REVIEW

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Circulating biomarker panels for targeted

therapy in brain tumors

Cristiana Tanase*,1, Radu Albulescu1,2, Elena Codrici1, Ionela Daniela Popescu1, Simona
Mihai1, Ana Maria Enciu1,3, Maria Linda Cruceru3, Adrian Claudiu Popa3, Ana Iulia
Neagu1,4, Laura Georgiana Necula1,4, Cristina Mambet1,4 & Monica Neagu1

ABSTRACT An important goal of oncology is the development of cancer risk-identifier

biomarkers that aid early detection and target therapy. High-throughput profiling represents
a major concern for cancer research, including brain tumors. A promising approach for
efficacious monitoring of disease progression and therapy could be circulating biomarker
panels using molecular proteomic patterns. Tailoring treatment by targeting specific
protein–protein interactions and signaling networks, microRNA and cancer stem cell
signaling in accordance with tumor phenotype or patient clustering based on biomarker
panels represents the future of personalized medicine for brain tumors. Gathering current
data regarding biomarker candidates, we address the major challenges surrounding the
biomarker field of this devastating tumor type, exploring potential perspectives for the
development of more effective predictive biomarker panels.

The most common and lethal primary type of brain tumors is represented by high-grade gliomas; KEYWORDS 
they present one of the highest rates of occurrence within the tumors of the CNS. Grade IV glioma • biomarker panels • brain
– glioblastoma (GBM) – represents the most common and aggressive type of brain tumor in adults tumors • circulating
with a poor prognosis of a 1–2-year survival rate after diagnosis [1] . biomarkers • proteomics
The treatment of GBM is a significant therapeutic challenge; it is obvious that new approaches • targeted therapy
regarding classical and new therapeutic targets involving angiogenic signals, signaling pathways,
protein–protein interactions, stem cell targets and crosstalk between all of them are still an unmet
need in this disease.
Due to the high complexity and heterogeneity of this type of cancer, conventional biochemical
methods can fail to notice important components and/or proteomic profiles of this disease. One of
the main goals of oncology is represented by biomarkers development, since it presents the potential
to identify cancer risks, as well as to improve early detection and targeted therapy. We are confident
that a biomarker panel would provide superior identifiers and/or predictors of a patient’s clinical
outcome. High-throughput proteomic profiling and multiplex analysis, such as xMAP and protein
arrays technologies, are becoming important approaches in GBM research. These technologies can
supply simultaneous analyses of biomarkers in a panel for improved diagnosis, patient stratification,
prognosis and drug screening. Biomarker discovery for brain tumors is an ongoing pursuit and the
search for the best molecule or combination of molecules is still unfolding [2] . In the last 5 years,
proteomics topics were a major presence in the literature, having a bulk of more than 5000 papers

1
Victor Babes National Institute of Pathology, Biochemistry-Proteomics Department, no 99–101 Splaiul Independentei, 050096 Sector
5 Bucharest, Romania
2
National Institute for Chemical Pharmaceutical R&D, 112 Calea Vitan, 031299 Sector 3, Bucharest, Romania
3
Carol Davila University of Medicine & Pharmacy, Cell Biology & Histology Department, no 8 B-dul Eroilor Sanitari, 050474 Sector 5
Bucharest, Romania
4
Stefan S Nicolau Institute of Virology, Bucharest, Romania
*Author for correspondence: bioch@vbabes.ro

10.2217/FON.14.238 © 2015 Future Medicine Ltd Future Oncol. (2015) 11(3), 511–524 ISSN 1479-6694 511
Review  Tanase, Albulescu, Codrici et al.
focusing on proteomics in cancer; around half
of these papers report biomarker candidates in
intracranial tumors. In spite of this abundance,
clinicians still do not count on a specific tumor
marker for brain tumors.
Therefore, this review, along with our hands-
on experience, emphasizes the latest knowledge
in circulatory biomarkers, the tumor-derived
blood-based biomarkers, circulating tumor cells,
circulating nucleic acids and circulating proteins
in patients with malignant gliomas.
Molecular classification of GBM
Histological and molecular analysis of CNS
tumors can identify approximately 120 sub-
types. Peripheral blood scans for predictive
biomarkers in CNS indicated that certain brain
tumors are indeed associated with distinct pro-
files of circulating factors, such as proteins (e.g.,
glial fibrillary acidic protein), DNA fragments
[3] or miRNAs (e.g., miRNA-21) [4] .
So far, the only subclassification of GBM
that also inf luences the therapeutic deci-
sion is the detection of O-methylguanine-
DNA methyltransferase (MGMT ) promoter
hypermethylation.
Recent progress in the molecular research of
GBM has led to the identification of other mark-
ers useful in either the diagnosis or prognosis of
this fatal disease. Mutation in isocitrate dehy-
drogenase genes, notably for isoform 1 (IDH1),
seems to bear favorable prognostic value for GBM
patients [5] . Furthermore, The Cancer Genome
Atlas Research Network showed evidence for a
CpG island methylator phenotype in GBMs,
associated with IDH1 mutations [6] . The newly
emerged data involving IDH mutations and epi-
genetic modifications prompted further inquir-
ies regarding the molecular mechanisms that
occur up- and down-stream from these events.
RBP1 promoter hypermethylation is found in
nearly all IDH1 and IDH2 mutant gliomas, and
is associated with improved patient survival [7] .
TET enzymes that catalyze oxidation of 5mC to
5hmC and epigenetically modify gene transcrip-
tion are mutated in several types of cancer, affect-
ing their activity and likely altering genomic
5hmC and 5mC patterns [8] . Modified expres-
sion of TET genes seems to have a significance
in risk stratification of GBM patients, along with
APOBEC deaminase genes [9] . Promoter hyper-
methylation of p16INK4, p14ARF, RB, PTEN
and p53 were also reported to play a role in the
epigenetic mechanisms of GBM pathology [10] .
512 Future Oncol. (2015) 11(3)
Loss of heterozygosity (LOH) on chromosome
1p/19q and chromosome 10 may help to predict
patient outcomes [11] .
The assessment of these specific alterations
has been encumbered by the scarceness of biotic
material; therefore, their detection in the cir-
culation would decrease diagnostic time and
expedite the therapeutic decision. Apart from
genetic circulating tumor DNA analysis, which
emulates tumor assessment for genetic and epi-
genetic aberrations, serum profiling for protein
biomarkers has also been proven useful as a diag-
nostic and/or prognostic tool for GBM. It is now
commonly accepted that a panel of biomarkers
shows better predictive power over a single bio-
marker, and recently, due to the development
of proteomic techniques, panel attainment has
become a feasible task [12] . Hence, a number of
proteins has been identified as potential diag-
nostic markers (A2M, factor VII, MDC, SCF
[13] and staging biomarkers [haptoglobin, plas-
minogen precursor, apolipoprotein A-1 and M,
and transthyretin]) [14] . A systematic review of
multiple independent proteomic analyses of
glioma, carried out by Deighton et al., demon-
strated alterations in 99 different proteins, ten of
which were repeatedly reported (PHB, Hsp20,
serum albumin, EGF receptor, EA-15, RhoGDI,
APOA1, GFAP, HSP70 and PDIA3) [15] .
Circulating tumor cells in malignant
gliomas
Circulating tumor cells (CTCs), originating
both from primary and metastatic lesions, can
be detected in the peripheral blood of patients
with different solid tumors [16] . Unlike normal
blood cells, CTCs are present in very low counts,
up to a few hundred per milliliter, depending on
the CTC definition and the methods used for
their detection and isolation [17] .
Several clinical studies performed in patients
with metastatic breast, prostate, colorectal and
non-small-cell lung cancer (NSCLC) showed
a correlation between CTC count and clinical
outcome, and suggested that CTC enumera-
tion may represent a noninvasive adjuvant tool
for prognosis, recurrence and therapy response
monitoring [18] .
However, it will be very useful in clinical
practice to detect CTCs in patients with early-
stage cancer in order to benefit from more
adequate risk stratification and personalized
therapy. This approach is limited by the fact
that most of the current methods are not able to
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 Circulating biomarker panels for targeted therapy in brain tumors  Review
accurately establish the CTC numbers in early-
stage tumors, and further investigation is needed
for this purpose.
Regarding malignant gliomas, although this
type of tumor is thought to rarely disseminate
outside the brain, there are reports in the lit-
erature of lung, liver, lymph node and bone
metastasis occurring in patients with GBM
multiforme [19] . Moreover, some cases of GBM
transmitted through organ transplantation from
affected donors are well documented [20] . These
data provide indirect evidence that CTCs are
present in patients with malignant gliomas,
although thus far they have not been ­successfully
detected [21] .
Due to the fact that brain tumors, such as
GBM, tend to lack cancer cell surface biomark-
ers such as EpCAM on which current methods
of CTC detection are based, in a very recent
paper Macarthur KM et al. proposed a new
strategy to detect CTCs. They used a telomer-
ase-specific adenoviral agent to assess telomerase
activity, which is increased in nearly all tumor
cells, but not in normal cells. The assay proved
to successfully detect CTCs in patients with
brain tumors, thus providing a promising tool
for monitoring treatment response [22] .
High-grade gliomas are characterized by
increased neovascularization and recruitment
of endothelial progenitor cells (EPCs); this rep-
resents one of the mechanisms involved in new
vessel formation. Related to this topic, Corsini
E et al. studied the effect of surgical and post-
surgical treatment at the level of EPCs in glioma
patients and their correlation with VEGF. They
found significantly decreased levels of EPCs in
all treated patients compared with untreated
ones. VEGF registered decreased levels only
after surgery, but not after chemotherapy; no
correlation was found between VEGF and EPCs
levels. Further studies are needed to assess the
usefulness of EPCs as markers of angiogenesis
monitoring in high-grade gliomas [23] .
Oncosomes as future circulating
biomarkers carriers
The complex interactions between CNS cell
types and distinctive cellular milieu, gener-
ates a particular biology for brain tumors,
which impacts on their sensitivity to treatment.
Oncosomes, or membrane-derived extracellu-
lar vesicles (EVs) secreted by cancer cells, can
transport proteins and nucleic acids from one
cell to the other, thus being active participants
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in the oncogenic transformation of various cells.
It has been recently noted that oncosomes can
contain several types of molecules involved in
pathogenesis pathways, such as tumor prolif-
eration, angiogenesis and invasion. Oncosomes
can contain proteins, transcripts, DNA and
miRNAs that ‘hijack’ the recipient cell’s physi-
ology and cell microenvironment. Oncogenic
EGF receptor (EGFRvIII), tumor suppressors
(PTEN) and oncomirs (miR-520g) were found
in the oncosomes identified in blood circulation
and the cerebrospinal fluid (CSF) of patients
diagnosed with brain tumors. These new ‘cir-
culatory’ biomarkers could be useful biomark-
ers in patient stratification and/or therapeutic
efficacy predictors [24] . As we are focusing on
circulatory biomarker candidates, the role of
exosomes/oncosomes in cancer, hence a circula-
tory particle capable of transferring tumor cell-
derived genetic material and signaling proteins
from one cell to another and thus linking tumo-
rigenesis, angiogenesis and metastasis, is even
more intriguing. In 2013 it was reported that
internalization of exosomes was derived from
GBM cells where the protein CAV1 negatively
regulates the uptake of exosomes. Moreover,
exosomes can induce activation of ERK1/2 and
HSP27, opening new opportunities for therapy
targets [25] .
Thus, oncosomes have come into view in
brain tumor research domain as important vehi-
cles of intercellular communication, providing a
new field of molecular biomarkers investigation.
miRNAs as molecules linking tissue
& circulating biomarkers
The discovery of miRNA in tissues, as well as
in body fluids together with their altered pro-
file in various pathological conditions, offers
a new perspective on the use of extracellular
miRNAs as informative biomarkers of disease
[26,27] . Similarly to other types of tumors, GBM
multiforme was associated with numerous miR-
NAs. Such miRNAs display either oncogenic or
tumor-suppressive properties, and are therefore
attractive therapeutic targets for future miRNA-
based therapies. Moreover, some studies have
suggested that miRNAs may be used as nonin-
vasive diagnostic and prognostic biomarkers due
to their release in the circulation [28] .
In our previous paper, we also highlighted the
fact that miRNA expression profiles in GBM
are better predictors of clinical outcome than
mRNA profiles [29] .
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Review  Tanase, Albulescu, Codrici et al.
In order to select candidate miRNA biomark-
ers for malignant astrocytomas, Yang et al. per-
formed genome-wide miRNA screening of serum
samples from astrocytoma patients and matched
controls. After subsequent validation of the
obtained results, seven miRNAs were found to be
significantly decreased in patients versus controls:
miR-15b, miR-23a, miR-133a, miR-150, miR-
197, miR-497 and miR-548b-5p. Moreover, these
identified miRNAs exhibited a marked increase
after surgery. As a conclusion, this miRNA pro-
file may be a potential noninvasive biomarker
panel for malignant astrocytoma [30] .
Different studies have demonstrated abnor-
mal miRNA expression profiles in GBM tis-
sues; some miRNAs were found to be upreg-
ulated (e.g., miR-17, miR-21, miR-93 and
miR-221/222), while others were downregulated
(miR-7, miR-34a, miR-128 and miR-137) [31] .
Lu et al. found an overexpression of miRNA-
17 in glioma tissues that was associated with
advanced pathological stage and poor overall
survival [32] , while in a previous study conducted
on glioma cell lines, Malzkorn et al. showed that
miR-17 inhibition reduces cell viability and
increases apoptotic activity [33] .
In vivo and in vitro studies on glioma cells
pointed out that miR-93-overexpressing cells
promoted endothelial cell spread, growth,
migration and tube formation supporting neo-
angiogenesis and tumor growth. This effect is at
least partly explained by the fact that miRNA-93
targets and silences integrin-β8, whose expres-
sion is associated with decreased cell growth [34] .
Significant overexpression of miR-221/222
was detected in high-grade gliomas compared
with low-grade gliomas, related to increased cell
invasion and poor prognosis by directly targeting
TIMP3 [35] .
Recent work reported decreased expression
of miR-128, which correlates with aggressive
human glioma subtypes; miRNA-128 exerts
tumor suppression effects by targeting RTK
signaling [36] . Although miRNA-128 was found
to be downregulated in GBM tissue compared
with normal brain tissue, Roth et al. detected
increased expression levels of miRNA-128 in
blood cells of GBM patients [37] .
Kefas B et al. found a significantly reduced
expression of miR-7 in human GBM tissue and
GBM cell lines, showing that miR-7 directly
inhibits EGFR expression and suppresses Akt
pathway activation, and thus decreases the
­viability and invasiveness of GBM cells [38] .
514 Future Oncol. (2015) 11(3)
Another miRNA found to be downregulated
in GBM is miR-34a; its expression has an anti-
tumor effect suppressing cell proliferation, G1/S
cell cycle progression, cell survival, cell migra-
tion and cell invasion in glioma cell lines. These
properties are due to inhibition of c-Met, Notch-1
and Notch-2 [39] .
Proteomic profiling for circulating
biomarkers discovery
During the last decade, a wide range of tech-
nologies has been employed in molecular and
genetic profiling studies for biomarker discovery
in brain tumors. However, a limited number of
biomarkers has been identified and none has
yet attained broad application for use in clinical
gliomas prognosis, therapeutic target selection
or molecular classification [40] .
Proteomic profiling offers large-scale analysis
of protein expression, post-translational modi-
fication and protein–protein interactions. The
ideal platforms for biomarker discovery include
genomic and metabolomic analyses, and have
the ultimate goal of unifying the information
into protein networks [41] .
While advanced proteomics, based on combi-
nations of techniques such as 2D or 2D-DIGE
and mass spectrometry, are the solution of choice
for detailed studies that involve sequencing,
high-throughput techniques, such as SELDI-
TOF and MALDI-TOF, are used for differen-
tial protein signatures. The major advantage of
SELDI-TOF mainly lies in the fast output of
molecular signatures, yet it has to be usually fol-
lowed by more in-depth studies (based on more
accurate MS/MS technology) in order to more
accurately identify the proteins with modified
expression [42] .
Identification of potential neoplasia diagnos-
tic, prognostic, predictive or treatment-assessing
biomarkers could allow differential proteomic
profiling of brain tumor versus disease-free state
for the detection and monitoring of pathology-
related changes. Specifically, proteomic analysis
of different fluids is less invasive than biopsy
and could provide potentially informative
causes of origin and progression of brain tumor
­pathologies [40] .
A number of studies have reported different
individual serum biomarkers for GBM, such as
YKL-40 [43] , GFAP [44] , MMP-9 [45] , EGFR [46]
and CD14 [47] .
Reyens et al. reported elevation of several
inflammatory proteins (C-reactive protein, IL-6,
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 Circulating biomarker panels for targeted therapy in brain tumors  Review
TNF-α and sialic acid), coagulation factors
(fibrinogen, endogen thrombin generation, pro-
thrombin fragments 1 and 2, and tissue factor)
and angiogenic factors (VEGF, soluble VEGF
receptor 1 and thrombospondin-1) in the plasma
of GBM patients [48] .
SELDI-TOF MS technology has been utilized
in the protein profiling analysis of a variety of
specimens derived from patients with brain
tumors for the discovery of biomarkers that
facilitate early diagnosis, establish tumor grade
and predict therapeutic outcome.
Kumar et al., based on a combination 2-DE/
MS approach, observed ten differentially
expressed proteins in the sera of patients with
GBM and validated haptoglobin a2 as serum
marker associated with tumor growth and
migration in GBM [49] .
In another study based on SELDI-TOF MS,
Petrik et al. discovered and validated AHSG as
a predictive and prognostic biomarker for GBM.
Thus, low serum levels of AHSG were correlated
with a short median survival rate (<3 months),
with normal levels in serum being associated
with prolonged survival (>2 years) [50] .
In a preliminary study [51] using SELDI-TOF
MS on serum samples, we identified a number of
11 clusters with significant differences between
GBM and controls (p < 0.05) out of a total of
152 clusters with m/z values 2–55 kDa; six clus-
ters were overexpressed and five underexpressed
in GBM patients compared with control.
Gautam et al. [52] , using an iTRAQ-based
LC-MS/MS approach in GBM patients plasma,
have identified a total of 296 proteins, out of
which 61 exhibited increased levels in the
patient group. Altered levels of FTL, S100A9
and CNDP1 were validated by ELISA. These
proteins may form useful starting points for the
development of plasma-based biomarker panels
in clinical investigations for GBM [52] .
Soluble biomarkers – main actors in panel
set up
While mass spectrometry-based approaches are
undoubtedly some of the most effective tools
in the de novo discovery of biomarkers, other
approaches are available for sorting out biomark-
ers from known molecules, such as cytokines,
chemokines or growth factors via protein arrays
or, more effective, xMAP multiplex technology.
While protein arrays are generally more pow-
erful in terms of the total number of proteins
that can be simultaneously detected, xMAP
future science group
arrays are more suitable to perform quantitative
analysis in high-throughput conditions [2] .
Reliable circulating biomarkers could sup-
port the management of gliomas by facilitating
neuroradiological differential diagnosis at initial
presentation, planning of surgical interventions
or monitoring of the disease course.
Tumor initiation and progression represent a
complex process involving genomic mutations,
microenvironmental factors and inflammatory
mediators. An active role in determining the
malignancy phenotype is owned by the tumor
microenvironment. Both host cells and cancer
cells crosstalk via a large variety of soluble fac-
tors, whose effects determine the final outcome
of the tumorigenic process [53] .
Inflammatory cells, cytokines/chemokines
and their receptors existing in tumors contrib-
ute to tumor growth, progression, metastasis
and immunosuppression. They regulate tumor
growth either directly by transformation, sur-
vival, proliferation and migration of cancer
cells, or indirectly by enhancing angiogenesis
or recruiting leukocytes [54] . The “match that
lights the fire” of cancer is genetic damage; in
this context, some types of inflammation may
provide the “fuel that feeds the flames” [55,56] .
Matrix metalloproteases (MMPs), important
components of the tumor microenvironment and
secreted by glioma cell lines, are responsible for
sVE release. Soluble VE-cadherin in the blood
might reflect VEGF activity at the tumor site.
Analysis of glioma patient sera confirmed the
presence of sVE in the bloodstream, and sVE
levels were significantly predictive of overall sur-
vival, irrespective of the histopathological grade
of tumors [57] .
Plasma MMP-2 and VEGF were identified
in the study of Xu et al. as potential biomarkers
that accurately distinguished high-grade glioma
patients from controls [13] . MMP-2 and VEGF
have also been investigated in urine samples
as diagnostic biomarkers for brain tumors [58] .
MMP-2 promotes cell invasion, angiogenesis,
activation of growth factors, and is involved in
the development of human glioma microves-
sels, facilitating glioma cell invasion [59] . VEGF
can promote tumorigenesis and angiogenesis of
human GBM stem cells, and VEGF expression is
significantly increased in high-grade ­astrocytomas
compared with low-grade astrocytomas [13] .
Serum S100B and NSE were considered
markers of CNS damage, yet further studies
were required to establish whether S100B is an
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Review  Tanase, Albulescu, Codrici et al.
independent prognostic biomarker in glioma
patients [60] . Subsequent studies have showen
that biomarkers such as S100B, NPY and SCGN
were markedly elevated in plasma of patients
with malignant glioma 1 year before clinical
manifestation [61] .
Changes in serum levels of PlGF were associ-
ated with overall survival in patients with recur-
rent GBM [62] . Plasma concentrations for PlGF
were correlated to neuropathological or neuro-
radiological features. In addition, IL-8, BDNF
and GDNF were detectable in different concen-
tration levels in serum/plasma. Measurement of
circulating GFAP and PlGF are potentially use-
ful as clinical biomarkers that may support dif-
ferential diagnosis of GBM versus intracerebral
metastasis [63] .
Serum IL-10 levels have been described as
significantly increased in high-grade glioma
patients compared with nontumor control
patients [55,64] . Increased levels of IL-10 in
GBM patients are consistent with the findings
that Th2 cytokines are elevated in patients with
advanced tumors [13] . IL-10 was identified as a
serum biomarker that accurately differentiates
GBM patients from controls [13] .
A2M has been upregulated in migrating
human glioma cells compared with nonmigrat-
ing glioma cells [65,66] . Neuronal and glioma-
derived SCF can induce angiogenesis within the
brain [67] . Expression of tissue factor, the cell
surface receptor for factor VII, correlates with
histologic grade of human glioma malignancy
and vascularity [68] . In addition to the accurate
classification of malignant glioma patients, cir-
culating factor VII was also found to have prog-
nostic significance. MDC was underexpressed
in plasma/serum samples from high-grade
glioma patients. MDC and its receptor CCR4
both show decreased expression in human grade
III astrocytoma and grade IV glioma cells [69] .
Knowing that these biomarkers have differential
expression levels in high-grade glioma tissues,
they were also identified in glioma patients’
blood using multiplex assay [13] . Thus, A2M, fac-
tor VII, MDC and SCF were identified as bio-
markers in patients with malignant glioma, both
in plasma and serum specimens, with signifi-
cant differences in the protein expression levels
between patients and controls. These molecules
represent protein biomarkers that may identify
patients with malignant glioma in independent
cohorts with similar accuracy in either plasma
or serum specimens [13] .
516 Future Oncol. (2015) 11(3)
An important issue regarding antiangio-
genic treatment discontinuation and patient
morbidity remains the toxicity derived from
antiangiogenic therapy, thus serum/plasma bio-
marker panels that correlate with drug toxicity
and changes in cytokine and angiogenic fac-
tors would be of great value. Changes in IL-13
from baseline to 24 h predicted on-target tox-
icities. Changes in IL-6, IL-10 and IL-13 were
frequently correlated with toxicity. Profiling of
IL-13 as a surrogate for endothelial dysfunc-
tion could individualize patients at risk during
antiangiogenic therapy [70] .
In our previous study [2] , a panel of cytokines
with modified expression was detected in GBM
patients’ sera using multiplex assay. Our results
indicate significant dysregulation in serum lev-
els of cytokines and angiogenic factors, three-
fold upregulation for IL-6, IL-1β, TNF-α and
IL-10, and twofold upregulation of VEGF,
FGF-2, IL-8, IL-2 and GM-CSF were noticed
(Figure 1) . Cytokines expression was strongly cor-
related with tumor grade, proliferation markers
and clinical aggressiveness in GBMs [2] .
A circulating biomarker-based signature is an
important approach for the clinical assessment
of malignant glioma, with the abovementioned
biomarkers facilitating accurate diagnosis and
therapy monitoring, and assessing survival rate
prognosis [40] .
Assessing circulating biomarkers through the
rapid and efficient method represented by array
technology can be a reliable tool for the diagno-
sis of brain tumors and for the discovery of new
potential therapeutic targets or therapy monitor-
ing. Biomarker profiling, obtained via proteomic
high-throughput technologies, could offer major
support to identify unique differences within an
individual tumor or between tumors of the same
histological grade [71] .
Signaling molecules profile as potential
biomarkers & therapy targets
As stated in the previous sections, the aggres-
siveness and therapeutic resistance of GBM also
represents the base for the search for signaling
dysregulation.
The intracellular pathways and the molecules
that append to these pathways could assume
both the biomarker and future therapy target
role [72] . GBM shows dysregulation in various
signaling pathways, including the G1/S cell cycle
checkpoint and the MAPK and PI3K effector
arms of RTK signaling [73] .
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 Circulating biomarker panels for targeted therapy in brain tumors  Review

Modulation of serum cytokine levels

in glioblastoma patients

IL-6
IL-1β
TNF-α
VEGF
FGF-2
IL-8
IL-2
GM-CSF
IFN-γ
IL-4
IL-12
0 1 2 3 4 5 6
Fold modification versus control

Figure 1. Modulation of serum cytokine levels in glioblastoma patients. The data represent group
averages of fold modification versus controls.

Dysregulations of PI3K/Akt/mTOR signaling PI3K inhibitor we have recorded upon cellular

were reported in GBM and induce RTK overac-
tivity (EGFR, PDGFR, mesenchymal–epithelial
transition factor), mutated PI3K subunits and/or
loss of tumor suppressor activity, namely PTEN
[74] . Indeed, 40% of GBM have a loss of PTEN,
triggering excessive PI3K signaling, while other
GBMs exhibit post-translational modifications
inducing inactivation of PTEN. A therapeuti-
cally wise approach and more specific to person-
alized medicine can benefit from the identifica-
tion of signaling dysregulation and the definition
of novel targets [75] . The PI3K signaling pathway
has been investigated as an important target for
the treatment of GBM [76] , seeking PI3K inhibi-
tors as therapeutic agents in GBM [77] . According
to one of our recently published papers, by
inhibiting the PI3K pathway in patient-derived
GBM cells, the expression of signaling proteins
belonging to various pathways can be altered,
and hence their tumor cell proliferation capacity
can be reduced. Our recent study has revealed
that treating GBM cell cultures isolated from
patient tumors with PI3K inhibitors induced
a significant decrease in the expression level of
several key signaling molecules involved in cell
survival (p38), proliferation (ERK1/2, IκBα,
p38 MAPK CREB), differentiation (ERK1/2,
CREB), migration (ERK1/2, CREB) and apop-
tosis (ERK1/2, P70S6K, IκBα, JNK, CREB) [2] .
We found that PI3K inhibitors reduced tumor
cell proliferation, as in similar reports for other
types of tumors [77] . The maximal efficacy of
responses is coherent with other experimen-
tal results pointing out the major relevance of
PI3K signaling in GBM and furthermore that
PI3K inhibitors can add to, or even increase, the
limited success of EGFR inhibitors in clinical
trials [78] . P70S6K, a serine/threonine kinase,
can be activated by both the PI3K and ERK
pathways. This multiple involvement supports
the idea of the simultaneous usage of PI3K and
MAPK inhibitors [79] . In accordance with our
results, a similar interference between PI3K and
MEK/ERK signaling pathways was reported by
Sunayama et al. [80] .
Pan PI3K inhibitor 2-(4-morpholinyl)-8-phe-
nyl-4H-1 benzopyran-4-one (LY294002) can
induce important antitumoral overall effects in
experimental models, but the LY294002 com-
pound has poor pharmacologic variables of insol-
ubility and a short half-life. Starting from this
structure, a novel RGDS-conjugated LY294002,
named SF1126, was designed. The goal was to
exhibit increased solubility and bind to specific
intratumor integrins, thus an cause enhanced
delivery of the active compound. Using the
U87MG GBM cell line SF1126 had an enhanced
efficacy due to the RGDS integrin (alpha v beta
3/alpha 5 beta 1) binding component. SF1126
has both antitumor and antiangiogenic activity,
recommending it as a viable pan PI3K inhibitor
for Phase I clinical trials [81] .
In GBM tumor samples the axonal guidance
protein, netrin-1, is overexpressed. Last year it

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Review  Tanase, Albulescu, Codrici et al.
was reported that, using tandem affinity puri-
fication and mass spectrometry technologies,
netrin-1 forms a complex with Notch2 and
Jagged1. This co-localization was present on
the cell surface, while in intracellular vesicles
netrin-1 was present only with Jagged1, but not
with Notch2. Netrin-1 induced Notch signaling
and consequent GBM invasion. Interestingly,
besides a candidate biomarker netri-1 can also
be a therapeutic target since the central domain
of netrin-1 counteracts the effects of netrin-1,
meaning that it inhibits GBM cell invasion and
Notch activation. This astonishing finding was
that Notch signaling complex remained at the
cell surface [82] . This year, a series of early-phase
clinical trials were reported where Notch sign-
aling was the therapy target using agents that
either obstructed Notch receptor cleavages such
as γ-secretase inhibitors (GSIs) or interfered with
the Notch ligand–receptor interaction, which
occurs in several solid tumors, including intrac-
ranial ones [83,84] . This report again underlies
the urgent need for efficacy biomarkers to sup-
port the development of this class of drugs [85] .
Niclosamide induced the cytostatic, cytotoxic
and antimigratory effects of GBM in in vitro
and in vivo models, and moreover reduced the
capacity of multipotent/self-renewing cells in
vitro. By analyzing the pathways triggered by
this drug, simultaneous inhibition of WNT/
CTNNB1-, NOTCH-, mTOR- and NF-κB
cascades are revealed. The authors report that in
GBM biological samples a heterozygous deletion
of the NFκBIA locus was found, thus besides
a possible genomic biomarker, this could serve
as an explanation for predicting the synergistic
activity of niclosamide with temozolomide, the
current standard in GBM [75] .
RTK-targeted therapy was investigated using
imatinib, sunitinib and cediranib in GBM mod-
els. In a panel of ten GBM cell lines, cediranib
proved to have the most potent antitumoral
effect, while cediranib and sunitinib sensitizes
the cells to the classic temozolomide. Cediranib
inhibited MAPK and AKT pathways, but the
authors did not find any correlation between
KIT, PDGFRA and VEGFR2 expression, and
therapy responses to any of the RTK inhibitors.
RTK therapy can rely upon future biomarkers
for therapy response in GBM [86] .
As already stated in the previous section,
circulating exosomes can induce the activa-
tion of ERK1/2 and HSP27 via CAV1; this
recent report opens new possibilities for therapy
518 Future Oncol. (2015) 11(3)
targets through CAV1 expression and ERK1/2
­signaling [87] .
Intracellular signaling molecules that basi-
cally depict the molecular heterogeneity of the
tumor are certainly important biomarkers in
predicting responsiveness and disease outcome
upon therapy. This assertion was sustained by
less favorable clinical trial results when mono-
therapy was used, therapy overlooking the com-
plexity of intracellular network of individual
tumors. Thus, intracellular key signaling mol-
ecules and the actual panel of these molecules are
to become important target therapies and valu-
able biomarkers, especially for therapy ­efficacy
monitoring.
The simultaneous detection of several mol-
ecules involved in various signaling pathways has
not been previously reported in GBM. Further
studies are needed in order to assess whether
this regulation is transcriptional or post-tran-
scriptional. Keeping in mind that personalized
medicine should be tailored to the patient’s
tumor particularities, screening cell behav-
ior and expression levels of signaling proteins
within tumor cells can highlight the important
differences between primary tumors of similar
histological type, and/or allow comparisons of
primary and relapse tumor samples.
Cancer stem cells as potential biomarkers
for GBM aggressiveness
Cancer stem cells (CSCs), identified in a myriad
of human solid tumors including brain tumors
[88] , have generated a new field of research where
a clear distinction should be made between can-
cer-initiating cells and cells that allow propaga-
tion of the tumor [89] . Nowadays, genomics and
epigenetic techniques have proven that cancer
takes control of specific genetic and epigenetic
programs from embryonic development circuits.
Two recognized models regarding the genera-
tion and function of GBM CSCs (GCSCs),
the stochastic and the hierarchical models, are
­currently recognized (Figure 2) .
According to the stochastic model, differenti-
ated or committed cells acquire genetic muta-
tions towards immortal proliferative capacity,
leading to cancer stem cells (GCSC in our
case), in contrast with the hierarchical model
where a stem cell or a glial precursor undergoes
a neoplastic transformation, leading to glioma
initiation and development [90] . Properties such
as extensive self-renewal ability and multipotency
are common for neural stem cells (NSCs) and
future science group
 Circulating biomarker panels for targeted therapy in brain tumors  Review

T
NSC CSC
Muta
tion

CSC
on
Mutati
NSC PC Mutati
on

CPC

DC

Several mutation events

Figure 2. Acknowledged theories regarding cancer stem cell generation – stochastic and
hierarchical theories. (A) Normal neural stem cells (NSCs) give rise to progenitors cells (PCs) with
limited proliferative capacity that further leads to differentiated normal cells (DCs). CSCs can form as
a result of abnormal differentiation of a normal NSC or neural progenitor cell (PC). CSC can develop
cancer progenitor cells (CPCs) that further are able to develop the actual tumor (T). (B) CSCs can
result from terminally differentiated cells, which acquire several genetic mutations.

GCSCs, and have been included in the defini-
tion of cancer stem cells. It has recently been
shown that chemokine CXCL12 and its recep-
tor CXCR4 can control proliferation, invasion
and angiogenesis in GBM cell lines and primary
cultures. In GCSCs an increased CXCR4 expres-
sion was found, as well as release of CXCL12
in vitro. As also found by us, there is a clear het-
erogeneity of GBMs that induce different levels
of both expression and secretion in individual
cultures, again an important argument for per-
sonalized therapy. CXCL12 activation-induced
Akt-mediated reduced apoptosis and self-renewal
activities, but less proliferation, while CXCR4
antagonist AMD3100 reduced self-renewal and
survival. In in vitro differentiated cells derived
from the same GBMs, the abovementioned inhi-
bition through AMD3100 was not obtained, this
finding being a strong argument for the coupled
CXCL12/CXCR4 interactions that are specific
for CSC in GBM mediating survival and self-
renewal, therefore representing a promising
future for therapeutic targets [91] . We point out
that this finding can be also extrapolated to cir-
culatory biomarkers, as CXCL12 is secreted by
GCSCs, enhancing the possibility to find this
biomarker in patients’ circulation.
As mentioned in the previous section, the
Notch pathway is highly activated in this type of
solid tumor. Moreover, it has recently been shown
in an animal model that mice constitutively
expressing the activated intracellular domain
of Notch2 display neurogenic niche hyperpla-
sia and reduced neuronal lineage entry. When
authors isolated neurospheres from this model,
an increased proliferation, survival and resistance
to apoptosis was obtained. In human GBM cell
lines, the Notch2 pathway induces an increased
proliferation and resistance to apoptosis. When
assessing gene expression in GBM patient tumor
samples, a positive correlation of Notch2 tran-
scripts with the transcripts that control antia-
poptotic processes, stemness and astrocyte fate
was found. In the meantime, Notch2 transcripts
were negatively correlated with gene transcripts
controlling the proapoptotic process and oligo-
dendrocyte fate. The Notch2 pathway in NSCs
can drive to GCSCs and can induce astrocytic
lineage entry and brain tumor ­development [92] .
GCSC autophagy was reported to be induced
by suberoylanilide hydroxamic acid (SAHA),
a histone deacetylase inhibitor, as an effect of
late-phase apoptosis. In vivo xenografts have
shown that SAHA reduced tumor growth and

future science group www.futuremedicine.com 519

Review  Tanase, Albulescu, Codrici et al.

determined autophagy. Actually, the authors
report that SAHA’s action is induced by down-
regulation of AKT/mTOR signaling. Taken
together, this recent study shows that in GCSC
therapeutic approaches, SAHA can be a capable
agent for autophagy induction [93] .
This year, in brain tumor initiating cells
(BTICs), neurotrophin receptors (p75NTR,
TrkA, TrkB and TrkC) and their ligands (NGF,
BDNF and NT3) were reported. Moreover,
BTICs secrete NGF and, thus, exogenouous
NGF induced BTIC proliferation. The authors
note that the intracellular domain of p75NTR is
to be found in GBM biological specimens, sug-
gesting that the receptor is activated and cleaved
in patient tumors. These results open a new

EXECUTIVE SUMMARY
Molecular classification of glioblastoma
●● Histologic and molecular analysis of CNS tumors can identify approximately 120 subtypes.
●● Recent progress in molecular research of GBM has led to the identification of novel biomarkers useful in the diagnosis
and prognosis of this fatal disease.
Circulating tumor cells in malignant gliomas
●● Circulating tumor cells originating from both primary and metastatic lesions can be detected in the peripheral blood
of patients with different solid tumors.
●● Several clinical studies performed in patients with different types of tumors, including brain tumors, showed a
correlation between CTCs and clinical outcome, and suggested that CTC detection may represent a noninvasive
adjuvant tool for evaluating prognostic value, recurrence capacity and therapy response monitoring.
Oncosomes as a future circulating biomarkers carrier
●● Oncosomes, or membrane-derived extracellular vesicles (EVs), secreted by cancer cells can transport proteins and
nucleic acids from one cell to the other, thus being active participants in the oncogenic transformation of various cells.
●● Oncosomes come into view in brain tumor research domains as an important vehicle of intercellular communication,
providing a new field of molecular biomarkers investigation.
miRNAs as molecules linking tissue & circulating biomarkers
●● Tumor-specific miRNAs are promising tools for the early detection of cancer and the development of personalized
therapies.
Proteomic profiling for circulating biomarkers discovery
●● A wide range of technologies have been employed in molecular profiling studies for biomarker discovery in brain
tumors. Proteomic profiling offers large-scale analysis of protein expression, post-translational modification and
protein–protein interactions.
●● SELDI-TOF-MS technology has been utilized in protein profiling analysis for the discovery of biomarkers facilitating
diagnosis, predicting therapeutic response and establishing tumor grade.
Signaling molecule profile with biomarker potency & future therapy targets
●● GBM shows dysregulation in various signaling pathways including the G1/S cell cycle checkpoint and the MAPK and
PI3K effectors of RTK signaling.
●● Deregulations of PI3K/Akt/mTOR signaling were reported in GBM and induce RTK overactivity (EGFR, PDGFR), mutated
PI3K subunits and/or loss of tumor suppressor activity, namely PTEN.
Cancer stem cells as potent biomarkers for GBM aggressiveness
●● Cancer stem cells, identified in a myriad of human solid tumors including brain tumors, have generated a new field of
research where a clear distinction should be made between cancer-initiating cells and cells that allow propagation of
the tumor.
●● Several molecules have been identified as candidate biomarkers for glioblastoma, but a more suitable approach for
efficacious therapy would rely on the use of personalized proteomic profiles.

520 Future Oncol. (2015) 11(3) future science group

 Circulating biomarker panels for targeted therapy in brain tumors  Review

door in the BTIC research domain for a novel
­potential clinical target [94] .
Conclusion & future perspective
Although there are many proteins described
as ‘potential biomarkers’ and although predic-
tive biomarkers have not been yet validated for
patients with GBM, a useful serum marker panel
of brain tumors is urgently needed. Discovering
a single biomarker that would be both sensi-
tive and specific for cancer might be more dif-
ficult than discovering a panel of biomarkers.
Although some molecules have been proposed
as potential GBM biomarkers, it is the current
opinion that a more adequate approach would
be to compare the proteomic profile of an indi-
vidual with the values recorded in healthy indi-
viduals (panel of biomarkers).
We believe that in the very near future a panel
comprising cytokines, chemokines and angio-
genic circulating biomarkers can offer an insight
regarding the diagnostic and invasive potential
of GBM. This front panel will be followed by
the identification of CTC along with miRNA
panels that depict disease progression, relapse
and therapy monitoring. In the long term, it is
a probability that intracellular signaling path-
ways elucidation accompanied by circulating
oncosomes will be developments in the bio-
markers field, providing this pathology with
new therapy targets.
GBM patients will benefit more from person-
alized therapy by tailoring their treatment and
aiming specific protein–protein interactions and
signaling networks according to tumor pheno-
type or patient clustering based on biomarker
panels. New, promising fields for GBM personal-
ized medicine are microRNA and CSC signaling.
Future therapies could emerge from the coopera-
tion between cancer stem cells and their niches,
which will improve the maintenance of their
characteristic features and behavior, with the
concomitant activation of differentiation signal-
ing pathways, such as RTKs–Akt, Notch, BMPs,
Hedgehog, Wnt-β-catenin, and so on.
New protocols based on a combination of
chemotherapy and immunotherapy are probably
a new approach for achieving therapeutic syn-
ergy and more suitability for brain tumors. The
cancer stem cell paradigm might become the
cornerstone for novel cancer research approaches
in the following years.
In this review, we have addressed the major
challenges surrounding biomarker development
in this particular type of devastating tumors; by
gathering the current data regarding biomarker
candidates, we can explore potential routes for
the development of a more effective predictive
biomarkers panel.
Financial & competing interests disclosure
This paper is partly supported by the Sectorial Operational
Programme Human Resources Development (SOPHRD),
and financed by the European Social Fund and the Romanian
Government under the contract number POSDRU 141531.
This work was partially supported by grants PN 09.33-04.15,
PN 09.33-03.10 and PN 09.33-01.01. The authors would
like to thank Irina Radu, certified translator in Medicine
– Pharmacy, certificate credentials: series E no. 0048, for
professional linguistic assistance. The authors have no other
relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial
conflict with the subject matter or materials ­discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.

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