Expression of SOX9 Is Related to Prognosis in Patients with Oesophageal Squamous Cell Carcinoma

Materials and Methods

Study groups. The study included 175 consecutive patients with ESCC who underwent curative surgery at Kagoshima University Hospital between 1996 and 2006. The patients ranged in age from 43 to 87 years (mean=63.3 years). All patients underwent oesophagectomy with lymph node dissection. None underwent endoscopic mucosal resection, palliative resection, preoperative chemotherapy, or radiotherapy, and none of them had synchronous or metachronous cancer in other organs. Specimens of cancer tissues and non-cancerous adjacent tissue were collected from the patients after informed consent had been obtained, in accordance with the institutional guidelines of our hospital. The specimens were classified according to the International Union against Cancer tumour-node-metastasis (TNM) system (10). All M1 classifications were due to distant lymph node metastases. All patients were followed-up after discharge with a radiographic examination every 1-3 months, computed tomography every 3-6 months, and ultrasonography every 6 months. Follow-up data were available for all patients, with a median follow-up period of 43 months (range=2-181 months).

Immunohistochemistry. Tumour samples were fixed with 10% formaldehyde in phosphate-buffered saline (PBS), embedded in paraffin, and sectioned into 4-μm-thick slices. After deparaffinization of the sections, endogenous peroxidase was blocked by immersing the slides in a 0.3% hydrogen peroxidase–methanol solution for 30 minutes at room temperature. For staining with SOX9 antibodies, sections were pre-treated with citrate buffer for 10 min at 100°C in a microwave oven. The sections were washed three times with PBS for 5 min, and then blocked by treatment with PBS containing 3% skim milk for 30 min at room temperature. The blocked sections were incubated overnight at 4°C with primary antibody: SOX9 (AB5535, Merck Millipore, Darmstadt Germany), diluted with PBS 1:500 before staining with a streptavidin-biotin peroxidase kit (Nichirei, Tokyo, Japan). The sections were washed three times in PBS for 5 min, and the immune complex was visualized by incubating the sections with diaminobenzidine tetrahydrochloride. They were rinsed briefly in water, counterstained with haematoxylin, and mounted. The normal oesophageal epithelium tissues were used as the positive control, and negative controls were generated by replacing the primary antibodies with PBS.

Immunohistochemistry was independently evaluated by two investigators (N.H. and H.O.). To evaluate SOX9 expression, 10 fields of the tumour were selected, and expression was evaluated in 1,000 tumour cells (100 cells/field) using high-power (×200) microscopy. For nuclear expression of SOX9, immunostaining of each section was scored on a 4-tiered scale for intensity: absent: 0% (Figure 1A), weak: less than 5% (Figure 1B), moderate: 5-50% (Figure 1C), and strong: 51-100% (Figure 1D). The protein expression was defined as positive when the intensity was moderate or strong.

Statistical analysis. Statistical analysis of group differences was performed using the chi-squared test and the t-test. The Kaplan–Meier method was used for survival analysis, and differences in survival were estimated using the log-rank test. The prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). The p-values in this study are from two-sided tests, and a p-value of less than 0.05 was considered significant. All statistical analyses were performed using the SPSS Statistics Software Package (SPSS Inc., Chicago, IL, USA).