Epstein-Barr Virus DNA Load Research Articles

Virological Markers in Epstein–Barr Virus-Associated Diseases

Epstein-Barr virus (EBV) is an oncogenic virus infecting more than 95% of the world's population. After primary infection-responsible for infectious mononucleosis in young adults-the virus persists lifelong in the infected host, especially in memory B cells. Viral persistence is usually without clinical consequences, although it can lead to EBV-associated cancers such as lymphoma or carcinoma. Recent reports also suggest a link between EBV infection and multiple sclerosis. In the absence of vaccines, research efforts have focused on virological markers applicable in clinical practice for the management of patients with EBV-associated diseases. Nasopharyngeal carcinoma is an EBV-associated malignancy for which serological and molecular markers are widely used in clinical practice. Measuring blood EBV DNA load is additionally, useful for preventing lymphoproliferative disorders in transplant patients, with this marker also being explored in various other EBV-associated lymphomas. New technologies based on next-generation sequencing offer the opportunity to explore other biomarkers such as the EBV DNA methylome, strain diversity, or viral miRNA. Here, we review the clinical utility of different virological markers in EBV-associated diseases. Indeed, evaluating existing or new markers in EBV-associated malignancies or immune-mediated inflammatory diseases triggered by EBV infection continues to be a challenge.

Characteristics of immunological events in Epstein-Barr virus infection in children with infectious mononucleosis.

Epstein-Barr virus (EBV) infection occurs commonly in children and may cause acute infectious mononucleosis (AIM) and various malignant diseases. Host immune responses are key players in the resistance to EBV infection. We here assessed the immunological events and laboratory indicators of EBV infection, as well as determined the clinical usefulness of evaluating the severity and efficacy of antiviral therapy in AIM patients. We enrolled 88 children with EBV infection. The immune environment was defined by immunological events such as frequencies of lymphocyte subsets, phenotypes of T cells, and their ability to secrete cytokines, and so on. This environment was analyzed in EBV-infected children with different viral loads and in children in different phases of infectious mononucleosis (IM) from disease onset to convalescence. Children with AIM had higher frequencies of CD3+ T and CD8+ T cells, but lower frequencies of CD4+ T cells and CD19+ B cells. In these children, the expression of CD62L was lower and that of CTLA-4 and PD-1 was higher on T cells. EBV exposure induced granzyme B expression, but reduced IFN-γ secretion, by CD8+ T cells, whereas NK cells exhibited reduced granzyme B expression and increased IFN-γ secretion. The frequency of CD8+ T cells was positively correlated with the EBV DNA load, whereas the frequencies of CD4+ T cells and B cells were negatively correlated. During the convalescent phase of IM, CD8+ T cell frequency and CD62L expression on T cells were restored. Moreover, patient serum levels of IL-4, IL-6, IL-10, and IFN-γ were considerably lower throughout the convalescent phase than throughout the acute phase. Robust expansion of CD8+ T cells, accompanied by CD62L downregulation, PD-1 and CTLA-4 upregulation on T cells, enhanced granzyme B production, and impaired IFN-γ secretion, is a typical characteristic of immunological events in children with AIM. Noncytolytic and cytolytic effector functions of CD8+ T cells are regulated in an oscillatory manner. Furthermore, the AST level, number of CD8+ T cells, and CD62L expression on T cells may act as markers related to IM severity and the effectiveness of antiviral treatment.

Synergistic Association of Hepatitis B Surface Antigen and Plasma Epstein-Barr Virus DNA Load on Distant Metastasis in Patients With Nasopharyngeal Carcinoma

Hepatitis B surface antigen (HBsAg) reportedly increases the risk of distant metastasis among patients with nasopharyngeal carcinoma (NPC). However, the associated potential interaction and changes in hazard ratios (HRs) between HBsAg and different plasma Epstein-Barr (EBV) DNA levels are unknown. Moreover, the potential HBsAg-positive-associated NPC metastatic mechanism remains unclear. To investigate the prognostic value and biological associations of HBsAg and plasma EBV DNA levels on distant metastasis in patients with NPC. Retrospective cohort study performed at Sun Yat-sen University Cancer Center between January 2010 and January 2013. A total of 792 patients with nonmetastatic NPC were enrolled. The median (range) follow-up time was 62.1 (1.4-83.4) months. Of these patients, 17.8% presented with HBsAg positivity. Cytological experiments were performed to evaluate the role of HBsAg in the invasion and migration of EBV-positive NPC cells. Data analysis was performed from July 2020 to April 2021. The primary end point was distant metastasis-free survival. Association rules were used to identify new rules related to distant metastasis. Interaction plots, univariate and multivariate Cox regression analyses, stratification analysis, and quantification using HRs were conducted. Additionally, cell migration and invasion assays, as well as Western blotting, were performed in the cytological validation. Among the 792 patients, 576 (72.7%) were male, with a median (IQR) age of 45 (38-53) years. The HBsAg-positive group exhibited a significant interaction and increased risk of distant metastasis when plasma EBV DNA cutoff levels were 1.5 × 1000 copies/mL or greater. The HR was 9.16 (95% CI, 2.46-34.14) when the plasma EBV DNA load reached 6 × 1000 copies/mL, which was higher than that in patients with stage IV disease (HR, 2.01; 95% CI, 1.13-3.56; P = .02). In cytological experiments, HBsAg promoted epithelial-mesenchymal transition by upregulating vimentin and fibronectin in EBV-positive NPC cells in vitro, thereby promoting invasion and migration of EBV-positive NPC cells. In this cohort study, the observed synergistic association between HBsAg and plasma EBV DNA load represented a novel potential mechanism underlying the increased risk of distant metastasis in patients with NPC. Hence, attention should be paid to patients with NPC with HBsAg positivity, especially when the plasma EBV DNA level is 6 × 1000 copies/mL or greater. Consideration of this synergistic association will contribute to more accurate individualized management.

Clinical relevance of plasma EBV DNA as a biomarker for nasopharyngeal carcinoma in non-endemic areas: A multicenter study in southwestern China

BackgroundNumerous clinical studies have validated plasma EBV DNA as a reliable biomarker for nasopharyngeal carcinoma (NPC) screening, tumor load monitoring, and prognosis prediction in endemic regions. However, the clinical relevance of plasma EBV DNA as a biomarker for NPC in non-endemic areas is still unclear. MethodThe pretreatment plasma EBV DNA of 1405 newly diagnosed NPC patients from three major regional hospitals in non-endemic areas were analyzed retrospectively. The medical records of 244 age- and gender-matched healthy individuals were reviewed. EBV DNA was detected using Polymerase Chain Reaction (PCR). Based on the baseline of 400 and 0 copies/mL, the distribution characteristics of the pretreatment EBV DNA load in different clinical stages and geographic regions were analyzed. The diagnostic value of pretreatment plasma EBV DNA for NPC with two baselines was evaluated using the ROC curve. ResultsNPC patients had a significantly higher pretreatment EBV DNA level than healthy controls (P＜0.001). Pretreatment EBV DNA was closely associated with clinical and TNM stages in non-endemic areas, as it was in endemic areas. However, when 400 copies/mL set as the detection baseline, the sensitivity and specificity for NPC diagnosis were 40.8 % and 100 %, respectively (AUC = 0.704, cut off = 200.5 copies/mL). This sensitivity was lower than that reported in endemic regions (41.5 % − 97.1 %). Lower sensitivity may result in false negatives, missing diagnoses during NPC screening. Further investigation revealed that 39.7 % (558/1405) of NPC patients had detectable EBV DNA and S amplification curves. Optimizing the detection limit to 0 copies/mL, the sensitivity could be improved to 80.5 % (AUC = 0.901). ConclusionsIn non-endemic areas, the clinical significance of plasma EBV DNA as a biomarker for NPC was restricted due to the low detection limit of 400 copies/mL. More efficient nucleic acid extraction and detection methods are needed to optimize the detection limit and increase the clinical application of plasma EBV DNA for NPC.

Epstein-Barr Viral Load Monitoring Strategy and the Risk for Posttransplant Lymphoproliferative Disease in Adult Liver Transplantation : A Cohort Study.

Primary infection with or reactivation of Epstein-Barr virus (EBV) can occur after liver transplant (LT) and can lead to posttransplant lymphoproliferative disease (PTLD). In pediatric LT, an EBV-DNA viral load (EBV VL) monitoring strategy, including the reduction of immunosuppression, has led to a lower incidence of PTLD. For adult LT recipients with less primary infection and more EBV reactivation, it is unknown whether this strategy is effective. To examine the effect of an EBV VL monitoring strategy on the incidence of PTLD after LT in adults. Cohort study. Two university medical centers in the Netherlands. Adult recipients of first LT in Leiden between September 2003 and January 2017 with an EBV VL monitoring strategy formed the monitoring group (M1), recipients of first LT in Rotterdam between January 2003 and January 2017 without such a strategy formed the contemporary control group (C1), and those who had transplants in Leiden between September 1992 and September 2003 or Rotterdam between 1986 and January 2003 formed the historical control groups (M0 and C0, respectively). Influence of EBV VL monitoring on incidence of PTLD. After inverse probability of treatment weighting of the 4 groups to achieve a balance among the groups for important patient characteristics, differences within hospitals between the historical and recent era in cumulative incidences-expressed as the number of events per 1000 patients measured at 5-, 10-, and 15-year follow-up-showed fewer events in the contemporary era in both centers. This difference was considerably larger in the monitoring center, whereas the 95% CI included the null value of 0 for point estimates. Retrospective, low statistical power, and incompletely balanced groups, and non-EBV PTLD cannot be prevented. Monitoring EBV VL may reduce PTLD incidence after LT in adults; larger studies are warranted. None.

The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Moppert J, Pawłowska M, Domagalski K, Szwed-Kowalska A. The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study. Pediatria Polska - Polish Journal of Paediatrics. 2023. doi:10.5114/polp.2023.126111. APA Moppert, J., Pawłowska, M., Domagalski, K., & Szwed-Kowalska, A. (2023). The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study. Pediatria Polska - Polish Journal of Paediatrics. https://doi.org/10.5114/polp.2023.126111 Chicago Moppert, Justyna, Małgorzata Pawłowska, Krzysztof Domagalski, and Aleksandra Szwed-Kowalska. 2023. "The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study". Pediatria Polska - Polish Journal of Paediatrics. doi:10.5114/polp.2023.126111. Harvard Moppert, J., Pawłowska, M., Domagalski, K., and Szwed-Kowalska, A. (2023). The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study. Pediatria Polska - Polish Journal of Paediatrics. https://doi.org/10.5114/polp.2023.126111 MLA Moppert, Justyna et al. "The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study." Pediatria Polska - Polish Journal of Paediatrics, 2023. doi:10.5114/polp.2023.126111. Vancouver Moppert J, Pawłowska M, Domagalski K, Szwed-Kowalska A. The concentration of IL-6, TNF-α, s-ICAM-1, and EBV DNA load – predictive factors of hepatological complications in children with infectious mononucleosis. A pilot study. Pediatria Polska - Polish Journal of Paediatrics. 2023. doi:10.5114/polp.2023.126111.

Epstein-Barr virus-associated lymphoproliferative disorders after BNT162b2 mRNA COVID-19 vaccination

Epstein-Barr virus-associated lymphoproliferative disorders (EBV-LPD) is a rare disease characterized by persistent or recurrent inflammation accompanied by EBV infection of T or NK cells that is not self-limiting, and it is fatal, if untreated. After receiving the first dose of the BNT162b2 mRNA COVID-19 vaccine, a 79-year-old male presented to the hospital with a 2-week history of fever. Laboratory results indicated pancytopenia, elevated liver transaminase levels, hyperferritinemia, and hypofibrinogenemia. Computed tomography revealed hepatosplenomegaly, but lymphadenopathy was not observed. A bone marrow biopsy, a random skin biopsy, and a liver biopsy revealed no malignancy, but an infectious evaluation revealed EBV viremia (5.19 Log IU/ml). Flow cytometry and RT-PCR revealed that the EBV genome was localized in NK cells, suggesting the diagnosis of EBV-NK-LPD. We administered prednisolone, intravenous immunoglobulin, and etoposide, but the EBV-DNA load failed to decrease, and he died 2 months later. Recently, case reports of COVID-19 vaccination-related hemophagocytic lymphohistiocytosis have been published. Although the mechanisms and risk factors for EBV-LPD after BNT162b2 mRNA COVID-19 vaccination remain unknown, it is important to note the possibility of reactivation of EBV after COVID-19 vaccination to initiate early and targeted therapy.

The Dynamic Change in Plasma Epstein-Barr Virus DNA Load over a Long-Term Follow-Up Period Predicts Prognosis in Nasopharyngeal Carcinoma.

The current study was designed to investigate the changes in the circulating Epstein-Barr virus DNA load (EBV DNA) at various time points before and after treatment and its clinical significance in nasopharyngeal carcinoma (NPC). A total of 142 patients with NPC were prospectively enrolled in this study. The plasma EBV DNA concentration was measured before and after treatment using qPCR. The prognostic values of the EBV DNA load were analyzed using the Kaplan-Meier and Cox regression tests. Following multivariate analysis, our data showed that high pre-EBV DNA loads were associated with significantly poorer distant metastasis free survival (DMFS) and progression free survival (PFS); detectable end-EBV DNA loads were associated with significantly worse loco-regional recurrence free survival (LRRFS) and PFS, and the detecTable 6 months-post-EBV DNA loads were associated with significantly poorer overall survival (OS), DMFS and PFS (p < 0.05). Additionally, combining the pre-EBV DNA load and the stage of the disease, our results showed that patients at stage III-IVA with a low pre-EBV DNA load had similar survival rates as patients at stage II with a low or high pre-EBV DNA load, but had better survival rates than those at stage III-IVA with a high pre-EBV DNA load. Taken together, we showed that the change of the EBV DNA load measured at several time points was more valuable than at any single time point for predicting patients' survival for NPC. Furthermore, combining the pre-EBV DNA load and the TNM classification could help to formulate an improved prognostic model for this cancer.

Clinical outcomes of non-nasopharyngeal lymphoepithelial carcinoma treated with a combined modality approach: A single-institution study.

This study presents a summary of the clinical characteristics of non-nasopharyngeal lymphoepithelial carcinoma (NNPLEC), effects of combined modality treatment and prognostic value of plasma Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) load, with the aim of providing a reference framework for optimizing treatment practices and outcomes. Patients with NNPLEC treated by our center between January 2000 and December 2020 were retrospectively reviewed. In total, 728 patients were included. The lung was identified as the most common primary tumor site (64.0%), followed by the salivary gland (19.2%). A total of 539 (74.0%) patients underwent surgery, 459 (63.0%) received chemotherapy, and 361 (49.6%) were subjected to radiotherapy. The median follow-up time was 45 months (range, 6-212 months) and 5-year overall survival (OS) was 79.1%. Increased plasma EBV-DNA load of >513.5 copies/mL was predictive of disease progression, with a specificity of 98.1% and a sensitivity of 98.9%. In multivariate Cox analysis, N stage, surgery, and radiotherapy were independent prognostic factors for both OS and PFS. Radiotherapy significantly improves OS in comparison with no radiotherapy group for salivary LEC, while surgery significantly improves OS for pulmonary LEC. Based on our analysis, surgery and radiotherapy are associated with better OS and PFS for NNPLEC. Radiotherapy could be recommended for salivary LEC, while surgery remains the primary treatment strategy for pulmonary LEC patients. An increased plasma EBV-DNA load of >513.5 copies/mL is strongly predictive of disease progression, supporting the importance of regular evaluation of plasma EBV-DNA as part of the diagnostic routine.

Phase II Study of a JAK1/2 Inhibitor Ruxolitinib for Systemic Chronic Active Epstein-Barr Virus Disease: An Investigator-Initiated Trial

Background: Systemic chronic active Epstein-Barr virus disease (sCAEBV) is a rare EBV-positive T- or NK-lymphoproliferative disease. Although the only curative treatment for sCAEBV available now is allogeneic hematopoietic stem cell transplantation (allo-HSCT), the presence of disease activity, persistent inflammation, at the time of allo-HSCT is associated with poor outcomes (Am J Hem.2022, 97, p780). STAT3 is constitutively activated contributing to cytokine production and cell survival of EBV-infected cells in sCAEBV (Oncotarget. 2019, 9, p31077). Thus, STAT3 inhibition by ruxolitinib, a JAK1/2 inhibitor, may be effective against disease activity of sCAEBV. Methods: We conducted an investigator-initiated trial of open-label, phase 2, prospective and multicenter to assess the efficacy and safety of ruxolitinib in sCAEBV patients with disease activity. The diagnostic criteria were as follows: elevated EBV-DNA load in the peripheral blood (PB) (>102.5 copies/µg DNA); EBV infected T or NK cells detected in affected tissues or PB; systemic inflammatory symptoms persisting for more than three months; exclusion of other possible diagnoses, such as immune deficiencies, autoimmune diseases, or lymphoma (Blood adv. 2021, 4, p2918). sCAEBV patients ≥13 y.o. with fever (≥ 37.5 ºC), and/or liver dysfunction (ALT ≥ 2.0 × ULN) were enrolled. Anti-VCA-IgM-positive patients were excluded. The initiation dose of ruxolitinib was 5-20 mg twice a day determined by the platelet count in PB. The treatment effects were classified as complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). CR was defined as the disappearance of all the elements of disease activity such as fever, liver dysfunction, uveitis, progressive skin lesions, and vasculitis. PR was defined as the disappearance of at least one of the elements listed above. PD was defined as the conversion to active disease, exacerbation of active disease, or the development of overt T- or NK-cell lymphoma. SD was defined as presenting no progression. Ruxolitinib was administered for eight weeks. If CR was observed and allo-HSCT was scheduled, ruxolitinib was terminated early even if these events had occurred within less than eight weeks after initiating investigational drug administration. The primary endpoint of efficacy assessments was CR rate at eight weeks after the initiation of drug administration or at the time of early termination. Safety was assessed based on the frequency of adverse events defined by Common Terminology Criteria for Adverse Events, version 4.0. Results: Nine patients were enrolled between January 2019 and August 2021; five males and four females aged 13-64 y.o.; median age of 40 y.o. The types of EBV-infected cells were as follows: four were CD4, one was CD8, and four were CD56. Seven patients completed the study. Five among them received ruxolitinib for eight weeks and were treated as outpatients. Two patients fulfilled the criteria of early termination and each was treated for 35 and 12 days. The responses are shown in the figure. CR rate and OR rate after eight weeks or at early termination, the primary and the secondary endpoints were 28.6% (2/7) and 42.9% (3/7), respectively. No one showed progressive disease. One patient developed a grade 4 adverse event of oral bleeding during the treatment and resulted in discontinuing the administration of ruxolitinib on day 13. Seven patients received allo-HSCT. Five patients achieved undetectable level of EBV-DNA PB after allo-HSCT. Conclusion: The results indicate that ruxolitinib has demonstrated encouraging effects and is a potent therapeutic reagent against sCAEBV with active disease. Further analysis shall complete its assessment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparing BamHI-W and CE-marked assays to detect circulating Epstein-Barr Virus (EBV) DNA of nasopharyngeal cancer patients in a non-endemic area

ObjectivesPlasma Epstein-Barr Virus (EBV)-DNA is a well-established prognostic biomarker in nasopharyngeal carcinoma (NPC). Different methods for assessment include single-copy gene targeted, European Conformity (CE)-marked assays, which are mostly employed in non-endemic settings, vs multiple-copy gene targeted, in-house BamHI-W based assays, which currently represent the most widely used method for EBV-DNA quantification. To date, evidence concerning the commutability of these different assays is still limited. Materials and methodsFrom August 2016 to March 2018, 124 plasma and 124 whole blood (WB) samples from 93 NPC patients were collected at different time-points for each patient. EBV-DNA viral load was quantified in pre- (n = 12) and post-treatment (n = 9), follow-up (n = 53), and recurrent/metastatic (R/M) (n = 50) phase. For each sample, one in-house BamHI-W vs three different CE-marked plasma assays were compared; the performance of plasma vs WB matrix was also assessed. Quantitative agreement of EBV-DNA values was evaluated by linear correlation and Bland-Altman analysis. ResultsA statistically significant (p = 0.0001) agreement between all CE-marked and the BamHI-W assays was found using plasma matrix, regardless of clinical phase. The results obtained in copies/ml were comparable to those expressed in IU/ml. When using WB matrix, the number of positive detections increased in the post-treatment phase. ConclusionsOur retrospective comparison supported an agreement between Plasma BamHI-W and CE-marked assays in measuring EBV-DNA for non-endemic NPC patients. There were no significant interferences from different measurement units (IU/ml vs copies/ml). Further evaluations are needed to better clarify the role of WB.

Association of delayed chemoradiotherapy with elevated Epstein-Barr virus DNA load and adverse clinical outcome in nasopharyngeal carcinoma treatment during the COVID-19 pandemic: a retrospective study

Background:To summarize the impact of radiotherapy (RT) and chemotherapy delays on patients with nasopharyngeal carcinoma (NPC) during the COVID-19 pandemic.Methods:We retrospectively included 233 patients with stage II-IVa NPC treated with RT and chemotherapy between December 11, 2019 and March 11, 2020. The outcomes were elevation in the EBV DNA load between two adjacent cycles of chemotherapy or during RT, and 1-year disease-free survival (DFS).Results:RT delay occurred in 117 (50%) patients, and chemotherapy delay occurred in 220 (94%) patients. RT delay of ≥ 6 days was associated with a higher EBV DNA elevation rate (20.4% vs. 3.6%, odds ratio [OR] = 6.93 [95% CI = 2.49–19.32], P < 0.001), and worse 1-year DFS (91.2% vs. 97.8%, HR = 3.61 [95% CI = 1.37–9.50], P = 0.006), compared with on-schedule RT or delay of < 6 days. Chemotherapy delay of ≥ 10 days was not associated with a higher EBV DNA elevation rate (12.5% vs. 6.8%, OR = 1.94 [95% CI = 0.70–5.40], P = 0.20), or worse 1-year DFS (93.8% vs. 97.1%, HR = 3.73 [95% CI = 0.86–16.14], P = 0.059), compared with delay of < 10 days. Multivariable analyses showed RT delay of ≥ 6 days remained an independent adverse factor for both EBV DNA elevation and DFS.Conclusion:To ensure treatment efficacy for patients with nonmetastatic NPC, initiation of RT should not be delayed by more than 6 days; the effect of chemotherapy delay requires further investigation.

Chronic Active Epstein-Barr Virus Infection With Central Nervous System Involvement in Children: A Clinical Study of 22 Cases.

To analyze the clinical features, treatment, and prognosis of chronic active Epstein-Barr virus infection (CAEBV) with central nervous system (CNS) involvement in children. Patients with CAEBV admitted to Beijing Children's Hospital, Capital Medical University, were enrolled in this study from January 2017 to December 2020. They were divided into a CNS group and a non-CNS group based on the presence of CNS involvement. Twenty-two patients developed CNS disease, accounting for 23.9% (22/92) of CAEBV patients in the same period. Of these, only 2 of 22 patients presented initially with neurologic symptoms in the CNS group, and they all improved after treatment. Cerebrospinal fluid (CSF) examination demonstrated normal protein concentration and cell number in all patients with CNS involvement. Only 7 patients were positive for CSF EBV-DNA. Twenty-one patients had neuroimaging abnormalities, such as white matter signal abnormalities, encephalography or calcification. In the CNS group, 7 (31.8%) patients died, including 5 who died of active hemophagocytic lymphohistiocytosis, 1 died of unrelated causes, and 1 died of respiratory failure caused by pulmonary lymphoproliferative disease progression after transplantation. The 3-year overall survival was lower in the CNS group than in the non-CNS group (63.6% ± 11.9% versus 86.9% ± 4.1%, P = 0.027). Hemophagocytic lymphohistiocytosis (HLH) is an independent risk factor for CNS involvement in patients with CAEBV (OR = 2.946, 95% CI: 1.042-8.335, P = 0.042). Compared with the non-CNS group, blood EBV-DNA loads and CD4+/CD8+ ratio of T lymphocytes in the CNS group were higher (P < 0.001), while fibrinogen levels and natural killer (NK)-cell activity were lower (P = 0.047). Children with CAEBV were more likely to develop CNS diseases with low NK-cell activity (NK-cell activity < 14.00%, P = 0.023) or high alanine aminotransferase (ALT) levels (ALT levels > 40 U/L, P = 0.032). CAEBV with CNS involvement has nonspecific clinical manifestations, laboratory data, neuroimaging but has a worse prognosis. Blood fibrinogen levels and NK-cell activity in CAEBV children with CNS involvement are lower than in those without CNS involvement. In contrast, blood EBV-DNA loads and CD4+/CD8+ ratio of T lymphocytes are higher. Children with CAEBV who presented with HLH, NK-cell activity <14.00%, serum ALT >40 U/L and high-blood EBV-DNA loads are prone to develop CNS diseases.

Development and validation of a joint model for dynamic prediction of overall survival in nasopharyngeal carcinoma based on longitudinal post-treatment plasma cell-free Epstein-Barr virus DNA load

ObjectivesTo develop and validate a joint model for dynamic prediction of overall survival (OS) in nasopharyngeal carcinoma (NPC) based on longitudinal post-treatment plasma cell-free Epstein-Barr virus (cfEBV) DNA load. Patients and methodsWe analyzed 695 patients with non-metastatic NPC and detectable post-treatment cfEBV DNA load who did not receive adjuvant therapy. We fitted the trajectories of post-treatment cfEBV DNA load as a function of time into a linear mixed-effect model and fitted a Cox regression model with covariates including age, T and N stages, and lactate dehydrogenase level. Finally, we combined both via joint modeling to develop and validate our dynamic model. ResultsA strong positive correlation was found between the individual longitudinal post-treatment cfEBV DNA load and the risk of death from any cause (P < 0.001). We developed a joint model capable of providing subject-specific dynamic prediction of conditional OS based on the evolution of the individual plasma cfEBV DNA load trajectory. The joint model showed reliable performance in both training and validation cohorts, with a large area under the curve (interquartile range [IQR]: training cohort, 0.775–0.850; validation cohort, 0.826–0.900) and low prediction errors (IQR: training cohort, 0.017–0.078; validation cohort, 0.034 –0.071). An increasing amount of data on cfEBV DNA load was associated with better model performance. ConclusionOur model provided reliable subject-specific dynamic prediction of conditional OS, which could help guide individualized post-treatment surveillance, risk stratification, and management of NPC in the future.

High plasma EBV-DNA load and positive EBER status associated with viral recurrence and persistent infection in early treatment of lymphoma.

The relationship between plasma EBV-DNA load (PEDL) and Epstein-Barr virus (EBV)-encoded small RNA (EBER) during the early treatment of lymphoma remains unclear. We explored discrepancies in PEDL and variables associated with EBER and evaluated the consistency between EBER and qualitative analysis of PEDL (qPEDL). Serial measurements of PEDL were performed to determine the dynamic changes of PEDL in early treatment of lymphoma. As a result, the median PEDL of non-Hodgkin's lymphoma NKT cell subtype (NHL-NKT) was higher than that of non-Hodgkin's lymphoma B cell subtype (NHL-B), the median PEDL of extranodal NK/T cell lymphoma (ENKTCL) was higher than that of diffuse large B cell lymphoma (DLBCL), and the median PEDL of EBER positive was higher than that of EBER negative. Age, Ki-67 ≧ 80%, Bcl-2 ≧ 80%, p53, and qPEDL were related to EBER. The PEDL could distinguish NHL-B, DLBCL, NHL-NKT, and ENKTCL from other lymphoma subtypes. EBER-positive patients spent more time with viral "turn negative (TN)" and "continuous positive (CP)" and less time with viral "continuous negative (CN)." The median PEDL of CP was higher than that of TN. In conclusion, although EBER affects the levels of PEDL in general, it has poor concordance with qPEDL. Our results show, for the first time, that high PEDL and positive EBER present a strong association with viral recurrence and persistent infection in the early treatment of lymphoma.

Epstein-Barr virus central nervous system involvement in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a common subtype of secondary HLH. EBV plays an important part in the course. EBV can cause central nervous system (CNS) infections, and there are few clinical studies on EBV-CNS infection in EBV-HLH patients. All patients who were diagnosed as EBV-HLH and underwent cerebrospinal fluid testing admitted to our center from January 2018 to December 2019 were retrospectively analyzed. Summarized the clinical data, evaluated treatment efficacy after intrathecal injection, and investigated the correlation between EBV-CNS infection with prognosis in EBV-HLH patients. Of 37 of 57 (64.9%) EBV-HLH patients has EBV-CNS infection. The survival of EBV-HLH patients without EBV-CNS infection was significantly better than that in EBV-CNS infection patients (P = 0.018). There were no statistically significant differences in sCD25, ferritin, ALT, AST, LDH, TB, WBC, Hb, and PLT counts between two groups (all P-values > 0.05). Higher EBV-DNA load in peripheral blood was correlated with EBV-CNS infection (P < 0.001). EBV-CNS infection is an independent risk factor affecting the survival of patients (P = 0.004). The CSF cell load of patients with and without EBV-CNS infection groups was significantly different (P = 0.024). Intrathecal injection with methotrexate combined with dexamethasone can effectively decrease CSF EBV-DNA load (P = 0.017) and CSF cell load (P = 0.025). EBV-CNS infection is an independent risk factor affecting prognosis in EBV-HLH patients. Therefore, EBV-CNS infection should cause concern for EBV-HLH patients. Cerebrospinal fluid testing is necessary for all patients. Methotrexate combined with dexamethasone intrathecal injection can be an effective treatment for EBV-CNS infection.

Genomic and microbial factors affect the prognosis of anti-pd-1 immunotherapy in nasopharyngeal carcinoma.

Antibodies targeting the programmed cell death protein-1 (PD-1) molecule have been reported to hold promising antitumor activities in patients with nasopharyngeal carcinoma (NPC). However, only a small subset of NPC patients benefits from the anti-PD-1 monotherapy and factors that affect the treatment response need further investigation. This study aimed to examine the impact of multiple genetic and environmental factors on outcome of anti-PD-1 immunotherapy by identifying tumor size, tumor mutation burden (TMB) based on whole exon sequencing, human leukocyte antigen class I (HLA-I) homo-/heterozygosity and supertypes, blood Epstein-Barr virus (EBV) DNA load, T cell proportions, and interferon-γ(IFN-γ) levels in a cohort of 57 NPC patients that received Nivolumab or Camrelizumab treatment. Moreover, we profiled the longitudinal changes in gut microbiota composition using shotgun metagenomics sequencing. We observed that high TMB combined with HLA-I heterozygosity was associated with improved clinical outcomes. In agreement with previous studies, we found that patients with higher plasma EBV DNA load showed worse progression-free survival. We found no evidence for an effect of gut bacterial diversity on the treatment response, but identified a higher abundance of seven specific gut bacteria at baseline of non-responders, including Blautia wexlera and Blautia obeum, as well as four other bacteria belonging to the Clostridiales order, and one Erysipelatoclostridium. Combined, this study provides insight into the influence of several genetic and environmental factors on anti-PD-1 immunotherapy responses in NPC patients.

Clinical Analysis of Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation in Severe Aplastic Anemia Patients

To analyze the clinical characteristics of SAA patients with post-transplantation lymphoproliferative disease (PTLD) after allogeneic hematopoietic stem cell transplantation, and to improve diagnosis and treatment of PTLD. The clinical data of 192 patients with SAA patients who underwent HSCT in a single center from September 2010 to September 2017 were analyzed retrospectively. All patients were received antithymocyte globulin（ATG） conditioning regimen and mesenchymal stem cell(MSC) infusion. Among 192 cases, PTLD occurred in 14 cases, the incidence was 7.29％， 9 of them were diagnosed by pathology， and 5 were diagnosed clinically. EBV infection occurred with a median time of 72（35-168） days, Viral load higher than 1×104 copies/ml occured in all PTLD patients. The incidence of probable PTLD in patients ≤12 years old and ＞12 years old was 11.11%, 2.38%, respectively (P<0.01）. Univariate and multivariate analysis that the EBV infection, patients age≤12 years old, HLA-mismatch in URD-HSCT, grade II to IV aGVHD were the independent risk factors for PTLD. All PTLD patients were treated with rituximab(RTX) when EBV-DNA load higher than 1×104 copies/ml, or reducted the use of immunosuppression(RIS), patients with poor therapeutic effect were treated combined with EBV-specific CTLs(EBV-CTL) and chemotherapy. All patients were treated effectively, and the total effective rate was 100％. The median follow-up time was 65(62-115) months, and the overall survival rate was 92.85%. One patients died of cerebral hemorrhage, 7 months after PTLD curred. The incidence of PTLD after HSCT with SAA who used ATG and MSC in conditioning regimen closely relates to EBV infection, age of patients≤12 years, HLA-mismatch in URD-HSCT, grade II to IV GVHD. Rituximab combined with RIS may reduce the incidence of PTLD, combined EBV-CTL and chemotherapy may be the useful and most important treatment for PTLD.

P1238: EFFICACY ANALYSIS OF PEGASPARGASE, GEMCITABINE, OXALIPLATIN, DEXAMETHASONE COMBINED WITH TISLELIZUMAB IN FIRST-LINE TREATMENT OF PLASMA EBV-DNA POSITIVE EXTRANODAL NK/T CELL LYMPHOMA

Background: Positive circulating EBV-DNA is associated with a very poor outcome in Natural killer/T cell lymphoma (NKTCL). Efforts to further improve treatment of plasma EBV positive NKTCL are urgently needed. Aims: To investigate the clinical efficacy, plasma EBV-DNA load and safety of P-GEMOXD combined with anti-programmed death 1 (anti-PD-1) antibody Tislelizumab in first-line treatment of plasma EBV-DNA positive NKTCL. Methods: In this prospective, single-arm, phase 2 trial, we recruited patients aged 18–70 years with newly diagnosed NKTCL who were EBV-DNA positive in plasma in Henan People’s Hospital. Patients received four courses of P-GEMOXD plus Tislelizumab treatment (pegaspargase 2500U/m2 [day3], gemcitabine 800mg/m2 [day1 and 8]; oxaliplatin100 mg/m2[day1], dexamethasone 20mg/d [day1-4]; Tislelizumab 200mg /d [day9] every 21days). Thirteen patients were enrolled from January 2018 to August 2021 December, the median follow-up was 26 (12-46) months. The short-term and long-term clinical efficacy, load of EBV-DNA and adverse reactions were observed. Results: Among the 13 patients, there were 9 males and 4 females, the median age was 54 years old (range 25-64). After 4 courses of treatment, 10 patients (76.9%) had complete response, 3 patients (23.1%) had partial response, and the objective response rate (ORR) was 100%. The 2-year progress-free survival (PFS) rate was 76.9%, and the 2-year overall survival (OS) rate was 85.7%. Among them, there were 6 patients with advanced stage (III and IV), 4 patients (66.7%) had complete response, 2 patients (33.3%) had partial response, 5 patients (83.3%) plasma EBV-DNA converted negative; the 2-year progression-free survival (PFS) rate was 66.7%, and the 2-year overall survival (OS) rate was 66.7%. Treatment-related AEs of any grade occurred in 84.6% (11/13) of all patients. The main adverse reactions were albumin reduction (8/13), fibrinogen reduction (9/13), autoimmune diseases (3 cases：1 case of hypopituitarism， 1 case of Hashimoto’s thyroiditis, and 1 case spondylitis). Summary/Conclusion: P-GEMOXD plus Tislelizumab can significantly improve the prognosis of patients with NKTCL, improve the rate of plasma EBV-DNA negative conversion, and the adverse reactions can be well tolerated.

Can Epstein-Barr virus-deoxyribonucleic acid load after induction chemotherapy combined with American Joint Committee on Cancer stage determine the chemotherapy intensity of locally advanced nasopharyngeal carcinoma?

Induction chemotherapy (IC) comprising docetaxel, cisplatin, and fluorouracil (TPF), combined with concurrent chemoradiotherapy (CCRT) effectively improves the survival rate of locally advanced nasopharyngeal carcinoma (LA-NPC). Selecting patients whose risk of tumor recurrence and metastasis is high and the appropriate chemotherapy intensity is a concern. We combined tumor-node-metastasis staging with the load of Epstein-Barr virus (EBV) after IC to select the individualized chemotherapy strength. The clinical data and prognostic factors of patients with stage III-IV LA-NPC treated with TPF IC combined with CCRT were analyzed retrospectively. The conventional treatment group received the standard three cycles TPF IC combined with CCRT. For the new treatment group, the cycles of IC were determined according to whether the EBV-DNA disappeared completely after a certain course of IC, if so, subsequent IC was stopped and the chemoradiotherapy stage was entered. Propensity score matching (PSM) was performed at a ratio of 1:1 to balance baseline characteristics. Survival outcomes and adverse events between the conventional treatment group and the new method treatment group were compared. The study included 256 patients, among whom 192 were matched successfully into 96 pairs. The patients were followed up for a median of 51 months. The proportions of patients receiving three, two, and one cycle of IC after PSM in the routine and new treatment cohorts were 93.8%, 3.1%, 3.1% versus 21.9%, 49.0%, 24.0%, respectively. However, their 3-year distant metastasis-free survival, local recurrence-free survival, progression-free survival, and overall survival did not differ significantly. The incidence of grade 3-4 neutropenia toxicity in CCRT decreased significantly in patients receiving the new treatment method compared with that in the conventional treatment group (p=0.026). Combining TNM stage and EBV-DNA load after IC to determine the courses of IC in patients with LA-NPC did not alter the curative effect but decreased toxicity.