Abstract 3359: Expression of stem cell markers Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II tongue squamous cell carcinoma
Authors: Habu Noboru , Imanishi Yorihisa , Kameyama Kaori , Shimoda Masayuki , Tokumaru Yutaka , Sakamoto Koji , Fujii Ryoichi , Shigetomi Seiji , Otsuka Kuninori , Sato Yoichiro , Watanabe Yoshihiro , Ozawa Hiroyuki , Tomita Toshiki , Fujii Masato , Ogawa Kaoru 
Published: July 2016

Abstract
Background: The side population (SP) of cancer cells is reportedly enriched with cancer stem cells (CSCs), however, the functional role and clinical relevance of CSC marker molecules upregulated in the SP of head and neck squamous carcinoma (HNSCC) cells are yet to be elucidated. Patients with clinical stage I/II (T1-2N0M0) tongue squamous cell carcinoma (TSCC) typically undergo partial glossectomy alone; however, development of delayed neck metastasis (DNM) tends to reduce their survival. In the present study, we aimed to determine the CSC markers in the SP of HNSCC cells along with their functions in cellular behaviors, and to clarify the association of these markers with DNM.
Methods: Flow cytometry was applied to isolate SP from main population (MP) in HNSCC cells (SCC4, SAS, and HSC4). The expression of the CSC markers was examined by semi-quantitative RT-PCR and immunocytochemistry. In vitro proliferation, migration, and invasion assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Oct3/4 and Nanog were evaluated using surgical specimens from 50 patients with stage I/II TSCC who underwent partial glossectomy alone.
Results: SPs were isolated in all three cell lines examined. Expression levels of Oct3/4 and Nanog were higher in SP cells than MP cells. Additionally, cell migration and invasion abilities were higher in SP cells than MP cells, whereas there was no difference in proliferation. Univariate analysis showed that expression of Oct3/4 and Nanog, vascular invasion, muscular invasion, and mode of invasion were significantly correlated with DNM. Multivariate logistic regression revealed that Oct3/4 expression (risk ratio = 14.78, p = 0.002) and vascular invasion (risk ratio = 12.93, p = 0.017) were independently predictive of DNM. Regarding the diagnostic performance, Oct3/4 showed the highest accuracy, sensitivity, and negative predictive value of 82.0%, 61.5%, and 86.8%, respectively, while vascular invasion showed the highest specificity and positive predictive value of 94.6% and 71.4%, respectively.
Conclusion: These results suggest that Oct3/4 and Nanog represent probable CSC markers in HNSCC, which contribute to the development of DNM in part by enhancing cell motility and invasiveness. Moreover, along with vascular invasion, expression of Oct3/4 in the primary cancer cells can be considered a potential predictor for selecting patients at high risk of developing DNM.
Citation Format: Noboru Habu, Yorihisa Imanishi, Kaori Kameyama, Masayuki Shimoda, Yutaka Tokumaru, Koji Sakamoto, Ryoichi Fujii, Seiji Shigetomi, Kuninori Otsuka, Yoichiro Sato, Yoshihiro Watanabe, Hiroyuki Ozawa, Toshiki Tomita, Masato Fujii, Kaoru Ogawa. Expression of stem cell markers Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3359.