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Current Cancer Drug Targets, 2010, 10, ???-??? 1
1568-0096/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
CETUXIMAB: From Bench to Bedside
B. Vincenzi*, A. Zoccoli, F. Pantano, O. Venditti and S. Galluzzo
Deptartment of Medical Oncology, University Campus Bio-Medico, Rome, Itlay
Abstract: Cetuximab (IMC-C225, Erbitux ImClone Systems Inc, New York, NY) is a recombinant, human/mouse chi-
meric monoclonal antibody (MAb) that binds specifically to the extracellular domain of the human epidermal growth fac-
tor receptor (EGFR) on both normal and tumor cells, and competitively inhibits the binding of epidermal growth factor
(EGF) as well as other ligands.
Cetuximab binding to the EGFR blocks phosphorylation and activation of receptor-associated kinases and their associated
downstream signalling (MAPK, PI3K/Akt, Jak/Stat pathways) resulting in inhibition of many cellular processes such as
induction of apoptosis, cell growth, decreased Matrix Metallo-Proteinase (MMPs) and vascular endothelial growth factor
(VEGF) production. Cetuximab is also able to display cytotoxic effect through antibody-dependent cellular cytotoxicity
(ADCC).
In vitro and in vivo experiments elucidated a wide range of biological properties attributed to cetuximab, these include: di-
rect inhibition of EGFR tyrosine kinase activity, inhibition of cell cycle progression, inhibition of angiogenesis, invasion
and metastatization processes, activation of pro-apoptotic molecules and synergic cytotoxicity effect with chemotherapy
and radiotherapy.
Several studies have shown cetuximab is able to inhibit growth of EGFR-expressing tumor cells in vitro as well as in nude
mice bearing xenografts of human cancer cell lines.
Moreover, numerous clinical trials demonstrated cetuximab efficacy in different tumor types and it is approved by Food
and Drugs Administration (FDA) for use in the treatment of metastatic colorectal cancer (mCRC) as single agent or in
combination with chemotherapy, for locally/regionally advanced head and neck squamous cell carcinoma (HNSCC) in
combination with radiotherapy, and as monotherapy for recurrent/metastatic HNSCC after failing platinum-based chemo-
therapy.
This review will illustrate pre-clinical and clinical data on biological properties of cetuximab focusing on the predictive
markers of clinical response to this drug.
Keywords: Cetuximab, epidermal growth factor receptor, targeted therapy, predictive markers.
INTRODUCTION
With the emergence of new technologies and with an in-
creasing knowledge of the molecular mechanisms that gov-
ern carcinogenesis and tumor progression came the discov-
ery and the development of numerous new drugs designed to
specifically target the relevant molecular pathways involved.
The term "targeted therapy" refers to a new generation of
cancer drugs designed to interfere with a specific molecular
target (typically a protein) that is believed to have a critical
role in tumor growth or progression. The identification of
appropriate targets is based on a detailed understanding of
the molecular changes underlying cancer. This approach
contrasts with the conventional, more empirical approach
used to develop cytotoxic chemotherapeutics, the mainstay
of cancer drug development in past decades.
The epidermal growth factor receptor (EGFR) represents
one such target [1, 2]. Since one of the most studied molecu-
lar targets is the EGFR, a number of drugs targeting this re-
ceptor have been approved for cancer treatment, and others
are being evaluated in late phase clinical trials [3-6].
*Address correspondence to this author at the Deptartment Medical Oncol-
ogy, University Campus Bio-Medico, Via Alvaro del Portillo 200, Rome,
Italy; Tel: +39-(0)6-225411160; Fax: +39-(0)6-225411934;
E-mail: b.vincenzi@unicampus.it
Cetuximab is a chimeric anti-human EGFR monoclonal
antibody (MAb) approved by Food and Drugs Administra-
tion (FDA) for use in the treatment of metastatic colorectal
cancer (mCRC) as single agent or in combination with che-
motherapy, for locally/regionally advanced head and neck
squamous cell carcinoma (HNSCC) in combination with
radiotherapy, and as monotherapy for recurrent/metastatic
HNSCC after failing platinum-based chemotherapy.
This review will illustrate pre-clinical and clinical data
on biological properties of cetuximab focusing on the predic-
tive markers of clinical response to this drug.
CETUXIMAB: EGFR AS TARGET
Cetuximab is a chimeric anti-human EGFR MAb [7] that
has recently been approved for use in mCRC as single agent
or in combination with chemotherapy [8, 9], for lo-
cally/regionally advanced HNSCC in combination with ra-
diotherapy, and as monotherapy for recurrent/metastatic
HNSCC after failing platinum-based chemotherapy [10].
However, despite the recent success of targeted therapy in
certain clinical oncology trials, including those using
cetuximab, there still exist an urgent need for strategies to
improve efficacy, as well as to reduce toxicity.
Cetuximab (IMC-C225, Erbitux ImClone Systems Inc,
New York, NY) is a recombinant, human/mouse chimeric
2 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
MAb that binds specifically to the extracellular domain of
the human EGFR [11-13].
The first EGFR-targeting MAbs were produced by John
Mendelsohn and colleagues in 1983. M225, a MAb, was
found to block ligand binding to EGFR, it binds EGFR with
similar affinity to epidermal growth factor (EGF) and trans-
forming growth factor-alpha (TGF-alpha) and it inhibits the
proliferation of various epithelial tumor cell lines [12].
M225’s potential was demonstrated by a pre-clinical
study which reports that M225 and cisplatin used on their
own had inhibitory effect on tumor graft. However, when
both agents were administered in combination, a synergistic
effect was observed, with significant tumor shrinkage and
greatly increased survival [14].
Because of limitate MAb technology at that time, M225
was a murine antibody and thus Human Anti-Mouse Anti-
bodies development was expected in humans. To remedy
this, the antigen-recognition sites of M225 were transferred
into a human antibody backbone creating a new MAb called
cetuximab (Erbitux) with a very low incidence of hypersen-
sitivity reaction [15].
Cetuximab binds specifically the extracellular domain of
the EGFR on both normal and tumor cells [11-13], and com-
petitively inhibits the binding of EGF as well as other
ligands, such as TGF-alpha [7].
Cetuximab is produced in mammalian (murine myeloma)
cell culture and it is composed of the Fv regions of a murine
anti-EGFR antibody with human IgG1 heavy and kappa light
chain constant regions and has an approximate molecular
weight of 152 kDa [16]. The binding to EGFR is character-
ized by a higher affinity (Kd= 0.1-0.2 nM) than either EGF
or TGF-alpha [7]. The selective action of cetuximab ac-
counts for a minor toxicity instead experienced with other
EGFR inhibitors acting on the intracellular domain of EGFR
and on a vast number of tyrosine kinases.
Cetuximab binding to the EGFR blocks phosphorylation
and activation of receptor-associated kinases and their asso-
ciated downstream signalling (Mitogen-Activated Protein
Kinase (MAPK), Phosphoinositide 3-kinase/Akt (PI3K/Akt)
and the Janus kinases/Signal Transducers and Activator of
Transcription (Jak/Stat) pathways) interfering with many
cellular processes such as induction of apoptosis, inhibition
of cell growth and vascular endothelial growth factor
(VEGF) production, decreased matrix metalloproteinase
[17].
Furthermore, cetuximab induces receptor down-
regulation through its initial dimerization and internalisation
[18].
The EGFR is a member of type I tyrosine kinase receptor
(TKR) family including EGFR/human epidermal growth
factor receptor (HER)1-4. It is constitutively expressed in
many normal epithelial tissues and it is over-expressed in
several human cancers as well as those of the colon and rec-
tum.
The EGFR (c-ErbB-1; HER1 in humans) is a trans-
membrane receptor tyrosine kinase member that belongs to
the HER receptor family [19]. Receptors of HER family
structurally contain extracellular, transmembrane, and tyro-
sine kinase domains. Each domain is responsible for a differ-
ent HER mediated signalling pathway: the extracellular por-
tion contains a ligand-binding site, while the intracellular
portion contains the tyrosine kinase domain [20].
The HER family consists of 4 structurally related cellular
receptors, which interact in many ways: HER1, also known
as EGFR or ErbB1, HER2, also known as ErbB2, HER3,
also known as ErbB3, HER4 or ErbB4 [21- 23].
In mammals, canonical EGFR activation involves the
binding of seven peptide growth factors: EGF, TGF-alpha,
heparin-binding EGF-like growth factor (HBEGF), am-
phiregulin (AREG), betacellulin (BTC), epiregulin (EREG),
and epigen (EPGN) [24]. This bond results in homodimeri-
zation of two EGFR or heterodimerization through different
members of the human EGFR family [25].
These events activate EGFR intracellular region resulting
in initiating a cascade of events into the cell such as auto-
phosphorylation and signal transduction [26-29].
EGFR activation leads to recruitment of a range of adap-
tor proteins (such as Shc, GRB2) [30] and activates a series
of intracellular signalling cascades to affect gene transcrip-
tion, which in turn results in cancer cell proliferation, re-
duced apoptosis, invasion and metastasis and also stimulates
tumor-induced angiogenesis.
The pathways mediating EGFR downstream effects have
been well studied and three major signalling pathways have
been identified: the first involves RAS-RAF-MAPK pathway
[31, 32]: phosphorylated EGFR recruits the guanine-
nucleotide exchange factor via the GRB2 and Shc adapter
proteins, activating RAS and subsequently stimulating RAF
and the MAP kinase pathway to affect cell proliferation, tu-
mor invasion, and metastasis; the second involves
PI3K/AKT pathway [33], which activates the major cellular
survival and anti-apoptosis signals via activating nuclear
transcription factors such as nuclear factor-kappa B (NF-kB)
[34]; the third involves JAK/STAT pathway which is also
implicated in activating transcription of genes associated
with cell survival.
EGFR activation may also lead to hydrolysis of phos-
phatidylinositol 4,5 biphosphate (PIP2) into inositol 1,4,5-
triphosphate (IP3) and diacylglycerol (DAG), resulting in
activation of protein kinase C (PKC) and Ca2+/calmodulin-
dependent protein kinases (CaMK).
The activation of these systems starts several transcrip-
tional programs that mediate a variety of cellular responses,
including cell division, motility, invasion, adhesion, cellular
repair and survival (or death) [35]. After signal transduction,
ligand-receptor complex can be internalized and either
down-regulated or recycling on the cell surface [36].
EGFR is expressed on normal and malignant epithelial
cells and it plays an important role in tumor biology. In fact,
it promotes proliferation, metastatization, angiogenesis and
inhibition of apoptosis [37, 38]. The main signal transduction
pathways associated with EGFR activation are summarized
in Fig. (1).
The most common EGFR alteration in tumor cells is its
over-expression, that may lead to ligand-independent recep-
tor dimerization. EGFR is frequently over-expressed in hu-
CETUXIMAB: From Bench to Bedside Current Cancer Drug Targets, 2010, Vol. 10, No. 1 3
man tumors including breast cancer, lung cancer, glioblas-
toma, bladder carcinoma, head and neck cancer, ovarian car-
cinoma, colorectal cancer and prostate cancer [39]. Over-
expression correlates with disease progression, poor outcome
and low response to therapy and resistance to cytotoxic treat-
ments [40, 41].
In malignant cells there are other mechanisms related to
the activation of EGFR like an increased production of
ligands by the same tumor cells [41-43] or an expression of a
mutant variant of the receptor (EGFR vIII) able to activate
constitutively its own tyrosine kinase [44, 45].
Furthermore, metallo-proteinase-mediated cleavage of
precursor membrane-bound EGF ligands seems to activate
the EGFR through the stimulation of G-protein-coupled re-
ceptors [46].
Recently, a ligand-independent mechanism of EGFR ac-
tivation via the urokinase plasminogen receptor has been
identified [47].
CETUXIMAB: BIOLOGICAL PROPERTIES
As previously described, cetuximab binds to and inhibits
EGFR activation. Through this mechanism cetuximab dis-
plays its antitumor activity.
In vitro and in vivo experiments elucidated a wide range
of biological properties attributed to this MAb, these include
the following: direct inhibition of EGFR tyrosine kinase ac-
tivity [12], inhibition of cell cycle progression [48, 49], inhi-
bition of angiogenesis, invasion and metastatization proc-
esses [50], activation of pro-apoptotic molecules [48, 51] and
synergic cytotoxicity effect with chemotherapy and radio-
therapy [52]. Moreover, cetuximab is able to induce ADCC.
The mechanisms by which antitumor activity is displayed are
not completely clarified yet.
Different studies demonstrated that cetuximab mediates
cell cycle arrest in various tumor cell lines, leading in some
cases to apoptosis. It is known that cyclin dependent kinases
are fine regulators of cell cycle progression particularly of
G1 phase progression and subsequent S-phase entry. Moreo-
ver, S-phase progression is guided by growth factors expres-
sion.
Wu X et al. demonstrated that blockage of EGFR medi-
ated by cetuximab induces G1 phase cell cycle arrest in DiFi
human colon adenocarcinoma cells and that this blockage
involves a cyclin/cycline dependent kinase (CDK) inhibitor
called p27KIP1 a kinase inhibitory protein [53].
Another interesting data derived from Kiyota et al. [54]
that analysed the effect of mAb225 (an EGFR-blocking
MAb 225) on human oral squamous cell carcinoma (SCC)
cell lines. The results suggest that the antiproliferative effect
of EGFR blockade may be mediated by cell cycle inhibitors
p27KIP1 and p15INK4B with cell accumulation in G1 phase
and cell decrease in S phase. In particular, G1 arrest was
accompanied by a decrease in CDK2-, CDK4-, and CDK6-
associated histone H1 kinase activities, and an increase in
cell cycle inhibitors p27KIP1 and p15INK4B.
Peng D and colleagues [49] demonstrated that EGFR-
blocking antibody mAb225 inhibits proliferation of andro-
gen-independent DU145 prostatic cancer cells by arresting
cell cycle progression in G1 though inhibition of CDK2 ac-
Fig. (1). The main signal transduction pathways associated with EGFR activation.
EGFR: epidermal growth factor receptor, GRB2: growth factor receptor-bound protein 2, K: kinase, MAPK: mitogen activated protein
kinase, P: phosphate, PI3-K: phosphoinositide-3 kinase, R: receptor
4 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
tivity. This effect is attributable to p27KIP1 up-regulation as
demonstrated at mRNA and protein levels.
Experimental data have shown that cetuximab exerts a
pro-apoptotic effect. It is known that G1 arrest is followed
by apoptosis.
Wu X et al. reported that DiFi colorectal carcinoma cell
line, which expresses high levels of EGFR on plasma mem-
branes, can be induced to undergo G1 cell cycle arrest and
apoptosis when cultured with mAb 225 at concentrations that
saturate EGF receptors. Moreover, addition of Insulin-like
Growth Factor-1 (IGF-1) or high concentrations of insulin
can delay mAb C-225-induced apoptosis, while G1 arrest
cannot be reversed by either IGF-1 or insulin [48]. These
results indicate that EGF receptor activation is required both
for cell cycle progression and for prevention of apoptosis in
DiFi cells, and that a signal transduction pathway shared by
receptors for insulin/IGF-1 and EGF may be involved in
regulating apoptosis triggered by EGF receptor inhibitors.
Liu B et al. investigated the mechanisms by which fibro-
blast growth factor (bFGF) and IGF-1 modulate mAb 225-
induced G1 arrest and apoptosis in DiFi cells. They found
bFGF could strongly inhibit both the antibody-induced cell
cycle arrest in G1 phase and the subsequent apoptosis. In
contrast, although IGF-1 delayed the onset of apoptosis, it
did not prevent mAb 225-induced G1 cell cycle arrest. Inter-
estingly, either bFGF than IGF-1 acted on MEK pathway in
DiFi cells [51].
Explore the mechanisms by which mAb 225 induces
apoptosis in DiFi cells may be useful to identify new tar-
get(s) to maximize the antitumor effect of mAb 225 by in-
ducing apoptosis in a variety of cancer cells. The precise
molecular mechanisms by which mAb 225 is able to induce
apoptosis in these cells remain not completely clear.
Another study conducted by Sclabas and colleagues
demonstrated that EGFR blockage through IMC-C225 MAb
in MDA Panc-28 human pancreatic cancer cell line leads to a
marked decrease of constitutive NF-kB DNA binding activ-
ity and as consequence to a decrease in bcl-xl and bfl-1 tran-
scription which are bcl-2 family members and are regulated
by NF-kB itself [55]. These results suggest an important role
of EGFR in NF-kB activation and that EGFR blockade by
IMC-C225 may be effective in anti-apoptotic genes down-
regulation.
Extensive experimental evidences reported that cetuxi-
mab displays antiangiogenetic activity decreasing tumor cell
production of angiogenic growth factors such as VEGF,
bFGF and interleukin-8 (IL-8).
Petit and colleagues were the first to shed light on the
antiangiogenetic properties of cetuximab. The authors evalu-
ated whether EGFR or ErbB2/neu may contribute to tumor
angiogenesis by examining their effects on the expression of
VEGF/vascular permeability factor (VPF). EGFR-positive
A431 human epidermoid carcinoma cells, which are known
to be heavily dependent on VEGF/VPF, C225 anti-EGFR
neutralizing antibody treated down-regulate VEGF produc-
tion by tumor cells themselves. Similar result was obtained
from analysis conduct on other tumor cell lines [56].
Perrotte et al. evaluate whether EGFR-directed therapy
affects angiogenesis by in vitro exposure of human transi-
tional cell carcinoma (TCC) of the bladder cell line 253J B-
V to cetuximab. The effect was the inhibition of mRNA and
protein production of VEGF, IL-8 and bFGF by the cells,
moreover anti-EGFR MAb C225 inhibits growth and metas-
tasis of human TCCs established in the bladder wall of
athymic nude mice [50].
Huang and co-workers examined the impact of cetuxi-
mab on invasive and metastatic potential of head and neck
human squamous cell carcinoma (SCC) using in vitro and in
vivo model systems [57]. In vitro treatment of human um-
bilical vascular endothelial cells (HUVEC) with cetuximab
reduces cell-to-cell interaction and results in disruption of
tube formation. The effect of C225 was additionally exam-
ined using an in vivo tumor xenograft neovascularization
model of angiogenesis. Systemic treatment with C225 not
only reduced tumor growth and the number of blood capil-
laries but also hindered the growth of established vessels
toward the tumor.
These results provide evidence that C225 is capable to
inhibit tumor-induced neovascularization and metastasis in
SCC of the head and neck.
Endothelial cells within several neoplasms have been
shown to express EGFR [58]. These cells proliferate follow-
ing EGFR activation [59] and are instead induced to apopto-
sis and reduce neovascularization following EGFR blockade
[60].
VEGF and IL-8 can act as survival factors for immature
endothelial cells [61] and protect endothelial cells from
apoptosis [62], so that decreased production of VEGF in-
duced by cetuximab may enhance endothelial cell apoptosis,
contributing to the reduction in neovascularity.
The results suggest that cetuximab displays its antiangio-
genic effect by acting directly on endothelial cells.
Other studies supported the antiangiogenetic effect and
demonstrated a cytostatic role of cetuximab. Moreover, the
association with other biological or cytotoxic agents in-
creases significantly cetuximab antitumor activity.
Ciardiello and colleagues evaluated the antiangiogenic
and antitumoral activity of cetuximab alone and in combina-
tion with a human VEGF antisepses (AS) 21-mer phos-
phorothioate oligonucleotide (VEGF-AS) in human GEO
colon cancer cells [63].
Immunohistochemical analysis of GEO tumor xenografts
showed a significant reduction of VEGF expression after
treatment with VEGF-AS and a reduction of VEGF, bFGF,
TGF-alpha expression after treatment with cetuximab, both
determining a parallel reduction in microvessel count.
The combined treatment leads to a significant impact on
VEGF expression but a slight or no impact on microvessels
density.
In the clinical setting Vincenzi and colleagues investi-
gated the impact of a combined weekly treatment of cetuxi-
mab plus irinotecan on VEGF, as pro-angiogenic factor, and
interferon (IFN)-gamma, as anti-angiogenic factor, modifica-
tion in advanced colorectal cancer patients [64].
CETUXIMAB: From Bench to Bedside Current Cancer Drug Targets, 2010, Vol. 10, No. 1 5
Cytokine levels were assessed at different time points be-
fore and after the beginning of the treatment. The median
VEGF basal value showed a decrease that interestingly
reached the highest statistical significance at the last time
point with a reduction of 51.75%. Thus, cetuximab reduction
of VEGF serum levels is a sudden and long lasting phe-
nomenon.
The median IFN-gamma basal level significantly in-
creased 1 day after treatment and persisted after 21 days. A
precise analysis of this behaviour remains to be investigated.
A significant negative correlation between VEGF and
IFN-gamma values at 1, 21 and 50 days after the first treat-
ment cycle was shown by a linear regression model.
These data effort the concept of anti-angiogenetic role of
cetuximab in cancer treatment, underlining the need of new
antiangiogenic combination treatments with potential syner-
gic activity.
In vitro and in vivo experiments stated that cetuximab in-
hibits the invasive and metastatic ability of different tumor
types.
Perrotte et al. in an orthotopic bladder carcinoma
xenograft model of tumor-bearing mice confirm that sys-
temic treatment with a chimeric anti-EGFR MAb C225 re-
sults in tumor regression, inhibits tumor growth and prevents
metastases to the regional lymph nodes and lungs [50].
Metallo proteinases (MMPs), a zinc-dependent endopep-
tidases family, are released by cancer cells and act by proc-
essing extra-cellular matrix components, cell surface pro-
teins, and immune modulators. It is known that deregulation
of proteolytic mechanisms has been implicated in tumor in-
vasion and metastatic processes.
Cetuximab has been shown to inhibit the expression as
well as the activity of several MMPs including the gelatinase
MMP-9. Tumor metastasis represents a complex multistep
process that requires migration, invasion, and angiogenesis.
Huang et al. observed that anti-EGFR antibody C225
treatment attenuated SCC-1 tumor cells migration through a
chemotaxis chamber in a dose-dependent manner. Further-
more, in the presence of C225, SCC-1 ability to invade
across a layer of extracellular matrix was significantly inhib-
ited. Using an in vivo orthotopic floor-of-mouth xenograft
model locoregional tumor invasion of SCC-1 into muscle,
vessel, bone, and perineural tissues was inhibited in C225-
treated mice. This inhibition was additionally characterized
by down-regulation of MMP-9 expression. These data sug-
gest that inhibition of metastatic potential by C225 may be
mediated via decreased migration and invasion of SCC cells.
These results show the capacity of C225 to block MMP-9
induction which plays a major role in invasion and metasta-
tization tumor processes.
Experimental evidence demonstrated that cetuximab acts
also on immune system even if the effects and its mecha-
nisms have not been widely studied and understood yet.
The effects on immune system account for its indirect an-
titumor activity through cytotoxic effect mediated by effec-
tor cells such as monocytes and natural-killer cells and me-
diated by antibody-dependent cellular cytotoxicity (ADCC)
activity, as it has a human IgG1 backbone. However, except
for one study that showed cetuximab capacity to mediate
ADCC activity against a melanoma cell line [65], no pub-
lished studies have focused on its ADCC activity.
The study by Kurai et al. analyzes cetuximab-mediated
ADCC activity against lung cancer cell lines. In this study
fresh peripheral blood mononuclear cells exhibited cetuxi-
mab-mediated ADCC activity against lung cancer cell lines
at a low concentration of cetuximab and maximum ADCC
activity was induced by very weak EGFR expression levels,
which are faintly detectable by immunohistochemistry.
ADCC activity was enhanced by interleukin-2 (IL-2) and it
was less impaired by weekly chemotherapy with conven-
tional cytotoxic drugs than was NK activity in lung cancer
patients [66]. These data provide new insight into the possi-
ble antitumor mechanism of cetuximab and particularly re-
garding its immunomodulating effect.
Cetuximab ADCC activity has been described against
several tumor cell lines expressing wild-type or mutant
EGFR such as in HEK293 cells transfected with wild-type
EGFR (293W) and a deletional mutant of EGFR (293D) and
in the mock transfectant (293M). ADCC activity was de-
tected in cells transfected with wild-type and mutant EGFR,
in a dose-dependent manner, but not in the mock transfec-
tant.
These results indicate that cetuximab affinity for the ex-
tra-cellular domain of EGFR and not its mutation status play
a key role in triggering ADCC activity [67].
Moreover, the inhibition of transforming growth factor-
beta (TGF-beta) could enhance cetuximab mediated ADCC
against TGF-beta producing SCCs [68].
CHEMOTHERAPY AND RADIOTHERAPY ENH-
ANCE THE ANTI-TUMOR EFFECTS OF CETUXI-
MAB: PRECLINICAL AND CLINICAL EVIDENCES
Experimental data reported that different chemotherapeu-
tic agents can enhance EGFR phosphorylation and activation
status [69-71] and instead EGF has been shown to increase
sensitivity to doxorubicin and topoisomerase II inhibitors in
human solid cancer cell lines [72, 73]. According to these
results, EGFR inhibitors could increase chemotherapy-
induced killing.
Fan et al. described a substantial xenograft growth inhibi-
tion (A431 SCC) in nude mice when treated with cetuximab
and cisplatin in combination compared with a single modal-
ity [14].
Likewise, studies with doxorubicin in combination with
anti-EGFR monoclonal antibodies demonstrated marked
tumor regression of A431 and MDA-468 xenografts in
athymic mice and the treatment with either doxorubicin or
anti-EGF receptor MAb alone temporarily inhibited growth,
but the combination of both agents substantially enhanced
antitumor activity over that of doxorubicin [74].
In murine models of human colorectal carcinoma,
cetuximab and irinotecan (CPT-11) combined treatment sig-
nificantly inhibited cellular growth compared with either
CPT-11 or IMC-C-225 monotherapy. The same benefit de-
6 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
rived from combined therapy was described in a CPT-11
refractory tumor model [75].
Cetuximab ability to overturn CPT-11 resistance could
depend by inhibition of EGFR signalling pathways (MAPK
and PI3K/AKT) leading to upregualtion of proapoptotic
stimuli and thus facilitating tumor cells death.
In human ovarian, breast and colon cancer cell lines
which express functional EGFR the anti proliferative activity
of MAb C225 (anti-EGFR humanized chimeric mouse MAb)
in combination with topotecan was evaluated.
A dose-dependent supra additive increase of growth inhi-
bition in vitro was observed when cancer cells were treated
with topotecan and MAb C225 in a sequential schedule.
Treatment with MAb C225 also markedly enhanced
apoptotic cell death induced by topotecan.
Treatment of mice bearing established human GEO colon
cancer xenograft with topotecan or with MAb C225 deter-
mined a transient inhibition of tumor growth in contrast, an
almost complete tumor regression was observed in all mice
treated with the two agents in combination [52].
In vitro treatment of human pancreatic carcinoma L3.6pl
cells with cetuximab plus gemcitabine resulted in additive
cytotoxic effects that increased with increasing gemcitabine
concentrations. Dose-dependent decreases in expression of
the angiogenic factors VEGF and IL-8 (but not bFGF) were
observed in the cetuximab-treated cells (mRNA and protein
levels).
Moreover the combined treatment enhanced growth inhi-
bition, tumor regression and metastasis. Significant differ-
ences in microvessel density and a direct correlation with the
difference in percentage of apoptotic endothelial cells were
observed after cetuximab or combination treatments (but not
gemcitabine alone) [76].
Growth factors have been shown to mediate cellular re-
sponses to radiation in numerous tissues. For this reason
growth factor receptors as well as proteins involved in cell
signalling are the main targets investigated as radiosensitive.
Experimental data demonstrated that radiation is able to
upregulate EGFR expression on cancer cells, and EGFR sig-
nalling inhibition sensitises cells to radiation effects [77].
SCC tumor xenografts treated with cetuximab and radia-
tion induces complete tumor regression in athymic mice
[78]. In vivo and in vitro data obtained using different cell
types such as HNSCC, colon, ovarian, non small cell lung
cancer (NSCLC), and breast cancer lines demonstrated that
cetuximab leads to enhanced killing in response to radiation
[79-81].
The antitumor effect is comprehensive of different
mechanisms such as cetuximab-induced inhibition of DNA
damage repair, increased radio-sensitivity deriving from spe-
cific perturbations in cell cycle phase distribution, inhibition
of tumor angiogenesis and enhancement of radiation-induced
apoptosis.
As already described, cetuximab acts on cell cycle phases
increasing the number of cells in G1 phase, which are radio-
sensitive, and decreasing those in S phase, which is more
radioresistant. Moreover, cetuximab is able to augment ra-
dion-induced apoptosis increasing proaopoptotic (Bax) and
decreasing antiapoptotic (Bcl-2) protein expression [82].
EGFR inhibition has been demonstrated to inhibit DNA
repair mechanisms by acting on EGFR import into the nu-
cleus and on DNA-PK, a protein implied in DNA damage
repair [83].
These mechanisms could explain the in vitro sensitizer
effect of cetuximab.
Another effect of EGFR inhibition is on angiogenesis
process. Experimental data describe that radiation could in-
crease VEGF expression but decrease of VEGF expression
following radiation could enhance tumor control in vivo [84-
86].
Supported by these results, many clinical trials have been
conducted and other are ongoing.
Data from phase II and randomized phase II studies lead
to the approval of cetuximab for the treatment of EGFR ex-
pressing metastatic colorectal carcinoma in patients who are
refractory to irinotecan based chemotherapy either in combi-
nation with irinotecan or as monotherapy [8, 9, 87].
BOND trial demonstrated that cetuximab has clinically
significant activity when given alone or in combination with
irinotecan in patients with irinotecan-refractory colorectal
cancer confirming the results of phase 2 studies.
The combination-therapy group had a significantly
higher response rate and a significantly longer time to
progression than the monotherapy group, suggesting that the
combination of irinotecan and cetuximab should be preferred
for patients with irinotecan-refractory cancer.
Patients whose disease had progressed during or within
three months after treatment with an irinotecan-based regi-
men were randomly assigned to receive both cetuximab and
irinotecan or cetuximab monotherapy. The rate of response
in the combination-therapy group was significantly higher
than that in the monotherapy group (22.9 vs. 10.8%). The
median time to progression was significantly greater in the
combination-therapy group (4.1 vs. 1.5 months). The median
survival time was 8.6 months in the combination-therapy
group and 6.9 months in the monotherapy group. Toxic ef-
fects were more frequent in the combination-therapy group,
but their severity and incidence were similar to those that
would be expected with irinotecan alone.
Moreover, the number of previous treatment regimens
and previous use of oxaliplatin did not affect the efficacy of
the cetuximab plus irinotecan combination. Cetuximab
monotherapy also shows activity accompanied only by mild
toxicity. These results suggest cetuximab monotherapy as a
valuable alternative option for patients who are not consid-
ered candidates for further treatment with irinotecan-based
chemotherapy or who decline such treatment [4].
The multicenter, open-label, phase III EPIC trial demon-
strated that cetuximab added to irinotecan significantly im-
proved progression free survival (PFS) (median, 4.0 v 2.6
months) and response rate (RR) (16.4% vs 4.2%), and re-
sulted in significantly better scores in the quality of life
(QOL) analysis of global health status in colorectal cancer
patients refractory to oxaliplatin.
CETUXIMAB: From Bench to Bedside Current Cancer Drug Targets, 2010, Vol. 10, No. 1 7
Median overall survival (OS) was comparable between
treatments 10.7 months with cetuximab/irinotecan and 10.0
months with irinotecan alone [87].
Although cetuximab is approved in treatment of EGFR
expressing colon cancer, Chung et al. showed that also
EGFR-negative tumors have the potential to respond to
cetuximab-based therapies [88].
Four out of 16 chemotherapy-refractory EGFR-negative
colorectal cancer patients (14 treated with cetuximab plus
irinotecan, and 2 with cetuximab monotherapy) objective
responses in terms of response rate (25%; 95% CI, 4% to
46%) were seen.
As will be analysed, EGFR analysis by current immuno-
histochemical (IHC) techniques has low sensibility and thus
does not seem to have predictive value. For these reasons,
selection or exclusion of patients for cetuximab therapy on
the basis of currently available EGFR IHC does not seem
warranted.
Vincenzi et al. described VEGF modification in EGFR-
negative patients treated with weekly cetuximab plus irinote-
can and their correlation with clinical tumor response and
outcome. Patient with a substantial VEGF reduction showed
a response rate of 45.5% compared with 8.7% in the no re-
duced VEGF group, a median time to progression (TTP) of 6
versus 3.9 months and a greater OS (11.0 and 9.6 months,
respectively).These data, even if obtained in a small popula-
tion, suggest that cetuximab may induce VEGF modifica-
tions in EGFR-negative colorectal cancer patients and that
these changes might at least in part be responsible for
cetuximab efficacy observed in these patients. The impact on
angiogenesis described in EGFR-negative colorectal cancer
patients could lead to a reconsideration of cetuximab use in
this subset of patients [89, 90]. The phase III CRYSTAL
study was designed to explore the effectiveness of cetuximab
addition to FOLFIRI regimen compared with FOLFIRI alone
as first-line treatment of unselected mCRC patients express-
ing EGFR. The primary endpoint of the study was PFS. A
total of 1,217 patients have been enrolled and randomized to
receive FOLFIRI or FOLFIRI plus cetuximab. The subset of
patients receiving cetuximab had a significantly prolonged
PFS and increased RR (47% vs 39%) [91]. The large, ran-
domized phase II oxaliplatin and cetuximab (OPUS) in first-
line treatment of mCRC trial reported a 46% RR in patients
treated with combination therapy (cetuximab plus FOLFOX-
4) vs 36% RR in the control arm [92]. The RR percentages
of OPUS study are lower than those described in other stud-
ies and those for FOLFOX-4 regimen. The cross-study vari-
ability in RRs might depend on patient selection criteria
which are more stringent for small phase II studies than
those applied for larger randomized studies. The addition of
cetuximab to radiotherapy (RT) in locally advanced HNSCC
patients improves locoregional disease control and survival
(median survival of 20 months) [93]. These reported benefits
lead to approval of cetuximab use in combination with RT in
the treatment of unresectable HNSCC in the United States
and in Europe. Promising benefits have been reported in
terms of median TTP and OS following cetuximab mono-
therapy or in combination with a platinum agent in patients
with platinum-refractory recurrent or metastatic HNSCC [94,
95], thus cetuximab is also approved in the United States as
monotherapy in patients with metastatic head and neck can-
cer refractory to standard chemotherapy.
The use of cetuximab as monotherapy or in combination
with chemotherapy has been also analysed in advanced
NSCLC patients. Docetaxel plus cetuximab as second-line
treatment of advanced NSCLC patients showed a clinical
benefit (25% RR with median TTP of 2.9 months) [96,97].
Xiong et al. conducted a phase III study to determine the
response rate, time to disease progression, survival duration
and rate, and toxicity with the combination of cetuximab and
gemcitabine as I line therapy in patients with EGFR-
expressing locally advanced or metastatic pancreatic cancer.
The authors stated that cetuximab in combination with gem-
citabine showed promising activity against advanced pancre-
atic cancer (partial response was 12.2%, stable disease was
63.4%, the median time to disease progression was 3.8
months, and the median overall survival duration was 7.1
months) [98].
Preclinical data have suggested a synergistic effect of
cetuximab combined with gemcitabine and cisplatin and
clinical data have shown a substantial improvement in re-
sponse and survival when gemcitabine is combined with a
platinum analogue compared with gemcitabine alone.
Aimed from these data Cascinu et al. have conducted a
multicentre, randomised phase II trial to assess the activity
and feasibility of a combination of cetuximab with gemcit-
abine and cisplatin compared with use of gemcitabine and
cisplatin alone for the treatment of advanced pancreatic can-
cer. No significant difference was noted between the groups
both for objective response, for disease control, for median
progression-free survival or median overall survival. The
authors concluded that the addition of cetuximab to a combi-
nation of gemcitabine and cisplatin does not increase re-
sponse or survival for patients with advanced pancreatic can-
cer [99].
Another phase III study by Philip and colleagues analyz-
ing the combination of cetuximab to gemcitabine versus
gemcitabine alone did not demonstrated benefits in terms of
OS, PFS and response in advanced pancreatic cancer patients
[100].
EGFR STATUS AS A PROGNOSTIC FACTOR IN
COLORECTAL CANCER
EGFR pathway plays a key role in tumorigenesis and in
tumor progression, due to its importance its expression has
been analysed as a possible prognosis marker in patients
with colorectal cancer.
EGFR expression at variable degree was found in up to
82% of colorectal carcinomas [8, 13, 101] but its expression
has been assessed in tumor samples by different methods
that together with the heterogeneity in tumor biopsies and in
enrolled patient populations have caused some discrepancy
in the results of EGFR expression as a prognostic factor
[102].
At least two studies linked EGFR expression with prog-
nostic data including tumor staging, histology grade, vascu-
lar and lymphatic tumor cell invasion [21, 103, 104]. Moreo-
8 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
ver, EGFR overexpression and tumor invasion (TNM tumor
stage) were significantly associated in CRC [105].
In contrast, other studies have no reported a difference in
EGFR status and histological type or tumor grade, or stage
[106,107].
Based on literature data Nicholson et al. examined the re-
lationship between EGFR expression and cancer prognosis
More than 200 studies were considered to analyse relapse-
free-interval or survival data directly in relation to EGFR
levels in over 20000 patients. Analysis of the data showed
that 10 cancer types both express elevated levels of EGFR
relative to normal tissues. In colorectal cancer EGFR pro-
vided more modest prognostic information compared to oth-
ers cancers, correlating to poor survival rates in 52% of stud-
ies [108].
The lack of a clear relationship between EGFR expres-
sion and prognosis is expected given that cetuximab activity
and its influence on cancer cell survival can be amplified by
a number of mechanisms other than increased receptor ex-
pression.
MOLECULAR PREDICTORS OF EFFICACY
With the development of modern molecular techniques
and the increasing knowledge of chemotherapeutic mecha-
nism(s) of action, the evaluation of chemotherapy-associated
molecular markers is an approach most likely to supply
novel “predictive” candidates for improved individualized
patient management [109, 110].
The goal of this approach is to identify patients that will
respond to a particular treatment and patients that will fail to
respond to it.
Furthermore, since many anticancer drugs are associated
with adverse side effects the identification of predictive
markers could avoid unnecessary risk to those patients for
whom treatment is likely to fail. Moreover, whereas the use
of markers for assessing prognosis has been widely dis-
cussed in recent years there are few reviews on predictive
factors [111].
We will summarize what we know on predictive markers
of response to cetuximab.
Epidemiological data show that only 10-15% of patients
benefit from mAb anti-EGFR target therapy, whereas about
85% receive mAb anti-EGFR target therapy without any
advantage and with toxicity risk.
Clinical studies of cetuximab in mCRC failed to reveal
an association between clinical outcome and EGFR protein
expression as measured by immunohistochemistry [8, 9].
But, clinical responses have been demonstrated in pa-
tients with undetectable EGFR protein expression [88] and
somatic mutations in the EGFR tyrosine kinase domain are
associated with sensitivity to the tyrosine kinase inhibitors
(TKIs) but not to cetuximab [112, 113].
Different markers have been identified in tumor samples
from mCRC patients as potential predictors of response to
cetuximab, i.e. activated EGFR, EGFR amplification, ab-
sence of KRAS mutations, PTEN expression, low VEGF
receptor (VEGFR) expression and loss of p21 or very re-
cently the expression of EGFR ligands such as EREG and
AREG [114].
The following are the studied makers and the related
studies.
EGFR Status
As mentioned before, the use of cetuximab is currently
restricted to those patients whose tumors express EGFR.
Although tumor EGFR status can be quantified by several
techniques (IHC, EIA, FACS, PCR, and FISH) in practice,
IHC has by far been the most commonly used method, with
most clinical cetuximab trials reported to date requiring
EGFR IHC positivity for study entry.
This method is currently used despite continuous evi-
dences in literature report that EGFR determination by im-
munohistochemistry do not correlate with clinical response
to treatment and that EGFR expression on primary tumor
may differ from metastatic sites.
The lack of correlation between IHC and clinical out-
come could depend from different reasons some related to
the methodology (time storage, tissue fixation technique
used, storage conditions of biological material, scoring sys-
tem, etc) other related to EGFR biology such as different
EGFR expression on primary tumor from metastatic sites
with an alternative/non-homogeneous pattern [8, 115, 116].
EGFR Amplification
The presence of an increased number of copies of EGFR,
assessed by FISH, has been described in mCRC and it was
associated with a better response to anti-EGFR therapies [17,
117].
Moroni et al. reported a possible predictive role of EGFR
gene copy number in the treatment of mCRC with cetuximab
or panitumumab. EGFR gene amplification evaluated by
fluorescence in situ hybridization (FISH) was correlated with
the objective response to treatment [118].
Romagnani et al. showed in 27 patients with mCRC
treated with cetuximab that 6 out of 10 responder patients
had an increased EGFR copy number compared to 2 out of
17 non-responder patients [119].
Nevertheless, Lenz et al. conducted a phase II trial of
cetuximab in refractory mCRC showing that an EGFR gene
copy number did not relate to response or progression-free
survival but to survival. The evaluation was done by using
polymerase chain reaction (PCR) technique [120].
The described discrepancies have to be related to the dif-
ferent assay methods: FISH in Romagnani’s study and a
PCR-based method in that of Lenz but this technique, as
known, does not distinguish tumor from normal neighbour-
ing tissue.
EGFR Mutations
Lynch et al. identified a subset of patients affected by
non-small-cell lung cancer who exhibit EGFR gene muta-
CETUXIMAB: From Bench to Bedside Current Cancer Drug Targets, 2010, Vol. 10, No. 1 9
tions and the presence of these mutations predicted for the
response to EGFR-targeting therapies [121].
The presence of EGFR gene mutations was also reported
in colorectal cancer but they were very rare and failed in
predicting the response to anti-EGFR treatment [113, 122,
123].
In the Nagahara study, none of 11 colorectal carcinoma
cell lines exhibited somatic mutations although 4 of 33 clini-
cal tumors presented missense mutations clustered in the
EGFR kinase domain in exons 19 and 20 [123]. The study by
Ogino et al. confirms the rarity of activating EGFR mutation
in CRC [124].
These studies suggest that for mCRC the impact of
treatment with a TKI is likely to be limited [125].
Activated EGFR
A small subset of patients EGFR-positive mCRC refrac-
tory to irinotecan treated in the BOND study had acti-
vated/phosphorylated EGFR (pEGFR) expression as useful
predictor of response to cetuximab based therapy [126]. Dis-
ease control rates differed between patients with a pEGFR
immunohistochemical score with a trend toward higher dis-
ease control in patients with high levels of pEGFR who were
treated with cetuximab with or without irinotecan. These
preliminary data need further demonstrations in clinical tri-
als.
EGFR Polymorphisms
The EGFR gene contains a highly polymorphic sequence
in intron- 1, which consists of a variable number of CA
dinucleotide repeats ranging from 9 to 21 [127].
This sequence plays a key role in gene transcription as
demonstrated by subjects or cell lines with a greater number
of CA repeats that have lower levels of mRNA and protein
expression [128, 129].
Graziano et al. reported that EGFR intron-1 S/S poly-
morphism (lower number of CA repeats) in germ-line cells
of mCRC patients cetuximab treated was associated with
favourable overall survival and treatment response [130].
In the clinical setting, the EGFR intron-1 (CA)n could
represent an easy and reproducible marker to be assessed
even though its predictive role might be altered by genetic
changes in cancer cells.
EGFR Ligands
The EGF signal pathway is activated by several stimuli.
In particular, elevated expression of EREG and/or AREG
may play an important role in tumor growth and survival by
stimulating an autocrine loop through EGFR.
Khambata-Ford and colleagues reported a predictive role
of EGFR ligands EREG and AREG showing that patients
with a high versus low EREG expression in metastatic biop-
sies have superior progression-free survival rates and are
more likely to have disease control with cetuximab based
therapies [114].
Yamada et al. found that AREG expression in primary
lesions of colorectal cancer is also an important predictive
marker of liver metastasis [131].
A recent study by Mutsaers reported treatment-induced
circulating ligands changes during EGFR inhibitors therapy
and evaluated their potential as surrogate markers of the op-
timal biological dose. Data obtained showed that treatment-
induced increases in circulating ligands, particularly TGF-
alpha, should be serially assessed in clinical trials of anti-
EGFR therapeutic antibodies as potential biomarkers to aid
in determination of the optimal biological dose [132].
The RAS/RAF/MAPK and the PTEN-PI3K/Akt Signal-
ling Pathways and Related Mutations
KRAS, a human homologue of the Kirsten rat sarcoma 2
virus gene, encodes a downstream G-protein that influences
EGFR signalling cascade. In particular, it plays a crucial role
in the RAS/MAPK pathway, and is involved in cell prolif-
eration. When KRAS gene is mutated, the KRAS protein is
constitutively active regardless of whether the EGFR is
stimulated or not [133]. The presence of activating KRAS
mutations might circumvent cetuximab’s inhibitory activity.
Nowadays, several retrospective studies widely demon-
strated the high prognostic and predictive value of KRAS
mutations in mCRC patients treated with cetuximab.
A small retrospective study conducted by Lievre et al.
with 30 cetuximab plus irinotecan refractory patients showed
that 40% of the patients had a KRAS mutation. But patients
with wild-type KRAS had a higher response rate and a much
longer survival than patients with mutated KRAS [134].
Shirin Khambata-Ford et al. have conducted a study to
identify markers that are associated with disease control in
patients treated with cetuximab. They demonstrated that the
majority of patients who achieved disease control following
cetuximab monotherapy had wild-type KRAS [114].
Moreover, in another study they associated KRAS muta-
tion status of 113 patients with irinotecan refractory mCRC
treated with CTX in clinical trials to survival benefit which
is even more pronounced in those patients with early radio-
logical response. These characteristics may be exploited for
response prediction [135].
Similar results were obtained by Di Fiore et al. from an
analysis of 59 patients with chemotherapy-refractory mCRC
treated with cetuximab plus chemotherapy, who had objec-
tive clinical responses had KRAS wild-type [125].
Based on these results, KRAS mutation status is currently
the most important standard biomarker to predict response to
anti-EGFR-based therapy in patients with mCRC, although
its predictive value has to be confirmed by prospective stud-
ies since all existing data are from retrospective analyses.
Ongoing trials will provide new data.
Another potential biomarker predictive of response to
EGFR antibodies is lipid phosphatase and tensin homologue
(PTEN). It is a key tumor suppressor gene located on chro-
mosome 10 which regulates PI3K activation, a downstream
effector of EGFR cascade. Mutations resulting in PTEN loss
lead to uncontrolled activation of PI3K/AKT signalling
10 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
pathway that may result in resistance to EGFR-blockade,
whether inactivation of this pathway is sufficient for EGFR
resistance is not clear. Mutated form of PTEN is present in
about 20 % of sporadic CRC. Recently, expression of PTEN
and loss of gene function were analysed in CRC patients
[136-138].
Starting from the preclinical evidence that Akt activation
is an important resistance factor for anti-EGFR therapy
[139]. Frattini et al. evaluated PTEN protein tumor expres-
sion in 27 cetuximab-treated mCRC patients and demon-
strated for the first time that the loss of PTEN protein ex-
pression is associated with non responsiveness to cetuximab
[137].
BRAF
BRAF is a serine/threonine kinase that belongs to the
RAS/RAF/MEK/ERK/MAPK pathway, which is involved in
the transduction of mitogenic signals from the cell mem-
brane to the nucleus. BRAF activates MEK, which in turn
activates the extracellular signal-regulated kinase (ERK).
This kinase is another candidate biomarker for resistance in
colorectal cancer, while BRAF mutations less frequent than
KRAS mutations [138].
A recent study by Benvenuti et al. reported that BRAF
status (activation) is associated with the lack of response to
anti-EGFR mAb treatment in mCRCs patients [139]. These
preliminary interesting results need to be confirmed in large
clinical trials.
NF-kB
NF-kB is a protein complex that acts as a transcription
factor and it is involved in cellular responses to stimuli such
as stress, cytokines, growth factors, free radicals, ultraviolet
irradiation, oxidised LDL and bacterial or viral antigen. NF-
kB target genes promote tumor cell proliferation, survival,
migration, inflammation, and angiogenesis. Moreover, NF-
kB is specifically activated by EGFR signalling [140-142].
Scartozzi et al. analysed the NF-kB role in predicting the
efficacy of cetuximab therapy in advanced colorectal tumors.
NF-kB expression has been shown to be responsible for re-
sistance to antineoplastic agents and it also plays a role in
EGFR activation downstream signalling pathway in colorec-
tal tumors. The authors by retrospectively analysing the nu-
clear immunoreactivity (defined as only distinct nuclear
staining) for NF-kB in EGFR-positive advanced colorectal
cancer patients receiving cetuximab and irinotecan demon-
strated that the response rate, the median time to progression
and overall survival were significantly improved in NF-kB-
negative patients than in those positive [143]. Moreover,
these findings seem to suggest that tumors that constitutively
and aberrantly express NF-kB are more likely to be refrac-
tory to cetuximab and irinotecan than those that do not show
nuclear expression of this transcriptional factor.
Circulating Factors
Among circulating factors VEGF and magnesium are the
most studied as predictor factors of cetuximab therapy effi-
cacy.
Increasing evidence suggests that EGFR mediated path-
ways are linked to tumor angiogenesis through up-regulation
of VEGF and other mediators of angiogenesis. Research
continues to highlight the importance of VEGF in colorectal
cancer and to generate new hypotheses about its role in angi-
ogenesis throughout development of this cancer.
The importance of angiogenesis and its blockade by tar-
get therapies lead to an increasing interest in explaining the
relationship between cetuximab and VEGF.
The precise mechanisms by which EGFR signalling
pathways regulate VEGF are not completely clarified.
Perrotte and colleagues in 1999 identified, in an in vitro
model, the role of angiogenesis inhibition as a new mecha-
nism that contributes to the antitumor effect of EGFR block-
ade therapy with cetuximab. The authors observed that re-
duction in bladder cancer vascularization is secondary to
down-regulation of VEGF, IL-8 and bFGF expression by
EGFR blockade therapy with cetuximab [50].
Recently, Vallböhmer et al. reported the association be-
tween intratumoral VEGF gene expression (quantify by Real
Time PCR method) and clinical outcome in terms to re-
sponse to cetuximab in patients with mCRC cetuximab
treated and EGFR-expressing. This evidence confirms the
tight relationship between angiogenesis and EGFR pathway
[144].
Vincenzi et al. in 2007 published the first evidence of
VEGF reduction as predictive marker of efficacy of cetuxi-
mab plus weekly irinotecan therapy in heavily pretreated
advanced colorectal cancer patients.
Patients with reduced circulating levels of VEGF during
treatment showed a better response rate, a longer median
time to progression and a greater overall survival than those
without VEGF modifications [64, 89, 90].
Also magnesium has been studied as a circulating factor
which is affected by cetuximab therapy.
The first description of hypomagnesemia cetuximab re-
lated was in 2005 [145].
After that, other cases have been reported and a growing
interest about the role of magnesium (Mg) in cetuximab bio-
logical effects started to be investigated, by both retrospec-
tive and perspective clinical trials [146-148].
Several hypotheses have been done about the mecha-
nisms of cetuximab-induced hypomagnesaemia [149-151].
They are mainly based on the high EGFR expression in the
kidney, especially in the ascending limb of the loop of Henle
where 70% of filtered Mg++ is reabsorbed, so that EGFR
blockade could interfere with Mg++ transport.
An important study of monogenic disorders to understand
this process has been conducted by Groenestege and col-
leagues [152]. They defined EGF as an autocrine/ paracrine
magnesiotropic hormone that regulates renal Mg2+ reabsorp-
tion by regulating the activity of the Mg++-permeable chan-
nel TRPM6 (transient receptor potential cation channel, sub-
family M, member 6). The authors identified a point muta-
tion in pro-EGF that disrupts sorting of the protein to the
basolateral membrane of distal convoluted tubule cells in
kidney nephrons. As a consequence, inhibition of the EGFR
CETUXIMAB: From Bench to Bedside Current Cancer Drug Targets, 2010, Vol. 10, No. 1 11
by anti-EGFR antibodies led to suppressed activity of
TRPM6 and renal Mg++ wasting.
Moreover, they showed that colorectal cancer patients
treated with cetuximab, an antagonist of the EGFR, develop
hypomagnesaemia.
Schrag D et al. reported that patients with colorectal can-
cers treated with cetuximab may develop an Mg++ wasting
syndrome with hypomagnesaemia and inappropriate urinary
excretion [145]. Tejpar et al. confirmed these findings, re-
porting that EGFR targeting antibodies (cetuximab and pani-
tumumab) may lead to magnesium wasting in patients af-
fected by colon cancer [148].
Another study reported a retrospective analysis of mag-
nesium decreasing during cetuximab treatment and, in par-
ticular, a severe hypomagnesaemia was associated with
treatment duration [147].
Vincenzi B et al. confirmed the previous findings by
showing a progressive decrease in serum magnesium con-
centrations during a period of 92 days in patients affected by
mCRC treated with cetuximab + irinotecan. These results
offered the first evidence in considering magnesium decrease
as a new predictor factor of efficacy and outcome in colorec-
tal cancer patients treated with cetuximab + irinotecan [64].
Mg is involved in physiological enzymatic reactions as
nucleic acid metabolism, protein synthesis and energy pro-
duction. In cancer biology, it seems to be involved in the
regulation of oxidative stress, carcinogenesis, tumor progres-
sion and angiogenesis (by acting on endothelial cells).
Moreover, it has been supposed that the interaction be-
tween anti-EGFR agents and Mg++ homeostasis could be
partially responsible of the anticancer activity of these agents
[153].
Also the effect on cell motility is so striking that Mg++
has been proposed to serve as a chemotactic factor for endo-
thelial cells [154].
In conclusion, hypomagnesaemia could contribute to the
antitumoral effect of cetuximab both by a direct action on
endothelial cells and thus on angiogenesis and by an indirect
influence on EGFR signalling and production of angiogenic
molecules.
A complex cross-talk among magnesium, angiogenesis
and EGFR signalling pathway is already been described and
it need to be deeply analysed yet.
Among the available and previous presented molecular
and genetic predictive markers, only KRAS mutational status
seems to be the most accurate and reproducible predictive
marker.
However, these studies are mostly based on retrospective
analysis from non-randomized trials and/or small samples of
patients.
For example, it cannot been excluded that EGFR amplifi-
cation is in fact linked to a better outcome, independently of
administered therapies. This phenomenon has been described
for NSCLC where EGFR amplification has been associated
with better survival for patients receiving either TKI or pla-
cebo, or TKI or chemotherapy. In this setting, EGFR ampli-
fication should not been considered as a predictive factor but
in fact as an intrinsic prognostic factor. It will therefore be
essential to validate these results in large-scale clinical pro-
spective trials.
Moreover, the influence of the IGF-IR and PI3K/AKT
pathways on EGFR-targeted therapy remains to be evaluated
in CRC.
CONCLUSIONS
Anti-EGFR MAb cetuximab has been approved by FDA
in the treatment of several tumor types and in different set-
ting, both alone and in combination with chemo/radio-
therapy.
Consistent and numerous preclinical trials have examined
its effects in cancer cells from different tumors.
The mechanisms of action are multiple: inhibition of cell
cycle progression, angiogenesis, invasion and metastatiza-
tion, increase and activation of pro-apoptotic molecules and
synergic cytotoxicity with chemotherapy and radiotherapy.
However, the biological activity of this drug is not com-
pletely known.
The interaction with Mg homeostasis is a recent active
field of research and it may help to better understand the
complex biological mechanisms of this agent.
Probably the key to maximize the benefit from cetuximab
in the treatment of cancer is linked to find predictive factors
of response and to design appropriate clinical trials to ex-
plore its potential role in the different settings of patients.
ABBREVIATIONS
ERK = extracellular signal-regulated kinase
GRB2 = growth factor receptor-bound protein 2
IGF = Insulin-like Growth Factor-1
IGF-IR = Insulin-like Growth Factor-1 receptor
bFGF = fibroblast growth factor
MMP = Metallo proteinasi
TKI = tyrosine kinase inhibitor
TRPM6 = transient receptor potential cation channel,
subfamily M, member 6
TCC = transitional cell carcinoma
VPF = vascular permeability factor
PIP2 = phosphatidylinositol 4,5 biphosphate
IP3 = inositol 1,4,5-triphosphate
DAG = diacylglycerol
MEK = MAPK kinase
ERK = extracellular signal-regulated kinase
TKR = tyrosine kinase receptor
HER = human epidermal growth factor receptor
AREG = amphiregulin
12 Current Cancer Drug Targets, 2010, Vol. 10, No. 1 Vincenzi et al.
EREG = epiregulin
mCRC = metastatic colorectal cancer
HNSCC = head and neck squamous cell carcinoma
TGF = transforming growth factor
MAPK = Mitogen-Activated Protein Kinase
PI3K = Phosphoinositide 3-kinase
EGFR = epidermal growth factor receptor
EGF = epidermal growth factor
MMP = matrix metallo-proteinase
VEGF = vascular endothelial growth factor
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Received: August 25, 2009 Revised: January 07, 2010 Accepted: January 07, 2010
