Increased expression of flotillin-2 protein as a novel biomarker for lymph node metastasis in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor rare throughout most of the world but common in Southeast Asia, especially in Southern China. Flotillin-2 (Flot-2) is not only an important component of cellular membrane, but also involves in various cellular processes such as membrane trafficking, T cell and B cell activation, regulation of several signaling pathways associated with cell growth and malignant transformation, keeping structure and junction of epidermal cells and formation of filopodia. Although such molecular effects of Flot-2 have been reported, whether the expression of Flot-2 protein is associated with clinicopathologic implication for NPC has not been reported. The purpose of this research is to investigate the expression of Flot-2 protein in NPC and control nasopharyngeal epithelial tissues by immunohistochemistry and elucidate the association between the expression of Flot-2 protein and clinicopathological characteristics of NPC. The results showed that the positive percentage of Flot-2 expression in the NPC, nasopharyngeal epithelia with atypical hyperplasia and in the control nasopharyngeal mucosa epithelia was 88.8% (119/134), 76.9% (10/13) and 5.7% (5/88), respectively. There was significantly higher expression of Flot-2 protein in NPC and nasopharyngeal epithelia with atypical hyperplasia compared to the control nasopharyngeal mucosa epithelia (P<0.001, respectively). The positive percentage of Flot-2 protein expression in NPC patients with lymph node metastasis was significantly higher than those without lymph node metastasis. Increasing of Flot-2 expression was obviously correlated with clinical stages of NPC patients. The expression of Flot-2 was proved to be the independent predicted factor for lymph node metastasis by multivariate analysis. The sensitivity of Flot-2 for predicting lymph node metastasis of NPC patients was 93%. Taken together, our results suggest that the increased expression of Flot-2 protein is a novel higher sensitivity biomarker that can predict lymph node metastases in NPC.

Figure 1. Expression of Flot-2 protein in NPC, atypical hyperplasia epithelial cells and control nasopharyngeal epithelial cells was detected by IHC.

The expression of Flot-2 protein was detected by immunohistochemistry using specific antibody as described in the section of materials and methods. Strong positive expression of Flot-2 protein was found in NPC cell membranes (Fig 1A, 20×, IHC, DAB staining). Weak Flot-2 membrane-staining was showed in the atypical hyperplasia epithelial cells (Fig 1B, 20×, IHC, DAB staining). Negative staining of Flot-2 was showed that in the control nasopharyngeal epithelial cells (Fig 1C, 20×, IHC, DAB staining). Negative control showed no Flot-2 staining in the NPC cells (Fig 1D, 20×, IHC, DAB staining).

Figure 2. Expression of Flot-2 protein in NPC and atypical hyperplasia nasopharyngeal epithelia compared to the control nasopharyngeal mucosa epithelia.

Results showed that there were significant differences between the groups which were statistically evaluated by chi-square test.

Figure 3. Kaplan-Meier curve for overall survival of NPC patients with expression of Flot-2 and different clinicopathological characteristics.

Kaplan-Meier analysis to plot the overall survival curves of all 134 NPC patients with expression of Flot-2 and different clinicopathological characteristics and statistical significance was assessed using the long-rank test. Fig 3A Expression of Flot-2 protein in the NPC patients was no significantly related to their prognosis (P>0.05, two sided). Fig 3B NPC patients with lymph node metastasis were significantly related to poor prognosis compared to those patients without lymph node metastasis (P = 0.009, two sided). Fig 3C NPC patients with clinical stage III-IV were significantly related to poor prognosis compared to those patients with clinical stage I-II (P = 0.005, two sided).