J Mol Med (2006) 84: 671681

DOI 10.1007/s00109-006-0054-4

ORIGINA L ARTI CLE

Heini Lassus . Harri Sihto . Arto Leminen .

Heikki Joensuu . Jorma Isola . Nina N. Nupponen .
Ralf Butzow

Gene amplification, mutation, and protein expression of EGFR

and mutations of ERBB2 in serous ovarian carcinoma
Received: 28 November 2005 / Accepted: 8 February 2006 / Published online: 11 April 2006
# Springer-Verlag 2006

Abstract EGFR and erbB-2 are targets for specific cancer

therapy. The purpose of this study was to examine the
frequency and clinicopathological correlations of gene
amplification, protein expression, and mutations of EGFR
and ERBB2 in serous carcinoma, the most common and
aggressive type of ovarian cancer. Tissue microarray
constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH
were further analyzed by fluorescence in situ hybridization.
One hundred ninety-eight samples were analyzed for
mutations in exons 18, 19, or 21 of EGFR and in exon

H. Lassus . R. Butzow
Department of Obstetrics and Gynecology,
Helsinki University Central Hospital,
P.O. Box 700 (Haartmanink. 8),
FIN-00029 HUS, Helsinki Finland
H. Lassus
Department of Obstetrics and Gynecology, Jorvi Hospital,
Espoo Finland
H. Sihto . H. Joensuu . N. N. Nupponen
Laboratory of Molecular Oncology, Department of Oncology,
Helsinki University Central Hospital,
University of Helsinki, Biomedicum,
Helsinki Finland
J. Isola
Institute of Medical Technology, University of Tampere,
Tampere Finland
A. Leminen
Department of Obstetrics and Gynecology,
Helsinki University Central Hospital,
P.O. Box 700 (Haartmanink. 8),
FIN-00029 HUS, Helsinki Finland
R. Butzow (*)
Department of Pathology, University of Helsinki,
P.O. Box 21, Helsinki 00014, Finland
e-mail: ralf.butzow@hus.fi
Tel.: +358-9-19126855
Fax: +358-9-47171731

HEINI LASSUS
received her M.D. and Ph.D.
from the University of Helsinki,
Finland. She is presently a
resident physician in the
Department of Obstetrics and
Gynecology of the Helsinki
University Central Hospital.
Her research interests include
molecular pathogenesis and
clinical aspects of ovarian
cancer.

RALF BUTZOW
received his M.D. and Ph.D.
from the University of Helsinki,
Finland. He has specialty in
ObGyn and Pathology and is
presently Adjunct Professor and
Head of the Department of
Gynecological Pathology at
Helsinki University/Helsinki
University Central Hospital.
His research group carries out
research on molecular
pathogenesis and pathology
of ovarian carcinoma.

20 of ERBB2 using denaturating high-performance liquid

chromatography and direct sequencing. Amplification of
EGFR was present in 12% (41/333), low-level gain in 43%
(144/333), and protein overexpression in 17% (66/379) of
the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade,
greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome.
Furthermore, increased copy number of EGFR was
associated with increased copy number of ERBB2. No
mutations were identified in EGFR, whereas one tumor had
an insertion mutation in exon 20 of ERBB2. Both
amplification and protein overexpression of EGFR occur
in serous ovarian carcinoma, but EGFR copy number has a

672

stronger prognostic value. This makes EGFR amplification

a potentially useful criterion for selecting patients in
clinical trials testing the effect of EGFR inhibitors in serous
ovarian carcinoma.
Keywords Cystadenocarcinoma . Serous . erbB-1 .
erbB-2 . Gene amplification . Mutation

Introduction
The prognosis of ovarian carcinoma is poor, reflecting the
frequent finding of advanced disease at diagnosis. The
efficacy of the available treatment is limited by toxic side
effects and there is an urgent need for more specific and
effective treatment modalities [1]. Receptor and nonreceptor tyrosine and serine/threonine kinases have emerged as
promising targets for specific drug development because
overexpression or aberrant activation of these kinases often
play an important role in the molecular pathogenesis of
solid tumors. Further, the structural heterogeneity of these
kinases has permitted development of small compounds
that in a specific way inhibit their activity.
Human epidermal growth factor receptor family consists
of four transmembrane tyrosine kinase receptors, EGFR
(HER1, erbB-1), erbB-2 (HER2, HER/neu), HER3, and
HER4. Several inhibitors of HER-family were approved as
anticancer agents and many more are being tested in
clinical trials. These include EGFR-specific small-molecule tyrosine kinase inhibitors (TKIs), such as gefitinib and
erlotinib, and monoclonal antibodies that block ligand
binding to the extracellular part of the receptor (e.g.,
cetuximab). erbB-2-specific TKIs (e.g., CP-724, CP-713,
and TAK165) are in earlier stages of development but
erbB-2-targeting antibodies trastuzumab and pertuzumab
were widely studied and used mainly in breast carcinoma.
Canertinib and lapatinib are small molecules that inhibit
both EGFR and erbB-2 [2]. The efficacy of these and
respective drugs in treatment of ovarian carcinoma can be
resolved only by controlled clinical trials.
EGFR and erbB-2 are expressed in many solid tumors
and overexpression is often associated with poor prognosis.
Mechanisms resulting in EGFR activation include mutation, gene amplification, and protein overexpression.
Deletion of extracellular domain of EGFR (EGFRvIII)
causes constitutive activation of the receptor. This mutation
occurs frequently in gliomas [3, 4]. Mutations in the kinase
domain of EGFR causing ligand-independent activity of
the receptor were recently identified in a subset of nonsmall
cell lung cancer (NSCLC) [5, 6]. EGFR amplification is
detected in more than 40% of gliomas [3] and up to 30% of
NSCLCs [7]. A recent study reported mutations in catalytic
domain of EGFR in a small subset (3.5%, 2/57) of ovarian
cancers [8]. The frequency and significance of EGFR
amplification in ovarian carcinoma was not studied
extensively by sensitive methods such as in situ hybridization and the prognostic impact of EGFR overexpression
has remained controversial. In a few studies, EGFR

overexpression was associated with poor clinical outcome

[911], whereas others have shown no effect [1214].
The most frequent manifestation of erbB-2 aberration is
gene amplification and protein overexpression, which in
ovarian carcinoma, are seen in 5 to 7% and 17 to 20% of
the cases, respectively [1518]. In particular, increased
copy number associates with aggressive disease characteristics and is an independent prognostic factor for overall
survival [18]. Recent mutational screening revealed
ERBB2 kinase mutations in some solid tumors, including
ovarian carcinoma (1 of 27 cases) [19].
Molecularly targeted drugs differ from conventional
agents in that they affect only tumor types and individual
tumors where the particular gene has a pathogenetic role.
Ideally, only those cases where the targeted gene is likely to
have a pathogenetic role should be included into clinical
trials testing targeted treatments. Ovarian carcinoma
consists of several histological types. These, as a rule,
were considered as one entity, despite the fact that their risk
factors, molecular pathogenesis, and clinical behavior are
distinct [2022]. To find biologically and clinically relevant
disease entities, we have concentrated on serous histological type, which is the most common form (50 to 60%) and
accounts for most deaths from ovarian carcinoma.
Previously, we have reported the characteristics of
serous ovarian carcinomas with ERBB2 amplification and
protein overexpression [18]. In the present study, we
analyzed the frequency of copy number increase, mutations, and overexpression of EGFR and correlated the
results to clinicopathological parameters. Furthermore, we
tested the frequency of ERBB2 mutation and compared the
EGFR and ERBB2 profiles to one another.

Materials and methods

Tissue samples
Tumor samples were obtained from patients undergoing
primary surgery for ovarian carcinoma at the Department
of Obstetrics and Gynecology, Helsinki University Central
Hospital between 1980 and 2000. The permission for the
study was provided by the Institutional Review Board and
the National Authority for Medicolegal Affairs of Finland.
Consecutive patients treated for serous ovarian carcinoma
were searched according to pathological records. To be
included in the study, data of primary treatment and the
survival status of the patient and tumor cell percentage
over 60 (range 6095, median 75%) were required.
Borderline tumors were excluded from the study, but
otherwise, the cases were not selected for stage or grade.
The clinicopathological characteristics were available for
the following number of patients: International Federation
for Gynecology and Obstetrics (FIGO) stage, 398 patients;
grade, 396 patients; age at diagnosis, 398 patients; tumor
size, 388 patients; residual tumor size, 369 patients, and
ascites, 391 patients. In 320 (80%) of the 398 patients
included in the chromogenic in situ hybridization (CISH)
and immunohistochemical analysis, total abdominal hys-

673

terectomy and bilateral salpingoophorectomy were performed along with surgical removal of tumor masses; and
in 170 of these, pelvic and/or para-aortic lymphadenectomy was also performed. In 41 (10%) cases, uni- or
bilateral salpingo-ophorectomy was performed and in 37
(9%) cases, only biopsies were obtained in the primary
surgery. The tumor samples for the study were obtained
from the primary surgery before patients received any
chemotherapy. In 341 cases (86%), platinum-based
combination therapy was given as first-line chemotherapy
and in 101 of these cases, paclitaxel was given in
combination with platinum compound. In 15 (4%) cases,
the patient received other than platinum-based chemotherapy and/or radiotherapy and in 42 (10%) cases, no
adjuvant therapy was given. Response to therapy was
evaluated after initial six cycles of chemotherapy and in
cases where no chemotherapy was given, the evaluation
was performed 56 months after the surgery (the same
time interval from the surgery as in patients receiving
chemotherapy). In 184 (46%) cases, second-look laparotomy was performed and in these cases, the evaluation of
the response was based on pathological findings. In other
cases, the evaluation of the response was based on
gynecological examinations, pelvic ultrasonography,
CA-125 measurements, and radiologic findings. Ovarian
carcinoma-specific overall survival was calculated from
the date of diagnosis to death from ovarian carcinoma.
Patients who died of intercurrent causes or were alive at
follow-up were censored. Ovarian carcinoma disease-free
survival was calculated for patients who were disease-free
after the primary treatment (surgery and first-line chemotherapy, if given) and it was the time from the date of
diagnosis to the relapse of the disease. The median followup of patients alive at the end of the study period was
5.0 years (range 0.4 to 20.7 years). The 5-year overall
survival rate for the whole cohort was 52% (95% CI,
4657).
Mutation analysis was performed from 198 fresh-frozen
serous ovarian carcinoma samples. Tissue microarrays
were used for CISH and immunohistochemical analysis
and they were constructed as described previously [23].
Tissue specimens from 34 normal ovarian and 23 normal
fallopian tube samples and 401 serous ovarian carcinomas
were arranged in six recipient paraffin blocks. Four core
tissue biopsies were obtained from each specimen. Amplification of EGFR was first analyzed by CISH, which is
based on conventional brightfield microscopy, making it
possible to analyze a large number of tumors. Cases
showing amplification by CISH were further analyzed by
fluorescence in situ hybridization (FISH) when material
was available for preparation of interphase nuclei (19
carcinomas).
Immunohistochemistry
Sections (5-m-thick) were cut from each block on coated
slides. The sections were deparaffinized in xylene and
rehydrated through graded concentrations of ethanol. Anti-

gen retrieval was performed using an autoclave at 120C for

2 min in citrate buffer (pH 6.0). Immunostaining for EGFR
and for the truncated EGFR (EGFRvIII) were done using a
mouse monoclonal antibody NCL-EGFR (dilution 1:150,
Novocastra Laboratories, Newcastle, UK) and NCL-EGFRT
(1:200, Novocastra), respectively. The primary antibodies
were diluted in Powervision preantibody blocking solution
and incubated overnight at 4C. Binding of the primary
antibody was detected with a Powervision + Poly-HRP
histostaining kit (DPVB+110DAB, Immunovision Technologies, Daly City, CA, USA). The immunohistochemical
analysis was evaluated by a pathologist (RB) without
knowledge of the clinicopathological information. The
epithelium of fallopian tubes (the normal serous epithelium
of Mllerian origin) (n=23) and ovarian surface epithelium
(OSE) (n=34) were used as a reference of normal expression
for all proteins. Membrane staining of EGFR was taken into
account and scored according to the intensity as two groups:
negative to weak immunopositivity and strong positivity
(stronger than normal membranous staining).
Immunohistochemical findings for erbB-2, p53, Ki-67,
PDGFRA, and KIT were previously published [18, 24, 25].
The primary antibodies were as follows: a monoclonal
antibody against erbB-2 (1:2,000; code NCL-CB11;
Novocastra), a polyclonal antibody against Ki-67 (1:150,
clone N/A; code A0047; DAKO A/S, Glostrup, Denmark),
a monoclonal antibody against p53 (1:100; clone DO-7,
Dako A/S, Glostrup, Denmark), a polyclonal antibody
against KIT (dilution 1:100; DAKO, Carpinteria, CA,
USA), and a polyclonal antibody against synthetic peptide
derived from C-terminal of PDGFRA (dilution 1:150,
Neomarkers, Lab Vision, Fremont, CA, USA). Immunohistochemical staining for p53 was considered aberrant
when tumor cells showed excessive p53 (>50% of tumor
cells showed homogeneous moderate or strong nuclear
immunopositivity) or completely negative p53 (no staining
in any of the tumor cells). Tumors with weak immunostaining, similar to that found in normal epithelium, were
considered to show normal p53 expression [24]. Membrane
staining was taken into account for erbB-2 and both
membrane and cytoplasmic staining for KIT and PDGFRA
were scored according to the intensity as follows: erbB-2
weak, moderate, or strong; KIT negative, weak, or strong;
and PDGFRA weak, moderate, or strong. The growth
fraction of the tumors (percentage of tumor cells positive
for Ki67) was considered low, moderate, and high when
<10, 1025, and >25% of tumor cells, respectively, showed
positive staining.
DNA extraction and polymerase chain reaction
The genomic DNA was extracted using standard methods.
The polymerase chain reactions (PCRs) were done nested.
Fifty nanograms of the genomic DNA was amplified in the
first reaction containing 0.6 Platinium PCR Buffer
(Invitrogen, Carlsbad, CA, USA), 1.4 mmol/l of MgCl2,
160 mol/l of deoxynucleotide triphosphates (Clontech,
Palo Alto, CA, USA), 0.3 mol/l of forward and reverse

674

oligonucleotide primers, DNA polymerases AmpliTaq

Gold (1.25 U; Applied Biosystems, Branchburg, NJ,
USA), and Platinium Taq (1.25 U; Invitrogen, Carlsbad,
CA, USA) in a volume of 25 l. Nested PCR reactions
were done by using 1.0 l of amplification product from
the first PCRs. The forward and reverse oligonucleotide
primers used to amplify EGFR exons 18, 19, and 21 are
listed elsewhere [6]. For exon 20 of ERBB2: forward: 5tggtctcccataccctctca-3 and reverse: 5-tgtggacataggggtttgct3. Heteroduplex formation was created by denaturing the
PCR products for 5 min at 95C and then the samples were
allowed to reanneal by decreasing temperature by 1C/min
from 95 to 40C.
Denaturing high-performance liquid chromatography
The tumors were investigated for the known mutational hot
spots in the EGFR kinase domain (exons 18, 19, and 21),
and for exon 20 of ERBB2 using denaturing highperformance liquid chromatography (DHPLC). The
DHPLC procedure was carried out as described elsewhere
[26]. The DYS271 standard, which consists of a 209-base
pair fragment of double stranded DNA and heterozygous
with an A to G mismatch at position 168, was used as a
control. One hundred ninety-eight samples were analyzed.
DNA sequencing
Samples with an abnormal elution profile in DHPLC
compared with a control consisting of lymphocyte DNA

Fig. 1 Serous ovarian carcinomas showing normal copy number

(two copies) (a) and amplification (>5 copies) (b) of EGFR gene by
CISH and copy number increase of EGFR by FISH (eight copies of
EGFR and four copies of the centromere) (c). Normal OSE (d), tubal
epithelium (e), and a serous ovarian carcinoma (f) showing low
immunostaining of EGFR protein. A serous ovarian carcinoma (g)

were subjected to automate sequencing. The PCR products

were first purified using QIAquick PCR purification kit
(Qiagen, Valencia, CA, USA). Direct sequencing of PCR
products was performed using BigDye3 termination
chemistry (Applied Biosystems) and an ABI 3100 Genetic
Analyzer (Applied Biosystems) according to the instructions provided by the manufacturer. All mutations found
were confirmed by sequencing of another DNA sample
extracted from the same tumor.
Chromogenic in situ hybridization
CISH for EGFR gene was performed as previously
described [27]. A signal copy number of three to five per
one centromere was considered indicative for gain/low
copy number increase and a copy number over five as gene
amplification.
Fluorescence in situ hybridization
The chromosome 7 copy number was determined using a
chromosome 7 centromere-specific probe CEP7 (Spectrum
Green, Vysis, Downers Drive, IL, USA) and the EGFR
copy number using a bacterial artificial chromosome (clone
RP11-815K24) probe (Invitrogen, Paisley, UK). The
correct probe identity was confirmed using PCR and
EGFR specific primers. Bacterial artificial chromosome
DNA was isolated using standard techniques and labeled
with the DIG-Nick translation mix (Roche, Mannheim,
Germany). Dual-color hybridizations were performed as

showing strong membranous staining indicating overexpression of

EGFR protein. A DHPLC elution profile for HER2 exon 20 wildtype sample of HER2 and YVMA duplication mutant sample (h). A
Chromatogram of mutated HER2 sample is shown as a converted
sequence Sequencer(TM) (i)

675

previously described [28]. Digoxigenin-labeled probes

were detected using a sheep antidigoxigenin-rhodamine
antibody.
Interphase nuclei were prepared as described elsewhere
[28]. The EGFR and chromosome 7 centromere probes
were cohybridized and after hybridization, the probes were
detected with avidin-fluorescein isothiocyanate and antidigoxigenin rhodamine, counterstained with 4-6-diamidino-2-phenylindole, and viewed under a fluorescence
microscope equipped with an ISIS digital image analysis
systems (MetaSystems, Altlussheim, Germany). Signal

copy numbers were counted from 50 randomly chosen

nonoverlapping nuclei.
DNA ploidy analysis
Core tissue biopsy specimen (diameter 0.8 mm) was taken
from area representing carcinoma in paraffin tissue block.
The tissue cores were deparaffinized, rehydrated, and DNA
flow cytometry was performed as previously described
[29].

Table 1 Association of EGFR gene copy number with clinicopathological characteristics

Clinicopathological characteristics

EGFR copy number

2 (n=148)

FIGO stage
I
II
III
IV
Grade
1
2
3
Residual tumor
1 cm
>1 cm
Age
<57 years
>57 years
Tumor size
10 cm
>10 cm
Ascites
No
Yes
p53
Normal
Aberrant
ERBB2 gene copy number
2 copies
35 copies
>5 copies
cKIT expression
Negative
Low
High
PDGFRA expression
Decreased
Normal
Increased
Ki67
010%
1025%
>25%

35 (n=144)

>5 (n=41)

p value

36/148
13/148
87/148
12/148

18/144
13/144
92/144
21/144

5/41
5/41
24/41
7/41

NS, 0.091

80/147
30/147
37/147

34/143
38/143
71/143

3/41
12/41
26/41

<0.0001

84/136
52/136

60/138
78/138

14/40
26/40

0.0012

92/148
56/148

65/144
79/144

15/41
26/41

0.0017

57/147
90/147

58/139
81/139

14/38
24/38

NS, 0.81

58/144
86/144

35/143
108/143

10/40
30/40

NS, 0.10

81/147
66/147

41/144
103/144

2/39
37/39

<0.0001

134/144
6/144
4/144

93/135
29/135
13/135

23/41
11/41
7/41

<0.0001

135/147
10/147
2/147

117/143
21/143
5/143

28/40
11/40
1/40

0.0053

20/147
113/147
14/147

13/141
114/141
14/141

6/41
25/41
10/41

0.044

99/147
32/147
16/147

56/143
39/143
48/143

10/40
18/40
12/40

<0.0001

Statistical analysis

Associations between factors were analyzed with the 2

and Fishers exact tests. The overall and disease-free
survival curves were constructed according to the Kaplan
Meier method and compared with the logrank test. A p
value of 0.05 was adopted as the limit for statistical
significance.

Cumulative overall survival

676
EGFR gene copy number

2 copies (n=148)
3-5 copies (n=144)
>5 copies (n=41)

0.8
0.6
0.4
0.2

Results
Mutation analysis of EGFR and ERBB2

EGFR gene copy number by chromogenic in situ

hybridization
Gene copy number of EGFR was interpretable in 333
(84%) of 398 serous ovarian carcinomas (Fig. 1a,b). Two
copies of the gene were seen in 148 (44%) and low-level
gain (35 copies per cell) in 144 (43%) of the interpretable
cases. Amplification (>5 copies per cell) of the gene was
detected in 41 (12%) of the specimens. Stromal cells in the
carcinoma samples showed two copies of EGFR and thus
served as internal controls for normal copy number.
EGFR gene copy number and clinicopathological
characteristics
Increased copy number of EGFR was associated with high
tumor grade (p<0.0001), large residual tumor size
(p=0.012), and greater patient age (p=0.017), but not
with tumor stage (p=0.091), tumor size (p=0.81), or ascites
(p=0.10) (Table 1).
Increased copy number of EGFR was associated with
aberrant p53 status (p<0.0001), increased copy number of
ERBB2 (p<0.0001), high c-KIT expression (p=0.0053),
high PDGFRA expression (p=0.044), and high Ki-67
expression (p<0.0001) (Table 1).
EGFR copy number was not associated with primary
response to therapy in patients treated with cyclophosphamide/platinum-based (n=193) or paclitaxel/platinum-based
(n=91) combination therapy (p=0.24 and 0.44).
EGFR gene copy number and survival
Increased copy number of EGFR was associated with
shorter overall survival (Fig. 2a, p<0.0001). The 5-year

15

10
Years from diagnosis

B
Cumulative overall survival

DHPLC analysis was successful in 95% of 198 serous

ovarian carcinomas. All heteroduplex DHPLC profiles
were verified by performing new PCRs, reanalyzed, and
resequenced. No sequence alterations were found in exons
18, 19, or 21 of EGFR. One insertional mutation was
identified in exon 20 of ERBB2 (amino acids 772775
duplicated, YVMA) (Fig. 1h,i).

EGFR protein expression

low expression (n=313)
high expression (n=66)

1
0.8
0.6
0.4
0.2

10

15

20

Years from diagnosis

Fig. 2 Overall survival in relation to EGFR gene copy number

detected by CISH (a) and to EGFR expression detected by
immunohistochemistry (b) in patients with serous ovarian carcinoma

overall survival rates for patients with tumors showing two

copies, low copy number increase, and amplification of
EGFR gene were 66% (95% CI, 5875%), 39% (3048%),
and 28% (1046%), respectively. Increased copy number
of EGFR was also associated with disease-free survival
(p=0.0007). The 5-year disease-free survival rates for
patients with tumors showing two copies, low copy number
increase, and amplification of EGFR gene were 81% (95%
CI, 7390%), 56% (4270%), and 50% (2475%),
respectively.
Within the group of diploid tumors, those with low copy
number increase of EGFR were associated with shorter
overall survival than those with two copies of EGFR
(p<0.0001).
Of the clinicopathological characteristics, tumor
stage (p<0.0001), grade (p<0.0001), residual tumor size
(p<0.0001), age (p<0.0001), and presence of ascites
(p<0.0001), but not the size of the tumor (p=0.080), were
associated with shorter overall survival. When the material
was stratified according to the clinicopathological characteristics, statistically significant association of increased
copy number of EGFR with poor overall survival was
observed in all clinicopathological subgroups, except in
grade 23 carcinomas and carcinomas with residual tumor
over 1 cm (stage III and IIIIV, grade 1, residual tumor

677

1 cm, age <57 and 57 years, tumor size 10 and >10 cm,
and absence and presence of ascites).

able cases. None of the tumors presented with positive

EGFR-vIII immunostaining.

Protein expression of EGFR

EGFR protein expression and clinicopathological

characteristics

Weak to moderate membrane positivity of EGFR was

detected in normal epithelium of fallopian tubes and normal
OSE. EGFR immunostaining was interpretable in 379
(95%) of the 398 serous ovarian carcinomas (Fig. 1dg).
Low expression was detected in 313 (83%) and high
expression (overexpression) in 66 (17%) of the interpret-

Overexpression of EGFR was associated with high tumor

grade (p=0.0018), large residual tumor size (p=0.028), and
greater patient age (p=0.021), but not with tumor stage
(p=0.32), tumor size (p=0.76) or presence of ascites
(p=0.66) (Table 2).

Table 2 Association of EGFR expression with clinicopathological characteristics

Clinicopathological characteristics

EGFR expression
Low (n=313)

FIGO stage
I
II
III
IV
Grade
1
2
3
Residual tumor
1 cm
>1 cm
Age
<57 years
57 years
Tumor size
10 cm
>10 cm
Ascites
No
Yes
p53
Normal
Aberrant
ERBB2 gene copy number
2 copies
35 copies
>5 copies
cKIT expression
Negative
Low
High
PDGFRA expression
Decreased
Normal
Increased
Ki67
010%
1025%
>25%

High (n=66)

p value

63/313
31/313
187/313
32/313

9/66
8/66
38/66
11/66

NS, 0.32

129/311
71/311
111/311

12/66
22/66
32/66

0.0018

160/291
131/291

25/63
38/63

0.028

168/313
145/313

25/66
41/66

0.021

118/306
188/306

26/64
38/64

NS, 0.76

100/308
208/308

19/64
45/64

NS, 0.66

134/306
172/306

15/66
51/66

0.0015

239/300
43/300
18/300

49/65
8/65
8/65

NS, 0.20

267/312
38/312
7/312

55/66
8/66
3/66

NS, 0.57

42/308
238/308
28/308

6/66
15/66
45/66

0.0059

169/308
76/308
63/308

25/66
24/66
17/66

0.038

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Overexpression of EGFR was associated with aberrant

p53 status (p=0.0015), increased PDGFRA expression
(p=0.0059), and high Ki-67 expression (p=0.038), but not
with copy number or expression of ERBB2 (p=0.20 and
0.27) or c-KIT expression (p=0.57) (Table 2). Expression
of EGFR by IHC was not associated with copy number of
EGFR detected by CISH (p=0.19).
Expression of EGFR was not associated with primary
response to therapy in patients treated with cyclophosphamide/platinum-based (n=230) or paclitaxel/platinum-based
(n=90) combination therapy (p=0.17 and 0.25).
EGFR protein expression and survival
Overexpression of EGFR was associated with shorter
overall survival (Fig. 2b, p=0.027). The 5-year overall
survival rates for patients with tumors showing low and
high expression of EGFR were 54% (95% CI, 4860%)
and 40% (2754%). Overexpression of EGFR was also
associated with shorter disease-free survival (p=0.017).
The 5-year disease-free survival rates for patients with
tumors showing low and high expression of EGFR were
72% (95% CI, 6579%) and 55% (3674%).
EGFR and chromosome 7 copy number
by fluorescence in situ hybridization
In tumors showing amplification of EGFR by CISH, FISH
analysis for both the EGFR and chromosome 7 centromere
probe was successful in 14 out of 19 cases. Ten of these 14
cases showed tetrasomy of chromosome 7. Increased copy
number of EGFR was detected in all cases and amplification of EGFR relative to centromere was found in 11 of the
14 cases. In ten tumors, EGFR copy number was five or
more (Table 3 and Fig. 1c).

Discussion
EGFR amplification (>5 copies) was found in 12% and
overexpression of EGFR in 17% of serous ovarian
carcinomas. Both increased copy number and overexpression of EGFR were associated with high tumor grade,
greater patient age, large residual tumor size, high proliferation index and aberrant p53, and shorter overall and
disease-free survival. Copy number of EGFR and its
association to disease characteristics was not reported
earlier using in situ hybridization analysis of a large
ovarian carcinoma material. The amplifications of EGFR
were low-level (510 copies), which is in contrast to
ERBB2 in which also high-level amplifications are found in
ovarian carcinoma [18]. However, the overall frequency of
EGFR amplification (12%) was higher than the frequency
of ERBB2 amplification (7%) [18].
Low copy number increase of EGFR was found in 44%
of the tumors. The molecular background of low-level gain
may be diverse: low-level gain of the gene or a larger
chromosomal segment, isochromosome formation, increased number of the chromosome (polysomy), polyploidy of the tumor genome, or their combinations. A
proportion of tumors with low copy number increase were
aneuploid suggesting gross genomic changes as a cause for
increased copy number of EGFR. However, patients with
diploid tumors and 35 copies of EGFR also presented
with shorter overall survival as compared to patients with
tumors showing two copies of EGFR, suggesting that also
low-level gain of EGFR may confer negative prognostic
impact.
Previously, a few studies have reported a correlation of
EGFR overexpression with poor patient outcome [911],
whereas others have found no association [1214]. In these
studies, the rate of EGFR-positive cases has ranged from 9
to 77%. These discrepancies may be related to different
antibodies, staining methodology, or employed reference.

Table 3 Copy number of EGFR and chromosome 7, ploidy, expression of EGFR, Ki67 and p53, and copy number of ERBB2 in serous
ovarian carcinomas with amplification of EGFR by CISH
Case

Ploidy

Chromosome 7

EGFR FISH

EGFR IHC

Ki67

p53

erbB-2 CISH

27
106
226
846
851
981
1013
2258
2276
2280
2285
2293
2311
2320

Hyperdiploid (DI=1.73)
Hyperdiploid (DI=1.85)
Hyperdiploid (DI=1.45)
Hyperdiploid (DI=1.82)
Hyperdiploid (DI=1.61)
Diploid
58% diploid, 42% hyperdiploid (DI 1.73)
Hyperdiploid (DI=1.45)
Diploid
Tetraploid (DI=2.19)
Hyperdiploid (DI=1.22)
Tetraploid (DI=1.94)
Not available
Tetraploid (DI=2.07)

3
4
4
3
4
4
4/2
4
4
4
3/4
5
4
2

5
4
6
7
6
6
4/7
4
8
6
3/4
10
6
2/4

Low
Low
High
Low
Low
High
Low
NI
Low
High
Low
Low
Low
Low

Moderate
High
Moderate
Moderate
High
High
Moderate
Low
Moderate
Moderate
High
Moderate
Moderate
Moderate

Aberrant
Normal
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant
Aberrant

35
2
35
2
35
2
2
2
2
35
2
35
2
>5

NI = not informative

679

According to the prevailing view, ovarian carcinoma

originates from the surface epithelium of the ovary and
particularly from its invaginations that are remnants of past
ovulation sites. Their epithelium has often transformed
from flat mesothelium-like cells into ciliated cylindrical
cells resembling the serous-type epithelium seen in the
fallopian tubes. Both the metaplastic OSE and tubal
epithelium express some EGFR. Using this expression
level as a reference, we detected overexpression in 17% of
the tumors and this significantly associated with aggressive
disease characteristics.
According to our results, the prognostic value of EGFR
amplification is even stronger than that of EGFR overexpression. This finding was not expected because gene
amplification is considered to have an impact through
increased protein expression. However, the same was
observed in breast and ovarian carcinoma regarding
ERBB2 [18, 30]. Furthermore, in breast carcinoma,
ERBB2 amplification is a stronger predictor of response
to targeted treatment by trastuzumab than protein level [31]
and in NSCLCs amplification of EGFR, but not protein
expression status, predicted response to EGFR inhibitor
gefitinib [7]. Uneven degradation of the examined protein
in tissue samples and the subjective nature of immunohistochemical interpretation may partly explain the seeming
discrepancy. In phase II study testing gefitinib in ovarian
carcinoma, the only patient with objective response (1 of
27) had a mutation in the catalytic domain of EGFR. In the
same study, three patients presented with prolonged stable
disease, but no mutations of EGFR were found in their
tumors. However, EGFR amplification was not tested in
the tumors of the patients with stable disease [8].
Ovarian cancer cell lines stably transfected with an
antisense EGFR present reduced proliferation, induction of
differentiation, and reduction of markers for cell invasion
in vitro [32, 33] and suppression of ovarian cancer cell
tumorigenicity by a dominant-negative EGFR was demonstrated in vivo [34]. Our finding that increased copy
number and protein overexpression associate with aggressive tumor characteristics further strengthens the evidence
that activation of EGFR plays a part in the molecular
pathogenesis of ovarian carcinoma.
An increased copy number of EGFR was associated with
an increased copy number of ERBB2. A tendency for such
an association was previously reported in two immunohistochemical studies of ovarian carcinoma [13, 35]. In
cultured ovarian cancer cells, erbB-2 expression is ratelimiting for EGFR-mediated stimulation and high levels of
erbB-2 expression weaken the ability of EGFR inhibitors to
inhibit EGFR phosphorylation [36, 37], suggesting a
collaborative role for EGFRerbB-2 in ovarian carcinogenesis. In ovarian cancer cells, suppression of both EGFR
and erbB-2 results in regression of aneuploidy and genomic
imbalances and restores a more normal phenotype [38].
Dual-inhibitors of EGFR and erbB-2 inhibit both liganddependent and ligand-independent downstream signaling
in primary human breast cancer cell lines and some patients
with metastatic cancers [39, 40]. Ovarian carcinomas with
both EGFR and erbB-2 activation might be particularly

suitable targets for testing dual TKIs like lapatinib and

canertinib.
No mutations were found in EGFR. A recent study
reported EGFR mutations in a small number of ovarian
carcinomas (3.5%; 2/57) [8]. This discrepancy is most
likely due to chance or difference in the study materials
because the method employed in our work has proven
sensitive and we have used it successfully in detection of
EGFR mutations in lung adenocarcinomas and bronchioloalveolar carcinomas [26]. We found one ERBB2 mutation, insertion of 12 base pairs in exon 20 (amino acids
772775, YVMA). This case was grade 3, stage IIIc,
negative for both EGFR and ERBB2 amplification and, in
addition, erbB-2 protein level was not elevated. Despite the
undetectable expression in immunohistochemistry, a tyrosine kinase receptor may be constitutively activated and
mediate oncogenic signaling into the cell. As an example, a
small subgroup of gastrointestinal stromal tumors (GISTs)
present with kit mutations, but no KIT expression, and they
show response to imatinib, an inhibitor of cKIT [41]. It is
interesting to note that mutations of ERBB2 were identified
earlier from a small subset of lung adenocarcinomas, but
not from other histological types [19, 42]. All the mutations
in these two studies, similar to our finding, were
duplications/insertions in a small region of exon 20.
Molecularly targeted drugs differ from conventional
chemotherapeutic agents in that they affect tumor types and
individual tumors where the particular gene has a pathogenetic role. For example, in mammary carcinoma (ERBB2
amplification), GIST (KIT or PDGFRA mutation) and lung
carcinoma (EGFR mutation/amplification), significant results are obtained only in patients harboring amplification
or activating mutation of the respective gene [5, 7, 31, 43].
Ideally, only those cases where the targeted gene is likely to
have a pathogenetic role should be included into clinical
trials testing targeted treatments. Knowledge of the kind of
aberrations (gain-of-function mutation, amplification, or
protein overexpression) present on the tumor type of
interest facilitates the realization of such trials.
In summary, mutations of EGFR or ERBB2 were rare in
serous ovarian carcinoma. EGFR amplification and protein
overexpression were more common and both were
associated with aggressive disease characteristics and
poor patient outcome. However, EGFR copy number had
a stronger effect on patient outcome than protein overexpression. These findings make EGFR amplification a
potential criterion for selecting patients in clinical trials
testing the effect of EGFR inhibitors in ovarian carcinoma.
Acknowledgements This study is supported by grants from the
Cancer Society of Finland, Foundation for the Finnish Cancer
Institute, the Academy of Finland, and Helsinki University Central
Hospital.

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