The whole-blood Epstein-Barr virus DNA can serve as a valuable molecular marker for diagnosis and prognosis prediction of nasopharyngeal carcinoma

Abstract

The clinical significance and prognostic role of whole-blood EBV-DNA in EBV-associated nasopharyngeal carcinoma (NPC) have not been thoroughly investigated. This study aims to explore the diagnostic and prognostic value of EBV-DNA for NPC in a non-endemic region of China. We enrolled patients with chronic active EBV infection (CAEBV), nasopharyngitis (NA), extranodal NK/T-cell lymphoma, nasal type (ENKTCL-NT), and NPC. Demographic and clinical data were collected and the diagnostic and prognostic values of EBV-DNA were analyzed. Immunohistochemistry was performed to detect EBV-encoded small ribonucleic acids (EBER), as well as the expression of p53, Ki-67, and epidermal growth factor receptor (EGFR). The levels of pretreatment Epstein-Barr virus DNA (preEBV-DNA) in new NPC cases were found to differ from those in other diseases and exhibited varying age distributions. The threshold value of preEBV-DNA for distinguishing NPC from CAEBV and NA was determined. We confirmed that epistaxis, diabetes mellitus, T3N2 or T4N0-2 stage, and IgM positivity were associated with higher levels of preEBV-DNA, and identified risk factors associated with the prognosis of locoregionally advanced NPC (La-NPC). Patients with intermittently or persistently positive EBV-DNA (IPCP), higher preEBV-DNA levels, and positive Epstein-Barr virus-encoded small RNA (EBER) status (EBERpos) had worse survival. New cases of NPC with elevated levels of EBV in the whole-blood and positive EBER status were shown to have a poor prognosis upon progression to La-NPC. EBV-DNA was found to be an indicator for predicting prognosis in La-NPC and could also be used to distinguish new NPC cases.

Keywords: Epstein-Barr virus; NPC; nasopharyngeal carcinoma; whole-blood EBV-DNA.

Figure 1

The flowchart illustrates the experimental design of this study.

Figure 2

Difference and diagnostic performance at diagnosis in whole blood EBV-DNA. A. Box plot showing preEBV-DNA levels at diagnosis among patients with similar symptoms. B. Box plot showing preEBV-DNA levels at diagnosis between two groups of new NPC patients. C. Box plot showing preEBV-DNA levels at diagnosis among different age groups of new NPC patients. D. Receiver operating characteristic (ROC) curve analysis comparing NPC with CAEBV, ENKTCL-NT, and NA (n = 432). E. ROC curve analysis comparing NPC with CAEBV and NA using optimal cutoff values (n = 394). NPC: nasopharyngeal carcinoma; CAEBV: chronic active EBV infection; NA: nasopharyngitis; ENKTCL-NT: extranodal NK/T-cell lymphoma, nasal type.

Figure 3

Regression analysis for new EBV high-NPC patients and risk stratification for La-NPC patients. (A) Forest plot of univariate analysis and (B) multivariate regression analysis in the new NPC cohort (n = 256, odds ratio with 95% confidence interval). (C) Sankey diagram illustrating the risk stratification of La-NPC patients. Cus: comorbidities (epistaxis) or underlying diseases (diabetes mellitus and/or hypertension); EBERpos/EGFRpos/p53pos: positive EBER/EGFR/p53 status; EBVhigh: preEBV-DNA levels above the mean of 3,563 copies/ml; Ki-67high: Ki-67 index above 60%; T: tumor size; N: nearby lymph nodes with cancer (Nx, N0-2); IP: Intermittently Positive; CP: Consistently Positive.

Figure 4

EBV-DNA levels, nomogram construction and risk analysis. A. Box plot showing pre-treatment EBV-DNA levels at diagnosis for each group. B. Box plot showing pre-treatment EBV-DNA levels during treatment for each group. C. Box plot showing EBV-DNA levels for different treatment statuses. D. Box plot showing EBV-DNA levels for different IgM statuses. E. Nomogram for predicting 1-year, 2-year, and 3-year survival probabilities. F-H. Calibration curves of the nomogram in the La-NPC cohort. The x-axis represents the predicted probability of the nomogram and the y-axis represents the actual survival probability. TN: Turn Negative; preEBV-DNA: pretreatment EBV-DNA; posEBV-DNA: post-treatment EBV-DNA; IgMneg: negative IgM results; IgMpos: positive IgM results.

Figure 5

Overall survival analysis based on different factors for La-NPC patients. A. Overall survival comparison between CNTN and IPCP. B. Overall survival comparison between high and negative EBV-DNA levels. C. Overall survival comparison between positive and negative p53 status. D. Overall survival comparison between high and low Ki-67 index. E. Overall survival comparison between positive and negative EBER status. F. Overall survival comparison between positive and negative EGFR status. CN: consistently negative; EBVneg: negative preEBV-DNA; EBERneg/EGFRneg/p53neg: negative EBER/EGFR/p53 status; Ki-67low: Ki-67 index is below 60%.