The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 656-658
Copyright © 2001 by The Endocrine Society
Increased Expression of the Vascular Endothelial Growth Factor Is a Pejorative Prognosis Marker in Papillary Thyroid Carcinoma
M. Klein,
J.-M. Vignaud,
V. Hennequin,
B. Toussaint,
L. Bresler,
F. Plénat,
J. Leclère,
A. Duprez and
G. Weryha
Departments of Endocrinology (M.K., J.L., G.W.), Human Pathology
(J.-M.V., V.H., F.P., A.D.), Otorhinolaryngology (B.T.), and Surgery
(L.B.), Centre Hospitalier et Universitaire de Nancy, 54500
Vandoeuvre-les-Nancy, France
Address correspondence and requests for reprints to: M. Klein, M.D., Ph.D., Clinique Endocrinologique, Hôpitaux de Brabois, Centre Hospitalier et Universitaire de Nancy, 54500 Vandoeuvre-les-Nancy, France. E-mail: marc.olivier.klein@free.fr or
weryha{at}facmed.U-nancy.fr
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Abstract
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Vascular endothelial growth factor (VEGF) is a potent stimulator of
endothelial cell proliferation. It has been implicated in tumor growth
of human thyroid carcinomas. Using the VEGF immunohistochemistry
staining score, we correlated the level of VEGF expression with the
metastatic spread of 19 cases of thyroid papillary carcinoma. The VEGF
immunostaining score, ranging from 0–9, was determined as the
multiplication of a percentage of labeled thyrocytes score (0, no
labeling; 1, <30%; 2, 31–60%; 3, >61% of labeled thyrocytes) and
an intensity score (0, no staining; 1, weak; 2, mild; 3, strong
staining). The mean score ± SD was 5.74 ± 2.59
for all carcinomas. The mean score for metastatic papillary carcinoma
was 8.25 ± 1.13 vs. 3.91 ± 1.5 for
nonmetastatic papillary cancers (P < 0.001). By
discriminant analysis, we found a threshold value of 6.0, with a
sensitivity of 100% and a specificity of 87.5%. There were no
statistical differences between metastatic and nonmetastatic carcinomas
when age, tumor size, or thyroglobulin levels were considered.
The VEGF immunostaining score seems to be a helpful marker for
metastasis spread in differentiated thyroid cancers. An increased
production of VEGF could assess an aggressive disease and be the
hallmark of a trend to produce metastasis. We propose the VEGF
immunostaining score as a marker for the prognosis in differentiated
thyroid cancers. A value of 6 or more, should be considered as at high
risk for metastasis threat, prompting the physician to institute a
tight follow up of the patient.
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Introduction
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ANGIOGENESIS IS THE formation of new
blood vessels from preexisting microvasculature. It occurs in
physiological (1) and reparative conditions
(2). Angiogenesis is also implicated in several
pathological conditions like inflammation and tumor growth
(2). Because angiogenesis is of central importance in
tumor growth and progression, it has become a target of cancer therapy.
Vascular endothelial growth factor (VEGF), also known as vascular
permeability factor, is a potent stimulator of endothelial cell
proliferation in vitro and in vivo
(3, 4, 5). This heparin-binding homodimeric glycoprotein has
been implicated in tumor growth (6, 7, 8, 9) and has been
proposed as a prognosis marker in several neoplasm (10, 11). Recently, VEGF has been observed in thyroid carcinoma, and
VEGF production by malignant thyrocytes has been proven in
vitro and in vivo (12, 13, 14).
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Subjects and Methods
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Study population
Human thyroid tissue samples from 19 patients of both sexes,
18–72 yr of age, were obtained from the pathology archives of the
Laboratory of Human Pathology of our institution. All
patients included in this retrospective study are followed at our
institution. The follow-up is based on clinical examination; dosage of
free T4, TSH, thyroglobulin, aspartate
aminotransferase, alanine aminotransferase, alkaline
phosphatase, calcium, phosphate, and creatinine twice a year;
ultrasonography of thyroid area and neck once a year; and neck and
chest tomodensitometry once a year. All patients had undergone total
thyroidectomy plus radioiodine therapy (at least 90 mCi twice) as
treatment for a papillary thyroid carcinoma. None had a second known
carcinoma. The 19 specimens of thyroid papillary carcinoma included 8
specimens that developed lymph node or systemic metastasis and 11
without any metastasis (Table 1
). The
mean follow-up was 5.2 yr (range, 3.5–9.5).
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Table 1. Individual age, TNM status and VEGF immunostaining
score at the time of thyroidectomy, and thyroglobulin level before the
first radioiodine therapy in 19 cases of papillary cancer of the
thyroid
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Immunohistochemistry
Immunohistochemistry was performed on 5-µm paraffin tissue
sections. Paraffin sections were dewaxed with xylene and ethanol and
processed in a pressure cooker in 0.1 M citrate buffer (pH
6.0) for 10 min, washed under running tap water and rehydrated in
TBS-Tween [0.05 M Tris-HCl , 150 mM NaCl, and
0.1% Tween (pH 7.4)], and incubated with 3% albumin for 20 min.
Sections were incubated overnight at 4 C in a humidified chamber with a
rabbit antihuman VEGF antibody [VEGF(Ab-2); Oncogene Research
Products, Cambridge, MA], recognizing VEGF121,
165, 189, and
206, diluted 1:200 in TBS-Tween. This anti-VEGF
polyclonal antibody is generated by immunizing rabbits with a peptide
from the N terminus region of VEGF165. The
sections were washed twice in TBS-Tween for 5 min and overlaid with a
biotinylated goat antirabbit IgG (DAKO Corp., Copenhagen,
Denmark) diluted 1:75 for 30 min at room temperature. The sections were
washed again in TBS-Tween, and each section was incubated with
streptavidin-peroxidase complex (DAKO Corp. A/S) diluted
1:250 for 30 min. The sections were again washed thoroughly in
TBS-Tween, and each section was incubated with 0.6 mg/mL
3,3'-diaminobenzidine in 0.5 M Tris-HCl buffer (pH 7.6)
containing 0.01% hydrogen peroxide. Afterward, sections were washed
with water and counterstained with hematoxylin. Finally, the sections
were mounted, dried, and examined under the light microscope.
Controls
To test the specificity of the labeling protocols, the following
controls were carried out on selected sections for each antibody:
omission of the incubation step with the primary antibody; substitution
of a nonimmune serum in place of the primary antibody; and omission of
the incubation step with both primary and secondary antibodies. To test
the specificity of anti-VEGF antibody, before the immunochemical study,
a preincubation step of this antibody with an excess of recombinant
human VEGF165 (Pepro Tech, Rocky Hill, NJ) was
performed in all control tissue samples. Under these conditions, no
labeling was observed in all tissue samples analyzed.
VEGF immunostaining score
To determine the VEGF immunostaining score, we used the design
proposed elsewhere, with modifications (13). In brief,
this semiquantitative score was established as: each sample was scored
twice—1) for the percentage of labeled thyrocytes (0, absence of
labeling over thyroid cells; 1, <30% of thyrocytes are labeled; 2,
30–60%; 3, >60%); and 2) for the intensity of the immunostaining
(0, no staining; 1, weak; 2, mild; 3, strong staining). Multiplication
of both scores allowed the final quotation ranging from 0–9. A
double-blind analysis was performed by two independent pathologists
(M.K. and J.-M.V.).
Statistical analysis
Results are given as mean ± 1 SD. Comparisons
were performed using ANOVA and the nonparametric Mann-Whitney
U test. A discriminant analysis was performed to
determine a threshold value. Logistic regression was used to determine
the independent risk factors for metastatic disease.
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Results
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Immunohistochemistry showed cytoplasmic staining of normal and
malignant thyrocytes with the anti-VEGF antibody in all samples
analyzed. The staining was stronger within tumoral areas when compared
with normal thyroid tissue. A focal staining of endothelial cells was
noticed in several vessels. No staining was observed within other
structures. No staining was observed with negative controls. The VEGF
immunostaining score was significantly higher in papillary carcinoma
associated with metastasis (mean, 8.25 ± 1.5; range, 6–9)
than without metastasis (mean, 3.91 ± 1.5; range, 1–6;
P < 0.001). Two papillary carcinomas not associated
with metastasis showed a low VEGF immunostaining score (1 and 2,
respectively), whereas all carcinomas with metastasis had a score of 6
or more (Fig. 1).
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Figure 1. Distribution of the two populations of
thyroid papillary carcinomas (with or without metastasis) according to
their VEGF immunostaining score.
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By discriminant analysis we found a threshold value of 6.0, with a
sensitivity of 100% and a specificity of 87.5%. There were no
statistical differences between metastatic and nonmetastatic carcinomas
when age (P = 0.8), tumor size (P =
0.3), or thyroglobulin levels (P = 0.2) were
considered. The VEGF immunostaining score was not significantly
correlated with the tumor size (P = 0.13). Using the
threshold value of 6.0, the VEGF immunostaining score represented the
sole independent risk factor for metastatic disease by logistic
regression (P < 0.001).
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Discussion
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As early as 1971, Folkman (15) postulated that a
tumor would have a limited growth without the generation of
neovascularization initiating from the normal tissue areas that
surround the tumor. Since then, it has been reported that the levels of
VEGF are often higher in malignant tumors than in normal tissues
(7, 16, 17). An increased expression of VEGF by
malignancies has been correlated with poorer outcome or increased risk
for recurrence or metastasis in different cancers like breast carcinoma
(11, 18), ovarian cancer (19, 20), gastric
carcinoma (10), melanoma (21), or head and
neck cancers (22). The production of VEGF by malignant
thyroid cells has been demonstrated in a few studies (13, 14, 23). However, a high VEGF immunostaining score per se
is not diagnosis for thyroid carcinoma; high scores have been observed
in thyroiditis (13). Both VEGF protein and VEGF messenger
RNA have been detected in thyrocytes. Papillary, vesicular, and
undifferentiated thyroid carcinomas have been associated with increased
expression of VEGF. Because the highest levels of VEGF were found in
undifferentiated thyroid carcinomas, Viglietto et al.
(14) suggested that preferential VEGF overexpression
represents an important event in the transition from low-grade to
high-grade thyroid tumors. A recent study confirmed these results
(12). Nevertheless, Soh et al.
(23) did not find an increased expression of VEGF in lymph
node or pulmonary metastasis of thyroid carcinoma if compared with
primary tumor. Another recent study found a correlation between the
expression of VEGF and the size of tumor in children and young adults
(24). In our study, using another design and performed in
elderly patients, such a correlation was not found. We found that the
VEGF immunostaining score was an independent marker for metastasis
occurrence. A score of 6 or more was correlated with an increased risk
of lymph node or systemic metastasis. Because this score is easy to
perform, it could be helpful in the management of the thyroid carcinoma
medical supervision. Metastasis outside the neck occur in 10–15% of
patients with thyroid papillary carcinomas (25). An
intensive follow-up is often expensive and stressful for the patient,
while a leaky follow-up can be accompanied by a delayed metastasis
detection. The addition of the VEGF immunostaining score could be
helpful to detect the patients with the highest risk of metastatic
spread. We, therefore, propose to have intensive survey with patients
having a VEGF immunostaining score of 6 or more. This survey strategy
could consist of measuring thyroglobulin twice a year, ultrasonography
of thyroid gland and lymph node areas once a year, and tomodensitometry
of the neck and chest once a year. A less expensive medical
supervision, based on physical examination and thyroglobulin
determination, could be favored in papillary carcinomas with a low VEGF
immunostaining score. Nevertheless, our data should be corroborated by
a large prospective study.
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Acknowledgments
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We are grateful to J.-M. Virion, Ph.D., and P. Kaminsky, M.D.,
Ph.D., for statistical analysis.
Received April 3, 2000.
Revised September 25, 2000.
Accepted October 6, 2000.
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Top
Abstract
Introduction
