0.5 µg/ml, total 
flow rate 0.75 ml/h) was administered over 96 hours 
via 2-4 intraparenchymal catheters placed after 
tumour resection. The primary endpoint was overall 
survival from the time of randomisation. Secondary 
and tertiary endpoints were safety and health-related 
quality-of-life assessments. From March 2004 
to December 2005, 296 patients were enrolled at 
52 centres. Median survival was 36.4 weeks (9.1 
months) for the toxin group and 35.3 weeks (8.8 
months) for  the Gliadel group (P = 0.476). For the 
efficacy evaluable population, the median survival 
was 45.3 weeks (11.3 months) for toxin and 39.8 
weeks (10 months) for Gliadel (P = 0.310). The 
AE profile was similar in both arms, except that 



Clinical Development of Experimental Therapies for Malignant Glioma

16 | SQU Medical Journal, February 2011, Volume 11, Issue 1

first treatment. Tumour response appeared to 
be concentration and dose dependent. Median 
survival after treatment in the responder group 
was 74 weeks, with 3 of the responders still alive 
at 102–142 weeks after the first treatment. Non-
responders survived a median of 36 weeks. 
Intratumoural infusions of TransMIDTM with 
total volumes of 5–180 ml were well tolerated. 
There were no treatment related deaths or life-
threatening or irreversible toxicity. In this first 
clinical trial, the relationship between dose, 
drug concentration, and sustained neurotoxicity 
established a MTD of 26.8 µg (40 ml at 0.67µg/
ml). The trial results indicated that therapy with 
TransMIDTM can reduce the size of malignant 
brain tumours refractory to conventional therapy 
without producing severe neurologic or systemic 
toxicity.78 

An open-label, single-arm, multicentre 
phase 2 study investigated intratumoural CED 
infusion of TransMID in recurrent or progressive 
malignant glioma in adults.69 The primary study 
objective was evaluation of efficacy and safety of 
TransMIDTM, and the primary endpoint was a 50% 
reduction in tumour volume (measured by MRI) 
within 12 months after the second treatment. 
Patients received TransMIDTM (0.67 µg/ml) at an 
escalating rate up to 200 µl/h per catheter for 4–5 
days, until a total volume of 40 ml was delivered. 
Forty-four patients were enrolled in total and all 
received at least one TransMIDTM infusion. Of 
the 34 patients evaluable for analysis, there were 
a total of 5 complete responders and 7 partial 
responders (total of 35% response), which was 
a statistically significant result. Median survival 
time for all 44 patients was 37 weeks. Infusions of 
TransMIDTM within this clinical protocol resulted 
in symptomatic progressive brain oedema in 8 
of 44 patients (14%). The results of this phase 2 
clinical trial have confirmed the safety and tumour 
response data of the phase 1 trial.69

A multicentre randomised, open label, active 
control, parallel assignment, safety and efficacy  
phase 3 study (KSB311R/CIII/001 trial) was  
launched in 2004 in order to compare TransMIDTM 
with the best standard treatment available for 
patients with progressive and/or recurrent non-
resectable GBM.79,80 Best standard treatment 
involved a chemotherapeutic regimen considered 
to be the best standard of care at the respective 

target receptor in normal tissue remains, however, a 
major concern with TransMIDTM (and with all other 
targeted toxin based therapies). 

TransMIDTM has been thoroughly evaluated 
in cell culture75 and in vivo. The efficacy of locally 
administered TransMIDTM against human glioma 
in nude mice was demonstrated by Laske et al. 
in 1994.76 These authors studied TransMIDTM 
compared to 454A12-rRA, and administered 
toxins intratumourally in a subcutaneous model. 
Repeated intratumoural injections of 10 µg 
TransMIDTM, or 10 µg 454A12-rRA, were compared 
to equimolar doses of the untargeted native toxin 
CRM-107, 454A12 antibody, rRA, and phosphate-
buffered saline. TransMIDTM administration 
resulted in almost complete tumour regression 
(>95%), without evidence of recurrence by day 
30. Unconjugated toxin components (CRM-107, 
454A12, or rRA) caused significant, but less potent 
tumour growth inhibition than the conjugated 
toxin.76 In a more recent study in 2002, Engebraaten 
et al. compared the efficacy of TransMIDTM with 
that of PE conjugated with the 425.3 antibody 
directed against EGFR.77 Mice with subcutaneous 
gliomas or rats with intracranial gliomas received 
different doses (1-10 µg) of TransMIDTM, or 
425.3-PE, injected intratumourally in established 
tumours. Both toxins showed significant 
antitumour effects in subcutaneous tumours, but 
only 425.3-PE was effective in intracranial gliomas 
in rats. Intracerebral TransMIDTM was toxic in 
doses above 10 ng, while intracerebral 425.3-PE 
was tolerated up to a dose of 4 µg per animal. The 
authors concluded that both toxins have promising 
efficacy in brain tumour models, but that 425.3-PE 
is better tolerated and has a more specific activity 
at higher doses.77

In 1997, Laske et al. treated 18 patients with 
recurrent malignant glioma with intratumoural 
high flow interstitial microinfusion of TransMIDTM 
in a dose-escalating single arm phase 1 clinical 
trial.78 The drug was infused at a maximum flow 
rate of 4-10 µl/min at a toxin concentration of 
0.1 µg/ml. Nine of the 15 evaluable patients 
responded to treatment by at least 50% reduction 
in tumour volume on MRI, including 2 complete 
responses. Reduction in tumour volume occurred 
no earlier than 1 month after completion of the 
first toxin infusion. In 4 patients, the response 
was not maximal until 6–14 months after the 



Nikolai G Rainov and Volkmar Heidecke

review | 17

distribution of the large molecules of immunotoxin 
in the microenvironment of the tumour and/or 
brain, as well as non-specific associated toxicity to 
glial and neuronal cells.19 With the introduction of 
MRI-based functional imaging and 3D volumetric 
techniques in routine clinical practice, it will be 
possible to determine quantitatively brain tumour 
volumes and the volume of infusate in the tumour 
and normal brain.81,82 Diffusion tensor imaging 
(DTI) is a new MRI imaging technique sensitive 
to directional movements of water molecules 
induced by tissue barriers.83 CED makes use of the 
extracellular space of the brain as a natural pathway 
for the widespread distribution of agents infused in 
an aqueous solution, and therefore DTI could be 
used for imaging of CED delivery without the need 
for contrast agents. Further key developments in 
MR imaging and MR-related computer software as 
well as infusion catheter design and computerised 
simulation and delivery modelling within the brain 
may provide much needed new resources to be 
combined with the biologically targeted toxins.19 

Further improvements in the molecular diagnostics 
of malignant glioma with genetic profiling of 
individual patients and tumours should allow for 
selection of subgroups of cases with high expression 
of the target receptor, who are most likely to benefit 
from the administration of a targeted immunotoxin.  

Clinical protocols have explored several 
points of presumed significance, such as the use 

institution, and consisting of either nitrosoureas, 
platinum compounds, temozolomide, procarbazine, 
or PCV (procarbazine, CCNU [lomustine], 
vincristine). The primary endpoint of the study 
was overall survival time. Adult patients eligible 
for enrolment into this trial had to be diagnosed 
with histologically confirmed GBM and had to 
have undergone conventional treatment, including 
surgery (biopsy or debulking) and/or radiation 
therapy and/or chemotherapy. Patients were 
required to have a recurrent and/or progressive 
tumour ≥1.0 cm and ≤4.0 cm in diameter.79,80 The 
trial was however prematurely terminated in 2007 
after an interim conditional power analysis showed 
that it was extremely unlikely that TransMIDTM 
would meet the trial criteria for efficacy.80 No data 
from this clinical trial has yet been published.  

Conclusions and 
Future Developments -
Immunotoxins
Targeted toxins have shown considerable promise in 
phase 1 and 2 clinical trials with recurrent malignant 
gliomas. There are however some major obstacles 
that need to be overcome before targeted toxins may 
enter the mainstream of brain tumour therapies. 
The most important of them are the heterogeneity 
of target receptor expression in malignant glioma 
cells, the non-uniform and poorly predictable 

Figure 4a: Schematic diagrams of virus vectors. Schematic representation of a wild type retrovirus (RV) and a RV vector 
with the corresponding packaging cell line (VPC). Wild type RV has a double-stranded RNA genome which is converted 
to DNA by viral reverse transcriptase and then integrated into the host genome at non-specific sites. The genetically 
modified RV vector genome contains the cis-acting elements required for replication (long terminal repeats - LTR, 
packaging sequence - Ψ), but lacks the viral genes (capsid core proteins - gag, reverse transcriptase, integrase and protease 
- pol, and the envelope antigens - env), which are replaced by up to 8 kb of foreign coding sequences (transgene cassette, 
e.g. cDNA encoding HSV-tk). A selectable marker (e.g. neomycin resistance gene - neo) allows for antibiotic selection 
of cells expressing the RV vector. In order that RV vectors replicate, it is necessary to provide the missing viral genes 
in trans, e.g. expressed within a genetically engineered RV VPC. VPC are most frequently of murine fibroblast origin. 
Separation of the packaging function from the genetic material to be transferred ensures that a replication incompetent 
RV is generated and makes RV vectors biologically safe.



Clinical Development of Experimental Therapies for Malignant Glioma

18 | SQU Medical Journal, February 2011, Volume 11, Issue 1

as oncolytic viruses.85 Most of the viruses employed 
in clinical trials are derived from a retrovirus (RV), 
adenovirus (AV), or herpes simplex virus type 1 
(HSV1) [Figure 4a and b]. Other viruses, such as the 
Newcastle disease virus (NDV) or reovirus, have 
been also employed in clinical trials, but to a much 
lesser extent.19,20,86-88

The effects of virus-mediated local therapy of 
malignant gliomas have been investigated only 
since the 1990s. The initially favoured and best 
explored gene therapy approach included the use 
of a replication-disabled, genetically modified virus 
vector capable of insertion of a toxicity-inducing 
gene into tumour cells.89-91  Most frequently, the 
inserted gene (transgene) rendered the infected 
tumour cells and their clonal progeny differentially 
sensitive to treatment with a pro-drug.91,92 The 
gene/vector system most widely utilised in initial 
clinical trials was the HSV thymidine kinase (HSV-
tk) gene transferred by a replication-incompetent 
RV vector which was released in situ by fibroblast-
derived virus-producing cells (VPC) [Figure 5].93 
The most severe limitation of clinical gene therapy 
with non-replicating viruses was, however, their 
inability to achieve sufficiently high levels of gene 
expression in sufficiently large numbers of target 
tumour cells to result in a clinical benefit.19,94,95A 
modified approach was therefore introduced 
in order to improve anti-tumour toxicity and 
virus distribution within the tumour mass and 
the surrounding normal brain. It employed 
conditionally replicating oncolytic viruses with 
a lytic life cycle instead of their non-replicating 

of different numbers of catheters, positioning of 
catheters, surgical resection of the tumour before 
or after toxin infusion, and single versus repeated 
infusion, but there is no clear answer to any of 
these questions. It remains unknown whether there 
are benefits from combining targeted toxins with 
chemotherapy and/or with fractionated external 
radiation. Protocols have investigated patients 
with recurrent or progressing GBM, and the role 
of targeted toxin infusion in newly diagnosed 
malignant glioma remains to be determined. 
Evidence of prolonged survival and late local 
recurrences in treated glioma patients has raised 
the question of periodically repeated application of 
toxin for extended control of local recurrence and 
progression. The randomised clinical trials were 
able to provide some answers as to the efficacy of 
the studied toxins and clinical protocols; however, 
more clinical research is needed to address the 
above mentioned global issues.

g e n e t i c a l ly m o d i f i e d v i r u s e s
Wild type viruses have been long known for their 
capability to destroy malignant tumour cells upon 
infection and intracellular replication. Genetic 
engineering of some of these viruses was, however, 
only recently done in an attempt to improve their 
utility as targeted gene transfer anti-cancer agents.84 
Recombinant and usually non-replicating viruses 
transferring to tumour cells genes with toxicity-
inducing intracellular effects are known as virus 
vectors, while replicating viruses with a lytic life 
cycle selectively destroying tumour cells are known 

Figure 4b: Schematic representation of the genome of a wild type adenovirus (AV) and recombinant AV vectors. The 
wild type AV genome is flanked by the inverted terminal repeats (ITR) and divided into E1A, E1B, E2, E3, and E4 regions, 
whose genes are expressed in a defined temporal sequence. It contains 36 kb of double-stranded DNA, of which several 
regions can be deleted to accommodate up to 10 kb of foreign DNA (e.g. insertion of a therapeutic transgene cassette 
in the E1 region, deletion of E3 region). Replication-deficient AV vectors are generated by placing the AV genome in a 
plasmid and replacing E1 with a transgene (e.g. HSV-tk), then transfecting the plasmid into a packaging cell line (VPC) 
that provides E1 functions in trans.  



Nikolai G Rainov and Volkmar Heidecke

review | 19

the same method and RV-VPC preparation, Shand 
et al. treated 48 patients with recurrent GBM in 
a phase 1/2 study.105  The authors of both reports 
independently concluded that RV-VPC injections 
were safe. Gene therapy in both trials resulted in a 
prolonged delay of recurrence in individual cases. 

Izquierdo et al. conducted phase 1 studies in 7 
patients with recurrent GBM. Five patients received 
a single intratumoural RV-VPC injection and 2 
patients intraoperative RV-VPC injections, later 
followed by repeated RV-VPC applications into 
the tumour resection cavity.106,107 In 2003, Prados 
et al. treated 30 patients in a phase 1/2 study with 
recurrent GBM by repeated direct injection of RV-
VPC, initially immediately after tumour resection 
in the walls of the resection cavity, and a week later 
via an intracavitary catheter.108 Fifty per cent of the 
patients experienced serious side effects possibly 
related to RV-VPC therapy. The median survival 
of all patients in the treatment group was 8.4 
months. Puumalainen et al. showed low tumour 
transduction efficiency with a RV-VPC construct 
carrying the marker gene lacZ.109 The same study 
also evaluated AV-mediated marker gene transfer 
and found much higher transduction rates in some 

counterparts.96-100  

n o n-r e p l i c at i n g r e t r o v i r u s
Starting in the late 1990s, several clinical studies 
were published which investigated the use of 
RV-mediated HSV-tk gene transfer delivered by 
intratumoural injections of VPC and followed 
by systemic administration of ganciclovir 
(GCV).89,90,91,101 The earliest phase 1/2 clinical 
study was published by Ram et al. in 1997.93 These 
authors investigated, in 15 patients with recurrent 
malignant glioma, the tolerability and efficacy of 
RV-producing VPC (RV-VPC) delivered by a single 
stereotactic intratumoural injection. Treatment 
resulted in anti-tumour activity in 4 patients with 
relatively small tumours. One patient with GBM 
had a complete response with a recurrence-free 
survival of 50 months.93 The limited efficiency of 
histologically demonstrated RV-mediated gene 
transfer suggested the presence of a bystander effect 
conferring toxicity to non-transduced tumour 
cells.102,103 In 1998, Klatzmann et al. treated 12 
patients with recurrent GBM in a phase 1/2 study 
by local injections of RV-VPC into the margins of 
the tumour cavity after tumour debulking.104 Using 

Figure 5: Schematic representation of the mode of action of recombinant retrovirus (RV) vector-mediated suicide gene 
therapy in patients with malignant glioma. Up to 20 ml of a suspension of viable RV-producing cells (VPC) carrying the 
transgene coding for herpes simplex virus (HSV) thymidine kinase (HSV-tk) are manually injected into the walls of the 
tumour resection cavity at the end of the surgical removal of a human glioblastoma multiforme (GBM). RV vectors, 
which have been genetically engineered to become replication-deficient, are produced in high titres by the implanted 
VPC. GBM cells are highly mitotic and, after infection by RV, are able to produce most RV proteins, including HSV-
tk. This enzyme converts the low-toxicity prodrug ganciclovir (GCV) into highly toxic metabolites, which block DNA 
replication during mitosis and render the tumour cells apoptotic. After repeated i.v. application of GCV to patients, 
all cells expressing HSV-tk are killed, including VPC. Cells not expressing HSV-tk may also be killed by the so-called 
“bystander effect” - transfer of toxic GCV metabolites from HSV-tk-expressing to HSV-tk-negative cells by direct 
cellular contact. Up to 10 non-expressing cells may be killed by one HSV-tk-expressing cells in cell culture.



Clinical Development of Experimental Therapies for Malignant Glioma

20 | SQU Medical Journal, February 2011, Volume 11, Issue 1

The median survival of all patients was 10 months, 
while 5 patients were alive at 1 year and 1 patient at 
3 years after therapy.111 Germano et al. carried out 
a dose-escalating phase 1 study in 11 patients with 
recurrent GBM.112 

AV-HSV-tk doses of 2.5x1011-9x1011 virus 
particles were administered intraoperatively to 
the walls of the resection cavity immediately after 
tumour resection. The median survival of treated 
patients was 59 weeks. No major toxicities occurred, 
and the few serious AEs were not related to AV 
administration.112 Smitt et al. conducted another 
phase 1 dose escalation study in 14 patients with 
recurrent malignant glioma.113 Patients received 
4.6x108-4.6x1011 virus particles injected manually 
in the walls of the tumour resection cavity at the 
end of tumour removal. Median overall survival 
was 4 months, however 4 patients survived for 
longer than 1 year following therapy. The treatment 
was safe and well tolerated. 113

Some groups were able to report significantly 
better clinical outcomes using the AV-HSV-tk 
approach. Sandmair et al. enrolled 21 patients 
with primary or recurrent malignant glioma in a 
comparative phase 1/2 study employing HSV-tk 
gene transfer by RV-VPC or by AV and comparing 
these with a third group receiving AV carrying 
only a marker gene, lacZ.114 The mean survival in 
the AV-HSV-tk group was significantly longer (15 
months) than that of the other groups with mean 
survival of 7.4 months for RV-VPC and 8.3 months 
for AV-LacZ. No serious AE were reported in any 
of the groups. 

Building on these promising results, Immonen 
et al. conducted a randomised prospective phase 2 
study to evaluate further the efficacy and safety of 
AV-HSV-tk in patients with primary or recurrent 
malignant glioma.115 Seventeen of the 36 patients 
received intraoperative injections of AV-HSV-
tk (3x1010 pfu) into the walls of the surgically 
created resection cavity followed by GCV (5 mg/
kg twice daily for 14 days), and 19 patients had 
tumour surgery without AV injections. Standard 
radiotherapy was carried out in all patients 
with previously untreated tumours. The mean 
survival of patients in the AV-HSV-tk group was 
significantly longer (70.6 weeks) than in the control 
group (39.0 weeks) (P = 0.0095). An additional 
post hoc subgroup analysis excluding patients with 
anaplastic astrocytoma showed that there still was 

areas of the tumours. Harsh et al. performed a 
gene-marking and neuropathological study in 
5 patients with recurrent GBM who received 
multiple stereotactically guided intratumoural 
injections of RV-VPC during a single surgical 
session.94 This study showed an extremely low gene 
transduction efficiency of the RV-VPC used.

Finally, a large prospective randomised 
multicentre phase 3 clinical trial was carried out 
to allow definitive evaluation of RV-VPC treatment 
in patients with newly diagnosed GBM.101A total of 
248 patients with previously untreated GBM were 
randomised to two groups and treated using either 
standard surgical resection and radiotherapy (n = 
124) or standard therapy plus adjuvant RV-VPC 
injected in the walls of the resection cavity at 
tumour surgery (n = 124). Systemic administration 
of GCV (5 mg/kg i.v. twice daily) was started two 
weeks after surgery and continued for another 2 
weeks. Somewhat surprisingly, this study found 
no significant difference in the progression-free, 
median, or 12-month survival of both the standard 
and the gene-therapy groups, although the 
approach was proven exceptionally safe. Based on 
the results of this trial, local therapy of malignant 
glioma with RV-VPC expressing HSV-TK has been 
largely abandoned.101

n o n-r e p l i c at i n g a d e n o v i r u s 
Studies with AV vectors were conducted and 
published, mostly in the late 1990s. Trask et al. 
conducted a phase 1 study of AV-HSV-tk in 12 
patients with recurrent malignant glioma.110 A 
single stereotactic intratumoural injection was 
used to deliver AV doses of 2x109-2x1012 pfu. 
Adequate safety was reported with all doses except 
the highest, which caused significant CNS toxicity. 
Median survival in this study was only 4 months, 
however 3 patients showed long-term survival of 2 
years or longer, which was interpreted as evidence 
for some local tumour control. Judy and Eck treated 
13 patients with primary or recurrent malignant 
gliomas in a modified phase 1 study.111 Twelve 
patients received stereotactic intratumoural AV-
HSV-tk injections and a week later underwent 
tumour mass resection with additional AV 
injections in the walls of the resection cavity. Total 
virus doses of 2x108-2x1011 pfu were used. Transient 
side effects, such as increased intracranial pressure 
(ICP), were noted only at the highest dose level. 



Nikolai G Rainov and Volkmar Heidecke

review | 21

an acceptable safety profile.116 The results of the 
study have not been published yet, and final data 
still need to be provided. Preliminary information 
in the public space however states that the trial may 
be statistically underpowered and that Cerepro® 
has failed to show sufficient primary endpoint 
efficacy.117

o n c o ly t i c v i r u s e s
Genetically modified and conditionally replicating 
AV and HSV have been the most frequently used 
viruses in early clinical studies with oncolytic 
viruses.87,89,97,99  Four phase 1 trials in recurrent 
malignant glioma have used  intracerebrally 
inoculated G207 - a conditionally replicating HSV 
with defects in both ICP6 and ICP34.5 genes, 
which has demonstrated anti-tumour efficacy 
in preclinical studies in glioma.89,118,119 Markert 
et al. in 2000 carried out a dose escalation study 
treating 21 patients with recurrent malignant 
glioma using stereotactic intratumoural injections 
of G207 (up to 3x109 pfu total dose).89 MRI studies 
and neuropathological analysis suggested some 
anti-tumour activity of the treatment. The best 
responding 4 patients survived for a mean of 
12.8 months, while the rest of the group had a 
mean survival of 6.2 months after therapy. Most 
importantly, this early trial showed that inoculation 
of a genetically engineered oncolytic HSV virus in 
the human brain is safe, despite the concerns about 
possible encephalitis well known from its wild type 
counterpart.89 

Rampling et al. (2000) conducted a first phase 
1 dose escalation study using another selectively 
replicating HSV mutant with disrupted ICP34.5 
genes known as HSV1716.120 Nine patients with 
recurrent malignant glioma received stereotactic 
intratumoural injections of HSV1716 up to a 
dose of 1x105 pfu. Four patients were alive for 
14–24 months after virus injection. No signs of 
encephalitis or major neurological side effects 
were noted.120 Papanastassiou et al. (2002) used 
intratumoural injections of HSV1716 (1x105 
pfu) in another phase 1 study in 12 patients with  
malignant glioma.87 The authors were able to 
demonstrate replication of virus in tumours 
resected 4–9 days after the HSV1716 injection. 
No toxicity was apparent. Additional data from 
the above studies were published separately and 
suggested intratumoural persistence and replication 

a significant survival benefit in GBM patients (55.3 
versus 37.0 weeks, P = 0.0214). The treatment was 
well tolerated and showed no major side effects.115

A phase 3 randomised and standard care-
controlled multicentre pivotal trial (also known as 
study 904) using the above AV-HSV-tk vector115 
designated as sitimagene ceradenovec (Cerepro®, 
Arc Therapeutics Ltd., London, UK) has been 
carried out in patients with newly diagnosed 
malignant glioma.116 Patients were randomised 
to either standard care plus Cerepro® or standard 
care alone. Standard care was surgery and 
radiotherapy or surgery and radiotherapy followed 
by temozolomide, resulting in 4 treatment groups. 
This allowed comparison of the efficacy of Cerepro® 
and temozolomide in the same trial without 
denying patients the best possible standard care. 
The primary endpoint was survival, defined as time 
to death or re-intervention.116  

The overall primary endpoint analysis in the 
ITT population (n = 236) compared Cerepro® 
with and without TMZ against controls with and 
without TMZ. It showed a 42 day improvement in 
median survival (310 days versus 268 days) in the 
two groups receiving Cerepro®. The improvement 
over standard care reached statistical significance 
(P < 0.032). At the primary endpoint, the group 
with Cerepro® and TMZ showed an improvement 
of 68% in median survival time compared with 
standard care (surgery and radiotherapy) controls 
(350 days versus 208 days). Against the same 
controls, treatment with Cerepro® alone showed 
an improved median survival trend approaching 
50%, similar to those given treatment with TMZ 
alone after standard care (300 days and 307 days, 
respectively versus 208 days with standard care). 
Improvements in the combined Cerepro® and TMZ 
treatment group (n = 58) and TMZ alone group (n 
= 76) were significant (P <0.05). In the Cerepro® 
alone treatment group (n = 61), the effect was not 
statistically significant (P < 0.065). Of the total 53 
patients still to report an event, only 7 are in the 
surgery and radiotherapy control group and thus 
confidence intervals and statistical significance 
levels in all treatment groups might be expected to 
improve with time. 

Whilst increases were observed in hemiparesis, 
aphasia and pyrexia following therapy, the serious 
AE reports for Cerepro® were in line with those in 
the previous studies, indicating that the virus has 



Clinical Development of Experimental Therapies for Malignant Glioma

22 | SQU Medical Journal, February 2011, Volume 11, Issue 1

manual injection of virus into tumours or tumour-
invaded surrounding brain, as well as intrinsic 
barriers to intra- and peritumoural spread in a 
glioma-harbouring brain, such as cysts, fibrotic 
membranes, and tumour necrosis.84,97

Therapy with oncolytic viruses seems to hold 
more promise in the few early clinical trials than 
the therapy with non-replicating virus vectors, 
although both approaches were already proven to 
be safe and lacking major side effects.96-99, 119,122,126 
However, further major advancements in virus 
designs, application modalities, and understanding 
of the interactions of the host immune system with 
the virus are clearly needed before oncolytic virus 
therapy of malignant brain tumours may enter 
routine clinical use.

Final Conclusions and 
Future Developments of 
Drug Delivery Modalities 
to Brain Tumours
The therapeutic success of any tumour-targeting 
and locally delivered agent will depend not only on 
its ability to kill tumour cells selectively, but to a high 
degree also on the delivery mode and distribution 
throughout a tumour and the surrounding normal 
brain tissue.19,84 Diffusion of particles (e.g. viruses) 
or large molecules (e.g. recombinant toxins) in 
tissue is a rather inefficient way of distribution and 
will depend not only on the concentration of the 
compound, but also on its size, molecular weight, 
polarity, and its avidity for the target receptor.40,92,127 
To circumvent this limitation, distribution of agents 
by CED, a much more efficient and fast mode for 
interstitial delivery by high-flow infusion, has 
been studied in several animal models.100,127 Local 
delivery of targeted toxins by CED seems to be 
the best approach to circumvent the limitations 
of the blood-brain barrier (BBB) and to increase 
therapeutic efficacy by high local concentrations 
of drug. All clinical trials with targeted toxins have 
adopted CED as the delivery mode of choice.19 

Virus particles can also be delivered by CED, 
although this modality has been studied mainly 
in animal experiments,20,84,127,128  while all human 
clinical trials carried out so far have employed bolus 
injections of virus or carrier suspensions.91,94,101,124 
Intratumoural stereotactically guided injections 
can provide adequate virus delivery only to spatially 

of HSV1716, allowing the virus to kill tumour cells 
over extended periods of time. Analysis of tumour 
explants showed viral replication for up to 9 days 
after initial injection, and the amount of recovered 
virus exceeded the input dose in some tumour 
samples. In addition, 1 patient was free of tumour 
progression at nearly 3 years, and 2 patients were 
alive and stable after almost 4 years.121 Harrow et al. 
(2004) followed 12 patients with newly diagnosed 
or recurrent malignant glioma treated in a previous 
study.122 Three long-time survivors with GBM were 
clinically stable at 15–22 months following surgery 
and virus injection. No long-term toxicity was 
reported.122

ONYX-015, a naturally occurring AV 
mutant with deletion in the viral E1B gene, has 
been previously used in clinical trials for head 
and neck cancer and gastro-intestinal tract 
malignancies.97,123,124 The first multicentre phase 1 
study using ONYX-015 in patients with recurrent 
malignant glioma was published by Chiocca 
et al. in 2004.124 Twenty-four patients received 
intraoperative doses of ONYX-015 (1x107-1x1010 
pfu) injected manually in the walls of the surgical 
cavity immediately after tumour resection. The 
median survival time for all patients was 6.2 
months. No definitive anti-tumour effect could be 
demonstrated, however none of the patients, at any 
virus dose, experienced serious AE and there was 
no dose-limiting neurological toxicity.124

Conclusions and 
Future Developments -
Genetically Modified 
Viruses
The clinical efficacy of local therapy for malignant 
glioma with non-replicating viruses has been so far 
rather disappointing, possibly with the exception of 
the studies with AV-HSV-tk.115,116 No clinical studies 
using non-replicating virus vectors are currently 
enrolling patients. 

Encouraging anti-tumour activity has been 
demonstrated in the some of the studies in malignant 
glioma treated with local injections of oncolytic 
AV and HSV.97-99,122 Systemic chemotherapy has 
been found to potentiate the anti-tumour effect of 
virus mediated oncolysis in other cancer types.123,125 
Factors likely to be an issue with any type of 
oncolytic virus are the physical limitations of 



Nikolai G Rainov and Volkmar Heidecke

review | 23

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limited areas of tumour, since the number of 
injection sites is limited for practical reasons by 
tumour size, length of surgery and increasing risk of 
haemorrhage.94 Direct intratumoural injections into 
the walls of the tumour resection cavity, although 
they can be performed under direct visual control 
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have the same basic limitations as stereotactic 
procedures.93,101 Moreover, the depth of injection 
is limited to 10–15 mm from the visible resection 
border, which seems to be insufficient to reach 
tumour cells migrating away from the main mass.101

CED has been shown to improve significantly 
virus distribution in and around experimental 
brain tumours.127 Virus particles may be efficiently 
delivered by CED over large areas of tumour 
and surrounding brain and achieve widespread 
distribution and much larger coverage than with 
bolus injections.92,127,128 Since a large amount of 
clinical experience has become available in the 
clinical trials employing CED of targeted toxins, 
it seems a logical step that clinical protocols with 
CED of viruses should be implemented in future. 
Such trials may be able to combine the biological 
advantages of oncolytic viruses with the spatial 
distribution affordable by CED.

c o n f l i c t o f i n t e r e s t
NGR has received research grants from Neurocrine 
Inc. (San Diego, CA) and IVAX Inc. (Miami, FL). 
The authors have no financial interests in any of the 
biotechnology companies mentioned in the review.

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