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Overexpression of Epidermal Growth Factor Receptor Restricted to Macrophages in Uveal Melanoma
Andrea G. M. Scholes, PhD;
Suzanne Hagan, BSc;
Paul Hiscott, MRCPath;
Bertil E. Damato, PhD;
Ian Grierson, PhD
Arch Ophthalmol. 2001;119:373-377.
ABSTRACT
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Objective To determine whether expression of the epidermal growth factor receptor
(EGFR) is of prognostic value in uveal melanoma.
Methods Thirty consecutive patients treated for primary posterior uveal melanoma
by enucleation or local resection were studied. Tumors were examined for EGFR
and CD68 expression by immunohistochemistry on formalin-fixed, paraffin-embedded
sections. Extracted DNA from paired frozen tumor and blood samples was examined
for loss of heterozygosity on chromosome 3 using polymerase chain reaction–based
microsatellite analysis. Immunoreactivity for EGFR was correlated with clinicopathological,
chromosome 3, and follow-up data.
Results Immunoreactivity for EGFR was observed in 7 (23%) of 30 uveal melanomas,
but was restricted to solitary or small groups of cells with macrophage-like
morphology. Immunoreactive cells were confirmed as macrophages using an antibody
to the macrophage marker CD68. Chromosome 3 loss, epithelioid cells, and microvascular
loops were detected in 17 (57%), 22 (73%) and 19 (63%) of the 30 tumors, respectively.
Metastatic disease was detected in 5 patients (17%). No correlation was found
between any of these variables and EGFR positivity.
Conclusions The absence of EGFR immunoreactivity in tumor cells does not support
the use of EGFR expression as a prognostic indicator in patients with uveal
melanoma. Future EGFR studies in uveal melanoma should be interpreted with
caution in view of our findings that tumor-associated macrophages can express
this receptor.
INTRODUCTION
THE EPIDERMAL growth factor receptor (EGFR), a member of the ErbB family
of receptor tyrosine kinases, is of fundamental importance in the regulation
of epithelial differentiation and proliferation.1-2
The interactions of this receptor are complex. Epidermal growth factor receptor
can bind at least 6 growth factors, including EGF, transforming growth factor  ,
amphiregulin, heparin-binding EGF, betacellulin, and epiregulin.2
Binding of EGF results in receptor down-regulation, whereas transforming growth
factor prolongs EGFR expression and signalling.3
The EGFR appears to play a role in malignant transformation and may be a useful
target for selective antitumor therapy.4 Overexpression
of EGFR has been detected in many solid human tumors, including cancers of
the head and neck, breast, lung, and bladder,4
and has been associated with poor prognosis, most convincingly in squamous
cell carcinoma of the head and neck.5 Increased
immunoreactivity for EGFR has also been reported in cutaneous benign and malignant
cutaneous lesions of melanocytic origin.6-8
Uveal melanoma is the most common primary intraocular malignant neoplasm
in adults. The mortality rate for patients with large tumors is high because
of the propensity for these tumors to metastasize to the liver.9
After clinical diagnosis of hepatic metastases, life expectancy is extremely
poor, with a median survival time of only 7 months.10
Recently, in a nude mouse model, expression of EGFR was correlated with the
metastatic potential of uveal melanoma cell lines and an increased capacity
for these tumor cells to localize in the liver.11
These findings suggested that EGFR expression may be a valuable indicator
of metastatic potential in uncultured uveal melanoma. The expression of this
receptor, however, had not been described in uncultured uveal melanoma, and
the tumors from which the cell lines were derived were not tested.11 Therefore, we undertook a study to determine whether
assessment of EGFR is of prognostic value in uncultured primary uveal melanoma.
We correlated EGFR expression with clinicopathological variables, metastatic
disease, and genomic alterations, specifically chromosome 3 loss, which is
associated with a high probability of metastatic disease.12-14
PATIENTS, MATERIALS, AND METHODS
PATIENTS AND SPECIMENS
Thirty consecutive patients treated for choroidal and/or ciliary body
melanoma by enucleation or local resection formed the basis of this study.
Informed consent was obtained from all patients to collect tumor and blood
samples for experimental purposes. Part of each tumor was snap frozen in liquid
nitrogen and the remainder fixed with formalin and embedded in paraffin. Histological
features, including cell type (spindle, epithelioid, or mixed) and the presence
or absence of microvascular loops, were determined from paraffin-embedded
sections of tumor stained with hematoxylin-eosin and periodic acid–Schiff.
Clinical details and follow-up data were obtained from our ocular oncology
database, linked with the Office of National Statistics (Southport, England)
to ensure accurate mortality data.
IMMUNOHISTOCHEMISTRY
Formalin-fixed, paraffin-embedded tumor tissue was examined for EGFR
expression by immunohistochemistry, using a monoclonal anti-EGFR antibody
(clone EGFR 113; Novacastra Laboratories Ltd, Newcastle upon Tyne, England;
diluted 1:75) and a polyclonal anti-EGFR antibody (1005; Santa Cruz Biotechnology
Inc, Santa Cruz, Calif; diluted 1:100). Sections were obtained from 2 tumor
blocks for each patient. The titer of the primary antibody used was the serial
dilution that gave intense membranous staining with negligible background
in formalin-fixed, paraffin-embedded human placenta, which overexpresses EGFR.15 Tumor sections (4 µm thick) were dewaxed in
xylene, then hydrated through graded alcohols to water. Subsequently, sections
were submerged in 0.01-mol/L citric acid (pH, 6.0), microwaved for 18 minutes,
and left to stand in this solution for an additional 15 minutes. Sections
were then washed in Tris-buffered saline solution (TBS; pH, 7.6) for 5 minutes
and blocked using normal horse serum (Vector Laboratories Ltd, Peterborough,
England). Blocking serum was removed and the sections were incubated overnight
with the primary antibody at 4°C in a moist chamber. After washing in
TBS, sections were incubated with a biotinylated secondary antibody (Vector
Laboratories Ltd) for 30 minutes, then washed twice in TBS, and incubated
with streptavidin-biotin–alkaline phosphatase complex (Dako Ltd, High
Wycombe, England) for 30 minutes, all at room temperature. Sections were washed
in TBS, and alkaline phosphatase–fast red substrate solution (Vector
Red substrate and levamisole [Vector Laboratories Ltd] with 5 mg of fast red
TR salt [Merck Ltd, Lutterworth, England] in 5 mL of TBS [pH, 8.2]) was applied
for 25 minutes. Sections were washed in running tap water for 5 minutes, counterstained
with hematoxylin, and mounted with an aqueous mounting medium (Glycergel;
Dako Ltd). Human placenta sections were included as positive controls, and
sections with replacement of the primary antibodies with an isotype control
antibody or blocking peptide served as negative controls. A bleaching protocol16 with 3,3'-diaminobenzidine tetrahydrochloride
as chromogen (which is resistant to hydrogen peroxide) was used for 4 sections
that were heavily pigmented. Melanin was bleached by overnight incubation
of sections in 1% (weight-volume ratio) disodium hydrogen phosphate solution
containing 3% hydrogen peroxide. Confirmation of the identity of cells with
macrophage-like morphology was determined using an antibody to the macrophage
marker CD68 (Dako Ltd), as described for the anti-EGFR antibody. Dual labeling
with the polyclonal anti-EGFR and monoclonal anti-CD68 antibodies was performed
using an alkaline phosphatase technique. Red and blue chromogens were used
for EGFR and CD68, respectively (Vector Red and Vector Blue; Vector Laboratories
Ltd).
MICROSATELLITE ANALYSIS
The DNA was extracted from blood and frozen tumor sections using standard
procedures.17 One frozen section of each tumor
was stained with hematoxylin-eosin to confirm the histological features and
that the specimen was composed of at least 90% tumor cells. Chromosome 3 alterations
were detected by polymerase chain reaction–based microsatellite analysis,18 using the following 10 microsatellite markers on
the p and q arms of chromosome 3 (Research Genetics, Huntsville, Ala): D3S1038 (3p26.1-3p25.2), D3S1283
(3p25-3p24.2), D3S1619 (3p24.2-3p22), D3S1029 (3p21.3-3p21.2), D3S1210 (3p14.1-3p12), D3S1271 (3cen-q13), D3S1589 (3q21), D3S1605 (3q25.1-3q25.2), D3S1580
(3q27), and D3S1311 (3q27-qter). Loss of heterozygosity
(LOH) was recorded for informative markers if the intensity of a tumor allele
was reduced by at least 30% relative to normal DNA.
RESULTS
The posterior uveal melanomas studied consisted of 8 spindle cell and
22 mixed/epithelioid cell tumors. The largest basal diameter of these tumors
ranged from 10 to 20 mm (mean, 15.8 mm), and 4 tumors had ciliary body involvement.
Microvascular loops were detected in 19 tumors (63%). Chromosome 3 alterations
were identified in 17 tumors (57%). Metastases were detected in 5 patients
(17%) within 8 to 21 months of primary treatment (maximum patient follow-up,
27 months); 3 of these patients have died.
Immunoreactivity for EGFR, of similar intensity to that in placenta
(positive control; Figure 1,
A), was observed in 7 (23%) of 30 uveal melanomas. The proportion of EGFR-positive
tumors associated with particular clinical and histological variables was
as follows: 3 (38%) of 8 spindle cell and 4 (18%) of 22 mixed/epithelioid
cell tumors; 3 (16%) of 19 tumors with microvascular loops and 4 (36%) of
11 without microvascular loops; 3 (18%) of 17 tumors with LOH and 4 (31%)
of 13 without LOH on chromosome 3; 2 (22%) of 9 tumors with a basal tumor
diameter of less than 15 mm and 5 (24%) of 21 with a basal tumor diameter
of at least 15 mm; and 1 (25%) of 4 tumors with ciliary body involvement and
6 (23%) of 26 without ciliary body involvement. There was no statistically
significant correlation between detection of EGFR and tumor cell type, presence
of microvascular loops, chromosome 3 LOH, tumor size, or ciliary body involvement
(Fisher exact test). Only 1 (20%) of 5 tumors from patients with metastatic
disease showed EGFR immunoreactivity, whereas all 5 tumors showed chromosome
3 LOH.
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A, Placenta (positive control) showing immunoreactivity with a monoclonal
anti–epidermal growth factor receptor (EGFR) antibody (alkaline phosphatase–fast
red). B and C, Serial sections of a choroidal melanoma of mixed cell type
showing immunoreactivity in cells of macrophage-like morphology with anti-EGFR
(B) and anti-CD68 (macrophage marker, C) antibodies. Arrowheads indicate examples
of cells labeled with both antibodies (original magnification x250).
Brown pigment is melanin.
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Immunoreactivity for EGFR was restricted to solitary or small groups
of cells with morphology typical of macrophages rather than tumor cells (Figure 1, B). Immunoreactive cells were confirmed
as macrophages using an antibody to the macrophage marker CD68 on a section
adjacent to that used for EGFR detection (Figure 1, C) and by dual labeling, which demonstrated colocalization
of CD68 on EGFR-immunoreactive cells (not illustrated). More cells were reactive
with anti-CD68 than with anti-EGFR antibody.
COMMENT
Overexpression of EGFR has been found in a number of solid tumor types
and has been associated with poor prognosis.4
Recently, expression of EGFR was correlated with the hepatic spread of uveal
melanoma cell lines in a nude mouse model,11
raising the possibility that EGFR expression may be a valuable indicator of
metastatic potential in uncultured uveal melanoma. Expression of EGFR, however,
had not been described in uncultured uveal melanoma, and the tumors from which
the cell lines were derived were not tested.11
In this study, we examined the expression of EGFR in 30 uncultured primary
posterior uveal melanomas. Expression of EGFR was detected in 7 tumors; however,
the immunoreactive cells had morphologic features resembling macrophages rather
than tumor cells and were confirmed as macrophages using an anti-CD68 antibody.
This lack of detection of EGFR on uncultured tumor cells apparently
conflicts with previous findings on cell lines.11
The extent to which the cell lines used by Ma and Niederkorn11
represent the tumors from which they were derived, however, is not known.
Cultured cells can diverge significantly from the original tumor, eg, by accumulation
of genomic alterations,19 and expression of
EGFR can be modulated during culture.20 Alternatively,
a small proportion of uveal melanomas may overexpress EGFR, and these tumors
may be more readily established as cell lines. In breast cancer, for example,
the success rate of establishing cell lines is low, but tumors overexpressing
the growth factor receptor HER-2/neu (ErbB2) are more likely to develop into
continuous cell lines.21 High concentrations
of EGF assist establishment of cell lines of uveal melanoma.22
However, the proportion of cells expressing EGFR was less than 6% in 4 of
the 7 cell lines studied by Ma and Niederkorn.11
As the regulation of EGFR is complex, it is possible that prolonged culture
in growth factor–containing media may have affected receptor expression.
Cell lines composed purely of spindle cells had a higher percentage of EGFR-positive
cells (5.3%-20.3%) than the epithelioid cell lines (1.7%-2.5%), and the incidence
of metastasis in nude mice was lowest with the epithelioid cell lines.11 This is the opposite of what might be expected from
human studies of tumor cell type and prognosis in patients with uveal melanoma.23-24
It cannot be ruled out that occasional uncultured uveal melanoma tumor
cells express EGFR, as the proportion of cells expressing EGFR was low in
some of the cell lines studied by Ma and Niederkorn.11
In our study, to verify the lack of tumor cell immunoreactivity with the monoclonal
anti-EGFR antibody, we analyzed sections from a second block of each tumor
using an additional polyclonal anti-EGFR antibody. However, tumor cells expressing
EGFR were still not detected.
Although long-term survival data are not yet available, detectable metastases
developed in 5 of the patients studied, and 3 of these have died. Tumor cells
showing EGFR immunoreactivity were not detected in the uveal melanomas from
these patients, and only 1 lethal tumor showed EGFR immunoreactivity in macrophages.
The lack of detection of EGFR in tumor cells from these patients suggests
that expression of this receptor is not a sensitive prognostic indicator in
posterior uveal melanoma. Furthermore, it is expected that many other patients
in our study will ultimately die of metastatic disease. Microvascular loops
and chromosome 3 LOH, which are strongly associated with a poor prognosis,12-14,25-26
were detected in a high proportion of the tumors. Indeed, chromosome 3 LOH
was detected in 17 (57%) of 30 tumors, a prevalence similar to that reported
by other groups, and all of the tumors from which metastases were detected
showed this alteration, supporting previous findings that chromosome 3 loss
is a sensitive prognostic indicator.12-14
Macrophages immunoreactive for EGFR have been reported in other lesions
and may be involved in their pathogenesis.27-30
It appears that macrophages may influence angiogenesis, a process that is
critical for tumor growth and metastasis.31
Macrophage infiltration in breast tumors and gliomas has been associated with
angiogenesis and poor prognosis,32-33
and anti-EGFR antibody has been shown to inhibit angiogenesis in a model of
transitional cell carcinoma of the bladder.34
Activated macrophages may play a role in the neovascularization of cutaneous
melanoma, as a correlation between increased macrophage index, malignancy,
and high vascular grade has been reported in these tumors.31
In our study, microvascular density was not determined, but other workers,
using endothelial cell markers, previously have reported this variable to
be of prognostic significance in uveal melanoma.35-36
We are now performing further studies of macrophage and microvascular patterns
to determine the relationship between these variables in uveal melanoma. The
presence of microvascular loops, also an indicator of poor prognosis in uveal
melanoma,25-26 was assessed in
our study. There was no correlation between the presence of EGFR-positive
macrophages and microvascular looplike structures; however, it has been shown
that endothelial cells do not line these periodic acid–Schiff–positive
structures.37
In summary, the absence of EGFR immunoreactivity in tumor cells does
not support the use of EGFR expression as a prognostic indicator in patients
with uveal melanoma. In view of our findings that EGFR-immunoreactive cells
in uveal melanoma were macrophages, we suggest that EGFR studies in uveal
melanoma be interpreted with caution.
AUTHOR INFORMATION
Accepted for publication August 5, 2000.
This study was supported by a grant from the University of Liverpool
Research Development Fund, Liverpool, England.
Corresponding author: Andrea G. M. Scholes, PhD, Unit of Ophthalmology,
Department of Medicine, Duncan Building, University of Liverpool, Liverpool
L69 3GA, England.
From the Unit of Ophthalmology, Department of Medicine (Drs Scholes,
Hiscott, and Grierson and Ms Hagan), and the Department of Pathology (Dr Hiscott),
University of Liverpool, and the Ocular Oncology Service, St Paul's Eye Unit,
Royal Liverpool University Hospital (Dr Damato), Liverpool, England.
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