0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 2 Printed in U.S.A.
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, ´ NAT, J. LECLE ` RE, A. DUPREZ, AND G. WERYHA F. PLE 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 ABSTRACT 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 dis-
criminant 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. (J Clin Endocrinol Metab 86: 656 – 658, 2001)
A
NGIOGENESIS 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 (3–5). This heparin-binding homodimeric glycoprotein has been implicated in tumor growth (6 –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 (12–14).
notransferase, 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).
Immunohistochemistry Immunohistochemistry was performed on 5-m 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 antiVEGF 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.
Subjects and Methods 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 amiReceived April 3, 2000. Revision received September 25, 2000. Accepted October 6, 2000. Address correspondence and requests for reprints to: M. Klein, M.D., Ph.D., Clinique Endocrinologique, Hoˆpitaux de Brabois, Centre Hospitalier et Universitaire de Nancy, 54500 Vandoeuvre-les-Nancy, France. E-mail:
[email protected] or
[email protected].
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VEGF AS MARKER IN PAPILLARY THYROID CARCINOMA
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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 Cases
Case Case Case Case Case Case Case Case Case Case Case Case Case Case Case Case Case Case Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Age
57 27 81 81 25 15 57 45 40 31 78 35 62 70 54 52 62 55
Tumor size
2 1.5 1.5 4 3 3 6 2.2 4.5 0.4 2.5 3 1.5, multifocal 2 0.5 2 2
Metastasis
Thyroglobulin (ng/mL)
N(⫹) N(⫹) N(⫹), M(⫹), bone N(⫹), M(⫹), lung N(⫹) N(⫹), M(⫹), lung N(⫹), mediastinum N(⫹) N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0 N0, M0
12.7 55.9 173.1 714 4 N.A. N.A. N.A. 0.93 35.8 Antithyroglobulin antibodies N.A. 0.1 104 9 N.A. 19 36.6 Antithyroglobulin antibodies
VEGF immunostaining score
7.5 9 9 9 6 7.5 9 9 5 4 6 3 4 6 2 4 1 3 5
N0, No lymph node metastasis; N(⫹), presence of lymph node metastasis; M0, no systemic metastasis; M(⫹), presence of systemic metastasis (localization precised); N.A., not available.
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.
Results
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;
FIG. 1. Distribution of the two populations of thyroid papillary carcinomas (with or without metastasis) according to their VEGF immunostaining score.
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). 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). Discussion
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
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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. Acknowledgments We are grateful to J.-M. Virion, Ph.D., and P. Kaminsky, M.D., Ph.D., for statistical analysis.
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