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Lung Cancer (2008) 61, 328—339
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/lungcan
Increased epidermal growth factor receptor (EGFR)
gene copy number is strongly associated with EGFR
mutations and adenocarcinoma in non-small cell
lung cancers: A chromogenic in situ hybridization
study of 182 patients
John Wen-Cheng Changa, Hui-Ping Liub, Meng-Heng Hsiehc, Yueh-Fu Fangc,
Meng-Shu Hsiehd, Jia-Juan Hsieha, Yu-Ting Chiue, Hsien-Yu Tsaie,
Yi-Hsuan Chene, Ya-Ting Chene, Hui-Yu Hsu e, Ying-Tsong Chene,
Shih-Feng Tsaie, Yi-Rong Chen e, Bae-Li Hsie, Shiu-Feng Huang e,f,∗
aDepartment of Hematology and Oncology, Chang Gung Memorial Hospital, TaoYuan, Taiwan
bDepartment of Cardiovascular and Thoracic Surgery, Chang Gung Memorial Hospital, TaoYuan, Taiwan
cDepartment of Thoracic Medicine, Chang Gung Memorial Hospital, TaoYuan, Taiwan
dDepartment of Family Medicine, Chang Gung Memorial Hospital, TaoYuan, Taiwan
eDivision of Molecular and Genomic Medicine, National Health Research Institute, 35 Keyan Road, Zhunan, Miaoli 35053, Taiwan
fDepartment of Pathology, Chang Gung Memorial Hospital, TaoYuan, Taiwan
Received 5 November 2007; received in revised form 13 January 2008; accepted 14 January 2008
KEYWORDS
EGFR mutation;
Gene copy number;
Chromogenic in situ
hybridization;
Non-small cell lung
cancer;
Tyrosine kinase
inhibitor
Summary To evaluate the association of epidermal growth factor receptor (EGFR) gene copy
number with EGFR and k-ras mutation status and tyrosine kinase inhibitor (TKI) sensitivity in
non-small cell lung cancer (NSCLC), EGFR gene copy number of 182 NSCLC tumor specimens
were analyzed by chromogenic in situ hybridization (CISH). EGFR and k-ras mutation analyses
were also performed for, respectively, 176 and 157 of the 182 patients. Additionally, 36 patients
in this study had received TKI monotherapy. The tumor was considered to be CISH positive
if the gene copy number was ≥5 signals per nucleus in ≥40% of tumor cells. CISH-positive
tumors were strongly associated with adenocarcinoma (56.8%) compared with squamous cell
carcinoma (15.9%) (p< 0.0001). The CISH-positive tumors were also strongly associated with
EGFR mutations (78%) compared with wild type (20.2%) (p< 0.0001). Only six tumors had k-ras
mutations. None had EGFR mutation and only one was CISH positive. In the patients treated
∗Corresponding author at: Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan,
Miaoli 35053, Taiwan. Tel.: +886 37 246166x35315; fax: +886 37 586459.
E-mail address: sfhuang@nhri.org.tw (S.-F. Huang).
0169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.lungcan.2008.01.009
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EGFR CISH study for NSCLC 329
with TKI, EGFR mutation was strongly associated with TKI responsiveness (22/25 responders)
(p< 0.0001), but the CISH-positive tumors were only marginally significant (18/25 responders)
(p= 0.0665). Patients with EGFR mutations or CISH-positive tumors were all associated with longer
median survival, although not statistically significant. Our results suggest Increased EGFR copy
number was highly correlated with EGFR mutation in adenocarcinoma. Although it is less cor-
related with TKI responsiveness when compared with EGFR mutations, it still could be a good
alternative molecular predictive marker for TKI responsiveness, since CISH can be done on paraffin
section and is much quicker than DNA sequencing.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Lung cancer has become the leading cause of cancer death
worldwide due to its high mortality rate and poor response
to conventional chemotherapeutic agents [1—3]. Thus, it has
become one of the most important malignant tumors for the
development of new anti-cancer drugs. Among the various
new therapeutic agents, targeted cancer therapy by inhibi-
tion of activated oncoprotein kinases using small molecule
drugs or antibodies has become an effective approach for
cancer therapy [4—6]. The rapid and good therapeutic
response of the epidermal growth factor receptor (EGFR)
tyrosine kinase inhibitor (TKI), gefitinib, for 10.4—27.5% of
advanced-stage NSCLC patients in phase II trial has made
TKI an important therapeutic agent for the treatment of
NSCLC, especially in East Asia [7,8]. The discovery of muta-
tions in the ATP-binding site of the EGFR tyrosine kinase
domain disclosed the mechanism of gefitinib responsiveness
in patients with NSCLC [9,10]. Kris has collected a total of
86 such patients reported in the literature, 73 had a par-
tial or complete response, giving the extraordinarily high
response rate of 85% in NSCLC patients with EGFR muta-
tions (ranging between 76% and 92% among the eight studies)
[11]. The EGFR mutation is most commonly seen in East
Asian patients with pulmonary adenocarcinoma. An associa-
tion with high positive prediction rate (ranging between 65%
and 87%) and negative prediction rate (ranging between 69%
and 90%) was reported from Taiwan [12,13], Japan [14,15]
and Korea [16,17]. In a prospective study, a response rate of
75% was observed in NSCLC patients with exon 19 deletion
and exon 21 L858R mutation [18].
On the other hand, the large phase III clinical trial (BR21)
for erlotinib, another TKI, did not demonstrate a significant
association between EGFR mutation and TKI sensitivity or
longer overall survival [19]. In addition, they found that
high polysomy or amplification of EGFR gene was associ-
ated with significantly better erlotinib sensitivity and longer
survival. Four similar retrospective studies by Hirsch et al.
and Cappuzzo et al. also had the similar results, suggesting
EGFR gene copy number change was a better predictor for
TKI sensitivity than EGFR mutation [20—23]. But the above
results have been disputed by Japanese researchers, since
the data from Japan consistently revealed the high predic-
tive value of EGFR mutation for TKI sensitivity and better
overall survival [24].
It is postulated that the rapid anti-tumor response after
EGFR TKI treatment is achieved only when the tumor has the
phenomenon of oncogene addiction [25,26]. That means the
tumor is highly dependent on EGFR for the tumor growth.
The ‘oncogenic shock’, a mechanistic explanation of the
massive apoptosis, would occur following the treatment of
susceptible cells with kinase inhibitors. Mutation and ampli-
fication of EGFR gene both can cause activation of EGFR
tyrosine kinase, and trigger the downstream oncogenic path-
ways [27,28]. Thus, It appears reasonable to suggest that
each of these two phenomena may cause EGFR addiction of
the tumor, which will correlate with TKI sensitivity. It is up
until now unclear about the interaction between amplifica-
tion and mutation of EGFR. From review of the literature,
most of the relevant studies dealing with EGFR gene copy
number change have only compared, independently, the
predictive values for TKI sensitivity and overall survival
between EGFR mutation and gene copy number change in
NSCLC patients. Only a few studies have focused on the
direct correlation between EGFR mutation and gene copy
number change [15,21—23]. Cappuzzo et al. and Hirsch et
al. have shown increased EGFR gene copy number were asso-
ciated with presence of EGFR mutation by FISH [21—23]. But
the FISH(+) rate in the patients with EGFR mutation were
only around 51—65%.
In order to clarify the correlation between EGFR gene
copy number change and mutation in our population, we
have performed EGFR gene copy number study by chro-
mogenic in situ hybridization (CISH) on 182 NSCLC tumor
specimens. All except six tumors in this series also had EGFR
mutation analyses of tyrosine kinase region from exon 18 to
21. In addition, 36 patients in this series had received TKI
monotherapy. Thus, the predictive value for TKI sensitivity
and overall survival of these two molecular markers were
also evaluated.
2. Patients and methods
2.1. Patients received surgical excision and no
EGFR inhibitor treatment
One hundred and fourteen patients received surgical exci-
sion during 2002—2005, and 43 patients received surgical
excision in 1996 for non-small cell lung cancers in Chang
Gung Memorial Hospital were included in this study. All of
these patients had fresh frozen tumor tissue available in
the tissue bank of Chang-Gung Memorial Hospital. Among
them, the EGFR mutation data of 101 patients from the 114
patients had been reported previously [12].
2.2. Patients received EGFR TKI treatment
Thirty-six patients with advanced non-small cell lung can-
cer and received gefitinib or erlotinib monotherapy during
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330 J.W.-C. Chang et al.
2003—2006 were included in this study. All of them had
tumor DNA available for mutation analyses from CT-guided
biopsy or paraffin-embedded tissue and adequate paraf-
fin sections for CISH study. The inclusion criteria were:
informed consent obtained from patients and families, stage
IIIB or stage IV patients with pathologically proven non-
small cell carcinoma, and adequate clinical follow-up data
for evaluation of the treatment response. Among them,
31 patients received 250 mg gefitinib daily, and 5 patients
received erotinib 150 mg daily, which was continued until
disease progression, intolerable toxicity, or death. Two of
the five patients received erotinib had gefitinib first and
changed to erotinib after drug resistance. Twenty-one of
the 36 patients’ EGFR mutation and CISH study data had
been reported previously [29]. But the survival data has been
updated for the current study.
In total, 193 patients were included in the current inves-
tigation. This study was aimed to determine the correlation
of EGFR copy number with the EGFR mutation status, TKI
sensitivity, and other clinicopathological characteristics in
non-small cell lung cancer patients. The study protocol has
been reviewed and approved by the Institutional Review
Board of Chang-Gung Memorial Hospital and National Health
Research Institutes.
2.3. EGFR tyrosine kinase mutation analyses
DNA extraction from fresh frozen tumor tissue or paraffin
embedded tumor tissue was performed according to the
protocol as mentioned previously [12,29]. For EGFR muta-
tion, coding sequences from exon 18 to 21 were amplified
and subjected to direct sequencing as previously described
[12,29].
2.4. k-ras Mutation analysis
For analysis of the k-ras mutation, only coding sequences
of exons 1 and 2 were amplified and subjected to direct
sequencing. The PCR primers used were: E1F: 5-GGT ACT
GGT GGA GTA TTT GAT AG-3; E1R: 5-CAA AGA ATG GTC CTG
CAC CAG-3; E2F: 5-GGA GCA GGA ACA ATG TCT TTT C-3;
E2R: 5-GCA TGG CAT TAG CAA AGA CTC-3. PCR amplification
was carried out in a total volume of 25 l, containing 50 ng
of template DNA. After denaturing at 94 ◦C for 5 min, a total
of 40 thermal cycles were carried out at 94 ◦C for 30 s, 53 ◦C
(exon 1) or 50 ◦C (exon 2) for 45 s and 72◦C for 1 min. Forward
and reverse sequencing reactions were performed using
the same primers for PCR on an ABI3730 genetic analyzer
(Applied Biosystems, CA, USA). Sequence variations were
determined using the Seqscape software (Applied Biosys-
tems). The k-ras reference sequence is based on NM 004985
from the NCBI database. All sequence variations were re-
examined by a second independent PCR amplification and
repeated sequencing reactions.
2.5. Determination of EGFR gene copy number by
chromogenic in situ hybridization (CISH)
The CISH was done on 4-m-thick formalin-fixed paraffin-
embedded tissue sections on coating slides. The EGFR probe
was digoxigenin-labeled (Zymed Inc., South San Francisco,
CA). The procedures were the same as we have reported
previously [30]. Briefly, the unstained tissue sections were
pretreated with 100 mM Tris, 50 mM EDTA, pH 7.0 solution,
and heated to 92 ◦C for 15min, then washed with phosphate-
buffered saline (PBS), followed by enzymatic digestion with
300 l of Digest-all (Zymed Inc.) at 37 ◦C for 10 min. The
digestion was stopped by 10% phosphate buffered-formalin
at room temperature for 1 min. Ten microliter of EGFR probe
(Zymed) was applied to each dehydrated and air-dried sec-
tion, and denatured at 94 ◦C for 3min. The hybridization was
performed overnight at 37 ◦C in a humidified chamber. Post-
hybridization washes were done with 0.5×standard saline
citrate (SSC) at 72 ◦C for 5 min, and then rinsed in PBS with
0.25% Tween 20 (Sigma—Aldrich Co., St. Louis, MO). Detec-
tion was done with a CISH Detection Kit (Zymed) according to
the manufacture’s recommended protocol. The non-specific
staining was blocked with Cas-Block (Zymed) for 10 min, fol-
lowed by incubation with mouse anti-digoxigenin antibody
for 1 h, and then incubated with HRP conjugated goat anti-
mouse antibody (Zymed) for 30 min. The DAB was used as
chromogen for 20 min. Finally, the tissue sections were coun-
terstained with hematoxylin for 30 s.
The CISH study results were evaluated with a regular
light microscope (Nikon E600) and counted by two scien-
tists (SF Huang, BL Hsi). At least 100 non-overlapping and
intact tumor nuclei were evaluated. The tumor was consid-
ered as CISH positive (have significant genomic gain) if the
copy number was ≥5 signals per nucleus in more than 40%
of tumor cells, which was modified from the criteria set by
Hirsch et al. [20,21]. We have modified the criterion from
≥4 copies to ≥5 copies per nucleus, since 4 copies was very
easy to be confused with two partially overlapping disomy
signals of two nuclei, which was based on our previous expe-
riences of FISH or CISH studies on prostate adenocarcinoma,
hepatocellular carcinoma and neuroblastoma [30—33]. The
tumor was considered as CISH negative (have no significant
genomic gain), if the copy number of the EGFR gene was ≤4
signals per nucleus in more than 60% of tumor cells.
2.6. Definition of responsiveness to TKI treatment
For evaluation of the response, plain chest X-ray was taken
every week for 4 weeks, and at least monthly thereafter.
Computed tomography scan was performed at base line and
every 3 months. Response was evaluated according to the
Response Evaluation Criteria in Solid Tumors (RECIST) cri-
teria [34]. In addition to patients with partial or complete
responses, patients with stable disease lasting more than
3 months were classified as clinical benefit and were also
considered as responders. Patients with progressive disease
or stable disease shorter than 3 months were classified as
non-responders.
2.7. Statistical analysis
Fisher’s exact test or Pearson’s chi-square test were used to
examine differences in major clinicopathological features,
TKI responsiveness, and EGFR mutation patterns in patients
with CISH positive or CISH negative tumors. Survival was
assessed using the Kaplan—Meier product-limit method. The
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EGFR CISH study for NSCLC 331
Table 1 Clinicopathological features associated with increased EGFR gene copy numbers in 182 non-small cell lung cancer
patients
EGFR copy no. ≥5EGFR copy no. ≤4p-Value*
Patient no. 83 99
Gender
Male 41 64 0.0381
Female 42 35
Age (years) 0.5563
≥60 53 59
≤59 30 40
Smoking 0.0267
Never 63 58
Former 19 38
Stage 0.2523
I2843
II 7 12
III 25 27
IV 23 17
Histology type <0.0001
Adenocarcinoma 71 57
Non-adenocarcinoma 12 42
EGFR <0.0001
Mutation 60 17
Wild type 20 79
*p-Values by Pearson’s chi-square test.
log rank test was used to assess between-group differences.
A two-sided p-value less than 0.05 was considered statisti-
cally significant. All of the analyses were performed using
SAS Version 9.0 (SAS Institute Inc., Cary, NC).
3. Results
3.1. EGFR copy number and clinical characteristics
A total of 193 patients were included in this study. However,
CISH study failed in 11 cases. One from the 114 patients
received surgery during 2002—2005, and 10 from the 43
patients received surgery in 1996. Poor tissue DNA preserva-
tion is probably the main reason for failure of hybridization.
Thus, A total of 182 patients have EGFR gene copy number
data by CISH study. The clinicopathological characteristics
associated with EGFR gene copy number change are shown
in Table 1. The CISH positive tumors were significantly asso-
ciated with female gender (p= 0.0381) and never-smokers
(p= 0.0267), but not associated with age or disease stage.
The CISH positive tumors were also strongly associated
with adenocarcinoma (56.8%) compared with squamous cell
carcinoma (15.9%) (p< 0.0001), but had no significant differ-
ence with other histology types (p= 0.2136) (Table 2). The
patterns of EGFR gene copy number change in tumors are
quite variable (Fig. 1A—F). In addition to the typical pic-
tures of ≥5 signals per nucleus, some tumor cells revealed
only one large dot signal, which was much bigger than the
regular-sized signal for one copy of EGFR gene (using the
signal size of the adjacent benign stromal cells or lympho-
cytes as control). Thus, we also classified these tumors as
being CISH positive (Fig. 1E and F). Even in the same tumor,
the signal patterns could also be different. While tumor
cells in one area had ≥5 signals per nucleus, the tumor
cells in the adjacent area of the same tumor could have
only one large dot signal (Fig. 1G). Features of more than
>10 copies or large dot signal (amplification) was frequently
Table 2 The association of increased EGFR gene copy numbers with pathology type of 182 non-small cell lung cancers
Pathology diagnosis EGFR copy no. ≥5 CISH(+) EGFR copy no. ≤4 CISH (−)p-Value*p-Value**
Adenocarcinoma 71 (56.8%) 54 (43.2%) 0.1284
Squamous cell carcinoma 7 (15.9%) 37 (84%) <0.0001 <0.0001
Others 5 (38.5%) 8 (61.5%) 0.4054 0.2058
All p-values were by Pearson’s chi-square test.
*The p-value of CISH(+) versus CISH(−) in the same pathology type.
** The p-value of CISH(+) versus CISH(−) using adenocarcinoma group as a reference group.
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332 J.W.-C. Chang et al.
Fig. 1 EGFR gene copy number determined by Chromogenic in situ hybridization (CISH). (A) An adenocarcinoma with no EGFR
mutation and two signals per nucleus (640×). (B) A squamous cell carcinoma with no EGFR mutation and two signals per nucleus
(640×). (C) An adenocarcinoma with L858R mutation and ≥5 signals per nucleus (400×). (D) An adenocarcinoma with delE746 A750
mutation and EGFR amplification, numerous EGFR signals are seen in most of the tumor nuclei (400×). (E) An adenocarcinoma with
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EGFR CISH study for NSCLC 333
Table 3 The Association of Increased EGFR gene copy numbers with EGFR mutation type in 176 NSCLC
EGFR mutation EGFR copy no. ≥5EGFR copy no. ≤4p-Value*p-Value**
Wild type 20 (20.2%) 79 (79.8%) <0.0001
L858R 23 (79.3%) 6 (20.7%) 0.0016 <0.0001
Exon 19 deletion 23 (85.2%) 4 (14.8%) 0.0003 <0.0001
Others 14 (66.7%) 7 (33.3%) 0.1266 <0.0001
All p-values were by Pearson’s chi-square test.
*The p-value of CISH(+) versus CISH(−) in the same EGFR mutation type.
** The p-value of CISH(+) versus CISH(−) using wild type EGFR as a reference group.
seen in exon 19 deletion and L858R mutation, especially in
the former (data not shown). Tumor heterogeneity was seen
occasionally when the tumor had two or more histopatho-
logical patterns. One pattern might have less than 4 copies
per nucleus while the other had more than five per nucleus.
The tumor cells of the less differentiated histological type
usually had higher copy numbers.
CISH can have good preservation of tissue morphology
for differentiation between tumor and non-tumor tissue and
is very easy to check the copy number change under the
light microscope. This is the major advantage of CISH over
fluorescence in situ hybridization (FISH). In this study, when
the tumor cells had more than 10 copies per nucleus, it was
very easy to be identified even just under the 40×low power
field (Fig. 1H).
3.2. EGFR copy number and EGFR mutations
Among the 182 patients with CISH data, all except 6 patients
also had successful EGFR mutation analyses from exon 18 to
21. The CISH positive tumors were strongly associated with
EGFR mutations when compared with wild type (p< 0.0001)
(Tables 1 and 3). When we further compared the association
with different EGFR mutations, the L858R (79%) and exon
19 deletion (82.6%) had highest association (Table 3). For
other types of mutations or multiple mutations containing
L858R or exon 19 deletion, only 66.6% of tumor were CISH(+),
which remained significantly higher than wild type. On the
whole, there were still 25% of the CISH positive tumors with
no detectable EGFR mutations.
To understand whether the cut-off criteria for CISH(+)
would change the significance of association with EGFR
mutation or not, we have evaluated the association with
different cut-off criteria, i.e. ≥3 copies, ≥4 copies and ≥5
copies in >40% of tumor cells by chi-square test. We found
that their associations were all statistically significant. The
p-value were 0.0005, <0.0001 and <0.0001, for ≥3 copies,
≥4 copies and ≥5 copies, respectively. But the chi-square
value was highest for ≥5 copies (12.21, 20.46 and 56.46 for
≥3 copies, ≥4 copies and ≥5 copies, respectively). We then
used these 3 cut-off criteria to evaluate the association of
CISH(+) tumor with different pathology types. When we used
≥4 copies as cut-off, 28 of the 44 squamous cell carcino-
mas (63.64%) and 102 of the 125 adenocarcinomas (81.6%)
would become CISH(+). If we used ≥3 copies as cut-off, 34
of the 44 squamous cell carcinomas (77.27%) and 112 of the
125 adenocarcinomas (89.6%) would become CISH(+). Thus,
when using ≥3 copies or ≥4 copies as cut-off, the associa-
tion rate with CISH(+) would became similar for squamous
cell carcinoma and adenocarcinoma, which was dramatically
different from the result when using ≥5 copies as cut-off
(Table 2).
3.3. EGFR copy number and k-ras mutations
Among the 182 patients with CISH data, 157 cases had ade-
quate DNA and successful k-ras mutation analyses of exons
1 and 2. Totally only six patients had somatic mutations
of k-ras. Five had codon 12 mutation, and one had codon
19 mutation. Three were previous smoker and three were
never-smokers. Five were adenocarcinoma (two were G12D,
three were G12V), and one was squamous cell carcinoma
(L19F). None of them had EGFR mutation and only one ade-
nocarcinoma (G12V) was also CISH positive. This patient had
progressive disease to gefitinib treatment.
3.4. EGFR copy number and TKI responsiveness
Among the 36 patients received TKI treatment, 11 had
progressive disease, 20 had partial response, one had sero-
logical response, and 4 had clinical benefit. Three of the
four patients with clinical benefit survived more than 2 years
and were still alive during the last follow-up. The remaining
one survived 13.1 months. Thus, there were 25 responders
totally. EGFR mutations were detected in 24 patients. The
clinical characteristics, EGFR mutation and copy number
associated with TKI response is shown in Table 4. The TKI
responsiveness was strongly associated with EGFR mutation
(p< 0.0001), significantly associated with adenocarcinoma
(p= 0.0231), and only marginally significant in association
with increased EGFR gene copy numbers (p= 0.0665). The
clinical characteristics associated with EGFR gene copy num-
bers of this smaller series is shown in Table 5. Since 33 of the
delE746 A750 mutation and a giant dot signal in most of the tumor nuclei (400×). (F) A large cell carcinoma with no EGFR
mutation and a giant dot signal in ≥50% of the tumor nuclei (400×). (G) An adenocarcinoma with L858R mutation. Two
patterns of CISH signal are seen among the tumor cells in one tumor gland. One pattern is a large dot signal and another
is ≥5 signals per nucleus (400×). (H) An adenocarcinoma with delE746 A750 mutation and marked EGFR amplification. The
histopathology type of the lung cancer and the EGFR amplification can be easily identified under very low power field (40×).
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334 J.W.-C. Chang et al.
Table 4 Clinical features associated with EGFR TKI responsiveness in 36 non-small cell lung cancer patients
Responders Non-responders p-Value
Patient no. 25 11
Gender 0.7182**
Male 12 4
Female 13 7
Age (years) 0.7175*
≥60 12 6
≤59 13 5
Smoking 0.7148**
Never 16 8
Former 9 3
Performance 0.3628**
000
1197
263
300
401
Stage 0.5705**
IIIb 2 2
IV 23 9
Histology type 0.0231**
Adenocarcinoma 25 8
Non-adenocarcinoma 0 3
EGFR <0.0001**
Mutation 22 2
Wild type 3 9
EGFR copy no. 0.0665**
≥5184
≤477
*p-Value by Pearson’s chi-square test.
** p-Value by Fisher’s exact test.
36 cases were adenocarcinoma, only EGFR mutation was sig-
nificant association with increased EGFR gene copy number
(p= 0.0156). In addition, CISH positive tumors were also sig-
nificantly associated with better performance (p= 0.0146),
which would need more data to clarify its value.
3.5. EGFR copy number and survival after TKI
treatment
The overall survival was compared between responders
and non-responders, patients with and without EGFR muta-
tions, and CISH positive versus CISH negative tumors
(Fig. 2A—C). There was significantly better survival for
responders (p= 0.016). The median survival for responders
versus non-responders were 16 and 7.5 months, respectively.
For EGFR mutation patients, although the median survival
(16.0 months) was better than wild type (9.465 months),
but statistically non-significant (p= 0.3283). For CISH posi-
tive patients, they also showed longer median survival (13.1
months) than CISH negative patients (9.665 months), but
also statistically non-significant (p= 0.7201).
4. Discussions
To the best of our knowledge, there had been only 9 pub-
lished reports on EGFR gene copy number change related
to EGFR mutation and TKI sensitivity in NSCLC patients
[15,19,20—23,35—37]. The criteria of increased gene copy
number were not all the same and the correlation with TKI
sensitivity and overall survival were also quite different. We
have summarized the results of the nine studies and our
results in Table 6. In many of the previous reports, although
the patient number of the whole series was large, the num-
ber of positive case with mutation or increased gene copy
number were small, which was even smaller when eval-
uated for TKI response. This was mainly due to the low
EGFR mutation rate and low TKI response rate in NSCLC
patients of western countries. In our study series, totally
83 of the 182 tumors (45%) were CISH positive and 77 of
the 176 tumors (42.3%) with EGFR mutation analysis data
had EGFR mutation. The absolute numbers of the patients
with EGFR mutation or high gene copy number (CISH posi-
tive) were both the largest in all reported series due to high
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EGFR CISH study for NSCLC 335
Fig. 2 (A) Kaplan—Meier plot of overall survival for tyro-
sine kinase inhibitor responders versus non-responders. The
median survivals were 16 and 7.5 months, respectively. (B)
Kaplan—Meier plot of overall survival for patients with EGFR
mutations versus wild type. The median survivals were 16 and
9.465 months, respectively. (C) Kaplan—Meier plot of overall
survival for patients with CISH positive versus CISH negative
tumors. The median survivals were 13 and 9.665 months,
respectively.
EGFR mutation rate in our NSCLC patients (Table 6). Thus,
our patient collection should be quite ideal for studying the
association between EGFR mutation and gene copy number
change. For the TKI sensitivity study, although 36 patients
was a relatively small series, it included 24 patients with
EGFR mutation, 22 with increased gene copy number, and
25 with TKI response. The absolute number is, therefore,
comparable to those of previous reported series (Table 6) for
comparison between EGFR mutation and gene copy number
change regarding TKI sensitivity.
In this 182 cases series, we have demonstrated the high
correlation between EGFR mutation and increased gene
copy number (Table 1). From Table 6, it is shown that even
though the criteria for significant increase of EGFR gene
copy number were not all the same, their goals (significant
genomic gain) were similar and the positive rates were also
similar in most of the nine studies. The most commonly used
criterion were ≥4 copies in ≥40% tumor cells or amplifica-
tion (gene to chromosome level ≥2.1) by dual-colored FISH,
established by Hirsch et al. [20,36]. Five of the nine EGFR
gene copy studies were using this criterion. We have modi-
fied the criterion from ≥4 copies to ≥5 copies per nucleus,
since 4 copies was very easy to be confused with two par-
tially overlapping disomy signals of two nuclei. Interestingly,
our study results also supported this new criterion. We have
evaluated the association with EGFR mutation by different
cut-off criteria, i.e. ≥3 copies, ≥4 copies and ≥5 copies
in >40% of tumor cells. Even though the associations were
all statistically significant for these three cut-off criteria
(the p-value were 0.0005, <0.0001 and <0.0001, respec-
tively), the chi-square value (56.46) was much higher for
≥5 copies than the other two cut-off criteria (12.21 for ≥3
copies and 20.46 for ≥4 copies, respectively), suggesting
stronger association. In addition, if we used ≥3 copies and
≥4 copies as cut-off, the association rate with CISH(+) would
be markedly increased for squamous cell carcinoma (77.27%
and 63.64%, respectively), which was dramatically different
from the result when using ≥5 copies as cut-off (15.9%).
Since EGFR mutation is rare in squamous cell carcinoma
[12,14], increased percentage of squamous cell carcinoma in
CISH(+) tumor would definitely reduce the association with
EGFR mutation as shown by the chi-square value. This could
also explain why that none of the previous studies using ≥4
copies as cut-off have demonstrated a significant association
of increased EGFR gene copy number with adenocarcinoma
(Table 6).
There have been two studies using quantitative PCR (Q-
PCR) for detection of increased gene copy numbers. One
used >4-folds [35], the other used >3 copies [15]. Although
the criteria were quite different, they all show significant
correlation between increased EGFR gene copy number and
EGFR mutations similar to our results. In the study by Takano
et al., all tumors with >6 copies of EGFR gene per cell had
EGFR exon 19 deletion [15]. The above results would sug-
gest that, to compare the EGFR gene copy number change
between NSCLC with and without EGFR mutation, using
higher copy number per nucleus as cut off line would be
better. This issue is important since it may also affect the
correlation between EGFR gene copy number change and
TKI sensitivity. The study by Dziadziuszko et al. also used
Q-PCR to study EGFR gene dosage effect related to gefitinib
sensitivity [37], but they used a very low cut off line (gene
Author's personal copy
336 J.W.-C. Chang et al.
Table 5 Clinical features associated with increased EGFR gene copy numbers and gefitinib responsiveness in 36 non-small cell
lung cancer patients
EGFR copy no. ≥5EGFR copy no. ≤4p-Value
Patient no. 22 14
Gender 0.8785*
Male 10 6
Female 12 8
Age (years) 0.1715*
≥60 9 9
≤59 13 5
Smoking 0.7272**
Never 14 10
Former 8 4
Performance 0.0290**
000
1197
236
300
401
Stage 0.6340**
IIIb 2 2
IV 20 12
Histology type 0.5471**
Adenocarcinoma 21 12
Non-adenocarcinoma 1 2
EGFR 0.0286**
Mutation 18 6
Wild type 4 8
Responsiveness 0.0665**
Responder 18 7
Non-responder 4 7
*p-Value by Pearson’s chi-square test.
** p-Value by Fisher’s exact test.
dosage >0.36, median value of all tumors). It is expectable
why they found EGFR gene dosage could not correlate with
gefitinib response or FISH results at all, and concluded that
EGFR gene dosage was not a good TKI response predictor.
Since their standard for increased EGFR gene dosage was
much too low when compared with all of the other EGFR
gene copy number studies in NSCLC (Table 6).
Now that EGFR gene copy number determined by FISH,
CISH or Q-PCR all could show correlation with EGFR muta-
tion or TKI response by previous studies and our results,
dual-colored FISH detecting both EGFR gene and chromo-
some 7 centromere copy numbers at the same time would
appear not so necessary and CISH (detecting EGFR gene copy
number only) would be technically easier for evaluating the
gene copy number due to better histopathology background
morphology (Fig. 1A—H) and more convenience (using light
microscope in stead of fluorescence microscope).
In addition to the high correlation between EGFR muta-
tion and increased gene copy number, we also found only
L858R mutation (79.3%) and exon 19 deletion (85.2%) were
significantly associated with increased EGFR gene copy num-
ber by itself. Other types of EGFR mutations, including
those double or multiple mutations with an L858R mutation
or an exon 19 deletion, do not have significant correla-
tion with high EGFR gene copy number (p= 0.1266), even
though their association was still significantly higher than
wild type (Table 3). The above results would strongly sug-
gest that there was a causative linkage between these two
most common mutation types and increased gene copy num-
ber of EGFR, i.e. the mutation occurred first and induced
gene amplification. The few exceptions that had no increase
of EGFR gene copy number in our series might be the
effect of individual host’s genetic background. But for other
EGFR mutations, the consequences were more variable,
and not all of them were able to induce EGFR amplifica-
tion.
In this study, we only found 6 k-ras mutations in 157
patients (3.8%) and only one was CISH positive tumor, which
was a patient with gefitinib resistance. Since the incidence
of k-ras mutation was so low, it probably plays no impor-
tant role for TKI sensitivity or overall survival in our NSCLC
patients.
Author's personal copy
EGFR CISH study for NSCLC 337
Table 6 Comparison of the 10 EGFR gene copy number studies in non-small cell lung cancers
Author,year (ref. no.) Patient ethnicity Detection
method
Criteria for increased
gene copy number
Increased EGFR gene copy number (polysomy) EGFR mutation
Case no. (%)aTotalADC
(%)
Polysomy in
ADC (%)
Polysomy in
SCC (%)
Correlation
with responseb
Correlation
with OS
Case no.
(%)d
Correlation with
responsee
Correlation
with OS
Polysomy in
mutation (%)
Hirsch et al. 2003 [36] U.S. FISH Gene to chromosome
level ≥2.1
16/183 (9%) 79 (48%) 3/79 (4%) 10/89 (11%) NA NA NA NA NA NA
Cappuzzo et al. 2005 [23] Italy FISH ≥4Copies in ≥40%
tumor cells or
amplification
33/102 (33%) 63(61.7%) 22/63 (33.3%) 9/26 (34.6%) (+), p<0.001, 12/33
versus 2/69
(+), p=0.03 15/89(17%) (+), p< 0.001 8/15
versus 4/74
(−)f,p=0.09 9/14 (63%)
Hirsch et al. 2005 [20] U.S. FISH Same as above 26/81 (32%) 81 (100%) 26 (32%) NA (−), p= 0.14, 5/19
versus 4/36
(+), p=0.04 NA NA NA NA
Hirsch et al. ISEL 2006 [21] Multi-Asian 3% FISH Same as above 114/370 (30.8%) 156 (42.2%) 52/156 (33.3%) 62/214 (29%) in
non-ADC
(+), p=0.001, 11/67
versus 5/155
(−)c,p=0.067 26/215 (12.1%) (+),p= 0.0001 6/16
versus 3/116
NA 13/20 (65%)
Hirsch et al. 2007 [22] U.S. and Italy FISH Same as above 59/183 (31.7%) 143 (78%) 47 (32.8%) 9/26 (34.6%) (+),p< 0.001, 19/59
versus 8/124
(+), p=0.002 43/157 (27.3%) (+), p< 0.001 17/43
versus 8/114
(−), p=0.19 18/35 (51%)
Tsaoet al. BR21 2005 [19] Multi-Asian 6% FISH Sameas above 56/125 (45%) 67 (54%) 48% 41% in non-ADC (+), p=0.03, 5/25
versus 1/41
(+), p=0.008 40/177 (23%) (−), p= 0.37 3/19
versus 6/81
(−), p=0.45 NA
Dziadziuszko, et al. 2006 [37] Italy Q-PCR Gene dosage >0.36 41/82 (50%) 53 (65%) 27 (51%) 14/29 (48.3%) in
non-ADC
(−), p=0.78, 4/41
versus 5/39
(−), p=0.89 13/82 (16%) NA NA NA
Bell et al. IDEAL Multi-Asian 28.9% IDEAL 7/90 (8%) 55 (61.1%) 14/79 (18%)
INTACT 2005 [35] Multi-Asian 2.9% Q-PCR ≥4-Fold INTACT 33/453 (7%) 220(48.6%) 19/275 (6.9%) 21/267(7.8%) in
non-ADC
(−), p=0.319, 2/7
versus 12/79
(−)c32/312 (10%) (+), p= 0.005 6/13
versus 6/61
(−)fNA
Takanoet al. 2005 [15] Japan Q-PCR ≥3 Copies 29/66(44%) 62 (93%) NA NA (+),p= 0.005, 21/29
versus 14/37
(−), p=0.33 39/66 (59%) (+), p< 0.0001
32/39 vs3/27
(+), p=0.0001 22/39 (56%)
Chang et al. this study Taiwan CISH ≥5 Copies in ≥40%
tumor cells
83/182 (45%) 125(68.6%) 71 (56.8%) 7/44 (15.9%) (−), p=0.0665,
18/22 versus 7/14
(−)c,p=0.72 77/176 (43.1%) (+),p< 0.0001
22/24 versus 3/12
(−)f,p=0.328 60/77 (78%)
Ref. no., reference number; ADC, adenocarcinoma; SCC, squamous cell carcinoma; OS, overall survival; FISH, fluorescence in situ hybridization; CISH, chromogenic in situ hybridization; Q-PCR, quantitative PCR; Multi-, patients from multiple countries; NA, not available; ISEL, INTACT, IDEAL: three
phase III clinical trial for gefitinib; BR21, phase III clinical trial for erlotinib.
aThe number of patients with increased EGFR gene copy number over the total number of patient with EGFR gene copy number data in the study and its percentage.
bThe p-value and the original case number (the number of patients with TKI response and increased EGFR gene copy number over the number of total patients with increased EGFR gene copy number versus the number of patients with TKI response but no increased EGFR gene copy number
over the number of total patients with no increased EGFR gene copy number).
cPatients with increased EGFR gene copy number had longer median survival than patients without increased EGFR gene copy number,but statistically non-significant.
dThe number of patients with EGFR mutation over the total number of patients with EGFR sequence data in the study and its percentage.
eThe p-value and the original case number (the number of patients with TKI response and EGFR mutation over the number of total patients with EGFR mutation versus the number of patients with TKI response but no EGFR mutation over the number of total patients with no EGFR mutation).
fPatients with EGFR mutation had longer median survival than patients without EGFR mutation, but statistically non-significant.
Author's personal copy
338 J.W.-C. Chang et al.
In this study, we also demonstrated that CISH-positive
tumors were strongly associated with adenocarcinoma
(56.8%) compared with squamous cell carcinoma (15.9%)
(p< 0.0001), but no significant difference with other histol-
ogy types (Table 2). Since we had found a high correlation of
the CISH-positive tumors with EGFR mutations, it would be
easy to suggest that the association was due to high EGFR
mutation rate in adenocarcinoma. However, Totally, 7 of the
44 squamous cell carcinomas were also CISH positive, but
only one of the 7 tumors had mutation (delE746 A750). Thus,
in squamous cell carcinoma, the mechanism of increased
EGFR gene copy number was not related to EGFR mutation
in most cases.
We also analyzed the correlation of EGFR copy number
change with TKI response and overall survival in 36 patients.
Our results demonstrated that increased EGFR copy num-
ber was less correlated with TKI responsiveness and overall
survival when compared with EGFR mutations (Table 4 and
Fig. 2). Our survival data of CISH positive tumor was similar
to the three big clinical trials (INTACT, IDEAL and ISEL) for
gefitinib [21,32], and the study from Japan [15], but quite
different from the other five studies (Table 6).
The differences of the clinical characteristics for the 36
cases series between CISH positive and CISH negative tumors
(Table 5) were similar to the 182 case series (Table 1), except
that gender and smoking history became non-significant.
Since all except 3 of the 36 patients received TKI treatment
had adenocarcinoma, this would suggest that the associa-
tion with female gender and never smoker for CISH positive
tumors found in the 182 patient series (Table 1) was related
to adenocarcinoma, because male patients and previous
smokers were more likely to develop squamous cell car-
cinoma [1]. In our previous studies of EGFR mutation and
gefitinib responsiveness, we also found that the frequent
association with female gender and non-smoker was actually
secondarily related to adenocarcinoma [12,29].
In four patients, multiple tumor specimens (two to four
surgical samples) from different time periods (primary and
recurrent) or different sites (primary and metastatic organs)
were available for CISH study. Interestingly, the CISH pat-
terns of different specimens were always consistent for
the same patient, which is similar to the N-myc ampli-
fication in neuroblastomas [38]. These four patients all
had CISH positive tumors, including one squamous cell
carcinoma with no EGFR mutation, two adenocarcinomas
with exon 19 deletions, and another one adenocarci-
noma with L858R mutation. Only one patient had EGFR
sequence analysis of the recurrent tumors. This patient
had a delE746 A750 mutation in his primary lung can-
cer and was an erlotinib-responder. He developed drug
resistance after 6.33 months of erlotinib treatment. A
T790M mutation was found in additional to the original
delE746 A750 mutations in the recurrent tumor. But the
CISH studies on primary and recurrent tumors revealed
totally the same patterns with >10 copies signals per
nucleus or giant dot signals. The above findings suggest
that once the polysomy or amplification of EGFR has devel-
oped, it would persist even new mutations developed.
Tokumo et al. had reported two patients with double
EGFR mutations (L858R+ D761Y and L858R+ T790M, respec-
tively) and high polysomy or amplification of EGFR [39].
Both patients were non-responsive to TKI therapy. Appar-
ently, in the above three patients, increased EGFR gene
copy number alone was not related to TKI sensitivity.
The EGFR mutations and the patient’s own genetic back-
ground were more important in determining the response to
TKI.
In summary, our study has demonstrated that increased
EGFR copy number (≥5 copies per nucleus) was highly
correlated with EGFR mutation in NSCLC patients, but
less correlated with TKI responsiveness and the overall
survival when compared with EGFR mutations. On the
other hand, our study also demonstrated the convenience
and good quality of EGFR gene copy number determi-
nation by CISH on paraffin sections. Thus, even though
the EGFR gene copy number change is less correlated
with TKI responsiveness compared with EGFR mutations,
it still could be a good alternative molecular predictive
marker for TKI responsiveness, since CISH can be done on
paraffin section and is much quicker than DNA sequenc-
ing.
Conflict of interest
The authors declare that there is no any kind of conflict of
interest involved in this article.
Acknowledgements
This work was supported by grants from National Health
Research Institutes (NHRI 95-A1-MG-PP-05, NHRI 96-
A1-MG-PP-04), and Chang-Gung Medical Research Fund
(CMRPG350031).
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