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Hepatology Research 2015; 45: 1014–1022
doi: 10.1111/hepr.12431
Original Article
Expression of amino acid transporters (LAT1, ASCT2 and xCT) as clinical significance in hepatocellular carcinoma Masashi Namikawa,1,2* Satoru Kakizaki,1* Kyoichi Kaira,1,3* Hiroki Tojima,1 Yuichi Yamazaki,1 Norio Horiguchi,1 Ken Sato,1 Noboru Oriuchi,4 Hideyuki Tominaga,5 Yutaka Sunose,6 Shushi Nagamori,7 Yoshikatsu Kanai,7 Tetsunari Oyama,8 Izumi Takeyoshi6 and Masanobu Yamada1 Departments of 1Medicine and Molecular Science, 3Oncology Clinical Development, 4Diagnostic Radiology and Nuclear Medicine, 6Thoracic and Visceral Surgery, and 8Diagnostic Pathology, Graduate School of Medicine, Gunma University, Gunma, 5Advanced Clinical Research Center, Fukushima Medical University, Fukushima, 2 Department of Internal Medicine, Kiryu Kosei General Hospital, Kiryu, and 7Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan Aim: Amino acid transporters play an important role in tumor progression and survival of cancer cells. However, the prognostic significance of L-type amino acid transporter 1 (LAT1), system ASC amino acid transporter-2 (ASCT2) and xCT expression in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of this study is to investigate the clinicopathological significance of these amino acid transporters in patients with HCC.
(55/84), respectively. Positive LAT1 expression was significantly associated with 4F2hc expression, Ki-67 and the serum albumin. By univariate analysis, LAT1 expression, disease stage and albumin had a significant relationship with overall survival. Tumor size, disease stage, portal vein invasion, albumin and α-fetoprotein had a significant relationship with progression-free survival. Multivariate analysis confirmed that LAT1 expression is an independent and significant prognostic factor for predicting worse outcome after surgery.
Methods: We examined 84 patients with surgically resected HCC. Tumor sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67 and microvessel density (MVD) determined by CD34.
Conclusion: LAT1 can serve as a significant prognostic marker for predicting negative prognosis after surgery.
Results: LAT1, 4F2hc, ASCT2 and xCT were positively
Key words: ASCT2, hepatocellular carcinoma, LAT1, prognostic factor, xCT
expressed in 61% (50/84), 77% (65/84), 63% (53/84) and 65%
INTRODUCTION
H
EPATOCELLULAR CARCINOMA (HCC) is the fifth most common type of human cancer and is associated with high prevalence of hepatitis B or C virus
Correspondence: Dr Kyoichi Kaira, Department of Medicine and Molecular Science and Department of Oncology Clinical Development, Graduate School of Medicine, Gunma University, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan. Email: [email protected] *These authors equally contributed to this article. Conflict of interest: None of the authors have financial or personal relationships with other people or organizations that could inappropriately influence this work. Received 7 July 2014; revision 30 September 2014; accepted 4 October 2014.
1014
infection. Even when the appropriate treatment such as surgery has been provided, the outcome of patients with HCC still remains dismal. Disease staging and grading by tumor differentiation are considered as the conventional prognostic factors in HCC.1 The prognostic factors to improve the outcome after treatment are not yet available. Thus, we should discover an established biomarkers for predicting the prognosis of HCC. Amino acid transporters play an important role in the cell growth, proliferation and survival of normal cells and transformed cells.2 Of these amino acids transporters, L-type amino acid transporter 1 (LAT1), system ASC amino acid transporter-1 (ASCT2) and xCT are identified to be significantly linked to the tumor pathogenesis and carcinogenesis.3,4 These amino acid transporters were highly expressed in human cancers.5–7 LAT1 transports large neutral amino acids, such as
© 2014 The Japan Society of Hepatology
Hepatology Research 2015; 45: 1014–1022
leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine, and requires a covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional expression in the plasma membrane.4,8 ASCT2 is a major glutamine transporter and transports neutral amino acids such as glutamine, leucine and isoleucine.9 System xc− is a cystine–glutamate exchanger transporter, composed of a light chain (xCT) and heavy chain (4F2hc).10,11 The expression of xCT has been reported to play a crucial role in the tumor progression and growth of cancer and glutathione-based drug resistance.12 The overexpression of LAT1, ASCT2 and xCT is shown to be a significant pathological predictor related to worse outcome in various types of human cancer.3,5–7,11 However, the clinicopathological significance of these amino acid transporters (LAT1, ASCT2 and xCT) in patients with HCC remains unknown. In the present study, therefore, we assessed the protein expression of LAT1, ASCT2 and xCT in the resected tissue specimen, and the expression level of these markers was correlated with pathological variables and outcome after surgery. Additionally, the expression of Ki-67 labeling index and angiogenic markers such as microvessel density (MVD) determined by CD34, were also examined by immunohistochemistry.
METHODS Patients
B
ETWEEN APRIL 1999 and July 2011, we analyzed 89 consecutive patients with HCC who received surgical resection at Gunma University Hospital. Five patients were excluded because these tumor specimens were not be available. Therefore, 84 patients were included and analyzed in this study. The study was approved by the institutional review board. The median age was 62 years, ranging 29–81 years. None of the patients had received neoadjuvant chemotherapy. All surgical specimens were reviewed and classified according to the World Health Organization classification by an experienced pathologist who was unaware of clinical or imaging findings. Pathological tumor–node–metastasis stages were established using the International System for Staging adopted by the American Joint Committee on Cancer and the Union Internationale Centre le Cancer. The day of surgery was considered the starting day for measuring postoperative survival. The follow-up duration ranged 97–3521 days (median, 1009).
Amino acid transporter in HCC 1015
Immunohistochemical staining LAT1 expression was determined by immunohistochemical staining with a murine antihuman LAT1 monoclonal antibody 4A2 (provided by Dr H. Endou [J-Pharma, Tokyo, Japan], 2 mg/mL, 1:3200 dilution).13 The production and characterization of the LAT1 antibody has been described previously.5–7 The anti-CD98 antibody is an affinity purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100 dilution) raised against a peptide mapping at the carboxy terminus of CD98 of human origin. CD98 clearly detects 4F2hc and this antigen is SLC3A2/4F2hc/ CD98hc, therefore, we used this antibody. Anti-ASCT2 and anti-xCT antibodies are affinity-purified rabbit polyclonal antibody (1:300 dilution) and affinitypurified murine polyclonal antibody (Santa Cruz Biotechnology; 1:200 dilution), respectively. The detailed protocol for immunostaining was also published elsewhere.5–7 Briefly, deparaffinized and rehydrated sections were treated with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min to block endogenous peroxidase activity. To expose the antigens, sections were autoclaved in ethylenediaminetetraacetic acid (pH 8.0) for 5 min and cooled for 30 min. After rinsing in phosphate-buffered saline, the sections were incubated with anti-LAT1 antibodies (1:3200), anti-ASCT2 antibodies (1:300) and anti-xCT antibodies (1:200) overnight followed by immunohistochemical staining with a Histofine Simple Stain MAX PO (MULTI) kit (Nichirei, Tokyo, Japan). The peroxidase reaction was carried out using 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl (pH 7.4). Negative control tissue sections were stained as described above, except that the primary antibody was omitted. The LAT1, 4F2hc, ASCT2 and xCT expression scores were assessed by the extent of obvious staining as follows: 0, less than 5% of tumor area stained; 1, 5–10% stained; 2, 11–25% stained; 3, 26–50% stained; and 4, 51% or more stained. The tumors in which stained tumor cells were scored as more than 1 were defined as positive expression. For CD34 and Ki-67, immunohistochemical staining was performed according to the procedures described in previous reports.5–7 The following antibodies were used: murine monoclonal antibodies against CD34 (Nichirei; 1:800 dilution) and Ki-67 (Dako, Glostrup, Denmark; 1:40 dilution). The number of CD34 positive vessels was counted in four selected hot spots in a ×400 field (0.26 mm2 field area). MVD was defined as the mean count of microvessels per 0.26 mm2 field area. The
© 2014 The Japan Society of Hepatology
1016 M. Namikawa et al.
median numbers of CD34 positive vessels were evaluated, and the tumors in which stained tumor cells made up more than each median value were defined as high expression. For Ki-67, a highly cellular area of the immunostained sections was evaluated. All epithelial cells with nuclear staining of any intensity were defined as high expression. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of Ki-67-stained nuclei (Ki-67 labeling index) in the sample. The median value of the Ki-67 labeling index was evaluated, and the tumor cells with greater than the median value were defined as high expression. The sections were assessed by light microscopy in a blinded fashion by at least two of the authors.
Statistical analysis Probability values of less than 0.05 indicated a statistically significant difference. Fisher’s exact test was used to examine the association of two categorical variables. Results are expressed as the mean 1 standard deviation. The significance of differences was determined by Student’s t-test. The correlation between different variables was analyzed using the non-parametric Spearman’s rank correlation coefficient test. Follow up for these 84 patients was conducted using the patient medical records. The Kaplan–Meier method was used to estimate survival as a function of time, and survival differences were analyzed by the log–rank test. Overall survival (OS) was determined as the time from tumor resection to death from any cause. Recurrence-free survival (RFS) was defined as the time between tumor resection and the first disease progression or death. Multivariate analyses were performed using the stepwise Cox proportional hazards model to identify independent prognostic factors. Statistical analysis was performed using JMP version 8 (SAS Institute, Cary, NC, USA) for Windows.
RESULTS Immunohistochemical staining and patients
T
HE IMMUNOHISTOCHEMICAL ANALYSIS of the biomarkers was performed on the 84 primary lesions with HCC. Figure 1 shows the representative immunohistochemical staining of LAT1, ASCT2 and xCT expression. The immunostaining of LAT1, ASCT2 and xCT was detected in carcinoma cells in tumor tissues and localized predominantly on their plasma membrane. These amino acid transporters are partially expressed as island shape in tumor tissues. All positive cells revealed strong membranous immunostaining. In
© 2014 The Japan Society of Hepatology
Hepatology Research 2015; 45: 1014–1022
total patients, a positive LAT1, 4F2hc, ASCT2 and xCT expression was recognized in 61% (50/84), 77% (65/ 84), 63% (53/84) and 65% (55/84), respectively. The average score of LAT1, 4F2hc, ASCT2 and xCT expression was 0.6 1 0.5, 0.8 1 0.5, 1.7 1 0.6 and 1.6 1 0.6 respectively, on a scale of 0–4. The scoring of LAT1 was significantly lower than that of 4F2hc (P = 0.007), ASCT2 (P < 0.001) and xCT (P < 0.001), demonstrating no statistical significance between ASCT2 and xCT (P = 0.467). The median number of CD34 was 14 (range, 2–29), and the value of 14 was chosen as a cut-off point. The median value of the Ki-67 labeling index was 21% (range, 5–72), and the value of 21% was chosen as a cut-off point. The high expression of CD34 and Ki-67 was recognized in 52% (44/85) and 51% (43/85), respectively. Table 1 shows patients’ demographics according to LAT1, ASCT2 and xCT expression. Positive LAT1 expression was significantly associated with 4F2hc expression, Ki-67 labeling index and the value of albumin. There was no significant relationship between ASCT2 expression and any clinicopathological marker. Positive xCT expression yielded a significant association with only tumor differentiation.
Correlation between LAT1 expression and different variables Using Spearman’s rank correlation, LAT1 had a statistically significant correlation with 4F2hc (r = 0.324, P = 0.003), serum albumin (r = −0.255, P = 0.022) and Ki-67 (cell proliferation) (r = 0.264, P = 0.015) (Table 2).
Different variables survival analysis The 5-year survival rates of OS and RFS for all patients were 62.9% and 17.2%, respectively. Tables 3 and 4 shows the results of univariate and multivariate analysis in OS and RFS. By univariate analysis, LAT1 expression, disease stage and albumin had a significant relationship with OS. Tumor size, disease stage, portal vein invasion, albumin and α-fetoprotein had a significant relationship with RFS. Figure 2 shows the Kaplan–Meier survival curve in patients with HCC according to the expression of LAT1. Based on the results of the univariate log–rank test, we screened prognostic factors with a cut-off of P < 0.05. We screened the three factors of LAT1, disease stage and albumin for multivariate analysis. Multivariate analysis confirmed that LAT1 expression, disease stage and albumin were independent prognostic factors for predicting worse OS.
Hepatology Research 2015; 45: 1014–1022
a
b
c
d
e
f
DISCUSSION
T
HIS IS A clinicopathological study to evaluate the expression of amino acid transporters (LAT1, ASCT2 and xCT) in patients with HCC. Of these amino acid transporters, LAT1 expression seemed to play a crucial role in the development and progression of HCC. Our study suggests that LAT1 is closely correlated with the expression of 4F2hc and cell proliferation by Ki-67 labeling index, and is an independent and significant prognostic marker for predicting negative outcome in patients with HCC. Moreover, the expression of LAT1 yielded a significant association with cell proliferation and angiogenesis. However, the expression of LAT1 in HCC was markedly lower among human neoplasms including lung cancer,5 pancreatic cancer,14 breast cancer,15 and head and neck cancer.7 It remains unclear why the expression degree of LAT1 in HCC is lower than
Amino acid transporter in HCC 1017
Figure 1 Representative immunohistochemical staining of a patient with hepatocellular carcinoma. Immunostaining of LAT1 (a), 4F2hc (b), ASCT2 (c) and xCT (d) displays a membranous immunostaining pattern. (e) Negative control of LAT1 immunostaining in patients with hepatocellular carcinoma. No immunostaining of these amino acid transporters (LAT1, ASCT2 and xCT) was observed in normal liver tissue (f). (Original magnifications: [a] ×400; [b] ×400; [c] ×400; [d] ×400; [e] ×400; [f] ×400.)
the other cancers. In the present study, we divided our study population into two groups with or without LAT1 expression, and compared the clinical and prognostic roles of LAT1 with those of ASCT2 and xCT in HCC. We could not find any relationship between LAT1 and the other transporters (ASCT2 and xCT), however, LAT1, ASCT2 and xCT were positively expressed in patients with HCC. Recently, Li et al. reported that LAT1 was overexpressed in HCC, having a significant association with tumor size, histological differentiation and tumor staging, and a high expression of LAT1 had a significantly increased risk of shortened survival time.16 The study also showed that LAT1 mRNA expression level was significantly higher in HCC tissues than in the corresponding non-cancerous tissues. This is almost corresponding to the results of our study. However, they did not investigate how the upregulation of LAT1 is affected
© 2014 The Japan Society of Hepatology
© 2014 The Japan Society of Hepatology 50/50 68/32 4/80/16 23/77 40/60 49/51 32/68 18/82 46/54 50/50 44/56 46/54 88/12 64/36 68/32 56/44 48/52
74/26 7/70/23 23/77 44/56 51/49 33/67 15/85 44/56 37/63 50/50 49/51 77/23 63/37 65/35 46/54 52/48
Positive (n = 50) (%)
46/54
Total (n = 84) (%)
56/44
33/67
62/38
62/38
62/38
56/44
59/41
26/74
41/59
13/87
35/65
55/45
50/50
24/76
12/56/32
82/18
41/59
Negative (n = 34) (%)
LAT1 expression
0.511
0.045
0.642
>0.999
0.007
0.472
0.193
0.027
0.823
0.546
0.816
0.656
53/47
55/45
72/28
–
77/23
40/60
49/51
42/58
45/55
13/87
34/66
47/53
47/53
11/89
>0.999 0.381
8/68/24
74/26
43/57
Positive (n = 50) (%)
0.061
0.206
0.506
P
48/52
32/68
55/45
–
77/23
58/42
55/45
39/61
39/61
19/81
32/68
55/45
42/58
23/77
6/74/20
74/26
65/35
Negative (n = 34) (%)
ASCT2 expression
0.821
0.069
0.115
58/42
45/55
–
69/31
78/22
>0.999 –
51/49
55/45
42/58
47/53
0.117
0.655
0.822
0.649
11/89
27/73
>0.999 0.536
56/44
51/49
22/78
0.651
0.657
0.215
5/69/26
76/24
>0.999 0.865
55/45
Positive (n = 50) (%)
0.073
P
38/62
48/52
–
52/48
76/24
38/62
45/55
38/62
34/66
24/76
45/55
38/62
34/66
3/97
10/72/18
69/31
45/55
Negative (n = 34) (%)
xCT expression
0.108
0.822
–
0.115
0.791
0.357
0.492
0.817
0.354
0.125
0.144
0.168
0.172
0.029
0.505
0.602
0.492
P
Bolding indicates statistical significance. AFP, α-fetoprotein; ASCT2, HBV, hepatitis B virus; HCV, hepatitis C virus; Ki-67 LI, Ki-67 labeling index; MD, moderately differentiated; PD, poorly differentiated; PIVKA-II, protein induced by vitamin K absence-II; WD, well differentiated.
Age 265/>65 years Sex Male/female Etiology HBV/HCV/others Differentiation WD/MD or PD Disease stage I or II/III or IV Tumor size
Hepatology Research 2015; 45: 1014–1022
doi: 10.1111/hepr.12431
Original Article
Expression of amino acid transporters (LAT1, ASCT2 and xCT) as clinical significance in hepatocellular carcinoma Masashi Namikawa,1,2* Satoru Kakizaki,1* Kyoichi Kaira,1,3* Hiroki Tojima,1 Yuichi Yamazaki,1 Norio Horiguchi,1 Ken Sato,1 Noboru Oriuchi,4 Hideyuki Tominaga,5 Yutaka Sunose,6 Shushi Nagamori,7 Yoshikatsu Kanai,7 Tetsunari Oyama,8 Izumi Takeyoshi6 and Masanobu Yamada1 Departments of 1Medicine and Molecular Science, 3Oncology Clinical Development, 4Diagnostic Radiology and Nuclear Medicine, 6Thoracic and Visceral Surgery, and 8Diagnostic Pathology, Graduate School of Medicine, Gunma University, Gunma, 5Advanced Clinical Research Center, Fukushima Medical University, Fukushima, 2 Department of Internal Medicine, Kiryu Kosei General Hospital, Kiryu, and 7Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan Aim: Amino acid transporters play an important role in tumor progression and survival of cancer cells. However, the prognostic significance of L-type amino acid transporter 1 (LAT1), system ASC amino acid transporter-2 (ASCT2) and xCT expression in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of this study is to investigate the clinicopathological significance of these amino acid transporters in patients with HCC.
(55/84), respectively. Positive LAT1 expression was significantly associated with 4F2hc expression, Ki-67 and the serum albumin. By univariate analysis, LAT1 expression, disease stage and albumin had a significant relationship with overall survival. Tumor size, disease stage, portal vein invasion, albumin and α-fetoprotein had a significant relationship with progression-free survival. Multivariate analysis confirmed that LAT1 expression is an independent and significant prognostic factor for predicting worse outcome after surgery.
Methods: We examined 84 patients with surgically resected HCC. Tumor sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67 and microvessel density (MVD) determined by CD34.
Conclusion: LAT1 can serve as a significant prognostic marker for predicting negative prognosis after surgery.
Results: LAT1, 4F2hc, ASCT2 and xCT were positively
Key words: ASCT2, hepatocellular carcinoma, LAT1, prognostic factor, xCT
expressed in 61% (50/84), 77% (65/84), 63% (53/84) and 65%
INTRODUCTION
H
EPATOCELLULAR CARCINOMA (HCC) is the fifth most common type of human cancer and is associated with high prevalence of hepatitis B or C virus
Correspondence: Dr Kyoichi Kaira, Department of Medicine and Molecular Science and Department of Oncology Clinical Development, Graduate School of Medicine, Gunma University, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan. Email: [email protected] *These authors equally contributed to this article. Conflict of interest: None of the authors have financial or personal relationships with other people or organizations that could inappropriately influence this work. Received 7 July 2014; revision 30 September 2014; accepted 4 October 2014.
1014
infection. Even when the appropriate treatment such as surgery has been provided, the outcome of patients with HCC still remains dismal. Disease staging and grading by tumor differentiation are considered as the conventional prognostic factors in HCC.1 The prognostic factors to improve the outcome after treatment are not yet available. Thus, we should discover an established biomarkers for predicting the prognosis of HCC. Amino acid transporters play an important role in the cell growth, proliferation and survival of normal cells and transformed cells.2 Of these amino acids transporters, L-type amino acid transporter 1 (LAT1), system ASC amino acid transporter-1 (ASCT2) and xCT are identified to be significantly linked to the tumor pathogenesis and carcinogenesis.3,4 These amino acid transporters were highly expressed in human cancers.5–7 LAT1 transports large neutral amino acids, such as
© 2014 The Japan Society of Hepatology
Hepatology Research 2015; 45: 1014–1022
leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine, and requires a covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional expression in the plasma membrane.4,8 ASCT2 is a major glutamine transporter and transports neutral amino acids such as glutamine, leucine and isoleucine.9 System xc− is a cystine–glutamate exchanger transporter, composed of a light chain (xCT) and heavy chain (4F2hc).10,11 The expression of xCT has been reported to play a crucial role in the tumor progression and growth of cancer and glutathione-based drug resistance.12 The overexpression of LAT1, ASCT2 and xCT is shown to be a significant pathological predictor related to worse outcome in various types of human cancer.3,5–7,11 However, the clinicopathological significance of these amino acid transporters (LAT1, ASCT2 and xCT) in patients with HCC remains unknown. In the present study, therefore, we assessed the protein expression of LAT1, ASCT2 and xCT in the resected tissue specimen, and the expression level of these markers was correlated with pathological variables and outcome after surgery. Additionally, the expression of Ki-67 labeling index and angiogenic markers such as microvessel density (MVD) determined by CD34, were also examined by immunohistochemistry.
METHODS Patients
B
ETWEEN APRIL 1999 and July 2011, we analyzed 89 consecutive patients with HCC who received surgical resection at Gunma University Hospital. Five patients were excluded because these tumor specimens were not be available. Therefore, 84 patients were included and analyzed in this study. The study was approved by the institutional review board. The median age was 62 years, ranging 29–81 years. None of the patients had received neoadjuvant chemotherapy. All surgical specimens were reviewed and classified according to the World Health Organization classification by an experienced pathologist who was unaware of clinical or imaging findings. Pathological tumor–node–metastasis stages were established using the International System for Staging adopted by the American Joint Committee on Cancer and the Union Internationale Centre le Cancer. The day of surgery was considered the starting day for measuring postoperative survival. The follow-up duration ranged 97–3521 days (median, 1009).
Amino acid transporter in HCC 1015
Immunohistochemical staining LAT1 expression was determined by immunohistochemical staining with a murine antihuman LAT1 monoclonal antibody 4A2 (provided by Dr H. Endou [J-Pharma, Tokyo, Japan], 2 mg/mL, 1:3200 dilution).13 The production and characterization of the LAT1 antibody has been described previously.5–7 The anti-CD98 antibody is an affinity purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100 dilution) raised against a peptide mapping at the carboxy terminus of CD98 of human origin. CD98 clearly detects 4F2hc and this antigen is SLC3A2/4F2hc/ CD98hc, therefore, we used this antibody. Anti-ASCT2 and anti-xCT antibodies are affinity-purified rabbit polyclonal antibody (1:300 dilution) and affinitypurified murine polyclonal antibody (Santa Cruz Biotechnology; 1:200 dilution), respectively. The detailed protocol for immunostaining was also published elsewhere.5–7 Briefly, deparaffinized and rehydrated sections were treated with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min to block endogenous peroxidase activity. To expose the antigens, sections were autoclaved in ethylenediaminetetraacetic acid (pH 8.0) for 5 min and cooled for 30 min. After rinsing in phosphate-buffered saline, the sections were incubated with anti-LAT1 antibodies (1:3200), anti-ASCT2 antibodies (1:300) and anti-xCT antibodies (1:200) overnight followed by immunohistochemical staining with a Histofine Simple Stain MAX PO (MULTI) kit (Nichirei, Tokyo, Japan). The peroxidase reaction was carried out using 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl (pH 7.4). Negative control tissue sections were stained as described above, except that the primary antibody was omitted. The LAT1, 4F2hc, ASCT2 and xCT expression scores were assessed by the extent of obvious staining as follows: 0, less than 5% of tumor area stained; 1, 5–10% stained; 2, 11–25% stained; 3, 26–50% stained; and 4, 51% or more stained. The tumors in which stained tumor cells were scored as more than 1 were defined as positive expression. For CD34 and Ki-67, immunohistochemical staining was performed according to the procedures described in previous reports.5–7 The following antibodies were used: murine monoclonal antibodies against CD34 (Nichirei; 1:800 dilution) and Ki-67 (Dako, Glostrup, Denmark; 1:40 dilution). The number of CD34 positive vessels was counted in four selected hot spots in a ×400 field (0.26 mm2 field area). MVD was defined as the mean count of microvessels per 0.26 mm2 field area. The
© 2014 The Japan Society of Hepatology
1016 M. Namikawa et al.
median numbers of CD34 positive vessels were evaluated, and the tumors in which stained tumor cells made up more than each median value were defined as high expression. For Ki-67, a highly cellular area of the immunostained sections was evaluated. All epithelial cells with nuclear staining of any intensity were defined as high expression. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of Ki-67-stained nuclei (Ki-67 labeling index) in the sample. The median value of the Ki-67 labeling index was evaluated, and the tumor cells with greater than the median value were defined as high expression. The sections were assessed by light microscopy in a blinded fashion by at least two of the authors.
Statistical analysis Probability values of less than 0.05 indicated a statistically significant difference. Fisher’s exact test was used to examine the association of two categorical variables. Results are expressed as the mean 1 standard deviation. The significance of differences was determined by Student’s t-test. The correlation between different variables was analyzed using the non-parametric Spearman’s rank correlation coefficient test. Follow up for these 84 patients was conducted using the patient medical records. The Kaplan–Meier method was used to estimate survival as a function of time, and survival differences were analyzed by the log–rank test. Overall survival (OS) was determined as the time from tumor resection to death from any cause. Recurrence-free survival (RFS) was defined as the time between tumor resection and the first disease progression or death. Multivariate analyses were performed using the stepwise Cox proportional hazards model to identify independent prognostic factors. Statistical analysis was performed using JMP version 8 (SAS Institute, Cary, NC, USA) for Windows.
RESULTS Immunohistochemical staining and patients
T
HE IMMUNOHISTOCHEMICAL ANALYSIS of the biomarkers was performed on the 84 primary lesions with HCC. Figure 1 shows the representative immunohistochemical staining of LAT1, ASCT2 and xCT expression. The immunostaining of LAT1, ASCT2 and xCT was detected in carcinoma cells in tumor tissues and localized predominantly on their plasma membrane. These amino acid transporters are partially expressed as island shape in tumor tissues. All positive cells revealed strong membranous immunostaining. In
© 2014 The Japan Society of Hepatology
Hepatology Research 2015; 45: 1014–1022
total patients, a positive LAT1, 4F2hc, ASCT2 and xCT expression was recognized in 61% (50/84), 77% (65/ 84), 63% (53/84) and 65% (55/84), respectively. The average score of LAT1, 4F2hc, ASCT2 and xCT expression was 0.6 1 0.5, 0.8 1 0.5, 1.7 1 0.6 and 1.6 1 0.6 respectively, on a scale of 0–4. The scoring of LAT1 was significantly lower than that of 4F2hc (P = 0.007), ASCT2 (P < 0.001) and xCT (P < 0.001), demonstrating no statistical significance between ASCT2 and xCT (P = 0.467). The median number of CD34 was 14 (range, 2–29), and the value of 14 was chosen as a cut-off point. The median value of the Ki-67 labeling index was 21% (range, 5–72), and the value of 21% was chosen as a cut-off point. The high expression of CD34 and Ki-67 was recognized in 52% (44/85) and 51% (43/85), respectively. Table 1 shows patients’ demographics according to LAT1, ASCT2 and xCT expression. Positive LAT1 expression was significantly associated with 4F2hc expression, Ki-67 labeling index and the value of albumin. There was no significant relationship between ASCT2 expression and any clinicopathological marker. Positive xCT expression yielded a significant association with only tumor differentiation.
Correlation between LAT1 expression and different variables Using Spearman’s rank correlation, LAT1 had a statistically significant correlation with 4F2hc (r = 0.324, P = 0.003), serum albumin (r = −0.255, P = 0.022) and Ki-67 (cell proliferation) (r = 0.264, P = 0.015) (Table 2).
Different variables survival analysis The 5-year survival rates of OS and RFS for all patients were 62.9% and 17.2%, respectively. Tables 3 and 4 shows the results of univariate and multivariate analysis in OS and RFS. By univariate analysis, LAT1 expression, disease stage and albumin had a significant relationship with OS. Tumor size, disease stage, portal vein invasion, albumin and α-fetoprotein had a significant relationship with RFS. Figure 2 shows the Kaplan–Meier survival curve in patients with HCC according to the expression of LAT1. Based on the results of the univariate log–rank test, we screened prognostic factors with a cut-off of P < 0.05. We screened the three factors of LAT1, disease stage and albumin for multivariate analysis. Multivariate analysis confirmed that LAT1 expression, disease stage and albumin were independent prognostic factors for predicting worse OS.
Hepatology Research 2015; 45: 1014–1022
a
b
c
d
e
f
DISCUSSION
T
HIS IS A clinicopathological study to evaluate the expression of amino acid transporters (LAT1, ASCT2 and xCT) in patients with HCC. Of these amino acid transporters, LAT1 expression seemed to play a crucial role in the development and progression of HCC. Our study suggests that LAT1 is closely correlated with the expression of 4F2hc and cell proliferation by Ki-67 labeling index, and is an independent and significant prognostic marker for predicting negative outcome in patients with HCC. Moreover, the expression of LAT1 yielded a significant association with cell proliferation and angiogenesis. However, the expression of LAT1 in HCC was markedly lower among human neoplasms including lung cancer,5 pancreatic cancer,14 breast cancer,15 and head and neck cancer.7 It remains unclear why the expression degree of LAT1 in HCC is lower than
Amino acid transporter in HCC 1017
Figure 1 Representative immunohistochemical staining of a patient with hepatocellular carcinoma. Immunostaining of LAT1 (a), 4F2hc (b), ASCT2 (c) and xCT (d) displays a membranous immunostaining pattern. (e) Negative control of LAT1 immunostaining in patients with hepatocellular carcinoma. No immunostaining of these amino acid transporters (LAT1, ASCT2 and xCT) was observed in normal liver tissue (f). (Original magnifications: [a] ×400; [b] ×400; [c] ×400; [d] ×400; [e] ×400; [f] ×400.)
the other cancers. In the present study, we divided our study population into two groups with or without LAT1 expression, and compared the clinical and prognostic roles of LAT1 with those of ASCT2 and xCT in HCC. We could not find any relationship between LAT1 and the other transporters (ASCT2 and xCT), however, LAT1, ASCT2 and xCT were positively expressed in patients with HCC. Recently, Li et al. reported that LAT1 was overexpressed in HCC, having a significant association with tumor size, histological differentiation and tumor staging, and a high expression of LAT1 had a significantly increased risk of shortened survival time.16 The study also showed that LAT1 mRNA expression level was significantly higher in HCC tissues than in the corresponding non-cancerous tissues. This is almost corresponding to the results of our study. However, they did not investigate how the upregulation of LAT1 is affected
© 2014 The Japan Society of Hepatology
© 2014 The Japan Society of Hepatology 50/50 68/32 4/80/16 23/77 40/60 49/51 32/68 18/82 46/54 50/50 44/56 46/54 88/12 64/36 68/32 56/44 48/52
74/26 7/70/23 23/77 44/56 51/49 33/67 15/85 44/56 37/63 50/50 49/51 77/23 63/37 65/35 46/54 52/48
Positive (n = 50) (%)
46/54
Total (n = 84) (%)
56/44
33/67
62/38
62/38
62/38
56/44
59/41
26/74
41/59
13/87
35/65
55/45
50/50
24/76
12/56/32
82/18
41/59
Negative (n = 34) (%)
LAT1 expression
0.511
0.045
0.642
>0.999
0.007
0.472
0.193
0.027
0.823
0.546
0.816
0.656
53/47
55/45
72/28
–
77/23
40/60
49/51
42/58
45/55
13/87
34/66
47/53
47/53
11/89
>0.999 0.381
8/68/24
74/26
43/57
Positive (n = 50) (%)
0.061
0.206
0.506
P
48/52
32/68
55/45
–
77/23
58/42
55/45
39/61
39/61
19/81
32/68
55/45
42/58
23/77
6/74/20
74/26
65/35
Negative (n = 34) (%)
ASCT2 expression
0.821
0.069
0.115
58/42
45/55
–
69/31
78/22
>0.999 –
51/49
55/45
42/58
47/53
0.117
0.655
0.822
0.649
11/89
27/73
>0.999 0.536
56/44
51/49
22/78
0.651
0.657
0.215
5/69/26
76/24
>0.999 0.865
55/45
Positive (n = 50) (%)
0.073
P
38/62
48/52
–
52/48
76/24
38/62
45/55
38/62
34/66
24/76
45/55
38/62
34/66
3/97
10/72/18
69/31
45/55
Negative (n = 34) (%)
xCT expression
0.108
0.822
–
0.115
0.791
0.357
0.492
0.817
0.354
0.125
0.144
0.168
0.172
0.029
0.505
0.602
0.492
P
Bolding indicates statistical significance. AFP, α-fetoprotein; ASCT2, HBV, hepatitis B virus; HCV, hepatitis C virus; Ki-67 LI, Ki-67 labeling index; MD, moderately differentiated; PD, poorly differentiated; PIVKA-II, protein induced by vitamin K absence-II; WD, well differentiated.
Age 265/>65 years Sex Male/female Etiology HBV/HCV/others Differentiation WD/MD or PD Disease stage I or II/III or IV Tumor size
