World J Surg DOI 10.1007/s00268-015-3023-7
ORIGINAL SCIENTIFIC REPORT
Overexpression of Ku80 Suggests Poor Prognosis of Locally Advanced Esophageal Squamous Cell Carcinoma Patients Shuai Wang • Zhou Wang • Xiangyan Liu Yu Liu • Yang Jia
•
Ó Socie´te´ Internationale de Chirurgie 2015
Abstract Background Recent studies have shown that Ku80 expression was implicated in development and progression of malignant tumors. In the present study, we analyzed for the first time the expression of Ku80 in locally advanced esophageal squamous cell carcinoma (ESCC) and its correlation with clinicopathologic features and patient survival. Methods The expression profile of Ku80 was analyzed in 126 cases of locally advanced ESCC and 79 cases of normal subjects as control using immunohistochemistry and Western blot. The associations of Ku80 expression with clinicopathological features were estimated by v2 test. We further performed univariate and multivariate analyses to identify prognostic factors for overall survival (OS) of patients. Results Immunohistochemistry and Western blot analyses both showed the Ku80 protein expression was significantly higher in ESCC than normal esophageal mucosa and corresponding healthy esophageal mucosa. Statistical analysis suggested a significant correlation of Ku80 overexpression with the tumor size (p = 0.037), differentiation degree (p = 0.018), depth of invasion (p = 0.020), lymph node metastasis (p = 0.045), clinicopathological staging (p = 0.001), and tumor recurrence (p = 0.011) in locally advanced ESCC patients. Moreover, overexpression of Ku80 was associated with reduced OS of patients after surgery (p = 0.001). Multivariate analysis with a Cox proportional hazards model further suggested that Ku80 expression was an independent prognostic indicator for patients’ OS (p = 0.029). Conclusions Ku80 was a predictor of tumor’s progression and prognosis of locally advanced ESCC patients. All of these results indicate that assessment of Ku80 level could improve stratification of locally advanced ESCC patients.
Introduction Although diagnostic and therapeutic techniques have improved over the past decade, the prognosis of esophageal S. Wang Z. Wang (&) X. Liu Y. Jia Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, People’s Republic of China e-mail: [email protected] Y. Liu Department of Pathology, Provincial Hospital Affiliated to Shandong University, No. 324, Jingwu Road, Jinan 250021, Shandong, People’s Republic of China
squamous cell carcinoma (ESCC) patients remains poor and the 5-year overall survival (OS) rate is only still below 35 % [1, 2]. The prognosis of ESCC is tumor-nodemetastasis (TNM) staging specific, however, TNM staging is not very accurate and sensitive to predict the recurrence and prognosis of ESCC patients [3, 4]. Our previous studies indicated there were large differences in the prognosis of patients with local advanced ESCC (pT2-4aN0-2M0), even they were in same TNM stage [5, 6]. So it is of great valuable to further stratify local advanced ESCC patients according to different clinical outcomes. Many reports had studied molecular predictors of prognosis of ESCC patients [7, 8]. However, there is no specific molecular biomarker
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accepted commonly and used routinely in ESCC until now. Therefore, more reliable biomarkers that can precisely predict prognosis of ESCC patients and stratify patients risk early are urgently needed. The carcinogenesis of ESCC is characterized by severe DNA damage caused by various factors, including high temperature of food and beverage, poor nutrition, infection, and heredity [9]. Ku80 is best known for its crucial role in non-homologous end joining (NHEJ) DNA repair. Ku80 could bind to the DNA double-strand broken ends, attract DNA-dependent protein kinases and trigger NHEJ DNA repair irrespective of sequence. Besides its pivotal role in DNA repair, many reports have demonstrated that Ku80 is involved in other cellular processes, including telomere maintenance, antigen receptor gene arrangements, regulation of cell cycle, colony formation, and apoptosis [10]. Recent studies show that Ku80 could act as an adhesion molecular and play a role in cell adhesion, migration, and invasion [11, 12]. Ku80 also regulates metalloproteinase-9mediated remodeling of extracellular matrix that appear at first glance irrelevant to its main function [13]. Moreover, Ku80 overexpression significantly associates with the tumorigenesis and progression of bladder carcinoma, gastric carcinoma, colorectal carcinoma, and breast carcinoma [14–17]. Yang et al. had reported that Ku80 plays an important role in radioresistance and chemoresistance in several esophageal cancer cell lines [18]. However, the clinicopathological significance and prognostic value of Ku80 in ESCC is unknown. To address this issue, we analyzed the clinical relevance of Ku80 expression with clinicopathologic features and the OS of patients with local advanced ESCC.
Materials and methods Ethics statement The study protocol was approved by the Research Ethic Committee of Provincial Hospital Affiliated to Shandong University. All the participants provided their written informed consents. Patient and tissue specimens From January 2007 to March 2008, patients with midthoracic ESCC who underwent surgery in our department were retrospectively studied. The inclusion criteria were as follows: (1) ESCC was confirmed via pathological examination. (2) According to 2009 Union for International Cancer Control (UICC) standard for midthoracic ESCC, esophagogastrectomy with lymph node dissection were done to achieve complete resection and pathological
123
examination of surgical specimens showed no residual cancer cells under the upper and lower cutting edge and lateral margin without residual focus (R0) [19]. (3) Average lymph node dissection was more than 12. (4) According to 2009 UICC TNM stage system, patients were restaged and patients with stage pT2-4aN0-2M0 were included in this study. In control group, 79 volunteers (31 women and 48 men, mean age: 61.3 ± 11.5 years) were screened from Provincial Hospital Affiliated to Shandong University as normal subjects without any malignant disease. Review of medical records in all before mentioned subjects indicated the absence of drug, smoking, and alcohol abuse. All individuals were Han people in China without consanguineous relationship. Questionnaires were administered to study subjects in-person in detail. The questionnaire collected individual information including medical history, family history, the use of over-the-counter medications and individual exposure to dietary carcinogens. There were no significant differences between the ESCC patients and volunteers (e.g., by age, gender, medical and family history, smoking status, exposure to dietary carcinogens, and dietary habit). Seventy nine normal esophageal mucosal samples from control group were harvested through electrical gastroscopy. In ESCC group, one pair of samples were harvested through electrical gastroscopy before chemotherapy or radiotherapy from each patient. Each pair of samples consisted of ESCC tissue and corresponding healthy mucosa. The corresponding healthy esophageal mucosa (CHEM) was harvested at a distance of more than 5 cm from margin of ESCC. There were no signs of deterioration, necrosis and free of tumor in normal esophageal mucosa (NEM) and CHEM through light microscope examination. All fresh tissue samples were rinsed in cold 0.9 % NaCl, and immediately stored at -80 °C until further use. Immunohistochemistry Immunohistochemical staining for Ku80 was performed on 4 lm tissue sample sections using ultravision quanto detection system (Thermo scientific, Fremont, CA, USA) following manufacturer’s instructions. Briefly, the sections were incubated with ultravision hydrogen peroxide block for 10 min to block endogenous peroxidase. Then the sections were incubated with ultravision protein block for 5 min to reduce nonspecific background staning. After the specimens were incubated with the rabbit monoclonal antihuman Ku80 primary antibody (1:500; Abcam Ltd., Cambridge, UK) overnight at 4 °C, they were washed with PBS, followed by incubation with primary antibody amplifier quanto for 10 min and HRP polymer quanto for
World J Surg Table 1 Correlation of Ku80 expression with clinicopathological features of locally advanced ESCC patients Characteristics
Cases
Ku80 expression level
(126)
Low (54)
High (72)
91
42
49
35
12
23
C60
67
31
36
\60
59
23
36
Gender Male Female Age (years)
p
0.228
11–25 %; 3, 26–50 %; 4, 51–75 %; 5, [75 %). 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 9 intensity score. Two experienced experts who were blinded to the patient data independently scored the samples and reach agreement by reanalysis and discussion.
0.41
Tumor size
Receiver operating characteristics (ROC) curve 0.037
C50 mm
74
26
48
\50 mm
52
28
24
Differentiation degree
0.018
Low
45
13
32
Mid-high
81
41
40
T2
44
25
19
T3 ? T4a
82
29
53
Depth of invasion
0.02
Lymph node metastasis
a
Western blot analyses 0.045
N2
28
10
21
N1 N0
59 39
21 23
35 16
II
41
26
15
III
85
28
57
Yes
56
17
39
No
70
37
33
TNM stage
0.001
Recurrence
The cut-off score for Ku80 overexpression was screened based on ROC curve. Raw data of Ku80 expression levels in ESCC group and control 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 cut-off score.
0.011
Statistical analysis was performed using the Chi-squared test p values \0.05 in bold font were considered significant a
The number of lymph node metastasis was classified as N2, N1, and N0 based on 2009 Union for International Cancer Control (UICC) standard [19]
10 min. After the sections were washed with PBS and DI water, they were incubated with 3,3-diaminobenzidine and counterstained with hematoxylin. The specificities of primary antibody were confirmed before, and further validated as positive control in HeLa cells, as previous studies have indicated Ku80 overexpression in HeLa cells [20]. The control sections incubated with PBS instead of the primary antibodies were used as negative control. The immunohistochemical scoring of Ku80 was performed using a semiquantitative system as we reported previously [6]. The specimens were examined under a light microscope. In five randomly selected fields per-section, the immunoreactive cells among 100 cells were assessed and quantified by percentage. Then, the average percentage of the five fields was used to assess the proportion score in six-category grading system (0, negative; 1, 1–10 %; 2,
The protein was extracted from tissue samples and protein concentration was determined using the bicinchoninic acid assay. Equal amounts of protein (40 lg) was 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 b-actin (1:1,000 dilution; Abcam, MA, USA). Following washing, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) anti-rabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein levels were quantified by an enhanced chemiluminescence (ECL) detection system (LAS 4000 mini system; General Electric, Fairfield, CT, USA). Follow-up Patients were examined regularly every 3–6 months, including thorough physical examination, chest and upper abdomen contrast-enhanced CT scan, abdominal ultrasound, positron emission tomography, bone scintigraphy, cerebral CT. Biopsy was performed based on specific symptomatology and clinical examination. The follow-up was ended in June 2013 and the complete 5-year follow-up data were reviewed. Statistical analysis The Mann–Whitney U test was used to determine the Ku80 expression differences in immunohistochemistry. In
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World J Surg Table 2 Treatment modalities of locally advanced ESCC TNM stage
Total n ( %)
II
III
Cases
41
85
126
Preoperative chemotherapy
19
85
104 (82.5)
None
10
–
10 (7.9)
Chemotherapy alone
16
21
37 (29.4)
Chemotherapy plus radiotherapy
–
48
48 (38.1)
Radiotherapy alone
15
16
31 (24.6)
Postoperative therapy
Western blot, data were presented as the mean ± SD, and the one-way analysis of variance was used to examine differences among different groups. Associations between categorical variables were analyzed using the Chi-squared test. The OS time was calculated from surgery to death from ESCC progression or related reasons. 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 analysis was performed using SPSS.17.0 software (SPSS, Chicago, IL, USA).
Results Clinical features and treatment modalities The total number of patients with midthoracic esophageal cancer who received surgery was 204, of whom 172 had a pathological confirmation of ESCC. 139 of these had received a clinicopathological diagnosis of stage pT2-4aN02M0. Of the 139 patients, 2 patients did not have R0 resection. Eleven cases of patients who had dissected lymph node fewer than 12 were also excluded. According to inclusion criteria, 126 patients in stage pT2-4aN0-2M0 were enrolled in this study. The median age of the patients was 62 years (from 45 to 78 years), and 91 (72.2 %) cases were men. The detailed clinicopathologic data for the 126 patients are outlined in Table 1. If preoperative evaluation indicated that the tumor might possibly be completely resected, complete resection of tumor with lymph node dissection was chosen as the first choice of treatment to locally advanced ESCC. Surgical procedure had been reported in our previous studies in detail [5, 6, 21]. Otherwise, the patients were treated with neoadjuvant chemotherapy first. The treatment modalities are shown in Table 2. Neoadjuvant chemotherapy was
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given to 104 patients using two cycle of cisplatin-based regimens; PF regimen [cisplatin (7 mg/m2 over 2 h) and 5-FU (350 mg/m2 over 24 h)] [22]. Of these 104 patients, 19 with stage II disease and 85 with stage III experienced a decrease in tumor volume. Postoperative adjuvant treatment was given routinely in a limited time to ESCC patients in stage IIB and III. The decision to use postoperative chemotherapy or (and) radiotherapy was individualized for each patient (Table 2). Adjuvant chemotherapy was performed in 85 cases using PF regimen [22], including 48 cases was treated along with adjuvant radiotherapy. Adjuvant radiotherapy was performed with involved fields that covered the supraclavicular to lower mediastinal lymph node and cardiac lymph node areas and perigastric lymph node areas were additionally irradiated for lower tumors with margins of approximately 1–2 cm. A total dose of 50–60 Gy was administered for patients with complete resection. In this study group, 56 patients (44.4 %) had recurrence during follow-up period. The median recurrence time was 21 ± 5.2 months. Mode of recurrence of ESCC included 43 cases of locoregional recurrence (35/126, 27.8 %), 12 cases of hematogenous metastasis (12/126, 9.5 %), and 9 cases of locoregional recurrence as well as hematogenous metastasis (9/126, 7.1 %). The expression of Ku80 in normal and neoplastic esophageal tissue By immunohistochemistry analysis, the positive expression of Ku80 showed as yellow or brownish yellow stain in the nucleus of malignant cells. The positive Ku80 staining strongly was readily identified in ESCC, whereas negative or low staining was usually observed in NEM and CHEM (Fig. 1). The immunohistochemical scoring of Ku80 in NEM, CHEM, and ESCC are shown in Fig. 2a. The protein level of Ku80 in ESCC was obvious higher than NEM and CHEM (p \ 0.001 and p \ 0.001). However, there was no obvious difference in Ku80 expression between CHEM and NEM (p = 0.541). According to the ROC curve (Fig. 2b), threshold value of 8 was the closest to the point with both maximum sensitivity (58.4 %) and specificity (92.0 %), and thereby selected as the cut-off value. The area under the curve was 0.855 [95 % confidence interval (CI): 0.781–0.911; p \ 0.001]. So, samples with the immunohistochemical score above 8 were identified as Ku80 high expression, others were Ku80 low expression. Patients were thus divided into two groups, namely high expression group (n = 72, 57.1 %) and low expression group (n = 54, 42.9 %). To validate the results of immunohistochemistry, we further analyzed Ku80 expression levels in different tissues by Western blot (Fig. 2c). The expression level of the Ku80 was 0.482 ± 0.064, 0.491 ± 0.081, and 1.017 ± 0.095
World J Surg Fig. 1 Immunohistochemical staining of Ku80 in esophageal cancer. a and b Representative negative expression of Ku80 in normal esophageal mucosa (NEM). c and d Representative low expression of Ku80 in corresponding healthy esophageal mucosa (CHEM). e and f Representative high expression of Ku80 in esophageal squamous cell carcinoma (ESCC) (low differentiation degree). 9200 and 9400: original magnification. Nuclei are counterstained with hematoxylin
in NEM, CHEM, and ESCC, respectively. ESCC displayed the greater expression level compared with NEM and CHEM (p = 0.002 and p = 0.002), which was consistent with the results of the immunohistochemistry. Association between Ku80 expression and clinicopathologic variables v2 analysis indicated that Ku80 expression was not associated with gender (p = 0.228) and age (p = 0.410), and was significantly associated with tumor size (p = 0.037), tumor differentiation degree (p = 0.018), depth of invasion (p = 0.020), lymph node metastasis (p = 0.045), TNM stage (p = 0.001), and tumor recurrence (p = 0.011) (Table 1). Taken as a whole, the overexpression of Ku80 was positively correlated with aggressive clinicopathological features of patients with locally advanced ESCC.
Association between Ku80 expression and patient survival The 1-, 3-, and 5-year OS rate of 126 patients was 90.5, 48.4, and 37.3 %. The median survival time was 35.0 months (95 % CI: 25.6–44.4 months). As shown in Table 3, univariate analysis revealed that tumor differentiation degree (p = 0.002), depth of invasion (p = 0.010), lymph node metastasis (p = 0.002), TNM stage (p = 0.001), and Ku80 expression level (p = 0.001) were significant prognostic factors. However, gender (p = 0.353), age (p = 0.305), and tumor size (p = 0.161) didn’t reach the statistical significance. Mid-high tumor differentiation degree, low aggressive local invasion, negative lymph node metastasis, early TNM stage, and low Ku80 expression showed significantly better prognosis. To rule out confounding factors, we performed Cox proportional hazards model analysis.
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Ku80 expression level (p = 0.029) were both independent and significant prognostic factors. To validate the prognostic value of Ku80, we further analyzed the 48 patients with same treatment mode (preoperative chemotherapy—surgery— postoperative chemotherapy plus radiotherapy) in stage III. As shown in Table 4, univariate analysis revealed that tumor differentiation degree (p = 0.001), depth of invasion (p = 0.002), and Ku80 expression level (p \ 0.001) were significant prognostic factors. Multivariate analysis suggested that tumor differentiation degree (p = 0.022), depth of invasion (p = 0.030), and Ku80 expression level (p = 0.001) were both independent and significant prognostic factors. Fig. 2 Ku80 expression in different tissues. a The immunohistochemical scores (IHS) of Ku80 in normal esophageal mucosa (NEM), corresponding healthy esophageal mucosa (CHEM), and esophageal squamous cell carcinoma (ESCC). Data are represented as a box and whisker plot and analyzed using Mann–Whitney U test. b ROC analysis of Ku80 expression and the selection of cut-off score. c The gel is representative of Western blot analyses. Lane 1, 2, and 3: Ku80 expression in NEM, CHEM, and ESCC, respectively; Lane 4, 5, and 6: b-actin expression as control in NEM, CHEM, and ESCC, respectively
Multivariate analysis revealed that tumor differentiation degree (p = 0.035), depth of invasion (p = 0.033), lymph node metastasis (p = 0.020), TNM stage (p = 0.017), and
Discussion In this study, we explored the clinicopathological significance of Ku80 in locally advanced (pT2-4aN0-2M0) ESCC. We first found the expression of Ku80 was increased in ESCC tissues compared with CHEM and NEM by immunohistochemistry and Western blot analyses. Our results were consistent with those of Yang et al. [18], which demonstrated the overexpression of Ku80 in ESCC lines. We further divided the 126 patients with locally advanced ESCC into two groups: high expression group and low expression group. Researches might face a tricky
Table 3 Univariate and multivariate analyses of overall survival for 126 cases of locally advanced ESCC patients Variable
Univariate analysis
Multivariate analysis
HR
95 % CI
p
HR
95 % CI
p
1.255
0.777–2.029
0.353
–
1.260
0.810–1.960
0.305
–
1.388
0.877–2.194
0.161
–
2.368
1.514–3.705
0.002
1.706
1.038–2.804
0.035
1.927
1.167–3.180
0.010
1.749
1.047–2.922
0.033
2.409
1.389–4.179
0.002
1.944
1.108–3.410
0.020
2.587
1.491–4.489
0.001
1.995
1.134–3.510
0.017
1.971
1.514–3.970
0.001
1.764
1.061–2.931
0.029
Gender Male versus female Age C60 versus \ 60 years Tumor size C50 mm versus \50 mm Differentiation degree Low versus mid-high Depth of invasion T2 versus T3 ? T4a Lymph node metastasis Positive versus negative TNM stage II versus III Ku80 expression level Low versus high
Statistical analysis was performed using the proportional hazard model (Cox). Data considered significant (p \ 0.05) in the univariate analyses were examined in the multivariate analyses HR hazard ratio, CI confidence interval p values \0.05 in bold font were considered significant
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World J Surg Table 4 Univariate and multivariate analyses of overall survival for 48 cases with same treatment mode Variable
Univariate analysis
Multivariate analysis
HR
95 % CI
p
HR
95 % CI
p
1.375
0.862–2.053
0.181
–
1.389
0.901–2.224
0.154
–
1.542
1.057–2.428
0.106
–
2.579
1.483–4.337
0.001
1.923
1.245–2.990
0.022
2.386
1.292–4.025
0.002
1.847
1.176–2.716
0.030
2.812
1.699–5.010
ORIGINAL SCIENTIFIC REPORT
Overexpression of Ku80 Suggests Poor Prognosis of Locally Advanced Esophageal Squamous Cell Carcinoma Patients Shuai Wang • Zhou Wang • Xiangyan Liu Yu Liu • Yang Jia
•
Ó Socie´te´ Internationale de Chirurgie 2015
Abstract Background Recent studies have shown that Ku80 expression was implicated in development and progression of malignant tumors. In the present study, we analyzed for the first time the expression of Ku80 in locally advanced esophageal squamous cell carcinoma (ESCC) and its correlation with clinicopathologic features and patient survival. Methods The expression profile of Ku80 was analyzed in 126 cases of locally advanced ESCC and 79 cases of normal subjects as control using immunohistochemistry and Western blot. The associations of Ku80 expression with clinicopathological features were estimated by v2 test. We further performed univariate and multivariate analyses to identify prognostic factors for overall survival (OS) of patients. Results Immunohistochemistry and Western blot analyses both showed the Ku80 protein expression was significantly higher in ESCC than normal esophageal mucosa and corresponding healthy esophageal mucosa. Statistical analysis suggested a significant correlation of Ku80 overexpression with the tumor size (p = 0.037), differentiation degree (p = 0.018), depth of invasion (p = 0.020), lymph node metastasis (p = 0.045), clinicopathological staging (p = 0.001), and tumor recurrence (p = 0.011) in locally advanced ESCC patients. Moreover, overexpression of Ku80 was associated with reduced OS of patients after surgery (p = 0.001). Multivariate analysis with a Cox proportional hazards model further suggested that Ku80 expression was an independent prognostic indicator for patients’ OS (p = 0.029). Conclusions Ku80 was a predictor of tumor’s progression and prognosis of locally advanced ESCC patients. All of these results indicate that assessment of Ku80 level could improve stratification of locally advanced ESCC patients.
Introduction Although diagnostic and therapeutic techniques have improved over the past decade, the prognosis of esophageal S. Wang Z. Wang (&) X. Liu Y. Jia Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, People’s Republic of China e-mail: [email protected] Y. Liu Department of Pathology, Provincial Hospital Affiliated to Shandong University, No. 324, Jingwu Road, Jinan 250021, Shandong, People’s Republic of China
squamous cell carcinoma (ESCC) patients remains poor and the 5-year overall survival (OS) rate is only still below 35 % [1, 2]. The prognosis of ESCC is tumor-nodemetastasis (TNM) staging specific, however, TNM staging is not very accurate and sensitive to predict the recurrence and prognosis of ESCC patients [3, 4]. Our previous studies indicated there were large differences in the prognosis of patients with local advanced ESCC (pT2-4aN0-2M0), even they were in same TNM stage [5, 6]. So it is of great valuable to further stratify local advanced ESCC patients according to different clinical outcomes. Many reports had studied molecular predictors of prognosis of ESCC patients [7, 8]. However, there is no specific molecular biomarker
123
World J Surg
accepted commonly and used routinely in ESCC until now. Therefore, more reliable biomarkers that can precisely predict prognosis of ESCC patients and stratify patients risk early are urgently needed. The carcinogenesis of ESCC is characterized by severe DNA damage caused by various factors, including high temperature of food and beverage, poor nutrition, infection, and heredity [9]. Ku80 is best known for its crucial role in non-homologous end joining (NHEJ) DNA repair. Ku80 could bind to the DNA double-strand broken ends, attract DNA-dependent protein kinases and trigger NHEJ DNA repair irrespective of sequence. Besides its pivotal role in DNA repair, many reports have demonstrated that Ku80 is involved in other cellular processes, including telomere maintenance, antigen receptor gene arrangements, regulation of cell cycle, colony formation, and apoptosis [10]. Recent studies show that Ku80 could act as an adhesion molecular and play a role in cell adhesion, migration, and invasion [11, 12]. Ku80 also regulates metalloproteinase-9mediated remodeling of extracellular matrix that appear at first glance irrelevant to its main function [13]. Moreover, Ku80 overexpression significantly associates with the tumorigenesis and progression of bladder carcinoma, gastric carcinoma, colorectal carcinoma, and breast carcinoma [14–17]. Yang et al. had reported that Ku80 plays an important role in radioresistance and chemoresistance in several esophageal cancer cell lines [18]. However, the clinicopathological significance and prognostic value of Ku80 in ESCC is unknown. To address this issue, we analyzed the clinical relevance of Ku80 expression with clinicopathologic features and the OS of patients with local advanced ESCC.
Materials and methods Ethics statement The study protocol was approved by the Research Ethic Committee of Provincial Hospital Affiliated to Shandong University. All the participants provided their written informed consents. Patient and tissue specimens From January 2007 to March 2008, patients with midthoracic ESCC who underwent surgery in our department were retrospectively studied. The inclusion criteria were as follows: (1) ESCC was confirmed via pathological examination. (2) According to 2009 Union for International Cancer Control (UICC) standard for midthoracic ESCC, esophagogastrectomy with lymph node dissection were done to achieve complete resection and pathological
123
examination of surgical specimens showed no residual cancer cells under the upper and lower cutting edge and lateral margin without residual focus (R0) [19]. (3) Average lymph node dissection was more than 12. (4) According to 2009 UICC TNM stage system, patients were restaged and patients with stage pT2-4aN0-2M0 were included in this study. In control group, 79 volunteers (31 women and 48 men, mean age: 61.3 ± 11.5 years) were screened from Provincial Hospital Affiliated to Shandong University as normal subjects without any malignant disease. Review of medical records in all before mentioned subjects indicated the absence of drug, smoking, and alcohol abuse. All individuals were Han people in China without consanguineous relationship. Questionnaires were administered to study subjects in-person in detail. The questionnaire collected individual information including medical history, family history, the use of over-the-counter medications and individual exposure to dietary carcinogens. There were no significant differences between the ESCC patients and volunteers (e.g., by age, gender, medical and family history, smoking status, exposure to dietary carcinogens, and dietary habit). Seventy nine normal esophageal mucosal samples from control group were harvested through electrical gastroscopy. In ESCC group, one pair of samples were harvested through electrical gastroscopy before chemotherapy or radiotherapy from each patient. Each pair of samples consisted of ESCC tissue and corresponding healthy mucosa. The corresponding healthy esophageal mucosa (CHEM) was harvested at a distance of more than 5 cm from margin of ESCC. There were no signs of deterioration, necrosis and free of tumor in normal esophageal mucosa (NEM) and CHEM through light microscope examination. All fresh tissue samples were rinsed in cold 0.9 % NaCl, and immediately stored at -80 °C until further use. Immunohistochemistry Immunohistochemical staining for Ku80 was performed on 4 lm tissue sample sections using ultravision quanto detection system (Thermo scientific, Fremont, CA, USA) following manufacturer’s instructions. Briefly, the sections were incubated with ultravision hydrogen peroxide block for 10 min to block endogenous peroxidase. Then the sections were incubated with ultravision protein block for 5 min to reduce nonspecific background staning. After the specimens were incubated with the rabbit monoclonal antihuman Ku80 primary antibody (1:500; Abcam Ltd., Cambridge, UK) overnight at 4 °C, they were washed with PBS, followed by incubation with primary antibody amplifier quanto for 10 min and HRP polymer quanto for
World J Surg Table 1 Correlation of Ku80 expression with clinicopathological features of locally advanced ESCC patients Characteristics
Cases
Ku80 expression level
(126)
Low (54)
High (72)
91
42
49
35
12
23
C60
67
31
36
\60
59
23
36
Gender Male Female Age (years)
p
0.228
11–25 %; 3, 26–50 %; 4, 51–75 %; 5, [75 %). 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 9 intensity score. Two experienced experts who were blinded to the patient data independently scored the samples and reach agreement by reanalysis and discussion.
0.41
Tumor size
Receiver operating characteristics (ROC) curve 0.037
C50 mm
74
26
48
\50 mm
52
28
24
Differentiation degree
0.018
Low
45
13
32
Mid-high
81
41
40
T2
44
25
19
T3 ? T4a
82
29
53
Depth of invasion
0.02
Lymph node metastasis
a
Western blot analyses 0.045
N2
28
10
21
N1 N0
59 39
21 23
35 16
II
41
26
15
III
85
28
57
Yes
56
17
39
No
70
37
33
TNM stage
0.001
Recurrence
The cut-off score for Ku80 overexpression was screened based on ROC curve. Raw data of Ku80 expression levels in ESCC group and control 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 cut-off score.
0.011
Statistical analysis was performed using the Chi-squared test p values \0.05 in bold font were considered significant a
The number of lymph node metastasis was classified as N2, N1, and N0 based on 2009 Union for International Cancer Control (UICC) standard [19]
10 min. After the sections were washed with PBS and DI water, they were incubated with 3,3-diaminobenzidine and counterstained with hematoxylin. The specificities of primary antibody were confirmed before, and further validated as positive control in HeLa cells, as previous studies have indicated Ku80 overexpression in HeLa cells [20]. The control sections incubated with PBS instead of the primary antibodies were used as negative control. The immunohistochemical scoring of Ku80 was performed using a semiquantitative system as we reported previously [6]. The specimens were examined under a light microscope. In five randomly selected fields per-section, the immunoreactive cells among 100 cells were assessed and quantified by percentage. Then, the average percentage of the five fields was used to assess the proportion score in six-category grading system (0, negative; 1, 1–10 %; 2,
The protein was extracted from tissue samples and protein concentration was determined using the bicinchoninic acid assay. Equal amounts of protein (40 lg) was 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 b-actin (1:1,000 dilution; Abcam, MA, USA). Following washing, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) anti-rabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein levels were quantified by an enhanced chemiluminescence (ECL) detection system (LAS 4000 mini system; General Electric, Fairfield, CT, USA). Follow-up Patients were examined regularly every 3–6 months, including thorough physical examination, chest and upper abdomen contrast-enhanced CT scan, abdominal ultrasound, positron emission tomography, bone scintigraphy, cerebral CT. Biopsy was performed based on specific symptomatology and clinical examination. The follow-up was ended in June 2013 and the complete 5-year follow-up data were reviewed. Statistical analysis The Mann–Whitney U test was used to determine the Ku80 expression differences in immunohistochemistry. In
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World J Surg Table 2 Treatment modalities of locally advanced ESCC TNM stage
Total n ( %)
II
III
Cases
41
85
126
Preoperative chemotherapy
19
85
104 (82.5)
None
10
–
10 (7.9)
Chemotherapy alone
16
21
37 (29.4)
Chemotherapy plus radiotherapy
–
48
48 (38.1)
Radiotherapy alone
15
16
31 (24.6)
Postoperative therapy
Western blot, data were presented as the mean ± SD, and the one-way analysis of variance was used to examine differences among different groups. Associations between categorical variables were analyzed using the Chi-squared test. The OS time was calculated from surgery to death from ESCC progression or related reasons. 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 analysis was performed using SPSS.17.0 software (SPSS, Chicago, IL, USA).
Results Clinical features and treatment modalities The total number of patients with midthoracic esophageal cancer who received surgery was 204, of whom 172 had a pathological confirmation of ESCC. 139 of these had received a clinicopathological diagnosis of stage pT2-4aN02M0. Of the 139 patients, 2 patients did not have R0 resection. Eleven cases of patients who had dissected lymph node fewer than 12 were also excluded. According to inclusion criteria, 126 patients in stage pT2-4aN0-2M0 were enrolled in this study. The median age of the patients was 62 years (from 45 to 78 years), and 91 (72.2 %) cases were men. The detailed clinicopathologic data for the 126 patients are outlined in Table 1. If preoperative evaluation indicated that the tumor might possibly be completely resected, complete resection of tumor with lymph node dissection was chosen as the first choice of treatment to locally advanced ESCC. Surgical procedure had been reported in our previous studies in detail [5, 6, 21]. Otherwise, the patients were treated with neoadjuvant chemotherapy first. The treatment modalities are shown in Table 2. Neoadjuvant chemotherapy was
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given to 104 patients using two cycle of cisplatin-based regimens; PF regimen [cisplatin (7 mg/m2 over 2 h) and 5-FU (350 mg/m2 over 24 h)] [22]. Of these 104 patients, 19 with stage II disease and 85 with stage III experienced a decrease in tumor volume. Postoperative adjuvant treatment was given routinely in a limited time to ESCC patients in stage IIB and III. The decision to use postoperative chemotherapy or (and) radiotherapy was individualized for each patient (Table 2). Adjuvant chemotherapy was performed in 85 cases using PF regimen [22], including 48 cases was treated along with adjuvant radiotherapy. Adjuvant radiotherapy was performed with involved fields that covered the supraclavicular to lower mediastinal lymph node and cardiac lymph node areas and perigastric lymph node areas were additionally irradiated for lower tumors with margins of approximately 1–2 cm. A total dose of 50–60 Gy was administered for patients with complete resection. In this study group, 56 patients (44.4 %) had recurrence during follow-up period. The median recurrence time was 21 ± 5.2 months. Mode of recurrence of ESCC included 43 cases of locoregional recurrence (35/126, 27.8 %), 12 cases of hematogenous metastasis (12/126, 9.5 %), and 9 cases of locoregional recurrence as well as hematogenous metastasis (9/126, 7.1 %). The expression of Ku80 in normal and neoplastic esophageal tissue By immunohistochemistry analysis, the positive expression of Ku80 showed as yellow or brownish yellow stain in the nucleus of malignant cells. The positive Ku80 staining strongly was readily identified in ESCC, whereas negative or low staining was usually observed in NEM and CHEM (Fig. 1). The immunohistochemical scoring of Ku80 in NEM, CHEM, and ESCC are shown in Fig. 2a. The protein level of Ku80 in ESCC was obvious higher than NEM and CHEM (p \ 0.001 and p \ 0.001). However, there was no obvious difference in Ku80 expression between CHEM and NEM (p = 0.541). According to the ROC curve (Fig. 2b), threshold value of 8 was the closest to the point with both maximum sensitivity (58.4 %) and specificity (92.0 %), and thereby selected as the cut-off value. The area under the curve was 0.855 [95 % confidence interval (CI): 0.781–0.911; p \ 0.001]. So, samples with the immunohistochemical score above 8 were identified as Ku80 high expression, others were Ku80 low expression. Patients were thus divided into two groups, namely high expression group (n = 72, 57.1 %) and low expression group (n = 54, 42.9 %). To validate the results of immunohistochemistry, we further analyzed Ku80 expression levels in different tissues by Western blot (Fig. 2c). The expression level of the Ku80 was 0.482 ± 0.064, 0.491 ± 0.081, and 1.017 ± 0.095
World J Surg Fig. 1 Immunohistochemical staining of Ku80 in esophageal cancer. a and b Representative negative expression of Ku80 in normal esophageal mucosa (NEM). c and d Representative low expression of Ku80 in corresponding healthy esophageal mucosa (CHEM). e and f Representative high expression of Ku80 in esophageal squamous cell carcinoma (ESCC) (low differentiation degree). 9200 and 9400: original magnification. Nuclei are counterstained with hematoxylin
in NEM, CHEM, and ESCC, respectively. ESCC displayed the greater expression level compared with NEM and CHEM (p = 0.002 and p = 0.002), which was consistent with the results of the immunohistochemistry. Association between Ku80 expression and clinicopathologic variables v2 analysis indicated that Ku80 expression was not associated with gender (p = 0.228) and age (p = 0.410), and was significantly associated with tumor size (p = 0.037), tumor differentiation degree (p = 0.018), depth of invasion (p = 0.020), lymph node metastasis (p = 0.045), TNM stage (p = 0.001), and tumor recurrence (p = 0.011) (Table 1). Taken as a whole, the overexpression of Ku80 was positively correlated with aggressive clinicopathological features of patients with locally advanced ESCC.
Association between Ku80 expression and patient survival The 1-, 3-, and 5-year OS rate of 126 patients was 90.5, 48.4, and 37.3 %. The median survival time was 35.0 months (95 % CI: 25.6–44.4 months). As shown in Table 3, univariate analysis revealed that tumor differentiation degree (p = 0.002), depth of invasion (p = 0.010), lymph node metastasis (p = 0.002), TNM stage (p = 0.001), and Ku80 expression level (p = 0.001) were significant prognostic factors. However, gender (p = 0.353), age (p = 0.305), and tumor size (p = 0.161) didn’t reach the statistical significance. Mid-high tumor differentiation degree, low aggressive local invasion, negative lymph node metastasis, early TNM stage, and low Ku80 expression showed significantly better prognosis. To rule out confounding factors, we performed Cox proportional hazards model analysis.
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World J Surg
Ku80 expression level (p = 0.029) were both independent and significant prognostic factors. To validate the prognostic value of Ku80, we further analyzed the 48 patients with same treatment mode (preoperative chemotherapy—surgery— postoperative chemotherapy plus radiotherapy) in stage III. As shown in Table 4, univariate analysis revealed that tumor differentiation degree (p = 0.001), depth of invasion (p = 0.002), and Ku80 expression level (p \ 0.001) were significant prognostic factors. Multivariate analysis suggested that tumor differentiation degree (p = 0.022), depth of invasion (p = 0.030), and Ku80 expression level (p = 0.001) were both independent and significant prognostic factors. Fig. 2 Ku80 expression in different tissues. a The immunohistochemical scores (IHS) of Ku80 in normal esophageal mucosa (NEM), corresponding healthy esophageal mucosa (CHEM), and esophageal squamous cell carcinoma (ESCC). Data are represented as a box and whisker plot and analyzed using Mann–Whitney U test. b ROC analysis of Ku80 expression and the selection of cut-off score. c The gel is representative of Western blot analyses. Lane 1, 2, and 3: Ku80 expression in NEM, CHEM, and ESCC, respectively; Lane 4, 5, and 6: b-actin expression as control in NEM, CHEM, and ESCC, respectively
Multivariate analysis revealed that tumor differentiation degree (p = 0.035), depth of invasion (p = 0.033), lymph node metastasis (p = 0.020), TNM stage (p = 0.017), and
Discussion In this study, we explored the clinicopathological significance of Ku80 in locally advanced (pT2-4aN0-2M0) ESCC. We first found the expression of Ku80 was increased in ESCC tissues compared with CHEM and NEM by immunohistochemistry and Western blot analyses. Our results were consistent with those of Yang et al. [18], which demonstrated the overexpression of Ku80 in ESCC lines. We further divided the 126 patients with locally advanced ESCC into two groups: high expression group and low expression group. Researches might face a tricky
Table 3 Univariate and multivariate analyses of overall survival for 126 cases of locally advanced ESCC patients Variable
Univariate analysis
Multivariate analysis
HR
95 % CI
p
HR
95 % CI
p
1.255
0.777–2.029
0.353
–
1.260
0.810–1.960
0.305
–
1.388
0.877–2.194
0.161
–
2.368
1.514–3.705
0.002
1.706
1.038–2.804
0.035
1.927
1.167–3.180
0.010
1.749
1.047–2.922
0.033
2.409
1.389–4.179
0.002
1.944
1.108–3.410
0.020
2.587
1.491–4.489
0.001
1.995
1.134–3.510
0.017
1.971
1.514–3.970
0.001
1.764
1.061–2.931
0.029
Gender Male versus female Age C60 versus \ 60 years Tumor size C50 mm versus \50 mm Differentiation degree Low versus mid-high Depth of invasion T2 versus T3 ? T4a Lymph node metastasis Positive versus negative TNM stage II versus III Ku80 expression level Low versus high
Statistical analysis was performed using the proportional hazard model (Cox). Data considered significant (p \ 0.05) in the univariate analyses were examined in the multivariate analyses HR hazard ratio, CI confidence interval p values \0.05 in bold font were considered significant
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World J Surg Table 4 Univariate and multivariate analyses of overall survival for 48 cases with same treatment mode Variable
Univariate analysis
Multivariate analysis
HR
95 % CI
p
HR
95 % CI
p
1.375
0.862–2.053
0.181
–
1.389
0.901–2.224
0.154
–
1.542
1.057–2.428
0.106
–
2.579
1.483–4.337
0.001
1.923
1.245–2.990
0.022
2.386
1.292–4.025
0.002
1.847
1.176–2.716
0.030
2.812
1.699–5.010
