Clinical Translational Research Received: January 8, 2014 Accepted after revision: January 23, 2014 Published online: June 4, 2014
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Epidermal Growth Factor Receptor-GEP100-Arf6 Axis Affects the Prognosis of Lung Adenocarcinoma Soichi Oka a Hidetaka Uramoto a Hidehiko Shimokawa a Sohsuke Yamada b Fumihiro Tanaka a a
Second Department of Surgery and b Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
Key Words Lung cancer · Adenocarcinoma · Epidermal growth factor receptor · GEP100 · Arf6 · Prognosis
the prognosis of patients with primary lung adenocarcinoma. The combination of p-EGFR, GEP100, and Arf6 staining can predict the prognosis of patients after surgery. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0030–2414/14/0866–0263$39.50/0 E-Mail [email protected] www.karger.com/ocl
Introduction
Lung cancer is the leading cause of cancer-related death in the majority of countries worldwide [1], and the proportion of adenocarcinoma is notably increasing [2]. TNM staging indicates the level of disease progression and the malignant potential of primary lung cancer [3]. However, even patients with disease at the same stage exhibit wide variations in their prognosis after curative resection. Therefore, the current TNM staging system, which is based on clinical and pathological findings, may have reached the limit of its usefulness [4]. Accurately predicting the cases in which disease is likely to recur can help guide the administration of adjuvant therapies, not only to those most likely to benefit from them, but also to prevent harmful treatment [4, 5]. In addition to the TNM classification, tumor markers, such as CEA, have proven to be independent factors predicting the risk of recurrence as clinical parameters [6, 7]. Despite a great deal of Hidetaka Uramoto Second Department of Surgery, School of Medicine University of Occupational and Environmental Health 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan) E-Mail hidetaka @ med.uoeh-u.ac.jp
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
Abstract The overexpression of Arf6 and GEP100 is responsible for the invasive activity that is crucial for the activation of the epidermal growth factor receptor (EGFR) signaling pathways in human cancer. However, whether or not the expression of the EGFR-GEP100-Arf6 axis can be used as a biomarker for the prognosis of lung cancer has yet to be fully determined. Tumor specimens were collected from 182 patients who underwent a complete resection for lung adenocarcinoma. We analyzed phospho-EGFR (p-EGFR), GEP100, and Arf6 expression levels in the primary tumor by immunohistochemical analysis. The expression of p-EGFR, GEP100, and Arf6 was observed in 65 (35.7%), 95 (52.2%), and 20 (11.0%) patients, respectively. Significant associations between p-EGFR and GEP100 expression and vessel invasion were identified. The expression of these individual molecules was not associated with any statistically significant differences in survival. However, triple positive expression of p-EGFR, GEP100, and Arf6 was significantly associated with an increased risk of death based on the multivariate analysis. The EGFR-GEP100-Arf6 axis affected
Patients and Methods
vals thereafter. The evaluations included a physical examination, chest roentgenography, analysis of blood chemical parameters, and measurements of tumor marker levels. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected. A follow-up was conducted in all patients. The median follow-up period was 41.3 months. Twenty-eight (15.4%) patients received adjuvant chemotherapy as follows: carboplatin plus paclitaxel (n = 18), carboplatin plus gemcitabine (n = 7), and tegafur-uracil (n = 3) [5, 15]. At the last follow-up examination, 152 patients were alive and free of cancer, 10 patients had died of other causes without evidence of cancer, 12 patients were alive with recurrent cancer, and 17 patients had died of cancer. In total, 28 (15.4%) of the 182 patients demonstrated disease recurrence after surgery. The majority of the sites of tumor recurrence were hematogenous metastases. Twentyfive and 6 cases had hematogenous (brain = 9, lung = 10, bone = 5, and adrenal metastasis = 1) and locoregional (lymph node metastasis = 4, and pleural dissemination = 2) recurrences, respectively. Two subjects had recurrent tumors in both the brain and bone, brain and adrenal gland, and bone and lymph nodes, respectively [16]. IHC Staining of Paraffin-Embedded Tumor Samples IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods [8, 17]. Briefly, all tissue specimens were formalin-fixed and processed similarly according to the standard histology practices. A 3-μm-thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin-eosin for the histological diagnosis. The sections were briefly immersed in citrate buffer [antigen retrieval solution (pH 9.0; Nichirei, Tokyo, Japan)] for phospho-EGFR (p-EGFR) and 0.01 mol/l citric acid (pH 6.0) for Arf6 and GEP100, and then incubated twice for 10 min at 121 ° C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with an antibody against p-EGFR (Tyr1068) (1H12 mouse monoclonal antibody; Cell Signaling Technology, Tokyo, Japan) diluted at 1:2,000, GEP100 (GeneScience, Tokyo, Japan) diluted at 1: 200 in phosphate-buffered saline overnight and 4 ° C, and Arf6 (3A-1 mouse monoclonal antibody; Santa Cruz Biotechnology, Dallas, Tex., USA) [13] diluted at 1:100 in phosphate-buffered saline for 60 min at room temperature [17, 18]. Thereafter, IHC staining was performed by the labeled polymer method (Histofine Simple Stain MAX-PO kit; Nichirei, Tokyo, Japan) according to the manufacturer’s instructions [17]. The positive controls for p-EGFR and GEP100 were samples of breast cancer tissue and MDA-MB-231 cells, respectively [13]. The controls for Arf6 were samples of HeLa cells, which were used according to the manufacturer’s instructions. The negative controls used mouse IgG (Dako) instead of the primary antibody for p-EGFR and Arf6, and phosphate-buffered saline for GEP100.
Patients, Clinical Features, and Follow-Up The institutional review board of our university approved this study and informed consent for the use of the tumor specimens was obtained from all the patients or their legal guardians. Tumor samples were obtained from 296 patients with primary lung adenocarcinoma who had undergone a surgical resection between 2003 and 2007 in our department. Nine of these patients had stage IV disease, and 25 underwent an incomplete resection. The tumor samples from 80 patients were too small to evaluate by immunohistochemical (IHC) staining for the EGFR, GEP100, and Arf6 status. As a result, 113 patients were excluded from the further analysis. Therefore, 182 tumor specimens were evaluated. All of the patients were Japanese, consisting of 100 males and 82 females in this series, with a median age of 68.5 years (range: 23–88). There were 75 never smokers, 50 former smokers, and 57 current smokers. Former smokers were defined as those who quit smoking at least 3 years before the time of surgery. The tumor stage was classified according to the TNM classification for lung cancer [14]. According to the pathological stage, 105 patients had tumors of stage IA, 39 of IB, 13 of IIA, 6 of IIB, 16 of IIIA, and 3 of stage IIIB. Vessel invasion was defined as blood vessel invasion or lymphatic vessel invasion in the surgical specimens. The patients were basically followed up every month within the first postoperative year and at approximately 2- to 4-month inter-
264
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Evaluation of the Stained Specimens Following the IHC detection of protein expression in each specimen, the percentage of immunoreactive tumor cells in five 400× fields selected randomly on one slide was recorded, and then the final value of positive tumor cells was determined as the average of the positively immunostained cells. To evaluate any correla-
Oka /Uramoto /Shimokawa /Yamada / Tanaka
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
effort that has been made to identify prognostic markers following surgery, there is currently no consensus in clinical practice. Molecular techniques provide powerful tools that have drastically changed our understanding of entire categories of lung cancer [8–10]. An overexpression of epidermal growth factor receptor (EGFR) signaling is the most frequently affected molecule in non-small cell lung cancer [9], and is often correlated with recurrent distant metastasis [11, 12]. An overexpression of Arf6 and GEP100, a guanine nucleotide-exchanging factor for Arf6, is responsible for the invasive activity that is crucial for the impact of the EGFR signaling pathways in lung adenocarcinoma [13]. However, the biological significance of these molecules in clinical specimens still remains largely unclear. We hypothesized that a deregulated EGFRGEP100-Arf6 axis can indicate aggressive behavior in lung adenocarcinoma. If the hypothesis is correct, the detection of this status might allow for the identification of not only patients at a high risk for recurrence, but can also identify candidate patients for adjuvant chemotherapy. Therefore, this study aimed to evaluate the prevalence and prognostic implications of the immunoreactivity for a representative EGFR-GEP100-Arf6 axis in a retrospective series of 182 resected patients with adenocarcinoma of the lung.
Color version available online
a
b
c
Fig. 1. The results of IHC analysis of p-EGFR, GEP100, and Arf6 staining: positive p-EGFR expression (a), positive GEP100 expression (b), and positive Arf6 expression (c). Scale bars = 50 μm.
(fig. 1). The expression of p-EGFR, GEP100, and Arf6 was observed in 65 (35.7%), 95 (52.2%), and 20 (11.0%) patients, respectively. No significant association between these molecules and the clinicopathological characteristics was found except for vessel invasion (tables 1–3). A significant association between the expression of p-EGFR and GEP100 and vessel invasion was identified. A significant association between p-EGFR and GEP100 expression was also identified.
Detection of p-EGFR, GEP100, and Arf6 Molecules and Correlations with the Clinicopathological Factors Positive reactions were mainly localized in the cell membrane and cytoplasm, for the p-EGFR and GEP100 expression, and in the cytoplasm for Arf6 expression
The Influence of the p-EGFR, GEP100, and Arf6 Molecules on Overall Survival The 5-year overall survival (OS) rates in patients positive and negative for p-EGFR expression were 82.5 and 80.6%, respectively (p = 0.873). The 5-year OS rates in patients positive and negative for GEP100 expression were 85.0 and 76.2%, respectively (p = 0.272). The 5-year OS rates in patients positive and negative for Arf6 expression were 80.0 and 81.6 %, respectively (p = 0.450). Thus, the Kaplan-Meier survival curves demonstrated that the individual proteins were not associated with any statistically significant differences in survival for lung adenocarcinoma patients (fig. 2). However, the combination of positive p-EGFR and positive Arf6 staining was associated with statistically significant differences in survival. The 5-year OS rates in patients with and without double positive expression of p-EGFR and Arf6 were 62.5 and 82.3%, respectively (p = 0.028; fig. 3a). When the analysis was conducted in only patients with the triple positive expression of p-EGFR, GEP100, and Arf6, the survival data also showed significant differences. The 5-year OS rates in patients with and without triple positive expression of these markers were 57.1 and 82.4%, respectively (p = 0.011; fig. 3b). In addition, triple positive expression
EGFR-GEP100-Arf6 Axis and Prognosis of Lung Adenocarcinoma
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Statistical Analyses Statistical significance was evaluated using the χ2 test or Fisher’s exact test. The Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was then performed according to Cox’s proportional hazards model. The odds ratio and 95% CI were calculated for each variable. Differences were considered to be statistically significant for p < 0.05. The data were analyzed using the Stat View software package (Abacus Concepts Inc., Berkeley, Calif., USA).
Results
265
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
tions with the clinicopathological characteristics, these protein expression scores were divided into positive or negative groups. Positive staining was judged to be present for p-EGFR when more than 5% of the tumor cells had moderate complete membranous and cytoplasmic staining [19]. The staining of GEP100 was classified as follows: 0 = negative or weak, 1+ = stained as strongly, and 2+ = stained more strongly. Positive staining was considered to be present for samples that scored 1 or 2 [13]. The staining of Arf6 was divided as follows: 0 = no cytoplasmic staining, 1+ = faint or barely perceptible incomplete cytoplasmic staining, 2+ = 10–30% of tumor cells with strong complete staining or >10% tumor cells with moderate complete staining, and 3+ = >30% tumor cells with strong complete staining. Positive staining was judged to be present for scores of 2 and 3. The slides were independently examined by two of the investigators (S.O. and H.S.) who were blinded to the clinicopathological data. When there was a discrepancy between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope.
Table 1. Relationship between p-EGFR expression and the clinicopathological characteristics
Variables
Gender Age, years p stage T status N status Smoking status Differentiation CEA Vessel invasion GEP100 Arf6
Category
male female
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Epidermal Growth Factor Receptor-GEP100-Arf6 Axis Affects the Prognosis of Lung Adenocarcinoma Soichi Oka a Hidetaka Uramoto a Hidehiko Shimokawa a Sohsuke Yamada b Fumihiro Tanaka a a
Second Department of Surgery and b Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
Key Words Lung cancer · Adenocarcinoma · Epidermal growth factor receptor · GEP100 · Arf6 · Prognosis
the prognosis of patients with primary lung adenocarcinoma. The combination of p-EGFR, GEP100, and Arf6 staining can predict the prognosis of patients after surgery. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0030–2414/14/0866–0263$39.50/0 E-Mail [email protected] www.karger.com/ocl
Introduction
Lung cancer is the leading cause of cancer-related death in the majority of countries worldwide [1], and the proportion of adenocarcinoma is notably increasing [2]. TNM staging indicates the level of disease progression and the malignant potential of primary lung cancer [3]. However, even patients with disease at the same stage exhibit wide variations in their prognosis after curative resection. Therefore, the current TNM staging system, which is based on clinical and pathological findings, may have reached the limit of its usefulness [4]. Accurately predicting the cases in which disease is likely to recur can help guide the administration of adjuvant therapies, not only to those most likely to benefit from them, but also to prevent harmful treatment [4, 5]. In addition to the TNM classification, tumor markers, such as CEA, have proven to be independent factors predicting the risk of recurrence as clinical parameters [6, 7]. Despite a great deal of Hidetaka Uramoto Second Department of Surgery, School of Medicine University of Occupational and Environmental Health 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan) E-Mail hidetaka @ med.uoeh-u.ac.jp
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
Abstract The overexpression of Arf6 and GEP100 is responsible for the invasive activity that is crucial for the activation of the epidermal growth factor receptor (EGFR) signaling pathways in human cancer. However, whether or not the expression of the EGFR-GEP100-Arf6 axis can be used as a biomarker for the prognosis of lung cancer has yet to be fully determined. Tumor specimens were collected from 182 patients who underwent a complete resection for lung adenocarcinoma. We analyzed phospho-EGFR (p-EGFR), GEP100, and Arf6 expression levels in the primary tumor by immunohistochemical analysis. The expression of p-EGFR, GEP100, and Arf6 was observed in 65 (35.7%), 95 (52.2%), and 20 (11.0%) patients, respectively. Significant associations between p-EGFR and GEP100 expression and vessel invasion were identified. The expression of these individual molecules was not associated with any statistically significant differences in survival. However, triple positive expression of p-EGFR, GEP100, and Arf6 was significantly associated with an increased risk of death based on the multivariate analysis. The EGFR-GEP100-Arf6 axis affected
Patients and Methods
vals thereafter. The evaluations included a physical examination, chest roentgenography, analysis of blood chemical parameters, and measurements of tumor marker levels. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected. A follow-up was conducted in all patients. The median follow-up period was 41.3 months. Twenty-eight (15.4%) patients received adjuvant chemotherapy as follows: carboplatin plus paclitaxel (n = 18), carboplatin plus gemcitabine (n = 7), and tegafur-uracil (n = 3) [5, 15]. At the last follow-up examination, 152 patients were alive and free of cancer, 10 patients had died of other causes without evidence of cancer, 12 patients were alive with recurrent cancer, and 17 patients had died of cancer. In total, 28 (15.4%) of the 182 patients demonstrated disease recurrence after surgery. The majority of the sites of tumor recurrence were hematogenous metastases. Twentyfive and 6 cases had hematogenous (brain = 9, lung = 10, bone = 5, and adrenal metastasis = 1) and locoregional (lymph node metastasis = 4, and pleural dissemination = 2) recurrences, respectively. Two subjects had recurrent tumors in both the brain and bone, brain and adrenal gland, and bone and lymph nodes, respectively [16]. IHC Staining of Paraffin-Embedded Tumor Samples IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods [8, 17]. Briefly, all tissue specimens were formalin-fixed and processed similarly according to the standard histology practices. A 3-μm-thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin-eosin for the histological diagnosis. The sections were briefly immersed in citrate buffer [antigen retrieval solution (pH 9.0; Nichirei, Tokyo, Japan)] for phospho-EGFR (p-EGFR) and 0.01 mol/l citric acid (pH 6.0) for Arf6 and GEP100, and then incubated twice for 10 min at 121 ° C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with an antibody against p-EGFR (Tyr1068) (1H12 mouse monoclonal antibody; Cell Signaling Technology, Tokyo, Japan) diluted at 1:2,000, GEP100 (GeneScience, Tokyo, Japan) diluted at 1: 200 in phosphate-buffered saline overnight and 4 ° C, and Arf6 (3A-1 mouse monoclonal antibody; Santa Cruz Biotechnology, Dallas, Tex., USA) [13] diluted at 1:100 in phosphate-buffered saline for 60 min at room temperature [17, 18]. Thereafter, IHC staining was performed by the labeled polymer method (Histofine Simple Stain MAX-PO kit; Nichirei, Tokyo, Japan) according to the manufacturer’s instructions [17]. The positive controls for p-EGFR and GEP100 were samples of breast cancer tissue and MDA-MB-231 cells, respectively [13]. The controls for Arf6 were samples of HeLa cells, which were used according to the manufacturer’s instructions. The negative controls used mouse IgG (Dako) instead of the primary antibody for p-EGFR and Arf6, and phosphate-buffered saline for GEP100.
Patients, Clinical Features, and Follow-Up The institutional review board of our university approved this study and informed consent for the use of the tumor specimens was obtained from all the patients or their legal guardians. Tumor samples were obtained from 296 patients with primary lung adenocarcinoma who had undergone a surgical resection between 2003 and 2007 in our department. Nine of these patients had stage IV disease, and 25 underwent an incomplete resection. The tumor samples from 80 patients were too small to evaluate by immunohistochemical (IHC) staining for the EGFR, GEP100, and Arf6 status. As a result, 113 patients were excluded from the further analysis. Therefore, 182 tumor specimens were evaluated. All of the patients were Japanese, consisting of 100 males and 82 females in this series, with a median age of 68.5 years (range: 23–88). There were 75 never smokers, 50 former smokers, and 57 current smokers. Former smokers were defined as those who quit smoking at least 3 years before the time of surgery. The tumor stage was classified according to the TNM classification for lung cancer [14]. According to the pathological stage, 105 patients had tumors of stage IA, 39 of IB, 13 of IIA, 6 of IIB, 16 of IIIA, and 3 of stage IIIB. Vessel invasion was defined as blood vessel invasion or lymphatic vessel invasion in the surgical specimens. The patients were basically followed up every month within the first postoperative year and at approximately 2- to 4-month inter-
264
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Evaluation of the Stained Specimens Following the IHC detection of protein expression in each specimen, the percentage of immunoreactive tumor cells in five 400× fields selected randomly on one slide was recorded, and then the final value of positive tumor cells was determined as the average of the positively immunostained cells. To evaluate any correla-
Oka /Uramoto /Shimokawa /Yamada / Tanaka
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
effort that has been made to identify prognostic markers following surgery, there is currently no consensus in clinical practice. Molecular techniques provide powerful tools that have drastically changed our understanding of entire categories of lung cancer [8–10]. An overexpression of epidermal growth factor receptor (EGFR) signaling is the most frequently affected molecule in non-small cell lung cancer [9], and is often correlated with recurrent distant metastasis [11, 12]. An overexpression of Arf6 and GEP100, a guanine nucleotide-exchanging factor for Arf6, is responsible for the invasive activity that is crucial for the impact of the EGFR signaling pathways in lung adenocarcinoma [13]. However, the biological significance of these molecules in clinical specimens still remains largely unclear. We hypothesized that a deregulated EGFRGEP100-Arf6 axis can indicate aggressive behavior in lung adenocarcinoma. If the hypothesis is correct, the detection of this status might allow for the identification of not only patients at a high risk for recurrence, but can also identify candidate patients for adjuvant chemotherapy. Therefore, this study aimed to evaluate the prevalence and prognostic implications of the immunoreactivity for a representative EGFR-GEP100-Arf6 axis in a retrospective series of 182 resected patients with adenocarcinoma of the lung.
Color version available online
a
b
c
Fig. 1. The results of IHC analysis of p-EGFR, GEP100, and Arf6 staining: positive p-EGFR expression (a), positive GEP100 expression (b), and positive Arf6 expression (c). Scale bars = 50 μm.
(fig. 1). The expression of p-EGFR, GEP100, and Arf6 was observed in 65 (35.7%), 95 (52.2%), and 20 (11.0%) patients, respectively. No significant association between these molecules and the clinicopathological characteristics was found except for vessel invasion (tables 1–3). A significant association between the expression of p-EGFR and GEP100 and vessel invasion was identified. A significant association between p-EGFR and GEP100 expression was also identified.
Detection of p-EGFR, GEP100, and Arf6 Molecules and Correlations with the Clinicopathological Factors Positive reactions were mainly localized in the cell membrane and cytoplasm, for the p-EGFR and GEP100 expression, and in the cytoplasm for Arf6 expression
The Influence of the p-EGFR, GEP100, and Arf6 Molecules on Overall Survival The 5-year overall survival (OS) rates in patients positive and negative for p-EGFR expression were 82.5 and 80.6%, respectively (p = 0.873). The 5-year OS rates in patients positive and negative for GEP100 expression were 85.0 and 76.2%, respectively (p = 0.272). The 5-year OS rates in patients positive and negative for Arf6 expression were 80.0 and 81.6 %, respectively (p = 0.450). Thus, the Kaplan-Meier survival curves demonstrated that the individual proteins were not associated with any statistically significant differences in survival for lung adenocarcinoma patients (fig. 2). However, the combination of positive p-EGFR and positive Arf6 staining was associated with statistically significant differences in survival. The 5-year OS rates in patients with and without double positive expression of p-EGFR and Arf6 were 62.5 and 82.3%, respectively (p = 0.028; fig. 3a). When the analysis was conducted in only patients with the triple positive expression of p-EGFR, GEP100, and Arf6, the survival data also showed significant differences. The 5-year OS rates in patients with and without triple positive expression of these markers were 57.1 and 82.4%, respectively (p = 0.011; fig. 3b). In addition, triple positive expression
EGFR-GEP100-Arf6 Axis and Prognosis of Lung Adenocarcinoma
Oncology 2014;86:263–270 DOI: 10.1159/000360089
Statistical Analyses Statistical significance was evaluated using the χ2 test or Fisher’s exact test. The Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was then performed according to Cox’s proportional hazards model. The odds ratio and 95% CI were calculated for each variable. Differences were considered to be statistically significant for p < 0.05. The data were analyzed using the Stat View software package (Abacus Concepts Inc., Berkeley, Calif., USA).
Results
265
Downloaded by: Siriraj Medical Library, Mahidol University 198.143.39.97 - 1/19/2016 1:03:35 AM
tions with the clinicopathological characteristics, these protein expression scores were divided into positive or negative groups. Positive staining was judged to be present for p-EGFR when more than 5% of the tumor cells had moderate complete membranous and cytoplasmic staining [19]. The staining of GEP100 was classified as follows: 0 = negative or weak, 1+ = stained as strongly, and 2+ = stained more strongly. Positive staining was considered to be present for samples that scored 1 or 2 [13]. The staining of Arf6 was divided as follows: 0 = no cytoplasmic staining, 1+ = faint or barely perceptible incomplete cytoplasmic staining, 2+ = 10–30% of tumor cells with strong complete staining or >10% tumor cells with moderate complete staining, and 3+ = >30% tumor cells with strong complete staining. Positive staining was judged to be present for scores of 2 and 3. The slides were independently examined by two of the investigators (S.O. and H.S.) who were blinded to the clinicopathological data. When there was a discrepancy between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope.
Table 1. Relationship between p-EGFR expression and the clinicopathological characteristics
Variables
Gender Age, years p stage T status N status Smoking status Differentiation CEA Vessel invasion GEP100 Arf6
Category
male female
