Tumor Biol. DOI 10.1007/s13277-015-3302-9
RESEARCH ARTICLE
Prognostic significance of Ku80 in pT2N0M0 esophageal squamous cell carcinoma after Ivor-Lewis esophagectomy Shuai Wang & Zhou Wang & Xiangyan Liu & Yu Yang & Mo Shi & Zhenguo Sun
Received: 6 October 2014 / Accepted: 2 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Recent studies have shown that Ku80, a DNA repair protein, was involved in progression of malignant tumors. This study aimed to clarify the clinicopathological significance and prognostic value of Ku80 in pT2N0M0 esophageal squamous cell carcinoma (ESCC). We enrolled 217 patients with pT2N0M0 midthoracic ESCC who had undergone IvorLewis esophagectomy. The expression profile of Ku80 was examined by immunohistochemistry. The results were correlated with the clinicopathological variables, overall survival (OS) and disease-free survival (DFS), in pT2N0M0 ESCC patients. The expression of Ku80 were higher in ESCC tissues than the corresponding health esophageal mucosa (P75 %). The intensity score was estimated using a four-category grading system (0, negative; 1, weak staining; 2, moderate staining; 3, strong staining). The immunohistochemical score was defined as proportion score × intensity score. Two experienced experts who were blinded to
Tumor Biol.
the patient data independently scored the samples and reach agreement by re-analysis and discussion. Receiver operating characteristics curve The cutoff scores for Ku80 overexpression were screen based on receiver operating characteristics (ROC) curve. Raw data of Ku80 expression level in ESCC group and CHEM group were analyzed by a MedCalc statistical software package 13.0.2.0 (MedCalc Software bvba, Belgium). The score closest to the point with both maximum sensitivity and specificity was selected as the cutoff score leading to the greatest number of patients classified correctly as having or not having the Ku80 overexpression.
and the one-way analysis of variance was used to examine differences among different groups. Associations between categorical variables were analyzed using the chi-square test. The OS time was calculated from the date of the surgery to death from ESCC progression or related reasons, excluding any other reasons. The DFS was defined as the time from surgery to the date of the first recurrence or metastasis within the followup time. Survival curves were calculated by the Kaplan-Meier method. Univariate log-rank test and Cox regression model analysis were performed to identify prognostic factors. A significant difference was defined as a two-tailed P value of less than 0.05. All statistical analyses were performed using SPSS.17.0 software (SPSS, Chicago, IL, USA).
Western blotting
Results
The protein was extracted from ESCC and CHEM tissues. The protein concentration was assessed using the bicinchoninic acid assay. Equal amounts of protein (40 μg) were resolved on 12 % SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5 % non-fat dry milk containing 0.05 % Tween-20 and 1 % BSA and incubated overnight at 4 °C with primary antibodies against Ku80 and against βactin (1:1,000 dilution; Abcam, MA, USA). Following washing, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) antirabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein levels were evaluated by enhanced chemiluminescence detection system (LAS 4000 mini system; General Electric, Fairfield, CT, USA).
Patients characteristics
Follow-up Patients were examined regularly every 3–6 months after surgery, including thorough physical examination, chest and upper abdomen contrast-enhanced CT scan, ultrasonography of the abdomen, positron emission tomography (PET), bone scintigraphy, and cerebral CT. The biopsy was performed based on specific imageological and clinical examination. Postoperative enlargement of lymph nodes was diagnosed as lymph node recurrence, and if new lesions appeared in the other organs, we clinically diagnose it as metastatic ESCC after exclusion of the new primary tumor. The follow-up was ended in January 2014 and the longest follow-up period was 160 months with median time of 65 months. Statistical analysis The Mann-Whitney U test was used to determine the Ku80 expression differences in immunohistochemistry (IHC). In Western blotting, data were presented as the means ± SD,
The total number of midthoracic esophageal cancer patients who received Ivor-Lewis esophagogastrectomy was 1894, of whom 1705 had a pathological confirmation of ESCC. A total of 513 had received a clinicopathological diagnosis of T2. A total of 279 cases of patients who had lymph node metastasis were excluded. Of the 234 patients without lymph node metastasis, 2 did not have R0 resection, and a further 15 who had dissected lymph node fewer than 12 were also excluded. According to inclusion criteria, 217 patients in stage pT2N0M0 were enrolled in this study. All patients did not received chemotherapy or radiotherapy before surgery. The mean number of lymph node resection was 23.2 (range 13 to 59). The median age of the patients was 58 years (from 36 to 75 years), and 165 (76.0 %) cases were men. Of those 217 patients with median tumor size of 28 mm, 104 (47.9 %) patients had a tumor size greater than 30 mm and others had a tumor size of 30 mm or less. The clinical data from all 217 patients with stage pT2N0M0 ESCC are shown in Table 1. Overexpression of Ku80 in ESCC By IHC analysis, the positive expression of Ku80 protein showed as yellow or brownish yellow stain in the nucleus of malignant cells. The positive Ku80 staining strongly was readily identified in cancer cell nucleus, whereas negative or low staining was usually observed in CHEM (Fig. 1). The immunohistochemical scoring (IHS) of Ku80 in ESCC was obvious higher than CHEM (P
RESEARCH ARTICLE
Prognostic significance of Ku80 in pT2N0M0 esophageal squamous cell carcinoma after Ivor-Lewis esophagectomy Shuai Wang & Zhou Wang & Xiangyan Liu & Yu Yang & Mo Shi & Zhenguo Sun
Received: 6 October 2014 / Accepted: 2 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Recent studies have shown that Ku80, a DNA repair protein, was involved in progression of malignant tumors. This study aimed to clarify the clinicopathological significance and prognostic value of Ku80 in pT2N0M0 esophageal squamous cell carcinoma (ESCC). We enrolled 217 patients with pT2N0M0 midthoracic ESCC who had undergone IvorLewis esophagectomy. The expression profile of Ku80 was examined by immunohistochemistry. The results were correlated with the clinicopathological variables, overall survival (OS) and disease-free survival (DFS), in pT2N0M0 ESCC patients. The expression of Ku80 were higher in ESCC tissues than the corresponding health esophageal mucosa (P75 %). The intensity score was estimated using a four-category grading system (0, negative; 1, weak staining; 2, moderate staining; 3, strong staining). The immunohistochemical score was defined as proportion score × intensity score. Two experienced experts who were blinded to
Tumor Biol.
the patient data independently scored the samples and reach agreement by re-analysis and discussion. Receiver operating characteristics curve The cutoff scores for Ku80 overexpression were screen based on receiver operating characteristics (ROC) curve. Raw data of Ku80 expression level in ESCC group and CHEM group were analyzed by a MedCalc statistical software package 13.0.2.0 (MedCalc Software bvba, Belgium). The score closest to the point with both maximum sensitivity and specificity was selected as the cutoff score leading to the greatest number of patients classified correctly as having or not having the Ku80 overexpression.
and the one-way analysis of variance was used to examine differences among different groups. Associations between categorical variables were analyzed using the chi-square test. The OS time was calculated from the date of the surgery to death from ESCC progression or related reasons, excluding any other reasons. The DFS was defined as the time from surgery to the date of the first recurrence or metastasis within the followup time. Survival curves were calculated by the Kaplan-Meier method. Univariate log-rank test and Cox regression model analysis were performed to identify prognostic factors. A significant difference was defined as a two-tailed P value of less than 0.05. All statistical analyses were performed using SPSS.17.0 software (SPSS, Chicago, IL, USA).
Western blotting
Results
The protein was extracted from ESCC and CHEM tissues. The protein concentration was assessed using the bicinchoninic acid assay. Equal amounts of protein (40 μg) were resolved on 12 % SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5 % non-fat dry milk containing 0.05 % Tween-20 and 1 % BSA and incubated overnight at 4 °C with primary antibodies against Ku80 and against βactin (1:1,000 dilution; Abcam, MA, USA). Following washing, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) antirabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein levels were evaluated by enhanced chemiluminescence detection system (LAS 4000 mini system; General Electric, Fairfield, CT, USA).
Patients characteristics
Follow-up Patients were examined regularly every 3–6 months after surgery, including thorough physical examination, chest and upper abdomen contrast-enhanced CT scan, ultrasonography of the abdomen, positron emission tomography (PET), bone scintigraphy, and cerebral CT. The biopsy was performed based on specific imageological and clinical examination. Postoperative enlargement of lymph nodes was diagnosed as lymph node recurrence, and if new lesions appeared in the other organs, we clinically diagnose it as metastatic ESCC after exclusion of the new primary tumor. The follow-up was ended in January 2014 and the longest follow-up period was 160 months with median time of 65 months. Statistical analysis The Mann-Whitney U test was used to determine the Ku80 expression differences in immunohistochemistry (IHC). In Western blotting, data were presented as the means ± SD,
The total number of midthoracic esophageal cancer patients who received Ivor-Lewis esophagogastrectomy was 1894, of whom 1705 had a pathological confirmation of ESCC. A total of 513 had received a clinicopathological diagnosis of T2. A total of 279 cases of patients who had lymph node metastasis were excluded. Of the 234 patients without lymph node metastasis, 2 did not have R0 resection, and a further 15 who had dissected lymph node fewer than 12 were also excluded. According to inclusion criteria, 217 patients in stage pT2N0M0 were enrolled in this study. All patients did not received chemotherapy or radiotherapy before surgery. The mean number of lymph node resection was 23.2 (range 13 to 59). The median age of the patients was 58 years (from 36 to 75 years), and 165 (76.0 %) cases were men. Of those 217 patients with median tumor size of 28 mm, 104 (47.9 %) patients had a tumor size greater than 30 mm and others had a tumor size of 30 mm or less. The clinical data from all 217 patients with stage pT2N0M0 ESCC are shown in Table 1. Overexpression of Ku80 in ESCC By IHC analysis, the positive expression of Ku80 protein showed as yellow or brownish yellow stain in the nucleus of malignant cells. The positive Ku80 staining strongly was readily identified in cancer cell nucleus, whereas negative or low staining was usually observed in CHEM (Fig. 1). The immunohistochemical scoring (IHS) of Ku80 in ESCC was obvious higher than CHEM (P
