Abstract
Objectives
To examine expression of the connective tissue growth factor (CTGF) gene in human thyroid cancer and establish whether a correlation exists between the presence of CTGF protein and clinicopathological parameters of the disease.
Methods
CTGF protein expression was investigated retrospectively by immunohistochemical analysis of CTGF protein levels in thyroid tumour tissue. Associations between immunohistochemical score and several clinicopathological parameters were examined.
Results
In total, 131 thyroid tissue specimens were included. High levels of CTGF protein were observed in papillary thyroid carcinoma tissue; benign thyroid tumour tissue scored negatively for CTGF protein. In papillary thyroid carcinoma, there was a significant relationship between high CTGF protein levels and Union for International Cancer Control disease stage III–IV, and presence of lymph node metastasis. In papillary thyroid carcinomas, CTGF protein levels were not significantly associated with sex or age.
Conclusions
These findings suggest that the CTGF protein level is increased in papillary thyroid carcinoma cells compared with benign thyroid tumours. CTGF expression might play a role in the development of malignant tumours in the thyroid.
Introduction
Thyroid carcinoma is the most common malignancy of the endocrine system and accounts for ∼1% of newly diagnosed cancers. 1 Thyroid carcinoma can be divided into papillary, follicular, medullary and anaplastic types.2,3 Papillary is the most common thyroid carcinoma, accounting for >83% of all such malignancies.4,5 The prognosis for patients with thyroid malignancy has improved as a result of the standardization of surgical techniques and advances in chemotherapy, although the rates of early diagnosis remain poor. 6 An increased understanding of molecular diagnostic features in thyroid cancer may be useful in improving its early diagnosis.
Connective tissue growth factor (CTGF; also known as CCN2), is a member of the CCN protein family. This protein family includes cysteine-rich, angiogenic inducer, 61 (CYR61 or CCN1), nephroblastoma overexpressed (NOV or CCN3), wingless-type mouse mammary tumour virus integration site family member 1 (WNT1) inducible signalling pathway protein 1 (WISP1 or CCN4), WNT1 inducible signaling pathway protein 2 (WISP2 or CCN5) and WNT1 inducible signalling pathway protein 3 (WISP3 or CCN6).7,8 CNN proteins are involved in numerous cellular processes including proliferation, connection, migration and differentiation, and in broader biological processes including fibrosis, wound healing and carcinogenesis; they are also involved in tumour development and progression.9,10 One study showed that CTGF protein expression is closely related to the pathological grade of enchondroma, and could play an important role in the assessment of tumour grading and disease prognosis. 11 CTGF has been found to induce tumour cell apoptosis in the breast cancer cell line MCF-7. 12 In hepatocellular carcinoma and metastatic liver cancer, CTGF protein expression in tumour tissue was significantly higher than in tissue surrounding the tumour. 13 However, few studies have reported CTGF protein expression in thyroid carcinomas. 14
The present retrospective study examined CTGF protein expression levels in human thyroid carcinomas, using immunohistochemical analysis. Relationships between immunohistochemical scores and several clinicopathological parameters were examined to determine the association between CTGF and thyroid carcinoma.
Patients and methods
Study population and tissue samples
Tumour specimens were obtained from patients with thyroid tumours who had undergone surgery at the Department of Thyroid Surgery, The First Affiliated Hospital of Jilin University, Changchun, Jilin, China, between January 2006 and March 2009. Case information was retrieved from patients’ medical records. Thyroid cancers were classified according to the fifth edition of the tumour–node–metastasis classification of the Union for International Cancer Control (UICC). 15 There were no other specific inclusion or exclusion criteria. Thyroid tissue was removed during surgery, cut into 10–20-µm sections, immediately snap frozen in liquid nitrogen, then stored at −80℃ until use.
Written informed consent was obtained from all patients who participated in this study. The study protocol was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Jilin University.
Immunohistochemical staining
Frozen thyroid tissue samples were fixed in 4% paraformaldehyde, dehydrated and subjected to routine paraffin-wax embedding and sectioning at 4 µm. For immunohistochemical analysis, sections were deparaffinized with xylene and rehydrated through a graded ethanol series. Antigen retrieval was achieved by boiling the sections for 10 min in 0.01 M citrate buffer (pH 6.0) and endogenous peroxidase was blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Nonspecific binding was blocked by incubating slides with normal goat serum for 30 min at room temperature. Sections were incubated overnight at 4℃ with mouse antihuman CTGF primary antibody (1 : 50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing three times with 0.01 M phosphate-buffered saline (pH 7.2), sections were incubated with biotin-labelled rabbit antimouse antibody (1 : 200 dilution; ZSGB-BIO, Beijing, China) for 2 h at room temperature. After washing three times with 0.01 M PBS (pH 7.2), immunostaining was visualized using a streptavidin-peroxidase reaction system, then developed with diaminobenzidine-hydrogen peroxide (Wuhan Boster Biological Technology Ltd, Wuhan, China). The degree of immunoreactivity was assessed semiquantitatively, as described previously, 16 using a scale from 0 to 3, where 0 represents absolutely no immunostaining, 1 represents <25% immunoreactive cells, 2 represents 25–50% of immunoreactive cells and 3 represents >50% of immunoreactive cells. Values of 0 and 1 indicated negative and low staining scores, respectively; values of 2 and 3 indicated high staining scores.
Statistical analyses
Statistical analyses were performed using the SPSS® software package, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Analysis of variance was used to evaluate the difference in CTGT protein levels between different groups. A P-value <0.05 was considered to be statistically significant.
Results
Demographic and clinical data for patients undergoing surgery for thyroid tumours.
Data presented as n (%) of patients.
N0, tumour cells absent from regional lymph nodes; N1, regional lymph node metastasis present; UICC stage, Union for International Cancer Control tumour–node–metastasis classification. 15
Immunoreactivity to CTGF appeared as fine, granular to diffuse, cytoplasmic and membrane staining (Figure 1). The expression of CTGF protein varied among the different thyroid tumour types (Table 2). Benign thyroid tumours (nodular goitre, adenoma, nodular goitre with adenoma) were nonreactive (negative) for CTGF. For thyroid cancers (papillary, follicular and medullary), both low and high CTGF scores were observed, with no cases scoring negatively. There was a significant difference in CTGF protein expression between malignant and benign thyroid tumours (Table 2; P = 0.004).
Immunohistochemical staining for connective tissue growth factor in malignant and benign tumour tissue sections from patients undergoing surgery for thyroid tumours: (A) nodular goitre; (B) thyroid adenoma; (C) papillary thyroid carcinoma; (D) follicular thyroid carcinoma; (E) medullary thyroid carcinoma. Connective tissue growth factor protein expression in thyroid carcinoma and benign thyroid tumour tissue samples from patients undergoing surgery for thyroid tumours. Data presented as n (%) patient incidence. Thyroid carcinoma types versus benign thyroid tumour types; analysis of variance.
Relationship between intensity of connective tissue growth factor staining and clinicopathological characteristics, in papillary thyroid carcinoma tissue samples from patients (n = 109) undergoing surgery for thyroid tumours.
Data presented as n (%) patient incidence.
N0 versus N1; bUICC stage I – II versus UICC stage III – IV; analysis of variance.
NS, no statistically significant differences (P ≥ 0.05); N0, tumour cells absent from regional lymph nodes; N1, regional lymph node metastasis present; UICC stage, Union for International Cancer Control tumour–node–metastasis classification. 15
Discussion
Connective tissue growth factor is believed to be a multifunctional signaling modulator that is involved in a variety of biological or pathological processes such as angiogenesis, osteogenesis, fibrosis in the kidneys and skin, and tumour development.17–19 CTGF has been identified as an oncogene in a number of cancer types. 12,20–23 The CTGF gene is overexpressed in prostate cancer, 20 glioma, 21 breast cancer 12 and adult acute lymphoblastic leukaemia. 22 Increased CTGF protein expression is also associated with progression of cervical tumours 23 and oesophageal squamous cell carcinoma. 24 Conversely, in lung adenocarcinoma 25 and colorectal cancer, 26 overexpression of the CTGF gene inhibits invasion and metastasis of cancer cells, both in vitro and in vivo.
Although specific criteria are used to diagnose various thyroid neoplasms, it can be difficult to distinguish between carcinomas (such as papillary carcinomas) and adenomas.3,4 Thyroid cancer-related molecular diagnostic features that could assist in this distinction would have great clinical value. The present study demonstrated that CTGF protein expression differed significantly between benign thyroid tumours and thyroid carcinomas. All 13 cases of benign thyroid tumours were negative for CTGF. In papillary thyroid carcinoma, 63 cases showed low CTGF scores and 46 showed high CTGF scores. Moreover, four cases of follicular thyroid carcinoma and four cases of medullary thyroid carcinoma showed low CTGF scores. These data suggest that the level of CTGF protein expression may have clinical significance in the differential diagnosis of benign versus malignant thyroid tumours.
In conclusion, the present study demonstrated that CTGF protein expression was significantly upregulated in thyroid carcinoma tissue compared with benign thyroid tumour tissue, suggesting that CTGF may be involved in the development of malignant thyroid tumours. The number of patients studied was, however, relatively small. Further research, involving a larger number of patients, is needed in order to verify these findings.
Footnotes
Declaration of conflicting interest
The authors declare that there are no conflicts of interest.
Funding
The authors gratefully acknowledge the financial support provided by The Health Bureau of Jilin, grant No. 2008Z017.