Histopathology 2015, 66, 234–243. DOI: 10.1111/his.12464
Clinicopathological significance of ASC amino acid transporter-2 expression in pancreatic ductal carcinoma Kyoichi Kaira,1,2 Yutaka Sunose,3 Kazuhisa Arakawa,4 Noriaki Sunaga,1 Kimihiro Shimizu,3 Hideyuki Tominaga,5 Noboru Oriuchi,6 Shushi Nagamori,7 Yoshikatsu Kanai,7 Tetsunari Oyama2 & Izumi Takeyoshi3 1
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan, Department of Diagnostic Pathology, Gunma University Graduate School of Medicine, Gunma, Japan, 3Department of Thoracic and Visceral Surgery, Gunma University Graduate School of Medicine, Gunma, Japan, 4Department of Surgery, Maebashi Red Cross Hospital, Gunma, Japan, 5Department of Molecular Imaging, Gunma University Graduate School of Medicine, Gunma, Japan, 6Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, and 7Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan
2
Date of submission 12 February 2014 Accepted for publication 16 May 2014 Published online Article Accepted 21 May 2014
Kaira K, Sunose Y, Arakawa K, Sunaga N, Shimizu K, Tominaga H, Oriuchi N, Nagamori S, Kanai Y, Oyama T, Takeyoshi I (2015) Histopathology 66, 234–243. DOI: 10.1111/his.12464
Clinicopathological significance of ASC amino acid transporter-2 expression in pancreatic ductal carcinoma Aims: ASC amino acid transporter-2 (ASCT2) is highly expressed in cancer cells. However, the clinicopathological significance of ASCT2 expression in pancreatic cancer remains unclear. The aim of this study was to investigate the clinical significance of ASCT2 expression in pancreatic cancer. Methods and results: Ninety-seven patients with surgically resected pancreatic ductal adenocarcinoma were evaluated. Tumour sections were stained by immunohistochemistry for ASCT2, Ki67, CD34 (to determine microvessel density), phosphoAKT (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) expression. ASCT2 was ex-
pressed in 54% (52/97) of tumours. Statistically significant differences in patient age, T stage, N stage, lymphatic permeation, vascular invasion, Ki67, and CD34 and p-mTOR expression were observed between tumours with and without ASCT2 expression. Multivariate analysis confirmed that vascular invasion, ASCT2 expression and Ki67 expression were independent predictive factors for a poorer prognosis. Conclusions: ASCT2 expression plays an important role in tumour cell growth, and is a promising pathological marker for predicting a worse outcome in pancreatic cancer.
Keywords: amino acid transporter, ASCT2, pancreatic cancer, prognostic factor
Introduction Amino acid transporters are essential for the growth and proliferation of normal cells and transformed Address for correspondence: K Kaira, MD, PhD, Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 371-8511, Japan. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
cells.1 System ASC amino acid transporter-2 (ASCT2; SLC1A5) is a Na+-dependent transporter of neutral amino acids, including glutamine, leucine, and isoleucine.2 ASCT2 is a major glutamine transporter in human hepatoma cells,3 and is associated with tumour growth and proliferation of cancer cells.4 Previous studies have shown ASCT2 expression in many types of human neoplasm, such as hepatocellular car-
ASCT2 expression in pancreatic cancer
cinoma, colorectal and prostate cancer, and glioma.3–8 The ASCT2 expression level has been reported to be associated with a malignant phenotype and poor prognosis in patients with prostate cancer and colorectal carcinoma.7,8 ASCT2 provides cancer cells with the amino acids that are essential for protein synthesis, and coordinates tumour cell growth through activation of the mammalian target of rapamycin (mTOR).3 Glutamine has an important role in the proliferation of cancer cells, and has high affinity for ASCT2.9 Of all amino acid transporters, L-type amino acid transporter-1 (LAT1), ASCT2 and xCT have been reported to be closely linked to the pathogenesis of cancer.4,10,11 Several studies have demonstrated that LAT1 expression plays a crucial role in the development and metastasis of various human neoplasms, and is a significant indicator for predicting poor outcome.12–21 A recent review focused on two amino acid transporters, ASCT2 and LAT1, in human carcinogenesis.4 However, the clinicopathological role of ASCT2 expression in human cancers remains unclear. Pancreatic cancer has a dismal outcome. As there are no established biomarkers for the prediction of therapeutic response or patient outcome, further study is warranted to identify appropriate clinical markers for guiding individual therapy and to improve prognosis. Recently, we reported that LAT1 and heavy chain of 4F2 cell surface antigen (4F2hc) expression are promising pathological markers for predicting the prognosis of patients with pancreatic cancer.21 However, the clinicopathological significance of ASCT2 expression in pancreatic cancer remains unknown. In the present study, therefore, ASCT2 expression was assessed in resected tissue specimens, as described previously.21
Materials and methods PATIENTS
Between July 1995 and March 2011, 113 consecutive patients with pancreatic ductal adenocarcinoma received surgical treatment at our institutions. Sixteen patients were excluded because of induction therapy and inadequate tissue specimens. Therefore, 97 patients were eligible for this study. The authors’ approach to the evaluation and resection of these tumours has been described previously.21 The study protocol was approved by the institutional review board. All surgical specimens were reviewed and classified according to the WHO classification by an experi© 2014 John Wiley & Sons Ltd, Histopathology, 66, 234–243.
235
enced pathologist who was unaware of clinical or imaging findings. Pathological tumour-node-metastasis (TNM) stages were established according to the International System for Staging Pancreatic Cancer adopted by the American Joint Committee on Cancer and the Union Internationale Contre le Cancer.22 The median age was 67 years (range, 25–86 years). Of all patients, 18, 40, 32 and seven had stage I, II, III and IV tumours, respectively. Postoperative adjuvant chemotherapy with gemcitabine, S-1 (Taiho Pharmaceutical, Tokyo, Japan) and oral tegafur (a fluorouracil derivative drug) was administered to 31 patients, 15 patients, and one patient, respectively. None of the patients had received neo-adjuvant chemotherapy. Intraoperative therapy was not performed for any patient. Postoperative survival was measured from the day of surgery; the follow-up duration ranged from 6 to 164 months (median, 14 months). A control group of patients with benign pancreatic lesions was also analysed, and these tissue specimens have been previously reported.21 Immunohistochemical staining of samples from 18 patients with surgically resected benign pancreatic disease was performed and compared with that of the pancreatic cancer samples. IMMUNOHISTOCHEMICAL STAINING
The detailed protocol for immunostaining has been previously published elsewhere.20,21,23–26 Briefly, deparaffinized and rehydrated sections were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. To expose antigens, sections were autoclaved in EDTA (pH 8.0) for 5 min and cooled for 30 min. After being rinsed in phosphate-buffered saline (PBS), sections were incubated with primary antibody overnight. They were then incubated with Histofine Simple Stain Max PO (Multi) polymer peroxidase kit (Nichirei Corporation, Tokyo, Japan). The peroxidase reaction was performed using 0.02% 3,30 -diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05 M Tris–HCl buffer (pH 7.4). Negative control tissue sections were prepared by omitting the primary antibody. ASCT2 staining was carried out using an affinitypurified rabbit polyclonal anti-ASCT2 antibody (generated as described previously26) at a dilution of 1:300. Expression was considered to be positive only if distinct membrane staining was present. ASCT2 expression was assessed by the extent of staining as follows: i, ≤10% of the tumour area stained; ii, 11– 25% stained; iii, 26–50% stained; and iv, ≥51%
236
K Kaira et al.
stained. The tumours with staining scores of >2 were defined as having positive expression. LAT1 expression was determined by immunohistochemical staining with an anti-LAT1 antibody [2 mg/ml, anti-human monoclonal mouse antibody, 4A2, provided by H. Endou (J-Pharma, Tokyo, Japan), dilution 1:3200]. The production and characterization of the anti-LAT1 antibody have been described previously.17 Immunostaining for 4F2hc was carried out using an affinity-purified rabbit polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) raised against a peptide mapping to the C-terminus of 4F2hc of human origin; the detailed protocol has been published elsewhere.21 LAT1 and 4F2hc expression was considered to be positive only if distinct membrane staining was present. The LAT1 and 4F2hc expression scores were assessed by the extent of staining as follows: i, ≤10% of tumour area stained; ii, 11–25% stained; iii, 26– 50% stained; and iv, ≥51% stained. The staining intensity was not considered when staining was assessed. The tumours in which stained tumour cells were scored as 3 or 4 were defined as having high expression. Staining was also carried out using the following: mouse monoclonal antibodies against CD34 (Nichirei; 1:800 dilution) and Ki67 (1:40 dilution; Dako, Glostrup, Denmark); a rabbit polyclonal antibody against phospho-AKT (1:200 dilution; Abcam, Tokyo, Japan); and a rabbit monoclonal antibody against phospho-mTOR (1:80 dilution; Cell Signaling, Tokyo, Japan). The number of CD34-positive vessels was counted in four selected hot spots in a 9 400 field (field area, 0.26 mm2). Microvessel density (MVD) was defined as the mean count of microvessels per 0.26-mm2 field area, and tumours with MVD greater than the median were defined as having high expression. Regarding Ki67 expression, a highly cellular area of the immunostained sections was evaluated. All epithelial cells with nuclear staining of any intensity were defined as having high expression. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of Ki67-stained nuclei (Ki67 labelling index) in the sample. The median value for the Ki67 labelling index was evaluated, and the tumour cells with a Ki67 labelling index greater than the median value were defined as having high expression. Regarding p-AKT and p-mTOR, a semiquantitative scoring method was used: i, 65 years)
43/54
10/8
0.445
Gender (male/female)
48/49
7/11
0.451
Rate of high expression or positivity (%) in various biomarkers ASCT2 (positive/negative)
53.6 (52/45)
0 (0/18)
Clinicopathological significance of ASC amino acid transporter-2 expression in pancreatic ductal carcinoma Kyoichi Kaira,1,2 Yutaka Sunose,3 Kazuhisa Arakawa,4 Noriaki Sunaga,1 Kimihiro Shimizu,3 Hideyuki Tominaga,5 Noboru Oriuchi,6 Shushi Nagamori,7 Yoshikatsu Kanai,7 Tetsunari Oyama2 & Izumi Takeyoshi3 1
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan, Department of Diagnostic Pathology, Gunma University Graduate School of Medicine, Gunma, Japan, 3Department of Thoracic and Visceral Surgery, Gunma University Graduate School of Medicine, Gunma, Japan, 4Department of Surgery, Maebashi Red Cross Hospital, Gunma, Japan, 5Department of Molecular Imaging, Gunma University Graduate School of Medicine, Gunma, Japan, 6Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, and 7Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan
2
Date of submission 12 February 2014 Accepted for publication 16 May 2014 Published online Article Accepted 21 May 2014
Kaira K, Sunose Y, Arakawa K, Sunaga N, Shimizu K, Tominaga H, Oriuchi N, Nagamori S, Kanai Y, Oyama T, Takeyoshi I (2015) Histopathology 66, 234–243. DOI: 10.1111/his.12464
Clinicopathological significance of ASC amino acid transporter-2 expression in pancreatic ductal carcinoma Aims: ASC amino acid transporter-2 (ASCT2) is highly expressed in cancer cells. However, the clinicopathological significance of ASCT2 expression in pancreatic cancer remains unclear. The aim of this study was to investigate the clinical significance of ASCT2 expression in pancreatic cancer. Methods and results: Ninety-seven patients with surgically resected pancreatic ductal adenocarcinoma were evaluated. Tumour sections were stained by immunohistochemistry for ASCT2, Ki67, CD34 (to determine microvessel density), phosphoAKT (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) expression. ASCT2 was ex-
pressed in 54% (52/97) of tumours. Statistically significant differences in patient age, T stage, N stage, lymphatic permeation, vascular invasion, Ki67, and CD34 and p-mTOR expression were observed between tumours with and without ASCT2 expression. Multivariate analysis confirmed that vascular invasion, ASCT2 expression and Ki67 expression were independent predictive factors for a poorer prognosis. Conclusions: ASCT2 expression plays an important role in tumour cell growth, and is a promising pathological marker for predicting a worse outcome in pancreatic cancer.
Keywords: amino acid transporter, ASCT2, pancreatic cancer, prognostic factor
Introduction Amino acid transporters are essential for the growth and proliferation of normal cells and transformed Address for correspondence: K Kaira, MD, PhD, Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 371-8511, Japan. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
cells.1 System ASC amino acid transporter-2 (ASCT2; SLC1A5) is a Na+-dependent transporter of neutral amino acids, including glutamine, leucine, and isoleucine.2 ASCT2 is a major glutamine transporter in human hepatoma cells,3 and is associated with tumour growth and proliferation of cancer cells.4 Previous studies have shown ASCT2 expression in many types of human neoplasm, such as hepatocellular car-
ASCT2 expression in pancreatic cancer
cinoma, colorectal and prostate cancer, and glioma.3–8 The ASCT2 expression level has been reported to be associated with a malignant phenotype and poor prognosis in patients with prostate cancer and colorectal carcinoma.7,8 ASCT2 provides cancer cells with the amino acids that are essential for protein synthesis, and coordinates tumour cell growth through activation of the mammalian target of rapamycin (mTOR).3 Glutamine has an important role in the proliferation of cancer cells, and has high affinity for ASCT2.9 Of all amino acid transporters, L-type amino acid transporter-1 (LAT1), ASCT2 and xCT have been reported to be closely linked to the pathogenesis of cancer.4,10,11 Several studies have demonstrated that LAT1 expression plays a crucial role in the development and metastasis of various human neoplasms, and is a significant indicator for predicting poor outcome.12–21 A recent review focused on two amino acid transporters, ASCT2 and LAT1, in human carcinogenesis.4 However, the clinicopathological role of ASCT2 expression in human cancers remains unclear. Pancreatic cancer has a dismal outcome. As there are no established biomarkers for the prediction of therapeutic response or patient outcome, further study is warranted to identify appropriate clinical markers for guiding individual therapy and to improve prognosis. Recently, we reported that LAT1 and heavy chain of 4F2 cell surface antigen (4F2hc) expression are promising pathological markers for predicting the prognosis of patients with pancreatic cancer.21 However, the clinicopathological significance of ASCT2 expression in pancreatic cancer remains unknown. In the present study, therefore, ASCT2 expression was assessed in resected tissue specimens, as described previously.21
Materials and methods PATIENTS
Between July 1995 and March 2011, 113 consecutive patients with pancreatic ductal adenocarcinoma received surgical treatment at our institutions. Sixteen patients were excluded because of induction therapy and inadequate tissue specimens. Therefore, 97 patients were eligible for this study. The authors’ approach to the evaluation and resection of these tumours has been described previously.21 The study protocol was approved by the institutional review board. All surgical specimens were reviewed and classified according to the WHO classification by an experi© 2014 John Wiley & Sons Ltd, Histopathology, 66, 234–243.
235
enced pathologist who was unaware of clinical or imaging findings. Pathological tumour-node-metastasis (TNM) stages were established according to the International System for Staging Pancreatic Cancer adopted by the American Joint Committee on Cancer and the Union Internationale Contre le Cancer.22 The median age was 67 years (range, 25–86 years). Of all patients, 18, 40, 32 and seven had stage I, II, III and IV tumours, respectively. Postoperative adjuvant chemotherapy with gemcitabine, S-1 (Taiho Pharmaceutical, Tokyo, Japan) and oral tegafur (a fluorouracil derivative drug) was administered to 31 patients, 15 patients, and one patient, respectively. None of the patients had received neo-adjuvant chemotherapy. Intraoperative therapy was not performed for any patient. Postoperative survival was measured from the day of surgery; the follow-up duration ranged from 6 to 164 months (median, 14 months). A control group of patients with benign pancreatic lesions was also analysed, and these tissue specimens have been previously reported.21 Immunohistochemical staining of samples from 18 patients with surgically resected benign pancreatic disease was performed and compared with that of the pancreatic cancer samples. IMMUNOHISTOCHEMICAL STAINING
The detailed protocol for immunostaining has been previously published elsewhere.20,21,23–26 Briefly, deparaffinized and rehydrated sections were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. To expose antigens, sections were autoclaved in EDTA (pH 8.0) for 5 min and cooled for 30 min. After being rinsed in phosphate-buffered saline (PBS), sections were incubated with primary antibody overnight. They were then incubated with Histofine Simple Stain Max PO (Multi) polymer peroxidase kit (Nichirei Corporation, Tokyo, Japan). The peroxidase reaction was performed using 0.02% 3,30 -diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05 M Tris–HCl buffer (pH 7.4). Negative control tissue sections were prepared by omitting the primary antibody. ASCT2 staining was carried out using an affinitypurified rabbit polyclonal anti-ASCT2 antibody (generated as described previously26) at a dilution of 1:300. Expression was considered to be positive only if distinct membrane staining was present. ASCT2 expression was assessed by the extent of staining as follows: i, ≤10% of the tumour area stained; ii, 11– 25% stained; iii, 26–50% stained; and iv, ≥51%
236
K Kaira et al.
stained. The tumours with staining scores of >2 were defined as having positive expression. LAT1 expression was determined by immunohistochemical staining with an anti-LAT1 antibody [2 mg/ml, anti-human monoclonal mouse antibody, 4A2, provided by H. Endou (J-Pharma, Tokyo, Japan), dilution 1:3200]. The production and characterization of the anti-LAT1 antibody have been described previously.17 Immunostaining for 4F2hc was carried out using an affinity-purified rabbit polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) raised against a peptide mapping to the C-terminus of 4F2hc of human origin; the detailed protocol has been published elsewhere.21 LAT1 and 4F2hc expression was considered to be positive only if distinct membrane staining was present. The LAT1 and 4F2hc expression scores were assessed by the extent of staining as follows: i, ≤10% of tumour area stained; ii, 11–25% stained; iii, 26– 50% stained; and iv, ≥51% stained. The staining intensity was not considered when staining was assessed. The tumours in which stained tumour cells were scored as 3 or 4 were defined as having high expression. Staining was also carried out using the following: mouse monoclonal antibodies against CD34 (Nichirei; 1:800 dilution) and Ki67 (1:40 dilution; Dako, Glostrup, Denmark); a rabbit polyclonal antibody against phospho-AKT (1:200 dilution; Abcam, Tokyo, Japan); and a rabbit monoclonal antibody against phospho-mTOR (1:80 dilution; Cell Signaling, Tokyo, Japan). The number of CD34-positive vessels was counted in four selected hot spots in a 9 400 field (field area, 0.26 mm2). Microvessel density (MVD) was defined as the mean count of microvessels per 0.26-mm2 field area, and tumours with MVD greater than the median were defined as having high expression. Regarding Ki67 expression, a highly cellular area of the immunostained sections was evaluated. All epithelial cells with nuclear staining of any intensity were defined as having high expression. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of Ki67-stained nuclei (Ki67 labelling index) in the sample. The median value for the Ki67 labelling index was evaluated, and the tumour cells with a Ki67 labelling index greater than the median value were defined as having high expression. Regarding p-AKT and p-mTOR, a semiquantitative scoring method was used: i, 65 years)
43/54
10/8
0.445
Gender (male/female)
48/49
7/11
0.451
Rate of high expression or positivity (%) in various biomarkers ASCT2 (positive/negative)
53.6 (52/45)
0 (0/18)
