Mutations, gene copy numbers, and protein expression of EGFR in lung cancer

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Mutations, gene copy numbers, and protein expression of EGFR in lung cancer

Sofi Isaksson, Mats Jönsson,
Monica Haglund, Per Jönsson
,

Karin Jirström, Leif Johansson
,

Maria Planck




Department of Oncology, Clinical Sciences, Lund University, SE
-
221 85
Lund,

Sweden


Department of Pathology,
Skane
University Hospital
, Lund, S
-
221 85 Lund, Sweden


Department of Thoracic Surgery,
Skane
University Hospital
, Lund, S
-
221 85 Lund, Sweden



Correspondence to:
maria.planck@med.lu.se







Key words;
EGFR, lung cancer, biomarker, mutation specific antibody



Abstract

Background:
The epidermal growth factor receptor (EGFR) gene is a frequent oncogene in
lung cancer
.
Alterations of EGFR such
as
protein expression,
gene co
py numbers, mutations

and alterations in

molecules a
c
ting downstream of EGFR

might correlate in different ways
.
Methods:
Tissue

microarrays (TMAs) with three cores from each of 369 surgically treated
NSCLCs were

evaluated for EGFR protein expression

by immunohistochemistry (IHC) and
for EGFR gene

copy numbers by fluorescence in situ hybridising (FISH). The tumours were
scored as IHC 0
-
3+ and as FISH negative (non amplified) or positive (polysomy or
amplified). Both tests were

evaluable for 334 cases.

The two most frequent EGFR mutations,
exon 21 L858R and exon 19 E746
-
A750 deletion, were revealed with mutation
-
specific
antibodies on TMA and subsequently

direct DNA sequencing.
Results:
IHC; 0 = 132, 1+ =
63, 2+ = 67, 3+ = 72. FISH; non

amplified = 196,

polysomy = 115, amplified = 23. All cores
containing viable tumour tissue

could be evaluated for both tests. The correlation between
IHC and FISH was highly

significant (p = 0.0007). EGFR IHC 1
-
3 was an adverse factor for
survival (p=0.02) but not

EGFR FI
SH (p=0.35).
19 mutations consisting of 9 deletions and 10
substitutions were revealed with mutation
-
specific antibodies on TMA.
Conclusions:
Lung
cancer TMA is ideal for large biomarker studies

by FISH and IHC. Furthermore, this study
confirms EGFR IHC ov
er expression as a negative

prognostic factor for survival and
demonstrates a highly significant correlation between

EGFR FISH and IHC.







Introduction

Lung cancer is one of the most common malignancies and the leading cause of cancer death,

with

an overall 5
-
year survival rate of less than 15%. EGFR, an important marker for
metastatic propensity and proliferation in lung

cancer, is a 170 kDa transmembrane
glycoprotein and one of four members of the HER family

of cell surface receptors and the
pro
tein product of the oncogene HER1. Members of the HER

family have an extracellular
ligand
-
binding area (except HER2), a hydrophobic

transmembrane domain and an
intracellular region with tyrosine kinase activity. Ligands are

growth factors such as EGF,
TGF
-

, amphiregulin, betacellulin and epiregulin. The binding of

a ligand

results in
autophosphorylation and downstream intracellular signalling.

EGFR also plays a role in cell
motility, adhesion (through interaction of E
-
cadherin and the

actin cytoskeleton),
invasion and
angiogenesis (4).


EGFR is expressed or over expressed in a

wide variety of solid human tumours, including
glioblastoma, prostate, breast, gastric,

colorectal, head and neck, bladder and ovarian cancers,
and also in 50
-
70% of NSCLCs,

whereas
expression is weaker in normal lung tissue and in
small
-
cell lung cancer (5
-
7).


EGFR protein expression, EGFR gene copy number, occurrence of somatic EGFR mutations,

and alterations in genes acting downstream of EGFR in intracellular signalling have all b
een

suggested as predictive biomarkers for selecting patients to receive treatment with
EGFRTKIs,

but no consensus regarding which marker would be the preferred choice has yet
been

achieved (8
-
11).


We evaluated
and correlated important EGFR

alterations
i
n
334 surgically resected NSCLCs
.
More specifically we analyzed
EGFR

protein expression by IHC,
gene copy numbers by
FISH

and mutation status by IHC with mutation
-
specific antibodies detecting
exon 21 L858R
and exon 19 E746
-
A750 deletion.

To
optimize the
implementation of FISH (and IHC) on this
large series we performed high

throughput tissue micro array (TMA)
-
based analyses of all
tumours.

Tumours with mutations revealed with mutation

specific antibodies on IHC were
subsequently sequenced.


Materials an
d methods

The histopathological reports in two consecutive case series of patients, surgically treated for

Stage I

IIIA lung carcinoma at the University Hospital of Lund, were studied. The first series

of patients (n=187) was treated in 1981
-
83, the second

(n=187) in 1995
-
97. None of the

patients had received any systematic neoadjuvant or adjuvant therapy. There were 241 (64%)

males and 133 (36 %) women; 25 % in the 1981
-
83 and 46% in the 1995
-
97 series. As to

histopathological subtypes; 148 (40%) cases wer
e classified as squamous cell carcinoma, of

which 80% occurred in males; 135 (36%) cases were classified as adenocarcinoma, of which

53% occurred in males.


Immunohistochemistry

For this study the histopathological

slides were reviewed and appropriate blocks identified

for construction of the TMAs (369 cases). Three 1.0 mm tissue cores were collected from

each tumour block and arranged in a recipient block using a manual tissue arrayer (Beecher

Inc., Sun Prairie, WI
, USA).

Semi
-
thin sections were cut from the paraffin blocks and stained
with hematoxylin and eosin

(H&E) staining, and for EGFR IHC and FISH.

EGFR protein
expression was evaluated by IHC, antibody NovoCastra (dilution 1:40), diluted

in Ab
-
dilution (S2022,

Dako). Sections of 4
μ
m were cut and dried for 2 hours at 60°C and

stored in 4°C until stained. Tissue sections were then deparaffinized in xylene and rehydrated

through graded alcohol and deionised water. For antigen retrieval, HIER (Heat Induced

Antigen
Retrieval) was used, the sections were processed in an microwave oven, Milestone

TTMega, standard program for 80 slides (blank slides filled up to 80 when needed) in antigen

solution TRS pH 9,9 (S3307, Dako). Staining was performed in an automated immunost
ainer,

Techmate 500Plus (Ventana Biotek, Tucson, AZ) with standard procedure “ENVP”.

Visualization system was EnVision (art.no, K5007) from Dako, counterstaining with

Hematoxyline and dehydration in graded alcohol, xylene and mounted. Rinse buffer TBS, pH

7, 6 containing 0, 1% TWEEN (8.22184, Merck). Endogenous peroxidase was inactivated by

HP
-
Block (S 2023, Dako).

The staining was scored as 0 (negative), 1+ (weakly positive), 2+ (moderately positive) and

3+ (strongly positive). Positivity of any grade
required staining in > 10% of the cell

membranes. All specimens were evaluated by one reader (LJ), and some results were also

confirmed by another reader (MH).


FISH

Copy number of the EGFR gene was determined by FISH using a dual
-
colour EGFR probe

(Vysis
, Il USA) labelled with spectrum orange for EGFR (7p12) in combination with a

spectrum green for centromeric localisation (CEP7) as reference. Shortly, sections were

deparaffinized in xylen and 100% ethanol and air
-
dried, pretreated in pretreatment solutio
n

(Abbott Vysis 2J06
-
30) for 10 min in 80°C water bath, Gant CD100 från VWR, and then

rinsed in distilled water for 3 min. Then 150 mg protease (Abbott Vysis 6J93
-
01) diluted in 37

ml protease buffer (Abbott Vysis art.no, 2J07
-
30) for 40 min in 37°C and ri
nsed in distilled

water for 3 min. Slides were then immersed in graded alcohol series (70%, 95% and 100%)

and air dried for 15 min. For hybridization 10


15
μ
l probe (EGFR SO/CEP7 SG DNA,

art.no, 5J48
-
01, Abbott Vysis) was applied to the hybridization are
a, which was covered with

a cover slip and sealed with rubber cement. For hybridization a semi
-
automated hybridizer,

ThermoBrite S 500
-
24 from Abbott Scandivavia AB, was used, i.e. denaturation in 85°C for 1

min and then hybridization overnight (16


20 h)

at 37°C. Next morning the rubber cement

seal was stripped off and the cover slip removed. The specimens were washed in a
posthybridizing

buffer (2XSSC/0,3% Igepal, prewarmed to +72°C) 2 min and rinsed in
distilled

water. The slides were air dried in an up
right dark position for 15 min. Ca 10
-

15
μ
l

mounting medium, Vectashield H1000 and H12000 (with DAPI) was mixed 1:1 and applied

onto the specimen and the area was covered with a coverslip and sealed with nail lacquer.

The specimens were evaluated in a
Nikon Eclipse E80i fluorescence microscopes equipped

with 100 W mercury
-
arc lamp and fitted with high
-
quality objectives (10×, 40×, 60x, 100×) in

combination with dual (red/green) and single (blue) interference filters and an Olympus DP
-

40 digital camera
(1.5 milj pixel, 1392 x 1040).
To be sure that the right core on the TMA was

evaluated in the fluorescence microscope a comparison was done continuously with a virtual

H&E slide of the same TMA scanned with an Aperio ScanScope CS System (courtesy of LRI

Im
aging AB, Lund, Sweden). All specimens were evaluated by one reader (MH), and most

results were also confirmed by another reader (LJ). All difficult cases were discussed, and a

consensus was reached.


The results were scored,
with slight modifications
, acc
ording to the suggestions of Varella
-

Garcia (20) as negative; non amplified = usually 2
-
3 copies of the EGFR gene and

chromosome 7 or positive; amplified = number of EGFR gene copies/number of

chromosome 7 copies > 2, or polysomy >

3 copies (usually 4
-
12) of both the EGFR gene and

chromosome 7.


Statistical analysis

The Wilcoxon test was used for association between IHC and FISH. The Kaplan
-
Meier

analysis

and log rank test were used to illustrate differences in overall survival (OS) according

to EGFR IHC and FISH.


Mutation
-
specific antibodies and immunohistochemistry

For mutation analysis two rabbit monoclonal antibodies (Cell Signaling
, dilution 1:10
) wi
th
specificity for the exon 21 L858R and the exon 19 E746
-
A750 deletion respectively, were
used.
Analyses were done on paraffin
-
embedded tissue microarrays.


The tumours were considered positive if staining was detected in at least one of the cores.


DN
A extraction



Direct DNA sequencing

The tumours with positive

staining
as revealed with

mutation specific antibodies were
sequenced using direct DNA sequencing.

Mutations of exon 19 and

21 of the EGFR gene were
analysed by direct DNA sequencing using the
BigDye Terminator Cycle Sequencing Kit v1.1
(Applied Biosystems®). The samples were initially purified and subsequently amplified. PCR was
performed in 10µL volumes using 80 ng (4µL 20ng/µL) template, 1, 75 µL 5xbuffer, 4 µL deionized
water and 0, 5 µL Ter
minator reaction mix. The mutations in exons
19 and 21
were determined using
primers described in supplementary table
X
. PCR was run under the following conditions; 25 cycles of
denaturation at 96°C for 10 s, primer annealing at 50°C for 5 s and elongation

at 60°C for 1 min.
Sequencing products were separated by capillary electrophoresis by ABI 3130xl Genetic Analyzer
(Applied Biosystems®). Analysis of the sequencing was performed using the 3100 data collection
software (Gene Code Corporation®). The sequenc
es were compared with XXX

(genomisk EGFR).

Results

IHC and FISH

The results are given in table 1 and examples of positive staining in figure 1A
-
C. IHC staining

was negative (0+) in 132 (39.5%) cases, weakly positive (1+) in 63 (19%) cases, moderately

positive (2+) in 67 (20%) cases and strongly positive (3+) in 72 (21.5%) cases.


FISH was
negative (non amplified) in 196 cases (59 %) and positive in 138 cases; 117 (34%)

polysomy
(Figure 2A), 23 (7%) amplified (Figure 2B).


In 334 of the cases both tests

were evaluable. The correlation between the two tests was

highly significant (p = 0.0007). Not only could IHC be evaluated for all these cases, but also,

the hybridizing was successful with all cores in the TMAs that contained viable tumour tissue.

Of the

cases, 97 (29 %) were negative for both tests while 103 (31 %) were positive for both

tests.


Survival

Kaplan
-
Meier plots showed that EGFR IHC positive cases (1
-
3+) had a significantly
worse

survival compared to EGFR IHC negative cases (Figure 3A, p=0.02)
. When 3+ cases
were

tested against 0
-
2+ cases, and 2
-
3+ cases against 0
-
1+ cases, the difference was not
significant

(data not shown).
A significant d
ifference in survival between EGFR FISH
positive cases (polysomy, amplified) and

EGFR FISH negative cases

(non amplified)
could
not be shown
(Figure 4A, p=0.35).




Mutation
-
specific antibodies and IHC

Staining w
ith the mutation
-
specific
antibodies

revealed

in total
20

mutations

of which
10

were
exon 19 deletions and 10 exon 21 substitutions.
The tumours

with mutations are presented in
table X with FISH a
nd EGFR IHC status.


Confirmation of mutations by d
irect sequencing

Of the tumours
positive in immunohistochemical analysis with the monoclonal antibodies
directed at

the exon 21 L858R and the exon 19 E7
46
-
A750 deletion
, respectively…

… infoga mutationsresultat efter sekvensering
(Table x)


Discussion

Although the TMA technique has been applied in some studies of biomarkers in lung cancer

(18, 21), no systematic comparison between TMA and conventional
slides as regards IHC has

been carried out, apart from a few small studies comparing EGFR IHC in biopsies and

resected specimens (22
-
23). Also, to our knowledge, the TMA technique has not been used

for assessment of EGFR FISH in lung cancer. However, HER2
FISH has been validated for

breast cancer (24), ovarian cancer (25) and for inter laboratory validation of FISH testing for

HER2 in breast cancer, using the TMA technique (26). Thus, it is reasonable to extrapolate

results for HER2 into EGFR, as the probes

for HER2 and EGFR are very similar and, in this

case, from the same supplier. Finally, in our study, hybridizing was successful with all cores

in the TMAs that contained viable tumour tissue, which makes TMA an almost ideal

technique

for biomarker studies with FISH in lung cancer. Thus, from a technical point of

view, the TMA technique is feasible for biomarker studies in lung cancer.

In 334 of the cases in our study both tests were evaluable and the correlation between IHC

and FISH w
as highly significant (Table 1, p = 0.0007). A previous study, with only 27 cases,

reported that EGFR IHC results were correlated with FISH results (P = 0.0125), but not with

prognosis (27). An association between EGFR FISH and EGFR IHC was also found by

H
irsch et al (16). They found that both FISH+ and IHC+ correlated with an improved survival

and that in multivariate survival analysis EGFR FISH and IHC were independent predictive

markers. We found 31% of the cases to be FISH+/IHC+ and 29% of the cases to
be FISH
-

/IHC
-
, which is comparable to the 23% and 30% reported by Hirsch et al (16). It has been

shown herein that both methods are reliable and valid as all cores in the TMA that contained

tumour tissue were evaluable with IHC as well as with FISH. Thus,

from our study and from

previous studies it may be concluded that EGFR FISH and EGFR IHC can be reliably

performed and bear about the same kind of information. Thus, in analogy with the situation

for breast cancer and HER2, both may be included in a panel

of tests (involving screening for

EGFR and KRAS mutations, and in the future perhaps MET amplification, AKT

phosphorylation as well as other markers) to receive therapy with TKIs.


In our study Kaplan
-
Meier plots showed that EGFR IHC positive cases (1
-
3+)

had a

significantly worse survival compared to EGFR IHC negative cases (Figure 3A, p=0.02). A

significant difference in survival could not be found between EGFR FISH positive and

negative cases (Figure 4A, p=0.35). This is in agreement with Zheng et al (1
8), and with

Meert et al (5) who in a meta
-
analysis of 2,185 NSCLC patients found statistical significance

between EGFR expression and survival only in the subgroup of studies using IHC. However,

the result of another study of 262 cases has indicated that
also EGFR FISH predicted worse

survival (19). When the 1981
-
83 and 1995
-
97 series of patients in our study were tested

separately for EGFR IHC significance remained only for the 1981
-
83 series. Surprisingly, this

association

was also found for EGFR FISH in the 1981
-
83 series of patients. Thus, for the

1981
-
83 series of patients both EGFR IHC and FISH positivity was an adverse factor for

survival. The reason for this is not entirely clear, although the most likely explanation
is a

covariation with TNM stage. Similar results have been reported very recently in a congress

abstract (28), were EGFR FISH was performed with TMA technique in a study of 356

NSCLC patients. A correlation was found between EGFR and progression of the T f
actor and

between EGFR genetic gain and amplification and tumour grade. It is well known that due to

the development and use of new medical technology clinical staging in the 80ies was not as

good as in the 90ies, leading to clinical understaging and more
advanced stages treated by

8

surgery. The complete pTNM in our material is not known, and is presently not possible to

reconstruct.


When we tested males and females separately, males with EGFR IHC positivity had a

significantly worse survival (Figure 5, p
=0,009) while no such difference was found for

females (Figure 5, p=0.80). Moreover, when IHC positivity was related to both decade and

gender there was still significance for males in the 1981
-
83 series (p=0.04, supplemental data)

and a strong tendency in

the 1995
-
97 series (p=0.04, supplemental data). Thus, the adverse

effect of EGFR IHC positivity on survival in this study is confined to males. An explanation

might be that squamous cell carcinoma is overrepresented in males in our study (80%) and a

signi
ficant association between EGFR overexpression and lower differentiation grade and

node positivity has, for instance, been reported in head and neck squamous cell carcinomas

(29).


When males and females were tested separately for EGFR FISH positivity no d
ifference with

respect to survival, neither among males (p=0.88, supplemental data) nor females (Figure 8,

p=0.14), was found. However, when FISH was related to decade and gender, surprisingly

there

was a highly significant relation between FISH positivity and survival for females in the

1981
-
83 series (p=0.004, supplemental data) but not in the 1995
-
97 series (p=0.99,

supplemental

data). The reason for this is unclear; however, only 25% of the cases in the

1981
-
83 series are women.


Data on EGFR protein expression, EGFR gene copy numbers and other markers for

responsiveness to EGFR
-
TKIs are conflicting. In several studies, EGFR mut
ations have been

strongly correlated with increased response rate and progression free survival in response to

erlotinib and gefitinib (9
-
10, 15). A significantly increased survival after TKI treatment in

patients whose tumours harbour EGFR mutations has a
lso been reported by several

investigators, although a prognostic rather than predictive role for mutations has not been

ruled out (30). Furthermore, different localizations of mutations within the EGFR gene confer

different sensitivity to TKIs, with exon
19 deletions being associated with the best responses

and certain point mutations in exons 19 and 20 being linked to drug resistance (31). Thus,

additional prospective trials are needed to determine the positive and negative predictive

roles, respectively,

of different EGFR mutations and also the impact of KRAS mutations,

which are associated with resistance to EGFR
-
inhibitors (11).


In response to gefitinib and erlotinib, high EGFR gene copy number (FISH
-
positive status)

has been significantly associated w
ith increased tumour response rate, time to progression,

and better survival compared to patients without gain of EGFR and, furthermore, significantly

correlated with mutation status and clinical parameters that have been suggested previously as

predictors

for benefit from TKI treatment (32). Most importantly, in participants from the

BR.21 trial, a significant overall survival benefit for erlotinib versus placebo was seen in

patients with FISH
-
positive tumours, but not in the FISH
-
negative patients (33) .


Early studies found no relationship between EGFR protein expression and benefit from

treatment with EGFR
-
inhibitors (12
-
14). However, in the BR.21 study, a survival

improvement for erlotinib versus placebo was demonstrated for patients whose tumours

showed EGFR protein expression by IHC, whereas no significant difference could be

demonstrated in the IHC
-
negative group (34). In agreement with the results from the BR.21

(34) (57%), ISEL (36) (69,6%) we found 60% positive staining for EGFR IHC, using 1+

staining intensity and staining in > 10% of the cell membranes as cut
-
off. Moreover, 10% cut

off has been reported to provide the best discrimination between EGFR
-
positive and
EGFRnegative

patients for survival hazard ratios comparing gefitinib to placebo

(36).


In summary, we found a highly significant correlation between increased EGFR gene copy

number and EGFR protein expression. Indeed, the main clinical question is who will
not

benefit

from treatment with EGFR
-
inhibitors and several groups have suggested that tumours

negative for both EGFR protein expression by IHC and for EGFR copy numbers by FISH are

unlikely to respond. Thus, in this perspective, a good correlation between these two
methods

in our and other studies is of value since it admits a reliable identification of a group of

patients who will not benefit from treatment. The next group of lung cancer patients to be

excluded from treatment with EGFR
-
inhibitors is probably those w
hose tumours harbour
KRAS
-
mutations (11, 33), thus leaving only the relevant subgroup of cases that should be

selected for this treatment. Furthermore, the need for selecting NSCLC patients in large

clinical trials by biomarkers for EGFR status can be faci
litated by use of TMA for
highthroughput

analysis of protein expression by IHC or copy numbers by FISH in large
cohorts

of patients.



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13

Legends to the figures.

Figure 1A. Immunohistochemistry for EGFR. NSCLC with weak (1+) EGFR staining.

Figure 1B. Immunohistochemistry for EGFR. NSCLC with moderate (2+) EGFR staining.

Fi
gure 1C, Immunohistochemistry for EGFR. NSCLC with strong (3+) EGFR staining.

Figure 2A. Fluorescence in situ hybridising (FISH) with polysomy. Equal number of signals

for the EGFR gene (orange) and CEP7 (green). Nuclei stained with DAPI.

Figure 2B.
Fluorescence in situ hybridising (FISH) with amplification. Clusters of signals for

the EGFR gene (orange) and two signals for CEP7 (green). Nuclei stained with DAPI.

Figure 3. Survival, IHC positive compared to IHC negative. A all patients, B 1981
-
83 ser
ies,

C 1995


97 series.

Figure 4. Survival, FISH positive compared to FISH negative. A all patients, B 1981
-
83

series, C 1995


97 series.

Figure 5. Comparison between IHC/FISH and gender.

Figure 6 (supplemental data). IHC comparison decade/gender

Figure

7 (supplemental data). FISH comparison decade/gender


TABLE
x


Tumor

Histology

Mutation
-
specific IHC*

Direct DNA
sequencing (exon 19
and 21)**

IHC***


FISH*
***


1


delE746
-
A750


3

2

2


delE746
-
A750


1

1

3


delE746
-
A750


1

1

4


delE746
-
A750


1

1

5


delE746
-
A750


0

0

6


delE746
-
A750


1

1

7


delE746
-
A750


0

0

8


delE746
-
A750


2

0

9


delE746
-
A750


2

1

10


L858R


0

0

11


L858R


1

1

12


L858R


2

1

13


L858R


2

1

14


L858R


3

2

15


L858R


3

2

16


L858R


1

1

17


L858R


3

2

18


L858R


3

2

19


L858R


0

1