each paragraph contain 300 words at least

include 8-10 reference with Citing those in essay (be sure all references are in text writing cited )

Include Tables and Figures from Literature ( needed  3-4) with title  below

plagiarism must be 0%

here the  the topic of paragraph  u must be write about it and each one separate from others :

1-About EGFR and signaling with scheme of EGFR Signaling 

2-Write the EGFR mutation testing methods (Capillary or NGS)

3-Few company names ex., LabCorp, MAYO clinic etc.,

4-Show a scheme or histogram about mutations in Lung cancer

5-Write about mutation appears after the inhibitor therapy that is resistance mutations

6-Write about mutation present in EGFR codon 18-21 that kill cancer with inhibitor therapy that is sensitization mutations

the file i upload use it  for answers all this and short your time 🙂

Reading Materials

Román, M. et al. (2018) ‘KRAS oncogene in non-small cell lung cancer: Clinical perspectives on the treatment of an old target’, Molecular Cancer. Molecular Cancer, 17(1), pp. 1–14. doi: 10.1186/s12943-018-0789-x.
DONG, Y., REN, W., QI, J., JIN, B., LI, Y., TAO, H., XU, R., LI, Y., ZHANG, Q. & HAN, B. 2016. EGFR, ALK, RET, KRAS and BRAF alterations in never-smokers with non-small cell lung cancer. Oncology letters, 11, 2371-2378.
THOMPSON, J. C., YEE, S. S., TROXEL, A. B., SAVITCH, S. L., FAN, R., BALLI, D., LIEBERMAN, D. B., MORRISSETTE, J. D., EVANS, T. L. & BAUML, J. 2016. Detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next-generation sequencing of cell-free circulating tumor DNA. Clinical Cancer Research, 22, 5772-5782.
Yokota, T. (2012) ‘Are KRAS/BRAF Mutations Potent Prognostic and/or Predictive Biomarkers in Colorectal Cancers?’, Anti-Cancer Agents in Medicinal Chemistry, 12(2), pp. 163–171. doi: 10.2174/187152012799014968.
Ellison, G. et al. (2013) ‘EGFR mutation testing in lung cancer: A review of available methods and their use for analysis of tumour tissue and cytology samples’, Journal of Clinical Pathology, 66(2), pp. 79–89. doi: 10.1136/jclinpath-2012-201194.

Mutations in the EGFR Pathway | AACC.org. https://www.aacc.org/publications/cln/articles/2013/october/egfr-mutations. Accessed March 14, 2020.
EGFR and KRAS mutations as criteria for treatment with tyrosine kinase inhibitors: retro- and prospective observations in non-small-cell lung cancer – Annals of Oncology. https://www.annalsofoncology.org/article/S0923-7534(19)37561-1/fulltext. Accessed March 14, 2020.
KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target | SpringerLink. https://link.springer.com/article/10.1186/s12943-018-0789-x. Accessed March 14, 2020.
Lung Cancer With an EGFR Mutation: Diagnosis and Treatment. https://www.verywellhealth.com/lung-cancer-with-an-egfr-mutation-4097338. Accessed March 14, 2020.
Anti-EGFR Therapies: Clinical Experience in Colorectal, Lung, and Head and Neck Cancers | Cancer Network. https://www.cancernetwork.com/oncology-journal/anti-egfr-therapies-clinical-experience-colorectal-lung-and-head-and-neck-cancers. Accessed March 14, 2020.

Brambilla, E., and Gazdar, A. (2009). Pathogenesis of lung cancer signalling pathways: roadmap for therapies. Eur Respir J 33, 1485-1497.
Colombino, M., Paliogiannis, P., Cossu, A., Santeufemia, D.A., Sardinian Lung Cancer Study, G., Sini, M.C., Casula, M., Palomba, G., Manca, A., Pisano, M., et al. (2019). EGFR, KRAS, BRAF, ALK, and cMET genetic alterations in 1440 Sardinian patients with lung adenocarcinoma. BMC Pulm Med 19, 209.
Jorge, S.E., Kobayashi, S.S., and Costa, D.B. (2014). Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data. Braz J Med Biol Res 47, 929-939.
Stewart, E.L., Tan, S.Z., Liu, G., and Tsao, M.S. (2015). Known and putative mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations-a review. Transl Lung Cancer Res 4, 67-81.
SU, S. & WU, Y.-L. 2017. Clinical trials of tyrosine kinase inhibitors for lung cancer in China: a review. Journal of hematology & oncology, 10, 147.
SESHACHARYULU, P., PONNUSAMY, M. P., HARIDAS, D., JAIN, M., GANTI, A. K. & BATRA, S. K. 2012. Targeting the EGFR signaling pathway in cancer therapy. Expert opinion on therapeutic targets, 16, 15-31.

:

Nan, X., Xie, C., Yu, X., and Liu, J. (2017). EGFR TKI as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer. Oncotarget 8, 75712.
Citri, A., and Yarden, Y. (2006). EGF–ERBB signalling: towards the systems level. Nature reviews Molecular cell biology 7, 505-516.
Herbst, R.S. (2004). Review of epidermal growth factor receptor biology. International Journal of Radiation Oncology Biology and Physics 59, S21-S26.
Sharma, S.V., Bell, D.W., Settleman, J., and Haber, D.A. (2007). Epidermal growth factor receptor mutations in lung cancer. Nature Reviews Cancer 7, 169-181.
Mazza, V., and Cappuzzo, F. (2017). Treating EGFR mutation resistance in non-small cell lung cancer–role of osimertinib. The application of clinical genetics 10, 49.

AF Gazdar. Activating and resistance mutations of EGFR in non-small-cell lung cancer: role in clinical response to EGFR tyrosine kinase inhibitors. Oncogene. 2009 August ; 28(Suppl 1): S24–S31.doi:10.1038/onc.2009.198
AlyssaM.Krasinskas. EGFR Signaling in Colorectal Carcinoma. Pathology Research International Volume 2011, Article ID 932932, 6 pages doi:10.4061/2011/932932

Morgillo F, Della Corte CM, Fasano M, et al. Mechanisms of resistance to EGFR-targeted drugs: lung cancer. ESMO Open 2016;1: e000060. doi:10.1136/ esmoopen-2016-000060
Zhenfang Du and Christine M. Lovly. Mechanisms of receptor tyrosine kinase activation in cancer. Molecular Cancer (2018) 17:58 https://doi.org/10.1186/s12943-018-0782-4

Marta Román, Iosune Baraibar, Inés López, Ernest Nadal, Christian Rolfo, Silvestre Vicent and Ignacio Gil-Bazo. KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target. Molecular Cancer (2018) 17:33 https://doi.org/10.1186/s12943-018-0789-x

Ammad Ahmad Farooqia, Marc de la Rocheb, Mustafa B.A. Djamgozc,d, Zahid H. Siddike. Overview of the oncogenic signaling pathways in colorectal cancer: Mechanistic insights. Seminars in Cancer Biology, Volume 58, October 2019, Pages 65-79. https://doi.org/10.1016/j.semcancer.2019.01.001

Lung cancer is the leading cause of cancer death,
accounting for one third of all deaths from cancer
worldwide. Like most cancers, lung cancer is a con-
glomeration of diseases of diverse aetiology, broadly
divided into small-cell lung cancer (SCLC, comprising
20% of lung cancers), and non-small-cell lung cancer
(NSCLC, comprising 80% of lung cancers). SCLC is a
tumour of neural crest origin and initially responds well
to chemotherapy, but commonly recurs with resistant
disease. NSCLC is thought to originate in lung epithe-
lial cells, and comprises diverse histological subtypes
including adenocarcinoma, bronchioloalveolar, squa-
mous, anaplastic and large-cell carcinomas1. Most
patients with advanced NSCLC present with metastatic
disease and, if left untreated, have a median survival
after diagnosis of 4–5 months and a 1-year survival of
less than 10% (REF. 2). Combination cytotoxic chemo-
therapy, the treatment of choice in these cases, results
in a modest increase in survival at the cost of signifi-
cant toxicity to the patient3. The advent of molecular-
targeted therapeutics has therefore generated much
optimism, given the perception that the limits of
chemotherapy in NSCLC have been reached and that
further advances in the treatment of NSCLC will have
to involve radically different approaches (reviewed in
REF. 4). Against this backdrop, the approval of small-
molecule inhibitors of the epidermal growth factor
receptor (EGFR) kinase for the treatment of NSCLC
in 2003 was heralded with much fanfare, although
the limitations of their efficacy have become readily
apparent (reviewed in REF. 5).

The deregulation of EGFR in NSCLC
The receptor tyrosine kinase (RTK) super-family of cell-
surface receptors serve as mediators of cell signalling
by extra-cellular growth factors6. Members of the ErbB
family of RTKs, such as EGFR (also known as ERBB1
or HER1), ERBB2 (also known as HER2), ERBB3 (also
known as HER3) and ERBB4 (also known as HER4)
have received much attention, given their strong asso-
ciation with malignant proliferation (reviewed in REF. 7).
Increased levels of EGFR gene expression are observed
in cancers of the head and neck, ovary, cervix, bladder,
oesophagus, stomach, brain, breast, endometrium,
colon and lung, and frequently seem to confer an
adverse prognosis (reviewed in REFS 6,8). Extending
previous observations of almost two decades ago9,10,
recent retrospective analyses have reported EGFR over-
expression in 62% of NSCLC cases, and its expression
is correlated with a poor prognosis8,11,12. In some cases,
genomic analyses documented the amplification of
chromosomal region 7p12, where the EGFR gene is
located13. In addition to EGFR overexpression, its
cognate ligands, epidermal growth factor (EGF) and
transforming growth factor-α (TGFα) are also fre-
quently expressed in NSCLCs, and can establish autocrine
loops that lead to receptor hyperactivity14,15. The disrup-
tion of these autocrine loops is the primary rationale for
antibody-based EGFR-targeted therapeutics16.

Various strategies involving small-molecule inhibi-
tors have also been developed to target EGFR and/or
its family members, and these are in various stages
of clinical testing (reviewed in REF. 17). Gefitinib

*Massachusetts General
Hospital Cancer Center and
Harvard Medical School, 149
13th Street, Charlestown,
Massachusetts 02129, USA.
‡Present address: National
Human Genome Research
Institute, National Institutes
of Health, Bethesda,
Maryland 20892, USA.
Correspondence to D.A.H.
e-mail: haber@helix.mgh.
harvard.edu
doi:10.1038/nrc2088

Neural crest
A pluripotent, ectodermally
derived ridge-like cluster of
cells found on either side of the
neural tube in vertebrate
embryos.

Cytotoxic chemotherapy
Chemicals or drugs that kill
proliferating cells, especially
cancer cells. Their side effects
are typically related to the
inhibition of normal cell
proliferation, with a narrow
window of selectivity for
cancer cells.

Epidermal growth factor receptor
mutations in lung cancer
Sreenath V. Sharma*, Daphne W. Bell*‡, Jeffrey Settleman* and Daniel A. Haber*

Abstract | The development and clinical application of inhibitors that target the epidermal
growth factor receptor (EGFR) provide important insights for new lung cancer therapies, as
well as for the broader field of targeted cancer therapies. We review the results of genetic,
biochemical and clinical studies focused on somatic mutations of EGFR that are associated
with the phenomenon of oncogene addiction, describing ‘oncogenic shock’ as a
mechanistic explanation for the apoptosis that follows the acute treatment of susceptible
cells with kinase inhibitors. Understanding the genetic heterogeneity of epithelial tumours
and devising strategies to circumvent their rapid acquisition of resistance to targeted
kinase inhibitors are essential to the successful use of targeted therapies in common
epithelial cancers.

NATURE REVIEWS | C A N C E R VO LU M E 7 | M A R C H 2 0 0 7 | 169

REVIEWS

© 2007 Nature Publishing Group

Molecular-targeted
therapeutics
Chemicals or drugs that target
known proteins that are
important in cancer cell
proliferation or survival at the
same time as being
dispensable to normal cells.
Although side effects are
typically less severe than with
cytotoxic agents, the effective
inhibition of the target protein
might not translate into
generally effective therapies,
hence the importance of
reliable biomarkers.

Autocrine loop
A mode of cell signalling in
which soluble ligands released
by cells stimulate receptors on
their own cell surfaces.

Reversible inhibitors
Inhibitors that bind non-
covalently with biological
molecules and interfere with
their activity.

(Iressa; AstraZeneca) and erlotinib (Tarceva; OSI
Pharmaceuticals, Genentech), two small-molecule drugs
that specifically target the tyrosine kinase activity of
EGFR (EGFR-tyrosine kinase inhibitors (EGFR-TKIs)),
received fast-track approval from the US Food and Drug
Administration (FDA) in 2003 and 2004, respectively,
for patients with advanced NSCLC who had failed to
respond to conventional chemotherapy5. Both drugs are
reversible inhibitors of the EGFR kinase, designed to act as
competitive inhibitors of ATP-binding at the active site
of the EGFR kinase18,19. The observation that sensitivity
to gefitinib and erlotinib correlated very strongly with a
newly discovered class of somatic activating mutations
in the EGFR kinase domain20–22 explained the unique
subset of drug-responsive cases, notably those aris-
ing in non-smokers and more frequently in women,
individuals of Asian ethnic background and those with
adenocarcinoma and bronchioloalveolar histology (for
a review of the recent clinical literature see Sequist
et al.23). In addition to providing a genetic marker for
a highly EGFR-TKI-responsive subset of NSCLCs, this
correlation has also highlighted the crucial importance
of mutationally activated kinases as anticancer drug
targets (reviewed in REF. 24) (FIG. 1).

In unselected NSCLC samples, EGFR mutations are
present in ~10% of cases in North America and Western
Europe, but ~30–50% of cases in individuals of East Asian
descent, and are associated with most (over 50%) adeno-

carcinomas with bronchioloalveolar features that arise in
non-smokers25–34. EGFR kinase domain mutations target
four exons (18–21), which encode part of the tyrosine
kinase domain (the entire kinase domain is encoded by
exons 18–24) and are clustered around the ATP-binding
pocket of the enzyme25,35–39. Consistent with their pur-
ported role in the aetiology of NSCLC, recent studies
have shown that exon 19 deletions that involve the LREA
motif, L858R, G719S and ins 770(NPG)-mutated EGFR
proteins are oncogenic in both cell culture and transgenic
mouse studies40–42. These mutations also increase the
kinase activity of EGFR, leading to the hyperactivation
of downstream pro-survival pathways, and consequently
confer oncogenic properties on EGFR43–45.

Kinase domain mutations in EGFR are generally
referred to as activating mutations, as they seem to result
in the increased kinase activity of the receptor. However,
this does not imply that these mutated EGFRs are
necessarily constitutively or fully active, as their degree of
ligand independence might be a function of experimental
context40,43,44,46,47. These partially activated mutant EGFRs
can be rendered fully ligand-independent, and therefore
constitutively active, by second site substitutions in EGFR,
such as the T790M mutation in exon 20 (REF. 46). In vitro
biochemical studies using purified recombinant wild-type
and mutant (L858R and ∆E746–A750) EGFR cytoplasmic
domains have shown that mutants have increased kcat
values and an increased Km for ATP48,49. Moreover, as has
been observed in cell-based studies, the mutants show an
increased sensitivity to inhibition by erlotinib (reduced Ki)
in these in vitro kinase assays. The reduced ATP affinity
seen with mutant kinases most probably accounts for their
increased sensitivity to the selective EGFR-TKIs, which
compete with ATP for binding to the catalytic site. Another
study, in which the phage-display method was used to exam-
ine the interaction of a large panel of kinases with selective
inhibitors, concluded that EGFR mutations, includ-
ing ∆E746-A750, do not themselves affect the affinity
for gefitinib and erlotinib50.

EGFR-targeted therapy of NSCLC
For unknown reasons, EGFR kinase domain mutations
seem to be restricted to a subset of NSCLC, although
very rare mutations have also been reported in SCLC,
cholangiocarcinoma, ovarian, colorectal, head and neck,
oesophageal and pancreatic cancers51–56. This Review
discusses the genetic and biochemical determinants of
erlotinib and gefitinib sensitivity in NSCLC. In light
of the rapid acquisition of resistance to these EGFR-TKIs,
we discuss the mechanisms by which resistance might
occur and the possibilities for alternative therapeutics.

Genetic determinants of sensitivity to gefitinib and erlo-
tinib. Early NSCLC clinical trials with gefitinib and erlo-
tinib were modestly encouraging, with partial responses
observed in approximately 10% of treated patients with
NSCLC57–60. Most responses were seen in East Asians,
females or non-smoking patients with NSCLC. These
patients had a high frequency of adenocarcinoma
with bronchioloalveolar features, and many showed a
dramatic and lasting response to second- or third-line

At a glance

• Advanced non-small-cell lung cancer (NSCLC) is the leading cause of cancer-
related deaths in the world.

• Epidermal growth factor receptor (EGFR) is expressed in 50% of NSCLCs, and its
expression is correlated with poor prognosis. These two factors make EGFR and its
family members prime candidates for the development of targeted therapeutics.

• Two EGFR-targeting small-molecule inhibitors, gefitinib (Iressa: AstraZeneca,
approved in May 2003) and erlotinib (Tarceva: OSI-Genentech, approved in
November 2004) received fast-track approval from the US Food and Drug
Administration as treatment for patients with advanced NSCLC who had failed to
respond to conventional chemotherapy.

• Early clinical data showed that 10% of patients with NSCLC responded to
gefitinib or erlotinib. Although infrequent, the speed and magnitude of clinical
responses were unique, as was the fact that they were seen in specific subsets of
cases (non-smokers, women, East Asians and patients with adenocarcinomas with
bronchioloalveolar histology).

• Molecular analysis showed that in most instances, responders harboured specific
mutations in the gene that encodes EGFR. Exon 19 mutations characterized by
in-frame deletions of amino-acids 747–750 account for 45% of mutations, exon 21
mutations resulting in L858R substitutions account for 40–45% of mutations, and
the remaining 10% of mutations involve exon 18 and 20.

• EGFR kinase domain mutations hyperactivate the kinase and confer a dependence
on the mutated kinase for the survival of the NSCLC tumour cells.

• The treatment of sensitive cells with targeted therapeutics such as gefitinib and
erlotinib seems to trigger a form of ‘oncogenic shock’, which is postulated to result
from the differential decay of downstream signals leading to a temporary
predominance of apoptotic signals.

• Acquired resistance to gefitinib and erlotinib might involve the recurrent mutation
T790M which affects the gatekeeper residue in the catalytic domain of the kinase
that weakens the interaction of the inhibitor with its target. Resistance can be
overcome in vitro by irreversible inhibitors of EGFR

R E V I E W S

170 | M A R C H 2 0 0 7 | VO LU M E 7 w w w.nature.com/reviews/cancer
© 2007 Nature Publishing Group

∆E746-A750
∆E746-T751
∆E746-A750 (ins RP)
∆E746-T751 (ins A/I)
∆E746-T751 (ins VA)
∆E746-S752 (ins A/V)
∆L747-E749 (A750P)
∆L747-A750 (ins P)
∆L747-T751
∆L747-T751 (ins P/S)
∆L747-S752
∆L747-752 (E746V)
∆L747-752 (P753S)
∆L747-S752 (ins Q)
∆L747-P753
∆L747-P753 (ins S)
∆S752-I759

L858R

(40–45%)

N826S
A839T
K846R
L861Q
G863D

V765A
T783A

G719C
G719S
G719A
V689M
N700D
E709K/Q
S720P

68
8

72
8

72
9

76
1

76
2

82
3

82
4

87
5

Autophosphorylation

Exon 2 5 1613 17 18–21

28

EGF binding EGF binding Tyrosine kinase

7 22–24

Exon

18

(nucleotide-binding loop)

Exon 19 Exon 21
(activation loop)

Exon 20

D761Y

T790M (50%)*
D770_N771 (ins NPG)
D770_N771 (ins SVQ)
D770_N771 (ins G), N771T
V769L
S768I

TM

(5%)

(45%)

(<1%)

(40–45%)

(5%)(<1%)

Mutations associated
with drug sensitivity

Mutations associated
with drug resistance

Unselected patients
A cohort of patients identified
on the basis of tissue diagnosis
but not correlated with
biomarkers (that is, sequencing
of the EGFR gene was not used
as a selection criterion).

Ligand independence
The activation of a receptor in
the absence of interaction with
its cognate ligand.

Kcat
The overall catalytic rate of an
enzyme (that is, the number of
substrate molecules converted
to product by each catalytic
site per unit of time.

Km
The Michaelis–Menten
constant. Km is a measure of
the affinity of a substrate for an
enzyme, and is the substrate
concentration at half the
maximal velocity of an enzyme.

Ki
The dissociation constant for
the binding of an inhibitor to an
enzyme.

Phage-display method
A method in which proteins or
peptides are displayed on the
surface of filamentous
bacteriophages, which can
then be used to study the
interaction of the peptide with
other proteins or chemicals.

gefitinib or erlotinib monotherapy. The sequencing of
the EGFR gene in tumour samples from these responders
showed somatic gain-of-function mutations20–22 (FIG. 1).
Overall, the incidence of EGFR mutations in NSCLC
among clinical responders to gefitinib or erlotinib is 77%,
compared with 7% in NSCLC cases that are refractory
to gefitinib or erlotinib20–22,28,30,33,61–73. Additional studies
have shown some differences in the clinical outcomes
that are associated with different mutations27,30,74,75. For
example, NSCLCs that harbour exon 19 deletion muta-
tions seem to respond better to gefitinib and erlotinib
than tumours with point mutations in exon 21, such
as L858R30,74,75. So far, insertion mutations in exon 20
have never been found to confer gefitinib or erlotinib

sensitivity in vitro, nor have they ever been reported to
occur in responsive cases, despite the fact that, at least
in some instances (for example, ins 770 (NPG)), they
seem to activate EGFR to a similar degree as sensitizing
mutations in exons 19 or 21 (REF. 40).

Although EGFR mutations were present in most
cases of NSCLC that were identified by virtue of
their dramatic clinical response to TKIs, controversy
has surrounded the predictive value of EGFR muta-
tions in unselected patients31,32,61,69. Approximately
10–20% of patients who do show a partial response
to gefitinib do not have identifiable EGFR muta-
tions, indicating that EGFR mutations are not the sole
determinants of TKI response20,22,28,30,31,33,61–64,68–70,72,73,76.

Figure 1 | Gefitinib- and erlotinib-sensitizing mutations of EGFR in NSCLC. A cartoon representation of epidermal
growth factor receptor (EGFR) showing the distribution of exons in the extracellular domain (EGF binding),
transmembrane domain (TM) and intracellular domain (comprising the tyrosine kinase and autophosphorylation
regions). The cysteine-rich regions in the extracellular domain (EGF binding; purple shaded region) and the tyrosine
kinase region in the intracellular domain (cyan shaded region) are also represented. Exons 18–21 in the tyrosine kinase
region where the relevant mutations are located are expanded (represented by the cyan bar), and a detailed list of EGFR
mutations in these exons that are associated with sensitivity (magenta boxes) or resistance (yellow boxes) to gefitinib or
erlotinib is shown. The most prevalent of EGFR kinase domain mutations, accounting for 45% of EGFR mutations in non-
small-cell lung cancer (NSCLC), are in-frame deletions of exon 19, nested around the LREA string of amino-acids located
between residues 747–750 of the EGFR polypeptide175. Another recurrent mutation is the L858R substitution in exon 21,
within the activation loop of EGFR, which comprises approximately 40–45% of EGFR mutations. Nucleotide
substitutions in exon 18 (for example, G719C or G719S) account for another 5% of EGFR mutations, as do in-frame
insertions in exon 20. The most noteworthy, clinically relevant mutation in exon 20 is T790M, which is detected in 50%
of the cases (denoted by *) as a second site mutation associated with acquired gefitinib and erlotinib resistance25,35–39.
Recently, D761Y, a T790M-like secondary mutation in exon 19 of EGFR (at the border of exon 19 and exon 20), was also
reported to be associated with resistance to gefitinib and erlotinib in NSCLC cells that contain the L858R-EGFR
mutation71,176. Although the inclusion of most of these sensitizing mutations are based on their occurrence in drug
responders, increased biochemical and cellular activity of these mutations has been documented in some cases. The
main mutations in each class are shown in bold type. Data compiled from20–22,28,30,31,33,71,177.

R E V I E W S

NATURE REVIEWS | C A N C E R VO LU M E 7 | M A R C H 2 0 0 7 | 171
© 2007 Nature Publishing Group

a Drug dosing

b Patient selection and inclusion criteria

Gefitinib (ISEL)

Erlotinib (BR21)

Drug (study)

Response to chemotherapy
preceding entry into trial

Criteria for inclusion in ISEL and BR21
clinical trials

150

18

37

45

38

34

28

600 250

150

MTD (mg day–1) Trial dose (mg day–1)

0%
10%
20%
30%
40%
50%
60%
70%
80%
90%

100%

ISEL BR21

ISEL: development of progressive disease
within 90 days of the preceding round of
chemotherapy (early relapse)

BR21: no selection for early relapse

Progressive disease
Stable disease
Partial response

Other molecular abnormalities, including the amplifica-
tion of wild-type EGFR or alterations in other ErbB family
members have been detected, although it is unclear
whether they account for most gefitinib-responsive cases
that lack EGFR mutations31,61,69,77–79. In particular, the
amplification of EGFR has been difficult to interpret by
itself, because gene copy number alterations that affect
both mutant and wild-type EGFR alleles have not been
distinguished in most studies. In addition, inter-study
variability stemming from the different techniques used
to measure copy number, including quantitative PCR
(qPCR), which provides a ‘global’ copy number assess-
ment, and fluorescence in situ hybridization (FISH),
which evaluates copy number at the single cell level,
have yielded divergent results, possibly owing to the use
of different threshold measurements and the distinction
between specific amplification of the EGFR locus versus
more general alterations in gene copy numbers linked to
aneuploidy. Significantly, EGFR kinase mutations seem
to be highly correlated with clinical characteristics that
are predictors of TKI-responsive disease, whereas EGFR
gene amplification, as measured by qPCR, seems to be
more common in smoking-associated cancers, and does
not show the same predilection towards distinct ethnic
background and tumour histology61.

Most retrospective studies to date have reported that
50–80% of EGFR-mutant NSCLCs respond to gefitinib
or erlotinib, and more recent studies from Asia, where
EGFR mutant NSCLC is 2–3 times more prevalent,
have reported responses in more than 75% of NSCLC
cases with mutant EGFR67,80,81. Most significantly,
although improvement in overall survival for the small
fraction of EGFR-mutant cases treated with gefitinib
or erlotinib has not reached statistical significance in
US and European studies, this has been readily appar-
ent in Asian studies with larger fractions of mutant
cases28,31,33,62,69,73. Taken together, the data suggest that a
subset of cases, marked primarily by EGFR mutations
and in some cases EGFR amplification, show dramatic
responses to TKIs. These responses might occasionally
be durable (that is, >3 years), but in most cases they only
last for ~6–12 months before resistant disease recurs.
Given the low frequency of EGFR-mutant NSCLC, a
modest (2–3 months) improvement in overall survival
has been observed in US and European retrospective
trials, driven primarily by the stabilization of disease
rather than tumour shrinkage, which is not tightly
linked to the presence of EGFR mutations31. In these
studies, increased EGFR gene copy number and high
levels of aneuploidy, as measured by FISH, seemed to be
more predictive of disease stabilization after treatment
with TKIs59,82. The effect on overall survival in genotypi-
cally uncharacterized cases was observed with erlotinib
(BR21 trial), but not gefitinib (ISEL trial), contributing
to the withdrawal of gefitinib from the US and European
market and the approval of erlotinib as third-line therapy
in NSCLC irrespective of tumour genotype59,82. A closer
examination reveals differences in the dose of the two
agents, together with differences in the composition
of the patient population that might account for the
observed differences in outcome between gefitinib and
erlotinib83 (FIG. 2). Nonetheless, gefitinib (which is still
in use in Asia) and erlotinib are comparable in virtually
all laboratory analyses, and the appropriate clinical role
of EGFR mutation analysis in the treatment of NSCLC
remains an evolving question, awaiting prospective
studies with adequate tumour analysis.

Biochemical determinants of sensitivity to gefitinib
and erlotinib. Unpublished results from our laboratory
suggest that sensitivity to EGFR-TKIs is not simply
recapitulated by expressing the mutant constructs in
transfected cells, pointing to the importance of cell-
ular context in conferring dependency on the EGFR
pathway. Furthermore, caution should be exercised
in interpreting in vitro data using NSCLC cell lines
as surrogates for clinical responses (FIG. 3). However,
in vitro studies with NSCLC cell lines have highlighted
the fact that gefitinib- and erlotinib-sensitizing muta-
tions invariably hyperactivate the EGFR signalling
pathway and promote EGFR-mediated anti-apoptotic
and pro-survival signals through the Ras–Raf–MEK
(mitogen-activated and extracellular-signal regulated
kinase kinase)–ERK1 and ERK2 (extracellular-signal-
regulated kinase 1 and 2), PI3K–Akt (phosphatidyli-
nositol-3 kinase–Akt) and STAT3 and STAT5 (signal

Figure 2 | Why gefitinib failed in the United States. a | The maximum tolerated dose
(MTD) for gefitinib and erlotinib, and the dose of the two drugs used in the ISEL and
BR21 trials. Although erlotinib was used at its MTD in the BR21 trial, gefitinib was used
at the sub-MTD level of 250 mg a day. b | The composition of the patients and their
response to chemotherapy at the time of recruitment for the ISEL and BR21 trials. The
histogram represents patients with progressive disease (orange), stable disease (green)
and partial response (yellow). Note that the patient pool recruited to the ISEL trial had
a significantly larger percentage of individuals with progressive disease as compared
to patients recruited to the BR21 trial (45% versus 28%), and conversely had a lower
percentage of patients that had a partial response to chemotherapy (18% versus 38%).
Also shown are the inclusion criteria for patient enrollment in both of the trials. In the
ISEL trial, only patients who had progressive disease within 90 days of cessation of
chemotherapy were included, but no similar time-limited exclusion criterion was
implemented for the inclusion of patients in the BR21 trial83. The differences in dosage
used and the patient selection criteria might have contributed to the differences in
outcomes between the two trials.

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Hypersensitive SensitiveSensitivity

Clinical dose of gefitinib (G) or erlotinib (E)

250 mg day–1 150 mg day–1

Insensitive

In vitro
equivalent
(µM) 0.0001 0.001 0.01 0.1 1 10

NSCLC
cell lines

Genetic
signatures

NCI H2170
(200 nM)

NCI H2073
(250 nM)

NCI-H3255 (1 nM)
PC9 (4 nM)

NCI-H1975
(12 µM)

NCI-H1650
(9 µM)

NCI-H460
(20 µM)

EGFR: wild typeEGFR: L858R
EGFR: ∆(E746-A750)

EGFR: T790M
PTEN loss
KRAS

G E

Oncogenic shock
A mechanism to explain
oncogene addiction, in which
the acute inactivation of an
oncoprotein is associated with
differential attenuation rates of
pro-survival and pro-apoptotic
signals emanating from the
oncoprotein, such that
apoptotic signals become
predominant and kill the
cancer cell.

Differential signal decay
A signalling imbalance created
by the rapid decay of pro-
survival signals and
persistence of the relatively
long-lived pro-apoptotic
signals after acute oncogene
inactivation.

transducer and activator of transcription proteins 3
and 5) pathways such that cancer cells might become
dependent on a functional EGFR for their survival43,84–86.
Interestingly, these are the same pathways that are
activated after ligand engagement and are inhibited
by gefitinib, including the ERK pathway involved in
cell proliferation and the pro-survival Akt pathway87–89.
The obvious implication is that shutting off EGFR
with specific kinase inhibitors, antibodies or RNA
interference would extinguish these proliferative and
survival signals on which the tumour cell is dependent,
therefore resulting in tumour cell death. Normal cells
(or non-EGFR-dependent tumour cells that do not
respond to gefitinib or erlotinib) remain unaffected,
as their pro-survival signals are either driven by other
genes or can be compensated for by other RTKs in
the event of EGFR inhibition. This is consistent with the
observation that gefitinib and erlotinib response in sen-
sitive cells results in the downregulation of ERK, Akt and
STAT3 and STAT5, whereas a similar downregulation
is not evident in insensitive or resistant cells43,87–90.

Although these pro-survival signalling pathways are
probably controlled by many RTK outputs in normal
cells, their dependency on mutated and/or activated
EGFR in some NSCLC tumours and cell lines bears the
hallmark of oncogene addiction (BOX 1).

The molecular mechanisms that underpin onco-
gene addiction remain to be elucidated. As commonly
understood, alterations of the signal-transduction
pathways in cancer cells are thought to underlie drug
hypersensitivity91. Based on modelling studies in vitro,
we have recently proposed that unbalanced pro-apop-
totic and pro-survival signals lead to a phenomenon that
we refer to as oncogenic shock, and might account for the
observed apoptotic outcome following the acute inacti-
vation of a crucial oncogene in an addicted cancer cell92
(FIG. 4). According to this model, an addicting oncogene
gives rise to both pro-apoptotic and pro-survival signal
outputs. While the oncogene is active, the pro-survival
signals pre-dominate and keep the pro-apoptotic signals
in check, enabling the survival and proliferation of
the cancer cell. After acute oncogene inactivation, the
relatively short-lived pro-survival signals decay first,
whereas the longer-lasting pro-apoptotic outputs are
maintained during a crucial window of time. Therefore,
differential signal decay leading to a signal imbalance and
a temporary predominance in pro-apoptotic outputs sets
in motion the apoptotic cascade and commits the cell
irrevocably to apoptosis, even if the signalling imbal-
ance is subsequently redressed. In support of the onco-
genic shock model, the apoptotic response to oncogene
inactivation in oncogene-addicted cells is abrogated if
the disruption of oncogene-derived signals is extended
over a period of time, rather than being acute, or if pro-
survival signals are transiently applied during the crucial
window of time following acute withdrawal92. Therefore,
the cell is not hard-wired to depend on a given oncogene,
but rather it requires time to adapt to the loss of such
a signal, and is highly susceptible to apoptosis during
that window of time. The implications of this model
for clinical practice, if confirmed, are considerable, as
it would argue against the co-administration of TKIs
with chemotherapy drugs that, by virtue of their own
effects on DNA-damage checkpoints, might attenuate
the acute effect of growth factor signal withdrawal. For
RTKs like EGFR, it is also possible that the acute effect of
EGFR-TKIs in abrogating kinase activity might be quali-
tatively different from that of anti-receptor antibodies,
which might enable a more gradual signal attenuation,
therefore explaining the differential effect of these two
classes of agents on EGFR-mutant NSCLC86.

Implicit in the oncogenic shock model is the
paradoxical requirement that activated oncogenes gen-
erate pro-survival and pro-apoptotic signals simultane-
ously93. Such a coupling of antagonistic signals is well
documented for Ras94,95, Src96,97, BCR-ABL98, EGFR43,99,
MYC100,101 and even viral oncogenes such as adenoviral
E1A102. Taken together in the context of NSCLC, mutated
EGFR might represent the genetic lesion to which the
tumour is addicted, and the acute withdrawal of these
signals by EGFR-TKIs might trigger oncogenic shock
and tumour cell apoptosis.

Figure 3 | NSCLC cell lines: in vitro surrogates of in vivo drug sensitivity.
Understanding the biochemical basis of sensitivity to gefitinib (G) and erlotinib (E) has
been aided by the generation and use of human tumour-derived non-small-cell lung
cancer (NSCLC) cell lines that show varying degrees of sensitivity to these inhibitors,
ranging from hypersensitive (IC

50
in the low nM; graded magenta box), to sensitive (IC

50

in the high nM; graded orange box) to extremely insensitive (IC
50

in the high µM; graded
yellow box). Representative examples of NSCLC cell lines from each category, including
their distinguishing genetic features, are also shown. The hypersensitive cell lines NCI-
H3255 and PC9 harbour the EGFR tyrosine kinase domain mutations L858R and ∆E746-
A750, respectively. Insensitive cell lines such as NCI-H1975 and NCI-H1650, although
harbouring the same kinase domain mutations (L858R and ∆E746-A750), have
additional changes such as T790M (NCI-H1975), phosphatase and tensin homologue
(PTEN) loss (NCI-H1650) or KRAS mutations in NCI-H460 cells. Although these cell lines
have been used extensively, conclusions derived from such in vitro systems should be
interpreted with caution in view of the off-target effects seen with these inhibitors50,
especially at supra-physiological concentrations, in excess of 1 and 2.5µM for gefitinib
and erlotinib, respectively. The in vitro concentrations used in tissue culture roughly
correlate to the plasma concentrations of these drugs in patients treated with the
standard doses of these agents (250 mg a day of gefitinib and 150 mg a day of erlotinib),
and have been used by researchers as a useful threshold to distinguish sensitive from
insensitive and/or resistant cell lines90,112,178–180.

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Gatekeeper residue
Amino acids with small side
chains found at the catalytic
site of enzymes that, when
mutated to amino acids with
bulkier side chains, can
sterically impede the binding
of a drug at the active site of
the enzyme at the same time
as retaining substrate binding.

Resistance to EGFR-targeted therapy
Not all EGFR kinase mutations are associated with hyper-
sensitivity to gefitinib and erlotinib. An overarching
conclusion that has emerged from studies of primary
insensitivity to EGFR-TKIs is that most cells that
express EGFR will show effective attenuation of EGFR
activity, but only in EGFR-addicted cancers will this be
accompanied by tumour shrinkage. Tumours that fail
to respond to gefitinib or erlotinib despite the presence
of an EGFR mutation might have sustained additional
genetic lesions that relieve this addiction, a mechanism
that could also mediate acquired resistance in previously
sensitive tumours.

Primary resistance. Recent studies suggest that inser-
tion mutations in exon 20 of the EGFR gene might
render the receptor about 100-fold less sensitive to
EGFR-TKIs compared with other sensitizing EGFR
kinase mutations40. It is unclear whether these muta-
tions differ from classical activating mutations in their
downstream signals (therefore attenuating oncogene
addiction), or whether they do not share the dif-
ferential binding affinity to ATP and the inhibitors.
However, these mutations are relatively rare, and in
most cases of NSCLC that fail to respond to EGFR-
TKIs it is likely that genetic lesions other than EGFR
are driving tumorigenesis. In most instances the
T790M mutation is associated with acquired resistance.
However, it has also been linked to primary resistance,
occurring together with a sensitizing mutation in four
unresponsive cases of NSCLC36,39.

About 15–30% of NSCLCs harbour activating muta-
tions in codons 12 and 13 of the KRAS gene103,104. By and
large, KRAS and EGFR mutations seem to be mutually
exclusive in NSCLC, and define distinct subsets of
tumours, with EGFR mutations being characteristic
of tumours that arise in non-smokers 25, whereas KRAS
mutations are more common in smoking-associated
cancers105,106. Mutant EGFR and KRAS might also

have overlapping and/or redundant signalling roles
in NSCLC aetiology25,27,29,107, which might explain the
conspicuous absence of KRAS mutations in EGFR-
TKI-responsive tumours108. Mutations in KRAS have
been proposed as a mechanism of primary resistance to
gefitinib and erlotinib108, although KRAS mutations are
almost always found in NSCLCs with wild-type EGFR.
Therefore, it is difficult to unequivocally determine
whether insensitivity is due to the presence of mutated
KRAS or the absence of mutated EGFR.

Tumour cells that are sensitive to gefitinib and
erlotinib are characterized by a rapid decrease in Akt
activity in response to drug treatment87,88, and failure to
downregulate Akt is a hallmark of insensitivity to the
drugs43,90. The activation of Akt is indirectly regulated by
the tumour suppressor phosphatase and tensin homo-
logue (PTEN), which is frequently lost in human can-
cers109. Although genetic alterations in PTEN are found
in <10% of cases of NSCLC110, the absence of PTEN expression is evident in as many as 70%, and might be mediated by epigenetic mechanisms such as PTEN promoter methylation111. In some cell types, restoring PTEN expression is asso-ciated with increased sensi- tivity to gefitinib or erlotinib, suggesting that it might modulate sensitivity in vivo89,112. Insulin-like growth factor receptor 1 (IGFR1), ERBB3 or activated ERBB2 expression have also been proposed to have a role in mediating resistance to gefinitib113–115. However, recent studies of a large cohort of gefitinib-treated NSCLC cases failed to uncover a correlation between PTEN or IGFR1 status and response to gefitinib treatment, and have questioned the role of these proteins in mediating primary insensitivity to gefitinib116.

Acquired resistance. Despite dramatic responses in EGFR-
mutant cases of NSCLC treated with gefitinib or erlotinib,
the development of drug resistance within 6–12 months of
the initiation of therapy greatly limits the ability of these
drugs to significantly prolong patient survival. A deeper
understanding of the molecular and cellular basis of this
phenomenon is crucial to the future development of alter-
native therapies to overcome this resistance.

A single secondary mutation in EGFR exon 20, T790M,
is present in a subset of EGFR-mutant tumours that
recur after an initial response to gefitinib or erlotinib35–37.
Using allele-specific PCR, the T790M mutation is detect-
able in approximately 50% of patients with NSCLC who
relapse after an initial response to TKIs, although in
some cases the mutation seems to be underrepresented
in the tumour cell genome relative to the total number
of EGFR alleles37,117. This suggests that T790M might
either be present in only a subset of resistant cancer cells,
or might be present only in a minority of copies of the
EGFR gene in each tumour cell39,118. Some studies have
also shown that T790M mutations are present before the
patient is exposed to the drug25,38,56,119,120, thereby sug-
gesting that this mutation might confer some selective
advantage to tumour outgrowth and might be further
selected after the exposure of the tumour to TKIs39.
The T790M mutation in EGFR is structurally analogous
to the mutated gatekeeper residue T315I in BCR-ABL,

Box 1 | Oncogene addiction

The term oncogene addiction was first coined in 2000 by Bernard Weinstein91,168,169
to describe the phenomenon by which a tumour cell, despite many other genetic
alterations, can become completely dependent on a single oncogenic pathway for
its proliferation and/or survival. Implicit in this dependency is the fact that the
tumour cell should be exquisitely sensitive to the targeted inhibition of the
addicting oncogene. Beyond gefitinib- and erlotinib-responsive NSCLC, oncogene
addiction is thought to explain responses of chronic myeloid leukaemia,
gastrointestinal stromal tumours and chronic myelomonocytic leukaemia to
imatinib, which targets the BCR-ABL, c-Kit, and platelet derived growth factor
receptor-β (PDGFRβ) kinases170. Transgenic mouse tumour models have shown a
similar addiction phenomenon, although they are somewhat biased in that the
inducible expression of an oncogene is used to trigger the genesis of a tumour that
is then shown to be dependent on the continued expression of the transgene for its
survival (for example, HRAS in melanoma, KRAS in lung carcinoma and MYC in
lymphoma and leukaemia171–173 (reviewed in REF. 174)). An interesting parallel is the
observation that, in some cases, the continued expression of the transgene does
lead to the emergence of cells that have sustained additional somatic genetic
lesions and have consequently acquired independence from the triggering
oncogene173. A similar mechanism might occur in human cancers that show
resistance to TKIs, despite the presence of the mutated kinase.

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Signal
intensity

Time

Pro-survival signals

Adaptation and
drug resistance

Vulnerable window
of drug sensitivity

Baseline/cancer
cell survival

Oncogenic shock
Period during which
pro-apoptotic signals
exceed pro-survival
signals

Pro-apoptotic signals

Oncoprotein
disruption byTKI

Irreversible inhibitors
Inhibitors that bind covalently
with biological molecules and
interfere with their activity.

T670I in c-KIT and T674I in platelet-derived growth
factor receptor-α (PDGFRα) that weaken the interac-
tion of inhibitors with the kinase and that have previ-
ously been shown to confer resistance to targeted agents
such as imatinib and other ATP-mimicking kinase
inhibitors121–123. Besides T790M, the only other study
of acquired resistance in clinical samples suggests that
an A disintegrin and metalloproteinase 17 (ADAM17)-
mediated heregulin-dependent autocrine loop activates
both ERBB2 and ERBB3 signalling pathways in NSCLC
and mediates resistance to EGFR-TKIs124.

The phenomenon of acquired resistance to gefit-
inib has been modelled in vitro using highly sensitive
NSCLC cell lines with EGFR mutations125. Mechanisms
of acquired drug resistance have been defined in vitro,
including the acquisition of (or selection for) the
T790M mutation118 and altered EGFR trafficking37.
Other possible mechanisms that confer resistance
include amplification of the mutant EGFR or the
hyperactivation of downstream signalling components
that circumvent EGFR inhibition, causing the increased
expression of signal-attenuating molecules or cellular
changes that alter the bioavailability of the drug126.
Some studies have raised the possibility that the multi-
drug resistance protein ATP-binding cassette G2
(ABCG2) might actively pump gefitinib from cells and

therefore confer resistance to the drug127,128, although
others have suggested that gefitinib itself inactivates
the multi-drug transporters ABCG2 and the ABC
transporter P-glycoprotein129–133. These alternative
mechanisms of gefitinib and erlotinib resistance still
await validation in vivo — an issue confounded by the
limited amounts of clinical specimens from recurrent
tumours and the absence of defined genetic lesions that
can be detected in tissue sections.

Alternative EGFR-targeted therapeutics
The development of resistance to EGFR-TKIs calls for
alternative strategies that still target EGFR signalling
but circumvent the insensitivity to kinase inhibitors.
Mutations such as T790M might have far-reaching
implications in the context of various receptor and
non-receptor tyrosine kinases, and represent a general
problem that needs to be overcome in TK-targeted
therapy123. Therefore, one of the main challenges in
the treatment of NSCLC is to design inhibitors that
can overcome the steric interference to drug binding
conferred by the T790M mutation. Irreversible inhibitors
seem to show some promise in this regard (TABLE 1). In
most cases, irreversible inhibitors form a covalent bond
with crucial cysteine residues — Cys797 within EGFR
or Cys805 within ERBB2 — in the active site of the
respective enzymes134,135. Given the fact that only EGFR
and ERBB2 (as opposed to ERBB4) have cysteines at
these corresponding positions, irreversible ErbB inhibi-
tors show very high specificity for EGFR and ERBB2.
Previous studies from our laboratory have shown that
the irreversible dual EGFR and ERBB2 inhibitors,
HKI-272 (REF. 136) and HKI-357 (REF. 37), as well as the
irreversible EGFR inhibitor EKB-569 (REF. 137) were all
able to overcome gefitinib resistance owing to T790M
in cis with an L858R mutation in EGFR37,138.

Interestingly, resistance to irreversible dual inhibitors
is not achieved as rapidly as resistance to gefitinib and
erlotinib in the laboratory37. Similarly, other studies have
shown that the irreversible EGFR inhibitor CL-387,785
(REF. 139), and the irreversible pan-ErbB inhibitor CI-1033
(also known as canertinib)140 can overcome resist-
ance to L858R-mutated EGFR harbouring the T790M
resistance-conferring mutation, whereas the reversible
EGFR and ERBB2 inhibitor GW-572016 (also known as
lapatinib) was ineffective in this regard35,141. CL-387,785
is also able to overcome gefitinib and erlotinib resistance
mediated by in-frame insertions in exon 20 of EGFR40.
A small subset of NSCLCs harbour mutations in ERBB2
(but not EGFR), and tumour cells that harbour the G776
insVG/C in ERBB2, although insensitive to erlotinib,
are sensitive to the EGFR and ERBB2 dual irreversible
inhibitor, HKI-272 (REF. 142). Similarly, a small subset
of NSCLCs that express the EGFR mutant variant III
(EGFRvIII) are also insensitive to gefitinib and erlotinib
but show sensitivity to HKI-272 (REF. 143). HKI-272 is
currently being evaluated in multi-center clinical trials
in NSCLC patients. Therefore, several independent
lines of evidence underscore the use of irreversible erbB
inhibitors, especially for situations in which reversible
inhibitors of EGFR lose efficacy.

Figure 4 | The role of differential signal attenuation in inducing oncogenic shock.
The oncogenic shock model proposes that pro-survival (orange curve) and pro-
apoptotic (red curve) signals emanating from an active oncoprotein in a tumour cell
are normally balanced so that the survival output predominates and results in the
survival of the cancer cell. After the acute disruption of oncogene function by targeted
kinase inhibitors (TKIs), pro-survival signals dissipate very rapidly, whereas pro-
apoptotic signals are relatively longer lived. During this vulnerable window of drug
sensitivity, the longer-lived pro-apoptotic signals gain the upper hand and cause the
cells to irrevocably undergo apoptosis. One possible mechanism by which tumour cells
acquire resistance to a therapeutic target is that they are able to adapt to and
overcome oncogenic shock.

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Although the effectiveness of the irreversible inhibi-
tor HKI-272 against EGFR is assumed to be responsible
for its ability to suppress the proliferation of cells with
acquired resistance to gefitinib and erlotinib, its ability to
target ERBB2 is also of potential interest, given the role
of other ErbB family members in EGFR signalling. ErbB
family members undergo ligand-induced homo- and
hetero-dimerization as a prelude to signal transduction
after receptor activation, with each receptor dimer show-
ing distinct ligand specificity (reviewed in REF. 7). The
complex functional interactions among members of the
ErbB family, combined with the ability to target several
family members, might open new avenues for overcoming
resistance to EGFR inhibitors.

Another strategy to overcome acquired resistance
resulting from the T790M mutation is based on the obser-
vation that various EGFR mutants, including the double
mutant L858R/T790M, associate with the molecular
chaperone heat shock protein 90 (HSP90) (REF. 144). This
interaction can be very specifically disrupted by the ben-
zoquinone ansamysin, geldanamycin, which results in the
degradation of gefitinib- and erlotinib-resistant mutant
EGFR and leads to the apoptosis of EGFR-dependent
tumour cells that harbour the mutated receptors144,145. The
selective HSP90 inhibitor 17-(allylamino)-17-demeth-
oxygeldanamycin (17-AAG) and its derivative IPI-504

(REF. 146) are currently in clinical trials in patients with
advanced solid tumours, and might be useful therapeutic
alternatives for gefitinib- and erlotinib-resistant NSCLC.
By overcoming the gatekeeper-residue mutations, such a
strategy, if successful, might be of value for the treatment
of a broad range of mutant receptor-driven cancers.

Rational combinations: is there a rationale?
At present, gefitinib and erlotinib, either alone or in com-
bination with other regimens, are being evaluated in 157
clinical trials for various cancers (see the NCI clinical
trials website). Despite some caveats147, EGFR-targeted
therapy has not been shown to have any beneficial effects
in combination with standard chemotherapeutic regi-
mens, which begs the question of whether or not there
is a rationale for the combination of therapeutics in the
treatment of NSCLC.

Insights into EGFR-dependent signalling might provide
a first clue to the rational design of combination therapies.
Research over the past 40 years has uncovered some of the
crucial players in the EGFR signal-transduction pathway,
which can be roughly divided into two categories: the pro-
survival arm of the pathway comprising the PI3K–mTOR
(mammalian target of rapamycin)–Akt cascade, and the
proliferative arm consisting of the Ras–Raf–MEK–ERK
pathway148 (FIG. 5). This simplistic framework would

Table 1 | Targeted therapeutics currently approved or being evaluated for the treatment of NSCLC

Class Therapeutic Target Company Stage of development
(tumour type)

EGFR TKI
(single reversible)

Gefitinib (Iressa;
ZD-1839)

EGFR AstraZeneca

Approved (NSCLC)

Erlotinib (Tarceva;
OSI-774)

EGFR OSI, Genentech and
Roche

Approved (NSCLC)

EGFR TKI
(single irreversible)

EKB-569 EGFR Wyeth Phase II * (colorectal)

CL-387,785 EGFR Wyeth Preclinical *

ErbB family TKI
(multiple reversible)

Lapatinib
(GW572016; Tykerb)

EGFR, ERBB2 GlaxoSmithKline Phase III (breast)

ErbB family TKI
(multiple irreversible)

Canertinib (CI-
1033; PD183805)

EGFR, ERBB2,
ERBB4

Pfizer Phase II * (NSCLC, breast)

HKI-272 EGFR, ERBB2 Wyeth Phase I/II * (NSCLC, breast)

BIBW 2992 EGFR, ERBB2 Boehringer
Ingelheim

Phase I/II (breast, prostate,
ovarian)

HKI-357 EGFR, ERBB2 Wyeth Preclinical *

RTK family TKI
(multiple reversible)

ZD-6474 EGFR, ERBB2,
FLT1, KDR

AstraZeneca Phase III * (NSCLC, thyroid)

AEE 788 EGFR, ERBB2,
KDR

Novartis Phase I/II (glioblastoma)

XL647 EGFR, ERBB2,
KDR, EPHB4

Exelexis Phase II (NSCLC)

ErbB family
heterodimerization

BMS-599626 EGFR, ERBB2 Bristol-Myers Squibb Phase I (metastatic solid
tumours)

HSP90 IPI-504 Mutant EGFR Infinity
Pharmaceuticals

Phase I/II * (multiple
myeloma, GIST)

17-AAG Mutant EGFR Kosan Phase I/II * (solid tumours)
*Ability to overcome resistance to gefitinib or erlotinib. EGFR, epidermal growth factor receptor; FLT1, fms-like tyrosine kinase 1;
GIST, gastrointestinal stromal tumour; KDR, kinase domain region; NSCLC, non-small-cell lung cancer; TKI, tyrosine kinase inhibitor;
RTKI, receptor TKI.

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GAP
RAS

SOS

RAF

MEK

ERK mTOR

PTEN

SHIP

AKT

p110

AKT

PDK1

TSC2

PHLPP

GTP

GDP
PIP2 PIP3

Cell growth Protein synthesis Cell survival

ErbB family Extracellular

Intracellular

P
P

p85GRB2

suggest that inhibitors that target different key compo-
nents of this network (in combination with EGFR-TKIs)
might provide greater therapeutic efficacy, particularly
in a setting where EGFR-TKI monotherapy follows a
consistent pattern of diminishing returns and eventually
becomes ineffective. Preclinical studies in an NSCLC cell
line xenograft model have suggested that a PI3K inhibi-
tor, PX-866, sensitizes otherwise insensitive tumours
to gefitinib149 (TABLE 2). Efforts are currently underway to
develop PI3K inhibitors with greater specificity150. The
serine-threonine kinase mTOR lies downstream of PI3K,
and is inhibited by rapamycin and rapamycin analogues
(TABLE 2). Preclinical studies suggest that mTOR inhibitors
might also have synergistic effects when combined with
targeted EGFR inhibitors151,152. At present, combinations
of gefitinib or erlotinib with sirolimus, temsirolimus or
everolimus are undergoing phase I and II evaluation in
patients with advanced NSCLC, recurrent malignant
glioma, prostate cancer and metastatic breast cancer153.
However, despite their dramatic effects in some preclinical
studies (reviewed in REF. 154), monotherapy with mTOR
inhibitors have so far yielded disappointing results in clini-
cal trials155. The selective inhibition of mTOR might in fact
lead to the activation of the PI3K pathway and result in
feedback activation of the pro-survival mediator Akt156,157.
Therefore, recent studies have tried to overcome this

problem through the use of dual inhibitors of PI3K and
mTOR, which seem effective in preclinical studies of
glioma cell lines150. The evaluation of these dual PI3K and
mTOR inhibitors, either as monotherapy or in combina-
tion with targeted ErbB family inhibitors, in clinical studies
might therefore hold considerable promise.

The Ras–MAPK (mitogen-activated protein kinase)
pathway is another important cell-proliferation pathway
downstream of EGFR that is frequently activated in cancer.
Although mutations in Ras oncogenes do not seem
to coexist with EGFR mutations, the pathway might
be important in mediating EGFR-mutant signals, and
therefore the inhibition of Ras or Raf in combination
with gefitinib or erlotinib might have some benefit. The
MEK inhibitor PD-325901 is currently being evaluated
as a single agent in phase II clinical trials in patients
with advanced NSCLC. However, activation of the
Ras–MAPK pathway has not been as well correlated
with response to EGFR inhibitors as the PI3K–Akt
pathway158.

In addition to manipulating components of EGFR sig-
nalling pathways, complementary molecular therapeutic
approaches that involve simultaneously targeting distinct
pathways have potential benefit. Although most of these
approaches are empirical by nature, a rationale does exist
for targeting both the tumour and stromal components of

Figure 5 | Cell-survival pathways downstream of activated erbB receptor tyrosine kinases. Two important cell-
survival pathways that operate downstream of activated ErbB transmembrane receptor tyrosine kinases (represented
by pairs of yellow, and yellow and blue receptors to represent homo- and hetero-dimers, respectively), along with some
of the key constituent signalling molecules are shown. The Ras–Raf–MEK–ERK pathway is shown on the left, and the
phosphatidylinositol 3-kinase (PI3K)–Akt pathway is shown on the right. Key points along the pathway where targeted
inhibition seems to exert a blockade are indicated by red circles, showing the relevant proteins they target (specific
examples cited in TABLE 2). ERK, extracellular signal-regulated kinase; GRB2, growth factor receptor-bound protein 2;
mTOR, mammalian target of rapamycin; SOS, son of sevenless.

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© 2007 Nature Publishing Group

a tumour. Tumour vasculature is a particularly important
target for therapeutic intervention, and it has been the
basis for the development of dual inhibitors of EGFR
and ERBB2 and the vascular endothelial growth factor
(VEGF) receptors FLT1 (fms-like tyrosine kinase 1) and
KDR (kinase domain region). Consistent with this con-
cept, the dual EGFR and VEGFR inhibitor ZD6474159,160
has shown efficacy in tumour xenografts that are resistant
to cetuximab or gefitinib161,162. The use of the EGFR and
VEGFR dual inhibitors ZD6464, AEE788163 and XL647 is
currently being evaluated in clinical trials163,164.

The way forward
Experience with gefitinib and erlotinib has taught us
the following important lessons: first, RTKs can be very
useful targets for therapeutic intervention in epithelial
cancers. Second, the targeted inhibition of RTKs might
only be efficacious in a small subset of patients, and
mutations in RTKs might be one of the useful predic-
tors of response. Third, acquired TKI resistance substan-
tially limits the therapeutic efficacy of these agents. The
first lesson validates the usefulness of drug-discovery
programmes focused on screening for RTK inhibitors,
and reinforces the ongoing efforts of organizations
engaged in this endeavor. However, the second lesson
might point to the need for a reorientation of our tradi-
tional approach to drug discovery. The low frequency of
genetically-defined responsive patient subsets calls for
the consideration of a far broader sampling of individual
cancer types, so as to achieve a representation of genetic
diversity at all levels of analysis, from mutation detection
in RTK genes to identifying new drug targets through
functional assays and screening for efficacy in preclinical
experiments. For example, to detect the 10% response to
gefitinib or erlotinib typically seen in NSCLC patients,
a cell-based drug screen would require a minimum of
100, and ideally 1,000, different NSCLC-derived cell
lines, far beyond traditional cell-based screens. In fact,
given the current cell-based screening strategies that
generally involve a few cell lines representative of each

tumour type, it is likely that gefitinib or erlotinib would
never have been picked up as a ‘hit’. A similar rationale
for increased sampling size can be applied to genetic
analyses of tumour samples in order to detect mutations
in EGFR in an unselected cohort of NSCLC patients.

Recent genetic studies underscore the affect of large-
scale genomic analyses in highlighting the complexity
of the ‘cancer landscape’, and more importantly in pin-
pointing the specific genetic alterations that are key to
the genesis of these tumours. For example, by analysing
120 primary lung tumours, the Sanger Center cancer
kinome sequencing project showed that ERBB2 muta-
tions occur in 4% of lung tumours165. In a recent whole-
cancer-genome sequencing project at Johns Hopkins
University in the United States, the number of distinct
genetic lesions in an individual tumour were fewer than
might have been predicted, but few of these were found
to be recurrent, even among different cases of the same
type of cancer166, pointing to potentially small subsets
of genetically-defined tumours across various histolo-
gies (different needles in different haystacks). Achieving
the ambitious goals of the US National Institutes of
Health (NIH) Cancer Genome Atlas (TCGA), to com-
prehensively annotate all cancer-associated mutations,
might therefore require the analysis of many individual
tumours within each histological type. Complementary
functional approaches, including the use of short hairpin
RNA libraries to identify genes that are essential to cancer
cell viability167, would also need to be applied across a
broad spectrum of different cancer cell lines, each rep-
resenting a different genetic context and potential addic-
tion to a different oncogenic pathway. In summary, as
the lessons learned from EGFR inhibition and cancer
therapy continue to evolve, they have already provided a
powerful example of clinical therapeutic affect achieved
through an understanding of molecular abnormalities
in cellular signalling, at the same time as warning of
the genetic complexity in cancer that will require the
coupling of different therapeutic strategies to individual
genetic variation.

Table 2 | Targeted therapeutics used alone or in combination with EGFR-TKIs

Mode of action Therapeutic Target Company Stage of development
(tumour type)

p110α-specific
inhibition

PX-866 (combination with gefitinib) PI3K ProlX
Pharmaceuticals

Preclinical (NSCLC)

Rapamycin
analogues

Sirolimus (combination with
gefitinib)

mTOR Wyeth Phase I/II (NSCLC,
glioblastoma)

Temsirolimus (CCI-779; combination
with erlotinib)

mTOR Wyeth Phase I/II (glioblastoma)

Everolimus (RAD001; combination
with gefitinib or erlotinib)

FKBP12,
mTOR

Novartis Phase I/II (NSCLC,
glioblastoma, breast)

AP23573 mTOR Ariad Phase I/II (endometrial)

MAPK pathway Sorafenib (BAY49-9006; alone or in
combination with erlotinib)

Raf, (KDR,
p38α?)

Bayer Phase I/II (NSCLC,
glioblastoma)

PD-325901 (single agent) MEK Pfizer Phase II (NSCLC)
Representative examples of different classes of targeted inhibitors that are undergoing evaluation either alone or in combination
with EGFR-TKIs are indicated. KDR, kinase domain region; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated and
extracellular-signal regulated kinase kinase; mTOR, mammalian target of rapamycin; NSCLC; non-small-cell lung cancer; PI3K,
phosphatidylinositol 3-kinase.

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Acknowledgements
The authors would like to thank T.-M. Chin and L. Sequist Van
Dam for their helpful comments. We apologize to our many
colleagues whose work is not cited owing to space constraints.
Another notable omission is the very important area of anti-
body-based ErbB-targeted therapies, which is dealt with very
well in other reviews.

Competing interests statement
The authors declare no competing financial interests.

DATABASES
The following terms in this article are linked online to:
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
ABCG2 | ABL | ADAM17 | BCR | EGF | EGFR | ERBB2| ERBB3 |
ERBB4 | ERK1 | ERK2 | HSP90 | IGFR1 | KDR | KRAS | MEK |
mTOR | MYC | PDGFRα | PI3K | STAT3 | STAT5 | TGFα | VEGF
National Cancer Institute: http://www.cancer.gov
lung cancer

FURTHER INFORMATION
Massachusetts General Hospital Cancer Center:
http://www.massgeneral.org/cancer/
NCI clinical trials website: http://www.cancer.gov/
clinicaltrials
Access to this interactive links box is free online.

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• Epidermal growth factor receptor mutations in lung cancer

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