# Anti-EGFR Therapy: Mechanism and Advances in Clinical Efficacy in Breast Cancer

Authors: John F. Flynn; Christina Wong; Joseph M. Wu
Journal: Journal of Oncology
 (2009)

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## Abstract

This review will focus on recent advances in the application of antiepidermal growth factor receptor (anti-EGFR) for the treatment of breast cancer. The choice of EGFR, a member of the ErbB tyrosine kinase receptor family, stems from evidence pinpointing its role in various anti-EGFR therapies. Therefore, an increase in our understanding of EGFR mechanism and signaling might reveal novel targets amenable to intervention in the clinic. This knowledge base might also improve existing medical treatment options and identify research gaps in the design of new therapeutic agents. While the approved use of drugs like the dual kinase inhibitor Lapatinib represents significant advances in the clinical management of breast cancer, confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety, pharmacokinetics, and clinical efficacy.


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## Body

## 1. Introduction

Despite
the availability of a new array
of biomarkers and a widely adapted clinically relevant/treatment-oriented
approach of classifying breast cancer cases over the last decade,
categorization of breast cancer is an ongoing challenge which is being
revisited more frequently by the scientific community. The goal is to fine-tune
the diagnostic assignment of breast cancer cases with the hope that this will
adequately address and improve the effectiveness of selecting treatment
modalities, particularly in regard to the choice of use of monoclonal
antibodies (MoAbs) and small molecule
tyrosine kinase inhibitors (smTKIs) against EGFR, a clinical strategy collectively referred to as anti-EGFR
therapy. EGFR is a member of the ErbB/HER family of tyrosine kinase receptors,
which also includes its well-documented family member ErbB2, clinically
referred to as HER-2/neu. Anti-EGFR therapy has found  application for cases
from all three major breast cancer subclasses, respectively, the
hormone-sensitive/insensitive group, the ER+/− and HER-2/neu+/− groups, and the
basal-like/triple negative (−) group. Of
note, HER-2/neu may also be a genetic biomarker since it has a more significant
correlation with a selective HER-2 (+ve) population of breast cancer cases than EGFR. Preliminary studies show that
anti-EGFR therapy has moderate clinical efficacy not only on EGFR-expressing
cells, but on HER-2-expressing and -overexpressing cells as well, suggesting
that the treatment outcome may depend on the expression and responsiveness of the heterodimerization of HER-2 with EGFR.
Although both EGFR and HER-2 (+ve) are
favored biomarkers of efficacy in many ongoing anti-EGFR clinical studies,
their expression is not sufficiently robust as a prognosticator for clinical
outcomes and should not be singularly used as a criterion for evaluating the
responsiveness of breast cancer cases to anti-EGFR treatment regimens [1].
Tumor targets for anti-EGFR therapy include early and advanced stage, and
metastatic breast cancer as well as an array of other solid tumors that are not
part of this review; data from recent studies suggest that various
anti-EGFR/TKI combinations may not only treat but also lower progression rates
of these forms of cancer.The primary
focus of this article is to review and summarize recent advances in anti-EGFR
therapies in order to generate a clinically relevant profiling system; a
complementary objective is to relate the structure of EGFR with its downstream
signaling mechanisms particularly in the context of inhibition by administered
anti-EGFR therapies. Database search engines like MEDLINE, PubMed, Scopus, and
ENTREZ were used, and the articles were selected according to the criteria: (i) anti-EGFR therapy and clinical efficacy in breast
cancer, (ii) publications from 1998–2008, and (iii) using reviews/conferences/special
reports/randomized clinical trials/phase II and III trials/general research articles. It
is hoped that reviews like this can help to elucidate the mechanisms
involved in anti-EGFR therapy as well as define relationships between the overexpression
of EGFR and other biomarkers of breast cancer. Recent data regarding
responsiveness to combination and multiregiment chemotherapies may also provide
insight on the mechanism and activity of anti-EGFR therapies, specifically that
of the  dual  kinase  inhibitor, Lapatinib  (GW572016),
which is capable of targeting both the EGFR and HER-2/neu tyrosine kinases that
are often overexpressed in breast cancer cells [4].


## 2. EGFR and Its Role in Breast Cancer

EGFR is a member of the EGFR/ErbB/HER family
of Type I transmembrane tyrosine kinase receptors, which includes ErbB1/HER-1 (EGFR itself),
ErbB2/HER-2/neu, ErbB3/HER-3, and ErbB4/HER-4. The ErbB receptors play an
essential role in organ development and growth by regulating both the
differentiation and morphology of cells and tissues. However, specific members,
most notably EGFR, are frequently overexpressed, and this aberrant expression
and the signaling event it elicits induce erroneous development and
unrestricted proliferation in a number of human malignancies including breast
cancer [5]. Members of the ErbB gene family, respectively, ErbB1, ErbB3, and
ErbB4 can be activated by various growth factor ligands, for example, the
epidermal growth factor (EGF). In
contrast, no known ligand has been demonstrated for ErbB2/HER-2/neu, despite
that it still plays an integral role in several signaling pathways as well as
tumorigenesis. Activation of EGFR inevitably involves homo- or heterodimerization
of EGFR with another EGFR molecule, or a different member of the ErbB family
(e.g., HER-2), which in turn induces the amplified signaling cascade (Figure 1). Increased activation of EGFR and/or HER-2
will eventually result in uncontrolled proliferation, a hallmark of cancer
cells. Additionally, the cells harboring overexpressed EGFR or improper
regulation of EGFR activation may decrease apoptosis, increase metastasis and
even angiogenesis. Dysfunctional EGFR-signaling networks are reportedly present
in a cohort of breast carcinomas with poor prognosis [5, 6].Figure 1
The EGFR Signaling Pathway. (a) Upon EGF-ligand binding to the EGFR there is subsequent
dimerization (homo- or hetero-) and
tyrosine kinase residue auto-/transphosphorylation of dimer partners, which in
turn initiates the actual downstream signaling pathways. (b) Ras signaling cascade in tabulated form. (c) PI3K signaling cascade in
tabulated form.To
better understand the role of EGFR
in breast carcinogenesis, the aforementioned relationship will be analyzed in
several parts. First, it is important to thoroughly investigate thesignaling pathway and mechanism of EGFR
to properly examine the correlation that exists between breast cancer and
anomalous EGFR expression; in this case, scrutiny of the structure of EGFR and
the role it plays in cell signaling is imperative. Secondly, anti-EGFR
therapies for breast cancer either in ongoing clinical phase testing or already
FDA-approved are reviewed or summarized in the tables, with focus directed to
specific developments and progress in clinical efficacy in recent years.
Examples discussed in detail in Section3 include Cetuximab, a monoclonal antibody against EGFR and the most
widely used anti-EGFR therapy in solid tumor treatment regimens; Lapatinib, an
innovative small molecule tyrosine kinase inhibitor of EGFR with a unique dual-TKI inhibitory activity
against both EGFR and HER-2/neu, which shows improved clinical efficacy and has
heightened the expectation for new breast cancer therapies. Although many
recent studies have demonstrated a beneficial role of Lapatinib used in
combination with anti-EGFR therapy, only selected examples will be reviewed to
illustrate how Lapatinib may be strategically explored to improve our understanding of the synergy resulting from its use
associated with anti-EGFR therapy.


### 2.1. EGFR Structure and Signaling Pathway Mechanism

The EGFR and the activated signaling
cascades it elicits play an integral role in the mechanism and efficacy of
anti-EGFR therapies (Figure1).
Examination of the EGFR structure (Figure 2) provides a contextual framework for the inception and development of two major
strategies of anti-EGFR therapy, respectively, anti-EGFR monoclonal antibodies
and small molecule tyrosine kinase inhibitors (smTKIs).
Examples of monoclonal antibodies for EGFR are found in Cetuximab and
Trastuzumab. Anti-EGFR drugs belonging to smTKIs and in clinical trials include
Erlotinib, Lapatinib, and Gefitinib.Figure 2
(a) Basic Structure of EGFR demonstrating relevant domains.(I) The extracellular domains: (1) domain I: L1; (2)  domain II:
CR1; domain III: L2; domain IV: CR2. (II)
Transmembrane domains. (III) The intracellular domains (1) juxtamembrane domain;
(2) tyrosine kinase domain; (3) regulatory region domain. The phosphorylation
of several substrates by the tyrosine kinase domain of the EGFR receptor is
responsible for activating the various signaling cascades seen in Figure 1. (b) Structure of domains I–IV of EGFR (no ligand bound). Note the
“protruding loop” in domain II (CR1) directed away from the C-shaped region of the ligand-binding zone formed by domains I, II,
and III. (c) The tyrosine kinase domain of
EGFR showing the N-lobe and C-lobe flanking the activation loop and active site
cleft [2, 3].


#### 2.1.1. Molecular Analysis of EGFR Structure

(1) Ectodomain: Ligand-Binding Domain
Domains I–IV make up the EGFR ectodomain, a 621-kDa
structure responsible for ligand binding and dimerization, both of which are
considered molecular antecedents for the induced conformational changes
required for the activation of the internal tyrosine kinase. Although the EGFR
ectodomain was first crystallized in 1998, the detailed structure of the EGFR
ectodomain dimer bound to ligand EGF was not resolved until 2002 and provided
information on a completely novel and unexpected mode of activation for the
EGFR signaling pathway involving the dimerization process [2, 7–9].
Domain I or L1 (where L: leucine-rich
domain) shares sequence and structural homology with the Domain III or L2, both of which are involved in ligand
binding based on site directed mutagenesis and deletion mutation studies [10].
Domains II and IV, also referred to CR1 and CR2 reflecting their high content
of cysteine residues and the potential for forming intradomain disulfide bonds,
are important in facilitating the overall conformational change induced by
binding of the ligand to EGFR. The CR1 also contains a “protruding loop”
capable of bending in a relatively straight, ligand-binding site of EGFR
(Figure 2(b)). This flexible molecular feature presumably enables binding of
the ligand between the two L domains and at the same time permits contacts to be
made with the “protruding loops” in CR1.(2) Transmembrane and Juxtamembrane Domains
The transmembrane domain
consists of 23 amino acids and plays an important role in anchoring the
receptor to the lipid bilayer of the cell. More than 50% of the transmembrane
domain is localized in caveolae or lipid rafts [10], through posttranslational
modifications, such as N-linked glycosylation, resulting in enrichment of EGFR
in defined locality of the membrane and hence faster receptor dimerization
following binding of the ligand [11]. Adjacent to the transmembrane domain
facing intracellularly is the juxtamembrane domain which is believed to
regulate various functional aspects of EGFR including control of the tyrosine
kinase activity, downregulation of the EGFR, ligand internalization, and
receptor sorting. Of note, this domain also has binding motifs that allow it to
interact with second messengers like calmodulin [10].(3) Tyrosine Kinase Domain
The tyrosine kinase domain
(TKD) is essential for the functional
activation of the receptor and consequently the induction of the EGFR signaling
pathways for the control of cell division and proliferation. The TKD has a bilobate
arrangement marked by an N-lobe, an activation loop, and a C-lobe [3]. This
molecular configuration accommodates binding of the substrate and ATP at the
active site, enabling substrate phosphorylation to occur in concomitance with
the hydrolysis of ATP (Figure 2(c)). The TKD contains important tyrosine (Y) residues that can assume various states of
phosphorylation/dephosphorylation. Knockdown or deletion studies of the
ectodomain suggest that it regulates the dimerization as well as prevent constitutive
activation of the tyrosine kinase. Binding of the ligand to the ectodomain relieves
some of the steric hindrances normally imposed on the tyrosine kinase activity,
resulting in activation. Site-directed mutagenesis  or deletion analysis in the
TKD shows that it is involved in EGFR dimerization, auto- and transphosphorylation
and also activation of the signaling cascades, all of which have been exploited
in the development of small molecule tyrosine kinase inhibitors (smTKIs) for targeting EGFR in various cancer types
including breast cancer.(4) The Activation Loop, Active Site, and C-Terminal Tail Regions of EGFR
The activation loop of the tyrosine
kinase of EGFR is quite distinct from other receptors harboring tyrosine
kinases. Namely, whereas most receptor TKDs require phosphorylation for
tyrosine kinase activation, this does not appear to be the case in EGFR. For
example, the phosphorylation of Tyr845 has little affect on the EGFR kinase activity [3], possibly due to a
conformational arrangement that directs the activation loop away from the
active site rendering it refractory to the state of phosphorylation of the
receptor tyrosine kinase. Activation of the EGFR tyrosine kinase phosphorylates
numerous targets, including itself (autophosphorylation), a different EGFR (homodimerization), HER-2/neu of the ErbB gene family (heterodimerization), and nonreceptor substrates such
as Grb2/SOS, STATs, PLC, and/or PI3K, which in turn initiate the
signaling cascades of MAPK/ERK, STAT,
PIP2, and AKT,
respectively. Not surprisingly, therefore, mutations in this region can cause a
substantial decrease in kinase
activity, an outcome considered desirable in cancer therapy and may underlie
the therapeutic efficacy of smTKIs. By binding to the TKD of EGFR, smTKIs may
act by sterically interfering with the binding of both the substrate and ATP
necessary for phosphorylation, resulting in an overall decreased signaling
activity of the EGFR.
Lastly, it is important to mention that the tyrosine
kinase activity of EGFR is tightly regulated via its own internal regulatory
region located at the C-terminal tail of the structure, which involves the
tyrosine residue cluster with the potential of being transphosphorylated during
EGFR-dimerization. It is noteworthy that EGFR dimerization induces
phosphorylation of several tyrosine residues including Tyr1069 Tyr1092 Tyr1110 Tyr1116 Tyr1172 Tyr1197,
creating docking sites for the recruitment of other adaptor molecules and
signaling proteins. These attributes suggest that the tyrosine-rich C-terminal
tail is a phosphorylable, mobile structure connected to a relatively stationary
TKD.
In summary, the EGFR may be divided into two
functional substructures. The first one consists of the extracellular
ectodomain responsible for ligand binding, dimerization, and the initiation of
signal transduction. The ectodomain has been the thematic target of anti-EGFR
therapy, vis-à-vis, development of monoclonal antibody directed at the ligand
binding region, which inactivates EGFR through competitive inhibition of ligand
binding, as well as by inducing overall downregulation of EGFR through
increased receptor internalization. Examples include the monoclonal antibodies like Cetuximab
and Trastuzumab, which play an extremely critical role in anti-EGFR
therapy. Currently, both these two drugs and Panitumumab are the only anti-EGFR
monoclonal antibodies approved by the FDA for use in the clinic. The
second major functional substructure of EGFR is the tyrosine kinase domain
located on the intracellular side of the plasma membrane. This domain plays a
key role in the activation of signaling cascades involved in cell
proliferation, division, and differentiation; therefore, inhibition of the
tyrosine kinase enzymatic activity of EGFR using small molecule TKIs is a
clinically relevant treatment option for breast cancer patients.


### 2.2. Breast Cancer and the Signaling Mechanism of EGFR

As a member of the ErbB receptor family, the EGFR
plays important roles in cell signaling, proliferation, differentiation, and
apoptosis. Signaling is initiated by binding of ligands to the extracellular
domain of the EGFR. Six well-characterized ligands of EGFR have been
identified, respectively, EGF, transforming growth factor-α (TGFα), amphiregulin, heparin binding EGF-like growth
factor, betacellulin, and epiregulin. Ligand binding induces conformational
change resulting in heterodimerization and the activation of the major
signaling pathways seen in Figure 1.


#### 2.2.1. Statistics and Etiology of Breast Carcinogenesis

Breast cancer is the
most common cancer and a major cause of morbidity and premature loss of life in
women worldwide, accounting for approximately 7% of all cancer-related deaths
[12]. The highest rates of breast cancer in the world are seen in the United
States, where approximately 1 out of every 8 women will develop invasive breast
cancer, which is responsible for almost 3% of all deaths in American women
[13]. Given the grim statistics, the need for more sensitive and reliable
detection methods is obvious. Equally urgent are treatment modalities that are
modest in cost, easily compliant, effective, have low to no toxicities, and
capable of targeting the multifaceted and heterogeneous nature of breast
carcinogenesis.Currently, there is still
lack of understanding of the natural
history of breast cancer. It had been hypothesized that lobular carcinoma in situ (LCIS) represented a precursor lesion of invasive cancer, and, based on this,
mastectomy was initially recommended [14]. Later studies have shown that the
risk of subsequent breast cancer is bilateral. Moreover, it became evident that
LCIS is not a premalignant lesion, but rather a marker that identifies women at
an increased risk for subsequent development of invasive breast cancer, with
the risk remaining elevated even beyond two decades. In a large prospective
study from the National Surgical Adjuvant Breast and Bowel Project involving a
5-year follow-up of 182 women with LCIS managed with excisional biopsy alone,
eight women developed ipsilateral breast tumors (four
with invasive tumors), and three women developed contralateral
breast tumors (two with invasive tumors) [15]. Therefore, it remains unclear whether or not LCIS progresses to ductal
carcinoma in situ (DCIS) during breast carcinogenesis. On the other hand, DCIS is a bona fide precursor
for invasive ductal carcinoma and lacks estrogen receptor (ER) expression. Furthermore, DCIS frequently overexpresses mutated p53, HER-2/neu,
and EGFR, all of which
show some clinical correlation with resistance
to hormone therapy and increased risk for the development of invasive,
metastatic breast cancer. Patients with ER(+ve)/PR(+ve) disease usually respond more favorably to
hormonal therapy (as compared to individuals with
ER(−ve)/PR(−ve) status),
presumably in part due to the overexpression of HER-2 and EGFR in ER(−ve) cells that provide “acquired growth stimulation
autonomy.” These findings suggest that strategies cotargeting HER-2 and EGFR
expression or their functions might have therapeutic and preventive potentials
particularly in ER(−ve) breast carcinoma
cases.


#### 2.2.2. Expression/Function of HER-2/EGFR and Signaling in Breast Carcinogenesis

The EGFR gene is frequently altered by gene amplification or overexpression at the mRNA and
protein levels in sporadic breast cancer cases. Numerous polypeptide ligands
sharing an EGF-like motif have been identified and shown to be capable of
inducing EGFR dimerization with different kinetics, eliciting signals of
variable durations, and coupled signal transduction to specific sets of
cytoplasmic proteins. In principle, therefore, this “ligand-initiated receptor-mediated signaling-executed”
molecular relay system might generate a large combinatorial set of biological
readouts with enormous potential for diversification, fine tuning, and
stringent control of cellular functions and responses. Of note, the HER-2/EGFR
has been proposed to act as a master regulator of a signaling network that
drives breast carcinoma epithelial cell proliferation; HER-2 gene amplification
was observed in 92% of breast cancer specimens and overexpression of HER-2 at
the mRNA, and protein levels have been correlated with cancer virulence, resistance to therapy, and poor prognosis. As discussed,
each member of the EGFR gene family has a multifunction structural organization
comprised of an extracellular ligand-binding/interacting domain connected by a
transmembrane span to an intracellular kinase domain. In an uninduced state,
the EGFR is organized such that the autoinhibitory loops flanking the kinase
active site sterically inhibit
it from binding substrates. Binding to ligands induces EGFR homo- or heterodimerization
concomitant with its autoactivation by a transphosphorylation mechanism
involving specific tyrosine residues located in the intracellular domain. In
turn, phosphorylated EGFR undergoes conformational changes that create
additional docking sites for adaptor proteins, kinases, and intracellular
messengers. Therefore, a tightly regulated, dynamic equilibrium presumably
exists between inhibited, activated, monomeric, and dimeric EGFR in order for
proper cell signaling to ensue. If any one of these mechanisms goes awry, the
results can be fatal to the cell and often can be fatal to the organism. The
same considerations may well contribute to the observed clinical efficacy or
lack thereof in EGFR-targeted therapies.The realization that
HER-2 is a master regulator of a signaling network that drives epithelial cell
proliferation identifies this protein as a target for cancer therapy. When overexpressed,
the HER-2 protein may be constitutively active, that is, signaling from the
receptor occurs by a ligand-independent manner. Under these conditions, growth-promoting
signals may be continuously transmitted into the cells in the absence of
ligand. As a result, multiple intracellular signal transduction pathways become
activated, resulting in unregulated cell growth and, in some instances, oncogenic transformation. Figure1 depicts
some of the signaling pathways elicited in response to ligand binding to EGFR
and either EGFR/EGFR homodimerization or EGFR/HER-2 heterodimerization
reactions. It also demonstrates the cascade of events resulting in the
transmission of signals into the nucleus and subsequent cell proliferation and
gene activation. The intracellular signaling pathways of EGFR and HER-2 are
thought to involve Ras-MAPK, and PI3K-, PKC-, NFκB-mediated pathways. Many
clinical trials have observed a poor clinical outcome and shortened survival
time for women whose breast tumors have HER-2 amplification. An inverse
correlation of ER and HER-2 levels between ER(+ve) and ER(−ve) breast cancer cells has been
demonstrated, which probably accounts for the development of tamoxifen resistance in breast cancer cells.


#### 2.2.3. Signaling Cross-Talk and Acquisition of Endocrine Resistance

Multiple
lines of evidence implicate breast cancer development and progression as under
the control of steroid hormones, in particular estrogens, via their interaction
with estrogen receptors (ERs) and cross-talk of ER with receptors including
EGFR [16–18]. The classical mechanism of ER signaling involves binding of
estrogens to intracellular ER, triggering a multitude of events that culminate
in altered transcription of estrogen-responsive genes. In sequence, protein
synthesis occurs resulting in cell proliferation, angiogenesis, breast cancer
growth, progression, and metastasis [19–21]. The ER-induced signaling
mechanism coupled with the fact that well over two thirds of breast cancers
exhibit high expression of ER, have provided the rationale for preventing and
treating breast cancer by estrogen antagonism, highlighted by the discovery of
tamoxifen. By selectively modulating the ER, tamoxifen is considered the
mainstay of estrogen antagonist therapy and among the most effective systemic
treatmentfor women with ER-positive breast cancer at all stages
today [21]. A serious obstacle, however, is intrinsicor acquired
resistance to endocrine agents. Manypatients present with primary (de novo) resistance to endocrinetherapy,
despite high tumor levels of ER, and all patients withadvanced
disease eventually acquire resistance
[22].What underlies the
refractoriness to endocrine therapies? A number of possible explanations may be
considered. For example, in addition to the aforementioned activation of
intracellular ER for transcription, estrogens have also been shown to bind
membrane-associated ER [20]. Evidence also exists on ER activation by a
ligand-independent but growth factor-dependent kinase-mediated mechanism [16].
Important contributing factors for resistance
to endocrine therapy include the levels of both ER and ER coregulatory
proteins, amplified extra- and intracellular signaling from growth
factor-mediatedpathways, as well as cross-talk between the ER
pathwayand other growth factor and kinase networks [16–18].
Other mechanisms may involve amplification and/or mutations of key proteins
involved in cross-talk, as well induction of promiscuity and/or antagonism to
therapeutic agents through mutational and posttranslational modification events
[21]. It is possible that aberrations and dysfunctions in these and other
mechanisms may occur with increasing frequency during the development of the
endocrine-resistant phenotype.
Delineation of the interplay between the estrogens, ER, and ER cross-talk with
receptors like EGFR will be an important diagnostic and prognostic objective in
anti-EGFR therapy. Similarly, discovery and development of novel agents that
can reverse resistance by targeting
the ER and its downstream signaling events, or by selective modulation of the
ER:EGFR cross-talk might improve therapeutic response rates.In summary, identifying
the factors and pathways responsible for endocrine resistance
and defining ways to overcome it are research gaps in need of further study and
will remain important diagnostic and therapeutic challenges in the continuing
war to better manage and treat breast cancer.


#### 2.2.4. Breast Cancer Treatment Using Multitarget Strategy Related to HER-2 Signaling

The amplification of the HER-2 gene
and overexpression of the HER-2 protein is frequently observed (10–40%) in human
breast cancer patients [23] and has been suggested to associate with tumor
aggressiveness, prognosis, and responsiveness to hormonal and cytotoxic agents.
These observations suggest that HER-2 is an appropriate target for
tumor-specific therapies, some of which are listed as follows.(1)
A humanized monoclonal antibody against HER-2, rhuMAbHER-2 (Trastuzumab),
is already approved for clinical use in the treatment of patients with
metastatic breast cancer. Some forms of HER-2 overexpressing breast tumors can
be successfully treated using antireceptor monoclonal antibodies,for example, Herceptin. However, because
multiple proteins are involved in growth-signaling pathways, development of a
uniformly active therapy may be strategically challenging. Herceptin inhibited
the P13K-dependent pathway, and not the MAPK pathway. Also, blockade of HER-2
function alone without the interception of the committed, associated downstream
events may restrict the effectiveness of therapeutic interventions.(2)
Tyrosine kinase inhibitors, such as emodin, which block HER-2 phosphorylation
and its intracellular signaling.(3)
Heat shock protein Hsp90-associated signal inhibitors, which induce degradation
of tyrosine kinase receptors, such as HER-2.


## 2.1. EGFR Structure and Signaling Pathway Mechanism

The EGFR and the activated signaling
cascades it elicits play an integral role in the mechanism and efficacy of
anti-EGFR therapies (Figure1).
Examination of the EGFR structure (Figure 2) provides a contextual framework for the inception and development of two major
strategies of anti-EGFR therapy, respectively, anti-EGFR monoclonal antibodies
and small molecule tyrosine kinase inhibitors (smTKIs).
Examples of monoclonal antibodies for EGFR are found in Cetuximab and
Trastuzumab. Anti-EGFR drugs belonging to smTKIs and in clinical trials include
Erlotinib, Lapatinib, and Gefitinib.Figure 2
(a) Basic Structure of EGFR demonstrating relevant domains.(I) The extracellular domains: (1) domain I: L1; (2)  domain II:
CR1; domain III: L2; domain IV: CR2. (II)
Transmembrane domains. (III) The intracellular domains (1) juxtamembrane domain;
(2) tyrosine kinase domain; (3) regulatory region domain. The phosphorylation
of several substrates by the tyrosine kinase domain of the EGFR receptor is
responsible for activating the various signaling cascades seen in Figure 1. (b) Structure of domains I–IV of EGFR (no ligand bound). Note the
“protruding loop” in domain II (CR1) directed away from the C-shaped region of the ligand-binding zone formed by domains I, II,
and III. (c) The tyrosine kinase domain of
EGFR showing the N-lobe and C-lobe flanking the activation loop and active site
cleft [2, 3].


### 2.1.1. Molecular Analysis of EGFR Structure

(1) Ectodomain: Ligand-Binding Domain
Domains I–IV make up the EGFR ectodomain, a 621-kDa
structure responsible for ligand binding and dimerization, both of which are
considered molecular antecedents for the induced conformational changes
required for the activation of the internal tyrosine kinase. Although the EGFR
ectodomain was first crystallized in 1998, the detailed structure of the EGFR
ectodomain dimer bound to ligand EGF was not resolved until 2002 and provided
information on a completely novel and unexpected mode of activation for the
EGFR signaling pathway involving the dimerization process [2, 7–9].
Domain I or L1 (where L: leucine-rich
domain) shares sequence and structural homology with the Domain III or L2, both of which are involved in ligand
binding based on site directed mutagenesis and deletion mutation studies [10].
Domains II and IV, also referred to CR1 and CR2 reflecting their high content
of cysteine residues and the potential for forming intradomain disulfide bonds,
are important in facilitating the overall conformational change induced by
binding of the ligand to EGFR. The CR1 also contains a “protruding loop”
capable of bending in a relatively straight, ligand-binding site of EGFR
(Figure 2(b)). This flexible molecular feature presumably enables binding of
the ligand between the two L domains and at the same time permits contacts to be
made with the “protruding loops” in CR1.(2) Transmembrane and Juxtamembrane Domains
The transmembrane domain
consists of 23 amino acids and plays an important role in anchoring the
receptor to the lipid bilayer of the cell. More than 50% of the transmembrane
domain is localized in caveolae or lipid rafts [10], through posttranslational
modifications, such as N-linked glycosylation, resulting in enrichment of EGFR
in defined locality of the membrane and hence faster receptor dimerization
following binding of the ligand [11]. Adjacent to the transmembrane domain
facing intracellularly is the juxtamembrane domain which is believed to
regulate various functional aspects of EGFR including control of the tyrosine
kinase activity, downregulation of the EGFR, ligand internalization, and
receptor sorting. Of note, this domain also has binding motifs that allow it to
interact with second messengers like calmodulin [10].(3) Tyrosine Kinase Domain
The tyrosine kinase domain
(TKD) is essential for the functional
activation of the receptor and consequently the induction of the EGFR signaling
pathways for the control of cell division and proliferation. The TKD has a bilobate
arrangement marked by an N-lobe, an activation loop, and a C-lobe [3]. This
molecular configuration accommodates binding of the substrate and ATP at the
active site, enabling substrate phosphorylation to occur in concomitance with
the hydrolysis of ATP (Figure 2(c)). The TKD contains important tyrosine (Y) residues that can assume various states of
phosphorylation/dephosphorylation. Knockdown or deletion studies of the
ectodomain suggest that it regulates the dimerization as well as prevent constitutive
activation of the tyrosine kinase. Binding of the ligand to the ectodomain relieves
some of the steric hindrances normally imposed on the tyrosine kinase activity,
resulting in activation. Site-directed mutagenesis  or deletion analysis in the
TKD shows that it is involved in EGFR dimerization, auto- and transphosphorylation
and also activation of the signaling cascades, all of which have been exploited
in the development of small molecule tyrosine kinase inhibitors (smTKIs) for targeting EGFR in various cancer types
including breast cancer.(4) The Activation Loop, Active Site, and C-Terminal Tail Regions of EGFR
The activation loop of the tyrosine
kinase of EGFR is quite distinct from other receptors harboring tyrosine
kinases. Namely, whereas most receptor TKDs require phosphorylation for
tyrosine kinase activation, this does not appear to be the case in EGFR. For
example, the phosphorylation of Tyr845 has little affect on the EGFR kinase activity [3], possibly due to a
conformational arrangement that directs the activation loop away from the
active site rendering it refractory to the state of phosphorylation of the
receptor tyrosine kinase. Activation of the EGFR tyrosine kinase phosphorylates
numerous targets, including itself (autophosphorylation), a different EGFR (homodimerization), HER-2/neu of the ErbB gene family (heterodimerization), and nonreceptor substrates such
as Grb2/SOS, STATs, PLC, and/or PI3K, which in turn initiate the
signaling cascades of MAPK/ERK, STAT,
PIP2, and AKT,
respectively. Not surprisingly, therefore, mutations in this region can cause a
substantial decrease in kinase
activity, an outcome considered desirable in cancer therapy and may underlie
the therapeutic efficacy of smTKIs. By binding to the TKD of EGFR, smTKIs may
act by sterically interfering with the binding of both the substrate and ATP
necessary for phosphorylation, resulting in an overall decreased signaling
activity of the EGFR.
Lastly, it is important to mention that the tyrosine
kinase activity of EGFR is tightly regulated via its own internal regulatory
region located at the C-terminal tail of the structure, which involves the
tyrosine residue cluster with the potential of being transphosphorylated during
EGFR-dimerization. It is noteworthy that EGFR dimerization induces
phosphorylation of several tyrosine residues including Tyr1069 Tyr1092 Tyr1110 Tyr1116 Tyr1172 Tyr1197,
creating docking sites for the recruitment of other adaptor molecules and
signaling proteins. These attributes suggest that the tyrosine-rich C-terminal
tail is a phosphorylable, mobile structure connected to a relatively stationary
TKD.
In summary, the EGFR may be divided into two
functional substructures. The first one consists of the extracellular
ectodomain responsible for ligand binding, dimerization, and the initiation of
signal transduction. The ectodomain has been the thematic target of anti-EGFR
therapy, vis-à-vis, development of monoclonal antibody directed at the ligand
binding region, which inactivates EGFR through competitive inhibition of ligand
binding, as well as by inducing overall downregulation of EGFR through
increased receptor internalization. Examples include the monoclonal antibodies like Cetuximab
and Trastuzumab, which play an extremely critical role in anti-EGFR
therapy. Currently, both these two drugs and Panitumumab are the only anti-EGFR
monoclonal antibodies approved by the FDA for use in the clinic. The
second major functional substructure of EGFR is the tyrosine kinase domain
located on the intracellular side of the plasma membrane. This domain plays a
key role in the activation of signaling cascades involved in cell
proliferation, division, and differentiation; therefore, inhibition of the
tyrosine kinase enzymatic activity of EGFR using small molecule TKIs is a
clinically relevant treatment option for breast cancer patients.


## 2.1.1. Molecular Analysis of EGFR Structure

(1) Ectodomain: Ligand-Binding Domain
Domains I–IV make up the EGFR ectodomain, a 621-kDa
structure responsible for ligand binding and dimerization, both of which are
considered molecular antecedents for the induced conformational changes
required for the activation of the internal tyrosine kinase. Although the EGFR
ectodomain was first crystallized in 1998, the detailed structure of the EGFR
ectodomain dimer bound to ligand EGF was not resolved until 2002 and provided
information on a completely novel and unexpected mode of activation for the
EGFR signaling pathway involving the dimerization process [2, 7–9].
Domain I or L1 (where L: leucine-rich
domain) shares sequence and structural homology with the Domain III or L2, both of which are involved in ligand
binding based on site directed mutagenesis and deletion mutation studies [10].
Domains II and IV, also referred to CR1 and CR2 reflecting their high content
of cysteine residues and the potential for forming intradomain disulfide bonds,
are important in facilitating the overall conformational change induced by
binding of the ligand to EGFR. The CR1 also contains a “protruding loop”
capable of bending in a relatively straight, ligand-binding site of EGFR
(Figure 2(b)). This flexible molecular feature presumably enables binding of
the ligand between the two L domains and at the same time permits contacts to be
made with the “protruding loops” in CR1.(2) Transmembrane and Juxtamembrane Domains
The transmembrane domain
consists of 23 amino acids and plays an important role in anchoring the
receptor to the lipid bilayer of the cell. More than 50% of the transmembrane
domain is localized in caveolae or lipid rafts [10], through posttranslational
modifications, such as N-linked glycosylation, resulting in enrichment of EGFR
in defined locality of the membrane and hence faster receptor dimerization
following binding of the ligand [11]. Adjacent to the transmembrane domain
facing intracellularly is the juxtamembrane domain which is believed to
regulate various functional aspects of EGFR including control of the tyrosine
kinase activity, downregulation of the EGFR, ligand internalization, and
receptor sorting. Of note, this domain also has binding motifs that allow it to
interact with second messengers like calmodulin [10].(3) Tyrosine Kinase Domain
The tyrosine kinase domain
(TKD) is essential for the functional
activation of the receptor and consequently the induction of the EGFR signaling
pathways for the control of cell division and proliferation. The TKD has a bilobate
arrangement marked by an N-lobe, an activation loop, and a C-lobe [3]. This
molecular configuration accommodates binding of the substrate and ATP at the
active site, enabling substrate phosphorylation to occur in concomitance with
the hydrolysis of ATP (Figure 2(c)). The TKD contains important tyrosine (Y) residues that can assume various states of
phosphorylation/dephosphorylation. Knockdown or deletion studies of the
ectodomain suggest that it regulates the dimerization as well as prevent constitutive
activation of the tyrosine kinase. Binding of the ligand to the ectodomain relieves
some of the steric hindrances normally imposed on the tyrosine kinase activity,
resulting in activation. Site-directed mutagenesis  or deletion analysis in the
TKD shows that it is involved in EGFR dimerization, auto- and transphosphorylation
and also activation of the signaling cascades, all of which have been exploited
in the development of small molecule tyrosine kinase inhibitors (smTKIs) for targeting EGFR in various cancer types
including breast cancer.(4) The Activation Loop, Active Site, and C-Terminal Tail Regions of EGFR
The activation loop of the tyrosine
kinase of EGFR is quite distinct from other receptors harboring tyrosine
kinases. Namely, whereas most receptor TKDs require phosphorylation for
tyrosine kinase activation, this does not appear to be the case in EGFR. For
example, the phosphorylation of Tyr845 has little affect on the EGFR kinase activity [3], possibly due to a
conformational arrangement that directs the activation loop away from the
active site rendering it refractory to the state of phosphorylation of the
receptor tyrosine kinase. Activation of the EGFR tyrosine kinase phosphorylates
numerous targets, including itself (autophosphorylation), a different EGFR (homodimerization), HER-2/neu of the ErbB gene family (heterodimerization), and nonreceptor substrates such
as Grb2/SOS, STATs, PLC, and/or PI3K, which in turn initiate the
signaling cascades of MAPK/ERK, STAT,
PIP2, and AKT,
respectively. Not surprisingly, therefore, mutations in this region can cause a
substantial decrease in kinase
activity, an outcome considered desirable in cancer therapy and may underlie
the therapeutic efficacy of smTKIs. By binding to the TKD of EGFR, smTKIs may
act by sterically interfering with the binding of both the substrate and ATP
necessary for phosphorylation, resulting in an overall decreased signaling
activity of the EGFR.
Lastly, it is important to mention that the tyrosine
kinase activity of EGFR is tightly regulated via its own internal regulatory
region located at the C-terminal tail of the structure, which involves the
tyrosine residue cluster with the potential of being transphosphorylated during
EGFR-dimerization. It is noteworthy that EGFR dimerization induces
phosphorylation of several tyrosine residues including Tyr1069 Tyr1092 Tyr1110 Tyr1116 Tyr1172 Tyr1197,
creating docking sites for the recruitment of other adaptor molecules and
signaling proteins. These attributes suggest that the tyrosine-rich C-terminal
tail is a phosphorylable, mobile structure connected to a relatively stationary
TKD.
In summary, the EGFR may be divided into two
functional substructures. The first one consists of the extracellular
ectodomain responsible for ligand binding, dimerization, and the initiation of
signal transduction. The ectodomain has been the thematic target of anti-EGFR
therapy, vis-à-vis, development of monoclonal antibody directed at the ligand
binding region, which inactivates EGFR through competitive inhibition of ligand
binding, as well as by inducing overall downregulation of EGFR through
increased receptor internalization. Examples include the monoclonal antibodies like Cetuximab
and Trastuzumab, which play an extremely critical role in anti-EGFR
therapy. Currently, both these two drugs and Panitumumab are the only anti-EGFR
monoclonal antibodies approved by the FDA for use in the clinic. The
second major functional substructure of EGFR is the tyrosine kinase domain
located on the intracellular side of the plasma membrane. This domain plays a
key role in the activation of signaling cascades involved in cell
proliferation, division, and differentiation; therefore, inhibition of the
tyrosine kinase enzymatic activity of EGFR using small molecule TKIs is a
clinically relevant treatment option for breast cancer patients.


## 2.2. Breast Cancer and the Signaling Mechanism of EGFR

As a member of the ErbB receptor family, the EGFR
plays important roles in cell signaling, proliferation, differentiation, and
apoptosis. Signaling is initiated by binding of ligands to the extracellular
domain of the EGFR. Six well-characterized ligands of EGFR have been
identified, respectively, EGF, transforming growth factor-α (TGFα), amphiregulin, heparin binding EGF-like growth
factor, betacellulin, and epiregulin. Ligand binding induces conformational
change resulting in heterodimerization and the activation of the major
signaling pathways seen in Figure 1.


### 2.2.1. Statistics and Etiology of Breast Carcinogenesis

Breast cancer is the
most common cancer and a major cause of morbidity and premature loss of life in
women worldwide, accounting for approximately 7% of all cancer-related deaths
[12]. The highest rates of breast cancer in the world are seen in the United
States, where approximately 1 out of every 8 women will develop invasive breast
cancer, which is responsible for almost 3% of all deaths in American women
[13]. Given the grim statistics, the need for more sensitive and reliable
detection methods is obvious. Equally urgent are treatment modalities that are
modest in cost, easily compliant, effective, have low to no toxicities, and
capable of targeting the multifaceted and heterogeneous nature of breast
carcinogenesis.Currently, there is still
lack of understanding of the natural
history of breast cancer. It had been hypothesized that lobular carcinoma in situ (LCIS) represented a precursor lesion of invasive cancer, and, based on this,
mastectomy was initially recommended [14]. Later studies have shown that the
risk of subsequent breast cancer is bilateral. Moreover, it became evident that
LCIS is not a premalignant lesion, but rather a marker that identifies women at
an increased risk for subsequent development of invasive breast cancer, with
the risk remaining elevated even beyond two decades. In a large prospective
study from the National Surgical Adjuvant Breast and Bowel Project involving a
5-year follow-up of 182 women with LCIS managed with excisional biopsy alone,
eight women developed ipsilateral breast tumors (four
with invasive tumors), and three women developed contralateral
breast tumors (two with invasive tumors) [15]. Therefore, it remains unclear whether or not LCIS progresses to ductal
carcinoma in situ (DCIS) during breast carcinogenesis. On the other hand, DCIS is a bona fide precursor
for invasive ductal carcinoma and lacks estrogen receptor (ER) expression. Furthermore, DCIS frequently overexpresses mutated p53, HER-2/neu,
and EGFR, all of which
show some clinical correlation with resistance
to hormone therapy and increased risk for the development of invasive,
metastatic breast cancer. Patients with ER(+ve)/PR(+ve) disease usually respond more favorably to
hormonal therapy (as compared to individuals with
ER(−ve)/PR(−ve) status),
presumably in part due to the overexpression of HER-2 and EGFR in ER(−ve) cells that provide “acquired growth stimulation
autonomy.” These findings suggest that strategies cotargeting HER-2 and EGFR
expression or their functions might have therapeutic and preventive potentials
particularly in ER(−ve) breast carcinoma
cases.


### 2.2.2. Expression/Function of HER-2/EGFR and Signaling in Breast Carcinogenesis

The EGFR gene is frequently altered by gene amplification or overexpression at the mRNA and
protein levels in sporadic breast cancer cases. Numerous polypeptide ligands
sharing an EGF-like motif have been identified and shown to be capable of
inducing EGFR dimerization with different kinetics, eliciting signals of
variable durations, and coupled signal transduction to specific sets of
cytoplasmic proteins. In principle, therefore, this “ligand-initiated receptor-mediated signaling-executed”
molecular relay system might generate a large combinatorial set of biological
readouts with enormous potential for diversification, fine tuning, and
stringent control of cellular functions and responses. Of note, the HER-2/EGFR
has been proposed to act as a master regulator of a signaling network that
drives breast carcinoma epithelial cell proliferation; HER-2 gene amplification
was observed in 92% of breast cancer specimens and overexpression of HER-2 at
the mRNA, and protein levels have been correlated with cancer virulence, resistance to therapy, and poor prognosis. As discussed,
each member of the EGFR gene family has a multifunction structural organization
comprised of an extracellular ligand-binding/interacting domain connected by a
transmembrane span to an intracellular kinase domain. In an uninduced state,
the EGFR is organized such that the autoinhibitory loops flanking the kinase
active site sterically inhibit
it from binding substrates. Binding to ligands induces EGFR homo- or heterodimerization
concomitant with its autoactivation by a transphosphorylation mechanism
involving specific tyrosine residues located in the intracellular domain. In
turn, phosphorylated EGFR undergoes conformational changes that create
additional docking sites for adaptor proteins, kinases, and intracellular
messengers. Therefore, a tightly regulated, dynamic equilibrium presumably
exists between inhibited, activated, monomeric, and dimeric EGFR in order for
proper cell signaling to ensue. If any one of these mechanisms goes awry, the
results can be fatal to the cell and often can be fatal to the organism. The
same considerations may well contribute to the observed clinical efficacy or
lack thereof in EGFR-targeted therapies.The realization that
HER-2 is a master regulator of a signaling network that drives epithelial cell
proliferation identifies this protein as a target for cancer therapy. When overexpressed,
the HER-2 protein may be constitutively active, that is, signaling from the
receptor occurs by a ligand-independent manner. Under these conditions, growth-promoting
signals may be continuously transmitted into the cells in the absence of
ligand. As a result, multiple intracellular signal transduction pathways become
activated, resulting in unregulated cell growth and, in some instances, oncogenic transformation. Figure1 depicts
some of the signaling pathways elicited in response to ligand binding to EGFR
and either EGFR/EGFR homodimerization or EGFR/HER-2 heterodimerization
reactions. It also demonstrates the cascade of events resulting in the
transmission of signals into the nucleus and subsequent cell proliferation and
gene activation. The intracellular signaling pathways of EGFR and HER-2 are
thought to involve Ras-MAPK, and PI3K-, PKC-, NFκB-mediated pathways. Many
clinical trials have observed a poor clinical outcome and shortened survival
time for women whose breast tumors have HER-2 amplification. An inverse
correlation of ER and HER-2 levels between ER(+ve) and ER(−ve) breast cancer cells has been
demonstrated, which probably accounts for the development of tamoxifen resistance in breast cancer cells.


### 2.2.3. Signaling Cross-Talk and Acquisition of Endocrine Resistance

Multiple
lines of evidence implicate breast cancer development and progression as under
the control of steroid hormones, in particular estrogens, via their interaction
with estrogen receptors (ERs) and cross-talk of ER with receptors including
EGFR [16–18]. The classical mechanism of ER signaling involves binding of
estrogens to intracellular ER, triggering a multitude of events that culminate
in altered transcription of estrogen-responsive genes. In sequence, protein
synthesis occurs resulting in cell proliferation, angiogenesis, breast cancer
growth, progression, and metastasis [19–21]. The ER-induced signaling
mechanism coupled with the fact that well over two thirds of breast cancers
exhibit high expression of ER, have provided the rationale for preventing and
treating breast cancer by estrogen antagonism, highlighted by the discovery of
tamoxifen. By selectively modulating the ER, tamoxifen is considered the
mainstay of estrogen antagonist therapy and among the most effective systemic
treatmentfor women with ER-positive breast cancer at all stages
today [21]. A serious obstacle, however, is intrinsicor acquired
resistance to endocrine agents. Manypatients present with primary (de novo) resistance to endocrinetherapy,
despite high tumor levels of ER, and all patients withadvanced
disease eventually acquire resistance
[22].What underlies the
refractoriness to endocrine therapies? A number of possible explanations may be
considered. For example, in addition to the aforementioned activation of
intracellular ER for transcription, estrogens have also been shown to bind
membrane-associated ER [20]. Evidence also exists on ER activation by a
ligand-independent but growth factor-dependent kinase-mediated mechanism [16].
Important contributing factors for resistance
to endocrine therapy include the levels of both ER and ER coregulatory
proteins, amplified extra- and intracellular signaling from growth
factor-mediatedpathways, as well as cross-talk between the ER
pathwayand other growth factor and kinase networks [16–18].
Other mechanisms may involve amplification and/or mutations of key proteins
involved in cross-talk, as well induction of promiscuity and/or antagonism to
therapeutic agents through mutational and posttranslational modification events
[21]. It is possible that aberrations and dysfunctions in these and other
mechanisms may occur with increasing frequency during the development of the
endocrine-resistant phenotype.
Delineation of the interplay between the estrogens, ER, and ER cross-talk with
receptors like EGFR will be an important diagnostic and prognostic objective in
anti-EGFR therapy. Similarly, discovery and development of novel agents that
can reverse resistance by targeting
the ER and its downstream signaling events, or by selective modulation of the
ER:EGFR cross-talk might improve therapeutic response rates.In summary, identifying
the factors and pathways responsible for endocrine resistance
and defining ways to overcome it are research gaps in need of further study and
will remain important diagnostic and therapeutic challenges in the continuing
war to better manage and treat breast cancer.


### 2.2.4. Breast Cancer Treatment Using Multitarget Strategy Related to HER-2 Signaling

The amplification of the HER-2 gene
and overexpression of the HER-2 protein is frequently observed (10–40%) in human
breast cancer patients [23] and has been suggested to associate with tumor
aggressiveness, prognosis, and responsiveness to hormonal and cytotoxic agents.
These observations suggest that HER-2 is an appropriate target for
tumor-specific therapies, some of which are listed as follows.(1)
A humanized monoclonal antibody against HER-2, rhuMAbHER-2 (Trastuzumab),
is already approved for clinical use in the treatment of patients with
metastatic breast cancer. Some forms of HER-2 overexpressing breast tumors can
be successfully treated using antireceptor monoclonal antibodies,for example, Herceptin. However, because
multiple proteins are involved in growth-signaling pathways, development of a
uniformly active therapy may be strategically challenging. Herceptin inhibited
the P13K-dependent pathway, and not the MAPK pathway. Also, blockade of HER-2
function alone without the interception of the committed, associated downstream
events may restrict the effectiveness of therapeutic interventions.(2)
Tyrosine kinase inhibitors, such as emodin, which block HER-2 phosphorylation
and its intracellular signaling.(3)
Heat shock protein Hsp90-associated signal inhibitors, which induce degradation
of tyrosine kinase receptors, such as HER-2.


## 2.2.1. Statistics and Etiology of Breast Carcinogenesis

Breast cancer is the
most common cancer and a major cause of morbidity and premature loss of life in
women worldwide, accounting for approximately 7% of all cancer-related deaths
[12]. The highest rates of breast cancer in the world are seen in the United
States, where approximately 1 out of every 8 women will develop invasive breast
cancer, which is responsible for almost 3% of all deaths in American women
[13]. Given the grim statistics, the need for more sensitive and reliable
detection methods is obvious. Equally urgent are treatment modalities that are
modest in cost, easily compliant, effective, have low to no toxicities, and
capable of targeting the multifaceted and heterogeneous nature of breast
carcinogenesis.Currently, there is still
lack of understanding of the natural
history of breast cancer. It had been hypothesized that lobular carcinoma in situ (LCIS) represented a precursor lesion of invasive cancer, and, based on this,
mastectomy was initially recommended [14]. Later studies have shown that the
risk of subsequent breast cancer is bilateral. Moreover, it became evident that
LCIS is not a premalignant lesion, but rather a marker that identifies women at
an increased risk for subsequent development of invasive breast cancer, with
the risk remaining elevated even beyond two decades. In a large prospective
study from the National Surgical Adjuvant Breast and Bowel Project involving a
5-year follow-up of 182 women with LCIS managed with excisional biopsy alone,
eight women developed ipsilateral breast tumors (four
with invasive tumors), and three women developed contralateral
breast tumors (two with invasive tumors) [15]. Therefore, it remains unclear whether or not LCIS progresses to ductal
carcinoma in situ (DCIS) during breast carcinogenesis. On the other hand, DCIS is a bona fide precursor
for invasive ductal carcinoma and lacks estrogen receptor (ER) expression. Furthermore, DCIS frequently overexpresses mutated p53, HER-2/neu,
and EGFR, all of which
show some clinical correlation with resistance
to hormone therapy and increased risk for the development of invasive,
metastatic breast cancer. Patients with ER(+ve)/PR(+ve) disease usually respond more favorably to
hormonal therapy (as compared to individuals with
ER(−ve)/PR(−ve) status),
presumably in part due to the overexpression of HER-2 and EGFR in ER(−ve) cells that provide “acquired growth stimulation
autonomy.” These findings suggest that strategies cotargeting HER-2 and EGFR
expression or their functions might have therapeutic and preventive potentials
particularly in ER(−ve) breast carcinoma
cases.


## 2.2.2. Expression/Function of HER-2/EGFR and Signaling in Breast Carcinogenesis

The EGFR gene is frequently altered by gene amplification or overexpression at the mRNA and
protein levels in sporadic breast cancer cases. Numerous polypeptide ligands
sharing an EGF-like motif have been identified and shown to be capable of
inducing EGFR dimerization with different kinetics, eliciting signals of
variable durations, and coupled signal transduction to specific sets of
cytoplasmic proteins. In principle, therefore, this “ligand-initiated receptor-mediated signaling-executed”
molecular relay system might generate a large combinatorial set of biological
readouts with enormous potential for diversification, fine tuning, and
stringent control of cellular functions and responses. Of note, the HER-2/EGFR
has been proposed to act as a master regulator of a signaling network that
drives breast carcinoma epithelial cell proliferation; HER-2 gene amplification
was observed in 92% of breast cancer specimens and overexpression of HER-2 at
the mRNA, and protein levels have been correlated with cancer virulence, resistance to therapy, and poor prognosis. As discussed,
each member of the EGFR gene family has a multifunction structural organization
comprised of an extracellular ligand-binding/interacting domain connected by a
transmembrane span to an intracellular kinase domain. In an uninduced state,
the EGFR is organized such that the autoinhibitory loops flanking the kinase
active site sterically inhibit
it from binding substrates. Binding to ligands induces EGFR homo- or heterodimerization
concomitant with its autoactivation by a transphosphorylation mechanism
involving specific tyrosine residues located in the intracellular domain. In
turn, phosphorylated EGFR undergoes conformational changes that create
additional docking sites for adaptor proteins, kinases, and intracellular
messengers. Therefore, a tightly regulated, dynamic equilibrium presumably
exists between inhibited, activated, monomeric, and dimeric EGFR in order for
proper cell signaling to ensue. If any one of these mechanisms goes awry, the
results can be fatal to the cell and often can be fatal to the organism. The
same considerations may well contribute to the observed clinical efficacy or
lack thereof in EGFR-targeted therapies.The realization that
HER-2 is a master regulator of a signaling network that drives epithelial cell
proliferation identifies this protein as a target for cancer therapy. When overexpressed,
the HER-2 protein may be constitutively active, that is, signaling from the
receptor occurs by a ligand-independent manner. Under these conditions, growth-promoting
signals may be continuously transmitted into the cells in the absence of
ligand. As a result, multiple intracellular signal transduction pathways become
activated, resulting in unregulated cell growth and, in some instances, oncogenic transformation. Figure1 depicts
some of the signaling pathways elicited in response to ligand binding to EGFR
and either EGFR/EGFR homodimerization or EGFR/HER-2 heterodimerization
reactions. It also demonstrates the cascade of events resulting in the
transmission of signals into the nucleus and subsequent cell proliferation and
gene activation. The intracellular signaling pathways of EGFR and HER-2 are
thought to involve Ras-MAPK, and PI3K-, PKC-, NFκB-mediated pathways. Many
clinical trials have observed a poor clinical outcome and shortened survival
time for women whose breast tumors have HER-2 amplification. An inverse
correlation of ER and HER-2 levels between ER(+ve) and ER(−ve) breast cancer cells has been
demonstrated, which probably accounts for the development of tamoxifen resistance in breast cancer cells.


## 2.2.3. Signaling Cross-Talk and Acquisition of Endocrine Resistance

Multiple
lines of evidence implicate breast cancer development and progression as under
the control of steroid hormones, in particular estrogens, via their interaction
with estrogen receptors (ERs) and cross-talk of ER with receptors including
EGFR [16–18]. The classical mechanism of ER signaling involves binding of
estrogens to intracellular ER, triggering a multitude of events that culminate
in altered transcription of estrogen-responsive genes. In sequence, protein
synthesis occurs resulting in cell proliferation, angiogenesis, breast cancer
growth, progression, and metastasis [19–21]. The ER-induced signaling
mechanism coupled with the fact that well over two thirds of breast cancers
exhibit high expression of ER, have provided the rationale for preventing and
treating breast cancer by estrogen antagonism, highlighted by the discovery of
tamoxifen. By selectively modulating the ER, tamoxifen is considered the
mainstay of estrogen antagonist therapy and among the most effective systemic
treatmentfor women with ER-positive breast cancer at all stages
today [21]. A serious obstacle, however, is intrinsicor acquired
resistance to endocrine agents. Manypatients present with primary (de novo) resistance to endocrinetherapy,
despite high tumor levels of ER, and all patients withadvanced
disease eventually acquire resistance
[22].What underlies the
refractoriness to endocrine therapies? A number of possible explanations may be
considered. For example, in addition to the aforementioned activation of
intracellular ER for transcription, estrogens have also been shown to bind
membrane-associated ER [20]. Evidence also exists on ER activation by a
ligand-independent but growth factor-dependent kinase-mediated mechanism [16].
Important contributing factors for resistance
to endocrine therapy include the levels of both ER and ER coregulatory
proteins, amplified extra- and intracellular signaling from growth
factor-mediatedpathways, as well as cross-talk between the ER
pathwayand other growth factor and kinase networks [16–18].
Other mechanisms may involve amplification and/or mutations of key proteins
involved in cross-talk, as well induction of promiscuity and/or antagonism to
therapeutic agents through mutational and posttranslational modification events
[21]. It is possible that aberrations and dysfunctions in these and other
mechanisms may occur with increasing frequency during the development of the
endocrine-resistant phenotype.
Delineation of the interplay between the estrogens, ER, and ER cross-talk with
receptors like EGFR will be an important diagnostic and prognostic objective in
anti-EGFR therapy. Similarly, discovery and development of novel agents that
can reverse resistance by targeting
the ER and its downstream signaling events, or by selective modulation of the
ER:EGFR cross-talk might improve therapeutic response rates.In summary, identifying
the factors and pathways responsible for endocrine resistance
and defining ways to overcome it are research gaps in need of further study and
will remain important diagnostic and therapeutic challenges in the continuing
war to better manage and treat breast cancer.


## 2.2.4. Breast Cancer Treatment Using Multitarget Strategy Related to HER-2 Signaling

The amplification of the HER-2 gene
and overexpression of the HER-2 protein is frequently observed (10–40%) in human
breast cancer patients [23] and has been suggested to associate with tumor
aggressiveness, prognosis, and responsiveness to hormonal and cytotoxic agents.
These observations suggest that HER-2 is an appropriate target for
tumor-specific therapies, some of which are listed as follows.(1)
A humanized monoclonal antibody against HER-2, rhuMAbHER-2 (Trastuzumab),
is already approved for clinical use in the treatment of patients with
metastatic breast cancer. Some forms of HER-2 overexpressing breast tumors can
be successfully treated using antireceptor monoclonal antibodies,for example, Herceptin. However, because
multiple proteins are involved in growth-signaling pathways, development of a
uniformly active therapy may be strategically challenging. Herceptin inhibited
the P13K-dependent pathway, and not the MAPK pathway. Also, blockade of HER-2
function alone without the interception of the committed, associated downstream
events may restrict the effectiveness of therapeutic interventions.(2)
Tyrosine kinase inhibitors, such as emodin, which block HER-2 phosphorylation
and its intracellular signaling.(3)
Heat shock protein Hsp90-associated signal inhibitors, which induce degradation
of tyrosine kinase receptors, such as HER-2.


## 3. Classes of Anti-EGFR Therapy

### 3.1. Advances in the Clinical Efficacy of Anti-EGFR Therapies for Breast Cancer Treatment

The race for a successful breast cancer treatment
intensified during the late 1990s and 2000s, resulting in the development of
innovative anti-EGFR therapies in the last few years including both monoclonal
antibodies (MoAbs) and small molecule
tyrosine kinase inhibitors. To systematically analyze and summarize the
clinical outcome of these anti-EGFR therapies, it is useful to identify and
define key terms used in clinical trials. The relevant key terms can be found
in various tables presented below, as appropriate (see Table1).Table 1
Response criteria and evaluation ratings used in the classification of clinical
efficacy and safety/toxicity scoring of anti-EGFR therapies for solid tumors. General
classification schemes used in review of clinical efficacy and safety, WHO criteria
[24].
General classification schemes used in review of clinical efficacy and safety:Objective
response and tumor response were evaluated by the WHO Criteria [24].Adverse events
(AEs) were assessed at each cycle using the common toxicity criteria (CTC).Cardiac
failure/cardiac toxicity was graded based on the NYHA classification system.The Cardiac Review and Evaluation Committee (CREC) evaluates cardiac dysfunction.Factors for clinical efficacy of treatment:In an intent-to-treat (ITT) population, in order to evaluate the overall response rate of the
individual “patient-drug interaction.”Overall response (OR): complete response (CR) + partial response (PR)Clinical benefit (CB): complete response (CR) + partial response (PR) + stable disease (SD)≥ 6 monthsTime-to-disease-progression (TTP): the time from randomization (randomized initiation of drug/therapy.Treatment regimen to be tested) to “disease-progression” or death (whichever event occurs first).


### 3.2. Monoclonal Antibodies

#### 3.2.1. Cetuximab

Cetuximab is
the most commonly used anti-EGFR therapeutic agent for the treatment of solid
tumors. Originally developed for treating colorectal cancer (primary: CRC &
metastatic: mCRC) and squamous cell carcinoma of the head and neck
and not yet approved as therapy for breast cancer, Cetuximab does provide an
excellent model for the development of new MoAbs that may one day be used in
breast cancer therapy. As a humanized mouse MoAb similar to others currently in
development: for example, EMD72000 (Matuzumab) and hR3 (Nimotuzumab), Cetuximab differs
from fully humanized MoAbs like Panitumumab, which have a lower incidence of
adverse events (AEs) (e.g., rash,
diarrhea) [25]. In addition, although Panitumumab blocks ligand-binding to EGFR
and causes receptor internalization like humanized Cetuximab, it does not induce
degradation of the receptors [25].


#### 3.2.2. Trastuzumab

Trastuzumab is
an anti-HER-2 receptor humanized MoAb that has shown significant clinical
benefits for the treatment of HER-2/neu(+ve) metastatic breast cancer as a single agent [26]. Phase II study investigated
the clinical efficacy and safety of Trastuzumab monotherapy given as first-line
treatment once every 3 weeks in woman with HER-2(+ve) metastatic breast cancer (MBC). In 105
patients receiving five cycles of therapy, the overall response rate was 19%
and the clinical benefit rate was 33%. Median time-to-progression was 3.4
months (range, 0.6 to 23.6 months).In general, the monotherapy was well tolerated and no significant AEs
were reported. The most  common treatment-related AEs were only mild-to-moderate
rigors pyrexia, headaches, nausea, and fatigue. Tables 2(a) and 2(b) show the
clinical efficacy and common AEs of Trastuzumab  monotherapy [27].Table 2
Part A. Response to first-line
3-weekly Trastuzumab monotherapy from Baselga et al. [27]. CR: Complete response;
PR: Partial response; SD: Stable disease; CBR: Clinical benefit rate; PD: Progressive
disease; ORR: Overall response rate; ITT: Intent-to-treat population. Part B. Most
common treatment-related adverse events (n = 105), adapted from Baselga et al.
[27].
(a)
No. of patients(n = 105)*ResponseNo.%CR22PR1817SD5351†CRB (CR + PR + SD > 6 months)3533PD3029ORR2019†*Data missing for 2 patients.†One patient with best response of SD in the main
study period achieved CR in
the 12-month follow-up period. Therefore, in the follow-up
analysis, ORR was 20%.(b)
No. of
patientsAdverse eventNo.%Rigors1918Pyrexia1615Headache1110Nausea1010Fatigue1010Trastuzumab
has also been shown to improve survival rates after chemotherapy, specifically
in the Herceptin Adjuvant (HERA) study [6]. HERA is an international multicenter-randomized
trial comparing 1 or 2 years of Trastuzumab treatment with observation alone
after standard neoadjuvant or
adjuvant chemotherapy in women with HER-2(+ve) node positive or high-risk node negative breast cancer. In an
intention-to-treat analysis of a total of 5102 patients, the unadjusted hazard
ratio for the risk of death with Trastuzumab compared with observation alone
was 0.66 (95% CI 0.47–0.91; P = .0115). Overall, the hazard rates were
lower for Trastuzumab treatment group after 1 year compared to the observation
group [29]. After 1 year of Trastuzumab
treatment, there were 218 disease-free survival events and 59 deaths, whereas
321 disease-free survival events and 90 deaths occurred in the control group.
Patients with one or more grade 3 or 4 AEs were 11%, however, there were minimal cardiac AEs
in the 1 year Trastuzumab group with no reported deaths related to cardiac
failures. Therefore, one-year treatment of Trastuzumab after adjuvant
chemotherapy has significant overall survival rates and minimal AEs [29].Trastuzumab in
combination with chemotherapeutic agents, specifically paclitaxel and
anthracycline (Doxorubicin), has shown
significant increases in response rates and disease-free progression (See Table3) [27–29]. The combination prolonged the median time to disease progression
from 4.6 to 7.4 months, increased the overall response rate from 32 to 50%,
extended duration of response from 6.1 to 9.1 months, and improved 1-year
survival times from 68 to 79% compared with chemotherapy alone. The clinical
efficacy of Trastuzumab alone and in combination with other chemotherapy
options is shown in Table 3. The probability of survival was shown to increase
by 25% for 25.4 months with Trastuzumab-plus-chemotherapy
compared to just 20.3 months for chemotherapy alone. Therefore, it appears that
Trastuzumab may sensitize cancer cells to other forms of chemotherapy. AEs were
expectedly seen as in other chemotherapy-treated patients with MBC. Thus, the
combination of Trastuzumab with chemotherapy (anthracycline
or paclitaxel) was active for the treatment of patients with
HER-2(+ve) MBC who had not been
previously treated for metastatic disease. As expected, the clinical benefits
from the treatment with Trastuzumab only apply to HER-2/neu(+ve) breast cancer patients, future studies should be designed using new targeted
patient population.Table 3
Efficacy of Trastuzumab when given in combination with chemotherapy in
metastatic breast cancer from Slamon et al. [28].
Tratuzumab +   AC
(n = 143)AC alone
(n = 138)Trastuzumab +
paclitaxel   (n = 92)Paclitaxel alone   (n = 96)Trastuzumab +   chemotherapy
(n = 235)Chemotherapy alone
(n = 234)Median TTP (months)7.86.16.937.44.6(P = .0004)(P = .0001)(P = .0001)Response rate (%)564241175032(P = .0197)(P = .0002)(P < .0001)Median duration of response (months)9.16.710.54.59.16.1(P = .0047)(P = .00124)(P = .0002)Median TTF (months)7.25.65.82.96.94.5(P = .0014)(P = .0001)(P = .0001)1-year survival (%)837272607968(P = .0415)(P = .0975)(P = .008)Median survival (months)26.822.822.818.425.420.3(P = .025)AC: Anthracycline; TTP: Time to disease
progression; TTF: Time-to-treatment failure.


### 3.3. Tyrosine Kinase Inhibitors

#### 3.3.1. Lapatinib

Lapatinib is a
member of the orally active small molecules that reversibly inhibit both ErbB1
and ErbB2 tyrosine kinases, which consequently leads to the downregulation of
both the MAPK and PI3K signaling cascades responsible for cell proliferation
and survival, respectively. As a dual kinase inhibitor, Lapatinib has shown
activity in a number of different metastatic and advanced tumor cell lines as
well as xenografts and has recently shown positive results in clinical testing
as well (see Figure3 and Tables4(a) and4(b)). Inhibition of ErbB1/EGFR alone using Gefitinib and Erlotinib,
examples of anti-ErbB1 smTKIs, has shown mixed clinical efficacy results for MBC [31]. Recent
studies have demonstrated that it may be advantageous to inhibit ErbB1 and
ErbB2 simultaneously in those patients overexpressing the ErbB2/HER-2/neu gene,
which constitutes approximately 25% of all cases of primary breast cancer [32–35]. Interestingly, although both Lapatinib and Erlotinib bind the ATP-binding
site of EGFR, only Lapatinib displays the unique dual kinase inhibitory
activity. The molecular underpinning for the observed differences awaits
further research in the future [36].Table 4
Part A. Patient response rate:
Lapatinib dose cohort comparison adapted from Gomez et al. [30]. Stable disease
patients who had a best response of stable disease (i.e., stable disease documented for a
minimum of 7 weeks). Clinical benefit response rates
include only patients with a best response of CR, PR, or stable disease for at
least 24 weeks. Disease status was assessed by an independent panel using response
evaluation. Criteria in solid tumors (see
Section3.3). CR: Complete response; PR: Partial response. Part B. Patients with drug-related
adverse events that occurred in >10% of patients receiving Lapatinib, Adapted
from Gomez et al. [30].
(a)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Patient responseNo.%No.%No.%Best responseCR000000PR152218263324Stable
disease405831457151Progressive
disease81216232417Unknown6946107Response rate: CR or PR, %21.726.123.995% CI12.7 to 33.316.3 to 38.117.1 to 31.9Odds ratio0.895% CI0.3 to 1.9P.691(b)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Adverse event*No.%No.%No.%Diarrhea243525364936Grade
1-2233322324533Grade 3113443Rash192918263727Grade
1-2192917253626Grade 3001111Pruritus142011162518Grade
1-2142011162518Grade 3000000Nausea913571410Grade
1-291346139Grade 3001111*No grade 4 adverse events occurred for these conditions.Molecular and crystal structures of EGFR
inhibitor Lapatinib and Lapatinib bound and complexed to EGFR
ATP-binding pocket, respectively. (a) Molecular
structure of Lapatinib (CID208908), an EGFR-ErbB2 inhibitor. (b) Overlay
of EGFR in the Lapatinib and Erlotinib complexes. EGFR in the Lapatinib and Erlotinib structures
is shown as red and green ribbons, respectively. Lapatinib is shown as a yellow
space-filling model. The two proteins were overlaid based on residues in the
COOH-terminal domain of the kinase. The COOH-terminal in both structures is CT.
Disordered residues in the COOH-terminal tail of EGFR are indicated by a dashed
line. The figure was prepared using QUANTA (Accelrys), adapted from Wood et al.
[36].
(a)(b)Clinical
efficacy and safety of Lapatinib as a monotherapy has been recently tested for
HER-2-amplified locally advanced cases or MBC [30]. In a total of 138 patients
treated with Lapatinib for a median of 17.6 weeks, the overall response rate
was 24% and the clinical benefit was 31%. The median time to response was 7.9
weeks, and the progression-free survival rates at 4 to 6 months were 63% and
43%, respectively. Response rates and common AEs are reported in Table 4. The most common AEs were
diarrhea, rash, pruritus, and nausea, which were primarily grade 1 or 2 toxicities.
This study supports further use of Lapatinib in first-line and early-stage
HER-2-overexpressing breast cancer patients [30].Combination therapy involving
Lapatinib has had
mixed results as of 2008. For example, despite the fact that Lapatinib combination
therapy with Capecitabine has shown success in treatment for HER-2(+ve) advanced breast cancer treatment, a
subpopulation of patients often reported occurrences of grade 4 diarrhea, as
well as fatigue, headache, and dizziness [40]. The same study eventually
reported a discontinuation of treatment (of combined
Lapatinib-plus-Capecitabine) due to increased occurrence of AEs in 22 women in the combination-therapy group
(13%) [40]. However, it was also
reported that 18 women in the monotherapy group (12%),
also experienced this high frequency of AEs, which appears to be inconsistent
with the safety reports for Lapatinib in a number of other sources, which
report no reports of any drug-related grade 4 AEs Table 5 [37, 38, 40].Table 5
Clinical efficacy of Trastuzumab and Lapatinib as monotherapy agents for metastatic
breast cancer [26, 27, 30, 39].
StudyNo. of patientsInitial and following doseOR (%)Median TOP and range (months)TrastuzumabBaselga et al. [27]1058 mg/kg, 6 mg/kg triweekly193.4 (range 0.6–23.6)Cobleigh et al. [26]2224 mg/kg, 2 mg/kg weekly153.1 (range 0–≥28)Vogel et al. [39]1144 mg/kg, 2 mg/kg weekly263.8 (range 3.3–5.3)Or 8 mg/kg, 4 mg/kg weeklyLapatinibGomez et al. [30]691500 mg once daily244.4 (range 0.5–23)Or 500 mg twice dailyOR: Overall response rate; TOP: Time to progression.To date, most lapatinib
therapies are still in progress and currently being evaluated.Furthermore,
regarding Phase I safety reports for Lapatinib, there were also no reports of
drug-related interstitial pneumonitis or cardiac dysfunction that was normally
found to be associated with other forms of ErbB-targeted therapies [37, 38].
There was still a need for further investigations regarding the clinical
efficacy, safety, and pharmacokinetics of Lapatinib, and thus these were goals
for the Phase II/III clinical
trials for Lapatinib. The most commonly reported AEs were diarrhea (42%) and rash (31%);
diarrhea incidence increased with increasing dose, whereas rash incidence had
no correlation with dose regimen [37]. Lapatinib is well tolerated in doses from 500–1600 mg once
daily [37].Another
study from 2006 investigated the dual kinase inhibitor activity of Lapatinib in
HER-2-overexpressing breast cancer cells as well as responses of a panel of 31
characterized human breast cancer cell lines to treatment by Trastuzumab,
including the use of Trastuzumab-conditioned HER-2(+ve) cell lines [4]. These
studies demonstrated four key observations associated with Lapatinib treatment
in breast cancer. First, they documented that the anti-proliferative effects of
Lapatinib were in fact concentration dependent and were seen in all breast
cancer cell lines. Second, they also reported a range of half-maximal
inhibitory concentrations for Lapatinib (IC50),
however, the study demonstrated a significant amount of variation among these
values: IC50 = 0.010–18.6 μmol/L. Third, these preliminary data
were also representative of long-termin vivo Lapatinib treatment regiments
for breast cancer; this was ascertained using a 77 consecutive-day Lapatinib
treatment schedule in which there was a significant reduction in the volume of
human breast cancer xenografts in athymic mice compared with untreated controls
[4]. The reduction in tumor volume demonstrated in the aforementioned clinical
study with respect to Lapatinib is consistent with the results obtained in the laboratory
setting as well (see Figure4) [37, 38].
Lastly, they examined the synergistic effects of a combinatorial therapy of
Lapatinib-plus-Trastuzumabfor
which their results have indeed provided the preliminary data necessary to
support the rationale for continuing research regarding the potential of
Lapatinib as a combination anti-EGFR therapy with Trastuzumab in
HER-2-overexpressing breast cancer and in patients with clinical resistance to Trastuzumab [4]. Thus, this review, in
agreement with several earlier reports, supports further investigation of the
benefits of Lapatinib as a first-line treatment regimen for early-stage
HER-2-overexpressing breast cancer cell lines as well as its use in the
treatment of both metastatic and locally advanced breast cancer cases [40].Immunohistochemical staining demonstrating the
clinical efficacy of Lapatinib. Figure4 identifies inhibition of activated,
phosphorylated ErbB2/HER-2/neu (p-ErbB2) in a breast cancer patient responding to Lapatinib treatment. (a) Shows a
dermal-lymphatic invasion (magenta) that
is consistent with recurrent inflammatory breast cancer. (b) and (c) Show further
immunohistochemical staining for p-ErbB2 performed on tumor biopsy samples
obtained from patient X on days 0 (4B) and 21 (4C) of Lapatinib therapy; note
the change in positive staining (brownish-yellow).
There is a significant decrease in the activation of p-ErbB2 in response to
Lapatinib [37, 38].
(a)(b)(c)


#### 3.3.2. Erlotinib

Erlotinib
treatment is most commonly found in combination with other chemotherapeutic
agents, including Capecitabine and Docetaxel. A study researched the additive
efficacy of Erlotinib with Capecitabine and Docetaxel [41]. The combined treatment was administered every 3 weeks, with
a total of 24 women with MBC; the overall response rate was 67%. The most
common treatment-related AEs were skin toxicities and diarrhea. The severe AEs
were relatively low, but as the Capecitabine/Docetaxel doses were increased,
the rate of grade 3 events also increased. The tolerability of the regimen has
been measured and the group reported an established dosage of Erlotinib (100 mg/day), Capecitabine (825 mg/m2), and Docetaxel (75 mg/m2) in patients
with MBC [41].


#### 3.3.3. Gefitinib

Gefitinib has oncebeen approved by FDA as
monotherapy for patients with locally advanced or metastatic nonsmall-cell lung
cancer (NSCLC) [45]. However, more
recently, Gefitinib has been used for the treatment of MBC, including a Phase II
study of Gefitinib in combination with Docetaxel as first-line therapy in MBC
[42]. In 41 patients, a response rate of 54% (95%
CI 45–75%), a stable disease response of 14%, and a progressive
disease response of 32% were reported. Grade 3 or 4 toxicities that were
observed included neutropenia (49%),
diarrhea (10%), acne-like rash (5%), and anemia (2%).
Overall, the Gefitinib and Docetaxel combination demonstrated an active and
generally well-tolerated regimen in women with MBC who have not been previously
treated with metastatic disease [42]. Gefitinib seemed very
promising in early clinical phase testing for the treatment of a number of
solid tumors, including NSCLC. However, the FDA recently withdrew Gefitinib
from its list of clinically effective therapies for NSCLC, but is still
currently under critical review in Phase II/III clinical studies for breast cancer: primary, metastatic, and advanced forms.With respect
to ER-HER2/neu cross-talk in ER/HER2/neu(+ve) breast cancer, Gefitinib has demonstrated promising responses [31]. In a
tamoxifen-resistant, HER2-overexpressing
MCF-7 breast cancer cell line, designated MCF-7/HER2-18, Gefitinib pretreatment
was shown to block ER : EGFR receptor cross-talk, reestablish corepressor
complexes with tamoxifen-bound ER on target gene promoters, eliminate tamoxifen
agonistic effects, and restore tamoxifen antitumor activity both in vitro and in vivo [23].


### 3.4. Combinational Therapies

#### 3.4.1. Lapatinib and Anti-ErbB2 Inhibitors: Trastuzumab

The
results of Lapatinib and Trastuzumab monotherapy and combined therapy
approaches for treating breast cancer are  summarized in Tables6 and
7. A
particular combination therapy study involved the comparison of Lapatinib-plus-pAb (where
pAb is a rabbit polyclonal antisera generated by vaccination with a human
ErbB2 fusion protein) and Lapatinib-plus-Trastuzumab [46]. This study showed that Lapatinib-plus-Trastuzumab combination therapy had
enhanced clinical efficacy compared to both Lapatinib and Trastuzumab
monotherapies but had similar efficacy as the secondary combination cocktail of
Lapatinib-plus-pAb [46]. The
Lapatinib-plus-Trastuzumab
combination therapy also showed both a significant downregulation of
survivin, an important
prosurvival/antiapoptosis protein, as well as enhanced apoptosis [46]. These
conclusions along with the information regarding clinical efficacy and AEs in
Table 7 provide sufficient preliminary evidence supporting this synergistic
cooperation seen in this combination therapy. It appears that Lapatinib may in
fact sensitize the cells to further treatment with Trastuzumab thereby
enhancing the individual activity of both drugs.Table 6
Overall response for Trastuzumab, Lapatinib, Erlotinib, and Gefitinib
combination therapies with chemotherapeutic agents [34, 40–43].
StudyNo. of patientsChemotherapyDoseOR (%)TrastuzumabSlamon et al.
[34]143DoxorubicinTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)56Doxorubicin (60 mg/m2)92PaclitaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)41Paclitaxel (175 mg/m2)Marty et al.
[43]186DocetaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)34Docetaxel (100 mg/m2 triweekly)LapatinibGeyer et al.
[40]163CapecitabineLapatinib (1250 mg/day)22*Capecitabine
(2000 mg/m2)ErlotinibTwelves et al.
[41]24Capecitabine,
docetaxelErlotinib (100 mg/day)68Capecitabine
(825 mg/m2)Docetaxel (75 mg/m2)GefitinibCiardiello et
al. [42]41DocetaxelGeftinib (250 mg/day)54Docetaxel (75 mg/m2 or 100 mg/m2)OR: Overall
response rate.*Study was
performed in women with HER2-positive metastatic breast cancer that has progressed
after trastuzumab-based therapy.Table 7
Efficacy end points in intent-to-treat population, adapted from Geyer et al.
[40].
End pointLapatinib plus capecitabineCapecitabine aloneHazard ratioP-value(N = 163)(N = 161)(95% CI)Median time to progression—mo8.44.40.49 (0.34–0.71)<.001†Median progression-free survival—mo8.44.10.47 (0.33–0.67)<.001†Overall response—% (95% CI)22 (16–29)14 (9–21).09‡Complete response—no. (%)1 (<1)0 (0)Partial response—no. (%)35 (21)23 (14)Clinical benefit—no. (%)44 (27)29 (18)Death—no. (%)36 (22)35 (22)End Points are based on evaluation by the independent
review committee under blinded conditions.†The P-value
was calculated with the log-rank test.‡The P-value was calculated with Fisher’s exact test.


#### 3.4.2. Lapatinib and Capecitabine

Lapatinib
combination therapy with Capecitabine has also shown success in treatment for
HER-2(+ve) advanced breast cancer
treatment (see Tables6 and 7) [40].
Patients with HER-2(+ve) MBC who had
progressed after treatment with regimens
that included an anthracycline, a taxene, and Trastuzumab were randomly
assigned to receive either Capecitabine alone versus Capecitabine in
combination with Lapatinib. The hazard ratio for the independently
assessed time to progression was 0.49 (95% CI 0.34 to 0.71; P < .001);
the median time to progression was 8.4 months in the combination therapy group,
whereas 4.4 months in the monotherapy group. The most common AEs were diarrhea, the hand-foot syndrome, nausea,
vomiting, fatigue, and rash, varying from grades 1 to 3. In grade 4, diarrhea,
fatigue, headache, and dizziness were reported. Discontinuation of treatment due to AEs occurred in 22 women in the
combination-therapy group (13%) and in
18 women in the monotherapy group (12%) [40]. However, as previously
indicated, these reports are refuted by other sources, which claim no grade 4
AEs and only minor toxicity reports for Lapatinib monotherapy, but there are no
other reviews specifically regarding further investigation into the
relationship between combinational therapies of Lapatinib-plus-Capecitabine,
if any does in fact exist, positively or negatively correlated with improved
outcome.Therefore, in
concurrence with combinational therapies, a major need clearly exists for
further research to be done in this specific area of anti-EGFR therapy. There
is a significant deficit in the hypotheses and models that can critically
evaluate the data reported thus far on most combinational therapies.


## 3.1. Advances in the Clinical Efficacy of Anti-EGFR Therapies for Breast Cancer Treatment

The race for a successful breast cancer treatment
intensified during the late 1990s and 2000s, resulting in the development of
innovative anti-EGFR therapies in the last few years including both monoclonal
antibodies (MoAbs) and small molecule
tyrosine kinase inhibitors. To systematically analyze and summarize the
clinical outcome of these anti-EGFR therapies, it is useful to identify and
define key terms used in clinical trials. The relevant key terms can be found
in various tables presented below, as appropriate (see Table1).Table 1
Response criteria and evaluation ratings used in the classification of clinical
efficacy and safety/toxicity scoring of anti-EGFR therapies for solid tumors. General
classification schemes used in review of clinical efficacy and safety, WHO criteria
[24].
General classification schemes used in review of clinical efficacy and safety:Objective
response and tumor response were evaluated by the WHO Criteria [24].Adverse events
(AEs) were assessed at each cycle using the common toxicity criteria (CTC).Cardiac
failure/cardiac toxicity was graded based on the NYHA classification system.The Cardiac Review and Evaluation Committee (CREC) evaluates cardiac dysfunction.Factors for clinical efficacy of treatment:In an intent-to-treat (ITT) population, in order to evaluate the overall response rate of the
individual “patient-drug interaction.”Overall response (OR): complete response (CR) + partial response (PR)Clinical benefit (CB): complete response (CR) + partial response (PR) + stable disease (SD)≥ 6 monthsTime-to-disease-progression (TTP): the time from randomization (randomized initiation of drug/therapy.Treatment regimen to be tested) to “disease-progression” or death (whichever event occurs first).


## 3.2. Monoclonal Antibodies

### 3.2.1. Cetuximab

Cetuximab is
the most commonly used anti-EGFR therapeutic agent for the treatment of solid
tumors. Originally developed for treating colorectal cancer (primary: CRC &
metastatic: mCRC) and squamous cell carcinoma of the head and neck
and not yet approved as therapy for breast cancer, Cetuximab does provide an
excellent model for the development of new MoAbs that may one day be used in
breast cancer therapy. As a humanized mouse MoAb similar to others currently in
development: for example, EMD72000 (Matuzumab) and hR3 (Nimotuzumab), Cetuximab differs
from fully humanized MoAbs like Panitumumab, which have a lower incidence of
adverse events (AEs) (e.g., rash,
diarrhea) [25]. In addition, although Panitumumab blocks ligand-binding to EGFR
and causes receptor internalization like humanized Cetuximab, it does not induce
degradation of the receptors [25].


### 3.2.2. Trastuzumab

Trastuzumab is
an anti-HER-2 receptor humanized MoAb that has shown significant clinical
benefits for the treatment of HER-2/neu(+ve) metastatic breast cancer as a single agent [26]. Phase II study investigated
the clinical efficacy and safety of Trastuzumab monotherapy given as first-line
treatment once every 3 weeks in woman with HER-2(+ve) metastatic breast cancer (MBC). In 105
patients receiving five cycles of therapy, the overall response rate was 19%
and the clinical benefit rate was 33%. Median time-to-progression was 3.4
months (range, 0.6 to 23.6 months).In general, the monotherapy was well tolerated and no significant AEs
were reported. The most  common treatment-related AEs were only mild-to-moderate
rigors pyrexia, headaches, nausea, and fatigue. Tables 2(a) and 2(b) show the
clinical efficacy and common AEs of Trastuzumab  monotherapy [27].Table 2
Part A. Response to first-line
3-weekly Trastuzumab monotherapy from Baselga et al. [27]. CR: Complete response;
PR: Partial response; SD: Stable disease; CBR: Clinical benefit rate; PD: Progressive
disease; ORR: Overall response rate; ITT: Intent-to-treat population. Part B. Most
common treatment-related adverse events (n = 105), adapted from Baselga et al.
[27].
(a)
No. of patients(n = 105)*ResponseNo.%CR22PR1817SD5351†CRB (CR + PR + SD > 6 months)3533PD3029ORR2019†*Data missing for 2 patients.†One patient with best response of SD in the main
study period achieved CR in
the 12-month follow-up period. Therefore, in the follow-up
analysis, ORR was 20%.(b)
No. of
patientsAdverse eventNo.%Rigors1918Pyrexia1615Headache1110Nausea1010Fatigue1010Trastuzumab
has also been shown to improve survival rates after chemotherapy, specifically
in the Herceptin Adjuvant (HERA) study [6]. HERA is an international multicenter-randomized
trial comparing 1 or 2 years of Trastuzumab treatment with observation alone
after standard neoadjuvant or
adjuvant chemotherapy in women with HER-2(+ve) node positive or high-risk node negative breast cancer. In an
intention-to-treat analysis of a total of 5102 patients, the unadjusted hazard
ratio for the risk of death with Trastuzumab compared with observation alone
was 0.66 (95% CI 0.47–0.91; P = .0115). Overall, the hazard rates were
lower for Trastuzumab treatment group after 1 year compared to the observation
group [29]. After 1 year of Trastuzumab
treatment, there were 218 disease-free survival events and 59 deaths, whereas
321 disease-free survival events and 90 deaths occurred in the control group.
Patients with one or more grade 3 or 4 AEs were 11%, however, there were minimal cardiac AEs
in the 1 year Trastuzumab group with no reported deaths related to cardiac
failures. Therefore, one-year treatment of Trastuzumab after adjuvant
chemotherapy has significant overall survival rates and minimal AEs [29].Trastuzumab in
combination with chemotherapeutic agents, specifically paclitaxel and
anthracycline (Doxorubicin), has shown
significant increases in response rates and disease-free progression (See Table3) [27–29]. The combination prolonged the median time to disease progression
from 4.6 to 7.4 months, increased the overall response rate from 32 to 50%,
extended duration of response from 6.1 to 9.1 months, and improved 1-year
survival times from 68 to 79% compared with chemotherapy alone. The clinical
efficacy of Trastuzumab alone and in combination with other chemotherapy
options is shown in Table 3. The probability of survival was shown to increase
by 25% for 25.4 months with Trastuzumab-plus-chemotherapy
compared to just 20.3 months for chemotherapy alone. Therefore, it appears that
Trastuzumab may sensitize cancer cells to other forms of chemotherapy. AEs were
expectedly seen as in other chemotherapy-treated patients with MBC. Thus, the
combination of Trastuzumab with chemotherapy (anthracycline
or paclitaxel) was active for the treatment of patients with
HER-2(+ve) MBC who had not been
previously treated for metastatic disease. As expected, the clinical benefits
from the treatment with Trastuzumab only apply to HER-2/neu(+ve) breast cancer patients, future studies should be designed using new targeted
patient population.Table 3
Efficacy of Trastuzumab when given in combination with chemotherapy in
metastatic breast cancer from Slamon et al. [28].
Tratuzumab +   AC
(n = 143)AC alone
(n = 138)Trastuzumab +
paclitaxel   (n = 92)Paclitaxel alone   (n = 96)Trastuzumab +   chemotherapy
(n = 235)Chemotherapy alone
(n = 234)Median TTP (months)7.86.16.937.44.6(P = .0004)(P = .0001)(P = .0001)Response rate (%)564241175032(P = .0197)(P = .0002)(P < .0001)Median duration of response (months)9.16.710.54.59.16.1(P = .0047)(P = .00124)(P = .0002)Median TTF (months)7.25.65.82.96.94.5(P = .0014)(P = .0001)(P = .0001)1-year survival (%)837272607968(P = .0415)(P = .0975)(P = .008)Median survival (months)26.822.822.818.425.420.3(P = .025)AC: Anthracycline; TTP: Time to disease
progression; TTF: Time-to-treatment failure.


## 3.2.1. Cetuximab

Cetuximab is
the most commonly used anti-EGFR therapeutic agent for the treatment of solid
tumors. Originally developed for treating colorectal cancer (primary: CRC &
metastatic: mCRC) and squamous cell carcinoma of the head and neck
and not yet approved as therapy for breast cancer, Cetuximab does provide an
excellent model for the development of new MoAbs that may one day be used in
breast cancer therapy. As a humanized mouse MoAb similar to others currently in
development: for example, EMD72000 (Matuzumab) and hR3 (Nimotuzumab), Cetuximab differs
from fully humanized MoAbs like Panitumumab, which have a lower incidence of
adverse events (AEs) (e.g., rash,
diarrhea) [25]. In addition, although Panitumumab blocks ligand-binding to EGFR
and causes receptor internalization like humanized Cetuximab, it does not induce
degradation of the receptors [25].


## 3.2.2. Trastuzumab

Trastuzumab is
an anti-HER-2 receptor humanized MoAb that has shown significant clinical
benefits for the treatment of HER-2/neu(+ve) metastatic breast cancer as a single agent [26]. Phase II study investigated
the clinical efficacy and safety of Trastuzumab monotherapy given as first-line
treatment once every 3 weeks in woman with HER-2(+ve) metastatic breast cancer (MBC). In 105
patients receiving five cycles of therapy, the overall response rate was 19%
and the clinical benefit rate was 33%. Median time-to-progression was 3.4
months (range, 0.6 to 23.6 months).In general, the monotherapy was well tolerated and no significant AEs
were reported. The most  common treatment-related AEs were only mild-to-moderate
rigors pyrexia, headaches, nausea, and fatigue. Tables 2(a) and 2(b) show the
clinical efficacy and common AEs of Trastuzumab  monotherapy [27].Table 2
Part A. Response to first-line
3-weekly Trastuzumab monotherapy from Baselga et al. [27]. CR: Complete response;
PR: Partial response; SD: Stable disease; CBR: Clinical benefit rate; PD: Progressive
disease; ORR: Overall response rate; ITT: Intent-to-treat population. Part B. Most
common treatment-related adverse events (n = 105), adapted from Baselga et al.
[27].
(a)
No. of patients(n = 105)*ResponseNo.%CR22PR1817SD5351†CRB (CR + PR + SD > 6 months)3533PD3029ORR2019†*Data missing for 2 patients.†One patient with best response of SD in the main
study period achieved CR in
the 12-month follow-up period. Therefore, in the follow-up
analysis, ORR was 20%.(b)
No. of
patientsAdverse eventNo.%Rigors1918Pyrexia1615Headache1110Nausea1010Fatigue1010Trastuzumab
has also been shown to improve survival rates after chemotherapy, specifically
in the Herceptin Adjuvant (HERA) study [6]. HERA is an international multicenter-randomized
trial comparing 1 or 2 years of Trastuzumab treatment with observation alone
after standard neoadjuvant or
adjuvant chemotherapy in women with HER-2(+ve) node positive or high-risk node negative breast cancer. In an
intention-to-treat analysis of a total of 5102 patients, the unadjusted hazard
ratio for the risk of death with Trastuzumab compared with observation alone
was 0.66 (95% CI 0.47–0.91; P = .0115). Overall, the hazard rates were
lower for Trastuzumab treatment group after 1 year compared to the observation
group [29]. After 1 year of Trastuzumab
treatment, there were 218 disease-free survival events and 59 deaths, whereas
321 disease-free survival events and 90 deaths occurred in the control group.
Patients with one or more grade 3 or 4 AEs were 11%, however, there were minimal cardiac AEs
in the 1 year Trastuzumab group with no reported deaths related to cardiac
failures. Therefore, one-year treatment of Trastuzumab after adjuvant
chemotherapy has significant overall survival rates and minimal AEs [29].Trastuzumab in
combination with chemotherapeutic agents, specifically paclitaxel and
anthracycline (Doxorubicin), has shown
significant increases in response rates and disease-free progression (See Table3) [27–29]. The combination prolonged the median time to disease progression
from 4.6 to 7.4 months, increased the overall response rate from 32 to 50%,
extended duration of response from 6.1 to 9.1 months, and improved 1-year
survival times from 68 to 79% compared with chemotherapy alone. The clinical
efficacy of Trastuzumab alone and in combination with other chemotherapy
options is shown in Table 3. The probability of survival was shown to increase
by 25% for 25.4 months with Trastuzumab-plus-chemotherapy
compared to just 20.3 months for chemotherapy alone. Therefore, it appears that
Trastuzumab may sensitize cancer cells to other forms of chemotherapy. AEs were
expectedly seen as in other chemotherapy-treated patients with MBC. Thus, the
combination of Trastuzumab with chemotherapy (anthracycline
or paclitaxel) was active for the treatment of patients with
HER-2(+ve) MBC who had not been
previously treated for metastatic disease. As expected, the clinical benefits
from the treatment with Trastuzumab only apply to HER-2/neu(+ve) breast cancer patients, future studies should be designed using new targeted
patient population.Table 3
Efficacy of Trastuzumab when given in combination with chemotherapy in
metastatic breast cancer from Slamon et al. [28].
Tratuzumab +   AC
(n = 143)AC alone
(n = 138)Trastuzumab +
paclitaxel   (n = 92)Paclitaxel alone   (n = 96)Trastuzumab +   chemotherapy
(n = 235)Chemotherapy alone
(n = 234)Median TTP (months)7.86.16.937.44.6(P = .0004)(P = .0001)(P = .0001)Response rate (%)564241175032(P = .0197)(P = .0002)(P < .0001)Median duration of response (months)9.16.710.54.59.16.1(P = .0047)(P = .00124)(P = .0002)Median TTF (months)7.25.65.82.96.94.5(P = .0014)(P = .0001)(P = .0001)1-year survival (%)837272607968(P = .0415)(P = .0975)(P = .008)Median survival (months)26.822.822.818.425.420.3(P = .025)AC: Anthracycline; TTP: Time to disease
progression; TTF: Time-to-treatment failure.


## 3.3. Tyrosine Kinase Inhibitors

### 3.3.1. Lapatinib

Lapatinib is a
member of the orally active small molecules that reversibly inhibit both ErbB1
and ErbB2 tyrosine kinases, which consequently leads to the downregulation of
both the MAPK and PI3K signaling cascades responsible for cell proliferation
and survival, respectively. As a dual kinase inhibitor, Lapatinib has shown
activity in a number of different metastatic and advanced tumor cell lines as
well as xenografts and has recently shown positive results in clinical testing
as well (see Figure3 and Tables4(a) and4(b)). Inhibition of ErbB1/EGFR alone using Gefitinib and Erlotinib,
examples of anti-ErbB1 smTKIs, has shown mixed clinical efficacy results for MBC [31]. Recent
studies have demonstrated that it may be advantageous to inhibit ErbB1 and
ErbB2 simultaneously in those patients overexpressing the ErbB2/HER-2/neu gene,
which constitutes approximately 25% of all cases of primary breast cancer [32–35]. Interestingly, although both Lapatinib and Erlotinib bind the ATP-binding
site of EGFR, only Lapatinib displays the unique dual kinase inhibitory
activity. The molecular underpinning for the observed differences awaits
further research in the future [36].Table 4
Part A. Patient response rate:
Lapatinib dose cohort comparison adapted from Gomez et al. [30]. Stable disease
patients who had a best response of stable disease (i.e., stable disease documented for a
minimum of 7 weeks). Clinical benefit response rates
include only patients with a best response of CR, PR, or stable disease for at
least 24 weeks. Disease status was assessed by an independent panel using response
evaluation. Criteria in solid tumors (see
Section3.3). CR: Complete response; PR: Partial response. Part B. Patients with drug-related
adverse events that occurred in >10% of patients receiving Lapatinib, Adapted
from Gomez et al. [30].
(a)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Patient responseNo.%No.%No.%Best responseCR000000PR152218263324Stable
disease405831457151Progressive
disease81216232417Unknown6946107Response rate: CR or PR, %21.726.123.995% CI12.7 to 33.316.3 to 38.117.1 to 31.9Odds ratio0.895% CI0.3 to 1.9P.691(b)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Adverse event*No.%No.%No.%Diarrhea243525364936Grade
1-2233322324533Grade 3113443Rash192918263727Grade
1-2192917253626Grade 3001111Pruritus142011162518Grade
1-2142011162518Grade 3000000Nausea913571410Grade
1-291346139Grade 3001111*No grade 4 adverse events occurred for these conditions.Molecular and crystal structures of EGFR
inhibitor Lapatinib and Lapatinib bound and complexed to EGFR
ATP-binding pocket, respectively. (a) Molecular
structure of Lapatinib (CID208908), an EGFR-ErbB2 inhibitor. (b) Overlay
of EGFR in the Lapatinib and Erlotinib complexes. EGFR in the Lapatinib and Erlotinib structures
is shown as red and green ribbons, respectively. Lapatinib is shown as a yellow
space-filling model. The two proteins were overlaid based on residues in the
COOH-terminal domain of the kinase. The COOH-terminal in both structures is CT.
Disordered residues in the COOH-terminal tail of EGFR are indicated by a dashed
line. The figure was prepared using QUANTA (Accelrys), adapted from Wood et al.
[36].
(a)(b)Clinical
efficacy and safety of Lapatinib as a monotherapy has been recently tested for
HER-2-amplified locally advanced cases or MBC [30]. In a total of 138 patients
treated with Lapatinib for a median of 17.6 weeks, the overall response rate
was 24% and the clinical benefit was 31%. The median time to response was 7.9
weeks, and the progression-free survival rates at 4 to 6 months were 63% and
43%, respectively. Response rates and common AEs are reported in Table 4. The most common AEs were
diarrhea, rash, pruritus, and nausea, which were primarily grade 1 or 2 toxicities.
This study supports further use of Lapatinib in first-line and early-stage
HER-2-overexpressing breast cancer patients [30].Combination therapy involving
Lapatinib has had
mixed results as of 2008. For example, despite the fact that Lapatinib combination
therapy with Capecitabine has shown success in treatment for HER-2(+ve) advanced breast cancer treatment, a
subpopulation of patients often reported occurrences of grade 4 diarrhea, as
well as fatigue, headache, and dizziness [40]. The same study eventually
reported a discontinuation of treatment (of combined
Lapatinib-plus-Capecitabine) due to increased occurrence of AEs in 22 women in the combination-therapy group
(13%) [40]. However, it was also
reported that 18 women in the monotherapy group (12%),
also experienced this high frequency of AEs, which appears to be inconsistent
with the safety reports for Lapatinib in a number of other sources, which
report no reports of any drug-related grade 4 AEs Table 5 [37, 38, 40].Table 5
Clinical efficacy of Trastuzumab and Lapatinib as monotherapy agents for metastatic
breast cancer [26, 27, 30, 39].
StudyNo. of patientsInitial and following doseOR (%)Median TOP and range (months)TrastuzumabBaselga et al. [27]1058 mg/kg, 6 mg/kg triweekly193.4 (range 0.6–23.6)Cobleigh et al. [26]2224 mg/kg, 2 mg/kg weekly153.1 (range 0–≥28)Vogel et al. [39]1144 mg/kg, 2 mg/kg weekly263.8 (range 3.3–5.3)Or 8 mg/kg, 4 mg/kg weeklyLapatinibGomez et al. [30]691500 mg once daily244.4 (range 0.5–23)Or 500 mg twice dailyOR: Overall response rate; TOP: Time to progression.To date, most lapatinib
therapies are still in progress and currently being evaluated.Furthermore,
regarding Phase I safety reports for Lapatinib, there were also no reports of
drug-related interstitial pneumonitis or cardiac dysfunction that was normally
found to be associated with other forms of ErbB-targeted therapies [37, 38].
There was still a need for further investigations regarding the clinical
efficacy, safety, and pharmacokinetics of Lapatinib, and thus these were goals
for the Phase II/III clinical
trials for Lapatinib. The most commonly reported AEs were diarrhea (42%) and rash (31%);
diarrhea incidence increased with increasing dose, whereas rash incidence had
no correlation with dose regimen [37]. Lapatinib is well tolerated in doses from 500–1600 mg once
daily [37].Another
study from 2006 investigated the dual kinase inhibitor activity of Lapatinib in
HER-2-overexpressing breast cancer cells as well as responses of a panel of 31
characterized human breast cancer cell lines to treatment by Trastuzumab,
including the use of Trastuzumab-conditioned HER-2(+ve) cell lines [4]. These
studies demonstrated four key observations associated with Lapatinib treatment
in breast cancer. First, they documented that the anti-proliferative effects of
Lapatinib were in fact concentration dependent and were seen in all breast
cancer cell lines. Second, they also reported a range of half-maximal
inhibitory concentrations for Lapatinib (IC50),
however, the study demonstrated a significant amount of variation among these
values: IC50 = 0.010–18.6 μmol/L. Third, these preliminary data
were also representative of long-termin vivo Lapatinib treatment regiments
for breast cancer; this was ascertained using a 77 consecutive-day Lapatinib
treatment schedule in which there was a significant reduction in the volume of
human breast cancer xenografts in athymic mice compared with untreated controls
[4]. The reduction in tumor volume demonstrated in the aforementioned clinical
study with respect to Lapatinib is consistent with the results obtained in the laboratory
setting as well (see Figure4) [37, 38].
Lastly, they examined the synergistic effects of a combinatorial therapy of
Lapatinib-plus-Trastuzumabfor
which their results have indeed provided the preliminary data necessary to
support the rationale for continuing research regarding the potential of
Lapatinib as a combination anti-EGFR therapy with Trastuzumab in
HER-2-overexpressing breast cancer and in patients with clinical resistance to Trastuzumab [4]. Thus, this review, in
agreement with several earlier reports, supports further investigation of the
benefits of Lapatinib as a first-line treatment regimen for early-stage
HER-2-overexpressing breast cancer cell lines as well as its use in the
treatment of both metastatic and locally advanced breast cancer cases [40].Immunohistochemical staining demonstrating the
clinical efficacy of Lapatinib. Figure4 identifies inhibition of activated,
phosphorylated ErbB2/HER-2/neu (p-ErbB2) in a breast cancer patient responding to Lapatinib treatment. (a) Shows a
dermal-lymphatic invasion (magenta) that
is consistent with recurrent inflammatory breast cancer. (b) and (c) Show further
immunohistochemical staining for p-ErbB2 performed on tumor biopsy samples
obtained from patient X on days 0 (4B) and 21 (4C) of Lapatinib therapy; note
the change in positive staining (brownish-yellow).
There is a significant decrease in the activation of p-ErbB2 in response to
Lapatinib [37, 38].
(a)(b)(c)


### 3.3.2. Erlotinib

Erlotinib
treatment is most commonly found in combination with other chemotherapeutic
agents, including Capecitabine and Docetaxel. A study researched the additive
efficacy of Erlotinib with Capecitabine and Docetaxel [41]. The combined treatment was administered every 3 weeks, with
a total of 24 women with MBC; the overall response rate was 67%. The most
common treatment-related AEs were skin toxicities and diarrhea. The severe AEs
were relatively low, but as the Capecitabine/Docetaxel doses were increased,
the rate of grade 3 events also increased. The tolerability of the regimen has
been measured and the group reported an established dosage of Erlotinib (100 mg/day), Capecitabine (825 mg/m2), and Docetaxel (75 mg/m2) in patients
with MBC [41].


### 3.3.3. Gefitinib

Gefitinib has oncebeen approved by FDA as
monotherapy for patients with locally advanced or metastatic nonsmall-cell lung
cancer (NSCLC) [45]. However, more
recently, Gefitinib has been used for the treatment of MBC, including a Phase II
study of Gefitinib in combination with Docetaxel as first-line therapy in MBC
[42]. In 41 patients, a response rate of 54% (95%
CI 45–75%), a stable disease response of 14%, and a progressive
disease response of 32% were reported. Grade 3 or 4 toxicities that were
observed included neutropenia (49%),
diarrhea (10%), acne-like rash (5%), and anemia (2%).
Overall, the Gefitinib and Docetaxel combination demonstrated an active and
generally well-tolerated regimen in women with MBC who have not been previously
treated with metastatic disease [42]. Gefitinib seemed very
promising in early clinical phase testing for the treatment of a number of
solid tumors, including NSCLC. However, the FDA recently withdrew Gefitinib
from its list of clinically effective therapies for NSCLC, but is still
currently under critical review in Phase II/III clinical studies for breast cancer: primary, metastatic, and advanced forms.With respect
to ER-HER2/neu cross-talk in ER/HER2/neu(+ve) breast cancer, Gefitinib has demonstrated promising responses [31]. In a
tamoxifen-resistant, HER2-overexpressing
MCF-7 breast cancer cell line, designated MCF-7/HER2-18, Gefitinib pretreatment
was shown to block ER : EGFR receptor cross-talk, reestablish corepressor
complexes with tamoxifen-bound ER on target gene promoters, eliminate tamoxifen
agonistic effects, and restore tamoxifen antitumor activity both in vitro and in vivo [23].


## 3.3.1. Lapatinib

Lapatinib is a
member of the orally active small molecules that reversibly inhibit both ErbB1
and ErbB2 tyrosine kinases, which consequently leads to the downregulation of
both the MAPK and PI3K signaling cascades responsible for cell proliferation
and survival, respectively. As a dual kinase inhibitor, Lapatinib has shown
activity in a number of different metastatic and advanced tumor cell lines as
well as xenografts and has recently shown positive results in clinical testing
as well (see Figure3 and Tables4(a) and4(b)). Inhibition of ErbB1/EGFR alone using Gefitinib and Erlotinib,
examples of anti-ErbB1 smTKIs, has shown mixed clinical efficacy results for MBC [31]. Recent
studies have demonstrated that it may be advantageous to inhibit ErbB1 and
ErbB2 simultaneously in those patients overexpressing the ErbB2/HER-2/neu gene,
which constitutes approximately 25% of all cases of primary breast cancer [32–35]. Interestingly, although both Lapatinib and Erlotinib bind the ATP-binding
site of EGFR, only Lapatinib displays the unique dual kinase inhibitory
activity. The molecular underpinning for the observed differences awaits
further research in the future [36].Table 4
Part A. Patient response rate:
Lapatinib dose cohort comparison adapted from Gomez et al. [30]. Stable disease
patients who had a best response of stable disease (i.e., stable disease documented for a
minimum of 7 weeks). Clinical benefit response rates
include only patients with a best response of CR, PR, or stable disease for at
least 24 weeks. Disease status was assessed by an independent panel using response
evaluation. Criteria in solid tumors (see
Section3.3). CR: Complete response; PR: Partial response. Part B. Patients with drug-related
adverse events that occurred in >10% of patients receiving Lapatinib, Adapted
from Gomez et al. [30].
(a)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Patient responseNo.%No.%No.%Best responseCR000000PR152218263324Stable
disease405831457151Progressive
disease81216232417Unknown6946107Response rate: CR or PR, %21.726.123.995% CI12.7 to 33.316.3 to 38.117.1 to 31.9Odds ratio0.895% CI0.3 to 1.9P.691(b)
Dosing regimen1500 mg once daily500 mg twice dailyAll patients(n = 69)(n = 69)(N = 138)Adverse event*No.%No.%No.%Diarrhea243525364936Grade
1-2233322324533Grade 3113443Rash192918263727Grade
1-2192917253626Grade 3001111Pruritus142011162518Grade
1-2142011162518Grade 3000000Nausea913571410Grade
1-291346139Grade 3001111*No grade 4 adverse events occurred for these conditions.Molecular and crystal structures of EGFR
inhibitor Lapatinib and Lapatinib bound and complexed to EGFR
ATP-binding pocket, respectively. (a) Molecular
structure of Lapatinib (CID208908), an EGFR-ErbB2 inhibitor. (b) Overlay
of EGFR in the Lapatinib and Erlotinib complexes. EGFR in the Lapatinib and Erlotinib structures
is shown as red and green ribbons, respectively. Lapatinib is shown as a yellow
space-filling model. The two proteins were overlaid based on residues in the
COOH-terminal domain of the kinase. The COOH-terminal in both structures is CT.
Disordered residues in the COOH-terminal tail of EGFR are indicated by a dashed
line. The figure was prepared using QUANTA (Accelrys), adapted from Wood et al.
[36].
(a)(b)Clinical
efficacy and safety of Lapatinib as a monotherapy has been recently tested for
HER-2-amplified locally advanced cases or MBC [30]. In a total of 138 patients
treated with Lapatinib for a median of 17.6 weeks, the overall response rate
was 24% and the clinical benefit was 31%. The median time to response was 7.9
weeks, and the progression-free survival rates at 4 to 6 months were 63% and
43%, respectively. Response rates and common AEs are reported in Table 4. The most common AEs were
diarrhea, rash, pruritus, and nausea, which were primarily grade 1 or 2 toxicities.
This study supports further use of Lapatinib in first-line and early-stage
HER-2-overexpressing breast cancer patients [30].Combination therapy involving
Lapatinib has had
mixed results as of 2008. For example, despite the fact that Lapatinib combination
therapy with Capecitabine has shown success in treatment for HER-2(+ve) advanced breast cancer treatment, a
subpopulation of patients often reported occurrences of grade 4 diarrhea, as
well as fatigue, headache, and dizziness [40]. The same study eventually
reported a discontinuation of treatment (of combined
Lapatinib-plus-Capecitabine) due to increased occurrence of AEs in 22 women in the combination-therapy group
(13%) [40]. However, it was also
reported that 18 women in the monotherapy group (12%),
also experienced this high frequency of AEs, which appears to be inconsistent
with the safety reports for Lapatinib in a number of other sources, which
report no reports of any drug-related grade 4 AEs Table 5 [37, 38, 40].Table 5
Clinical efficacy of Trastuzumab and Lapatinib as monotherapy agents for metastatic
breast cancer [26, 27, 30, 39].
StudyNo. of patientsInitial and following doseOR (%)Median TOP and range (months)TrastuzumabBaselga et al. [27]1058 mg/kg, 6 mg/kg triweekly193.4 (range 0.6–23.6)Cobleigh et al. [26]2224 mg/kg, 2 mg/kg weekly153.1 (range 0–≥28)Vogel et al. [39]1144 mg/kg, 2 mg/kg weekly263.8 (range 3.3–5.3)Or 8 mg/kg, 4 mg/kg weeklyLapatinibGomez et al. [30]691500 mg once daily244.4 (range 0.5–23)Or 500 mg twice dailyOR: Overall response rate; TOP: Time to progression.To date, most lapatinib
therapies are still in progress and currently being evaluated.Furthermore,
regarding Phase I safety reports for Lapatinib, there were also no reports of
drug-related interstitial pneumonitis or cardiac dysfunction that was normally
found to be associated with other forms of ErbB-targeted therapies [37, 38].
There was still a need for further investigations regarding the clinical
efficacy, safety, and pharmacokinetics of Lapatinib, and thus these were goals
for the Phase II/III clinical
trials for Lapatinib. The most commonly reported AEs were diarrhea (42%) and rash (31%);
diarrhea incidence increased with increasing dose, whereas rash incidence had
no correlation with dose regimen [37]. Lapatinib is well tolerated in doses from 500–1600 mg once
daily [37].Another
study from 2006 investigated the dual kinase inhibitor activity of Lapatinib in
HER-2-overexpressing breast cancer cells as well as responses of a panel of 31
characterized human breast cancer cell lines to treatment by Trastuzumab,
including the use of Trastuzumab-conditioned HER-2(+ve) cell lines [4]. These
studies demonstrated four key observations associated with Lapatinib treatment
in breast cancer. First, they documented that the anti-proliferative effects of
Lapatinib were in fact concentration dependent and were seen in all breast
cancer cell lines. Second, they also reported a range of half-maximal
inhibitory concentrations for Lapatinib (IC50),
however, the study demonstrated a significant amount of variation among these
values: IC50 = 0.010–18.6 μmol/L. Third, these preliminary data
were also representative of long-termin vivo Lapatinib treatment regiments
for breast cancer; this was ascertained using a 77 consecutive-day Lapatinib
treatment schedule in which there was a significant reduction in the volume of
human breast cancer xenografts in athymic mice compared with untreated controls
[4]. The reduction in tumor volume demonstrated in the aforementioned clinical
study with respect to Lapatinib is consistent with the results obtained in the laboratory
setting as well (see Figure4) [37, 38].
Lastly, they examined the synergistic effects of a combinatorial therapy of
Lapatinib-plus-Trastuzumabfor
which their results have indeed provided the preliminary data necessary to
support the rationale for continuing research regarding the potential of
Lapatinib as a combination anti-EGFR therapy with Trastuzumab in
HER-2-overexpressing breast cancer and in patients with clinical resistance to Trastuzumab [4]. Thus, this review, in
agreement with several earlier reports, supports further investigation of the
benefits of Lapatinib as a first-line treatment regimen for early-stage
HER-2-overexpressing breast cancer cell lines as well as its use in the
treatment of both metastatic and locally advanced breast cancer cases [40].Immunohistochemical staining demonstrating the
clinical efficacy of Lapatinib. Figure4 identifies inhibition of activated,
phosphorylated ErbB2/HER-2/neu (p-ErbB2) in a breast cancer patient responding to Lapatinib treatment. (a) Shows a
dermal-lymphatic invasion (magenta) that
is consistent with recurrent inflammatory breast cancer. (b) and (c) Show further
immunohistochemical staining for p-ErbB2 performed on tumor biopsy samples
obtained from patient X on days 0 (4B) and 21 (4C) of Lapatinib therapy; note
the change in positive staining (brownish-yellow).
There is a significant decrease in the activation of p-ErbB2 in response to
Lapatinib [37, 38].
(a)(b)(c)


## 3.3.2. Erlotinib

Erlotinib
treatment is most commonly found in combination with other chemotherapeutic
agents, including Capecitabine and Docetaxel. A study researched the additive
efficacy of Erlotinib with Capecitabine and Docetaxel [41]. The combined treatment was administered every 3 weeks, with
a total of 24 women with MBC; the overall response rate was 67%. The most
common treatment-related AEs were skin toxicities and diarrhea. The severe AEs
were relatively low, but as the Capecitabine/Docetaxel doses were increased,
the rate of grade 3 events also increased. The tolerability of the regimen has
been measured and the group reported an established dosage of Erlotinib (100 mg/day), Capecitabine (825 mg/m2), and Docetaxel (75 mg/m2) in patients
with MBC [41].


## 3.3.3. Gefitinib

Gefitinib has oncebeen approved by FDA as
monotherapy for patients with locally advanced or metastatic nonsmall-cell lung
cancer (NSCLC) [45]. However, more
recently, Gefitinib has been used for the treatment of MBC, including a Phase II
study of Gefitinib in combination with Docetaxel as first-line therapy in MBC
[42]. In 41 patients, a response rate of 54% (95%
CI 45–75%), a stable disease response of 14%, and a progressive
disease response of 32% were reported. Grade 3 or 4 toxicities that were
observed included neutropenia (49%),
diarrhea (10%), acne-like rash (5%), and anemia (2%).
Overall, the Gefitinib and Docetaxel combination demonstrated an active and
generally well-tolerated regimen in women with MBC who have not been previously
treated with metastatic disease [42]. Gefitinib seemed very
promising in early clinical phase testing for the treatment of a number of
solid tumors, including NSCLC. However, the FDA recently withdrew Gefitinib
from its list of clinically effective therapies for NSCLC, but is still
currently under critical review in Phase II/III clinical studies for breast cancer: primary, metastatic, and advanced forms.With respect
to ER-HER2/neu cross-talk in ER/HER2/neu(+ve) breast cancer, Gefitinib has demonstrated promising responses [31]. In a
tamoxifen-resistant, HER2-overexpressing
MCF-7 breast cancer cell line, designated MCF-7/HER2-18, Gefitinib pretreatment
was shown to block ER : EGFR receptor cross-talk, reestablish corepressor
complexes with tamoxifen-bound ER on target gene promoters, eliminate tamoxifen
agonistic effects, and restore tamoxifen antitumor activity both in vitro and in vivo [23].


## 3.4. Combinational Therapies

### 3.4.1. Lapatinib and Anti-ErbB2 Inhibitors: Trastuzumab

The
results of Lapatinib and Trastuzumab monotherapy and combined therapy
approaches for treating breast cancer are  summarized in Tables6 and
7. A
particular combination therapy study involved the comparison of Lapatinib-plus-pAb (where
pAb is a rabbit polyclonal antisera generated by vaccination with a human
ErbB2 fusion protein) and Lapatinib-plus-Trastuzumab [46]. This study showed that Lapatinib-plus-Trastuzumab combination therapy had
enhanced clinical efficacy compared to both Lapatinib and Trastuzumab
monotherapies but had similar efficacy as the secondary combination cocktail of
Lapatinib-plus-pAb [46]. The
Lapatinib-plus-Trastuzumab
combination therapy also showed both a significant downregulation of
survivin, an important
prosurvival/antiapoptosis protein, as well as enhanced apoptosis [46]. These
conclusions along with the information regarding clinical efficacy and AEs in
Table 7 provide sufficient preliminary evidence supporting this synergistic
cooperation seen in this combination therapy. It appears that Lapatinib may in
fact sensitize the cells to further treatment with Trastuzumab thereby
enhancing the individual activity of both drugs.Table 6
Overall response for Trastuzumab, Lapatinib, Erlotinib, and Gefitinib
combination therapies with chemotherapeutic agents [34, 40–43].
StudyNo. of patientsChemotherapyDoseOR (%)TrastuzumabSlamon et al.
[34]143DoxorubicinTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)56Doxorubicin (60 mg/m2)92PaclitaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)41Paclitaxel (175 mg/m2)Marty et al.
[43]186DocetaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)34Docetaxel (100 mg/m2 triweekly)LapatinibGeyer et al.
[40]163CapecitabineLapatinib (1250 mg/day)22*Capecitabine
(2000 mg/m2)ErlotinibTwelves et al.
[41]24Capecitabine,
docetaxelErlotinib (100 mg/day)68Capecitabine
(825 mg/m2)Docetaxel (75 mg/m2)GefitinibCiardiello et
al. [42]41DocetaxelGeftinib (250 mg/day)54Docetaxel (75 mg/m2 or 100 mg/m2)OR: Overall
response rate.*Study was
performed in women with HER2-positive metastatic breast cancer that has progressed
after trastuzumab-based therapy.Table 7
Efficacy end points in intent-to-treat population, adapted from Geyer et al.
[40].
End pointLapatinib plus capecitabineCapecitabine aloneHazard ratioP-value(N = 163)(N = 161)(95% CI)Median time to progression—mo8.44.40.49 (0.34–0.71)<.001†Median progression-free survival—mo8.44.10.47 (0.33–0.67)<.001†Overall response—% (95% CI)22 (16–29)14 (9–21).09‡Complete response—no. (%)1 (<1)0 (0)Partial response—no. (%)35 (21)23 (14)Clinical benefit—no. (%)44 (27)29 (18)Death—no. (%)36 (22)35 (22)End Points are based on evaluation by the independent
review committee under blinded conditions.†The P-value
was calculated with the log-rank test.‡The P-value was calculated with Fisher’s exact test.


### 3.4.2. Lapatinib and Capecitabine

Lapatinib
combination therapy with Capecitabine has also shown success in treatment for
HER-2(+ve) advanced breast cancer
treatment (see Tables6 and 7) [40].
Patients with HER-2(+ve) MBC who had
progressed after treatment with regimens
that included an anthracycline, a taxene, and Trastuzumab were randomly
assigned to receive either Capecitabine alone versus Capecitabine in
combination with Lapatinib. The hazard ratio for the independently
assessed time to progression was 0.49 (95% CI 0.34 to 0.71; P < .001);
the median time to progression was 8.4 months in the combination therapy group,
whereas 4.4 months in the monotherapy group. The most common AEs were diarrhea, the hand-foot syndrome, nausea,
vomiting, fatigue, and rash, varying from grades 1 to 3. In grade 4, diarrhea,
fatigue, headache, and dizziness were reported. Discontinuation of treatment due to AEs occurred in 22 women in the
combination-therapy group (13%) and in
18 women in the monotherapy group (12%) [40]. However, as previously
indicated, these reports are refuted by other sources, which claim no grade 4
AEs and only minor toxicity reports for Lapatinib monotherapy, but there are no
other reviews specifically regarding further investigation into the
relationship between combinational therapies of Lapatinib-plus-Capecitabine,
if any does in fact exist, positively or negatively correlated with improved
outcome.Therefore, in
concurrence with combinational therapies, a major need clearly exists for
further research to be done in this specific area of anti-EGFR therapy. There
is a significant deficit in the hypotheses and models that can critically
evaluate the data reported thus far on most combinational therapies.


## 3.4.1. Lapatinib and Anti-ErbB2 Inhibitors: Trastuzumab

The
results of Lapatinib and Trastuzumab monotherapy and combined therapy
approaches for treating breast cancer are  summarized in Tables6 and
7. A
particular combination therapy study involved the comparison of Lapatinib-plus-pAb (where
pAb is a rabbit polyclonal antisera generated by vaccination with a human
ErbB2 fusion protein) and Lapatinib-plus-Trastuzumab [46]. This study showed that Lapatinib-plus-Trastuzumab combination therapy had
enhanced clinical efficacy compared to both Lapatinib and Trastuzumab
monotherapies but had similar efficacy as the secondary combination cocktail of
Lapatinib-plus-pAb [46]. The
Lapatinib-plus-Trastuzumab
combination therapy also showed both a significant downregulation of
survivin, an important
prosurvival/antiapoptosis protein, as well as enhanced apoptosis [46]. These
conclusions along with the information regarding clinical efficacy and AEs in
Table 7 provide sufficient preliminary evidence supporting this synergistic
cooperation seen in this combination therapy. It appears that Lapatinib may in
fact sensitize the cells to further treatment with Trastuzumab thereby
enhancing the individual activity of both drugs.Table 6
Overall response for Trastuzumab, Lapatinib, Erlotinib, and Gefitinib
combination therapies with chemotherapeutic agents [34, 40–43].
StudyNo. of patientsChemotherapyDoseOR (%)TrastuzumabSlamon et al.
[34]143DoxorubicinTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)56Doxorubicin (60 mg/m2)92PaclitaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)41Paclitaxel (175 mg/m2)Marty et al.
[43]186DocetaxelTrastuzumab (4 mg/kg initial dose, 2 mg/kg weekly)34Docetaxel (100 mg/m2 triweekly)LapatinibGeyer et al.
[40]163CapecitabineLapatinib (1250 mg/day)22*Capecitabine
(2000 mg/m2)ErlotinibTwelves et al.
[41]24Capecitabine,
docetaxelErlotinib (100 mg/day)68Capecitabine
(825 mg/m2)Docetaxel (75 mg/m2)GefitinibCiardiello et
al. [42]41DocetaxelGeftinib (250 mg/day)54Docetaxel (75 mg/m2 or 100 mg/m2)OR: Overall
response rate.*Study was
performed in women with HER2-positive metastatic breast cancer that has progressed
after trastuzumab-based therapy.Table 7
Efficacy end points in intent-to-treat population, adapted from Geyer et al.
[40].
End pointLapatinib plus capecitabineCapecitabine aloneHazard ratioP-value(N = 163)(N = 161)(95% CI)Median time to progression—mo8.44.40.49 (0.34–0.71)<.001†Median progression-free survival—mo8.44.10.47 (0.33–0.67)<.001†Overall response—% (95% CI)22 (16–29)14 (9–21).09‡Complete response—no. (%)1 (<1)0 (0)Partial response—no. (%)35 (21)23 (14)Clinical benefit—no. (%)44 (27)29 (18)Death—no. (%)36 (22)35 (22)End Points are based on evaluation by the independent
review committee under blinded conditions.†The P-value
was calculated with the log-rank test.‡The P-value was calculated with Fisher’s exact test.


## 3.4.2. Lapatinib and Capecitabine

Lapatinib
combination therapy with Capecitabine has also shown success in treatment for
HER-2(+ve) advanced breast cancer
treatment (see Tables6 and 7) [40].
Patients with HER-2(+ve) MBC who had
progressed after treatment with regimens
that included an anthracycline, a taxene, and Trastuzumab were randomly
assigned to receive either Capecitabine alone versus Capecitabine in
combination with Lapatinib. The hazard ratio for the independently
assessed time to progression was 0.49 (95% CI 0.34 to 0.71; P < .001);
the median time to progression was 8.4 months in the combination therapy group,
whereas 4.4 months in the monotherapy group. The most common AEs were diarrhea, the hand-foot syndrome, nausea,
vomiting, fatigue, and rash, varying from grades 1 to 3. In grade 4, diarrhea,
fatigue, headache, and dizziness were reported. Discontinuation of treatment due to AEs occurred in 22 women in the
combination-therapy group (13%) and in
18 women in the monotherapy group (12%) [40]. However, as previously
indicated, these reports are refuted by other sources, which claim no grade 4
AEs and only minor toxicity reports for Lapatinib monotherapy, but there are no
other reviews specifically regarding further investigation into the
relationship between combinational therapies of Lapatinib-plus-Capecitabine,
if any does in fact exist, positively or negatively correlated with improved
outcome.Therefore, in
concurrence with combinational therapies, a major need clearly exists for
further research to be done in this specific area of anti-EGFR therapy. There
is a significant deficit in the hypotheses and models that can critically
evaluate the data reported thus far on most combinational therapies.


## 4. Conclusion

Breast
cancer is a disease that is responsible for approximately 1% of the mortality
rate worldwide. The importance of developing new and improved therapies for its
treatment is therefore undisputable. Recently, advances in anti-EGFR therapy
have given hope to the development of new breast cancer therapies with improved
specificity, activity, and safety. Increasingly, there is recognition and
acceptance of the unique role anti-EGFR therapy plays in the armamentarium of
treatment options available to breast cancer patients. Recently, novel members
of this group, such as Lapatinib, have been brought to the forefront of this
research as it not only is an extremely effective drug in the clinical setting,
but it also serves as an excellent model for the development of future EGFR
and/or HER-2 inhibitors. The novel dual kinase inhibitor activity of Lapatinib,
which displays tyrosine kinase receptor inhibitory activity against both EGFR
and HER-2, is both exciting and intriguing. The unique activity of Lapatinib to
inhibit both mechanisms of signaling cascades should be studied extensively in
order to improve upon the current model of tyrosine kinase inhibition and its
role in anti-EGFR therapy. Other drugs with similar activities to Lapatinib,
such as CI-1033, a pan-ErbB tyrosine kinase inhibitor, should also be studied
thoroughly in order to identify any important similarities between them and to
determine how these crucial factors can perhaps be modified to enhance their
activity in future anti-EGFR drug prototypes. Other areas of anti-EGFR therapy
that should be investigated include the ability of the various anti-EGFR
therapeutic modalities to sensitize cancer cells to other forms of chemotherapy
originally considered refractory for an
individual patient. This is another extremely important avenue that should
be investigated exhaustively.Although there is much
improvement to be done,
the wealth of knowledge surrounding these therapies continues to grow (see Table8). This observation, along
with recent advances in crystallography and docking techniques, the development
of improved high-throughput
analyses for identifying novel anti-EGFR activity, as well as advances
in DNA/RNA-microarray technology
used for classification purposes and extremely useful in the clinical setting,
all continue to contribute to the overall understanding
and development of these new treatment regiments as well as treating breast
cancer as a whole. The design rationale of new anti-EGFR therapies lies in the
intimate relationship between the mechanisms of action of current forms of
treatment and the structure of the EGFR. We believe that meticulous inspection
of the unique intermolecular interactions of these drugs with this receptor and
its family members will not only lead to future accomplishments in anti-EGFR
therapy but will also increase
insight into chemotherapy as a whole for breast cancer.Table 8
Summary of Anti-EGFR therapy agents.
The5 anti-EGFR therapy drugs
discussed in this review: these 5 drugs are currently being used or in clinical
phase testing for anti-EGFR therapy of breast cancer. All of these agents are
either already being used in the clinical setting or are in Phase III clinical development.
Drug nameOther names for the drugClassification of drugTarget receptor(s) of drugSpecial cancer types and efficacyImportant commentsDrug manufacturerCetuximabErbitux (humanized form of the murine MoAb:
C225)MoAb (chimeric IgG1)Blocks EGFR;A large variety
of solid tumors: -CRC/mCRC; -SCCHN;Most widely
used anti-EGFR monoclonal antibody used in solid tumor therapy (07/2007)
[44];ImClone
Systems, Inc., Princeton, NJ. & NY, NY.TrastuzumabHerceptinMoAb (human IgG1)Blocks HER2/neu;Mostly widely
used MoAb in treating HER2+ -overexpressing
cases of BC;Extremely
important drug in breast cancer;F. Hoffmann-La
Roche Ltd, Basel, Switzerland.ErlotinibTarceva (OSI-774)smTKIInhibition of EGFR;Solid tumor
therapy: -Pancreatic
cancer; -NSCLC
(recent);Nothing unique;Genetech, Inc.,
South San Fransisco, CA.GefitinibIressa (ZD1839)smTKI -anilinoquinazolineInhibition of EGFR;Previously used
for NSCLC-currently w/d by FDA;Recently
withdrawn by FDA for treatment of NSCLC;AstraZeneca
Pharmaceuticals, Wilmington, DE.In clinical
phase testing for BC as well as other metastatic & advanced cancers;LapatinibTykerb (GW572016)smTKIBoth EGFR and HER2/neu; (Dual-TKI Action)Solid tumor
therapy: -BC.Extremely
important smTKI in current BC treatment.Glaxo-Smith-Kline,
Philadelphia, PA.MoAb:
Monoclonal antibody; EGFR: Epidermal growth factor receptor (ErbB1); HER-2/neu:
Human epidermal growth factor receptor 2; smTKI: Small molecule tyrosine kinase
inhibitor; w/d:  Withdrawn; NSCLC: Nonsmall
cell lung cancer; BC: Breast cancer; CRC: Colorectal cancer; mCRC: Metastatic colorectal
cancer; SCCHN: Squamous cell carcinoma of the head and neck.



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