EGFR Mutations and Lung Cancer

Extract from the article “ EGFR Mutations and Lung Cancer”

doi: 1553-4006/11/0228-0049$20.00

NSCLC: non–small cell Abstract

lung carcinoma

Mutation: a change in Epidermal growth factor receptor (EGFR) is a transmembrane protein

the DNA sequence of a with cytoplasmic kinase activity that transduces important growth
cell’s genome that factor signaling from the extracellular milieu to the cell. Given that
results from more than 60% of non–small cell lung carcinomas (NSCLCs) express
substitution, loss,
EGFR, EGFR has become an important therapeutic target for the
duplication, gain, or
treatment of these tumors. Inhibitors that target the kinase domain of
abnormal transfer of
nucleic acid bases.
EGFR have been developed and are clinically active. More
importantly, such tyrosine kinase inhibitors (TKIs) are especially
Tyrosine kinase (TK): effective in patients whose tumors harbor activating mutations in the
an enzyme that can tyrosine kinase domain of the EGFR gene. More recent trials have
phosphorylate (i.e., suggested that for advanced NSCLC patients with EGFR mutant
transfer a phosphate tumors, initial therapy with a TKI instead of chemotherapy may be the
group from ATP to) a
best choice of treatment. Therefore, mutation testing is mandatory to
tyrosine residue in a
identify these patients, given that selection based only on clinico-
protein
pathologic characteristics is inadequate. We review the role
Predictive markers: of EGFR mutations in the diagnosis and management of NSCLC.
clinical, cellular, and
molecular markers that
predict response of a
Keywords
tumor to treatment;
usually assessed by
non–small cell lung carcinoma, epidermal growth factor, receptor
tumor shrinkage or a
survival benefit from
tyrosine kinase, tyrosine kinase inhibitor, sensitizing
treatment mutation, oncogene addiction

TKI: tyrosine kinase

inhibitor
INTRODUCTION

RAS: rat sarcoma viral

oncogene homolog
Lung cancer is the leading cause of cancer-related mortality
worldwide, with an overall five-year survival rate of 15% . Non–small
RAF: v-raf murine cell lung carcinoma (NSCLC) constitutes approximately 75–80% of all
leukemia viral oncogene lung cancers. When the tumor is confined to the lung with minimal
homolog MEK: regional lymph node spread, the most effective treatment is surgery.
mitogenactivated However, ∼70% of patients present with locally advanced or
protein kinase kinase
metastatic disease at the time of diagnosis and are not eligible for
MAPK:
surgical resection.
mitogenactivated
protein kinase PI3K:
phosphoinositide 3- Significant advances in treatment were recently achieved with drugs
kinase designed specifically to target molecules that regulate critical growth
and/or survival pathways of cancer cells. Dramatic responses in
PTEN: phosphatase
and tensin homolog
approximately 10% of patients, who had demonstrated resistance to
AKT: v-akt murine one or more chemotherapy regimens, were reported in Phase I and II
thymoma viral trials of the epidermal growth factor receptor (EGFR) tyrosine kinase
oncogene homolog
1
inhibitors (TKIs) erlotinib (Tarceva®) and gefitinib (Iressa®). In 2004, the results of the
National Cancer Institute of Canada Clinical Trials Group BR.21 study led to the global
approval of erlotinib as the first targeted therapy for advanced NSCLC. At approximately
the same time, two groups reported the discovery of mutations in the tyrosine kinase
(TK) domain of the EGFR gene in NSCLC. Furthermore, the presence of these mutations
appeared to correlate with sensitivity to the EGFR inhibitor gefitinib. Because the patients
whose tumors harbored mutations shared certain characteristics with the patients who
responded to erlotinib in the BR.21 trial, it was postulated that EGFR TK domain mutations
would be an important molecular predictive marker for clinical benefit from EGFR TKIs.

This review summarizes the current knowledge of EGFR TK domain mutations and the
role they play in the management of NSCLC. It also highlights the importance
of EGFR mutation status in the selection of patients for EGFR TKI therapy.

EGFR

The EGFR gene is located on the short arm of chromosome 7 (7p11.2) and encodes a
170-kDa type I transmembrane growth factor receptor with TK activity. EGFR belongs to
the HER/erbB family of receptor tyrosine kinases (RTKs), which includes HER1
(EGFR/erbB1), HER2 (neu, erbB2), HER3 (erbB3), and HER4 (erbB4). These receptors
display similar molecular structures: They have an extracellular, cysteine-rich ligand-
binding domain; a single α-helix transmembrane domain; a cytoplasmic TK domain (in all
receptors except HER3); and a carboxy-terminal signaling domain. Homodimerization
and/or heterodimerization with other family members—most commonly HER2, which lacks
its own specific ligand—in response to ligand binding activates the TK.

A kinase is a type of enzyme that transfers phosphate groups from high-energy donor
molecules, such as ATP to specific target molecules (substrates); the process is
termed phosphorylation. The opposite, an enzyme that removes phosphate groups from
targets, is known as a phosphatase. Kinase enzymes that specifically phosphorylate
tyrosine amino acids are termed tyrosine kinases.

This process results in autophosphorylation of the cytoplasmic domain of the receptor and
enables it to interact with adaptor molecules, which couple the receptors to downstream
signaling pathways. Intracellular signaling is mediated mainly through the RAS-RAF-MEK-
MAPK pathway, the PI3K-PTEN-AKT pathway, and the signal transducer and activator of
transcription (STAT) pathway. Downstream EGFR signaling ultimately leads to increased
proliferation, angiogenesis, metastasis, and decreased apoptosis (Figure 1).

The TK activity of EGFR may be dysregulated by several oncogenic mechanisms,

including EGFR gene mutation, increased gene copy number, and EGFR protein
overexpression. The receptors and ligands of the EGFR family also mediate complex
interactions between tumor cells and the tumor microenvironment. Improper activation of
EGFR TK inhibits tumor cell apoptosis and contributes to tumor progression. EGFR may
also interact with the integrin pathway and activate matrix metalloproteinases to alter
cellular adhesion, stimulate cell motility and invasion, and promote metastasis. Gain-of-
function or activating mutations of the EGFR gene occur in some NSCLCs, leading to
2
constitutive TK activity. These findings make EGFR a rational target for therapeutic
intervention and support the development of novel anticancer agents that target EGFR.

Figure 1. Simplified schema of epidermal growth

factor receptor (EGFR)-induced signals that
regulate critical cellular functions relevant to
carcinogenesis. Abbreviations: ERK,
extracytoplasmic-regulated kinase; Grb-2, growth
factor receptor–bound protein 2; MAPK, mitogen-
activated protein kinase; MEK, MAPK kinase;
mTOR, mammalian target of rapamycin; PI3K,
phosphoinositide 3-kinase; PTEN, phosphatase
and tensin homolog; RAF, v-raf murine leukemia
viral oncogene homolog; RAS, rat sarcoma viral
oncogene homolog; SOS, sister of sevenless;
STAT, signal transducer and activator of
transcription.
EGFR TYROSINE KINASE DOMAIN MUTATIONS

Two independent studies first reported the existence of somatic mutations in the TK
domain of EGFR; the mutations are characterized by short deletions in exon 19
and point mutations (G719S, L858R, and L861Q) in exons 19 and 21 (Figure 2).

Figure 2. Amino acid and nucleotide sequence changes in exon 19 deletion and exon 21
L858R mutations involving the tyrosine kinase domain of epidermal growth factor receptor.

According to their nucleotide changes, the mutations have been classified into three types.
Class I mutations include short in-frame deletions that result in the loss of four to six amino
acids (E746 to S752) encoded by exon 19. Class II mutations are single-nucleotide

3
substitutions that may occur throughout exons 18 to 21. Class III mutations are in-frame
duplications and/or insertions that occur mostly in exon 20. Among all TK domain
mutations, 85–90% are exon 19 class I deletions and exon 21 L858R mutations. Although
initial reports suggested that there is an almost equal distribution of exon 19 deletions and
L858R mutations, more recent reports from clinical trials suggest a slightly higher
frequency of deletions versus point mutations. Recently, a rare exon 22 mutation (E884K)
that may confer differential sensitivity to different EGFR small-molecule inhibitors was
reported.

Figure 3. Structural models reflecting the kinase domain of epidermal growth factor
receptor (EGFR) that are (a) wild type and (b) L858R mutant, and (c) locations of identified
mutations. (b) In the kinase domain bearing an activated mutant (b), the activation loop is
in the open position, which normally occurs only when the receptor is activated by ligand.
The ligand-induced asymmetric dimerization of EGFR kinase domain permits head-to-tail
interaction between the N-lobe and the C-lobe of the two receptor kinase domains.

4
In its inactive form, the EGFR kinase domain assumes a structure that results in
autoinhibition of its activity. Mutation at the TK domain of EGFR results in the
destabilization of its domain conformation, constitutive activation of its kinase activity, and
activation of its downstream signaling pathways (Figure 3) . The latter include AKT and
STAT, which have crucial antiapoptosis functions for cell survival . The discovery that lung
cancers harboring constitutively active mutant EGFR are exquisitely sensitive to the
apoptotic or growth inhibitory activities of EGFR TKIs strongly supports the so-called
oncogene addiction theory, which dominates current views of the role of oncogenic
mutations in carcinogenesis and cancer treatment.

PATIENT CHARACTERISTICS ASSOCIATED WITH MUTATIONS

Clinico-pathological features that correlate with EGFR-activating mutations include East

Asian ethnicity, adenocarcinoma histology, female sex, and a history of never having
smoked. Mutations are more common in women than in men (42% versus 14%), in
patients who have never smoked than in patients who have smoked (51% versus 10%),
and in patients with adenocarcinoma than in those with other histologies (40% versus 3%).
Interestingly, the higher mutation rates among patients who have never smoked and
adenocarcinoma patients are consistent in both Asian and non-Asian patient populations.
The distinct association of mutations with women, patients who have never smoked, and
adenocarcinoma patients also suggests different etiologies and mechanisms for the
development of NSCLC in Asian and Caucasian patients.

EGFR INHIBITORS

The two major classes of agents designed to inhibit EGFR activity are small-molecule TKIs
and monoclonal antibodies. Monoclonal antibodies act by binding to the extracellular
region of the receptor and function as competitive antagonists to inhibit ligand binding.
Cetuximab (Erbitux®) is a humanized mouse monoclonal antibody developed against the
ligand-binding domain of EGFR. Two TKIs, erlotinib and gefitinib, are approved for the
treatment of NSCLC. These agents were designed to reversibly bind the ATP-binding site
of the EGFR kinase domain, thereby inhibiting its activity. Importantly, these agents
demonstrate higher binding affinity for EGFR with activating mutation than do the wild-type
receptors, consistent with the exquisite sensitivity of the mutant receptor for these drugs..
In contrast to erlotinib or gefitinib, irreversible inhibitors have demonstrated activity against
tumors that have developed secondary resistance mutations

EGFR MUTATIONS AND RESPONSE TO EGFR TYROSINE KINASE

INHIBITORS

In the original publications reporting the presence of EGFR TK domain mutations

in NSCLC, all 18 refractory patients had wild-type EGFR . Since then, the association
between the presence of activating EGFR mutations and high response rates to TKIs has
been confirmed in numerous studies ,which reported overall response rates of 50% to
100% among patients with EGFR mutant tumors; response rates among patients with
5
wild-type EGFR are 0% to 30%. The rates are not significantly different between East
Asian and Caucasian patients, which suggests that the response is directly related to
mutation status and not ethnicity .

Among all EGFR mutations, four types are strongly correlated with TKI sensitivity in vitro
and in vivo. These are point mutations in exons 18 (G719A/C) and 21 (L858R and L861Q)
and in-frame deletions in exon 19. Mutations in exon 19 of the EGFR gene appear to
confer sensitivity more often than do point mutations in exons 20 and 21. Other
mutations may be associated with greater sensitivity to inhibitors, but their rarity precludes
any firm conclusions .

EGFR MUTATIONS THAT PREDICT RESISTANCE

Although almost all EGFR TK domain mutations that have been detected and studied
functionally have been activating mutations, not all of them are associated with increased
sensitivity to TKIs. Functional studies of one of the exon 20 in-frame insertion mutations
using cell-culture models found it to be transforming yet insensitive to the inhibitory effect
of gefitinib and erlotinib . Studies on the responsiveness of several class II point
mutations (E709G, G719S, S768I, and L861Q) found variable responses to the inhibitory
activity of gefinitib on both the cellular viability and the EGFR activation of cells expressing
these mutants. Only limited information about the clinical response of these rarely reported
mutations to EGFR TKI is available. Whereas V689M, N700D, L718P, V765A, V783A,
A839T, and K846R are associated with response to gefitinib, E709Q/L, A763V, N826S,
and V752I are associated with lack of response .

Figure 4. For summary of somatic mutations found in EGFR. Mutations in green are
typically sensitive to EGFR TKIs, those in red are typically resistant. Approximate
frequency of occurrence in NSCLC patients of each mutation is shown in parentheses.

6
SUMMARY POINTS

1. EGFR is highly expressed in >60% of NSCLCs and plays an important role in

regulating the proliferation, survival, motility, and differentiation of the tumor cells.
2. Mutation in the TK domain of EGFR results in constitutive and oncogenic activation
of the receptor and dependency of the tumor cells in the EGFR signaling pathway.
3. Mutations may occur in many sites on exons 18 to 21 of EGFR, but >85% of such
mutations consist of short deletions in exon 19 and a L858R point mutation in exon
21. Receptors and cells that harbor these two groups of mutations are highly
sensitive to EGFR TKIs.
4. During EGFR TKI treatment of patients whose tumors have sensitizing mutations,
disease progression is commonly associated with the identification of
additional EGFR mutations associated with TKI resistance.
5. EGFR mutations define a new group of NSCLCs that can be treated more
effectively by specific targeted therapy.
6. Future diagnosis and classification of lung cancer will require incorporation of
molecular tests into the standard diagnostic workup.