ORIGINAL ARTICLE

CD98 as a novel prognostic indicator for patients with stage III/IV hypopharyngeal squamous cell carcinoma Miroru Toyoda, MD,1 Kyoichi Kaira, MD, PhD,2,5* Masato Shino, MD, PhD,1 Koichi Sakakura, MD, PhD,1 Katsumasa Takahashi, MD, PhD,1 Yukihiro Takayasu, MD, PhD,1 Hideyuki Tominaga, PhD,4 Noboru Oriuchi, MD, PhD,3 Osamu Nikkuni, MD, PhD,1,6 Masami Suzuki, MD,6 Misa Iijima, MD,7 Norifumi Tsukamoto, MD, PhD,5 Shushi Nagamori, PhD,8 Yoshikatsu Kanai, MD, PhD,8 Tetsunari Oyama, MD, PhD,2 Kazuaki Chikamatsu, MD, PhD1 1

Department of Otolaryngology–Head and Neck Surgery, Gunma University Graduate School of Medicine and Oncology Center, Gunma University Hospital, Gunma, Japan, 2Department of Diagnostic Pathology, Gunma University Graduate School of Medicine, Gunma University Hospital, Gunma, Japan, 3Department of Diagnostic Radiology and Nuclear Medicine and 4Department of Molecular Imaging, Gunma University Graduate School of Medicine and Oncology Center, Gunma University Hospital, Gunma, Japan, 5Oncology Center, Gunma University Hospital, Gunma, Japan, 6Department of Head and Neck Surgery, Gunma Prefectural Cancer Center, Gunma, Japan, 7Department of Pathology and Clinical Laboratories, Gunma Prefectural Cancer Center, Gunma, Japan, 8Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan.

Accepted 4 June 2014 Published online 1 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23797

ABSTRACT: Background. Both L-type amino acid transporter 1 (LAT1) and CD98 are strongly expressed in primary human cancer and play essential roles in tumor growth. We studied the clinicopathological significance of LAT1 and CD98 expression in hypopharyngeal squamous cell carcinoma (SCC). Methods. A total of 70 patients with stage III/IV disease were retrospectively reviewed. Immunohistochemical staining of tumor sections was used to examine LAT1, CD98, Ki-67, CD34, and p53. Results. High LAT1 and CD98 expression were noted in 60.0% and 47.1%, respectively (p 5 .174). A statistically significant correlation was recognized between LAT1 and CD98 expression and both expressions

INTRODUCTION Head and neck cancers include malignant neoplasms arising from a variety of sites in the upper aerodigestive tract, with the most common sites being the oropharynx, hypopharynx, larynx, and oral cavity.1 Primary surgery followed by chemoradiotherapy or concurrent platinumbased chemoradiotherapy constitutes the standard treatment for patients with locally advanced head and neck cancer.2 Among the types of head and neck cancer, hypopharyngeal squamous cell carcinoma (SCC) is highly aggressive disease characterized by invasion to

*Corresponding author: K. Kaira, Department of Diagnostic Pathology, Gunma University Graduate School of Medicine and Oncology Center, Gunma University Hospital, Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: [email protected] Contract grant sponsor: This work was supported in part by grant 23591750 (K. K.) and grant 23592523 (K. C.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and National Hospital Organization Policy Based Medical Services. Miroru Toyoda and Kyoichi Kaira contributed equally to this work. Additional Supporting Information may be found in the online version of this article.

were closely associated with tumor cell proliferation. Although LAT1 expression was not significantly associated with poor survival, multivariate analysis revealed high CD98 expression to be an independent prognostic factor for predicting a poor outcome. Conclusion. CD98 is a promising prognostic marker for predicting outcomes after surgical treatment in patients with advanced hypopharyngC 2014 Wiley Periodicals, Inc. Head Neck 37:1569-1574, 2015 eal SCC. V

KEY WORDS: hypopharynx, L-type amino acid transporter 1 (LAT1), CD98, immunohistochemistry, prognostic factor

adjacent structures and enlarged cervical lymph node deposits with a dismal prognosis. Unfortunately, current chemotherapeutic regimens have limited efficacy; therefore, new effective therapies are needed to improve the survival of patients with hypopharyngeal SCC. Furthermore, clinical markers that can predict the prognosis after treatment should be established to improve the outcome of therapy. Amino acid transporters play an essential role in the growth and proliferation of both normal cells and transformed cells.3,4 L-type amino acid transporter 1 (LAT1) transports large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine, and requires a covalent association with the heavy chain of 4F2 cell surface antigen (CD98) for its functional expression in the plasma membrane.5,6 Previous studies have demonstrated that LAT1 is highly expressed in proliferating tissues, many tumor cell lines, and primary human tumors.6–14 The expression of LAT1 is closely associated with cell proliferation, angiogenesis, cell cycle regulator, and also with the expression of CD98.14,15 Recent studies have shown that the expression of LAT1 and CD98 is a significant indicator for predicting poor prognosis in various human cancers, and HEAD & NECK—DOI 10.1002/HED

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several experimental studies have disclosed that transport of most of the amino acids into a human oral cancer cell line is mediated by LAT1 and associated CD98,8–16 and that the inhibition of LAT1 expression suppresses cell growth in human oral cancer cell lines.17 However, it remains to be assured whether the expression of LAT1 and CD98 is similarly critical in the pathogenesis of hypopharyngeal SCC in vivo. In order to elucidate the clinicopathological role of CD98 as well as LAT1 protein expression of LAT1 and CD98 was correlated with pathological markers, such as cell proliferation, p53, angiogenesis, and also with prognosis in patients with surgically resected locoregionally advanced (stage III/IV) hypopharyngeal SCC.

MATERIALS AND METHODS Patients We investigated 78 consecutive patients with pathologically confirmed hypopharyngeal SCC who underwent surgical resection at Gunma University Hospital and Gunma Prefectural Cancer Center between 2001 and 2012. Seven patients with pathological stage I or II disease and a patient without pathological specimen available were excluded from the study. Therefore, a total of 70 patients with stage III/IV disease were analyzed in the study. Formalin-fixed, paraffin-embedded primary tumor samples from the 70 patients were examined. Additionally, tissue specimens of cervical lymph node metastases from 57 patients were available, and 1 to 3 tumor-infiltrated lymph nodes were examined in each case. All surgical specimens were reviewed and classified according to the World Health Organization classification by an experienced pathologist who was unaware of the clinical or imaging findings. Pathologic TNM classifications were established using the Classification of Malignant Tumors by the International Union against Cancer and American Joint Committee on Cancer system. The study protocol was approved by the institutional review board.

Immunohistochemical staining LAT1 expression was determined by immunohistochemical staining with an antihuman LAT1 antibody (1.6 mg/mL, antihuman monoclonal mouse IgG1, KM3149, provided by Kyowa Hakko; dilution of 1:800). The specificity of the antibody was confirmed by Western blotting, as shown in Supplemental Figure S1, online only. A specific single band was detected with the antibody in HEK293 cells stably expressing human LAT1 but not in cells stably expressing human LAT2 (Supplemental Figure S1, online only). The anti-CD98 antibody used is an affinity-purified rabbit polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) raised against a peptide mapping to the carboxy terminus of human CD98. The detailed protocol for immunostaining has been published elsewhere.8,15 The LAT1 and CD98 expression scores were defined by the extent of staining as follows: 1 5 10% of tumor area stained; 2 5 11% to 25% stained; 3 5 26% to 50% stained; 4 5 51% to 75% stained; and 5 5 76%. The tumors in which the stained tumor cells were scored as 3, 4, or 5 were defined as pos1570

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itive; tumors scoring 4 or 5 were defined as having high expression. The immunohistochemical staining for CD34, Ki-67, and p53 was performed according to the procedures described in the previous reports.14 The following antibodies were used: mouse monoclonal antibodies against CD34 (1:800 dilution; Nichirei, Tokyo, Japan), Ki-67 (1:40 dilution; Dako, Glostrup, Denmark), and p53 (D07; 1:50 dilution; Dako). The number of CD34-positive vessels was counted in 4 selected hot spots in a 4003 field (field area of 0.26 mm2). Microvessel density was defined as the mean count of microvessels per 0.26 mm2 field area. The median numbers of CD34-positive vessels were evaluated, and the tumors in which the stained tumor cells comprised more than each median value were defined as positive. For p53, a microscopic examination of the nuclear reaction product was performed and scored. Based on a previous report,14 p53 expression greater than 10% of the tumor cells was defined as positive expression. A highly cellular area of the immunostained sections was evaluated for Ki-67, and the epithelial cells with nuclear staining of any intensity were defined as positive. Approximately 1000 nuclei were counted on each slide, and the proliferative activity was assessed as the percentage of Ki-67stained nuclei (Ki-67 labeling index) in the sample. The median Ki-67 labeling index value was determined, and high expression in the tumor cells was defined as a value greater than the median. The sections were assessed using light microscopy in a blinded fashion by 2 experienced authors.

Statistical analysis Probability values of < .05 indicated a statistically significant difference. Fisher’s exact test was used to examine the association of 2 categorical variables, and the correlation between different variables was analyzed using a nonparametric Spearman’s rank test. Follow-up of the 70 patients was conducted using the patients’ medical records. The Kaplan–Meier method was used to estimate survival as a function of time, and the survival differences were analyzed by a log-rank test. The day of surgery was defined as the starting day for measuring postoperative survival. Overall survival (OS) was determined as the time from tumor resection to death from any cause. Progression-free survival (PFS) was defined as the time between tumor resection and the first disease progression or death. Multivariate analyses were performed using a stepwise Cox proportional hazards model to identify independent prognostic factors. The median follow-up duration was 615 days (range, 30–3549 days). The statistical analysis was performed using GraphPad Prism 4 software (Graph Pad Software, San Diego, CA) and JMP 8 (SAS Institute, Cary, NC) for Windows.

RESULTS Patient demographics Table 1 provides the patient characteristics according to clinicopathological markers. The age of the patients ranged from 37 to 84 years, with a median age of 68 years. Thirteen patients (19%) had stage III disease, and

CD98

TABLE 1. Patient’s demographics. Variable

Age Sex Tumor size, mm T classification N classification Disease stage Pathological differentiation Lymphatic permeation Vascular invasion Resected status LAT1 CD98 Ki-67 CD34 p53

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Immunohistochemical analysis Hypopharynx (n 5 70)

65/>65 M/F 30/>30 T1–3/T4 N0/N1–3 III/IV WD/MD or PD

29/41(41%/59%) 61/9(87%/13%) 17/53(24%/76%) 39/31(56%/44%) 22/48(31%/69%) 13/57(19%/81%) 22/48(31%/39%)

Yes/no Yes/no R0/R1 Positive/negative Positive/negative Positive/negative Positive/negative Positive/negative

58/12(83%/17%) 49/21(70%/30%) 58/12(83%/17%) 60/10(86%/14%) 58/12(83%/12%) 35/35(50%/50%) 34/36(49%/51%) 39/31(56%/44%)

Abbreviations: WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; LAT1, L-type amino acid transporter 1.

57 patients (81%) had stage IV. Forty-nine patients (70%) received postoperative adjuvant chemotherapy with cisplatin, 5-fluorouracil, docetaxel, or S-1 (Taiho Pharmaceutical, Tokyo, Japan), oral administration of tegafur (a fluorouracil derivative drug), and/or radiotherapy.

An immunohistochemical analysis was performed on the 70 primary sites and 57 cervical lymph node metastatic sites. Figure 1 shows the immunohistochemical staining for LAT1 and CD98 expression, which were detected in carcinoma cells in the tumor tissues, predominantly localized at the plasma membrane. All positive cells demonstrated strong membranous LAT1 and CD98 immunostaining, whereas cytoplasmic staining was rarely observed. Positive LAT1 and CD98 expression were found in 85.7% (60 of 70) and 82.8% (58 of 70), respectively (p 5 .816), of the primary sites and 75.4% (43 of 57) and 64.9% (37 of 57), respectively (p 5 .306), of the metastatic lymph nodes. Moreover, high LAT1 and CD98 expression in the primary sites and resected lymph nodes was detected in 60.0% (42 of 70) and 47.1% (33 of 70), respectively (p 5 .174), and 49.1% (28 of 57) and 28.1% (16 of 57), respectively (p 5 .033). The average score of LAT1 and CD98 expression was 3.5 6 1.2 and 3.1 6 1.0, respectively, in all lesions (p 5 .021), 3.7 6 1.1 and 3.7 6 0.9, respectively, in the primary tumors (p 5 .073), and 3.3 6 1.3 and 2.9 6 1.2, respectively, in the lymph nodes (p 5 .131). Based on the analysis (n 5 127) of 70 primary sites and 57 metastatic lymph nodes, the median number of CD34-positive vessels was 13 (range, 2–28), and this value was chosen as a cutoff point. The median value of the Ki-67 labeling index was 38 (range, 3–90), and the value of 38% was chosen as a cutoff point. Positive p53 expression was observed in 55.7% (39 of 70) of the primary sites and 54.4% (31 of 57) of the metastatic lymph nodes. The clinicopathological features were compared with LAT1 and CD98 immunohistochemistry results. High LAT1 expression had only a significantly correlation with N factor, however, positive LAT1 and CD98 expression was not significantly associated with any other factors. The pathological markers were compared between the primary tumor and the resected lymph node. The score of CD98 expression was significantly higher in the primary tumors than in the metastatic lymph nodes.

Correlation between L-type amino acid transporter 1/CD98 expression and different variables LAT1 expression was significantly correlated with CD98, CD34, and the Ki-67 labeling index (Table 2). The expression of LAT1 yielded a statistically significant correlation with CD98 expression and cell proliferation, and the expression of CD98 was also significantly correlated with cell proliferation in the primary tumor.

Univariate and multivariate survival analyses FIGURE 1. Immunostaining of L-type amino acid transporter 1 (LAT1) (A) and CD98 (B) in a tissue from a 73-year-old man with hypopharyngeal squamous cell carcinoma (p stage IV) demonstrating a intense membranous immunostaining pattern with a staining score of 5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

The patients with hypopharyngeal cancer (n 5 70) showed a median survival time and 5-year survival rate for OS of 916 days and 23.8%, respectively, and the median survival time and 5-year survival rate for PFS were 898 days and 27.9%, respectively. Table 3 shows results of the univariate and multivariate analyses in all patients. The univariate analysis demonstrated that lymphatic permeation, vascular invasion, and CD98 expression were HEAD & NECK—DOI 10.1002/HED

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TABLE 2. Correlation between L-type amino acid transporter 1 expression and other biomarkers. Biomarkers

Spearman g

95% CI

p value

All lesions (n 5 127) CD98 0.342 0.173–0.491 CD34 0.259 0.084–0.419 Ki-67 0.309 0.137–0.463 Primary tumors (n 5 70) CD98 0.323 0.087–0.524 CD34 0.184 20.060 to 0.407 Ki-67 0.340 0.107–0.537 Resected lymph node metastatic lesions (n 5 57) CD98 0.301 0.036–0.527 CD34 0.292 0.026–0.519 Ki-67 0.347 0.087–0.563

< .001 .003 .004 .006 .127 .004 .023 .027 .008

Abbreviation: 95% CI, 95% confidence interval. All lesions include primary sites (n 5 70) and resected metastatic lymph nodes (n 5 57). Resected lymph node metastatic lesions consist of 57 resected lesions in 57 patients who received resected metastatic lymph nodes resection. The figures in bold indicate statistical significance.

significantly associated with poor OS, and significant prognostic markers for PFS included lymphatic permeation, vascular invasion, CD98, CD34, and p53. According to the results of the univariate log-rank test, we screened for

prognostic factors with a cutoff of p < .05. The multivariate analysis confirmed that lymphatic permeation and a high CD98 expression were independent prognostic factors for predicting poor OS, and vascular invasion, high CD98 expression, and p53 positivity for poor PFS. Figure 2 shows the Kaplan–Meier survival curve in patients with high and low CD98 expression. We also performed the survival analysis according to positive and negative LAT1 and CD98 expression; however, positive LAT1 and CD98 expression was not significantly associated with poor OS and PFS according to the univariate and multivariate analyses.

DISCUSSION This is the first study to evaluate the protein expression of LAT1 and CD98 in patients with advanced hypopharyngeal SCC. Positive LAT1 and CD98 expression (26% stained) in the primary sites was detected in 85.7% and 82.8% of cases, respectively (p 5 .816), and high LAT1 and CD98 expression (51% stained) was found in 60.0% and 47.1% of cases, respectively (p 5 .174). Semiquantitative analysis revealed that the LAT1 expression score was significantly higher than CD98, however, statistically significant correlation was recognized between LAT1 and CD98 expression, indicating that they are closely associated with tumor cell proliferation in patients

TABLE 3. Univariate and multivariate analysis in overall survival and progression-free survival. OS Univariate 5-y survival rate %

Variable

Age, y 65 / >65 Sex Male / female Disease stage III / IV Pathological differentiation WD / MD or PD Lymphatic permeation Yes / no Vascular invasion Yes / no Lymph node metastasis Yes / no Resected status R0 / R1 LAT1 High / low CD98 High / low Ki-67 Positive / negative CD34 Positive / negative p53 Positive / negative

PFS Multivariate

p value

95% CI (HR)

Univariate

p value

5-y survival rate %

p value

Multivariate

95% CI (HR)

p value

39 / 19

.089

42 / 16

.198

25 / 56

.986

23 / 77

.160

59 / 13

.077

56 / 19

.232

29 / 21

.921

36 / 22

.976

15 / 81

.007

1.070–5.208 (2.043)

.027

21 / 72

.030

0.168–2.997 (0.819)

.768

17 / 69

.004

0.192–0.933 (0.489)

.137

17 / 79

.002

1.103–12.20 (3.207)

.031

21 / 40

.282

27 / 25

.712

26 / 25

.350

29 / 21

.222

31 / 20

.125

34 / 20

.307

6 / 36

.012

9 / 42

.005

1.320–7.960 (2.716)

.034

25 / 17

.714

27 / 26

.915

9 / 39

.261

19 / 50

.013

0.737–3.116 (1.472)

.286

18 / 17

.280

11 / 55

.001

1.309–5.630 (2.617)

.006

1.026–2.019 (1.428)

.034

Abbreviations: OS, overall survival; PFS, progression-free survival; 95% CI, 95% confidence interval; HR, hazard ratio; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; LAT1, L-type amino acid transporter 1. The figures in bold indicate statistical significance.

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FIGURE 2. Prognosis after surgical resection shown by a Kaplan– Meier analysis of overall survival (OS) according to CD98 expression. A statistically significant difference was recognized between patients with high and low CD98 expression. A little table shows number of patients in this plot at 0, 500, 1000, and 2000 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

with hypopharyngeal SCC. If the cutoff point of LAT1 and CD98 expression was defined as more than a score of 3 (positive expression), the expression of these markers did not significantly correlate with the clinicopathological factors and prognosis. However, LAT1 expression with more than a score of 4 (high expression) demonstrated a close relationship with lymph node metastasis. High CD98 expression was shown to be an independent prognostic factor, although LAT1 was not identified as predictive of outcome. The positive rates of LAT1 expression in lung, pancreatic, breast, prostate, and gastric cancer have recently been reported to be 51%, 53%, 43%, 22%, and 41%, respectively.8,11–13,15 In patients with lung cancer, LAT1 expression in SCC (a positive rate of 91%) was significantly higher than adenocarcinoma (a positive rate of 29%), although the mechanism of different expression between SCC and adenocarcinoma remains unknown.8 The positive rate of LAT1 expression in hypopharyngeal SCC was almost similar to that of pulmonary SCC. However, the previous reported cases had undergone surgical resection of early stage diseases and evaluated the expression of LAT1 and CD98. Thus, it remains unknown about the detailed information of LAT1 and CD98 expression in the advanced (stage III/IV) diseases. It is also difficult to obtain adequate tumor specimens from these patients. Therefore, our present study was focused on the role of LAT1 and CD98 expression in patients with advanced disease. In our study, the expression of LAT1 was significantly associated with lymph node metastasis, cell proliferation, and angiogenesis, consistent with the results of previous reports.14,15 However, our study could not identify LAT1 as a potential prognostic factor in advanced

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hypopharyngeal SCC. Further study is warranted to investigate the role of LAT1 as a prognostic indicator in patients with early stage disease. CD98 consists of a type II single-pass transmembrane heavy chain (CD98hc) of approximately 80 to 85 kDa that is disulfide-linked to a multipass light chain of approximately 40 kDa.18 Many tumors express CD98, and its expression in the resected tissue is closely correlated with clinical aggressiveness and poor outcome in solid tumors.13–15 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.19 An in vitro study in human cell lines showed that CD98hc promotes malignant transformation and growth of tumors20 because the intracellular domain of CD98hc regulates integrin-dependent signals via interaction with integrin b subunit. CD98dependent integrin signaling promotes cellular proliferation and adhesion through proteins, such as mitogenactivated protein kinase (MAPK), phosphotidylinositol-3kinase (PI3-K)-Akt. LAT1 imports essential amino acids in exchange for the glutamine that has been imported by other transporters. This CD98-mediated exchange of glutamine in the presence of essential amino acids activates the mammalian target of rapamycin (mTOR) pathway, thereby blocking autophagy and promoting protein synthesis and cell growth. In this study, CD98 was found to be highly expressed in the tumor specimens of advanced hypopharyngeal SCC and was identified as an independent prognostic factor for predicting a poor outcome. Recent studies have demonstrated that CD98 (but not LAT1) expression is an independent predictive marker for patients with advanced non-small cell lung cancer (NSCLC) with lymph node metastasis21; on the other hand, the overexpression of LAT1 is a significant prognostic indicator in early stage NSCLC as compared to CD98.22 These findings indicate that the prognostic role of CD98 may become important from the early stage to the advanced stage in NSCLC. Although our study did not investigate the direct relationship between CD98 expression and signaling pathway of mTOR and integrin, the detailed investigation of mTOR and integrin signaling with the role of CD98 may add insight into tumorigenesis and proliferation in human neoplasms. Recent studies had documented that a high CD98 expression was significantly associated with worse outcome as compared to a low CD98 expression in patients with a human papillomavirus (HPV)-positive oropharyngeal SCC.23,24 Many studies demonstrated that the patients with HPV-positive oropharyngeal SCC respond better to treatment than those with HPV-negative. Cancer stem cells are implicated to play a role in resistance to anticancer therapy. It was reported that CD98 is a promising cancer stem cell enrichment marker in head and neck SCC.24 Previous studies showed the importance and possible new role of CD98 as a cancer stem cell marker.23,24 The results of these studies suggest that a high CD98 expression has a potential of the resistance to anticancer treatment, therefore, it is closely related to worse outcome after therapy.23,24 Although it has been described that HPV is closely associated with the development of oropharyngeal SCC, it remains unknown whether the high HEAD & NECK—DOI 10.1002/HED

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expression of CD98 could be a negative prognostic marker for patients with hypopharyngeal SCC with HPVpositivity. In addition, we have not investigated the relationship between CD98 expression and cancer stem cells in patients with hypopharyngeal SCC. However, CD98 expression has the potential relevance to predict the worse outcome regardless of HPV-positivity in patients with pharyngeal SCC. Laryngopharyngectomy with reconstruction of the pharynx has been an initial treatment modality for hypopharyngeal SCC; in advanced disease, most patients will be treated either with laryngopharyngectomy followed by chemoradiotherapy or induction chemoradiotherapy. Because a primary tumor with high CD98 expression has a potential dismal prognosis after surgery in advanced hypopharyngeal SCC, it may be useful to develop individualized treatment according to the expression level of CD98. We recently described that LAT1, CD98, Ki-67 (cell proliferation), and CD34 (angiogenesis) were highly expressed in the metastatic sites than in the primary tumors.25 In advanced hypopharyngeal SCC, however, the expression of these markers was not significantly different between the primary tumors and lymph node metastases, even though CD98 expression was higher in the primary tumor. Primary tumors with advanced disease have an aggressive malignant feature; therefore, planning of an intensive multimodal therapy aimed at local control with systemic treatment will be required. In conclusion, LAT1 and CD98 were found to be highly expressed in patients with hypopharyngeal SCC, and high CD98 expression proved to be predictive as an independent prognostic marker. Although a significant correlation was observed between LAT1 and CD98 expression, a further investigation is warranted to determine the therapeutic potential of CD98 inhibition in advanced hypopharyngeal cancer.

Acknowledgments

3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

The authors thank all staff members of the Department of Pathology and Clinical Laboratories at Gunma Prefectural Cancer Center for their technical assistance in the immunohistochemical analysis. Advanced research for medical products Mining Programme of the National Institute of Biomedical Innovation (NIBIO) is also acknowledged.

21. 22. 23.

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IV hypopharyngeal squamous cell carcinoma.

Both L-type amino acid transporter 1 (LAT1) and CD98 are strongly expressed in primary human cancer and play essential roles in tumor growth. We studi...
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