(Investigative Ophthalmology and Visual Science. 2000;41:2023-2027.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Expression of Epidermal Growth Factor Receptor: Risk Factor in Uveal Melanoma
H. Monique H. Hurks1,
Jessica A. W. Metzelaar-Blok1,
Ed R. Barthen1,
Aeilko H. Zwinderman2,
Didi De Wolff-Rouendaal1,
Jan E. E. Keunen1 and
Martine J. Jager1
1 From the Departments of Ophthalmology and
2 Medical Statistics, Leiden University Medical Center, The Netherlands.
 |
Abstract
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PURPOSE. To investigate the prognostic significance of the expression of
epidermal growth factor receptor (EGFR) in uveal melanoma. EGFR is a
transmembrane glycoprotein, and its expression has been correlated with
the development of metastases in various malignancies.
METHODS. Frozen sections from 22 primary uveal melanomas were examined for EGFR
expression by a three-step immunoperoxidase staining, using a mouse
anti-human EGFR IgG2b monoclonal antibody. The results were compared
with patient survival and clinical and histopathologic parameters.
RESULTS. EGFR expression could not be determined on one tumor due to excessive
pigmentation. Two patients died of causes unrelated to melanoma, and
two patients were lost to follow-up. Out of 21 tumors, six tumors
showed immunoreactivity for EGFR. Five of these six patients (83%)
died due to metastases, compared with 2 (17%) of 12 patients with no
EGFR expression (Kaplan–Meier analysis P = 0.0004).
EGFR-positive tumors tended to have a greater tumor prominence and a
higher mitotic rate.
CONCLUSIONS. The expression of EGFR was significantly correlated with death due to
metastatic disease and therefore can be regarded as an important
prognostic factor in human uveal melanoma.
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Introduction
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With an annual incidence of 6 to 8 cases per million persons in
white populations, uveal melanoma is the most common primary malignant
intraocular tumor in adults. The high mortality rate is related to the
development of metastases with a strong preference for the
liver.1
2
No effective therapy for the treatment or
prevention of these metastases is available. Several prognostic factors
have been identified for uveal melanoma, including histopathologic
factors (e.g., cell type, largest tumor diameter, SD of nucleolar
area), genetic factors (e.g., aneuploidy, chromosome 3 monosomy), and
immunologic factors (e.g., expression of human leukocyte antigens, T
cell infiltration).3
Recently, Ma and Niederkorn4
observed in murine studies
that expression of epidermal growth factor receptor (EGFR) by uveal
melanoma cells was significantly correlated with the development of
liver metastases and decreased survival. EGFR is a 170-kDa
transmembrane glycoprotein that is expressed on a wide variety of
normal and malignant cells. Binding of EGFR, by epidermal growth factor
(EGF), transforming growth factor (TGF)-
, or hepatocyte growth
factor, can induce cell differentiation, proliferation, and the
expression of oncogenes such as fos and
jun.5
Enhanced EGFR expression has been shown
to be an indicator of poor prognosis in various human
malignancies.6
7
8
9
The purpose of the present study was to investigate the prognostic
significance of EGFR expression in human uveal melanoma. Therefore, we
determined the EGFR expression on frozen sections of 22 primary uveal
melanomas and compared these data with survival and various clinical
and histopathologic parameters. The results showed that EGFR expression
was a significant marker of poor prognosis.
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Materials and Methods
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Patients and Tumors
Between 1987 and 1992, tumor specimens were obtained from 22
primary uveal melanomas immediately after enucleation. The patients
were not treated with radio- or thermotherapy. The research protocol
followed the tenets of the Declaration of Helsinki. A part of the tumor
was snap-frozen in liquid isopentane and stored at -70°C. The
remainder of each specimen was processed for histopathologic
examination by an ocular pathologist. Of the 22 patients studied, 10
were women and 12 were men. Age range was 38 to 91 years, with a mean
age at diagnosis of 66 years. Seven patients died of a tumor-related
cause, 2 died of other causes, 11 were still alive, and 2 patients were
lost to follow-up at the time of the study.
Histopathology
The tumors were histologically classified according to cell type.
Five (23%) tumors consisted of spindle cells, 3 (13%) tumors of
epithelioid cells, and 14 (64%) tumors of both cell types. All tumors
were localized in the choroid, with the exception of patient 2, in whom
the tumor was based in the ciliary body. The tumor of patient 13 was
localized in both the choroid and the ciliary body. Largest tumor
diameter and prominence of the tumor were measured in millimeters, and
the number of mitoses was counted in 15 high-power fields with a
magnification of x320 (Table 1)
.
Immunohistochemistry
Frozen sections, mounted on glass slides coated with
aminopropyltriethoxysilane (APES; Sigma, St. Louis, MO), were fixed in
acetone at 4°C for 10 minutes. After they were washed in
phosphate-buffered saline (PBS), the slides were preincubated with 40%
human serum in PBS with 3% bovine serum albumin (BSA) for 20 minutes.
The slides were washed again in PBS and incubated for 30 minutes with a
mouse anti-human EGFR IgG2b monoclonal antibody (R-1; Santa Cruz
Biotechnology, Santa Cruz, CA) at a dilution of 1:20 or with 1%
BSA-PBS as negative control. After they were washed, the slides were
then incubated with biotinylated antimouse immunoglobulins (LSAB-2 kit
from Dako, Glostrup, Denmark) for 10 minutes, washed again and
incubated in methanol with 0.3%
H2O2 for 30 minutes. After
another washing, the slides were incubated with horseradish
peroxidase–conjugated streptavidin (Dako) for 10 minutes. The
peroxidase reaction was developed using 5% 3-amino-9-ethyl-carbazole
(Sigma) in 0.1 M sodium acetate buffer (pH 5) containing 0.05%
H2O2. The sections were
counterstained with Mayer’s hematoxylin and finally embedded in
Kaiser’s glycerin. Sections of colon carcinoma were used as positive
control for the EGFR expression.
Statistical Analysis
The correlation between EGFR expression and melanoma-related death
and clinical and histologic parameters was assessed using the
Kaplan–Meier survival analysis, the log rank test, and Cox
proportional hazards regression model. The relationship between EGFR
expression and clinical and histopathologic parameters was determined
with the 
2 test or the nonpaired, two-sided
Student’s t-test, as appropriate.
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Results
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Immunohistochemistry
EGFR expression was determined on frozen sections of 22 primary
uveal melanomas by a three-step immunoperoxidase staining. One tumor
(tumor 4, Table 1
) could not be evaluated due to excessive
pigmentation. Six of the remaining 21 tumors showed immunoreactivity
for EGFR (Table 1)
. The staining pattern in these tumors was diffuse,
with EGFR-positive as well as EGFR-negative areas (Fig. 1)
. Although one EGFR-positive tumor was amelanotic, the
EGFR-positive fields in the other tumors were more often found in the
pigmented parts of the tumor. In addition, EGFR-positive cells were
frequently observed near blood vessels (Fig. 1)
.
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Figure 1. Expression of EGFR on frozen sections of colon carcinoma (A,
B) and primary uveal melanomas (C through
F). EGFR expression was determined by three-step
immunoperoxidase staining, using a mouse-anti-human EGFR IgG2b
monoclonal antibody. (A) Negative control; (B)
positive control for the EGFR expression on colon carcinoma tissue;
(C) positive EGFR staining of uveal melanoma cells;
(D) cluster of positive-staining cells near a blood vessel;
and (E, F) positive EGFR staining in a heavily
pigmented area (E) and an EGFR-negative field in a less
pigmented area (F) of the same tumor.
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EGFR Expression and Survival
In the 22 patients included in the study, the 5-year tumor-related
survival rate was 67%. Of the six patients with EGFR expression in the
tumor, five patients (83%) died within 5 years of uveal melanoma
metastases. One EGFR-positive patient was still alive 9 years after
enucleation. Of the 15 patients with EGFR-negative tumors, two patients
were lost to follow-up, and one patient died of a cause unrelated to
the tumor. Of the remaining 12 patients, 2 (17%) died of metastatic
disease, 5 and 7 years after enucleation. The median follow-up time of
the 10 patients still alive was 8.9 ± 0.4 years (SE). The
Kaplan–Meier survival curves (Fig. 2) show a clear significant difference (P = 0.0004)
between the survival rates of patients with EGFR-positive and
EGFR-negative tumors. The relative risk was 12.2, with a 95%
confidence interval (CI) of 2.17–68.1 (Cox proportional hazards
regression model). These data indicate that EGFR expression is an
important prognostic factor.
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Figure 2. Kaplan–Meier survival curves of 18 uveal melanoma patients after
primary enucleation, depicting the probability of death due to uveal
melanoma versus expression of EGFR. P = 0.0004 (log
rank test).
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EGFR Expression and Clinical and Histopathologic Parameters
Various histopathologic data were compared between EGFR-positive
and EGFR-negative tumors (Fig. 3)
. Although the differences were not significant, EGFR-positive tumors
had a greater tumor prominence (average, 7.3 mm versus 5.8 mm,
P = 0.19) and a higher mitotic rate (13.8 versus 9.3
mitoses per 15 high-power fields, P = 0.23). In
addition, patients with EGFR-positive tumors were older (average, 70
years versus 64 years, P = 0.33). No associations were
found between EGFR expression and tumor diameter, cell type,
pigmentation, or gender. In contrast to the EGFR-negative group,
Bruch’s membrane was broken in all EGFR-positive tumors (no
significance).
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Figure 3. Tumor diameter (A), prominence (B), and mitotic
rate (C) in EGFR positive and EGFR negative primary uveal
melanomas. The mitotic rate is expressed as the number of mitoses per
15 high-power fields. Magnification, x320. Each dot
represents a tumor. Averages are represented by horizontal
bars.
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In addition to EGFR, survival was significantly associated with age
(P = 0.038, relative risk = 1.07 per year; 95%
CI, 1.00–1.14). After adjustment for age, EGFR was still a significant
predictor of tumor-related survival (P = 0.01, multiple
Cox regression model). No correlations were found between survival and
the other histopathologic characteristics.
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Discussion
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EGFR expression has been correlated with the growth and
development of metastases of various neoplasms. In a number of cancers,
including breast cancer,6
gliomas,8
squamous
carcinoma,9
and laryngeal cancer,7
EGFR has
been shown to be a marker of poor survival. In other cases, such as
cutaneous melanoma, there is controversy about whether EGFR expression
is related to tumor progression.10
11
In the present
study, a clearly significant difference was found between the survival
rates of patients with EGFR-positive and EGFR-negative tumors,
indicating that, in uveal melanoma, EGFR expression is an important
prognostic factor.
Tumor expression of EGFR may enhance uveal melanoma metastasis by
different mechanisms. Binding of EGFR (e.g., by EGF or TGF-, both
present in the eye) results in the activation of several pathways that
may lead to increased cell growth, DNA synthesis, motility, chemotaxis,
secretion of proteases, cell adhesion, and the expression of
oncogenes.5
12
These effects all play an important role in
the metastatic cascade. An interesting question is whether EGFR is
involved in the preference of uveal melanoma cells to metastasize to
the liver. In human breast carcinoma, EGFR expression was correlated
with lymph node metastases,13
whereas in studies with
human cutaneous melanoma cell lines, a positive correlation was found
between EGFR expression and the potential of lung metastases to develop
after subcutaneous inoculation of he tumor cells in nude
mice.10
14
In colon carcinoma, EGFR expression was, as in
uveal melanoma, correlated with the development of liver
metastases.15
In the liver, high levels of hepatocyte
growth factor and TGF- which can bind to EGFR, are present. As also
has been suggested by Ma and Niederkorn,4
the
presence of these EGFR ligands in the liver may be involved in the
preference of uveal melanoma cells to metastasize to this organ.
However, ligands for EGFR (e.g., TGF- and EGF) have been found in
many other tissues and body fluids.16
In the nude mice
studies of Ma and Niederkorn,4
intravenously injected
human uveal melanoma cells were shown to accumulate predominantly
(70%) in the liver. Blocking EGFR with neutralizing antibodies reduced
liver metastasis and prolonged host survival. Of interest, anti-EGFR
antibody treatment reduced the number of EGFR-positive melanoma cells
in the liver, but did not affect the liver homing of EGFR-negative
tumor cells. Finally, EGFR has been shown to protect tumor cells
against the cytolytic effects of tumor necrosis
factor-4
and against killing by
interleukin-2–activated peripheral blood mononuclear
cells.17
Prevention of tumor lysis may be an additional
mechanism by which EGFR contributes to tumor progression.
In the present study, tumor EGFR expression tended to be correlated
with tumor prominence and mitotic activity. In several cancers,
a positive correlation between EGFR expression and proliferation has
been demonstrated.12
13
In our study, tumor diameter, cell
type, and mitotic rate, which are established prognostic factors in
uveal melanoma, were not correlated with survival. The relatively small
number of patients and the selection for large tumors (tumor material
was obtained from patients for whom eye-salvaging therapies were
inappropriate) may explain our findings.
The clear association between EGFR expression and increased risk of
metastatic disease offers the possibility of using EGFR for the
development of new therapeutic strategies in uveal melanoma. Ma and
Niederkorn4
have shown in a murine model that treatment
with anti-EGFR antibodies prolong host survival. In phase I trials of
lung carcinoma and gliomas, EGFR-blocking antibodies significantly
inhibited tumor growth.18
19
Ligands for EGFR can also be
used as carriers of cytotoxins or radioactive
isotopes.5
20
In addition, because tyrosine kinase is
important in the signal transduction pathway of EGFR, the use of
tyrosine kinase inhibitors may be a promising
strategy.5
21
These and other approaches with EGFR as a
therapeutic target are reviewed by Voldborg et al.5
To summarize, in agreement with the murine studies of Ma and
Niederkorn,4
the present study demonstrated that in
humans, EGFR expression was also correlated with death due to uveal
melanoma metastases. Most likely, the presence of EGFR-binding growth
factors in the liver provide the most optimal environment for the
embedding of uveal melanoma cells in this organ. Expression of EGFR may
be used as a prognostic factor for survival and for the development of
new therapeutic strategies in patients with uveal melanoma.
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Footnotes
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Supported in part by the Dutch Cancer Foundation (KWF).
Submitted for publication September 14, 1999; revised January 24, 2000; accepted February 2, 2000.
Commercial relationships policy: N.
Corresponding author: H. Monique H. Hurks, Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. mhurks{at}div3.azl.nl
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Abstract
Introduction
