Analysis of Plasma EBV-DNA and Soluble Checkpoint Proteins in Nasopharyngeal Carcinoma Patients after Definitive Intensity-Modulated Radiotherapy

Abstract

Background: Tumor immunotherapy and immunological checkpoint-related proteins are research hotspots. Intensity-modulated radiotherapy (IMRT) is the main treatment for nasopharyngeal carcinoma (NPC). Hence, the evaluation of its effect is very important. The aim of this study was to assess the relationship between the concentrations of soluble checkpoint proteins, plasma EBV-DNA, and cytokines in NPC patients treated with IMRT.

Methods: In this study, the plasma samples of 37 NPC patients and 40 healthy controls were collected. Luminex MAGPIX was used to detect the concentrations of 32 plasma targets, including soluble programmed cell death 1 (sPD-1). RT-qPCR was used to measure EBV-DNA.

Results: The concentrations of 33 plasma targets were detected in NPC patients before and after IMRT to explore the changes after IMRT. The results showed that IMRT could increase the expression of sPD-1 and significantly reduce the level of EBV-DNA in the plasma of NPC patients. The expression level of sPD-1 in TNM I/II patients was significantly higher than that in III/IV patients. Besides, the concentrations of 12 other targets were significantly different after IMRT, including LAG-3, PD-L1, TIM-3, IFN-γ, IL-12p70, IL-1β, IL-5, IL-6, TNF-α, IL-10, IL-17A, and IL-22. High sPD-1 patients had longer survival than those with low sPD-1. Also, patients with lower EBV-DNA and TNM grades I and II/III had longer survival than those with higher EBV-DNA or TNM IV.

Conclusions: This study demonstrated that the concentration of sPD-1 was significantly increased and EBV-DNA was significantly reduced in the NPC patients after IMRT. Plasma EBV-DNA level was a highly specific and sensitive biomarker for NPC diagnosis. Both sPD-1 expression and EBV-DNA concentration in plasma were related to the survival of patients.

Figure 1

Comparison of 32 plasma checkpoint protein expression in NPC patients before and after IMRT with controls (∗p <0.05, ∗∗p<0.01, and ∗∗∗p <0.001).

Figure 2

Comparison of EBV-DNA level in NPC patients before and after IMRT with controls (∗p <0.05, ∗∗p<0.01, and ∗∗∗p <0.001).

Figure 3

ROC curve analysis of EBV-DNA levels for distinguishing between NPC patients and controls. The area under the ROC curve is 0.912 (p≤0.001).

Figure 4

Kaplan-Meier survival analysis of NPC patients. (a) Comparison of the OS between patients with sPD-1 concentrations above and below the median (10.19 pg/ml, p=0.021). (b) Comparison of the OS between the patients with EBV-DNA levels above and below the median (1132.56 copies/ml, p=0.015). (c) Comparison of the OS between TNM I, II+III, and IV patients (p=0.026). Early stage, I; middle stage, II+III; late stage, IV.