MIB-1 index is unlikely to predict relapse-free survival in patients who underwent R0-esophagectomy for esophageal squamous cell carcinoma

MIB-1 index is unlikely to predict relapse-free survival in patients who underwent... Summary MIB-1 is a cell proliferation marker and has previously been investigated as a diagnostic or prognostic indicator of malignancy. Previous studies have investigated MIB-1 index and clinicopathological factors in relation to prognosis of patients with esophageal cancer, with conflicting results. The aim of this study is to assess the prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma. A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal squamous cell carcinoma were enrolled in this study. Preoperatively, 29 patients underwent chemotherapy, six underwent chemoradiotherapy, and the remaining did not undergo any preoperative therapy. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The mean MIB-1 index was 39.3 ± 21.0 (range: 0–91.3). There was no significant correlation between clinicopathological factors and MIB-1 index in the study patients, irrespective of whether they underwent preoperative therapy. Univariate analysis revealed no significant association between MIB-1 index and RFS. However, depth of tumor invasion, lymph node metastasis and stage, all showed a significant correlation to RFS. Multivariate analysis of RFS revealed that stage was the only significant factor. Conversely, MIB-1 index was not significantly related to RFS (p = 0.41). In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for esophageal cancer. INTRODUCTION Esophageal cancer (EC) is the eighth most common cancer worldwide. Specifically, in 2012, 3.2% of all new cases of cancer diagnosed were classified as EC (World cancer research fund international. http://www.wcrf.org/int/cancer-facts-figures/worldwide-data). A standard treatment for nonmetastatic EC is curative resection. Accurate prognostication facilitates the identification of patients at a high risk of recurrence. Such patients would benefit from adjuvant therapies and surveillance planning. Evaluation of TNM stage (tumor, nodes, and metastasis) is very important in predicting the prognosis of patients with EC; however, the discovery of a new prognostic parameter could change treatment strategies and follow-up plans. The nuclear antigen, Ki-67, is expressed in all cell cycle phases except G0. MIB-1 index using monoclonal antibody MIB-1 for recognizing the Ki-67 antigen has been investigated as a diagnostic or prognostic indicator of malignant tumors, suggesting cellular proliferation.1,2 Many studies have investigated the correlation between MIB-1 index, clinicopathological factors, and the prognosis of patients with EC; however, the results have been conflicting.3–14 The aim of this study is to assess the clinicopathological and prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma (SCC). MATERIALS AND METHODS A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal SCC between March 2006 and December 2014 were enrolled in this study. The study group comprised of 66 men and 12 women with a mean age of 65.4 ± 7.4 years (range: 45–79 years). Of the 78 tumors, 14 were located in the upper esophagus, 42 were located in the middle esophagus, and 22 were located in the lower esophagus. Preoperative chemotherapy using 5-fluorouracil (5-FU) and cisplatin (CDDP) was administered in 29 patients according to the JCOG9907 study.15 Preoperative chemoradiotherapy using 5-FU, CDDP, and 50–70 Gy, was administered in six patients with cT4 cancer. Surgical approach involved 76 patients undergoing right thoracotomy, one undergoing left thoracotomy and, one undergoing transhiatal esophagectomy. The use of a gastric tube was common for reconstruction (n = 66). Postoperative chemotherapy (5-FU and CDDP) was administered in 20 patients with positive lymph nodes and informed consent for adjuvant chemotherapy was obtained. Immunohistochemistry of Paraffin-embedded sections for MIB-1 (1 : 100 dilution, DAKO, Glostrup, Denmark) was performed by using Bond-MAX (Leica, Microsystem, Newcastle upon Tyne, UK) according to an instruction manual. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Due to the marked heterogeneity of proliferative activity within a tumor, the number of MIB-1 positive cells was counted in the area with the highest proliferative activity (Figs 1,2). Accordingly, MIB-1 index was commonly determined at the deepest part or the core of the SCC. Fig. 1 View largeDownload slide Immunostaining with monoclonal antibody MIB-1 in a case without preoperative therapy, staged T3, N0, M0, and II showing MIB-1 index 37.0%. Figure (a) loupe image, (b) magnified view (×20) of a closed square in Figure (a). Fig. 1 View largeDownload slide Immunostaining with monoclonal antibody MIB-1 in a case without preoperative therapy, staged T3, N0, M0, and II showing MIB-1 index 37.0%. Figure (a) loupe image, (b) magnified view (×20) of a closed square in Figure (a). Fig. 2 View largeDownload slide Hematoxylin and eosin staining and immunostaining with the monoclonal antibody MIB-1 in a case with preoperative therapy, staged T3, N0, M0, and III showing grade 2 histological effects of preoperative therapy and MIB-1 index 74.4%. Figure (a) loupe image, (b) magnified view (×10). Fig. 2 View largeDownload slide Hematoxylin and eosin staining and immunostaining with the monoclonal antibody MIB-1 in a case with preoperative therapy, staged T3, N0, M0, and III showing grade 2 histological effects of preoperative therapy and MIB-1 index 74.4%. Figure (a) loupe image, (b) magnified view (×10). The resected EC were histopathologically classified according to the seventh edition of the international union against cancer/American joint committee on cancer TNM classification.16 Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The clinicopathological factors investigated were age, sex, tumor location, tumor diameter, depth of tumor invasion (T), lymph node metastases (N), lymphatic invasion, venous invasion, stage, and histological differentiation. Histological effects of preoperative therapy were classified according to the 11th edition of the Japanese Classification of Esophageal Cancer.17 Our department follows a standardized surveillance protocol for postoperative patients who have undergone esophagectomy for EC, wherein patients are followed up clinically for a period of 5 years at 3-month intervals. Postoperative data included clinical assessment, laboratory tests, and computed tomography of the chest and abdomen. All evidences of recurrence were obtained from patients’ medical records. Follow-up information through to April 2016 was compiled for all survivors and RFS was calculated from the day treatment was initiated (the day when preoperative treatment began or the date of the esophagectomy), until the date of identification of disease recurrence. The study protocol was approved by the ethics committee of our hospital (No. 2017–047). Statistical analysis The correlation between MIB-1 index and clinicopathological factors was investigated using Mann–Whitney U test, Kruskal–Wallis test, or Spearman's rank correlation coefficient, as appropriate. Survival rates were calculated by the Kaplan–Meier method, and the log-rank test was employed to evaluate differences in RFS between groups in the univariate analysis. Multivariable analysis was performed using a Cox proportional hazard model, in which factors with p < 0.05 in the univariate analysis and MIB-1 index were included. Depth of tumor invasion, lymph node metastases, and stage were integrated to one factor (stage), as they had a close relationship. A p value less than 0.05 was considered to indicate statistical significance. All statistical analyses were performed using the JMP data analysis software, version 10.0 for Windows (SAS Institute Inc., Cary, NC, USA). RESULTS Patient demographics are shown in Table 1. The mean MIB-1 index was 39.3 ± 21.0 (range: 0–91.3). There was no significant correlation between MIB-1 index and clinicopathological factors in the study patients (n = 78), as well as in patients with (n = 35) or without (n = 43) preoperative therapy (Table 2). Five year RFS of the study patients was 58.9% with a median follow-up of 38.9 months (IQR: 17.4–77.5). The results of univariate analysis of RFS according to clinicopathological factors and MIB-1 index are shown in Table 3. When the differences in RFS were examined by bisecting MIB-1 index at an interval between 10 and 80, the difference of RFS between the two groups was widest at a MIB-1 index value of 40. There was no significant difference between MIB-1 index and RFS (p = 0.21, Fig. 3). However, depth of tumor invasion, lymph node metastases, lymphatic invasion, venous invasion, and stage were significantly correlated with RFS. Multivariate analysis of RFS revealed that stage was the only significant factor in determining RFS (Table 3), while MIB-1 index was not significantly related to RFS. MIB-1 index did not correlate with RFS in the subgroup analyses for patients with or without preoperative therapy (Fig. 4, Table 4). Although patients with grade two histological effects from preoperative therapy had a tendency for better RFS (p = 0.08), MIB-1 index was not significantly related to histological effects of preoperative therapy (Table 2). Fig. 3 View largeDownload slide Relapse-free survival of patients with MIB-1 index >40 and ≦40, showing no significant difference between the two groups. Fig. 3 View largeDownload slide Relapse-free survival of patients with MIB-1 index >40 and ≦40, showing no significant difference between the two groups. Fig. 4 View largeDownload slide Relapse-free survival of patients (a) with preoperative therapy, and (b) without preoperative therapy. Fig. 4 View largeDownload slide Relapse-free survival of patients (a) with preoperative therapy, and (b) without preoperative therapy. Table 1 Patients demographics Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 View Large Table 1 Patients demographics Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 View Large Table 2 Relationship between max MIB-1and clinicopathological factors Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 View Large Table 2 Relationship between max MIB-1and clinicopathological factors Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 View Large Table 3 Relapse-free survival (RFS) according to the clinicopathological factors Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 View Large Table 3 Relapse-free survival (RFS) according to the clinicopathological factors Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 View Large Table 4 Relapsee-free survival (RFS) according to the preoperative therapy Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 View Large Table 4 Relapsee-free survival (RFS) according to the preoperative therapy Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 View Large DISCUSSION This study showed that MIB-1 index did not correlate with clinicopathological factors in patients who underwent R0 resection for esophageal SCC. In addition, there was no relationship between MIB-1 index and RFS. This was the case in subgroup analyses for patients with and without preoperative therapy. Esophagectomy is a standard therapy for EC, and TNM stage is most important in predicting the prognosis of patients. However, some patients have early recurrence or long-term survival unassociated with TMN stage. Therefore, supplementary prognostic parameters have been investigated for precise prognostication. Tumor proliferation is considered a useful parameter for predicting clinical outcomes. One of the simplest methods for determining tumor proliferation is to count mitosis; however, mitotic counts are not completely reliable or reproducible.18 Nuclear Ki-67 antigen is a cell proliferative indicator, and expressed in all cell cycle phases except G0. The MIB-1 monoclonal antibody recognizing Ki-67 antigen was first discovered as a positive cell marker for proliferation in 1983 by Gerdes et al.1 Approximately 20 years ago, MIB-1 monoclonal antibody was first applied to paraffin-embedded tissue sections fixed in formalin, and MIB-1 index was regarded as a cell proliferative indicator.1,19 Many studies have investigated MIB-1 index with clinicopathological factors and prognosis in patients with not only EC but also in patients with lung, breast, colorectal, and gastric cancers; however, the results were inconsistent.20–27 Lam et al. showed that patients with stage III EC with MIB-1 index <300 per 1000 cells had longer survival rates than those with a score of 300 or above per 1000 cells.3 Likewise, Youssef et al. showed a significant correlation between MIB-1 index and overall survival (OS) in patients with esophageal SCC.4 On the contrary, Sarbia et al. showed that MIB-1 index had no correlation with depth of tumor invasion, lymph node metastases, tumor size, tumor grade, lymphatic or venous invasion, and OS in patients with esophageal SCC.5 The inconsistency of results among the various studies may be attributed to several factors. First, various methodologies were used for the different studies. In particular, each study used a different site for evaluating MIB-1 index. Sarbia et al. counted a minimum of 1000 nuclei per tumor in areas with the highest proliferative activity.5 Miyazaki et al. calculated MIB-1 index at the invasive front of the tumor in three consecutive high power fields.14 Kawamura et al. evaluated MIB-1 index at the site of the maximum number of positive nuclei in the tumors.6 Additionally, Chino et al. calculated MIB-1 index at the invasive tip or the nest of the tumor.7 The esophageal cancer usually undergoes differentiation and keratinization toward the center of the tumor focus, accordingly, the growth edge of the cancer tends to show high proliferative activity. Hence, it is necessary to consider the selected area used to count MIB-1 index while comparing study results. Second, MIB-1 index is usually high around the basal layer of the normal squamous epithelium. This may explain why MIB-1 index did not correlate with depth of tumor invasion in this study (Table 2), or in other studies.5,14 Third, proliferation rate comprises two parameters: (1) the fraction of proliferating cells assessable by MIB-1, and (2) cell cycle time not assessable by MIB-1. Hence, MIB-1 does not reflect full cell proliferation. Finally, EC frequently shows heterogeneous proliferation. Consequently, when MIB-1 index is evaluated in certain areas of a tumor, it does not reflect the entire tumor proliferation. Some studies have reported that MIB-1 index was significantly higher in the cases responding to preoperative therapy than in those failing to respond to preoperative therapy.28–30 In addition, other previous studies have reported that tumor degeneration grade was a significant prognostic factor in patients who underwent preoperative therapy.31–37 Our study revealed that histological effects had a marginal influence on RFS in the patients who had undertaken preoperative therapy. However, there was no correlation between histological effects and MIB-1 index, suggesting that MIB-1 index may be useful in evaluating proliferation rate of a particular area of a tumor but not the entire tumor. We acknowledge our study had some limitations. First, the study comprised of a limited number of patients and therefore, the statistical significance of the results may be relatively poor. Second, our study included patients with esophageal SCC, but did not include patients with adenocarcinoma. Consequently, interpretation and application of the results may be limited, especially in the western population. In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for patients undergoing R0 resection for esophageal SCC with or without preoperative therapy. Further study is needed to evaluate the prognostic use of MIB-1 index and to standardize MIB-1 index scoring method. Notes Specific author contributions: All authors of this research paper have directly participated in the planning, execution of this study. Norihiro Yuasa and Ayami Kiriyama performed collection of the data. Hideo Miyake, Eiji Takeuchi, Hidemasa Nagai, Masahumi Ito, and Kanji Miyata supervised this study. All authors of this paper have read and approved the final version submitted. References 1 Gerdes J , Schwab U , Lemke H , Stein H . Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation . Int J Cancer 1983 ; 31 : 13 – 20 . Google Scholar CrossRef Search ADS PubMed 2 Gerdes J , Li L , Schlueter C et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67 . Am J Pathol 1991 ; 138 : 867 – 73 . Google Scholar PubMed 3 Lam K Y , Law S Y , So M K , Fok M , Ma L T , Wong J . Prognostic implication of proliferative markers MIB-1 and PC10 in esophageal squamous cell carcinoma . Cancer 1996 ; 77 : 7 – 13 . Google Scholar CrossRef Search ADS PubMed 4 Youssef E M , Matsuda T , Takada N et al. Prognostic significance of the MIB-1 proliferation index for patients with squamous cell carcinoma of the esophagus . Cancer 1995 ; 76 : 358 – 66 . Google Scholar CrossRef Search ADS PubMed 5 Sarbia M , Bittinger F , Porschen R et al. The prognostic significance of tumour cell proliferation in squamous cell carcinomas of the oesophagus . Br J Cancer 1996 ; 74 : 1012 – 6 . 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Relationship between proliferative activity of cancer cells and clinicopathological factors in patients with esophageal squamous cell carcinoma . World J Gastroenterol 2005 ; 11 : 2956 – 9 . Google Scholar CrossRef Search ADS PubMed 10 Chen M , Huang J , Zhu Z , Zhang J , Li K . Systematic review and meta-analysis of tumor biomarkers in predicting prognosis in esophageal cancer . BMC Cancer 2013 ; 13 : 539 . Google Scholar CrossRef Search ADS PubMed 11 Ikeda G , Isaji S , Chandra B , Watanabe M , Kawarada Y . Prognostic significance of biologic factors in squamous cell carcinoma of the esophagus . Cancer 1999 ; 86 : 1396 – 405 . Google Scholar CrossRef Search ADS PubMed 12 Chino O , Makuuchi H , Shimada H , Machimura T , Mitomi T , Osamura R Y . Assessment of the proliferative activity of superficial esophageal carcinoma using MIB-1 immunostaining for the Ki-67 antigen . J Surg Oncol 1998 ; 67 : 18 – 24 . Google Scholar CrossRef Search ADS PubMed 13 Ikeguchi M , Saito H , Katano K , Tsujitani S , Maeta M , Kaibara N . Correlation between the lymphocytic infiltration of tumors and the proliferative activity of cancer cells from surgically treated esophageal carcinoma . Oncology 1997 ; 54 : 311 – 7 . Google Scholar CrossRef Search ADS PubMed 14 Miyazaki T , Kato H , Kimura H et al. Evaluation of tumor malignancy in esophageal squamous cell carcinoma using different characteristic factors . Anticancer Res 2005 ; 25 : 4005 – 11 . Google Scholar PubMed 15 Ando N , Kato H , Igaki H et al. A randomized trial comparing postoperative adjuvant chemotherapy with cisplatin and 5-fluorouracil versus preoperative chemotherapy for localized advanced squamous cell carcinoma of the thoracic esophagus (JCOG9907) . Ann Surg Oncol 2012 ; 19 : 68 – 74 . Google Scholar CrossRef Search ADS PubMed 16 Sobin LH , Gospodarowicz MK , Wittekind C , (eds). TNM Classification of Malignant Tumours , 7th edn . Chichester : Wiley , 2009 . 17 Japan Esophageal Society . Japanese Classification of Esophageal Cancer, 11th edn . Esophagus 2017 ; 14 : 1 – 65 . CrossRef Search ADS PubMed 18 Quinn C M , Wright N A . The clinical assessment of proliferation and growth in human tumours: evaluation of methods and applications as prognostic variables . J Pathol 1990 ; 160 : 93 – 102 . Google Scholar CrossRef Search ADS PubMed 19 Weidner N , Moore D H 2nd , Vartanian R . Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel "paraffin"-reactive MIB1 antibody . Hum Pathol 1994 ; 25 : 337 – 42 . Google Scholar CrossRef Search ADS PubMed 20 Scagliotti G V , Micela M , Gubetta L et al. Prognostic significance of Ki67 labelling in resected nonsmall cell lung cancer . Eur J Cancer 1993 ; 29A : 363 – 5 . Google Scholar CrossRef Search ADS PubMed 21 Ji Y , Zheng M , Ye S , Chen J , Chen Y . PTEN and Ki67 expression is associated with clinicopathologic features of non-small cell lung cancer . J Biomed Res 2014 ; 28 : 462 – 7 . Google Scholar PubMed 22 Gnant M , Harbeck N , Thomssen C . St. Gallen 2011: Summary of the Consensus Discussion . Breast Care (Basel) 2011 ; 6 : 136 – 41 . Google Scholar CrossRef Search ADS PubMed 23 Untch M , Gerber B , Harbeck N et al. 13th St. Gallen international breast cancer conference 2013: primary therapy of early breast cancer evidence, controversies, consensus - opinion of a German team of experts (zurich 2013) . Breast Care (Basel) 2013 ; 8 : 221 – 9 . Google Scholar PubMed 24 Melling N , Kowitz C M , Simon R et al. High Ki67 expression is an independent good prognostic marker in colorectal cancer . J Clin Pathol 2016 ; 69 : 209 – 14 . Google Scholar CrossRef Search ADS PubMed 25 Ma Y L , Peng J Y , Zhang P , Liu W J , Huang L , Qin H L . Immunohistochemical analysis revealed CD34 and Ki67 protein expression as significant prognostic factors in colorectal cancer . Med Oncol 2010 ; 27 : 304 – 9 . Google Scholar CrossRef Search ADS PubMed 26 Böger C , Behrens H M , Röcken C . Ki67-an unsuitable marker of gastric cancer prognosis unmasks intratumoral heterogeneity . J Surg Oncol 2016 ; 113 : 46 – 54 . Google Scholar CrossRef Search ADS PubMed 27 Li N , Deng W , Ma J et al. Prognostic evaluation of Nanog, Oct4, Sox2, PCNA, Ki67, and E-cadherin expression in gastric cancer . Med Oncol 2015 ; 32 : 433 . Google Scholar CrossRef Search ADS PubMed 28 Ressiot E , Dahan L , Liprandi A et al. Predictive factors of the response to chemoradiotherapy in esophageal cancer . Gastroenterol Clin Biol 2008 ; 32 : 567 – 77 . Google Scholar CrossRef Search ADS PubMed 29 Takeuchi H , Ozawa S , Ando N , Kitagawa Y , Ueda M , Kitajima M . Cell-cycle regulators and the Ki-67 labeling index can predict the response to chemoradiotherapy and the survival of patients with locally advanced squamous cell carcinoma of the esophagus . Ann Surg Oncol 2003 ; 10 : 792 – 800 . Google Scholar CrossRef Search ADS PubMed 30 Kitamura K , Saeki H , Kawaguchi H et al. Immunohistochemical status of the p53 protein and Ki-67 antigen using biopsied specimens can predict a sensitivity to neoadjuvant therapy in patients with esophageal cancer . Hepatogastroenterology 2000 ; 47 : 419 – 23 . Google Scholar PubMed 31 Tu C C , Hsu P K , Chien L I et al. Prognostic histological factors in patients with esophageal squamous cell carcinoma after preoperative chemoradiation followed by surgery . BMC Cancer 2017 ; 17 : 62 . Google Scholar CrossRef Search ADS PubMed 32 Meguid R A , Hooker C M , Taylor J T et al. Recurrence after neoadjuvant chemoradiation and surgery for esophageal cancer: does the pattern of recurrence differ for patients with complete response and those with partial or no response? J Thorac Cardiovasc Surg 2009 ; 138 : 1309 – 17 . Google Scholar CrossRef Search ADS PubMed 33 Dittrick G W , Weber J M , Shridhar R et al. Pathologic nonresponders after neoadjuvant chemoradiation for esophageal cancer demonstrate no survival benefit compared with patients treated with primary esophagectomy . Ann Surg Oncol 2012 ; 19 : 1678 – 84 . Google Scholar CrossRef Search ADS PubMed 34 Hölscher A H , Bollschweiler E , Bogoevski D , Schmidt H , Semrau R , Izbicki J R . Prognostic impact of neoadjuvant chemoradiation in cT3 oesophageal cancer—A propensity score matched analysis . Eur J Cancer 2014 ; 50 : 2950 – 7 . Google Scholar CrossRef Search ADS PubMed 35 Meredith K L , Weber J M , Turaga K K et al. Pathologic response after neoadjuvant therapy is the major determinant of survival in patients with esophageal cancer . Ann Surg Oncol 2010 ; 17 : 1159 – 67 . Google Scholar CrossRef Search ADS PubMed 36 Donahue J M , Nichols F C , Li Z et al. Complete pathologic response after neoadjuvant chemoradiotherapy for esophageal cancer is associated with enhanced survival . Ann Thorac Surg 2009 ; 87 : 392 – 8 . Google Scholar CrossRef Search ADS PubMed 37 Berger A C , Farma J , Scott W J et al. Complete response to neoadjuvant chemoradiotherapy in esophageal carcinoma is associated with significantly improved survival . J Clin Oncol 2005 ; 23 : 4330 – 7 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diseases of the Esophagus Oxford University Press

MIB-1 index is unlikely to predict relapse-free survival in patients who underwent R0-esophagectomy for esophageal squamous cell carcinoma

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus.
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1120-8694
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1442-2050
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10.1093/dote/dox145
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Abstract

Summary MIB-1 is a cell proliferation marker and has previously been investigated as a diagnostic or prognostic indicator of malignancy. Previous studies have investigated MIB-1 index and clinicopathological factors in relation to prognosis of patients with esophageal cancer, with conflicting results. The aim of this study is to assess the prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma. A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal squamous cell carcinoma were enrolled in this study. Preoperatively, 29 patients underwent chemotherapy, six underwent chemoradiotherapy, and the remaining did not undergo any preoperative therapy. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The mean MIB-1 index was 39.3 ± 21.0 (range: 0–91.3). There was no significant correlation between clinicopathological factors and MIB-1 index in the study patients, irrespective of whether they underwent preoperative therapy. Univariate analysis revealed no significant association between MIB-1 index and RFS. However, depth of tumor invasion, lymph node metastasis and stage, all showed a significant correlation to RFS. Multivariate analysis of RFS revealed that stage was the only significant factor. Conversely, MIB-1 index was not significantly related to RFS (p = 0.41). In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for esophageal cancer. INTRODUCTION Esophageal cancer (EC) is the eighth most common cancer worldwide. Specifically, in 2012, 3.2% of all new cases of cancer diagnosed were classified as EC (World cancer research fund international. http://www.wcrf.org/int/cancer-facts-figures/worldwide-data). A standard treatment for nonmetastatic EC is curative resection. Accurate prognostication facilitates the identification of patients at a high risk of recurrence. Such patients would benefit from adjuvant therapies and surveillance planning. Evaluation of TNM stage (tumor, nodes, and metastasis) is very important in predicting the prognosis of patients with EC; however, the discovery of a new prognostic parameter could change treatment strategies and follow-up plans. The nuclear antigen, Ki-67, is expressed in all cell cycle phases except G0. MIB-1 index using monoclonal antibody MIB-1 for recognizing the Ki-67 antigen has been investigated as a diagnostic or prognostic indicator of malignant tumors, suggesting cellular proliferation.1,2 Many studies have investigated the correlation between MIB-1 index, clinicopathological factors, and the prognosis of patients with EC; however, the results have been conflicting.3–14 The aim of this study is to assess the clinicopathological and prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma (SCC). MATERIALS AND METHODS A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal SCC between March 2006 and December 2014 were enrolled in this study. The study group comprised of 66 men and 12 women with a mean age of 65.4 ± 7.4 years (range: 45–79 years). Of the 78 tumors, 14 were located in the upper esophagus, 42 were located in the middle esophagus, and 22 were located in the lower esophagus. Preoperative chemotherapy using 5-fluorouracil (5-FU) and cisplatin (CDDP) was administered in 29 patients according to the JCOG9907 study.15 Preoperative chemoradiotherapy using 5-FU, CDDP, and 50–70 Gy, was administered in six patients with cT4 cancer. Surgical approach involved 76 patients undergoing right thoracotomy, one undergoing left thoracotomy and, one undergoing transhiatal esophagectomy. The use of a gastric tube was common for reconstruction (n = 66). Postoperative chemotherapy (5-FU and CDDP) was administered in 20 patients with positive lymph nodes and informed consent for adjuvant chemotherapy was obtained. Immunohistochemistry of Paraffin-embedded sections for MIB-1 (1 : 100 dilution, DAKO, Glostrup, Denmark) was performed by using Bond-MAX (Leica, Microsystem, Newcastle upon Tyne, UK) according to an instruction manual. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Due to the marked heterogeneity of proliferative activity within a tumor, the number of MIB-1 positive cells was counted in the area with the highest proliferative activity (Figs 1,2). Accordingly, MIB-1 index was commonly determined at the deepest part or the core of the SCC. Fig. 1 View largeDownload slide Immunostaining with monoclonal antibody MIB-1 in a case without preoperative therapy, staged T3, N0, M0, and II showing MIB-1 index 37.0%. Figure (a) loupe image, (b) magnified view (×20) of a closed square in Figure (a). Fig. 1 View largeDownload slide Immunostaining with monoclonal antibody MIB-1 in a case without preoperative therapy, staged T3, N0, M0, and II showing MIB-1 index 37.0%. Figure (a) loupe image, (b) magnified view (×20) of a closed square in Figure (a). Fig. 2 View largeDownload slide Hematoxylin and eosin staining and immunostaining with the monoclonal antibody MIB-1 in a case with preoperative therapy, staged T3, N0, M0, and III showing grade 2 histological effects of preoperative therapy and MIB-1 index 74.4%. Figure (a) loupe image, (b) magnified view (×10). Fig. 2 View largeDownload slide Hematoxylin and eosin staining and immunostaining with the monoclonal antibody MIB-1 in a case with preoperative therapy, staged T3, N0, M0, and III showing grade 2 histological effects of preoperative therapy and MIB-1 index 74.4%. Figure (a) loupe image, (b) magnified view (×10). The resected EC were histopathologically classified according to the seventh edition of the international union against cancer/American joint committee on cancer TNM classification.16 Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The clinicopathological factors investigated were age, sex, tumor location, tumor diameter, depth of tumor invasion (T), lymph node metastases (N), lymphatic invasion, venous invasion, stage, and histological differentiation. Histological effects of preoperative therapy were classified according to the 11th edition of the Japanese Classification of Esophageal Cancer.17 Our department follows a standardized surveillance protocol for postoperative patients who have undergone esophagectomy for EC, wherein patients are followed up clinically for a period of 5 years at 3-month intervals. Postoperative data included clinical assessment, laboratory tests, and computed tomography of the chest and abdomen. All evidences of recurrence were obtained from patients’ medical records. Follow-up information through to April 2016 was compiled for all survivors and RFS was calculated from the day treatment was initiated (the day when preoperative treatment began or the date of the esophagectomy), until the date of identification of disease recurrence. The study protocol was approved by the ethics committee of our hospital (No. 2017–047). Statistical analysis The correlation between MIB-1 index and clinicopathological factors was investigated using Mann–Whitney U test, Kruskal–Wallis test, or Spearman's rank correlation coefficient, as appropriate. Survival rates were calculated by the Kaplan–Meier method, and the log-rank test was employed to evaluate differences in RFS between groups in the univariate analysis. Multivariable analysis was performed using a Cox proportional hazard model, in which factors with p < 0.05 in the univariate analysis and MIB-1 index were included. Depth of tumor invasion, lymph node metastases, and stage were integrated to one factor (stage), as they had a close relationship. A p value less than 0.05 was considered to indicate statistical significance. All statistical analyses were performed using the JMP data analysis software, version 10.0 for Windows (SAS Institute Inc., Cary, NC, USA). RESULTS Patient demographics are shown in Table 1. The mean MIB-1 index was 39.3 ± 21.0 (range: 0–91.3). There was no significant correlation between MIB-1 index and clinicopathological factors in the study patients (n = 78), as well as in patients with (n = 35) or without (n = 43) preoperative therapy (Table 2). Five year RFS of the study patients was 58.9% with a median follow-up of 38.9 months (IQR: 17.4–77.5). The results of univariate analysis of RFS according to clinicopathological factors and MIB-1 index are shown in Table 3. When the differences in RFS were examined by bisecting MIB-1 index at an interval between 10 and 80, the difference of RFS between the two groups was widest at a MIB-1 index value of 40. There was no significant difference between MIB-1 index and RFS (p = 0.21, Fig. 3). However, depth of tumor invasion, lymph node metastases, lymphatic invasion, venous invasion, and stage were significantly correlated with RFS. Multivariate analysis of RFS revealed that stage was the only significant factor in determining RFS (Table 3), while MIB-1 index was not significantly related to RFS. MIB-1 index did not correlate with RFS in the subgroup analyses for patients with or without preoperative therapy (Fig. 4, Table 4). Although patients with grade two histological effects from preoperative therapy had a tendency for better RFS (p = 0.08), MIB-1 index was not significantly related to histological effects of preoperative therapy (Table 2). Fig. 3 View largeDownload slide Relapse-free survival of patients with MIB-1 index >40 and ≦40, showing no significant difference between the two groups. Fig. 3 View largeDownload slide Relapse-free survival of patients with MIB-1 index >40 and ≦40, showing no significant difference between the two groups. Fig. 4 View largeDownload slide Relapse-free survival of patients (a) with preoperative therapy, and (b) without preoperative therapy. Fig. 4 View largeDownload slide Relapse-free survival of patients (a) with preoperative therapy, and (b) without preoperative therapy. Table 1 Patients demographics Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 View Large Table 1 Patients demographics Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 Total (n = 78) Patients without preoperative therapy (n = 43) Patients with preoperative therapy (n = 35) Age (mean ± SD, range (y.o.)) 65.4 ± 7.4 (45–79) 66.5 ± 8.1 (45–79) 64.1 ± 6.4 (49–76) Sex (M : F) 66 : 12 35 : 8 31 : 4 Location (upper : middle : lower) 14 : 42 : 22 9 : 23 : 11 5 : 19 : 11 Tumor diameter (mean ± SD, range (mm)) 42 ± 23 (5–108) 49 ± 25 (15–108) 33 ± 17 (5–80) Depth of tumor invasion (T1a : T1b : T2 : T3 : T4a) 11 : 18 : 17 : 31 : 1 6 : 12 : 6 : 18 : 1 5 : 6 : 11 : 13 : 0 Lymph node metastasis (N0 : N1 : N2 : N3) 30 : 28 : 13 : 7 18 : 13 : 7 : 5 12 : 15 : 6 : 2 Lymphatic invasion (negative : positive) 29 : 49 13 : 30 16 : 19 Venous invasion (negative : positive) 58 : 20 30 : 13 28 : 7 Stage (I : II : III) 30 : 14 : 34 18 : 8 : 17 12 : 6 : 17 Histological differentiation (well : moderate : poor) 6 : 59 : 12 5 : 29 : 8 1 : 30 : 4 View Large Table 2 Relationship between max MIB-1and clinicopathological factors Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 View Large Table 2 Relationship between max MIB-1and clinicopathological factors Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) n MIB-1 (%) p n MIB-1 (%) p n MIB-1 (%) p Age  <65 38 35.6 ± 20.4 19 33.1 ± 14.3 19 38.1 ± 25.2 0.1317 0.4631 0.3538  ≧65 40 42.8 ± 21.2 24 40.1 ± 24.0 16 46.9 ± 16.1 Sex (M : F)  M 66 40.5 ± 21.4 35 37.4 ± 21.6 31 43.9 ± 21.1 0.2395 0.9627 0.1615  F 12 32.7 ± 17.9 8 34.9 ± 14.9 4 28.2 ± 24.8 Location  Ut 14 36.4 ± 16.7 9 32.3 ± 14.0 5 43.8 ± 20.3  Mt 42 40.2 ± 22.6 0.8484 23 40.3 ± 22.8 0.6544 19 40.0 ± 23.1 0.7756  Lt 22 39.4 ± 20.9 11 33.8 ± 19.8 11 45.0 ± 21.4 Tumor diameter (mm)  <40 41 39.8 ± 20.1 18 36.5 ± 19.9 23 42.3 ± 20.3 0.8225 0.9607 0.9032  ≧40 37 38.7 ± 22.3 25 37.3 ± 21.1 12 41.7 ± 25.1 Depth of tumor invasion  T1a 11 40.2 ± 21.6 6 39.7 ± 20.4 5 40.8 ± 25.4  T1b 18 38.0 ± 16.7 12 37.3 ± 18.3 6 39.5 ± 14.4 0.8232 0.8911 0.6268  T2 17 35.8 ± 27.5 6 34.5 ± 26.2 11 36.5 ± 29.4  T3, T4a 32 41.6 ± 19.7 19 36.7 ± 21.2 13 48.6 ± 15.3 Lymph node metastasis  pN0 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  pN1 28 37.2 ± 20.2 13 35.8 ± 19.7 15 38.5 ± 21.2 0.3016 0.5976 0.3144  pN2 13 31.6 ± 18.5 7 28.0 ± 17.4 6 35.8 ± 20.5  pN3 7 46.2 ± 21.2 5 38.9 ± 19.3 2 64.5 ± 16.8 Lymphatic invasion  Negative 29 41.5 ± 29.5 13 43.6 ± 23.9 16 39.7 ± 25.7 0.4871 0.2091 0.5401  Positive 49 38.0 ± 18.8 30 34.1 ± 18.4 19 44.2 ± 18.2 Venous invasion  Negative 58 39.3 ± 21.3 30 36.8 ± 19.5 28 42.1 ± 23.1 0.9676 0.9053 0.7415  Positive 20 39.1 ± 20.6 13 37.4 ± 23.1 7 42.2 ± 16.4 Stage  I 30 42.9 ± 22.4 18 40.8 ± 22.5 12 46.1 ± 22.8  II 14 31.1 ± 20.6 0.2244 8 36.7 ± 20.0 0.5698 6 23.8 ± 20.6 0.1046  III 34 39.4 ± 19.6 17 33.1 ± 18.6 17 45.8 ± 18.9 Histological differentiation  Well 6 51.7 ± 25.6 5 49.9 ± 28.2 1 60.2  Moderate 59 37.6 ± 20.8 0.2991 29 34.8 ± 18.9 0.2989 30 40.4 ± 22.5 0.4885  Poor 12 39.5 ± 19.1 8 34.0 ± 19.5 4 50.6 ± 14.1 Preoperative therapy  Yes 35 42.1 ± 21.7 0.2837  No 43 37.0 ± 20.4 Histological effects of preoperative therapy  Grade 0, 1 30 44.0 ± 19.1 0.2107  Grade 2 5 30.8 ± 34.1 View Large Table 3 Relapse-free survival (RFS) according to the clinicopathological factors Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 View Large Table 3 Relapse-free survival (RFS) according to the clinicopathological factors Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 Univariate analysis Multivariate analysis 5-year RFS (%) p Hazard ratio 95% confidence interval p Age  <65 (n = 38) 61.3 0.7954  ≧65 (n = 40) 57.1 Sex (M : F)  M (n = 66) 58.5 0.8897  F (n = 12) 59.7 Location  Ut (n = 14) 69.8  Mt (n = 42) 55.2 0.7593  Lt (n = 22) 59.3 Tumor diameter (mm)  <40 (n = 41) 69.6 0.1453  ≧40 (n = 37) 48.5 Depth of tumor invasion  pT1a (n = 11) 100  pT1b (n = 18) 80.9 0.0325  pT2 (n = 17) 44  pT3, T4a (n = 32) 42.9 Lymph node metastasis  pN0 (n = 30) 81.4  pN1 (n = 28) 66.8 0.0001  pN2 (n = 13 8.8  pN3 (n = 7) 42.9 Lymphatic invasion  Negative (n = 29) 86.3 1 0.0035  Positive (n = 49) 43.9 2.474 0.8788139–8.0855 0.0878 Venous invasion  Negative (n = 58) 67.2 1 0.0164  Positive (n = 20) 35.2 1.0062 0.4364–2.3224 0.9882 Stage  I (n = 30) 81.4 1  II (n = 14) 68.8 0.0013 1.0541 0.2516–4.1554 0.9394  III (n = 34) 38.2 3.0004 1.0915–9.7160 0.0325 Histological differentiation  Well (n = 6) 83.3  Moderate (n = 59) 57.9 0.3671  Poor (n = 12) 42.9 Preoperative therapy  Yes (n = 35) 59.7 0.6267  No (n = 43) 58.3 MIB-1  ≦40 (%) (n = 42) 49.7 1 0.2058  >40 (%) (n = 36) 69.6 0.7302 0.3314–1.5308 0.4101 View Large Table 4 Relapsee-free survival (RFS) according to the preoperative therapy Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 View Large Table 4 Relapsee-free survival (RFS) according to the preoperative therapy Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 Patients without preoperative Patients with preoperative therapy (n = 43) therapy (n = 35) 5-year RFS (%) p 5-year RFS (%) p Age  <65 (n = 19) 68.0 (n = 19) 53.4 0.374 0.6274  ≧65 (n = 24) 47.5 (n = 16) 67.7 Sex (M : F)  M (n = 35) 59.0 (n = 31) 58.2 0.6213 0.3098  F (n = 8) 53.6 (n = 4) 75.0 Location  Ut (n = 9) 64.8 (n = 5) 80.0  Mt (n = 23) 64.3 0.6599 (n = 19) 42.2 0.1991  Lt (n = 11) 45.5 (n = 11) 79.5 Tumor diameter (mm)  <40 (n = 18) 74.5 (n = 23) 66.4 0.1056 0.5107  ≧40 (n = 25) 47.6 (n = 12) 48.6 Depth of tumor invasion  pT1a (n = 6) 100.0 (n = 5) 100.0  pT1b (n = 12) 100.0 (n = 6) 44.4 0.0035 0.8105  pT2 (n = 6) 33.3 (n = 11) 53.0  pT3, T4a (n = 19) 36.8 (n = 13) 60.6 Lymph node metastasis  pN0 (n = 18) 77.4 (n = 12) 90.9  pN1 (n = 13) 69.2 (n = 15) 64.2 0.0211 0.0034  pN2 (n = 7) 14.3 (n = 6) 0.0  pN3 (n = 5) 40.0 (n = 2) 50.0 Lymphatic invasion  Negative (n = 13) 80.2 (n = 16) 90.9 0.1195 0.0009  Positive (n = 30) 50.6 (n = 19) 32.5 Venous invasion  Negative (n = 30) 67.9 (n = 28) 66.2 0.0529 0.0654  Positive (n = 13) 38.5 (n = 7) 26.8 Stage  I (n = 18) 77.4 (n = 12) 90.9  II (n = 8) 75.0 0.0195 (n = 6) 60.0 0.0723  III (n = 17) 35.3 (n = 17) 41.6 Histological differentiation  well (n = 5) 80.0 (n = 1) 100  moderate (n = 29) 53.2 0.7017 (n = 30) 62.2 0.0744  poor (n = 8) 48.6 (n = 4) 25.0 Histological effects of preoperative therapy  Grade 0, 1 (n = 30) 53.3 0.0835  Grade 2 (n = 5) 100 MIB-1  ≦40 (%) (n = 26) 46.6 (n = 16) 53.9 0.0984 0.8689  >40 (%) (n = 17) 74.5 (n = 19) 68.4 View Large DISCUSSION This study showed that MIB-1 index did not correlate with clinicopathological factors in patients who underwent R0 resection for esophageal SCC. In addition, there was no relationship between MIB-1 index and RFS. This was the case in subgroup analyses for patients with and without preoperative therapy. Esophagectomy is a standard therapy for EC, and TNM stage is most important in predicting the prognosis of patients. However, some patients have early recurrence or long-term survival unassociated with TMN stage. Therefore, supplementary prognostic parameters have been investigated for precise prognostication. Tumor proliferation is considered a useful parameter for predicting clinical outcomes. One of the simplest methods for determining tumor proliferation is to count mitosis; however, mitotic counts are not completely reliable or reproducible.18 Nuclear Ki-67 antigen is a cell proliferative indicator, and expressed in all cell cycle phases except G0. The MIB-1 monoclonal antibody recognizing Ki-67 antigen was first discovered as a positive cell marker for proliferation in 1983 by Gerdes et al.1 Approximately 20 years ago, MIB-1 monoclonal antibody was first applied to paraffin-embedded tissue sections fixed in formalin, and MIB-1 index was regarded as a cell proliferative indicator.1,19 Many studies have investigated MIB-1 index with clinicopathological factors and prognosis in patients with not only EC but also in patients with lung, breast, colorectal, and gastric cancers; however, the results were inconsistent.20–27 Lam et al. showed that patients with stage III EC with MIB-1 index <300 per 1000 cells had longer survival rates than those with a score of 300 or above per 1000 cells.3 Likewise, Youssef et al. showed a significant correlation between MIB-1 index and overall survival (OS) in patients with esophageal SCC.4 On the contrary, Sarbia et al. showed that MIB-1 index had no correlation with depth of tumor invasion, lymph node metastases, tumor size, tumor grade, lymphatic or venous invasion, and OS in patients with esophageal SCC.5 The inconsistency of results among the various studies may be attributed to several factors. First, various methodologies were used for the different studies. In particular, each study used a different site for evaluating MIB-1 index. Sarbia et al. counted a minimum of 1000 nuclei per tumor in areas with the highest proliferative activity.5 Miyazaki et al. calculated MIB-1 index at the invasive front of the tumor in three consecutive high power fields.14 Kawamura et al. evaluated MIB-1 index at the site of the maximum number of positive nuclei in the tumors.6 Additionally, Chino et al. calculated MIB-1 index at the invasive tip or the nest of the tumor.7 The esophageal cancer usually undergoes differentiation and keratinization toward the center of the tumor focus, accordingly, the growth edge of the cancer tends to show high proliferative activity. Hence, it is necessary to consider the selected area used to count MIB-1 index while comparing study results. Second, MIB-1 index is usually high around the basal layer of the normal squamous epithelium. This may explain why MIB-1 index did not correlate with depth of tumor invasion in this study (Table 2), or in other studies.5,14 Third, proliferation rate comprises two parameters: (1) the fraction of proliferating cells assessable by MIB-1, and (2) cell cycle time not assessable by MIB-1. Hence, MIB-1 does not reflect full cell proliferation. Finally, EC frequently shows heterogeneous proliferation. Consequently, when MIB-1 index is evaluated in certain areas of a tumor, it does not reflect the entire tumor proliferation. Some studies have reported that MIB-1 index was significantly higher in the cases responding to preoperative therapy than in those failing to respond to preoperative therapy.28–30 In addition, other previous studies have reported that tumor degeneration grade was a significant prognostic factor in patients who underwent preoperative therapy.31–37 Our study revealed that histological effects had a marginal influence on RFS in the patients who had undertaken preoperative therapy. However, there was no correlation between histological effects and MIB-1 index, suggesting that MIB-1 index may be useful in evaluating proliferation rate of a particular area of a tumor but not the entire tumor. We acknowledge our study had some limitations. First, the study comprised of a limited number of patients and therefore, the statistical significance of the results may be relatively poor. Second, our study included patients with esophageal SCC, but did not include patients with adenocarcinoma. Consequently, interpretation and application of the results may be limited, especially in the western population. In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for patients undergoing R0 resection for esophageal SCC with or without preoperative therapy. Further study is needed to evaluate the prognostic use of MIB-1 index and to standardize MIB-1 index scoring method. Notes Specific author contributions: All authors of this research paper have directly participated in the planning, execution of this study. Norihiro Yuasa and Ayami Kiriyama performed collection of the data. Hideo Miyake, Eiji Takeuchi, Hidemasa Nagai, Masahumi Ito, and Kanji Miyata supervised this study. All authors of this paper have read and approved the final version submitted. References 1 Gerdes J , Schwab U , Lemke H , Stein H . Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation . Int J Cancer 1983 ; 31 : 13 – 20 . 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Diseases of the EsophagusOxford University Press

Published: Dec 19, 2017

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