Plasma Epstein-Barr Virus Research Articles

Abstract CT049: Long term outcome of EBV-associated lymphoma patients treated with post-remission therapy using EBV LMP1 and LMP2a specific CTLs

Abstract Background: Conventional therapeutic approaches, such as chemotherapy and radiotherapy, have been limited in successfully achieving durable response in Epstein Barr virus (EBV)-associated malignancies causing frequent relapse, low overall survival rate and poor prognosis. EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach to treat EBV associated lymphoma by enhancing EBV-specific immunity. Methods: To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated ten EBV+ ENKTCL patients and two EBV+ PTLD patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation were eligible to receive eight doses of 2 x107 LMP1/2a CTLs/m2. Results: Following infusion, there were no immediate or delayed toxicities. The seven-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71·4% to 100%) for ENKTCL patients respectively with a median follow-up of 94 months. Similarly, PTLD patients showed long-term remission with a follow-up of up to 70 months. Overall, circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Conclusion: Adoptive transfer of LMP1/2a CTLs in patients is a safe and effective post-remission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of EBV-associated lymphoma. Citation Format: Nayoun Kim, Hyun-Jung Sohn, Keon-Il Im, Young-Woo Jeon, Young-Sun Nam, Yunejin Song, Jun Seok Lee, Tai Gyu Kim, Seok-Goo Cho. Long term outcome of EBV-associated lymphoma patients treated with post-remission therapy using EBV LMP1 and LMP2a specific CTLs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT049.

Plasma Epstein-Barr Virus Load as an Early Biomarker and Prognostic Factor of Human Immunodeficiency Virus-related Lymphomas.

Epstein-Barr virus (EBV) has been implicated in lymphomagenesis and can be found infecting tumor cells and in plasma at lymphoma diagnosis, especially in human immunodeficiency virus (HIV)-infected patients. Our aim was to evaluate the usefulness of plasma EBV load as biomarker and prognostic factor in HIV-positive patients with lymphomas. EBV loads were measured by polymerase chain reaction in plasma samples of 81 HIV-positive patients' lymphomas at different moments: within 1 year before lymphoma diagnosis, at diagnosis, and at complete response (CR). Control samples included HIV-negative patients with lymphomas and HIV-positive patients without neoplasia or opportunistic infections. HIV-positive patients with lymphomas had more frequently-detectable EBV load at lymphoma diagnosis (53%) than either HIV-negative patients with the same lymphoma type (16%; P < .001) or HIV-positive individuals without neoplasia or opportunistic infection (1.2%; P < .001). HIV-positive lymphoma patients with detectable EBV load in plasma at lymphoma diagnosis had statistically significant decrease of EBV load at CR. High EBV load (>5000 copies/mL) at lymphoma diagnosis was an independent negative prognostic factor for overall survival and progression-free survival in HIV-positive patients with lymphomas. Detectable plasma EBV loads identified HIV-positive subjects that would eventually develop lymphoma (area under the curve, 82%; 95% CI: 0.67-0.96). Plasma EBV load can be used as a biomarker and as a prognostic factor in HIV-positive patients with lymphomas. The presence of the EBV load in the plasma of an HIV-positive patient can be an early predictor of lymphoma development.

Plasma Macrophage Migration Inhibitory Factor and CCL3 as Potential Biomarkers for Distinguishing Patients with Nasopharyngeal Carcinoma from High-Risk Individuals Who Have Positive Epstein-Barr Virus Capsid Antigen-Specific IgA

PurposeThe purpose of this study was to identify novel plasma biomarkers for distinguishing nasopharyngeal carcinoma (NPC) patients from healthy individuals who have positive Epstein-Barr virus (EBV) viral capsid antigen (VCA-IgA).Materials and MethodsOne hundred seventy-four plasma cytokines were analyzed by a Cytokine Array in eight healthy individuals with positive EBV VCA-IgA and eight patients with NPC. Real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were employed to detect the expression levels of macrophage migration inhibitory factor (MIF) and CC chemokine ligand 3 (CCL3) in NPC cell lines and tumor tissues. Plasma MIF and CCL3 were measured by ELISA in 138 NPC patients, 127 EBV VCA-IgA negative (VN) and 100 EBV VCA-IgA positive healthy donors (VP). Plasma EBV VCA-IgA was determined by immunoenzymatic techniques.ResultsThirty-four of the 174 cytokines varied significantly between the VP and NPC group. Plasma MIF and CCL3 were significantly elevated in NPC patients compared with VN and VP. Combination of MIF and CCL3 could be used for the differential diagnosis of NPC from VN cohort (area under the curve [AUC], 0.913; sensitivity, 90.00%; specificity, 80.30%), and combination of MIF, CCL3, and VCA-IgA could be used for the differential diagnosis of NPC from VP cohort (AUC, 0.920; sensitivity, 90.00%; specificity, 84.00%), from (VN+VP) cohort (AUC, 0.961; sensitivity, 90.00%; specificity, 92.00%). Overexpressions of MIF and CCL3 were observed in NPC plasma, NPC cell lines and NPC tissues.ConclusionPlasma MIF, CCL3, and VCA-IgA combination significantly improves the diagnostic specificity of NPC in high-risk individuals.

Lymphoma associated hemophagocytic syndrome: A single-center retrospective study.

To improve the understanding of lymphoma associated hemophagocytic syndrome (LAHS) and find an effective treatment for this fatal disease, 57 patients with LAHS were retrospectively reviewed. The most common histopathological type was extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) (45.61%). Patients with B-cell LAHS were significantly older (P<0.001), and exhibited a higher triglyceride level (P=0.012), lower serum ferritin level (P=0.014) and lower plasma Epstein-Barr virus DNA (P<0.001) compared with patients with T/NK-cell LAHS. The median survival time of all patients was 43 days, and patients with B-cell (n=14) and T/NK-cell (n=43) LAHS had a median survival time of 55 and 40 days, respectively (P=0.797). Compared with patients who were treated based on HLH-2004 protocols combined with multidrug chemotherapy, those without chemotherapy had a reduced prognosis (P=0.002). The patients that underwent hematopoietic stem cell transplantation (HSCT) following chemotherapy had a significantly improved overall survival (OS) compared with patients that did not undergo HSCT (P=0.001). Patients with B-cell LAHS treated with rituximab (P=0.015) and patients with ENKL treated with L-asparaginase/pegaspargase (L-asp/peg) (P=0.009) had an improved prognosis compared with patients not treated with these drugs. In the T/NK-cell LAHS group, patients treated with chemotherapy containing gemcitabine did not exhibit an improved OS compared with those not treated with gemcitabine (P=0.326). Furthermore, multivariate analysis demonstrated that long diagnosis time and poor performance status were independent prognosis factors for all patients with LAHS. The present study indicated that survival time does not differ between patients with B-cell LAHS and patients with T/NK-cell LAHS. Early diagnosis and appropriate immunochemotherapy plus HSCT are essential to achieve improved outcomes. The outcome of patients with B-cell LAHS may be significantly improved following treatment with rituximab. L-asp/peg-containing regimens are promising treatments for patients with NK/T-cell LAHS.

Sequencing-based counting and size profiling of plasma Epstein–Barr virus DNA enhance population screening of nasopharyngeal carcinoma

Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.

The current status of clinical trials focusing on nasopharyngeal carcinoma: A comprehensive analysis of ClinicalTrials.gov database.

PurposeClinical Trials have emerged as the main force in driving the development of medicine. However, little is known about the current status of clinical trials regarding nasopharyngeal carcinoma (NPC). This study aimed at providing a comprehensive landscape of NPC-related trials on the basis of ClinicalTrials.gov database.Patients and methodsWe used the keyword “nasopharyngeal carcinoma” to search the ClinicalTrials.gov database and assessed the characteristics of these trials.ResultsUp to December 30, 2016, 462 eligible trials in total were identified, of which 222 (48.0%) recruited only NPC (NPC trials) and the other 240 (52.0%) recruited both NPC and other cancers (multiple cancer trials). Moreover, 47 (10.2%) were Epstein-Barr virus (EBV)-related trials and 267 (57.8%) focused on metastatic/recurrent disease. Compared with NPC trials, the multiple cancer trials had a higher percentage of phase 1 (26.7% vs. 6.7%, P < 0.001) studies and more patients with metastatic/recurrent disease (72.5% vs. 41.9%, P < 0.001). Notably, non-EBV trials had more phase 2 or 3 (78.4% vs. 48.8%, P < 0.001) and interventional studies (89.5% vs. 70.7%, P = 0.002) than EBV trials. Obviously, more phase 2/3 or 3 trials were conducted in patients with non-metastatic/recurrent disease (29.4% vs. 4.9%, P < 0.001); however, metastatic/recurrent trials were more likely to be anticancer (94.6% vs. 63.6%, P < 0.001).ConclusionsThe role of plasma EBV DNA in clinical trials is underestimated, and high-level randomized clinical trials should be performed for patients with metastatic/recurrent disease.

EPSTEIN-BARR VIRUS AND NASOPHARYNGEAL CARCINOMA: VIRAL MARKERS FOR DIAGNOSTICS AND ASSESSMENT OF CLINICAL STATUS OF PATIENTS.

The etiological role of the Epstein-Barr virus (EBV) in the development of an undifferentiated histological variant of nasopharyngeal carcinoma (uNPC) found for the first time in regions with a high incidence of this pathology, the Southern provinces of China and the countries of Southeast Asia, and later in the rest of the world, has served as a basis for the widespread use of EBV serological markers for the diagnosis of this form of tumor. In recent years, the use of a test based on the quantitative determination of the EBV DNA concentration in the blood plasma of uNPC patients for early detection and monitoring of the disease has become widespread in endemic regions. In non-endemic regions, such studies virtually have not been carried out, and moreover, the comparative evaluation of the significance of two viral markers, serological and EBV DNA load in the bloodstream of uNPC patients, for diagnostics and evaluation of the therapeutic effect was not investigated. The aim of this study was to compare the clinical value of two serological markers and plasma EBV DNA load in uNPC patients from non-endemic region (Russia). The obtained results indicate that IgA antibodies to the viral capsid antigen (IgA/VCA) and plasma EBV DNA concentration can be successfully used for the diagnosis of uNPC, while IgG/VCA antibodies have no practical significance as an uNPC marker. In addition, it was found that plasma EBV DNA load is more sensitive marker of uNPC than IgA/VCA titers because DNA copy numbers reflect more accurately the effect of the therapy and the clinical state of patients at the stages of remission or relapse. It was shown for the first time that in the non-endemic region the simultaneous evaluation of IgA/VCA antibody levels and the plasma EBV DNA loads are the most effective markers for the diagnostics of uNPC. However, we believe, that it is more practical to use IgA/VCA antibody levels for uNPC screening, and plasma EBV DNA copies - for monitoring of the disease.

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.

Dynamic changes in plasma Epstein-Barr virus DNA load during treatment have prognostic value in nasopharyngeal carcinoma: a retrospective study.

Circulating plasma Epstein–Barr virus DNA (EBV DNA) is related to tumor recurrence and metastasis and has potential as a dynamic, sensitive, and specific marker in nasopharyngeal carcinoma (NPC). We investigated the clinical significance of assessing plasma EBV DNA load at various time points during treatment. Patients with NPC (n = 949) for whom plasma EBV DNA load was measured by real‐time quantitative polymerase chain reaction (RT‐qPCR) before treatment (pre‐EBV) and at midtreatment (mid‐EBV), end of treatment (end‐EBV), and 3 months after completing treatment (3 m‐EBV) were retrospectively assessed. Receiver operating characteristic (ROC) curve analysis was used to identify the optimal EBV DNA cutoff point for each time point. Overall survival (OS), distant metastasis‐free survival (DMFS), and progression‐free survival (PFS) were compared using Kaplan–Meier estimates. High pre‐EBV, high mid‐EBV, high end‐EBV, and high 3 m‐EBV were all associated with significantly poorer OS, DMFS, and PFS in the entire cohort. Detectable end‐EBV and 3 m‐EBV was associated with significantly poorer OS, DMFS, and PFS. Among patients with detectable end‐EBV, adjuvant therapy significantly improved OS (HR 2.419; 95% CI 1.297–4.51, P = 0.03) and DMFS (HR 2.45; 95% CI 1.243–4.828, P = 0.04), but not PFS (P = 0.17). EBV DNA represents a dynamic biomarker for monitoring treatment and predicting survival in NPC. Assessing plasma EBV DNA before, during, and after chemoradiotherapy could be clinically valuable and enable selection of patients most likely to benefit from additional therapy and improve assessment of treatment response and disease surveillance. Further multicenter prospective investigations are warranted.

Evaluation of performance and application of three nucleic acid extraction methods for quantification of plasma Epstein-Barr virus DNA

Objective To evaluate and compare the analytical performances and application values of three nucleic acid extraction methods for quantification of plasma Epstein-Barr Virus (EBV) DNA. Methods It used silica membrane spin column, boiling and automated magnetic bead method to extract viral nucleic acid in parallel, and combined real-time fluorescence quantitative PCR assays for quantitative EBV-DNA quantification.The performances of three methods were determined and compared by using the third-party reference materials, and the clinical values were analyzed by pairing detecting 100 NPC patients and 100 healthy subjects in pair. Results The accuracy and imprecision of three methods were all in line with requirements, and the results of clinical samples were linearly correlated. But actually the reproducibility and intermediate imprecision of the magnetic bead method were smaller and stable than those of the spin column method and the boiling method (all <3%); the limit of detection for the magnetic bead method was 3.334×101 IU/ml, better than that of spin column method (4.159×101 IU/ml) and boiling method (8.511×101 IU/ml); the linear range of the magnetic bead method was 5.4×101-5.4×105 IU/ml, slightly wider than that of the boiling method(5.4×102-5.4×105 IU/ml); the ability of anti - Hb interference ability of magnetic bead method is better than that of boiling method; and the positive rate and the mean viral load of the NPC samples measured with the magnetic bead method were significantly higher (95%, 8.342×103 IU/ml) than those measured with the spin column method (84%, 4.707×103 IU/ml) and the boiling method (78%, 2.571×103 IU/ml) (P all<0.05). Conclusion The automated magnetic bead nucleic acid extraction method offered better analytical performance and higher clinical value for EBV DNA quantification in plasma.(Chin J Lab Med, 2018, 41: 59-65) Key words: Herpesvirus 4, human; DNA, viral; Centrifugation; DNA cleavage; Automation, laboratory; Clinical laboratory techniques; Evaluation studies

Chemokine CCL27 is a novel plasma biomarker for identification the nasopharyngeal carcinoma patients from the Epstein-Barr virus capsid antigen-specific IgA seropositive population

BackgroundTo investigate the predictive value of chemokine CCL27 for identifying early stage nasopharyngeal carcinoma (NPC) patients within a population seropositive for Epstein-Barr virus (EBV) capsid antigen-specific IgA (VCA-IgA).MethodsCCL27 in plasma samples from 104 NPC patients, 112 VCA-IgA–positive healthy donors, and 140 VCA-IgA–negative normal subjects was measured by ELISA. Expression of CCL27 in nasopharyngeal tissue from 20 VCA-IgA–positive healthy donors and 20 NPC patients was examined by immunohistochemical staining.ResultsLevels of CCL27 in the plasma of VCA-IgA–positive healthy donors (607.33 ± 218.81 pg/ml) were significantly higher than the levels in all NPC patients (437.09 ± 217.74, P = < 0.0001) and in the subset of patients with early stage NPC (463.85 ± 226.17, P = 0.0126). Plasma CCL27 levels were significantly lower in the VCA-IgA–negative normal subjects (358.22 ± 133.15 pg/ml) than in either the VCA-IgA–positive healthy donors (P < 0.0001) or the NPC patients (P = 0.0113). CCL27 protein was detected in 16 of 20 (80%) nasopharyngeal tissue samples from VCA-IgA–positive healthy donors and in 3 of 20 (15%) tumor tissue samples from NPC patients. There was no relationship between CCL27 levels and VCA-IgA titers or plasma EBV DNA content. Receiver operating characteristic (ROC) curves demonstrated that plasma CCL27 levels had a sensitivity of 67.00%, a specificity of 73.10%, and an area under the ROC of 0.725 (95% confidence interval [CI]: 0.657–0.793) for distinguishing between NPC patients and VCA-IgA–positive healthy donors. Further analysis showed that CCL27 levels could distinguish between early stage NPC patients and VCA-IgA–positive healthy donors with an area under the ROC of 0.712 (95% CI: 0.560–0.865), a sensitivity of 59.80%, and a specificity of 84.60%.ConclusionsChemokine CCL27 could successfully identify NPC patients within a VCA-IgA–positive population.

The plasma Epstein–Barr virus DNA level guides precision treatment for nasopharyngeal carcinoma in the intensity-modulated radiotherapy era: a large population-based cohort study from an endemic area

Background:In the intensity-modulated radiotherapy (IMRT) era, the survival benefit of concurrent chemotherapy for locoregionally advanced nasopharyngeal carcinoma (LA-NPC) remains undetermined. This study aimed to evaluate the benefits of IMRT with concurrent chemotherapy compared with IMRT alone for LA-NPC patients with different plasma Epstein–Barr virus (EBV) DNA levels.Methods:Patients were identified from a prospectively maintained database in an endemic area between November 2002 and December 2013. Cox proportional hazards models, propensity score matching, and inverse probability weighting models were established for survival analysis. Stratification analysis was performed based on interaction effects analysis. Finally, sensitivity analysis was performed considering unmeasured confounders.Results:A total of 1357 eligible patients were enrolled (median follow up 62.4 months; range 3.5–155.8 months). No significant survival differences were observed between groups in the entire cohort. Notably, a significant interaction effect was observed between treatment regimens and EBV DNA levels. In patients with high EBV DNA levels (>4000 copies/ml), all three models showed that IMRT with concurrent chemotherapy significantly improved overall survival [hazard ratio (HR) 2.521, 95% confidence interval (CI) 1.218–5.216], disease-free survival (HR 2.168, 95% CI 1.349–3.483), and distant metastasis-free survival (HR 2.331, 95% CI 1.194–4.551) compared with IMRT alone. No differences were found in patients with low EBV DNA levels. Sensitivity analysis confirmed the robustness of the results.Conclusion:In the IMRT era, concurrent chemotherapy treatment of LA-NPC patients with high EBV DNA levels is reasonable. However, the optimal regimen for LA-NPC patients with low EBV DNA levels needs further validation in randomized clinical trials.

Epstein-Barr Virus in Patients with Classical Hodgkin’s Lymphoma

Background. A close relationship between Epstein-Barr virus (EBV) and classical Hodgkin’s lymphoma (cHL) has been established in approximately 1/3 patients. EBV-positive lymphomas are characterized by increased level of EBV specific antibodies emerging long before tumor symptoms, аs well as a high plasma EBV DNA concentration. These viral markers normally correlate with clinical manifestations and the outcome of treatment performed. In patients with EBV-negative lymphomas, however, there has been no attempt to assess the clinical significance of either humoral response to EBV or EBV DNA concentration in plasma. Aim. To evaluate diagnostic and prognostic significance of EBV markers in patients with EBV-negative lymphomas. Methods. The clinical trial included 13 cHL-patients admitted at the Department of chemotherapy of hemoblastoses of NN Blokhin National Medical Cancer Research Center. The male to female ratio was 1: 1.3, the median age was 26.4 years. Leukocyte and lymphocyte counts were evaluated in all the patients before, during, and after treatment as well as throughout the follow-up period. The same indicators were analysed in the control group which contained 80 healthy persons (with the median age of 29.4 years, male to female ratio 1.5: 1). The study was based on serologic test for EBV antibodies and quantitative analysis of the viral DNA copy number in plasma. Results. The obtained data show a low immunie response to EBV and its diminishment after several polychemotherapy treatment cycles, correlating with decreased leukocyte and lymphocyte levels. As opposed to levels of virus-specific antibodies which do not reflect the efficacy of anticancer therapy, plasma EBV DNA concentration in 2 patients decreased to 0 after remission had been achieved. Conclusion. Although the number of observations is limited, one could suggest that viral load values in plasma of patients with EBV-negative lymphomas can prove to be a useful marker of anticancer therapeutic effect. Additional studies of these markers are required.

Surrogate endpoints shortening the therapeutic evaluation duration for different subgroups of patients with nasopharyngeal carcinoma receiving intensity-modulated radiotherapy: A retrospective analysis of 830 patients stratified by the 8th edition of the UICC/AJCC staging system and plasma Epstein-Barr viral.

Purpose: Investigating surrogate endpoints shortening the time of therapeutic evaluation in nasopharyngeal carcinoma (NPC) after radical treatment.Patients and Methods: We retrospectively analyzed 830 patients receiving intensity-modulated radiotherapy (IMRT) from 2008 to 2010 and being stratified by the 8th edition of UICC/AJCC staging system and the plasma Epstein-Barr virus DNA (EBV DNA). The annual rates of overall survival (OS), progression-free survival (PFS), loco-regional recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) were sequentially calculated using the life table and compared by the McNemar method.Results: The time of shortening therapeutic evaluation by surrogate endpoints: OS, PFS, LRFS and DMFS could be shortened to 1-year (100% vs 100%, P=1) in patients with stage I; OS, PFS, LRFS and DMFS could be shortened to 3-year (96.9% vs 96.1%, P = 1; 94.6% vs 92.2%, P = 0.125; 96.9% vs 95.3%, P = 0.5) and 4-year (92.2% vs 91.2%, P = 0.125) in stage II; In the high EBV DNA group , OS and DMFS could be shortened to 1-year (100% vs 100%, P = 1;100% vs 100%, P = 0.25) in stage II; OS and PFS could be shortened to 3-year (94.3% vs 91.4%, P = 1;82.9% vs 74.3%, P = 0.25) in stage III; OS could be shortened to 4-year (75% vs 72.7%, P = 1) in stage IVA.Conclusions: The time of therapeutic evaluation could be shortened to <5-year in stages I-II patients. The year of surrogate endpoints could be ahead in stages II-IVA with high EBV DNA.

Interplay of Tumor Spread, Volume and Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma: Feasibility of An Integrative Risk Stratification Scheme.

Purpose: To investigate the inter-correlation of tumor spread, volume and quantitative plasma Epstein-Barr virus DNA level (pEBV DNA), and to further assess the prognostic efficacy of a novel risk stratification combining anatomic, volumetric and biological features in nasopharyngeal carcinoma (NPC).Methods and Materials: One hundred and twelve patients with non-metastatic NPC were prospectively enrolled. Correlation of pEBV DNA with tumor invasiveness, lymph node (LN) metastasis, tumor volume and classification was tested by univariate and multivariate analyses. 5-year distant metastasis free survival (DMFS) was evaluated using Kaplan-Meier method and Cox proportional hazards model.Results: Tumor volume, TNM stage and pEBV DNA were strongly inter-correlated to each other. Nodal volume, skull base invasion and LN metastasis to supraclavicular fossa were determined to be independent predictors for pEBV DNA level. To exclude collinearity, a risk stratification based on combination of EBV DNA, nodal volume and anatomic features was established, offering significant distinguishing ability in 5-year DMFS. Further multivariate Cox regression analysis found the novel stratification to be independent predictor of DMFS.Conclusions: Both anatomic spread and tumor volume contribute to pEBV DNA level, leading to strong inter-correlation between NPC stage, volume and EBV DNA. The proposed risk stratification combining anatomic, volumetric and biological features showed potential in refining DMFS prediction.

The predictive value of pre- and post-induction chemotherapy plasma EBV DNA level and tumor volume for the radiosensitivity of locally advanced nasopharyngeal carcinoma.

This study was dedicated to investigate the predictive value of pre- and post-induction chemotherapy plasma EBV (Epstein-Barr Virus) DNA level and tumor volume for the radiosensitivity of locally advanced NPC. 129 previously untreated locally advanced NPC patients were enrolled. Plasma EBV-DNA copy number and tumor volume was detected before and after induction chemotherapy. The tumor volume was also measured after radiotherapy. Among 129 patients, 98 were positive for EBV DNA. The residual gross target volume of the primary tumor (GTVnx) and GTVnd after radiotherapy was positively correlated with post-induction chemotherapy EBV copy number (rho=0.357, P<0.001; rho=0.356, P<0.001, respectively). Univariate logistic regression analyses showed that the AUC of ROC curves of post-induction chemotherapy tumor volume, tumor regression rate before and after induction chemotherapy, post-induction EBV copy number, EBV decrease rate for predicting no residual nasopharyngeal tumor were 0.859, 0.782, 0.678 and 0.657, respectively. Multivariate logistic analyses showed that T stage, post-induction chemotherapy EBV copy number and tumor volume were independent predictors for no residual nasopharyngeal tumor after radiotherapy. The changes in plasma EBV DNA and tumor volume during treatment could be used to predict the sensitivity of locally advanced NPC patients in response to intensity-modulated radiation therapy (IMRT).

Plasma EBV microRNAs in paediatric renal transplant recipients.

Epstein-Barr virus (EBV) was the first human virus identified to express microRNA (miRNA). To date, 44 mature miRNAs are encoded for within the EBV genome. EBV miRNAs have not been profiled in paediatric renal transplant recipients. In this study, we investigated circulating EBV miRNA profiles as novel biomarkers in paediatric renal transplant patients. Forty-two microRNAs encoded within 2 EBV open reading frames (BART and BHRF) were examined in renal transplant recipients who resolved EBV infection (REI) or maintained chronic high viral loads (CHL), and in non-transplant patients with acute infectious mononucleosis (IM). Plasma EBV-miR-BART2-5p was present in higher numbers of IM (7/8) and CHL (7/10) compared to REI (7/12) patients. A trend was observed between the numbers of plasma EBV miRNAs expressed and EBV viral load (p < 0.07). Several EBV-miRs including BART7-3p, 15, 9-3p, 11-3p, 1-3p and 3-3p were detected in IM and CHL patients only. The lytic EBV-miRs, BHRF1-2-3p and 1-1, indicating active viral replication, were detected in IM patients only. One CHL patient developed post-transplant lymphoproliferative disease (PTLD) after several years and analysis of 10 samples over a 30-month period showed an average 24-fold higher change in plasma EBV-miR-BART2-5p compared to the CHL group and 110-fold higher change compared to the REI group. Our results suggest that EBV-miR-BART2-5p, which targets the stress-induced immune ligand MICB to escape recognition and elimination by NK cells, may have a role in sustaining high EBV viral loads in CHL paediatric kidney transplant recipients.

Clinical Significance of Plasma Epstein-Barr Virus DNA in Pulmonary Lymphoepithelioma-like Carcinoma (LELC) Patients

Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a histologically distinctive subtype of NSCLC and an Epstein-Barr virus (EBV)-associated epithelial neoplasm. We investigated the clinical significance of plasma concentrations of EBV DNA in patients with pulmonary LELC. Two independent sets of plasma samples from a total of 429 patients with patients with pulmonary LELC (287 initial and 142 confirmatory) were available for EBV DNA determination. Plasma samples from the patients were subjected to a real-time quantitative polymerase chain reaction before treatment and 3 months after radical resection. Cutoff points were determined for pretreatment plasma EBV DNA concentration (low <4000 copies/mL versus high ≥4000 copies/mL) on the basis of a measure of heterogeneity with the log-rank test statistic with respect to overall survival (OS). The Kaplan-Meier method and Cox regression were used to evaluate the relationship between plasma EBV DNA concentrations and clinical outcome. Among patients with advanced-stage pulmonary LELC who underwent sequential blood draws, we evaluated the relationship between change in disease status and change in EBV DNA concentrations by using nonparametric tests. High EBV DNA concentration was associated with shorter OS in the initial, confirmatory, and combined data sets (combined data set hazard ratio= 3.67, 95% confidence interval: 2.72-4.38, p < 0.001). These findings persisted after multivariable adjustment. Compared with low EBV DNA concentration, high EBV DNA concentration was associated with shorter OS in patients with any stage of disease. High EBV DNA concentration was also associated with shorter disease-free survival (DFS) in patients with stage I/II disease. Patients with persistently detectable plasma EBV DNA had significantly poorer OS (p < 0.001) and DFS (p < 0.001) than did patients with undetectable EBV DNA 3 months after radical resection. In patients who underwent sequential evaluation of EBV DNA, an association was identified between an increase in EBV DNA concentration and a poor response to treatment and disease progression of pulmonary LELC. High baseline EBV DNA concentration is an independent poor prognostic marker in patients with pulmonary LELC. These results should be confirmed in larger prospective trials.

Target-induced proximity ligation triggers recombinase polymerase amplification and transcription-mediated amplification to detect tumor-derived exosomes in nasopharyngeal carcinoma with high sensitivity

Tumor-derived exosomes (TEXs) are extracellular vesicles that are continuously released into the blood by tumor cells and carry specific surface markers of the original tumor cells. Substantial evidence has implicated TEXs as attractive diagnostic markers for cancer. However, the detection of TEXs in blood at an early tumor stage is challenging due to their very low concentration. Here, we established a method called PLA-RPA-TMA assay that allows TEXs to be detected with high sensitivity and specificity. Based on two proximity ligation assay (PLA) probes that recognize a biomarker on a TEX, we generated a unique surrogate DNA signal for the specific biomarker, which was synchronously amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA), and then the products of the RPA-TMA reaction were quantitatively detected using a gold nanoparticle-based colorimetric assay. We established proof-of-concept evidence for this approach using TEXs from nasopharyngeal carcinoma (NPC) cells, with a detection limit of 102 particles/mL, and reported the measurement of plasma Epstein-Barr virus latent membrane protein 1 (LPM1)-positive (LMP1+, accuracy: 0.956) and epidermal growth factor receptor (EGFR)-positive (EGFR+, accuracy: 0.906) TEXs as potent early diagnostic biomarkers for NPC.

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.