Upregulation of cyclin D1 can act as an independent prognostic marker for longer survival time in human nasopharyngeal carcinoma

Abstract Background Cyclin D1 is an essential part of oncogenic transformation. We previously proved that cyclin D1 was upregulated in nasopharyngeal carcinoma (NPC) and promoted the NPC cell proliferation. But the association between cyclin D1 and the clinical outcome of NPC has not yet been determined. The study explores the possible relevance between the cyclin D1 expression and clinical parameters and its predictive value of prognosis in NPC patients. Methods We analyzed the clinical data from 379 NPC patients and 112 non‐NPC patients in our previous study, which made further statistics. Receiver operating curve (ROC) was applied to select the optimal cutoff points. By analyzing the clinical data from 101 NPC patients using Chi‐squared test, we estimated the relationship between the cyclin D1 expression level and clinicopathological parameters. We also used Kaplan‐Meier method and log‐rank test assess and compared the disease‐free survival (DFS) rate and overall survival (OS) rate. The Cox proportional hazards model was adopted to perform the univariate and multivariate analyses. Result Receiver operating curve analysis reported that cyclin D1 was used to differentiate between NPC patients and non‐NPC patients (P < .001, sensitivity: 53.6%, specificity: 85.7%, AUC = 0.752). Cyclin D1 was positively correlated with lymph node metastasis (P = .015). A survival analysis of the 101 NPC patients indicated that the positive expression of cyclin D1 was predictive of a good prognosis (DFS: P = .010, OS: P = .019). Multivariate analysis showed that cyclin D1 could be used independently to predict NPC patients' prognosis (DFS: P = .038). Conclusion The overexpression of cyclin D1 is a good prognostic marker for NPC.


| INTRODUC TI ON
Nasopharyngeal carcinoma (NPC) is one of the most commonly seen head and neck malignancy with high prevalence in Asia, especially in China. 1,2 Such unique ethnic and geographical distribution of NPC suggests that the causes of this disease may be linked to the genetic and environment factors. 3,4 Although great progress has been made in early diagnosis and treatment for NPC, the mortality rates still remain high as a result of local recurrences and distant metastasis. [5][6][7][8] Moreover, many biomarkers of NPC are undetected, and the factors related to prognosis of NPC are still unclear. Thus, the discovery of new biomarkers for NPC is helpful to the development of prognosis and treatment strategies.
The abnormal G1-S phase in a cell cycle is one of the significant characteristics of cancer. The G1/S abnormality usually occurs in most epithelial tumors, which may lead to growth advantages and increase the occurrence of tumors. 9,10 Cyclin D1 is encoded by CCND1, which is located on chromosome 11q13. As a known regulator, cyclin D1 binds with cyclin-dependent kinase (cdk 4/6) and inhibits the activation of retinoblastoma (Rb) protein, thus regulating the process from G1 to S phase. Accumulating researches have reported that cyclin D1 involved in cell cycle control, transcription regulation, and DNA repair. 11,12 The overexpression of Cyclin D1 may, directly and indirectly, affect the other cellular processes, thus promote the development and progression of cancers. Previously, we have proved that cyclin D1 was evidently upregulated in NPC and positively related with its progression. 13 However, the relationship between the expression of cyclin D1 and clinical outcome of NPC is still not clear. Thus, in this research, we analyzed the clinical parameters and outcome of NPC, and provided a theoretical basis for the prognosis evaluation of NPC by analyzing the prognostic factors.

| Survival analysis
Valid follow-up data of the 101 NPC patients was collected on June 30, 2019 and the correlation between the expression of cyclin D1 and clinicopathological parameters is presented in Table 1. The longest survival time was 182 months. Overall survival (OS) refers to the time from the first diagnosis to the death of any cause. Disease-free survival (DFS) refers to the time from the diagnosis to the first treatment failure. We used Kaplan-Meier method to estimate the OS and DFS, and the log-rank test to compare the differences. If P < .05, the results analyzed in the log-rank test were considered statistically significant. Cox regression analysis, which could be used to detect independent predictors of survival using a stepwise enter method, was carried out to examine the effect of age, gender, TNM stage, lymph node metastasis, and the expression of cyclin D1 on the clinical outcome. In a two-tailed test, the result was considered statistically significant when P < .05.

| Statistical analysis
Chi-squared test was used to examine the relationship between the expression of cyclin D1 and clinicopathological parameters. Receiver operating curve (ROC) showed the sensitivity and specificity of a  Table 2 showed the relationship between clinicopathological parameters of NPC and the expression of cyclin D1. Cyclin D1 expression was positively correlated with lymph node metastasis (P = .015).

| Correlations between the expression of cyclin D1 and clinicopathological parameters in NPC patients
However, no association between the cyclin D1 expression and age, sex, histological type, tumor stage, and clinical stage was found.

| Cyclin D1 could be used to distinguish NPC and non-NPC patients
The value of cyclin D1 expression in distinguishing between NPC and non-NPC patients was assessed. An optimal cutoff value was set to test some important date points, and then the area under the curve (AUC), sensitivity, and specificity were calculated. The ROC curve for the scoring systems for all NPC and non-NPC patients was shown in Figure 1. The AUC for cyclin D1 expression in the prediction of NPC patients was 0.752 (95% confidence interval = 0.705-0.798, P < .001), with a sensitivity of 53.6%, and specificity of 85.7%.   (Table 4). In contrast, studies found that increased levels of cyclin D1 expression was associated with positive prognosis in breast cancer. 27,28 These researches indicated that cyclin D1 may play different roles in the prognosis of cancer. But the mechanism of cyclin D1 survival prognosis is unknown.

| D ISCUSS I ON
Our previous research had demonstrated that cyclin D1 was overexpressed in NPC. However, the association between the expression of cyclin D1 and clinicopathological parameters was not clearly. In this study, we analyzed the association between cyclin D1 and clinicopathological parameters, and extended the follow-up period of NPC patients. No significant correlation between cyclin D1 expression and patients' age gender, histological type, tumor stage, and clinical stage was observed in this study, and similar results were reported by many previous studies. 29,30 However, a significant positive correlation between cyclin D1 overexpression and lymph node metastasis was found in our study, which was consistent with other studies. 31  In conclusion, this study proves that cyclin D1 expression had the strongest correlation with lymph node metastasis and could help to distinguish NPC and non-NPC patients. Furthermore, the overexpression of cyclin D1 in NPC is closely association with good prognosis in NPC patients. However, the specific mechanism of how cyclin D1 expression relates to prognosis in NPC patients has not been elucidated. Thus, we need to explore this mechanism in further studies.

ACK N OWLED G M ENTS
This research was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
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