Post-treatment Epstein-Barr Virus DNA Research Articles

Integrating Baseline Nutritional and Inflammatory Parameters with Post-Treatment EBV DNA Level to Predict Outcomes of Patients with De Novo Metastatic Nasopharyngeal Carcinoma Receiving Chemotherapy Combination PD-1 Inhibitor.

To develop and validate a prognostic nomogram based on baseline nutritional and inflammatory parameters for risk stratification in patients with de novo metastatic nasopharyngeal carcinoma (dmNPC) receiving chemotherapy combination programmed death-1 (PD-1) inhibitor. This retrospective study analyzed 131 patients with dmNPC (88 and 43 in the training and validation cohorts, respectively) between March 2017 and November 2020. All these patients received chemotherapy combined with PD-1 inhibitor treatment. We identified independent risk factors using univariate and multivariate Cox regression analyses and established a nomogram to predict the progression-free survival (PFS). The predictive accuracy of the nomogram was evaluated and independently validated. Baseline nutritional risk index (NRI), prognostic nutritional index (PNI), systemic immune-inflammation index (SII), uric acid (UA), and post-treatment Epstein-Barr virus (EBV) DNA were used to develop a nomogram that could divide patients into favorable- and unfavorable-prognosis groups. The median PFS (mPFS) was significantly longer in the favorable-prognosis group compared to the unfavorable-prognosis group (35.10 months [95% CI: 27.36-42.84] vs. 7.23 months [95% CI: 6.50-7.97]; p = 0.001). All results were confirmed in the validation cohort. The proposed model improved the prognostic risk stratification for patients with dmNPC undergoing chemotherapy combined with PD-1 inhibitor treatment.

Impact of EBV positivity on clinical outcomes in nasopharyngeal cancer.

e18021 Background: Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique geographical distribution. Epstein Barr virus (EBV) plays a key role in the pathogenesis of NPC, with pre and post-treatment serum EBV DNA level as an established prognostic indicator. However, evidence regarding the impact of the tumor cells’ EBV positivity on clinical outcomes is inconsistent. Methods: After IRB approval, we performed a retrospective chart review on NPC patients aged above 18 years seen at Corewell Health William Beaumont University Hospital between July 2015 and December 2020. The Epstein Barr encoding region in-situ hybridization from the biopsy sample determined EBV positivity. We compared progression-free survival (PFS) and overall survival (OS) based on EBV positivity status. Results: A total of 60 NPC patients were included: 19 were EBV-positive, 20 were EBV-negative, and 21 were EBV-unknown. The median age at diagnosis was 50.7 years, and 58.3 years in EBV-positive and negative groups respectively. Most patients in both groups were male (68.4% vs 65%) and had advanced (stage III or stage IV) disease (84.2% vs 75%). The median follow-up duration was 37.3 months. Median PFS and OS were 41 months and 114 months respectively in EBV-negative patients, while median PFS and OS were not reached in EBV-positive patients. There was no significant difference in PFS (0.944) and OS (0.117) among the EBV-positive and negative groups. Among advanced-stage patients, the median PFS was 33 months and 31 months in EBV-negative and EBV-positive groups, respectively, while median OS was not reached in both groups; the difference in PFS (p = 0.821) and OS (0.240) was statistically non-significant. When compared among patients with EBV-positive, negative, and unknown status, PFS was statistically non-significant (p = 0.347) but there was a statistical difference in OS (0.027). Conclusions: Our analysis suggests that the EBV status of the tumor cells does not predict clinical outcomes (PFS or OS) in NPC patients. Even though the OS trend favored EBV-positive patients overall and among those with advanced disease, it was not statistically significant. A difference in OS was seen when patients with unknown status were included and analyzed together with EBV-positive and negative patients, which raises the question of whether the outcome would be different if the majority of patients with unknown status belonged to either the EBV-positive or negative group. With studies in the literature reporting disparate findings, larger prospective studies are needed to better illustrate the impact of tumor cells' EBV positivity on clinical outcomes in NPC.

Prognostic significance of circulating Epstein-Barr virus DNA in pulmonary lymphoepithelioma-like carcinoma: A meta-analysis and validation study.

In small-scale studies, circulating Epstein-Barr virus (EBV) DNA levels have prognostic value in patients with pulmonary lymphoepithelioma-like carcinoma (LELC). Therefore, we performed a comprehensive meta-analysis to evaluate the prognostic significance of circulating EBV DNA levels in patients with pulmonary LELC. Studies that discussed the prognostic significance of circulating EBV DNA detection in pulmonary LELC were eligible for inclusion in this study. The overall survival (OS) and progression-free survival (PFS) were the primary outcomes. Pooled hazard ratio (HR), 95% confidence intervals (CIs), and pvalue were calculated to estimate the prognostic significance of EBV DNA levels. Additionally, we conducted a further observation using an independent cohort. The pooled HR and 95% CI of pretreatment EBV DNA levels for OS and PFS were 3.63 (95% CI: 2.90-4.55) and 2.88 (95% CI: 1.90-4.38), respectively. The pooled HR and 95% CI for Posttreatment EBV DNA levels for OS and PFS were 3.77 (95% CI: 2.96-4.80) and 3.52 (95% CI: 1.91-6.51, p < 0.001), respectively. The independent cohort showed similar results that patients with high pretreatment EBV DNA or positive posttreatment EBV DNA had significantly inferior PFS. Circulating EBV DNA levels provide prognostic values of survival and treatment response in pulmonary LELC patients.

Prognostic value of Epstein-Barr virus biomarkers for nasopharyngeal carcinoma staging and post-treatment surveillance

: The relationship between Epstein-Barr virus (EBV) infection and endemic nasopharyngeal carcinoma (NPC) has facilitated more than 40 years of biomarker-inspired translational research. Serologic and nucleic acid EBV biomarkers span the spectrum of this disease from population-level early detection, pre-treatment prognostication, response-adapted therapy, and long-term surveillance. Plasma EBV DNA remains the cornerstone of biomarker prognostication and surveillance for NPC, and there is increasing high-quality evidence that it merits inclusion in future staging systems. Recently-completed and ongoing biomarker-adapted clinical trials will determine whether biomarker-adapted management will become the standard of care. The HKNPCSG-0502 randomized trial demonstrated that post-treatment EBV DNA is prognostic but not predictive for response to adjuvant chemotherapy (AC), while the ongoing NRG-HN001 randomized trial may ultimately support the omission of AC in most patients. The next generation of biomarker-informed clinical trials may integrate early response to induction chemotherapy and/or immunotherapy. In this review, we discuss the clinical role and prognostic performance of EBV-based biomarkers for pre-treatment staging and post-treatment surveillance. In particular, we synthesize the available evidence which suggests that biomarker-informed staging systems might improve upon anatomic staging, but highlight the challenges in inter-laboratory reproducibility inherent to diagnostic assays without international standardization. Thereafter, we review a breadth of evidence which supports that undetectable post-treatment EBV biomarkers are highly specific for long-term cure. Finally, we contextualize emerging biomarkers that may further improve prognostication. Although these novel biomarkers have yet to supersede plasma EBV DNA in clinical performance, they may complement EBV DNA and identify the subset of patients at highest risk for clinical relapse.

Identifying optimal clinical trial candidates for locoregionally advanced nasopharyngeal carcinoma: Analysis of 9468 real-world cases and validation by two phase 3 multicentre, randomised controlled trial

Background and purposeThis study aims to identify the optimal high-risk candidates for clinical trials in locoregionally advanced nasopharyngeal carcinoma (NPC). Materials and methodsNon-metastatic NPC patients (n = 9,468) were included. Recursive partitioning analyses (RPA) were performed to generate risk stratification. Receiver operating characteristics curve was used to determine the cut-off value of pre-treatment Epstein-Barr virus (EBV) DNA for progression-free survival (PFS). Individual-level data from two clinical trials were used for validation. ResultsAnatomic stratification based on T and N category (eighth edition TNM, TNM-8) classified the N2-3 or T4 as an anatomic high-risk group with 5-year PFS of 69% (95% confidence interval: 68–71%). Prognostic stratification identified patients with pre-treatment EBV DNA ≥4000 copies/mL as a prognostic high-risk group with 5-year PFS of 69% (67–70%). The c-index was significantly higher for anatomic stratification (0.621, p < 0.001) and prognostic stratification (0.585, p < 0.001) compared with existing TNM-8 stage groups (0.562). The validation cohorts based on clinical trials data showed greater PFS benefit than the results of the original trials [Hazard ratio: NCT01245959, 0.64 vs. 0.67; NCT01872962, 0.42 vs. 0.52]. Moreover, detectable post-treatment EBV DNA indicated a high risk of progression with 5-year PFS of 38.7% and was the most adverse independent factor for all endpoints. ConclusionsN2-3 or T4 NPC patients were ideal candidates for multicenter clinical trials in locoregionally advanced NPC. Patients with detectable post-treatment EBV DNA are suitable candidates for adjuvant trials.

Prognostic Value of Plasma Epstein-Barr Virus DNA Levels Pre- and Post-Neoadjuvant Chemotherapy in Patients With Nasopharyngeal Carcinoma.

BackgroundIn this study, we evaluated the prognostic value of the plasma levels of Epstein-Barr virus (EBV) DNA in patients with nasopharyngeal carcinoma (NPC) at different treatment stages.MethodsWe retrospectively analyzed the Data of 206 patients with NPC. Pre-neoadjuvant chemotherapy (pre-NACT), post-NACT, post-radiotherapy, and post-treatment plasma EBV DNA levels were used to establish prognostic nomograms. The concordance index (C-index) and calibration curves were used to compare the prognostic accuracy of the nomograms. The results were confirmed in a validation cohort consisting of patients who were tested for EBV DNA levels at all four stages of treatment. The Kaplan-Meier method was used to calculate the progression-free survival (PFS) and overall survival (OS). Survival differences were calculated using the log-rank test.ResultsEBV DNA-positive patients had worse 3-year PFS and 5-year OS than EBV DNA-negative patients; this was true for pre-NACT (PFS: 82.7% vs. 57.3%, P < 0.001; OS: 90.9% vs. 68.7%, P = 0.08) and post-NACT (PFS: 85.0% vs. 50.6%, P < 0.001; OS: 91.7% vs. 65.7%; P = 0.001) EBV DNA levels but not for post-radiotherapy (PFS: 72.2% vs. 60.9%, P = 0.192; OS: 73.1% vs. 77.2%, P = 0.472) or post-treatment (PFS: 77.3% vs. 59.2%, P = 0.063; OS: 77.5% vs. 79.7%, P = 0.644) levels. Nomograms combining pre-NACT and post-NACT EBV DNA levels had a superior prognostic ability than those of post-radiotherapy and post-treatment EBV DNA levels.ConclusionPre-NACT EBV DNA levels combined with post-NACT EBV DNA levels can more reliably predict survival outcomes in patients with NPC.

Prognostic model for risk stratification of de novo metastatic nasopharyngeal carcinoma patients treated with chemotherapy followed by locoregional radiotherapy

BackgroundThere is no clinically applicable prognostic model designed for patients with de novo metastatic nasopharyngeal carcinoma (mNPC) treated with chemotherapy followed by locoregional radiotherapy (LRRT). We sought to develop a predictive tool of overall survival for individualized prediction and risk stratification in this heterogeneous patient population.Patients and methodsA total of 244 eligible patients with de novo mNPC, who were treated with platinum-based first-line chemotherapy followed by LRRT, were included in this retrospective study. We divided patients into the training and validation sets based on the date of initial treatment, with 152 patients treated between 2008 and 2013 comprising the training set for model development and 92 patients treated at a later time (2014 to 2015) forming the validation set. We applied Cox proportional hazards model to examine factors associated with overall survival (OS). We developed and subsequently validated a prognostic model to predict OS. We assessed the performance of this prognostic model and stratified patients based on prognostic scores obtained from this proposed model.ResultsThe median OS of the entire cohort was 60.9 months. C-creative protein, number of metastatic sites, liver metastasis, post-treatment Epstein–Barr virus DNA, and response of metastasis were significantly associated with OS. A prognostic model for individual survival prediction was developed and graphically represented as a nomogram. The model showed favorable discrimination (C-index: 0.759), predictive accuracy [time dependent area under the curve (tAUC) at 5 years: 0.800], and calibration, and was further validated in an independent dataset. A risk stratification derived from the model can stratify these patients into three prognostic subgroups with significantly different survival.ConclusionWe developed and validated a prognostic model that exhibited adequate performance in individualized prediction and risk stratification for patients with de novo mNPC treated with chemotherapy followed by LRRT.

Potential pitfalls in incorporating plasma Epstein-Barr virus DNA in the management of nasopharyngeal carcinoma.

This study identifies potential pitfalls in incorporating plasma Epstein-Barr virus (EBV) DNA into the management of nasopharyngeal carcinoma (NPC). A total of 208 NPC patients without distant metastasis who received radical treatment and had measurements of EBV DNA at baseline, 8 weeks and 26 weeks postradiotherapy were analyzed. Prognostic and predictive values at each time-point were compared. Risk stratification by pretreatment level failed to identify a poor prognostic group. Detectable EBV DNA at 8 weeks and 26 weeks postradiotherapy were both associated with significantly poorer 5-year disease-free survival (HR 0.30, P < .001 and HR 0.03, P < .001, respectively) and overall survival (HR 0.27, P = .009 and HR 0.03, P < .001, respectively). Eighty percentage had detectable EBV DNA at recurrence (53.3% for local only, 100% for regional only, and 100% for distant failure). Posttreatment EBV DNA, particularly at 26 weeks post-radiotherapy, has high prognostic and predictive values. Surveillance endoscopy/imaging are recommended for the detection of local recurrence.

Prognostic value of Epstein-Barr virus DNA level for nasopharyngeal carcinoma: a meta-analysis of 8128 cases.

Plasma levels of Epstein-Barr virus (EBV) DNA have been employed to predict survival outcomes of patients with nasopharyngeal carcinoma (NPC). However, the prognostic value of subsequent EBV DNA levels (mid or post treatment) for NPC is needed to identify by a large cohort of patients. We performed a meta-analysis of studies including data from 8128 patients to evaluate the prognostic value of EBV DNA in NPC patients. We searched PubMed, Web of Science, and the Cochrane library for prospective and retrospective studies. Hazard ratios (HRs) and confidence intervals (CIs) were extracted from the studies or calculated and pooled to assess the association between EBV DNA levels pre-treatment (pre-DNA), mid-treatment (mid-DNA), and post-treatment (post-DNA) on clinical outcomes. A total of 22 studies with 8128 patients was included for analysis. Pre-DNA levels predicted overall survival, progression-free survival, distant metastasis-free survival, and local-regional failure survival with HRs (95% CIs) of 2.70 (2.06, 3.54), 2.70 (2.12, 3.44), 3.49 (2.35, 5.17), and 2.00 (1.45, 2.76), respectively, and the corresponding HRs for post-DNA levels were 4.86 (3.30, 7.17), 6.29 (3.41, 11.60), 5.68 (2.71,11.93), respectively. Mid-DNA levels predicted overall survival and progression-free survival with an HR (95% CI) of 3.02 (1.54, 5.29) and 3.15 (2.05, 4.83). Subgroup analysis showed that the HR of post-DNA wasn't influenced by different detection time of post-DNA (P = 0.22, I2 = 33.2%). The EBV DNA levels have a significant prognostic impact in patients with NPC. The effect of post-treatment EBV DNA level dominated that of pre-DNA and mid-DNA levels.

Molecular Prognostic Value of Circulating Epstein-Barr Viral DNA in Nasopharyngeal Carcinoma: A Meta-Analysis of 27,235 Cases in the Endemic Area of Southeast Asia.

Aims: This meta-analysis evaluated the value of using Epstein-Barr virus (EBV) DNA titers as a predictive factor in assessing the clinical course of nasopharyngeal carcinoma (NPC) patients in Southeast Asia. Methods: A systematic search was performed using PubMed, the Cochrane Library, and Embase online databases. Eligible studies with complete baseline information and extractable hazard ratios (HRs), 95% confidence intervals (CIs), and other details were included in this analysis. All pooled statistics were calculated using RevMan 5.3 software with inverse variance methods, forest plots, or funnel plots directly; the results were analyzed both in toto and by subgroups using meta-regression and sensitivity analyses. Results: Forty studies involving 27,235 subjects were included in this meta-analysis. When the recommended EBV DNA viral cutoff values of 2,000, 0, and 0 copies/mL were used for the pretreatment, mid treatment, and post treatment groups, respectively, all values above these cutoffs were associated with at least a 2.5-fold increased risk of death compared with patients with lower levels of EBV DNA (HRs and 95% CIs were 2.47 [2.10-2.89], 2.67 [1.50-4.75], and 5.25 [3.58-7.71], respectively, p < 0.05). This finding could also explain heterogeneities among the studies. Conclusion: Higher pretreatment, mid-treatment, and post-treatment EBV DNA levels were all significantly correlated with poor outcomes for patients afflicted with NPCs. Further investigations of EBV DNA levels, combined with addressing tumor size, lymph nodes, metastases stages, are needed to determine whether they can be used as clinical guidelines for diagnosis and treatment.

Persistent peripheral blood EBV-DNA positive with high expression of PD-L1 and upregulation of CD4 + CD25 + T cell ratio in early stage NK/T cell lymphoma patients may predict worse outcome.

Although gemcitabine, oxaliplatin and L-asparaginase/pegylated asparaginase (P-GEMOX) treatment for early-stage extranodal natural killer/T cell lymphoma (ENKTL) is effective, some patients die within 1year of diagnosis. We attempted to determine an optimal biomarker for identifying such patients. We enrolled 71 patients with ENKTL who received P-GEMOX between January 2011 and January 2014. We classified the patients according to the outcome into worse (died within 1year) or better groups (survival time ≥ 3, 4 or 5years). The area under the curve (AUC) was determined to identify the optimal biomarker for differentiating the groups. The AUC was highest in patients who were plasma Epstein-Barr virus (EBV) DNA-positive post-treatment. The AUC was 0.82, 0.86 and 0.86 when the worse group was compared to the better group, respectively. Among the post-treatment EBV DNA-positive patients, as compared to EBV DNA-negative patients, pre-treatment EBV DNA-positive patients had a higher proportion of CD4 + CD25 + T cells. There was higher programmed cell death protein ligand-1(PD-L1) expression in post-treatment EBV DNA-positive patients. Post-treatment positive EBV DNA status maybe a useful biomarker of worse outcomes in early stageENKTL.

Optimal plasma pretreatment EBV DNA cut-off point for nasopharyngeal cancer patients treated with intensity modulated radiation therapy.

Plasma Epstein-Barr virus (EBV) DNA concentration at the time of diagnosis (pre-EBV) can be used to stratify risk for nasopharyngeal cancer (NPC) patients. However, pre-EBV cut-off values vary among studies. This was a post hoc analysis of 208 NPC patients from a phase II/III study comparing sequential (SEQ) vs. simultaneous integrated boost (SIB) intensity modulated radiation therapy. The objective was to identify the optimal pre-EBV cut-off value to predict overall survival (OS), progression free survival (PFS) and distant metastatic free survival (DMFS) rates. The pre-EBV and post-treatment EBV DNA (post-EBV) were detectable in 59.1% and 3.8% of the patients, respectively. A new pre-EBV cut-off value of 2300 copies/ml was identified by the receiver operating characteristics analysis. This cut-off value showed 82% sensitivity, 59% specificity and 31.7% positive and 93.5% negative predictive values in predicting OS. The 3-year OS, PFS and DMFS were 95.6 vs. 73.8%, 89.8 vs. 55.3% and 93 vs. 70.1% for pre-EBV < vs. ≥2300 copies/ml, respectively. Older age group (≥45 years), high pre-EBV and detectable post-EBV concentration were independent predictors for OS, PFS and DMFS in a multivariate analysis. When the stage grouping and pre-EBV value were combined, a subgroup of patients with stage II-III and pre-EBV values <2300 copies/ml. had the best survival outcomes, while the worst survival subgroup was the patients with stage III-IVb with pre-EBV values ≥2300 copies/ml. Pre-EBV cut-off of 2300 copies/ml is an optimal value predicting OS, PFS and DMFS.

Prognostic value of plasma Epstein\u2013Barr virus DNA level during posttreatment follow-up in the patients with nasopharyngeal carcinoma having undergone intensity-modulated radiotherapy

BackgroundThe value of Epstein–Barr virus (EBV) DNA assay during posttreatment follow-up of the patients with nasopharyngeal carcinoma (NPC) presenting with different pretreatment plasma EBV DNA levels remains unclear. In the present study, we aimed to evaluate the prognostic value of plasma EBV DNA assay during posttreatment follow-up in the patients with NPC who have undergone intensity-modulated radiotherapy.MethodsThe medical records of 385 NPC patients treated with intensity-modulated radiotherapy between November 2009 and February 2012 were reviewed. All patients underwent plasma EBV DNA assays before treatment, within 3 months after treatment, and then every 3–12 months during posttreatment follow-up period. The recurrence rates for patients with different pretreatment and posttreatment follow-up plasma EBV DNA levels were analyzed.ResultsOf the 385 patients, 267 (69.4%) had detectable pretreatment plasma EBV DNA (> 0 copy/mL) and 93 (24.2%) had detectable posttreatment EBV DNA during a median follow-up of 52.8 months (range 9.3–73.8 months). Detectable EBV DNA during posttreatment follow-up was found in 14.4% (17/118) and 28.5% (76/267) of patients with undetectable and detectable pretreatment EBV DNA, respectively, and was significantly associated with tumor recurrence in both patient groups. EBV DNA was detectable in 12.8% (40/313) of patients who remained disease-free, 56.4% (22/39) of patients with locoregional recurrence alone, and 93.9% (31/33) of patients with distant metastasis as the first recurrence event (P < 0.001); 6.5% (19/292) of patients with undetectable EBV DNA and 57.0% (53/93) of patient with detectable EBV DNA during posttreatment follow-up experienced tumor recurrence. Compared with other cut-off values, the cut-off value of 0 copy/mL for EBV DNA during posttreatment follow-up had the highest area under the ROC curve (AUC) value (0.804, 95% confidence interval 0.741–0.868) for predicting tumor recurrence (sensitivity, specificity, and accuracy: 73.6%, 87.2%, and 84.7%, respectively).ConclusionPlasma EBV DNA level during posttreatment follow-up is a good marker for predicting distant metastasis but not locoregional recurrence in the patients with NPC irrespective of the pretreatment EBV DNA levels.

Treatment response and prognosis according to pre and post treatment Ebstein-Barr virus DNA level in nasopharyngeal carcinoma patients.

e17544 Background: To evaluate pre and post-treatment Epstein-Barr virus (EBV) DNA copy numbers, to investigate changes with treatment and the possibility of using it as a follow-up marker in nasopharyngeal carcinoma (NPC) patients. Methods: Blood samples were obtained before (0-3 weeks) and after treatment (mean 6 weeks) and number of EBV DNA copies were determined by PCR . Treatment was administered as concomittant chemoradiotherapy ± induction chemotherapy. MRI and PET-CT were performed prior (0-4 weeks) and after (6-16 weeks) treatment. Mann Whitney test was used for 2 group comparisons of parameters with no normal distribution and comparison of quantitative data. Fisher-Freeman-Halton and Wilcoxon Signed Ranks tests were used for comparison of qualitative data and intra-group comparison of parameters with non-normal distribution. Kaplan Meier Survival analysis was used to assess survival. Results: The study included 50 NPC patients treated between 2011 and 2015. Median follow-up is 25.3 (27.5 ± 14) months. 12 patients had residual-recurrent or metastatic (3 patients initially had distant metastasis) disease. Mean survival is 49.77 ± 2.4 months. Median posttreatment EBV DNA level is lower than pretreatment EBV DNA level (p = 0.001). Pretreatment EBV DNA copy numbers are higher in patients with residual-recurrent or metastatic disease (RRM) than those without RRM (p=0.05). Mortal patients had higher pre (p = 0.015) and post (p = 0.043) treatment EBV DNA levels than those who survived. Overall survival (OS) is higher in patients with undetectable (n:41) than those with detectable (n:9) EBV DNA levels after treatment (p = 0.02). Conclusions: The OS is lower in patients with higher pretreatment and detectable posttreatment EBV DNA levels. This study implies that pre and post treatment serum EBV DNA levels can be used as prognostic indicators and also be beneficial as a follow up marker in NPC patients. [Table: see text]

Salivary Epstein-Barr virus DNA level in patients with nasopharyngeal carcinoma following radiotherapy

Nasopharyngeal carcinoma (NPC) is a solid tumor closely associated with Epstein-Barr virus (EBV) infection. The purpose of this investigation was to detect and quantify the EBV DNA level in salivary samples of NPC patients following treatment using real-time PCR. A total of 175 consecutive newly diagnosed NPC patients' whole saliva samples were collected before treatment, and the EBV DNA level was measured by real-time PCR, with the primers and probe targeting the BamHI-W region of the EBV genome. The post-treatment salivary EBV DNA level was also assessed in 46 patients. The change of EBV DNA level before and after treatment and relationship of EBV DNA level to demographic data and tumor staging were tested by Wilcoxon signed-rank test and Mann-Whitney U test, respectively with the level of significance set at 0.05. The EBV detection rate of pre-treatment saliva samples was 80%. The EBV DNA level of post-treatment saliva samples was significantly higher than the pre-treatment ones (P<0.01). There is a trend that patients with advanced-stage showed a higher EBV DNA level than patients with early-stage. The detection of EBV DNA in saliva using real-time PCR might be a feasible and non-invasive method for early diagnosis of NPC.

Different Clinical Significance of Pre- and Post-treatment Plasma Epstein–Barr Virus DNA Load in Nasopharyngeal Carcinoma Treated with Radiotherapy

Aims To correlate the pre-treatment plasma Epstein–Barr virus (EBV) DNA with tumour burden and to explore the prognostic implications of pre- and post-treatment plasma EBV DNA load in nasopharyngeal carcinoma patients treated with radiotherapy. Materials and methods Plasma EBV DNA load was measured using a real-time quantitative polymerase chain reaction assay in 69 patients with nasopharyngeal carcinoma before and after radiation treatment and correlated with tumour volume and treatment outcome. Tumour volume was calculated by multiplying the sum of the areas of gross extent of the primary tumour and regional lymph nodes shown by computed tomography images and/or magnetic resonance imaging. Prognostic models for distant metastasis and overall survival were constructed using a multivariable fractional polynomial algorithm. Results The pre-treatment plasma EBV DNA concentration was significantly associated with tumour volume (Spearman correlation coefficient, 0.61; P < 0.001). The multivariable fractional polynomial algorithm selected post-treatment EBV DNA and administration of chemotherapy as prognostic factors for distant metastasis ( P < 0.001, P = 0.021, respectively), as well as for overall survival ( P < 0.001, P = 0.018, respectively). Conclusions Pre- and post-treatment plasma EBV DNA load have important clinical significance. Pre-treatment plasma EBV DNA concentration reflects tumour burden, whereas clearance of circulating plasma EBV DNA after treatment predicts the risk of distant metastasis and overall survival.

Long-Term Prognostic Effects of Plasma Epstein-Barr Virus DNA by Minor Groove Binder-Probe Real-Time Quantitative PCR on Nasopharyngeal Carcinoma Patients Receiving Concurrent Chemoradiotherapy

To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U), (2) high pretreatment EBV DNA (> or =1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT.

A Comparison Study of Different PCR Assays in Measuring Circulating Plasma Epstein-Barr Virus DNA Levels in Patients with Nasopharyngeal Carcinoma

To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance. Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables. Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002). The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.

Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) DNA can be detected and quantified in the plasma of patients with EBV-related tumors, such as nasopharyngeal carcinoma (NPC). Although NPC at early stages can be cured by radical radiotherapy, there is a high recurrence rate in patients with advanced NPC. The pretreatment level of circulating EBV DNA is a prognostic factor for NPC, but the prognostic value of post-treatment EBV DNA has not been studied. We designed a prospective study in Hong Kong, China, to investigate the value of plasma EBV DNA as a prognostic factor for NPC. One hundred seventy NPC patients, without metastatic disease at presentation, were treated with a uniform radiotherapy protocol. Circulating EBV DNA was measured by real-time quantitative polymerase chain reaction before treatment and 6-8 weeks after radiotherapy was completed. Risk ratios (RRs) were determined with a Cox regression model, and associations of various factors with progression-free and overall survival and recurrence rates were determined with a stepwise Cox proportional hazards model. All statistical tests were two-sided. Ninety-nine percent of patients achieved complete clinical remission. Levels of post-treatment EBV DNA dominated the effect of levels of pretreatment EBV DNA for progression-free survival. The RR for NPC recurrence was 11.9 (95% confidence interval [CI] = 5.53 to 25.43) for patients with higher post-treatment EBV DNA and 2.5 (95% CI = 1.14 to 5.70) for patients with higher pretreatment EBV DNA. Higher levels of post-treatment EBV DNA were statistically significantly associated with overall survival (P<.001; RR for NPC recurrence = 8.6, 95% CI = 3.69 to 19.97). The positive and negative predictive values for NPC recurrence for a higher level of post-treatment EBV DNA were 87% (95% CI = 58% to 98%) and 83% (95% CI = 76% to 89%), respectively. Levels of post-treatment plasma EBV DNA in patients with NPC appear to strongly predict progression-free and overall survival and to accurately reflect the post-treatment residual tumor load.