Author Affiliations: Department of Medicine and Molecular Science (Kaira K), Department of Thoracic and Visceral Surgery (Sunose Y and Takeyoshi I), and Department of Diagnostic Radiology and Nuclear Medicine (Oriuchi N), Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma, Japan; Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan (Kanai Y)

Corresponding Author: Kyoichi Kaira, MD, Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, and Oncology Center, Gunma University Hospital, Showa-machi, Maebashi, Gunma 371-8511, Japan (Tel: +81-27-2208136; Email: kkaira1970@yahoo.co.jp)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60278-2

Published online July 17, 2014.

 

Contributors: KK designed the study, acquired patient data, and drafted the manuscript. SY collected and reviewed patient information. ON provided advice on patient management. KY and TI made critical revisions to the manuscript. All authors read and approved the final manuscript. KK is the guarantor.

Funding: This study was supported by a grant from the Advanced Research for Medical Products Mining Programme of the National Institute of Biomedical Innovation (NIBIO).

Ethical approval: This study was approved by the institutional review board of Gunma University Hospital (ethical committee for clinical studies-Gunma University faculty of Medicine) and written informed consent was obtained from all of the patients who participated in this study or their families.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

ABSTRACT: CD98 has been described to play a crucial role in tumor progression and survival. However, the role of CD98 in biliary tract cancer remains unclear. We found that 36.7% of all patients with biliary tract cancer had a high CD98 expression. Statistical analysis using Spearman's rank correlation showed that CD98 was significantly correlated with L-type amino acid transporter 1 (LAT1, r=0.562, P<0.001), Ki-67 (r=0.230, P=0.006) and CD34 (r=0.290, P=0.005). Multivariate analysis confirmed that a high CD98 expression was an independent prognostic factor for predicting poor outcome. CD98 is closely associated with tumor growth, biological aggressiveness, and survival of patients. With these data we proposed that CD98 expression is necessary for the development and pathogenesis of biliary tract cancer.

 

(Hepatobiliary Pancreat Dis Int 2014;13:654-657)

KEY WORDS: CD98; biliary tract cancer; amino acid transporter; prognostic factor

Bliary tract cancer is one of the dismal diseases, however, no biomarker has been established to predict the therapeutic response or outcome after surgery. We have recently reported that L-type amino acid transporter 1 (LAT1) could be an independent prognostic marker for predicting poor outcome based on the clinicopathological analysis of tissue specimens in 139 patients with biliary tract adenocarcinoma, as well as in vitro and in vivo antitumor effect by the inhibition of LAT1 in a mouse model.^[1] LAT1 is one of the L-type amino acid transporters, and is highly expressed in various human neoplasms.^{[2, 3]} LAT1 requires covalent association with the heavy chain of 4F2 cell surface antigen (CD98) for its functional expression.^[2] LAT1 and CD98 are coordinately expressed in human cancers, therefore, the level of CD98 expression is closely correlated with that of LAT1 expression.^{[4, 5]} CD98 is a significant predictor for patients with advanced lung cancer^[5] and the co-expression of LAT1 and CD98 has been described to play a crucial role in the tumor survival and development in patients with gastric and pancreatic cancer. The role of CD98 expression has been investigated in other digestive tract cancers such as colorectal cancer, colitis-associated cancer and liver cancer.^[6-8] However, it remains unclear about the clinicopathological significance of CD98 expression in biliary tract cancer, although CD98 is known to promote transformation and growth of tumor.^{[4, 5]} Here, we evaluated the expression of CD98 and its correlation with the disease, and explored the usefulness of CD98 as a new marker for biliary tract cancer.

 

 

We analyzed 139 consecutive patients with biliary tract adenocarcinoma who underwent surgical resection between September 2000 and October 2011. We evaluated the CD98 expression in resected tissue specimens, as described previously.^[1] The study protocol was approved by the institutional review board.

 

The anti-CD98 antibody is an affinity purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., USA, 1:100 dilution) raised against a peptide mapping at the carboxy terminus of CD98 of human origin. LAT1 expression was determined by immunohistochemical staining with LAT1 antibody [2 mg/mL, anti-human monoclonal mouse antibody, 4A2, provided by Dr H. Endou (J-Pharma, Tokyo, Japan), 1:3200 dilution]. The detailed protocol for immunostaining was published elsewhere.^[3] LAT1 and CD98 expression scores were assessed by the extent of staining: 1, ≤10% of tumor area stained; 2, 11%-25% stained; 3, 26%-50% stained; 4, >50% stained (3 or 4 was defined as high expression). CD34 (Nichirei, Tokyo, Japan, 1:800 dilution) and Ki-67 (Dako, Glostrup, Denmark, 1:40 dilution) were also examined according to the previous study.^[1]

 

Statistical analysis was performed using JMP 8 (SAS, Institute Inc., Cary, NC, USA) for Windows. Fisher's exact test was used to examine the association of two categorical variables. The correlation between different variables was analyzed using the nonparametric Spearman's rank-order correlation coeffecient test. A P value of <0.05 indicated a statistically significant difference.

 

 

In the current study, 139 patients with cholangiocarcinoma (CC) comprised 89 patients with extrahepatic CC (EHCC), 20 with intrahepatic CC (IHCC) and 30 with gallbladder carcinoma (GBC). The median age of these patients was 71 years (range 42-86), and most of the patients (90.6%, 126/139) had a pathological stage of I to III. A high CD98 expression was recognized in 36.7% (51/139) of all patients and the average score (mean±SD) of CD98 was 2.04±0.97. The analysis according to the location of CC demonstrated that a high CD98 expression was 34.8% (31/89) in EHCC, 30.0% (6/20) in IHCC, and 46.7% (14/30) in GBC. No statistically significant difference in the ratio of high CD98 expression was observed among EHCC, IHCC and GBC. We also analyzed the pathological markers (LAT1, cell proliferation by Ki-67 labeling index, angiogenic markers such as microvessel density determined by CD34) and postoperative survival according to the CD98 expression. Previously, we reported the immunohistochemical data for LAT1, Ki-67 and CD34 in the same patients with biliary tract cancer.^[1] The present study showed that high LAT1 was observed in 89 (64.0%) of the 139 patients, of whom 59 (66.3%) suffered from EHCC, 12 (60.0%) from IHCC and 18 (60.0%) from GBC. The median CD34-positive vessel was 21% (range 4%-52%) and the median value of the Ki-67 labeling index was 35% (range 2%-76%). High Ki-67 and CD34 were 44.6% (62/139) and 49.6% (69/139), respectively (Fig. 1). The correlation analysis showed that CD98 had a significant correlation with LAT1 (r=0.562, P<0.001), Ki-67 (r=0.230, P=0.006) and CD34 (r=0.290, P=0.005, Table 1).^[1] Survival analyses showed that CD98 had a negative correlation with overall survival and progression-free survival of patients with biliary tract cancer (Table 2 and Fig. 2). Univariate analysis confirmed that resected status, tumor differentiation, lymphatic permeation, vascular invasion, lymph node metastasis, tumor stage, and CD98 expression were prognostic factors for overall survival and progression-free survival. Multivariate analysis demonstrated that lymphatic permeation and a high CD98 expression were independent prognostic factors for predicting poor overall survival, and lymphatic permeation and vascular invasion for poor progression-free survival.

 

 

To our knowledge, this is a first preliminary study on the correlation between CD98 expression and biliary tract cancer, we found that the expression of CD98 was closely associated with tumor cell proliferation, angiogenesis and worse outcome in patients with biliary tract cancer. CD98 as a disulfide-linked 125-kDa heterodimeric membrane glycoprotein has been reported to be associated with cellular proliferation, transformation and adhesion, and also the LAT system.^{[2, 4]} CD98 is expressed at high levels on the surface of various neoplasms.^{[4, 5]} We found that CD98 is a promising pathological marker for predicting worse outcome in cholangiocarcinoma patients. This finding is corresponding to that of the previous studies, and CD98 expression has been identified as a significant worse predictor in human cancers of the lung, pancreas, breast, and urinary tract.^{[3, 5]} Moreover, CD98 expression is also shown to be significantly correlated with the LAT1 expression, cell proliferation, and angiogenesis in various human neoplasms.^[3] Therefore, our data implied that both LAT1 and CD98 play an important role in the development and metastasis of human cancers.

 

The roles of CD98 in cancer have been thought to be closely associated with the functions of amino acid transport and integrin signaling, which promote tumor growth and metastasis.^[4] CD98-mediated exchange of glutamine in the presence of essential amino acids activates the mammalian target of rapamycin pathway, thereby promoting protein synthesis and cell growth.^[2] The CD98 expression is paralleled with the progression of tumors.^[4] Therefore, CD98 may be necessary for the tumor cell growth in biliary tract cancer patients with aggressive features. Our study suggested that CD98 could be a novel prognostic indicator for patients with biliary tract cancer. Further study is warranted to elucidate the detailed mechanism of how CD98 activates the signaling pathway of mTOR and integrin, and to examine the potential of CD98 inhibition as a therapeutic target in patients with cholangiocarcinoma.

 

 

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2 Kanai Y, Segawa H, Miyamoto Ki, Uchino H, Takeda E, Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 1998;273:23629-23632. PMID: 9726963

3 Kaira K, Sunose Y, Arakawa K, Ogawa T, Sunaga N, Shimizu K, et al. Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer. Br J Cancer 2012;107:632-638. PMID: 22805328

4 Cantor JM, Ginsberg MH. CD98 at the crossroads of adaptive immunity and cancer. J Cell Sci 2012;125:1373-1382. PMID: 22499670

5 Kaira K, Oriuchi N, Imai H, Shimizu K, Yanagitani N, Sunaga N, et al. CD98 expression is associated with poor prognosis in resected non-small-cell lung cancer with lymph node metastases. Ann Surg Oncol 2009;16:3473-3481. PMID: 19777189

6 Kaira K, Oriuchi N, Imai H, Shimizu K, Yanagitani N, Sunaga N, et al. L-type amino acid transporter 1 and CD98 expression in primary and metastatic sites of human neoplasms. Cancer Sci 2008;99:2380-2386. PMID: 19018776

7 Nguyen HT, Dalmasso G, Torkvist L, Halfvarson J, Yan Y, Laroui H, et al. CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice. J Clin Invest 2011;121:1733-1747. PMID: 21490400

8 Ohkame H, Masuda H, Ishii Y, Kanai Y. Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models. J Surg Oncol 2001;78:265-272. PMID: 11745822