Prognostic significance of Oct4 and Sox2 expression in hypopharyngeal squamous cell carcinoma

This study was approved by the Institutional Review Board and Human Ethics Committee of Sun Yet-sen University Cancer Center. A total of 85 patients were included with histologically confirmed squamous cell carcinoma of the hypopharynx who were treated from 2002 to 2004 at the Sun Yet-sen University Cancer Center. Relevant clinical pathologic features (Table 1) were obtained from the patients' files and/or by telephone interviews with the patient or their relatives. Tumor types and histological-grade classifications were designated according to World Health Organization classification of tumors: pathology and genetics of head and neck tumors [23].

Immunohistochemistry was performed on 4-μm-thick routinely processed paraffin sections. Oct4 was detected using a rabbit polyclonal anti-Oct4a antibody (Cell signaling, #2890, UK, dilution 1:100). Sox-2 was detected using a rabbit polyclonal anti-Sox antibody (Cell signaling, #3579, UK, dilution 1:100). A total of 85 formalin-fixed, paraffin-embedded hypopharyngeal squamous cell carcinoma tissue samples were dried overnight at 56°C. After deparaffinization and rehydration, sections were heat-pretreated in a citrate buffer (92°C in microwave oven) and incubated in 3% H₂O₂ to block endogenous peroxidase activity. Then the sections were examined by immunostaining using the primary antibodies overnight at 4°C in a humidity chamber. The avidin-biotin technique was applied using DAB for visualization and hematoxylin for nuclear counterstaining. Negative controls were prepared by omitting the primary antibody. Histological and IHC evaluation were independently performed by two pathologists without knowledge of the clinicopathological outcomes of the patients. Slides with indeterminate evaluation were re-evaluated, and a consensus was reached. Briefly, each slide was examined in its entirety under a light microscope, and an initial score was assigned which represented the estimated proportion of positive tumor cells (0: none; 1: < 1/4; 2: 1/4 to 1/2; 3: 1/2 to 3/4; and 4: > 3/4). Next, an intensity score was assigned which represented the average intensity of staining of the positive tumor cells (0, none; 1, weak; 2, intermediate; and 3, strong). The proportion and intensity scores were then added to obtain a total score, which ranged from 0 to 7. Specimens were categorized into one of two groups according to their overall scores: (1) low expression, < 4 points; (2) high expression, 4-7 points.

Statistical analysis was performed using the SPSS 17.0 software package for Windows. The χ² test was used to evaluate categorical variables. Associations between clinicopathological features and immunohistochemical Oct4 or Sox2 expression were analyzed using the logistic regression model with the presence of overall survival as the dependent variable. Multivariate survival analyses were performed with the Cox regression model. Overall survival (OS) was measured from the onset of treatment to the date of death or the survival status at the last date of follow-up. The loco-regional control (LRC) was the interval from the onset of treatment to the date of recurrence. Recurrence was defined as local tissue invasion by the primary tumor or regional lymph node involvement. Disease-free survival (DFS) was defined as the interval between the onset of treatment and the date when recurrence or metastasis was diagnosed. OS, LRC and DFS probabilities were estimated by the Kaplan-Meier method and the significance of differences were assessed by the log-rank test. A P-value < 0.05 was considered statistically significant, and a P-value < 0.01 was considered strongly statistically significance.

Table 1 presents a summary of sex, age, tumor stage, histological grade, and the status of SCCA (Squamous Cell Carcinoma Antigen) as well as TSGF (Tumor Supplied Group of Factor). In this study, there were 85 hypopharyngeal carcinoma patients consisting of 84 males and 1 female. The median age was 60 years (range: 37-82 years). According to the 6th Edition of the International Union Against Cancer (UICC) TNM classification system, there were 7 Stage II patients, 24 Stage III patients, and 54 Stage IV patients, as shown in Table 2. Recurrences were confirmed by histopathology or visual examination and were found to have occurred in 72 patients. The median time to recurrence was 5.5 months (range 1-50 months). Seventy cancer-related deaths were reported. The median time to death was 17 months (range 0.16-74 months). The reasons for death were local recurrence (62 patients), pulmonary metastases (3 patients), hepatic or abdominal cavity metastases (3 patients) and mediastinal metastases (2 patients).

The last follow-up date is Sep. 29^th, 2009, with a median follow-up time 52 months (range 7-69.5 months). The 1-, 3- and 5-year overall survival rates (OS) were 64.7%, 22.4%, 18.8%, respectively; disease-free survival (DFS) was 24.7%, 15.3%, 12.9%, respectively. The local-regional control rates were 24.7%, 16.5%, 15.3%, respectively.

Positive staining for Oct4 and Sox2, mainly localized in the nucleus, were observed in the cancer cells of tumor tissues (Fig. 1). The distribution of immunostaining scores is listed in the Table 1. The highest expression rate of Oct4 was 9.4% (8 of 85), whereas that of Sox2 was 71.8% (61 of 85). Furthermore, the expression Oct4 is correlated with the cervical lymph node metastasis (p = 0.007) whereas the expression of Sox2 is correlated with the histological grade (p = 0.03), as in Table 1.

Univariate analyses showed no significant association between OS, DFS or LRC and T stage, cervical lymph node metastasis, TNM stage, age, or histological grade (Table 2, 3). Patients with high Oct4 expression had a significantly better prognosis, including longer survival (p = 0.000) and lower recurrence rate (p = 0.000). Even though Sox2 expression showed no association with prognosis, the highest overall survival rate was documented in the high Oct4 expression/high Sox2 expression subgroup. The 5-years overall survival rate was 61.5% for this group (p = 0.000) (Fig. 2).

A multivariate survival analysis was performed with the Cox regression model for each predictor of prognosis to calculate odds ratios, as well as 95% confidence intervals. The model was simplified in a stepwise fashion by removing variables that had a p value ≥0.05. Only three variables remained statistically significant as independent predictors of OS and LRC in the multivariate analysis. Also, because the variable Oct4 & Sox2 expression consisted of Oct4 expression and Sox2 expression, this variable is replaced by Sox2 expression (Table 4). The results indicate that the expression status of Oct4 (p = 0.004) was an independent predictive factors for prognoses of hypopharyngeal squamous cell carcinoma patients.