Epidermal Growth Factor Receptor Messenger RNAExpression,
Gene Dosage, and Gefitinib Sensitivity in Non^Small
Cell Lung Cancer
Rafal Dziadziuszko,1,2Samir E.Witta,1Federico Cappuzzo,1,3Seongjin Park,4Koji Tanaka,5
Peter V. Danenberg,6Anna E. Baro ¤ n,1Lucio Crino,3Wilbur A. Franklin,1Paul A. Bunn, Jr.,1
MarileilaVarella-Garcia,1Kathleen D. Danenberg,7and Fred R. Hirsch1
Purpose: Epidermal growth factor receptor (EGFR) mRNA expression and EGFR gene dosage
by quantitative PCR in tumor samples obtained from patients with gefitinib-treated non^small
cell lung cancer were analyzed in order to determine the association with treatment outcome,
clinical, and biological features [EGFR copy number by fluorescent in situ hybridization (FISH),
EGFR tyrosine kinase mutations, and EGFR protein expression].
transcription-PCRin 64 patients, and EGFR gene dosage was analyzed by real-time quantitative
PCRin 82 patients from paraffin-embedded specimens.
Results: EGFR mRNA expression was higher in responders to gefitinib as compared with non-
responders (P = 0.012). Patients with high EGFR mRNA expression (>5.01) had 43% response
probability, whereas patients with low EGFR mRNA expression had 8% response probability
(P = 0.006). Patients with high EGFR mRNA expression had longer median progression-free
(5.3 versus 2.8 months, P = 0.028) but not overall survival (13.8 versus10.9 months, P = 0.87).
EGFR mRNA expression was higher in FISH-positive patients (P = 0.001) and in patients with
positive EGFR immunostaining (P < 0.001) but not in patients with EGFR mutations (P = 0.19).
EGFR gene dosage did not predict response (P = 0.54), progression-free (P = 0.73), or overall
survival (P = 0.89). EGFR gene dosage was not associated with FISH positivity (P = 0.15),
relative mRNA expression (P = 0.27), EGFR mutation status (P = 0.39), and EGFR protein
expression (P = 0.35).
Conclusion: EGFR mRNA expression is a predictive biomarker for response to gefitinib and to
progression-free survival after gefitinib treatment. EGFR gene dosage is neither predictive for
response nor progression-freenor overall survival.
Novel therapeutic options in non–small cell lung cancer
(NSCLC) include epidermal growth factor receptor (EGFR)
tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib
(1, 2). Erlotinib produced a significant survival advantage
compared with placebo in a randomized phase III trial, but
30% of patients in each arm died in the first 3 months with no
evidence for benefit in at least this fraction of patients (3).
Thus, the search for an optimal selection of patients who will
benefit from these treatments remains important. Patient
selection based on clinical favorable variables is not sufficient,
as some survival benefit was observed in groups with low
response rates such as males and squamous cell histologies
(3, 4). Thus, selection based on molecular markers may be
EGFR gene copy number, EGFR mutation status, and protein
expression have been evaluated for their prediction of response
to EGFR TKIs. In previous studies from our group, high EGFR
copy number evaluated by fluorescent in situ hybridization
(FISH) correlated with improved response and survival of
NSCLC patients treated with gefitinib (5, 6). These findings
were confirmed in subgroup analyses of two phase III clinical
studies, comparing erlotinib (BR.21 trial; ref. 7) or gefitinib
(ISEL trial; ref. 8) with placebo in second- or third-line
treatment of advanced NSCLC. In the NCIC BR.21 trial, the
positive treatment effect of erlotinib was confined to the EGFR
FISH-positive as compared with the FISH-negative group of
patients, both in terms of response rate (20% versus 2%,
respectively) and survival (hazard ratios of 0.44 versus 0.85,
of Colorado Cancer Center, Aurora, Colorado;2Medical University of Gdansk,
Gdansk, Poland;3Bellaria Hospital, Bologna, Italy;4Tokyo Medical and Dental
University,Tokyo;5Mie University School of Medicine, Mie, Japan;6University of
Received1/17/06; revised 3/12/06; accepted 3/14/06.
Grant support: National Cancer Institute grants Cancer Center Core Grant 2P30-
CA46934 and Specialized Program of Research Excellence P01-CA58187. R.
Dziadziuszko was supported by a National Cancer Institute-International Union
AgainstCancerTranslational Cancer Research Fellowship, fundedby National Cancer
Institute. F. Cappuzzo was supported by the Department of Oncology, Bellaria
The costs of publicationof this article were defrayedinpart by the payment of page
charges.This article must therefore be hereby marked advertisement in accordance
Requestsfor reprints: FredR. Hirsch, UniversityofColorado Cancer Center, P.O.
Box 6511, Mail Stop 8111, Aurora, CO 80045. E-mail: Fred.Hirsch@UCHSC.edu.
F2006 American Association for Cancer Research.
www.aacrjournals.orgClin Cancer Res 2006;12(10) May15, 20063078
dosage is larger in Japanese versus Western NSCLC patients
(14, 16), and thus progression-free advantage could more easily
be detected in patients with high EGFR gene dosage from Far
East. In the study of Bell and colleagues (16), high EGFR gene
dosage was not predictive of gefitinib treatment benefit, but
indicated better prognosis of patients regardless of treatment
received. Much larger sample size would be needed in our study
to detect a survival difference of this magnitude.
We did not find a significant correlation between FISH
positivity according to University of Colorado Cancer Center
scoring criteria and EGFR gene dosage by qPCR. The two
measurements are related to the same process—EGFR gene
quantification, but their results yield different information and
cannot be interchangeably used. Potential reasons for this
discrepancy include the direct measurement of gene copy
numbers in single tumor cells by FISH, which cannot be
achieved by qPCR even when microdissection is done, and
relative measurement of EGFR gene dosage to the reference
gene, with no guarantee that the reference gene is always
diploid and not amplified or deleted.
It can be concluded from the current study that high EGFR
mRNA expression analyzed by qRT-PCR is associated with
increased response to gefitinib and prolonged progression-free
survival. Our study supports further evaluation of EGFR mRNA
expression as a biomarker of sensitivity for EGFR TKIs in larger
data sets. In contrast to EGFR gene copy number assessed by
FISH, we could not show any predictive value of EGFR gene
dosage by qPCR for response or survival.
Cancer Therapy: Clinical
www.aacrjournals.org Clin Cancer Res 2006;12(10) May15, 2006 3084
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