A Historical Perspective and Modern Techniques in Pulmonary Pathology
Moderators: Dr. Henry D. Tazelaar, Dr. Ming S. Tsao and Dr. Brendan Mullen
Section 4 -
Targeted Therapies for Lung Cancer - EGFR Inhibitors
Wilbur A. Franklin
University of Colorado Denver Health Sciences Center
EGF was one of the first epithelial cell signaling molecules to be isolated and
characterized . In the 45 years since this original observation, what has become the EGFR (or
ErbB) pathway has been explored and characterized in minute detail and has been found particularly active
in lung. The recent introduction of agents that block EGFR signaling has spurred interest in the
pathway. This review will provide a summary of the current understanding of the role of epidermal growth
factor receptor (EGFR) and its related signaling pathway in lung cancer biology and treatment.
EGFR Structure and Function
The EGFR family consists of four homologous tyrosine kinase proteins that are present at the surface
of lung cancer cells. These receptors include EGFR, HER2/neu, ErbB-3 and
ErbB-4. Each of the proteins with the exception of ErbB3 is composed of three functional
domains--extracellular, transmembrane and intracytoplasmic. ErbB3 lacks a functional intracellular
domain. Binding to EGF, TGFa or to any of several other ligands activates the receptors. On ligand
binding, receptors form either homodimers or heterodimers at the cell surface. The formation of the
dimers permits phosphorylation of serine residues of the intracytoplasmic domains of the now adjacent
dimer partners (trans autophosphorylation). Phosphorylation can activate
several downstream signaling cascades. These include the formation of a complex that includes src2, GRB2, SH3, and SOS complex with activated receptor. The formation of this
complex results in the sequential phosphorylation (activation) of ras, raf, MAP kinase, Akt and finally
in gene activation. Molecular lesions that could lead to ErbB pathway activation in lung cancer are
EGFR Expression in Lung Carcinogenesis
In normal lung tissue EGFR is strongly expressed by basilar cells of the respiratory mucosa and in
most cases by pneumocytes lining the peripheral airways. In the earliest histologically recognizable
proliferative lesions in the bronchial epithelium, basal cell hyperplasia, increased thickness of the
basilar cell layer is associated with corresponding increase in the expression of EGFR. In progressively
more advanced lesions ranging from squamous metaplasia through carcinoma in situ there is consistent and
heavy overexpression of EGFR. The consistent expression of EGFR in premalignant epithelium suggests that
EGFR blocking agents may be useful for chemoprevention in high-risk patients with recognizable lesions in
the lower airways but to date no clinical trials have been successfully mounted to test this hypothesis
and most attention has been focused on EGFR in invasive lung cancer.
EGFR Expression in Lung Cancer
For several years it has been known that EGFR is overexpressed in lung carcinoma. The first
demonstration of EGFR overexpression in lung cancer in situ was a 1984 study
that used a radiolabeled antibody and autoradiography to localize the receptor to tumor
cells . Since that initial report there have been many studies documenting expression in situ
by immunohistochemical (IHC) methods in both frozen and paraffin sections. Despite variations in
detection techniques, there is general agreement that EGFR is expressed at highest levels and most
frequently (80%) in squamous cell carcinoma while it is expressed less frequently in adenocarcinomas and
large cell carcinomas (about 50%) . The protein is expressed along the plasma membranes and
in the cytoplasm of tumor cells. The prognostic significance of EGFR expression is still controversial
with some reports indicating a correlation with poor prognosis  and others suggesting no
effect on prognosis . These variations may be due to inconsistency in testing methods or to
variations in the patient populations evaluated
Assessment of prognostic significance of
EGFR is complicated by the diverse effects of EGFR and related signaling molecules as described below.
Genetic changes in EGFR
Although overexpression of EGFR has been observed for some time, it has only been recently that
attention has been focused on genetic mechanisms that might explain this overexpression. Changes in
expression levels and activation status may be affected by two mechanisms that are often observed in the
same tumors: increased gene copy number and mutation.
Increase Gene Copy Number (High Polysomy and True Amplification) in Lung Cancers
EGFR is encoded on chromosome 7. Increase in EGFR gene copy number has been evaluated by DNA blotting
methods such as Southern blot or array CGH and by FISH. Blotting methods have the disadvantage that
cellular localization of signal cannot be assessed and quantification of amplification in individual
cells is not possible. The most accurate method for determining gene copy number at the cellular level
is FISH. The quantification of FISH however, has not been well standardized in the past and is
complicated by heterogeneity among tumor cells. Dr. Garcia at the University of Colorado has proposed a
quantification method in which tumors have been divided into two categories based on the presence or
absence of >3 copies in 40% of tumor cells. Using this criterion, we initially found that 25% of
NSCLC were FISH positive and that high EGFR protein expression levels were more frequent in FISH positive
tumors than in FISH negative tumors . We could detect no prognostic significance from
amplification or overexpression.
The availability of drugs that block EGFR heightened interest in these results. Several studies have
now shown that a positive FISH test as defined above is associated with response to EGFR
Increased copy number appears to drive overexpression of EGFR and tumors that
are sensitive to EGFR blockers may be dependent on (addicted to?) this pathway for survival and growth
while resistant tumors may be driven by any of myriads of other signaling abnormalities that known to
occur in lung cancer.
Mutations of the EGFR1 Gene in Lung Cancer
In addition to EGFR gene copy number and expression abnormalities, it is now evident that EGFR may be
affected by somatic mutations in the EGFR gene. The best defined and probably most frequent mutations
are those in exons that code for the tyrosine kinase domain. In 2004, two Boston groups almost
that NSCLC contained in frame deletions in exon 19 and point
mutations of exon 21 that cluster around the ATP binding pocket of the tyrosine kinase domain. Tumors
that contain these mutations tended to occur in non-smoking Asian females and to be associated with
superior response to EGFR blockade. Since these initial reports these results have been verified in many
However, few studies, have reported improved overall survival in patients with mutations. One
possible explanation for this is that not all mutations may equally affect outcome. It has been recently
reported in abstract form that response to EGFR blockade and survival may be significantly better in
tumors with exon 19 deletion than in those with point mutation. This is presumably due to enhanced
effect on receptor phosphorylation and enhanced downstream signaling in tumors with deletion. An
acquired mutation in exon 20 (T790M) that imparts resistance to receptor blockade has also been
described . This mutation may not be present in primary tumor and may thus be acquired in
response to EGFR blocking treatment. Finally, it should be noted that EGFR mutations are not the only
mutations that may drive tumor cells and that Ki-ras mutations occur tumors
from smokers and may drive part of the same downstream pathway activated EGFR itself. EGFR and Ki-ras
mutations have been reported to occur separately with little if any overlap in individual tumors and
demographic groups. A small group of lung carcinomas may also be HER2/neu
and/or amplified . Taken together these results suggest that
complex and inconsistent genetic rearrangements may be responsible for the malignant phenotype of lung
Currently the precise role for mutational analysis as a predictor of response to EGFR blockers is
unsettled. One problem is selection of the optimal method for mutation testing. Accurate assessment of
mutation in clinical samples can be technically difficult. In most institutions, frozen samples of tumor
are not routinely available and this has lead to reliance on DNA from fixed tissue. Several testing
variables may affect results of testing DNA from formalin-fixed, paraffin-embedded material including the
amount of tumor sample, the use of microdissection to isolate tumor cells from stroma, deamination of
adenine and cytosine in formalin fixed DNA , the particular Taq polymerase used for
sequencing and the primer design employed for PCR in cases where only small amounts of DNA are
available. Standards for EGFR mutational testing will have to be adopted for meaningful comparisons to
be made across studies.
Correlations Among Markers
It has been found that 8 of 9 EGFR mutant cell lines also have high gene copy (either high polysomy or
amplification). There is thus reason to suspect that there may be considerable overlap in marker
status. Several prognostic studies have shown that the effect of mutation, gene copy number and
overexpression by IHC are additive . These studies suggest that high gene copy number and
protein overexpression enhance the effect of mutation alone. The constellation of mutational status,
gene copy number and protein overexpression may have the optimal predictive power for predicting response
to EGFR blockers. In future clinical trials it will be important to collect data on all three these EGFR
biomarkers to precisely identify patients who might benefit from EGFR blockade.
EGFR Genetic Changes in Premalignancy
Chromosomal gain is frequently present in premalignant bronchial epithelium. Recent studies have
confirmed that chromosome 7 is of one of the most frequently gained chromosomes in central airway
premalignancy. The functional consequences of this gain are not known. To date, the presence of
chromosome 7 gain has not been shown to correlate with level of protein expression in premalignant
bronchial mucosa. To exert a strong influence on overall cellular physiology, it may be necessary that
multiple genetic events occur that would effectively transform the cell. That mutation may be one of
these events is suggested by the work of Wistuba who found EGFR mutation in benign epithelium of patients
with mutant tumor elsewhere in the lung . That the multihit phenomenon is at work here is
suggested by a recently encountered patient with Li-Fraumeni syndrome (hit 1) who at the age of 34
developed an EGFR-mutant (hit 2) lung carcinoma.
The amount of available information on EGFR genetic status is still small and anecdotal. In view of
the magnitude of lung cancer problem, it will be of considerable importance to better define the role of
the EGFR pathway in subjects without invasive carcinoma but at risk who may benefit from EGFR blockade.
The clinical testing of bioactive compounds targeting specific cell signaling pathways has stimulated
the need for more accurate information regarding the identity and level of expression of the specific
targets as well as intermediates that may be affected by the targets. Current interest is focused
primarily on RTK surface proteins. Experience gained in the assessment of this pathway may be applicable
to a number of other pathways that are being rapidly defined through application of tumor profiling
methods made possible by high throughput genomic and proteomic technologies. In the next few years,
these methods can be expected to result in accelerated identification of potentially useful molecular
targets in both malignant and premalignant epithelium. Bioactive drugs aimed at these targets hold the
promise of reducing the terrible morbidity and mortality from this most lethal of tumors and for
permitting a shift in the focus of treatment from advanced tumors to early invasive or preinvasive
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