Abstract
OBJECTIVE To investigate the significance of vascular endothelial growth factor (VEGF) and nuclear factor-kappaB (NF-κB) expression in thyroid carcinoma.
METHODS The expression of NF-κB and VEGF was determined by immunohistochemistry in formalin-fixed and paraffin-embedded specimens obtained from 10 normal thyroid tissues (NT), 12 cases of thyroid adenoma (TA) and 68 cases of thyroid carcinoma (TC).Differences in expression between NT, TA and TC were statistically analyzed. In addition, in cases of TC, the relationship of NF-κB and VEGF expression with various clinicopathological factors, including histological typing, clinical staging and lymph node metastasis, as well as the correlation between NF-κB and VEGF expression was examined.
RESULTS In contrast to the negative immunoreactivity for VEGF in NT, there was a significantly higher positive incidence (PI) in TA (41.7%, P= 0.040) and TC (75.0%, P<0.001), and a significant difference between TA and TC (P=0.036). Immunoreactivity for NF-κB in NT was negative and significantly higher in TC (63.2%, P<0.001), but not in TA (16.7%, P= 0.481). However the PI difference between TA and TC (P=0,003) was significant. Between the histological types of TC, a significantly higher PI was found in undifferentiated thyroid carcinoma (UTC),namely,100% for VEGF and 90.0% for NF-κB. We also found significant positive relationships of VEGF and NF-κB expression with the clinical stage and lymph node metastasis. Furthermore, a significant positive correlation between VEGF and NF-κB expression in TC was observed.
CONCLUSION Our data showed that the expression of VEGF and NF-κB/P65 was greater in TC and UTC, and documented their significant positive correlations with the clinical stage and lymph node metastasis in TC. In addition there was a significant positive relationship between their expression, suggesting that they have important roles in TC and that they may be potential targets for gene therapy in TC patients.
keywords
Vascular endothelial growth factor (VEGF), a dimeric 42-kd protein, is a multifunctional cytokine that plays a key role in both physiological and pathological angiogenesis.[1] Angiogenesis is known to be a prerequisite for tumor growth and metastasis.VEGF has been recognized as an autocrine regulator of angiogenesis in several thyroid cancer cell lines, and experimental and clinical studies have suggested that VEGF is the dominant angiogenic factor in thyroid cancer.[2]
Nuclear factor-kappa B (NF-κB) is a family of homo- or heterodimeric transcription factors formed by proteins of the Rel family, including NF-κB 1 (p50), NF-κB 2 (p52), RelA (p65),RelB and c-Rel. [3] The heterodimer composed of NF-κB 1 (p50) and RelA (p65) is the most common and active form of NF-κB.[4] NF-κB activation has been connected with multiple aspects of oncogenesis, such as apoptotic resistance, transformation, growth, metastasis and angiogenesis.[5] Recently, it has been suggested that NF-κB plays an important role in thyroid carcinogenesis.[6]
In this study, we investigated the significance of VEGF and NF-κB expression and their correlation in patients with thyroid carcinoma (TC) including papillary, follicular, medullary and undifferentiated TC.
MATERIALS AND METHODS
Clinical specimens
Stored paraffin embedded tissues of normal thyroid (NT, n=10), thyroid adenoma (TA, n=12) and thyroid carcinoma (TC, n=68) were retrieved from storage at the Department of Pathology, China Medical University,Shenyang,China. All the NT, TA and TC specimens were obtained from surgical resections between 1999 and 2005. None of the patients had received chemotherapy or radiotherapy prior to their operation. Among the 68 patients with TC, 48 were female and the average age was 43.8 years (range, 23~71). Tumors were evaluated by one pathologist, and verified by another investigator for histological typing, clinical staging and lymph node metastasis. Based on WHO criteria of histological classification for TC, 34 were papillary, 14 follicular, 10 medullary and 10 undifferentiated. According to AJCC criteria for clinicopathological classification of TC, 27 were Stage I, 19 Stage II, 13 Stage III, and 9 Stage IV. Based on the clinicopathological documents for the TC cases lymph node metastasis were positive for 27 cases and negative for 41 cases. The project was approved by the ethics committees of the hospital and informed consent was obtained from the patients or their family.
Antibodies and reagents
Rabbit antihuman NF-κB/P65 monoclonal antibody, rabbit antihuman VEGF polyclonal antibody and a streptavidin biotin peroxidase complex (SABC) kit as well as 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reagents were all purchased from Boster corporation (Wuhan, Hubei Province, China).
Immunohistochemistry for VEGF and NF-κB/P65
Serial 4 micron sections were made from paraffin-embedded tissues and deparaffinized after being mounted on slides. The slides were heated for three 5-min cycles in a microwave oven at 500 W using 0.01 mol/L pH 6.0 citrate buffer to unmask the antigens. The slides were then immersed in a 3% solution of hydrogen peroxide in methanol to block endogenous peroxidase activity. After washing with phosphate buffer saline (PBS), the slides were incubated in a humid chamber overnight with the primary antibody against VEGF or NF-κB/P65 at a dilution of 1/100 at 4°C, for 60 min with the biotin conjugated second antibody, and for 10 min with the third antibody streptavidinperoxidase at room temperature. Then the immunoreactive products were visualized with the DAB/hydrogen peroxide solution and lightly counterstained with hematoxylin. PBS was used instead of the primary antibodies for negative controls. The sections were examined under light microscope.
Evaluation of VEGF and NF-κB/P65 immunoreactivity
Cells were considered to be positive for VEGF or NF-κB/P65 when immunoreactivity was clearly observed in the cytoplasm or nucleic. Expression of VEGF and NF-κB/P65 was evaluated based on the percentage of stained cells and staining intensity. At least 5 representative areas were randomly selected at 200 x magnification under a light microscope. Points were allocated based on the percentage of positive cells as follows: 0 points, ≤ 1% positive cells; 1 point, 1~25% positive cells; 2 points, 25-50% positive cells; 3 points, 50~75% positive cells; 4 points, >75% positive cells. The staining intensity was classified as follows: 1 point, weak intensity; 2 points, moderate intensity; 3 points, strong intensity. Points for the percentage and intensity of positive cells were multiplied to get the overall score (OS) for each specimen. According to the OS, specimens were defined as negative (OS<1) or positive (OSe ≥ 1).
Statistical analysis
The data were analyzed by SPSS 13.0 for windows. A two tailed P<0.05 was taken as statistically significant. Chi square and Fisher’s exact tests were used for comparison between groups for the expression of VEGF or NF-κB/P65.The Kendall’s tau-b test was used to identify the correlation between VEGF and NF-κB P65 expression in TC cases.
Results
Expression of VEGF in NT, TA and TC
VEGF immunostaining was mainly cytoplasmic with the exception of a little weak concomitant nuclear staining in a few cases. VEGF immunoreactivify was found in NT tissues, in contrast to TA and TC cases, including PTC, FTC, MTC and UTC (Fig.l). TC showed a significantly higher PI for VEGF immunoreactivity compared to either NT (P<0.001) or TA (P = 0.036), and there was also a marked difference in PI between NT and TA (JM).O4O, Table 1). Furthermore, among the TC cases, UTC showed a significantly higher PI compared with PTC, FTC and MTC, but no remarkable difference in PI was observed between PTC, FTC and MTC (Table 2). Additionally, a significant positive correlation was observed between VEGF expression and other clinicopathological parameters, such as clinical staging (P=0.007) and lymph node metastasis (P=0.032).
Representative cases of immunostaining for VEGF from (A) NT, and (B) TA, (C) PTC, (D) FTC, (E) MTC and (F) UTC. NT, normal thyroid; TA, thyroid adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; UTC, undifferentiated thyroid carcinoma. Original magnification, × 400.
Incidence of VEGF expression in NT, TA and TC
Relationships between VEGF expression and clinicopathological parameters in TC
Expression of NF-κB/P65 in NT, TA and TC
NF-κB/P65 immunoreactivity was observed in both the cytoplasm and nucleas in most of the TC tissues including PTC, FTC, MTC and UTC, and in a small minority of TA cases with a little weak cytoplasmic staining, but not in the NT or negative controls (2). The incidence of NF-κB/P65 expression in NT, TA and TC is summarized in (Table 3). Statistical analysis demonstrated a significantly higher PI of NF-κB/P65 in TC compared with NT (P<0.001) or TA (P=0.003), whereas no significant difference in the PI was found between NT and TA. In the TC, NF-κB/P65 expression was much more frequently observed in UTC than in PTC (P=0.027) or FTC (p=0.033), without a significant difference found between PTC, FTC and MTC as well as between UTC and MTC (Table 4). We also observed a significantly positive correlation among NF-κB/P65 expression with other clinicopathological parameters, such as clinical staging (p=0.001) and lymph node metastasis (P=0.002).
Representative cases of immunostaining for NF-κB/P65 from (A) NT, and (B) TA, (C) PTC, (D) FTC, (E) MTC and (F) UTC. NT, normal thyroid; TA, thyroid adenoma; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; UTC, undifferentiated thyroid carcinoma. Original magnification, × 400.
Incidence of NF-κB/P65 expression in NT, TA and TC
Relationships between NF-κB/P65 expression and clinicopathologieal parameters in TC
Correlation between VEGF and NF-κB/P65 expression in TC
In a total of 68 cases of TC, VEGF immunoreactivity was negative in 17 cases, of which 14 (82.3%) were also negative for NF-κB/P65 expression. On the other hand, NF-κB/P65 expression was positive in 43 cases, of which 40 (93.0%) were also positive for VEGF expression. Therefore, we further examined the co-expression relationship of NF-κB/P65 with VEGF through statistical analysis. A significant positive correlation between NF-κB/P65 and VEGF was revealed by the Kendall’s tau-b test (P<0.001, r=0.550, Table 5).
Relationships between VEGF expression and clinicopathologieal parameters in TC
Discussion
Thyroid tumors are one of most common endocrine tumors, comprised of both benign (e.g. adenomas), and malignant tumors, which include papillary, follicular, medullary, and undifferentiated carcinomas. These various types constitute about 60%, 20%, 5% and 10% of thyroid malignant tumors, respectively. Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are generally well differentiated and least aggressive, but when they show anaplastic transformation (dedifferentiation), they will become more progressive; medullary thyroid carcinoma (MTC) is somewhat more aggressive; undifferentiated thyroid carcinoma (UTC) is most dedifferentiated and aggressive. Thyroid adenoma (TA) as well as PTC and FTC arise from follicular epithelial cells, while MTC arises from parafollicular cells.
It is known that growth and metastasis of tumors are dependent on angiogenesis, including vasculoangiogenesis, lymphangiogenesis, and vasoangiogenesis. Vasculoangiogenesis (new blood vessel formation) or lymphangiogenesis (new lymphatic vessel formation) is thought to be based on differentiation of blood vascular or lymphatic endothelia from angioblasts or lymphangioblasts, respectively. Vasoangiogenesis is a novel type of new blood vessel formation, whose vascular channels are lined partially or completely by tumor cells. The VEGF family has been known to be vital for these angiogenic processes, indicating its important role in tumor evolution.[7-9] In the present study, VEGF expression was demonstrated to be greater in TC than in TA and NT, and showed significant positive correlations with dedifferentiation, clinical stage, and lymph node metastasis in TC cases. These results provide strong evidence for the concept that VEGF is involved in TC progression, and that VEGF may be a potential prognostic factor in TC.
Up to the present, studies on NF-κB in TC have been few and only limited to thyroid cancer cell lines. The studies have documented the participation of NF-κB activation in apoptotic resistance and oncogenesis of TC. [10,12] In our clinical investigation, we demonstrated for the first time that NF-κB/P65 expression showed a higher frequency in TC and a highest frequency in UTC, and that its expression significantly correlates with the clinical stage and lymph node metastasis. These findings suggest that NF-κB/P65 is involved in TC progression and that it may have potential clinical value, such as a prognostic factor or as a gene therapy target for TC. A significantly positive correlation between NF-κB and VEGF has been reported in colorectal adenocarcinoma. [13] Consistent with this finding, we also found this correlation in TC, although we can not establish a cause and effect linkage between NF-κB and VEGF by means of our immunohistochemical results. However it does suggest that NF-κB could promote angiogenesis associated with VEGF. Several recent studies have implicated the role of NF-κB in regulating VEGF expression. [14,15] Cox-2, an angiogenic factor, has been demonstrated to be one of the target genes of NF-κB. [16,17] The possibility exists that VEGF may also be one of the targets of NF-κB, a possibility which needs to be clarified.
In summary, our data demonstrated a higher frequency of VEGF and NF-κB/P65 expression in TC and their highest frequencies in UTC, and documented their significant correlations with the clinical stage and lymph node metastasis in TC. In addition there was a significant positive relationship between their expressions, suggesting they have important roles in TC and that they may be potential targets for gene therapy in TC patients.
- Received April 17, 2006.
- Accepted June 7, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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