Phosphorylated p70S6K expression is an independent prognosticator for patients with esophageal squamous cell carcinoma Shau-Hsuan Li, MD,a Chang-Han Chen, PhD,b Hung-I. Lu, MD,c Wan-Ting Huang, MD,d Wan-Yu Tien, MSc,a Ya-Chun Lan, MSc,a Ching-Chang Lee, MD,e Yen-Hao Chen, MD,a Hsuan-Ying Huang, MD,c Alice Y. W. Chang, PhD,b and Wei-Che Lin, MD, PhD,f Kaohsiung, Taiwan, ROC
Background. Although marked improvements have been made in surgical technique and chemoradiotherapy, the prognosis for patients with esophageal squamous cell carcinoma (ESCC) is still unsatisfactory. The mammalian target of rapamycin (mTOR) and its downstream signaling, p70 ribosomal S6 protein kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), seem to play central roles in the regulation of cancer cell proliferation and survival. The significance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC remains uncertain, but this pathway is of particular concern because effective inhibitors are already available. Methods. Immunohistochemistry performed to evaluate the expression of phosphorylated mTOR (pmTOR), phosphorylated p70S6K (p-p70S6K), phosphorylated 4E-binding protein 1 (p-4E-BP1), and Ki67 using 105 surgically resected ESCC correlated with treatment outcome. The effect of the mTOR signaling pathway inhibitor everolimus on ESCC cell lines were investigated in vitro by the 3-(4.5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and in vivo by a nude mouse xenograft model. Results. Univariate analysis showed that p-mTOR overexpression (P = .022), p-p70S6K overexpression (P = .002), and Ki-67 labeling index >50% (P = .045) were associated with inferior overall survival (OS). In a multivariate comparison, p-p70S6K overexpression (P = .001; hazard ratio, 2.247) remained independently associated with worse OS. In cell lines and the xenograft model, everolimus significantly inhibited ESCC growth. Conclusion. Overexpression of p-p70S6K is associated independently with a poor prognosis among patients with ESCC. The mTOR signaling pathway inhibitor everolimus can inhibit ESCC growth in vitro and in vivo. Our findings suggest that inhibition of mTOR signaling pathway may be a promising novel target for ESCC. (Surgery 2015;157:570-80.) From the Department of Hematology-Oncology,a Center for Translational Research in Biomedical Sciences,b Departments of Thoracic & Cardiovascular Surgery,c Pathology,d and Diagnostic Radiology,f Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine; and the Department of Gastroenterology,e Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, ROC
Conflict of Interest: None declared. This work was supported in part by grants from the National Science Council, Taiwan (NSC 103-2314-B-182A-069-MY3) and Chang Gung Memorial Hospital (CMRPG8B1262). Accepted for publication October 23, 2014. Reprint requests: Shau-Hsuan Li, MD, Department of Hematology-Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niaosung Hsiang, Kaohsiung Hsien, Taiwan, ROC. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.10.014
570 SURGERY
DESPITE IMPROVEMENTS IN SURGICAL TECHNIQUES, perioperative management, and surgery combined with chemotherapy and radiotherapy, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) remains unsatisfactory.1-3 Even after extensive resections, patients continue to develop recurrences.1-3 Therefore, identification of prognostic biomarkers for ESCC could improve risk-adapted treatment strategies and may offer useful insight that aid the development of novel, targeted, therapeutic strategies. The mammalian target of rapamycin (mTOR), also known as FK506 binding protein
Surgery Volume 157, Number 3
12-rapamycin-associated protein 1, is a serine/ threonine protein kinase that supports cell growth, cellular metabolism, cell proliferation, cell motility, cell survival, protein synthesis, and transcription initiating angiogenesis and autophagy.4 mTOR is found in 2 different complexes within the cell, mTORC1 and mTORC2. Currently, the best-understood roles of mTOR are those mediated by mTORC1, which is sensitive to rapamycin.4 Rapamycin exerts its biologic effect by binding to FK506-binding protein-12; this complex then binds to mTOR, inhibiting its kinase activity and subsequent signaling to downstream targets.5 mTORC1 is activated by phosphorylation of Ser2448 through the phosphatidylinositol 3-kinase/AKT signaling pathway and receives inputs from a variety of biochemical pathways, including hypoxia, changes in intracellular energy, growth factor signaling molecules, hormones, and cytokines.4 The best-described targets for mTOR are its downstream markers p70 ribosomal S6 protein kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). p70S6K, a mitogen-activated Ser/Thr protein kinase, plays an important role in the regulation of cell growth, G1 cell-cycle progression, and cell proliferation by enhancing the translation of components required for protein synthesis. This kinase is controlled by multiple phosphorylation events located within the catalytic, linker, and pseudosubstrate domains and subsequently phosphorylates specifically ribosomal protein S6. Phosphorylation of Thr389 in the linker domain is critical for kinase function.6 Inhibition of p70S6K activity inhibits the entry into S phase of the cell cycle and exhibits cell-cycle arrest at G0/G1 phase, suggesting that the activation of p70S6K plays an obligatory role in mediating mitogenic signals during cell activation.6 The mammalian eIF4E-binding proteins 1, 4EBP1, harbor a conserved eIF4E-binding motif and phosphorylation sites critical to the regulation of cap-dependent translation. eIF4E is the first translation factor shown to cause malignant transformation when overexpressed in cultured rodent fibroblasts.7 eIF4E may transform cells by promoting the translation of specific mRNAs, such as hypoxia-inducible factor-1 alpha, vascular endothelial growth factor, and cyclin D1.8 These downstream targets are responsible for the activation of various intracellular processes, leading to the activation of cell proliferation and induction of angiogenesis or metastasis. eIF4E levels in the cell are controlled tightly by the translational
Li et al 571
repressors, 4E-BP1, with assembly of the eIF4E initiation complex being inhibited by 4E-BP1. Hypophosphorylated 4E-BP1 interacts strongly with eIF4E, whereas the hyperphosphorylated form binds weakly.9 Previous studies on several types of cancers have shown that the aberrant activation of mTOR and its downstream signaling, p70S6K and 4E-BP1, is associated with poor prognosis in cancer patients.4,9 The mTOR signaling pathway inhibitors and rapamycin analogs such as Everolimus (Novartis, Basel, Switzerland) and Temsirolimus (Wyeth-Ayerst, Radnor, PA), display potential activity against a wide panel of cancers in preclinical and clinical settings.9,10 The clinical importance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC remains largely undefined, but this pathway is of particular concern, because effective inhibitors are already available. The aim of this study is to evaluate the significance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC. MATERIALS AND METHODS Patient population. Patients with ESCC who underwent surgical resection at Kaohsiung Chang Gung Memorial Hospital were reviewed retrospectively. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Patients with synchronous cancers in other organs and patients receiving preoperative chemoradiotherapy, preoperative chemotherapy, or preoperative radiotherapy were excluded. We identified 105 patients with available paraffin blocks and followup. Patients underwent a radical esophagectomy with cervical esophagogastric anastomosis (McKeown procedure) or an Ivor Lewis esophagectomy with intrathoracic anastomosis, reconstruction of the digestive tract with gastric tube, and pylorus drainage procedures. Two-field lymph node dissection was performed in all patients. The pathologic TNM stage was determined according to the 7th American Joint Committee on Cancer (AJCC) staging system.11 After the operation, patients were followed at 3-month intervals for 2 years, 6-month intervals up to year 5, and annually thereafter. Overall survival (OS) was calculated from the time of operation to death as a result of all causes. Disease-free survival (DFS) was computed from the time of operation to the recurrence or death from any cause without evidence of recurrence. Immunohistochemistry. Immunohistochemical (IHC) staining was performed using an immunoperoxidase technique. Staining was performed on
572 Li et al
slides (4 mm) of formalin-fixed, paraffinembedded tissue sections with primary antibodies against p-mTOR (Ser2448, Clone 49F9, 1:50, Cell Signaling Technology, Boston, MA, USA), p-p70S6K (Thr389, Clone 1A5, 1:75, Cell Signaling Technology), p-4E-BP1 (Thr37/46, Clone 236B4, 1:150, Cell Signaling Technology), and Ki-67 (MIB1, 1:100; Dako, Glostrup, Denmark). Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (958C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for $18 hours at 48C overnight. Immunodetection was performed using the LSAB2 kit (Dako, Carpinteria, CA) followed by 3-39-diaminobenzidine for color development and hematoxylin for counterstaining. For p-mTOR, incubation without the primary antibody and normal esophageal squamous epithelium12 were used as a negative control, and normal submucosa and gastric glands12 were used as positive controls. For p-p70S6K and p-4EBP1, incubation without the primary antibody was used as a negative control, and breast carcinoma13 known for its p-p70S6K and p-4E-BP1 positivity was used as a positive control. The staining assessment was carried out independently by 2 pathologists (S.L.W. and W.T.H.) blinded to the clinicopathologic features or prognosis. We followed the previously published method to score the expression of p-mTOR, p-p70S6K, and p-4E-BP1.12,14,15 The fraction of tumor cells with noticeable staining (0%–100%) was recorded, and the average value of 2 pathologists was calculated in each patient. Whenever the 2 pathologists disagreed (eg, difference >10% or discordant scores), the slides were reevaluated and discussed, and a consensus was reached by 2 pathologists. IHC expression levels of p-mTOR, p-p70S6K, and p-4E-BP1were scored using the following 3-tier system: score 0, 50% were identified (for p-mTOR) in 59 (56%), including 30 with IHC score 1 and 29 with IHC score 2; 61 (58%) patients (for p-p7056K), including 32 with IHC score 1 and 29 with IHC score 2; 47 (45%) patients (for p-4EBPI), including 24 patients with IHC score 1 and 23 patients with IHC score 2; and 50 (48%) patients (for Ki-67; Fig 1). The correlations between clinicopathologic parameters and IHC expression of p-mTOR, p-p70S6K, p-4E-BP1, and Ki-67 labeling index are summarized in Table I. Expression of p-mTOR was associated with p-p70S6K expression (P < .001), and Ki-67 labeling index (P = .007). Expression of p-p70S6K was associated with N stage (P = .039), p-4E-BP1 expression (P = .008), and Ki-67 labeling index (P < .001); in
574 Li et al
Surgery March 2015
Fig 1. Immunohistochemical staining of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated p70S6K (p-p70S6K), phosphorylated 4E-binding protein 1 (p-4E-BP1), and Ki-67. (A) Low expression of p-mTOR. (B) Overexpression of p-mTOR. (C) Low expression of p-p70S6K. (D) Overexpression of p-p70S6K. (E) Low expression of p-4E-BP1. (F) Overexpression of p-4E-BP1. (G) Low expression of Ki-67. (H) Overexpression of Ki-67. Original magnification, 3200.
contrast, p-mTOR expression was not associated with p-4E-BP1 expression. Survival analyses. Correlations of clinicopathologic parameters, p-mTOR, p-p70S6K, p-4E-BP1, and Ki-67 labeling index with OS and DFS are shown in Table II. Univariate analyses demonstrated that 7th AJCC stage III+IV (P < .001), T3+4 disease (P < .001), positive regional lymph
node (P < .001), grade 3 (P = .003), positive surgical margin (P = .009), p-mTOR overexpression (P = .022, Fig 2, A), p-p70S6K overexpression (P = .002, Fig 2, B), and Ki-67 labeling index >50% (P = .045) were associated with inferior OS. Additionally, 7th AJCC stage III+IV (P < .001), T3+4 disease (P = .003), positive regional lymph node (P < .001), upper third
Li et al 575
Surgery Volume 157, Number 3
Table I. Associations between immunohistochemical markers and clinicopathologic factors p-mTOR expression Parameters
Low
Age (y) 50% 15
p-p70S6K expression
p-4E-BP1expression
Ki-67 labeling index
Over
P value
Low
Over
P value
Low
Over
P value
#50%
>50%
P value
32 27
.08
19 25
30 31
.54
28 30
21 26
.71
26 29
23 27
.90
59 0
.19
43 1
60 1
.99
58 0
45 2
.20
53 2
50 0
.50
25 34
.74
19 25
24 37
.69
21 37
22 25
.27
23 32
20 30
.85
26 33
.21
27 17
25 36
.039
27 31
25 22
.50
30 25
22 28
.28
24 35
.24
24 20
24 37
.12
24 34
24 23
.32
27 28
21 29
.47
44 15
.86
32 12
47 14
.61
45 13
34 13
.54
39 16
40 10
.28
8 51
.34
3 41
8 53
.35
8 50
3 44
.34
4 51
7 43
.26
44 15
.12
37 7
47 14
.37
48 10
36 11
.43
12 43
9 41
.63
50 9
.51
36 8
55 6
.22
49 9
42 5
.47
48 7
43 7
.85
13 46
Background. Although marked improvements have been made in surgical technique and chemoradiotherapy, the prognosis for patients with esophageal squamous cell carcinoma (ESCC) is still unsatisfactory. The mammalian target of rapamycin (mTOR) and its downstream signaling, p70 ribosomal S6 protein kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), seem to play central roles in the regulation of cancer cell proliferation and survival. The significance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC remains uncertain, but this pathway is of particular concern because effective inhibitors are already available. Methods. Immunohistochemistry performed to evaluate the expression of phosphorylated mTOR (pmTOR), phosphorylated p70S6K (p-p70S6K), phosphorylated 4E-binding protein 1 (p-4E-BP1), and Ki67 using 105 surgically resected ESCC correlated with treatment outcome. The effect of the mTOR signaling pathway inhibitor everolimus on ESCC cell lines were investigated in vitro by the 3-(4.5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and in vivo by a nude mouse xenograft model. Results. Univariate analysis showed that p-mTOR overexpression (P = .022), p-p70S6K overexpression (P = .002), and Ki-67 labeling index >50% (P = .045) were associated with inferior overall survival (OS). In a multivariate comparison, p-p70S6K overexpression (P = .001; hazard ratio, 2.247) remained independently associated with worse OS. In cell lines and the xenograft model, everolimus significantly inhibited ESCC growth. Conclusion. Overexpression of p-p70S6K is associated independently with a poor prognosis among patients with ESCC. The mTOR signaling pathway inhibitor everolimus can inhibit ESCC growth in vitro and in vivo. Our findings suggest that inhibition of mTOR signaling pathway may be a promising novel target for ESCC. (Surgery 2015;157:570-80.) From the Department of Hematology-Oncology,a Center for Translational Research in Biomedical Sciences,b Departments of Thoracic & Cardiovascular Surgery,c Pathology,d and Diagnostic Radiology,f Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine; and the Department of Gastroenterology,e Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, ROC
Conflict of Interest: None declared. This work was supported in part by grants from the National Science Council, Taiwan (NSC 103-2314-B-182A-069-MY3) and Chang Gung Memorial Hospital (CMRPG8B1262). Accepted for publication October 23, 2014. Reprint requests: Shau-Hsuan Li, MD, Department of Hematology-Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niaosung Hsiang, Kaohsiung Hsien, Taiwan, ROC. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.10.014
570 SURGERY
DESPITE IMPROVEMENTS IN SURGICAL TECHNIQUES, perioperative management, and surgery combined with chemotherapy and radiotherapy, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) remains unsatisfactory.1-3 Even after extensive resections, patients continue to develop recurrences.1-3 Therefore, identification of prognostic biomarkers for ESCC could improve risk-adapted treatment strategies and may offer useful insight that aid the development of novel, targeted, therapeutic strategies. The mammalian target of rapamycin (mTOR), also known as FK506 binding protein
Surgery Volume 157, Number 3
12-rapamycin-associated protein 1, is a serine/ threonine protein kinase that supports cell growth, cellular metabolism, cell proliferation, cell motility, cell survival, protein synthesis, and transcription initiating angiogenesis and autophagy.4 mTOR is found in 2 different complexes within the cell, mTORC1 and mTORC2. Currently, the best-understood roles of mTOR are those mediated by mTORC1, which is sensitive to rapamycin.4 Rapamycin exerts its biologic effect by binding to FK506-binding protein-12; this complex then binds to mTOR, inhibiting its kinase activity and subsequent signaling to downstream targets.5 mTORC1 is activated by phosphorylation of Ser2448 through the phosphatidylinositol 3-kinase/AKT signaling pathway and receives inputs from a variety of biochemical pathways, including hypoxia, changes in intracellular energy, growth factor signaling molecules, hormones, and cytokines.4 The best-described targets for mTOR are its downstream markers p70 ribosomal S6 protein kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). p70S6K, a mitogen-activated Ser/Thr protein kinase, plays an important role in the regulation of cell growth, G1 cell-cycle progression, and cell proliferation by enhancing the translation of components required for protein synthesis. This kinase is controlled by multiple phosphorylation events located within the catalytic, linker, and pseudosubstrate domains and subsequently phosphorylates specifically ribosomal protein S6. Phosphorylation of Thr389 in the linker domain is critical for kinase function.6 Inhibition of p70S6K activity inhibits the entry into S phase of the cell cycle and exhibits cell-cycle arrest at G0/G1 phase, suggesting that the activation of p70S6K plays an obligatory role in mediating mitogenic signals during cell activation.6 The mammalian eIF4E-binding proteins 1, 4EBP1, harbor a conserved eIF4E-binding motif and phosphorylation sites critical to the regulation of cap-dependent translation. eIF4E is the first translation factor shown to cause malignant transformation when overexpressed in cultured rodent fibroblasts.7 eIF4E may transform cells by promoting the translation of specific mRNAs, such as hypoxia-inducible factor-1 alpha, vascular endothelial growth factor, and cyclin D1.8 These downstream targets are responsible for the activation of various intracellular processes, leading to the activation of cell proliferation and induction of angiogenesis or metastasis. eIF4E levels in the cell are controlled tightly by the translational
Li et al 571
repressors, 4E-BP1, with assembly of the eIF4E initiation complex being inhibited by 4E-BP1. Hypophosphorylated 4E-BP1 interacts strongly with eIF4E, whereas the hyperphosphorylated form binds weakly.9 Previous studies on several types of cancers have shown that the aberrant activation of mTOR and its downstream signaling, p70S6K and 4E-BP1, is associated with poor prognosis in cancer patients.4,9 The mTOR signaling pathway inhibitors and rapamycin analogs such as Everolimus (Novartis, Basel, Switzerland) and Temsirolimus (Wyeth-Ayerst, Radnor, PA), display potential activity against a wide panel of cancers in preclinical and clinical settings.9,10 The clinical importance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC remains largely undefined, but this pathway is of particular concern, because effective inhibitors are already available. The aim of this study is to evaluate the significance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC. MATERIALS AND METHODS Patient population. Patients with ESCC who underwent surgical resection at Kaohsiung Chang Gung Memorial Hospital were reviewed retrospectively. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Patients with synchronous cancers in other organs and patients receiving preoperative chemoradiotherapy, preoperative chemotherapy, or preoperative radiotherapy were excluded. We identified 105 patients with available paraffin blocks and followup. Patients underwent a radical esophagectomy with cervical esophagogastric anastomosis (McKeown procedure) or an Ivor Lewis esophagectomy with intrathoracic anastomosis, reconstruction of the digestive tract with gastric tube, and pylorus drainage procedures. Two-field lymph node dissection was performed in all patients. The pathologic TNM stage was determined according to the 7th American Joint Committee on Cancer (AJCC) staging system.11 After the operation, patients were followed at 3-month intervals for 2 years, 6-month intervals up to year 5, and annually thereafter. Overall survival (OS) was calculated from the time of operation to death as a result of all causes. Disease-free survival (DFS) was computed from the time of operation to the recurrence or death from any cause without evidence of recurrence. Immunohistochemistry. Immunohistochemical (IHC) staining was performed using an immunoperoxidase technique. Staining was performed on
572 Li et al
slides (4 mm) of formalin-fixed, paraffinembedded tissue sections with primary antibodies against p-mTOR (Ser2448, Clone 49F9, 1:50, Cell Signaling Technology, Boston, MA, USA), p-p70S6K (Thr389, Clone 1A5, 1:75, Cell Signaling Technology), p-4E-BP1 (Thr37/46, Clone 236B4, 1:150, Cell Signaling Technology), and Ki-67 (MIB1, 1:100; Dako, Glostrup, Denmark). Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (958C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for $18 hours at 48C overnight. Immunodetection was performed using the LSAB2 kit (Dako, Carpinteria, CA) followed by 3-39-diaminobenzidine for color development and hematoxylin for counterstaining. For p-mTOR, incubation without the primary antibody and normal esophageal squamous epithelium12 were used as a negative control, and normal submucosa and gastric glands12 were used as positive controls. For p-p70S6K and p-4EBP1, incubation without the primary antibody was used as a negative control, and breast carcinoma13 known for its p-p70S6K and p-4E-BP1 positivity was used as a positive control. The staining assessment was carried out independently by 2 pathologists (S.L.W. and W.T.H.) blinded to the clinicopathologic features or prognosis. We followed the previously published method to score the expression of p-mTOR, p-p70S6K, and p-4E-BP1.12,14,15 The fraction of tumor cells with noticeable staining (0%–100%) was recorded, and the average value of 2 pathologists was calculated in each patient. Whenever the 2 pathologists disagreed (eg, difference >10% or discordant scores), the slides were reevaluated and discussed, and a consensus was reached by 2 pathologists. IHC expression levels of p-mTOR, p-p70S6K, and p-4E-BP1were scored using the following 3-tier system: score 0, 50% were identified (for p-mTOR) in 59 (56%), including 30 with IHC score 1 and 29 with IHC score 2; 61 (58%) patients (for p-p7056K), including 32 with IHC score 1 and 29 with IHC score 2; 47 (45%) patients (for p-4EBPI), including 24 patients with IHC score 1 and 23 patients with IHC score 2; and 50 (48%) patients (for Ki-67; Fig 1). The correlations between clinicopathologic parameters and IHC expression of p-mTOR, p-p70S6K, p-4E-BP1, and Ki-67 labeling index are summarized in Table I. Expression of p-mTOR was associated with p-p70S6K expression (P < .001), and Ki-67 labeling index (P = .007). Expression of p-p70S6K was associated with N stage (P = .039), p-4E-BP1 expression (P = .008), and Ki-67 labeling index (P < .001); in
574 Li et al
Surgery March 2015
Fig 1. Immunohistochemical staining of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated p70S6K (p-p70S6K), phosphorylated 4E-binding protein 1 (p-4E-BP1), and Ki-67. (A) Low expression of p-mTOR. (B) Overexpression of p-mTOR. (C) Low expression of p-p70S6K. (D) Overexpression of p-p70S6K. (E) Low expression of p-4E-BP1. (F) Overexpression of p-4E-BP1. (G) Low expression of Ki-67. (H) Overexpression of Ki-67. Original magnification, 3200.
contrast, p-mTOR expression was not associated with p-4E-BP1 expression. Survival analyses. Correlations of clinicopathologic parameters, p-mTOR, p-p70S6K, p-4E-BP1, and Ki-67 labeling index with OS and DFS are shown in Table II. Univariate analyses demonstrated that 7th AJCC stage III+IV (P < .001), T3+4 disease (P < .001), positive regional lymph
node (P < .001), grade 3 (P = .003), positive surgical margin (P = .009), p-mTOR overexpression (P = .022, Fig 2, A), p-p70S6K overexpression (P = .002, Fig 2, B), and Ki-67 labeling index >50% (P = .045) were associated with inferior OS. Additionally, 7th AJCC stage III+IV (P < .001), T3+4 disease (P = .003), positive regional lymph node (P < .001), upper third
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Surgery Volume 157, Number 3
Table I. Associations between immunohistochemical markers and clinicopathologic factors p-mTOR expression Parameters
Low
Age (y) 50% 15
p-p70S6K expression
p-4E-BP1expression
Ki-67 labeling index
Over
P value
Low
Over
P value
Low
Over
P value
#50%
>50%
P value
32 27
.08
19 25
30 31
.54
28 30
21 26
.71
26 29
23 27
.90
59 0
.19
43 1
60 1
.99
58 0
45 2
.20
53 2
50 0
.50
25 34
.74
19 25
24 37
.69
21 37
22 25
.27
23 32
20 30
.85
26 33
.21
27 17
25 36
.039
27 31
25 22
.50
30 25
22 28
.28
24 35
.24
24 20
24 37
.12
24 34
24 23
.32
27 28
21 29
.47
44 15
.86
32 12
47 14
.61
45 13
34 13
.54
39 16
40 10
.28
8 51
.34
3 41
8 53
.35
8 50
3 44
.34
4 51
7 43
.26
44 15
.12
37 7
47 14
.37
48 10
36 11
.43
12 43
9 41
.63
50 9
.51
36 8
55 6
.22
49 9
42 5
.47
48 7
43 7
.85
13 46
