The Correlations Between Expressions Of STAT3 And CyclinD1 And Clinical Features As Well As Prognosis Of Patients With Esophageal Squamous Cell Carcinoma

Objective:Recently, in the worldwide, especially China, there has been a dramatic rise in the incidence and mortality due to esophageal adenocarcinoma and squamous cell carcinoma [1, 2]and esophageal squamous cell carcinoma(ESCC) occurs at a high frequency in China(13 per100 000) [3]. As the dominant type of esophageal cancer in China, ESCC has a generally poor prognosis due to the lack of effective clinical methods for its early detection [4]. There is a pressing need for reliable biomarkers to identify the subset of patients with a high risk of poor survival outcomes and guide treatment.Signal transducer of activator of transcription 3(STAT3) and cyclin D1 are overexpressed in various human cancers, and their overexpression positively correlates to tumor progression and poor prognosis. However, the clinical significance of dual high expression of these two proteins in ESCC has yet to be determined.Methods:1. We collected clinical data includes paraffin-embedded ESCC samples and adjacent noncancerous tissues from 82 patients who underwent curative surgery between June 2003 and June 2007 in Guangzhou Cancer Hospital, China. Demographic and clinicopathological data of ESCC patients were collected from medical records. None of the patients received radiotherapy or chemotherapy prior to curative surgery. The following clinicopathological parameters were recorded: age, sex, histological grade, tumor size, depth of invasion, and clinical stage.2. Consecutive sections measuring 4 μm from TMA were placed on pathological slides for immunohistochemistry(IHC) staining. The protein expression levels of STAT3 and cyclin D1 in cancer tissue or adjacent non-cancerous tissue were evaluated by microscopic examination of stained tissue sections, and determined by integrating the percentage of positive tumor cells and the intensity of positive staining. The association between STAT3/cyclin D1 protein expression of cancer tissue and adjacent non-cancerous tissue was analyzed by Chi-square test.3. The data were analyzed with SPSS software version 19.0. The association between STAT3/cyclin D1 protein expression and clinicopathological features was analyzed by Chi-square test.4. The data were analyzed with SPSS software version 19.0. Kaplan-Meier and log-rank tests were used to analyze the association between overall survival rates and the expression of STAT3/cyclin D1 or clinicopathological features of ESCC patients.5. The data were analyzed with SPSS software version 19.0. Cox regression analysis was used for the multivariate analysis and searched out the most dangerous factors of ESCC patients from tumor stage, high expression of STAT3 or cyclin D1 and dual high expression of STAT3 and cyclin D1.Results:1. According to IHC staining, the cancer cells were found to be relatively homogenous within a tumor. Both the expression of STAT3 and cyclin D1 were found to be mainly located in cytoplasm/nucleus and nucleus, respectively. Brown cytoplasm and nucleus immunoreactivity for the STAT3 and cyclin D1 were recognized as positive staining. We found high expression of STAT3 and cyclin D1 were detected in 54 and 43 out of 82 TMA cancer tissues(65.9% and 52.4%) while the remaining cancer tissues and most of the adjacent non-cancerous tissues showed low expression of STAT3 and cyclin D1. Both STAT3 and cyclin D1 protein expression levels in the ESCC tissues were significantly higher than those in the adjacent non-cancerous tissues2. As shown in chi-square test, High expression of STAT3 was found to significantly correlate with advanced tumor stage(P=0.047) and high expression of cyclin D1 was found to significantly correlate with lager tumor size(P=0.009) and advanced tumor stage(P=0.012). Interestingly, the expression of STAT3 was positively associated with the expression of cyclin D1(P=0.025). No significant difference in STAT3 or cyclin D1 expression was observed with gender, age at surgery, histological grade, and depth of invasion(P>0.05).3. In univariate survival analyses, Kaplan-Meier survival curves were employed and the statistics were carried out by log-rank method. Kaplan-Meier analysis demonstrated that tumor stage had a significant impact on overall survival as a well-known clinicopathological prognostic factor(P=0.016). What is more, overall survival in patients with high expression of STAT3/cyclin D1 was significantly shortened than in patients with low expression of STAT3/cyclin D1. The mean value of overall survival time was 27.67 months or 23.09 months in patients with low expression of STAT3 or cyclin D1 compared with 17.48 months and 21.81 months in patients with high expression of STAT3 or cyclin D1, respectively(P=0.044, 0.032).4. Univariate analyses also indicated that the difference of survival time among the ESCC patients with dual and single high expression and dual low expression of STAT3 or cyclin D1 was significant(P=0.008), suggesting that combining with STAT3 and cyclin D1 maybe more favourable for predicting the outcome of the patients with ESCC(Table 3).Furthermore, we compared the survival time of three groups of ESCC patients with dual low expression of STAT3 and cyclin D1(n=20), high expression of STAT3 and low expression(n=20)/high expression of cyclin D1 and low expression of STAT3(n=11) and dual high expression of STAT3 and cyclin D1(n=31), respectively. And we found that the prognosis of patients with dual high expression of STAT3 and cyclin D1 was the poorest(P=0.013, 0.045).There is no significance of survival time(P=0.980) between the patients with high expression of STAT3 and low expression of cyclin D1(n=20) and high expression of cyclin D1 and low expression of STAT3(n=11).5. Since variables observed to have a prognostic influence by univariate analysis may covariate, the expression of STAT3 and cyclin D1 protein expression and those clinicopathological parameters that were significant in univariate analysis were further examined in multivariate analysis using Cox regression model. The results showed that high expression of STAT3 or cyclin D1 protein were both independent prognostic factors for overall survival(STAT3 hazard ratio: 2.687, 95% confidence interval: 1.091~6.619, P=0.032;cyclin D1 hazard ratio: 2.115, 95% confidence interval: 1.029~4.347, P=0.042). More importantly, dual high expression of STAT3 and cyclin D1 protein was found to be the most significant independent adverse prognostic factor for overall survival(hazard ratio: 3.223,95% confidence interval: 1.417~7.330, P=0.005). With regard to clinicopathological parameters, tumor stage remained to be an independent prognostic factor for overall survival(hazard ratio: 2.499, 95% confidence interval: 1.030~6.053, P=0.043).Conclusions:1. Both STAT3 and cyclin D1 protein expression levels in the 82 cases of ESCC tissues were significantly higher than those in the adjacent non-cancerous tissues.2. High expression of STAT3 was found to significantly correlate with advanced tumor stage and high expression of cyclin D1 was found to significantly correlate with lager tumor size and advanced tumor stage in 82 cases of ESCC patients.3. High expression of STAT3 or cyclinD1 protein and tumor stage were both independent prognostic factors for overall survival of 82 ESCC patients.4. Dual high expression of STAT3 and cyclin D1 protein was found to be the most significant independent adverse prognostic factor for overall survival of 82 ESCC patients.