Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates from the superior mucosal epithelium of the nasopharyngeal cavity. The incidence of NPC is characterized by a distinct geographical distribution. In 2018, the International Agency for Research on Cancer (IRAC) estimated over 70% new cases occur in East and Southeast Asia.1 The estimated age-standardized incidence rate of NPC is about 0.4 new cases per 100,000 individuals in North America, while the incidence rate is less than 3.0 per 100,000 person-years in China.2 As the most common cancer in the head and neck regions, the main risk factors1 of NPC include environmental factors, history of Epstein-Barr (EBV) virus infection, smoking, drinking, habitual consumption of preserved foods, and genetic susceptibility.3–5 Intensity-modulated radiotherapy (IMRT) is still the main therapeutic approach for NPC, however, for patients with advanced stage disease, the 5 year survival rate is 50–60%.6 Aberrant gene expression has been associated with malignant progression and poor prognosis in patients with NPC.7–9
Human polycomb protein 2(hPC2) also known as Chromobox homolog 4 (CBX4), is a member of the polycomb repressive complex 1(PRC1). PcG-PRC1 complex, which acts by chromatin remodeling and histone modification, plays a pivotal role in the lineage differentiation of the embryonic mesoderm layer.10 CBX4 is a protein-coding gene with chromatin binding and protein ligase activity, and is involved in related signaling pathways including cell senescence and small ubiquitin-related modifiers(SUMOs).11 Accumulating evidence has demonstrated that dysregulation of hPC2 is involved in many malignancies. The expression of CBX4/hPC2 has also been correlated with the clinical prognosis of hepatocellular carcinoma, osteosarcoma, and breast cancer.12–14 However, the expression pattern and prognostic significance of CBX4/hPC2 remain unclear in NPC. Herein, we used Immunohistochemistry (IHC) to detect the expression of hPC2 and investigated its prognostic value in NPC.
Materials and Methods
Patients and Specimens
A total of 180 subjects were recruited from the Xinjiang Autonomous Region People’s Hospital from January 2000 to December 2013. The archived paraffin biopsy tissue specimens corresponding to the patients’ follow-up visits were collected and sectioned. The histological type was established for head and neck tumors according to the World Health Organization (WHO) 2006 classification, and the TNM stage of NPC was defined using the AJCC Cancer Staging Manual, 7th Edition.15 NPC patients were enrolled based on the following criteria: absence of distant metastasis at the first presentation, initial diagnosis histopathologically confirmed, and no history of anti-tumor treatments before diagnosis. The exclusion criteria were the presence of other malignant tumors, previous antitumor treatment, death from non-tumor-related reasons, and incomplete follow-up data. We calculated the overall survival (OS) from the end of radiotherapy until death or the last follow-up. Recurrence-free survival (RFS) was defined as the interval from the date of radiotherapy completion to the date of first recurrence or the last follow-up. Distant metastasis-free survival (DMFS) was defined as the interval from the end of radiotherapy to the date of first distant organ metastasis or end of follow-up. All recurrences or distant metastases were confirmed by nasal endoscopy, magnetic resonance imaging (MRI), or computed tomography (CT) imaging. The present study was approved by the Ethics Committee of Xinjiang Autonomous Region People’s Hospital. Informed, written consent was obtained from all participants and the entire study was performed according to the principles of the Declaration of Helsinki.
A total of 180 NPC tissue samples were collected. Briefly, the formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut into 4-μm paraffin sections were then dried in the oven at 60°C for 60min. Immunohistochemistry (IHC) staining was performed according to the streptavidin-peroxidase method, sections were dewaxed in preheated xylene and rehydrated through incubation in an ethanol gradient (100%, 95%, 85%, 75%),then immersed in 3% hydrogen peroxide (H2O2) for 15 min. Antigen retrieval was performed by heating in a pressure cooker with citrate buffer (pH 6.0) for 5 min, followed by recovery at room temperature (25°C),Non-specific binding was blocked with 5% non-immunologic goat serum (Zhongshan Golden bridge Biotechnology, Beijing China) for 30 min at room temperature and was followed by incubation with the rabbit polyclonal anti-hPC2 (Bethyl, Cat. No. IHC 00668,1:100 dilution) overnight at 4°C in a humidified chamber. After washing with PBS, a secondary antibody (Gene Tech, Cat. No 500710) was incubated at room temperature for 30min. Slides were rinsed in PBS and peroxidase substrate DAB was added for color development for 3min. The sections were counterstained with hematoxylin, dehydrated in a graded series of ethanol (75%, 85%, 95%, 100%),followed by xylene, and cover slipped. Known positive human breast cancer tissue slide was used as positive control, the primary antibody was replaced by IgG from normal goat serum as a negative control, and PBS was applied as the blank control.
The slides were evaluated independently by two pathologists blinded to the clinicopathological and follow-up information. A semiquantitative scoring criterion for IHC was used, in which both staining intensity and the percentage of positive cells were scored. The color score was based on the staining intensity (colorless: 0; mild brown: 1; moderate brown: 2; and strong brown: 3).Under a 100-fold upright optical microscope, five random visual fields were counted for each sample section, one score was given according to the percentage of positive staining cells in each field, with a range from 0–100 by 5 increments (0, 5, 10 … 100). Another score was given based on the staining intensity category, and varied from 0 to 3 (0, 1, 2, 3). The H-score in each field was calculated by multiplying the above two scores (H-score=1×I1+2×I2+3×I3), and the final H-score was obtained as the average H-score value ranging from 0 to 300.16 We used the ROC curve to determine the cut-off value17 of hPC2expression in NPC. According to the ROC curve analysis, the cut-off value 160 was used to divide the patients into two groups: samples with IHC score below or equal to the threshold were defined as low expression, while samples with IHC score above the threshold were defined as high-expression.
Statistical analysis was performed using SPSS Software, version 16.0 (SPSS Inc., Chicago, IL, USA). A receiver-operating characteristic (ROC) curve analysis was used to determine the immunohistochemical cut-off value for high or low expression. Survival curves were plotted using the Kaplan–Meier method and compared using the Log rank test. The Cox proportional hazards model was used for univariate and multivariate survival analysis. Significant variables in the univariate analysis were selected for the multivariate analysis. In all analyses, a 2-tailed, p-value<0.05 was considered statistically significant.
Expression of hPC2 In clinical NPC Samples and Cut-Off Value for hPC2 Expression
hPC2 was expressed in 91.7% (165/180) of NPCs; positive staining was mainly located in the nucleus (Figure 1). The number of samples with high expression and low expression were 48.3% (87/180) and 51.7% (93/180), respectively (Table 1). A ROC curve for the sensitivity and specificity of the clinicopathological parameters were plotted and 160 was chosen as the cut-off value for separating hPC2 expression levels, sensitivity and specificity was 91.3% and 87.5% respectively. The area under the curve (AUC) values for each variables were calculated (Figure 2).
Table 1 Correlation Between the hPC2 Expression and Clinicopathological Features in NPC
Figure 2 Receiver operator characteristic (ROC) curve analysis was used to determinate the cut-off values for hPC2 expression in NPC. Sensitivity and 1-specificity for each clinical parameter were…
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