We retrospectively collected clinical data from 145 patients with esophageal squamous cell carcinoma between January 2007 and December 2008. These patients had all undergone surgical treatment for esophageal cancer, were clearly diagnosed, and had not received chemotherapy or radiotherapy. In addition, pathological tissue samples from the 145 patients were collected to detect the expression of B7-H6 by immunohistochemistry. In addition, 7 non-malignant esophageal tissue samples from the non-malignant portion of the esophagus were collected and used as controls. The clinicopathological data of all patients were available and were included in the statistical analysis. This study was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center and written informed consent was provided by all patients based on the Declaration of Helsinki.
Antibodies and major reagents
A rabbit anti-human B7-H6 polyclonal antibody (ab121794) was purchased from Abcam (Cambridge, MA, USA; dilution 1/100), and a horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit secondary antibody was purchased from Dako (Glostrup, Denmark). A DAB colour developing kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China; cat. no. zli-9017).
Paraffin-embedded tissue samples were cut into 5 μm serial sections and baked for 30 minutes in a 60°C constant-temperature box. The sections were deparaffinized with xylene and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution for 10 minutes, and antigen retrieval was performed at 100°C for 20 minutes in a sodium citrate buffer solution (0.01 mmol/L, pH 6.0). After being soaked in distilled water for 10 minutes, the sections were incubated with 10% foetal bovine serum to block nonspecific binding. Next, the sections were incubated with the rabbit anti-human B7-H6 polyclonal antibody at 4°C overnight and then incubated with the HRP-conjugated goat anti-mouse/rabbit secondary antibody at room temperature for 60 minutes. The excess secondary antibody was removed by washing with TBS and developed with DAB colourant; the sections were stained with haematoxylin, dehydrated with an alcohol gradient, dried and sealed with neutral resin.
Evaluation of immunohistochemical (IHC) staining
The esophageal squamous cell carcinoma tissue samples were examined by two independent senior pathologists who were not informed of the patients’ clinicopathological characteristics. The B7-H6 immunohistochemical staining results were analyzed according to a previously described method: (10, 18) H-score = (% tumour cells unstained × 0) + (% tumour cells stained weakly × 1) + (% tumour cells stained moderately × 2) + (% tumour cells stained strongly × 3). The staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained), or “3” (strongly stained). The H-scores ranged from 0 (100% negative tumour cells) to 300 (100% strongly stained tumour cells). The results from the two pathologists were averaged and used in the statistical analyses.
Overall survival (OS), which was defined as the time from surgery to patient death or last follow-up, was used as a measure of prognosis. The final follow-up date was December 24, 2018. SPSS software (version 13.0; IBM Corp., Armonk, NY, USA) was used for data analysis. The correlation between the B7-H6 expression level and different clinicopathological parameters was analysed by the chi-square (χ2) test, and survival data were analysed by univariate and multivariate Cox regression analyses, receiver operating characteristic (ROC) curve analysis, and Kaplan-Meier analysis with the log-rank test. Statistical significance was defined at P<0.05.