Novel Therapies in Glioblastoma

Abstract

Conventional treatment of glioblastoma has advanced only incrementally in the last 30 years and still yields poor outcomes. The current strategy of surgery, radiation, and chemotherapy has increased median survival to approximately 15 months. With the advent of molecular biology and consequent improved understanding of basic tumor biology, targeted therapies have become cornerstones for cancer treatment. Many pathways (RTKs, PI3K/AKT/mTOR, angiogenesis, etc.) have been identified in GBM as playing major roles in tumorigenesis, treatment resistance, or natural history of disease. Despite the growing understanding of the complex networks regulating GBM tumors, many targeted therapies have fallen short of expectations. In this paper, we will discuss novel therapies and the successes and failures that have occurred. One clear message is that monotherapies yield minor results, likely due to functionally redundant pathways. A better understanding of underlying tumor biology may yield insights into optimal targeting strategies which could improve the overall therapeutic ratio of conventional treatments.

Full text

Hindawi Publishing Corporation
Neurology Research International
Volume 2012, Article ID 428565, 14 pages
doi:10.1155/2012/428565
Review Article
Novel Therapies in Glioblastoma
James Perry, Masahiko Okamoto, Michael Guiou, Katsuyuki Shirai, Allison Errett,
and Arnab Chakravarti
Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute,
The Ohio State University, Columbus, OH 43210, USA
Correspondence should be addressed to James Perry, james.perry@osumc.edu
Received 16 August 2011; Accepted 9 December 2011
Academic Editor: Stuart Burri
Copyright © 2012 James Perry et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Conventional treatment of glioblastoma has advanced only incrementally in the last 30 years and still yields poor outcomes. The
current strategy of surgery, radiation, and chemotherapy has increased median survival to approximately 15 months. With the
advent of molecular biology and consequent improved understanding of basic tumor biology, targeted therapies have become
cornerstones for cancer treatment. Many pathways (RTKs, PI3K/AKT/mTOR, angiogenesis, etc.) have been identified in GBM as
playing major roles in tumorigenesis, treatment resistance, or natural history of disease. Despite the growing understanding of
the complex networks regulating GBM tumors, many targeted therapies have fallen short of expectations. In this paper, we will
discuss novel therapies and the successes and failures that have occurred. One clear message is that monotherapies yield minor
results, likely due to functionally redundant pathways. A better understanding of underlying tumor biology may yield insights into
optimal targeting strategies which could improve the overall therapeutic ratio of conventional treatments.
1. Introduction
Glioblastoma (GBM) is a grade IV glioma and accounts
for approximately 75% of all high-grade gliomas with
approximately 9,000 new cases per year diagnosed in the
United States alone, making it the most common adult
brain tumor. GBM is the most aggressive glial neoplasm,
and despite advances in medical management, the out-
comes remain quite poor. The current standard of care for
high-grade glioma patients is maximum surgical resection
combined with radiation and concomitant and adjuvant
temozolomide (TMZ) therapy. The addition of radiotherapy
for the treatment of GBMs led to the first significant
improvement in patient survival starting in the late 1970s.
More recently, Stupp et al. have shown that the addition
of the chemotherapeutic agent TMZ can increase survival
further to approximately 15 months.
2. Development of Standard of Care (RT + TMZ)
Surgery is a critical component of standard of care, allowing
histological diagnosis, but more critically, tumor debulking.
This greatly reduces the number of cells to be killed by
radiation or chemotherapy. It also decreases intracranial
pressure which, depending on the location of the tumor, may
result in recovery of CNS function or decrease in usage of
corticosteroid. Recently, the effectiveness of aggressive surgi-
cal resection on survival was suggested by some prospective
analyses [1–3].
Unfortunately, most glioblastomas recur following
surgery. The efficacy of radiation therapy (RT) was reported
in the 1970s [4,5], and RT has become a standard adjuvant
therapy in patients with malignant glioma. In 2005, the
efficiency of concomitant and adjuvant TMZ was suggested
by a phase III study that was conducted by the European
Organization for Research and Treatment of Cancer
(EORTC) and the National Cancer Institute of Canada
(NCIC) [6]. In the EORTC/NCIC study, a total of 573
patients with newly diagnosed glioblastoma were enrolled.
The authors reported the combined therapy of TMZ and RT
increased median survival time (MST) when compared with
RT-alone (14.6 months versus 12.1 months, P<.001). At the
5-year analysis of this study, the 5-year overall survival rate
was 9.8% for the combination therapy group versus 1.9%

2Neurology Research International
for the RT alone group (P<.001), with a median follow-up
of 61 months [7]. With this strong evidence, combination
therapy with TMZ and RT is widely prescribed and currently
considered the standard treatment for patients with newly
diagnosed glioblastoma.
Some investigators had suggested that the epigenetic
silencing of a DNA repair enzyme named O-6-methylgua-
nine-DNA methyltransferase (MGMT) by promoter methy-
lation was associated with good prognosis for patients with
glioblastoma treated with alkylating agents such as TMZ
[8,9]. In agreement with previous studies, patients with
a methylated MGMT promoter had significantly improved
MST when compared with patients with an unmethylated
MGMT promoter (21.7 months versus 15.3 months, P<
.001) in the EORTC/NCIC trial [10]. Furthermore, this
study indicated that patients with an unmethylated MGMT
promoter received less benefit from the combined therapy.
MGMT promoter methylation status is widely used to
predict the efficacy for combination therapy of RT and TMZ
for newly diagnosed glioblastoma.
Although the combination therapy of RT and TMZ
has become standard, most patients will still eventually
recur. Thus the development of a new treatment strategy is
needed in order to overcome the resistance of glioblastoma
to current therapy. One strategy is increasing the intensity
of radiation dose. However, neither radiosurgery boost
[11] nor brachytherapy boost [12] shows improvement
in survival. Another strategy is the optimization of TMZ
usage by approaches such as dose-dense regimens. RTOG
0525/EORTC 26052-22053, a prospective randomized trial,
was conducted by the Radiation Therapy Oncology Group
International (RTOG) and EORTC. It aimed to determine
whether a dose-dense TMZ regimen is more effective than
the standard TMZ regimen in the adjuvant setting, and the
results of this study showed no significant difference between
the two arms [13]. Moreover, the adverse effects, especially
in the field of lymphopenia and fatigue, were significantly
increased in the dose-dense arm.
3. Technological Advancements in
GBM Therapy
Current research efforts in both the basic and clinical sciences
are improving clinicians’ ability to more accurately target and
treat GBM. In addition to targeted therapies, which will be
discussed later in this paper, there have been advancements
in the technological arena that are improving patient care.
3.1. Target Delineation. MRI remains the gold standard
for delineating tumor in both the pre- and postoperative
setting. Gross tumor volume is felt to be best represented
by areas of contrast-enhanced T1 signal while areas of
T2/FLAIR enhancement reflect regions of infiltrative tumor.
These volumes form the basis for radiotherapy target
delineation. A limitation of contrast-enhanced MRI is that
it relies on surrogate markers of tumor presence (tumor-
associated breakdown of the blood-brain barrier and cerebral
edema) versus a direct measure of actual gross tumor
mass and spread. Functional imaging techniques such as
positron emission tomography (PET) play a pivotal role
in the staging and planning of cancers in other parts
of the body. Unfortunately, the most widely used radio-
tracer [18F]-fluorodeoxyglucose is relatively insensitive at
delineating malignancy in the brain due to the high basal
metabolic rate of normal brain tissue [14]. Other clini-
cally available radiotracers such as L-methyl-11C-methionine
(MET) and O-(2-[18F]fluoroethyl)-L-tyrosine(FET) have
shown promise in localizing gliomas. Recent studies have
shown MET PET can more accurately identify areas of active
tumor versus traditional MRI [15,16]. MET is actively taken
up by gliomas but shows only low uptake in normal brain. In
a study of 14 patients with high-grade glioma, MET PET was
highly correlated with areas of endothelial proliferation and
mitotic activity [17], a more direct marker of tumor activity
that can be visualized using MRI. In a study of 26 patients
with GBM, Lee et al. [15] showed that 5 of 26 patients had
areas of MET PET positivity outside radiotherapy volumes
defined by MRI. All of these patients had noncentral failures.
In 14 patients where MET PET-positive areas were covered
in the high-dose radiotherapy volume, none had noncentral
failures. FET PET has also been shown to correlate with
areas of active tumor. In a study of 31 patients with GBM,
FET PET was highly correlated with areas of active tumor
on biopsy [18]. FET shows minimal uptake in macrophages
or inflammatory tissues, indicating that it may be superior
to MET in identifying areas of active tumor, especially in
the postoperative setting. In a study comparing FET PET to
MRI, Piroth et al. showed that areas active on FET PET not
covered by MRI-based radiotherapy target volumes predict
a shorter disease-free survival (5.1 versus 9.6 months) and
overall survival (6.9 versus 20 months) further supporting
the integration of metabolically based treatment planning
methods into radiation planning for GBM [19]. An emerging
technique that may also play an important role in target
delineation for GBM is diffusion tensor imaging (DTI). DTI
uses specialized MRI sequences to measure the diffusion of
water in the brain, the greatest values of which lie along white
matter tracts. Postmortem studies in GBM patients show
that glioma cells tend to migrate the greatest distance from
the primary site along these tracts. In a study of 14 patients
with recurrent GBM, Krishnan et al. were able to show a
strong relationship between the sites of recurrence and the
DTI maps emanating from the primary site [20]. While none
of the aforementioned imaging techniques have been tested
in a phase III study, they provide promising tools by which
clinicians cannot only more accurately identify areas of active
tumor but also potentially predict their most likely route of
spread to help refine radiotherapy treatment fields.
3.2. Disease Monitoring. Treatment-related effects such as
postoperative scarring and hemorrhage, peritumoral edema,
inflammation, and microvascular changes make radio-
graphic assessment problematic as these changes can mimic
progressive disease. This has been termed pseudoprogres-
sion. Additionally, given the high doses and often large vol-
umes required to treat GBM, up to 30% of patients develop
radionecrosis which can also mimic disease recurrence on

Neurology Research International 3
MRI [21]. The front-line treatment for radionecrosis is
steroids and time. Some patients require surgical resection
for more advanced cases resistant to steroid treatment. The
ability to diagnose true progression and initiate second-line
therapy is paramount in a patient population with such
poor disease-free and overall survival. Both PET imaging and
magnetic resonance spectroscopy (MRS) have been shown
to be superior to MRI in helping distinguish active disease
from pseudoprogression or radionecrosis. The specificity
of FET PET and MET PET to detect recurrent disease is
approximately 90% and 70%, respectively [19]. Areas active
on FET PET show a high correlation with biopsy-proven
areas of active disease [18], and regions of enhancing tissue
on MRI are often negative on FET PET. Numerous studies
of magnetic resonance spectroscopy (MRS), which measures
the differential concentrations of metabolites in the brain,
also show improved accuracy in distinguishing recurrent
disease versus MRI (71% versus 55%) [22]. Classic MRS
is hampered by poor spatial resolution and nonvolumetric
data; however, recent advances in technology now permit
volumetric MRS which may improve its diagnostic value.
Integration of these metabolically based imaging techniques
may help to improve our ability to detect disease progression
early and optimize 2nd-line therapy for patients.
3.3. Treatment in the Recurrent Setting. The vast majority
of patients with GBM recur within 8–12 months follow-
ing completion of therapy highlighting the importance of
developing efficacious treatments for patients with recurrent
disease. Studies examining reirradiation with or without
chemotherapy have shown the most promising results. Given
the fact that the majority of patients fail within 2 cm of
the primary site, numerous studies examining the efficacy
of stereotactic radiosurgery (SRS), as delivered by a variety
of different methods, have been completed [23–25]. SRS
provides a modest improvement in outcome but with
significant risk of radionecrosis requiring surgical resection.
Patients undergoing fractionated stereotactic radiotherapy
(FSRT) at the time of recurrence have median survival
ranging from approximately 7 to 14 months for all patients
but with reduced rates of morbidity [23,26–28]. Dosing
regimens with a cumulative dose exceeding 40Gy appear to
be associated with a greater degree of radiation damage [29].
Response on postprocedure MRI seems to be an important
prognostic factor for either SRS or FSRT. In a study of 36
patients (26 SRS, 10 FSRT) patients showing response on
posttreatment MRI had median survival of 15.8 months
versus 7.3 months for nonresponders [23].
Combination chemoradiotherapy has shown some effi-
cacy in the recurrent setting. In a study of 25 patients with
recurrent high-grade glioma (20 GBM, 4 anaplastic astro-
cytoma), treatment with FSRT (30 Gy in 5 fractions) plus
concurrent bevacizumab resulted in posttreatment median
survival of 12.5 months [30] with comparable toxicity
rates as reported by other studies of GBM patients treated
with bevacizumab. Three of 25 patients had to discontinue
treatment due to CNS toxicity, wound complications, and
bowel perforation. A recent phase I dose-escalation trial
of gefitinib, an epidermal growth factor receptor (EGFR)
tyrosine kinase inhibitor (TKI), plus FSRT (18 to 36 Gy in 3
fractions) in 15 patients (11 GBM, 4 anaplastic astrocytoma)
showed this combination was well tolerated at all dose levels.
Median progression-free survival was 7 months with a 6-
month progression-free survival of 63% and a 1-year overall
survival of 40%. Of course, given the mixture of grade III
and grade IV histologies, it is difficult to compare outcomes
with studies of GBM patients alone, but these initial results
show promise. Studies combining cytotoxic chemotherapy
with FSRT agents have shown similar response rates with
acceptable toxicity profiles [31–33].
Recurrent malignant gliomas can also be managed with
chemotherapy. Modifications in the dosing regimen of TMZ
show modest improvements in progression-free survival.
Standard dosing regimens of TMZ (200 mg/m2)producea6
month progression-free survival of 21% [34]. TMZ delivered
in a low-dose, protracted schedule (75 mg/m2for 21 days of
a 28-day cycle) in an attempt to deplete MGMT produced
PFS-6 of 30% [35].
4. Targeted Therapy
Considering glioblastoma treatment is the most expensive
cancer treatment per capita in the United States, and
outcomes are still so universally poor, there is a great need for
more effective therapeutic options. Targeted therapy and per-
sonalized medicine are currently two of the more aggressively
pursued ideas in cancer treatment. While targeted therapies
aim to affect a specific alteration, most chemotherapies
are generic, broad-based DNA-damaging agents that affect
all cells in a similar manner. Targeted therapies offer the
possibility of selectively killing cancer cells and sparing
normal tissue. An example of a commonly targeted pathway
in glioblastoma is the EGFR receptor tyrosine kinase.
5. Receptor Tyrosine Kinase (RTK) Inhibitors
5.1. EGFR. As mentioned above, EGFR is known to be
an important player in gliomagenesis and in the aggressive
and therapeutic-resistant phenotype demonstrated by this
tumor. In addition to its critical role in several survival
signaling pathways, alterations in EGFR are some of the most
common mutations found in GBM. EGFR is overexpressed
in approximately 50% of tumors, and of those nearly half
express the constitutively active EGFRvIII mutant [36].
These combined facts have made this growth factor receptor
a very popular target for molecular therapies. Many clinical
trials have examined the efficacy of EGFR inhibitors, and
to date there is little evidence to support their use in a
monotherapy setting. While gefitinib, a selective inhibitor
of EGFRs tyrosine kinase domain (Figure 1), is approved
for use in non-small-cell lung cancer, a recent phase II
clinical trial of gefitinib in newly diagnosed GBM patients
by the Mayo/North Central Cancer Treatment Group showed
no significant improvement in either overall survival or
progression-free survival [37]. Another recent phase II trial
studying the efficacy of erlotinib, which also inhibits the
tyrosine kinase domain of EGFR (Figure 1), and TMZ
with RT for newly diagnosed GBM had to be stopped

4Neurology Research International
RTK (EGFR, VEGFR, IGFR,
PDGFK, HER2, etc.)
RTKi
Gefitinib (EGFR)
Erlotonib (EGFR)
Lapatinib (EGFR/HER2)
Imatinib (PDGFR)
NVP AEW541 (IGFR)
Cell growth,
proliferation, survival
Monoclonal
antibodies
bevacizumab
Cal-101, BKM120
MK-2206
Temsirolimus (CCI-779)
Everolimus (RAD001)
Dual PI3K/mTOR
PI-103
PKI-548, PKI-402
AKT
Notch re ceptor
GSIs
Oncolytic virus
CTL
Cell death
Stem cell
maintenance,
differentiation,
proliferation
Dendritic cell (APC)
P
PI3K
MHC
Class I
Delivery: toxins ,
siRNA, target genes
mTOR
Cell lysis
PARP: inhibitors (DNA repair)
HDAC: inhibitors (vorinostat)
DNA
mRNA
miRNA
Nucleus
P
IL-13Rα2
EGFR VIII
γ-secretase
Figure 1: Novel therapies in GBM. RTKs and survival signaling pathways are major drug targets in GBM. Receptors have been targeted
extracellularly by monoclonal antibodies or intracellularly at the tyrosine kinase domain. Major nodes in survival signaling pathways
(P13K, AKT, mTOR) have been the focus of intense study and drug development. More recent approaches include stem-cell targeting
(GSIs), inhibition of DNA rapair (PARP inhibitors), and targeting a host of cellular pathways through microRNA manipulation. Novel
tumor cell killing approaches are also being studied. Oncolytic virus therapy, either alone or in combination with targeted agent delivery
and immunotherapy, are being employed to efficiently kill tumor cells while sparing normal tissue. RTK, receptor tyrosine kinase; EGFR,
epidermal growth factor receptor; VEGFR, vascular endothelial growth factor receptor; IGFR, insulin-like growth factor receptor 1; PDGFR,
platelet-derived growth factor receptor; mTor, mammalian target of rapamycin; GSI, gamma-secretase inhibitor; APC, antigen-presenting
cell; CTL, cytotoxic T lymphocyte, MHC class I, major histocompatibility complex I; PARP, poly(ADP-ribose) polymerase; HDAC, histone
deacetylase inhibitor.

Neurology Research International 5
short of full accrual due to the lack of benefit and the
unreasonable toxicity, including at least three treatment-
related deaths [38]. A different phase II trial of erlotinib in
the same setting showed less toxicity and even demonstrated
improved median survival (19.3 months versus 14.1 months)
compared to historical controls [39]. Interestingly, the first
study only escalated erlotinib dose to 150 mg/day while the
second study, which showed improved toxicities as compared
to historical controls, escalated to a maximum dose, in some
patients, of 300 mg/day. Overall results from multiple clinical
trials show monotherapy EGFR inhibition, or addition of
these inhibitors to standard-of-care treatments has shown
little benefit to patients and a dramatically increased toxicity
profile.
In terms of current multitherapy strategies, combin-
ing several RTK inhibitors, or attacking several important
glioma survival strategies, is likely to improve outcomes
over monotherapies, while also possibly reducing toxicity.
A recent study showed that EGFR expression in a GBM
xenograft model increased the efficacy of an anti-vascular
endothelial growth factor (VEGF) (vandetanib or cediranib)
therapy in combination with irradiation when compared
to xenografts lacking EGFR expression [40]. A preclinical
study of monoclonal antibody inhibition of both EGFR
and VEGFR-2 has demonstrated improved efficacy in an
orthotopic xenograft model [41]. This research was not
performed in the presence of IR and therefore may have
less relevance to glioblastoma therapy. However, it does
demonstrate the importance, as well as the complexity, of
designing combination therapies. In a similar vein, it was
recently shown that HER2 inhibition might help overcome
EGFR resistance and increase radiosensitivity in a GBM
cell model [42]. A different combination of EGFRvIII
inhibition with C-met inhibition showed synergy against
PTEN null/EGFRvIII positive tumors, a very aggressive
tumor population [43]. With the promise shown, even
novel inhibitors are being tested in combination. A recent
study reported that inhibition of autophagy was able to
enhance the cell-death-inducing capabilities of erlotinib in
a glioma cell model [44]. Lovastatin, a member of the
statin family (normally used to reduce cholesterol), was also
shown to increase the efficacy of EGFR inhibitor therapies
[45]. However, in a clinical phase I/II trial of lapatinib,
a dual EGFR/HER2 inhibitor, showed poor results [46].
Even rational combinations targeting this pathway have not
led to expected results, as shown by the underwhelming
results in a recent phase II trial examining erlotinib ther-
apy in combination with an mTOR inhibitor (sirolimus)
[47]. While EGFR inhibitor combination treatments have
produced improved results compared to monotherapy, we
are still a long way from being able to determine what
combinations will provide benefit for which patients.
5.2. Insulin-Like Growth Factor Receptor I (IGFR). While
EGFR has been the major focus of targeted receptor tyrosine
kinase therapies, there has been work into other known
survival signaling activating receptors such as IGFR. It has
previously been established that there is significant cross-talk
between IGFR and EGFR receptors, and the similar cellular
responses to signaling through these receptors could play
a large role in mediating resistance to anti-EGFR therapies
[48]. In 2002, Chakravarti et al. showed upregulation of the
IGFR gene in a GBM cell line resistant to AG1478, an EGFR
TKI. In this work, they demonstrated that upregulation of
IGFR in this resistant cell line correlated with sustained
activation of the PI3K pathway. Cotargeting of the IGFR
and the EGFR receptors led to increased apoptosis, as well
as a reduction in invasive potential [49]. A more recent
study demonstrated that combination of the IGFR inhibitor
NVP-AEW541 with dasatinib led to increased apoptosis in
GBM cell lines, but not in nonneoplastic human astrocytes,
and synergistically inhibited clonogenic survival [50]. These
studies highlight the possible efficacy of cotargeting IGF
and EGF receptors to overcome therapeutic resistance and
enhance therapeutic gain.
5.3. Platelet-Derived Growth Factor Receptor (PDGFR).
PDGF signaling is another commonly altered signaling path-
way in glioblastoma. A recent study in GBM cell lines showed
that varying concentration of imatinib, a PDGFR inhibitor,
had either cytostatic effects, at low concentrations, and pos-
sibly cytotoxic events at high concentrations [51]. Compar-
atively, another report in GBM cell lines showed treatment
with imatinib actually led to the activation and sustained
signaling through the ERK1/2, PI3K, and other important
cell survival signaling pathways [52]. Some reports have even
identified that PDGFR status was not predictive of imatinib
efficacy even though it was shown to be a prognostic marker
[53]. These results indicate that while PDGFR inhibition
might be an interesting target, much more study is needed.
6. PI3K/AKT/mTOR Inhibitors
In addition to EGFR and other RTK therapies, there has
been a major focus on inhibiting downstream survival
signaling pathways stimulated by these receptors. The two
most prominent and most studied pathways are the MAPK
signaling cascade and the PI3K/AKT/mTOR pathway. Ini-
tially it was believed PI3K signaling was responsible for
cell survival, while the MAPK pathway was involved in cell
proliferation. Now, these two pathways are thought to share
a significant amount of overlap and to both be involved in
cell growth, proliferation, and survival. As targeted therapies
have become a more important piece of the cancer treatment
arsenal, these pathways have been the focus of a significant
amount of research effort.
One of the initial works identifying PI3K inhibitors as
viable for the treatment of GBM was a paper by Kubota
et al. which showed the early PI3K inhibitor wortmannin
sensitized GBM cells to radiation regardless of p53 status
[54]. Wortmannin was then later shown to reverse the
growth advantage seen in GBM cells which both lacked
PTEN expression and overexpressed the EGFRvIII “always-
on” variant growth receptor [55]. However wortmannin,
while potent, has significant levels of nonspecific kinase inhi-
bition and is soluble in organic solvents, which has limited
its applicability for human clinical trials [56]. Following
the proof of principle of this novel targeting therapy, many

6Neurology Research International
new PI3K inhibitors were developed and used in clinical
trials. These include perifosine, cal101, px-866, pi-103, and
others with some PI3K inhibitors even being specifically
assessed in glioblastoma (XL765, XL147, and BKM 120)
(Figure 1). Results of many of these trials have been poor;
however, therapies using these drugs in combination with
other inhibitors have recently become a focus.
In addition to PI3K inhibition, small-molecule inhibitors
have been developed both upstream (i.e., AKT inhibitors)
and downstream (i.e., mTOR inhibitors) in this pathway.
AKT has been targeted because this kinase is the central node
in the RTK/PI3K/AKT/mTOR signaling cascade. Direct inhi-
bition of this molecule would prevent downstream signaling
similar to RTKi or PI3K inhibition. The importance of devel-
oping these novel inhibitors at different stages of the signal-
ing cascade has become even more obvious with the devel-
opment of RTKi refractory tumors. Only one AKT inhibitor,
mk-2206, has currently made it into phase II clinical trials.
Recently GlaxoSmithKline has begun phase I trials with
two different AKT inhibitors, with mixed results. The initial
phase I study of drug GSK690693 was withdrawn, and trials
of drug GSK2141795 are currently not recruiting patients.
Also, a phase II trial of MK-2206 had been planned in recur-
rent glioma; however, that trial has since been withdrawn.
However promising it has been preclinically, AKT inhibition
has proven difficult to translate into clinical efficacy.
Probably the most targeted member of this pathway is the
mammalian target of rapamycin (mTOR). In gliomas, it has
been observed that the mTORC2 complex promotes growth
and cell motility [57]. An early study demonstrating efficacy
of targeting this pathway showed increased radiosensitization
of a U87 xenograft [58]. Based on available data, it seems
likely that in order to effectively block mTOR activity in
cells, both mTORC1 and mTORC2 complexes will need to
be targeted [59]. These preclinical results have been critical
to planning the numerous clinical trials that have been
performed with mTOR inhibitors in glioblastoma. A phase
I trial of rapamycin in PTEN-deficient glioblastoma patients,
while showing some promising results, also demonstrated
the inherent difficulties of targeting this protein. In this
trial, multiple patients were observed who showed elevated
levels of pAKT following mTOR inhibition, which was
correlated with shorter time to progression [60]. The AKT
activation observed was likely due to alteration of signaling
feedback loops, again highlighting the complexity of targeted
therapy. Combination therapy to block these feedback loops
may also improve efficacy [61]. Despite this complexity,
promising results have pushed mTOR inhibitors to further
trials. Several of these mTOR inhibitors have been or are
currently being tested in the clinical trials setting specifically
in gliomas, including temsirolimus, everolimus (RAD001),
and sirolimus. Temsirolimus (CCI-779) has been the most
extensively studied drug in clinical trials. A phase I study
determined the clinically effective dose to be 250 mg IV
weekly [57]. Phase II trials with CCI-779 as a monotherapy in
recurrent GBM showed no effectiveness despite low toxicity
and initial disease stabilization [62]; however, a North
Central Cancer Treatment Group study showed a statistically
significant time to progression increase in temsirolimus
responders (5.4 months versus 1.9 months, 2.3 months
overall) [63].
Because of the promise of combination therapy, cur-
rently there is a significant emphasis on dual PI3K/mTOR
inhibitors. Several novel small-molecule inhibitors have been
developed that have dual specificity for these targets. XL765
has recently been shown to reduce cell viability in vitro
and in limited animal study showed a possible effectiveness
when combined with TMZ therapy [64]. Similarly PKI-
587 and PKI-402 were shown to have a strong in vitro
antitumorigenic effect across multiple cell types including
glioma cells, while also slowing tumor growth in xenograft
models [65,66]. Another dual PI3K/mTOR inhibitor, PI-
103, which is known to have monotherapy efficacy in glioma
[67] was recently shown to specifically reduce tumor volumes
in combination with NSC-delivered s-trail in an orthotopic
intracranial xenograft model [68]. PI-103 combination ther-
apy has also proven effective in sensitizing cells to both
chemotherapy [69] and radiation [70] through reducing
DNA damage repair. RAD001 is currently being used in
multiple combination treatment studies, including studies
employing an oncolytic virus [71], Raf inhibitors [72], and
VEGFR-2 [73]. In a GBM orthotopic xenograft model, it was
shown however that PTEN does not serve as a predictive
marker for RAD001 effectiveness, despite the importance of
PI3K/AKT signaling activation in mTOR upregulation [74].
There has also been evidence for targeting this signaling
pathway from both ends, with a report showing rapamycin
promotes a response to EGFR inhibitors in either PTEN-
sufficient or PTEN-deficient GBM cells by reducing tumor
cell growth. This treatment combination also results in
tumor cell death in PTEN-deficient cells [75]. While there
is hope clinical trials with these novel dual-targeting agents
will demonstrate better efficacy than current monotherapies,
there are still significant difficulties in trying to determine the
best role for these targeted therapies in cancer treatment.
7. Antiangiogenic Therapies
Antiangiogenic therapy, which has been well studied in
many types of cancer, has also emerged as a novel therapy
for glioblastoma. Glioblastoma is characterized by vascu-
lar proliferation or angiogenesis [76], and advances in
molecular biology have allowed us to target angiogenesis
of glioblastoma. VEGF, a critical mediator of angiogenesis,
is highly expressed in glioblastoma and regulates tumor
angiogenesis [77,78]. Preclinical studies have shown that
VEGF inhibitors inhibit the growth of glioma cells [79,80].
Antiangiogenic therapies for glioblastoma are currently the
most advanced of any targeted therapy, and many clinical
trials have demonstrated their efficacy. In fact, bevacizumab
has been approved by the US food and Drug Administration
in the setting of recurrent glioblastoma.
Bevacizumab is a humanized monoclonal antibody
against VEGF and prevents the activation of VEGF receptor
tyrosine kinases (Figure 1)[81]. This drug is considered a
well-established antiangiogenic therapy in several angiogenic
tumors [82]. In glioblastoma, a phase II study of the addition
of bevacizumab to the standard treatment of TMZ and

Neurology Research International 7
radiotherapy was conducted for 70 newly diagnosed patients
[83]. The median overall survival and PFS were 19.6 and 13.6
months, respectively. Another phase II study of additional
bevacizumab to standard therapy showed median PFS was
13.8 months in 125 newly diagnosed glioblastoma patients
[84]. Recently two clinical trials of bevacizumab have been
reported in 2011 ASCO annual meeting for newly diagnosed
glioblastoma. Vredenburgh et al. performed a phase II
study of bevacizumab, TMZ, and radiotherapy followed by
bevacizumab, TMZ, and oral topotecan [85]. The 6-month
event-free survival was 90%, and median overall survival has
not been reached. Although the regimen was tolerable, there
were 2 treatment-related deaths, including CNS hemorrhage
and pneumonitis. Omuro et al. conducted a phase II trial
of bevacizumab, TMZ, and hypofractionated stereotactic
radiotherapy for newly diagnosed glioblastoma patients with
tumor volume under 60 cc [86]. The median PFS was 11
months, and objective response rate was 90%. Furthermore,
1-year overall survival was 90% with median follow-up of
13 months. Despite a more aggressive radiotherapy schedule,
the regimen was well tolerated and had promising results.
Additional bevacizumab seems to have favorable effects;
however, it is still unclear whether this regimen can improve
overall survival. Two randomized phase III trials, RTOG
0825 and AVAGLIO, are ongoing to demonstrate the efficacy
and safety of combined therapy of bevacizumab, TMZ, and
radiotherapy for newly diagnosed glioblastoma [87,88].
The addition of bevacizumab to TMZ and radiotherapy is
expected to become frontline treatment for glioblastoma,
and is already thought of by some as nearly a part of standard
of care.
While bevacizumab has been thoroughly investigated
in clinical and preclinical studies, other drugs have also
been studied as antiangiogenic therapy for glioblastoma
patients [89]. Cilengitide, which is selective for αvβ3and
αvβ5 integrin receptors, is considered a novel antiangiogenic
therapy for glioblastoma. A phase II study of recurrent
glioblastoma treated by cilengitide showed that 6 month PFS
was 15%, and treatment was well tolerated [90]. Furthermore
a phase I/IIa study of cilengitide combined with TMZ
and radiotherapy was performed for 52 newly diagnosed
glioblastoma patients [91]. This regimen was well tolerated
and showed promising results with median overall survival
of 16.1 months. Currently, two randomized trials, CENTRIC
and CORE, are ongoing and are expected to show the benefits
of additional cilengitide to standard therapy for newly diag-
nosed glioblastoma patients [88,92]. Other antiangiogenic
therapies (e.g., VEGF receptor tyrosine kinase inhibitors)
have also been performed in clinical and preclinical studies
[89], although there have not been any drugs to show strong
antiglioma effects compared with bevacizumab. Further
investigation is warranted to establish the efficacy and safety
of novel antiangiogenic therapy for glioblastoma.
8. Novel Targeted Therapies
8.1. Notch Inhibitors. One of the most controversial topics in
cancer biology is the theory of cancer stem cells or tumor-
initiating cells. Despite the split opinions regarding the
existence of this cell population, mounting evidence has
spurred development of novel therapeutics to target this
proposed group of cells. While proper definition and
identification of this stem cell population is still ongoing,
researchers have used clues about pathways critical to
known stem cell populations to design novel therapies.
One such pathway is the notch signaling pathway, which
is important in both normal and neoplastic development
in the central nervous system by controlling proliferation,
apoptosis, stem cell maintenance, differentiation, and home-
ostasis [93]. Specifically in gliomas, notch has been linked
to overexpression of EGFR [94]; however, it is more likely
notch’s role in the maintenance of stem cell populations that
make it an interesting therapeutic target [95,96]. Gamma
secretase inhibitors (GSIs), which are known to inhibit
notch, have been shown to inhibit glioma stem cell growth
[97], while overexpression of notch induces tumor growth
and can be blocked by treatment with GSIs (Figure 1). While
notch inhibition is still very novel, it has shown efficacy
in preclinical models and shows a strong possibility for
combination therapy targeting both tumor cell bulk with
conventional therapies as well as the tumor-initiating cell
population.
8.2. Virotherapy/Gene Therapy for GBMs. Cancer therapy
using viruses comes in a variety of approaches including
direct viral cytotoxicity and targeted toxin delivery. The
use of viruses to deliver tumor suppressors, or siRNAs, to
knockdown oncogene expression, immune modulating com-
pounds, or antiangiogenic compounds, is also underway, yet
these attempts are often designed in combination, or as a
method to enhance oncolytic viruses (Figure 1).
The general goal behind direct viral cytotoxicity or
oncolytic virotherapy is to design a virus that specifically
and faithfully infects and replicates only in tumor cells. This
is usually accomplished by attenuating the virus to restrict
their replication to actively dividing cells (i.e., tumor cells)
while sparing normal, nonreplicating tissue. Viruses such
as herpes simplex virus 1 (HSV), adenovirus, and reovirus
have been attenuated so as to conditionally replicate within
cancer cells [98–100]. Oncolytic viral therapy has undergone
major changes, using the virus not only as a cytotoxic
therapy, but also as a delivery mechanism. Researchers
have used oncolytic HSV-1 to deliver vasculostatin, an
antiangiogenic compound [101], as well as chondroitinase
ABC I [102]. This method has demonstrated significantly
enhanced therapeutic efficacy over virus alone. Similar
approaches with adenoassociated virus (aav) particles [103]
have shown codelivery of oncolytic virus with antitumor
molecules has enhanced efficacy and improved survival in
xenograft models. Each of these oncolytic viruses has also
been examined in phase I clinical trials in glioma and have
been shown to be well tolerated [104–106].
Targeted toxin delivery is similar to oncolytic virus
therapy in that it is a virus-mediated cytotoxic therapy.
While oncolytic viruses are directly responsible for tumor cell
death, the strategy for this therapy is to use nonreplicating
virusparticles,suchasaav,todeliverapowerfultoxin,

8Neurology Research International
that is, Pseudomonas exotoxin (PE), specifically to cancer
cells by targeting preferentially expressed receptors. Some of
the receptors include the IL-13R variant α2, which varies
from the receptor expressed on normal brain cells [107], as
well as EGFR [108] or the EGFRvIII variant [109]. Clinical
trials have been performed with an IL-13Rα2 targeting virus
delivering cintredekin besudotox (CB). A phase III study of
this therapy compared to Gliadel Wafer administration at
first recurrence showed no survival benefit [110]. In general,
despite promising preclinical results with viral therapy for
gliomas, clinical trials resulting from this work have yet to
show any significant survival benefits. This pattern is not
uncommon in cancer therapy, yet it does point out the need
for more research. Despite the poor clinical trial results to
date, the positive data coming from viral therapy research
indicate that this form of therapy has significant future
potential.
8.3. Immunotherapy. One alternative to target gliomas with-
out affecting normal cells is by utilizing the body’s natural
defenses to kill tumor cells. There are currently two main
immunotherapy strategies being tested against gliomas:
adoptive immunotherapy and active immunotherapy.
Adoptive immunotherapy is the process of stimulating
immune cells ex vivo and then readministering them to the
patient in hope of therapeutic benefit. This can be done
either intravenously or directly into the tumor. The two
major cell types used are lymphocyte activated killer (LAK)
cells or cytotoxic T lymphocytes (CTLs). Recent reports have
identified a synergistic response between cytokine-induced
killer cells (CIKs) and TMZ [111]. Specific targeting of HER-
2 by T cells was shown to induce regression in HER-2-
positive autologous tumor xenografts as well as to target
the tumor-initiating cell (TIC) population, demonstrating a
targeted approach may prove to be more effective [111,112].
AsignificantnumberofphaseIorphaseI/IItrialshavebeen
performed using LAK cells or CTLs. These trials were largely
in the recurrent setting and demonstrated limited efficacy
combined with significant rates of toxicity for LAK cells
[113], and better tolerance and improved survival for CTLs
[113]. While responses to the adoptive therapies have shown
only limited efficacy to date, it is possible that combination
therapy or more targeted immunotherapeutic approaches
may be of use.
Active immunotherapy is similar to vaccination, the
idea being to stimulate the patient’s immune system by
using tumor-related sources of antigen (whole tumor cells,
tumor protein lysates, mRNA, synthetic peptides). These
sources of antigen can either be injected alone or coupled
to dendritic cells [113]. Dendritic cells are powerful antigen-
presenting cells, and dendritic cell therapy is designed to
increase antigen presentation by incubating tumor antigens
with these cells before injecting them back into the body
(Figure 1). This method of active tumor immunotherapy
has been the most extensively studied with widely varying
results. Some of the trials performed using this method
have shown promising results, while others demonstrated no
benefit [113]. A major problem in interpreting results from
these trials is the wide variation in protocols for everything
from acquiring cells, type of tumor antigen chosen, and
number of cells used. However, targeting this type of therapy
may lead to enhanced clinical benefit. One major trial
using patient tumor cell cultures infected with Newcastle
Disease virus followed by gamma irradiation demonstrated
significant increases in progression-free survival, 40 weeks
versus 26 weeks, as well as overall survival, 100 weeks versus
49 weeks [114]. This trial also reported increased 1-year
(91% versus 45%), 2-year (39% versus 11%), and long-term
survivors (4% versus 0%). These results are very promising;
however, the trial was nonrandomized and studied a limited
number of subjects (23 patients receiving immunotherapy
with 87 controls). Controls were also not treated using
current standard of care as this trial was performed in 2004
before the results of the Stupp trial were published. Human
cytomegalovirus (CMV) has been identified to be associated
with tumors in a significant proportion of glioblastoma
patients (50–90%) [115,116]. Currently two clinical trials are
ongoing at the Duke Brain Tumor Immunotherapy Program
attempting to utilize this knowledge. The Vaccine Therapy
in Treating Patients with Newly Diagnosed Glioblastoma
Multiforme (ATTAC) [117] and the Evaluation of Recovery
From Drug-Induced Lymphopenia Using Cytomegalovirus-
Specific T-Cell Adoptive Transfer (ERaDICATe) [118]trials
are either recruiting or in a data analysis phase, with results
likely to be reported soon.
8.4. DNA Damage. DNA damage repair is a double-edged-
sword in the cancer world. Lack of proper DNA repair
can lead to genomic instability and the generation of
cancer. However, once cancer is established, DNA repair
genes undermine many effective cancer therapeutics. Both
radiotherapy and chemotherapies are designed around a
DNA-damaging strategy designed to induce cell death and
tumor regression. In the presence of DNA repair proteins,
these therapies have reduced efficacy. By targeting DNA
repair proteins in cancer cells, we can again render them
sensitive to radiation and chemotherapies. PARP plays a role
in single stranded DNA, repair and inhibitors are currently
being tested in a number of cancer sites. If PARP is inhibited,
single-stranded nicks will not be repaired and will lead
to DNA strand breaks during replication. Double-stranded
DNA breaks are extremely toxic lesions to cells, and thereby
PARP inhibition should increase cell death in proliferating
cells (Figure 1). PARP inhibitors E7016 and AZD2281 have
been shown to radiosensitize GBM cells both in vitro and
in vivo [119,120], with an enhanced effect when combined
with heat shock protein 90 (HSP90) inhibition [121]. PARP
inhibitors have also been shown to increase the efficacy
of chemotherapies such as DNA topoisomerase I poisons,
TMZ, iriniotecan, or cilengitide [122–124]. It has also been
observed that PTEN loss can negatively affect homologous
recombination, thereby increasing the efficacy of PARP
inhibition as well as other DNA-damaging modalities [125].
In addition to PARP inhibition, other targets have been
identified to utilize DNA repair as a therapeutic strategy.
Inhibition of PP2A has been shown to augment DNA-
damaging agents by inducing Plk-1 and AKT activation
and decreasing p53 expression which has led to complete

Neurology Research International 9
remission or significant tumor regression, when combined
with TMZ or doxorubicin, in a large number of tumors in a
xenograft model [126].
8.5. Autophagy. Autophagy is an evolutionarily conserved
process through which the cell is able to degrade damaged
organelles and other cell components [127]. Reports identify
autophagy to serve a dual role in cancer. Limited and
controlled autophagy can be a survival method for cancer
cells which allows them to overcome current therapies
such as chemotherapy and radiation [128–130]. However,
autophagy has also been identified as a possible therapeutic
target because sustained and uncontrolled autophagy can
lead to cell death [131,132]. This process is interlinked
with several crucial cancer survival pathways including
p53 signaling and the PI3K pathway, as well as apoptotic
signaling molecules like Bif-1 [127]. Currently, attempts
are being made to control this switch and tip cells into
a pro-death state. This work is similar to the rationale
behind apoptotic research. Resistance to apoptosis is one
of the major hallmarks of many cancer types, and the
ability to regulate this cellular process would allow for
not only increased efficacy of current treatments but also
a novel target for future therapy. As with most therapies
discussed so far, combination therapy will likely play a
key role in future treatment plans. Recent studies have
demonstrated autophagy can be induced in glioma cells
by current standard-of-care therapy [130,133] and AKT
signaling plays a major role in the prosurvival effects of this
process [129,134]. This data indicates that cotargeting AKT
will be important in regulating and controlling the prodeath
role of autophagy in glioma.
8.6. HDAC Inhibitors. HDAC inhibitors have been studied
in the setting of GBM and shown positive indications in
both preclinical and clinical testing. In preclinical models,
HDACs have been shown to sensitize cells to chemotherapy
[135], to have antiproliferative activity by increasing PTEN
and AKT expression while reducing phosphorylation of the
proteins to their active forms [136], to increase apoptosis
in GBM cells through activation of the JNK pathway and
reductionintelomeraseactivity[137], and to sensitize cells
to radiation [138]. In addition, in a phase II clinical trial,
the potent HDAC inhibitor, vorinostat, was shown to have
modest single-agent efficacy and to be well tolerated [139].
8.7. MicroRNA. As microRNAs have become better studied,
the possible roles in therapeutic scenarios have increased.
Issues still remain in targeted delivery of these microRNA
constructs to tumor cells, but many possible targets have
been identified for almost all cancer types. In glioblas-
toma, some of these targets include miR-124 and miR-137,
which could target both tumor-initiating cells, by inducing
differentiation, and normal glioblastoma cells by arresting
cell growth [140]. The miR 302–367 cluster has also been
shown to induce TIC differentiationaswellastoreduce
the infiltrative properties of these cells [141]. A miR-21
inhibitor has been shown to sensitize the glioblastoma cell
line U251 to ionizing radiation [142]. miR-34-a has been
shown to inhibit glioblastoma growth by targeting the c-Met
and notch pathways, two well-known signaling pathways
linked to glioma pathogenesis [143]. miR-10b has been
linked to glioblastoma cell growth [144], and miR-124a has
been linked to migration and invasion [145]. This sampling
of microRNA targets linked to glioblastoma growth, survival,
TIC maintenance, and migration/invasion underscores the
possible therapeutic application of microRNAs to regulate
major pathways linked to disease severity.
9. Discussion
In this paper we have discussed RTK inhibition, angiogenesis
inhibitors, and the PI3K/Akt/mTOR inhibition in detail
due to the substantial amount of research conducted in
these areas and have also briefly discussed several very
novel areas of research including Notch inhibition, viral
and immunotherapies, and DNA repair pathways. Within
the heavily researched pathways, the overarching theme in
glioblastoma therapy is that monotherapies demonstrate
limited efficacy. Because single-agent therapies have shown
no significant benefit, it is critical to begin designing
rational combinations. Different RTK inhibitors combined
with PI3K/mTOR dual inhibitors or antiangiogenic agents
combined with Akt inhibition are already being examined.
It is likely that many of the novel therapies discussed in
this work will demonstrate greater efficacy when paired with
the more studied targeted therapies. Because many of these
targets are within the same signaling cascade, inhibiting
pathways horizontally rather than vertically should remove
some of the compensatory mechanisms glioblastomas use to
overcome treatment. It is also important to note that many
of the molecular biology advancements will be augmented
by advancements in current treatments. Improved tumor
border delineation or detection of microscopic disease will
enhance the efficacy of upfront surgical and radiotherapy
interventions while better methods for posttreatment image
surveillance will improve treatments in the recurrent setting.
Critically, it should be acknowledged that these therapies
will need to work in conjunction with the current standard
of care, highlighting treatments that can serve as radio-or
chemosensitizers. Glioblastoma carries a very poor progno-
sis, but with improved technology and novel, personalized,
rational, targeted therapies patient survival and quality of life
will be greatly improved.
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