Plasma EBV DNA Research Articles

Selection of induction chemotherapy cycles for stage N3 nasopharyngeal carcinoma based on pre-treatment plasma EBV DNA.

This study aimed to explore the selection of induction chemotherapy (IC) cycles for stage N3 nasopharyngeal carcinoma (NPC). We employed propensity score matching (PSM) to categorize patients into 3-cycle and 4-cycle IC groups (IC = 3 and IC = 4). The log-rank and chi-squared tests were used respectively to evaluate the differences in survival and acute toxicities. Survival outcomes including overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS), and locoregional relapse-free survival (LRRFS) were evaluated among the two groups. After PSM, each group comprised 99 patients. The IC = 4 group exhibited markedly improved survival outcomes compared with the IC = 3 group. Multivariate analysis revealed that pre-EBV DNA was an independent risk factor affecting PFS and DMFS. For high-risk patients with pre-EBV DNA ≥ 7800 copies/ml, the IC = 4 group demonstrated greater survival compared to the IC = 3 group. Among low-risk patients with pre-EBV DNA < 7800 copies/ml, both groups showed comparable survival outcomes. In terms of acute adverse reactions, the IC = 4 group experienced higher incidences, particularly with grade 2-4 alanine transaminase elevation and thrombocytopenia. For stage N3 NPC, pre-EBV DNA could be a powerful predictor for guiding the selection of IC cycles. The IC = 4 regimen is probably more beneficial to high-risk patients due to superior survival, while for low-risk patients, the IC = 3 regimen may be sufficient.

Analysis of the epidemiology and clinical characteristics of Epstein-Barr virus infection.

The Epstein-Barr virus (EBV) is responsible for a spectrum of human diseases and demonstrates a considerable prevalence among various populations. Advances in molecular epidemiological research have enhanced our comprehension of EBV-related pathologies. In this study, our objective was to examine the epidemiological profile and clinical features of EBV infection in Chongqing, China. We enrolled patients suspected of EBV-related diseases who were admitted to the First Affiliated Hospital of Chongqing Medical University between May 2013 and November 2022. Inclusion criteria were based on those who underwent EBV-specific immunofluorescence or plasma EBV-DNA testing. Among 13 584 inpatients, the overall seropositivity rates for EBNA-1-IgG, EBV-VCA-IgM, EBV-EA-IgG, EBV-EA-IgA, EBV-VCA-IgA, and EBV-DNA were 91.89%, 7.22%, 18.00%, 16.19%, 30.78%, and 18.00%, respectively. The seropositivity rate for EBNA-1-IgG steadily increased with age. The seropositivity rate for VCA-IgM, an indicator of acute EBV infection, was highest in patients aged 11-20 years at 26.41%, decreasing to 2%-6% in older patients. Additionally, among 205 outpatients, the EBV-DNA positivity rate was 14.15%. In 3670 individuals from health check-up centers, the seropositivity rates for EBV-EA-IgA and EBV-VCA-IgA were 11.96% and 28.09%, respectively, and the EBV-DNA positivity rate was 11.92%, all of which were lower than those in inpatients. Among the 762 EBV-DNA positive inpatients, adults aged 31-40 years were the least affected, with a seropositivity rate of 12.00%, which increased with age. The most common diseases associated with primary EBV infection were infectious mononucleosis (IM) (35.49%), followed by EBV infection (14.15%) and pneumonia (7.19%). The most common diseases associated with EBV reactivation were pneumonia (16.80%), nasopharyngeal carcinoma (NPC) (11.02%), and autoimmune diseases (7.04%). Patients with hemophagocytic lymphohistiocytosis (HLH) had the highest viral load, significantly higher than those with NPC, pneumonia, and liver cirrhosis. This large-scale retrospective study explores the epidemiological characteristics and disease spectrum of EBV infection across all age groups. The findings contribute to the improvement of diagnostic and management strategies for EBV infection.

Treatment of pediatric refractory or relapsed Epstein-Barr virus-associated hemophagocytic syndrome with PD-1 inhibitors.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a type of pediatric HLH that occurs frequently in Asia. Although immunochemotherapy based on etoposide and hormone has improved survival rates, there are still about 30% of HLH patients that do not respond. The objective of the article is to examine the efficacy and safety of programmed cell death protein 1 (PD-1) inhibitors for children with relapsed/refractory (r/r) EBV-HLH. A retrospective case note review of four pediatric patients with r/r EBV-HLH who were treated with PD-1 inhibitors at Sun Yat-sen Memorial Hospital, Sun Yat-sen University. All four patients responded to PD-1 inhibitors and achieved partial response after their first infusion. Plasma EBV DNA copy number and HLH-related monitoring indicators decreased in all of these patients. All patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), and two were still alive at the last follow-up on December 30, 2022. Two patients died because of transplantation-related complications. Serious side effects included increased liver enzymes and edema in two patients. PD-1 inhibitors are an effective salvage therapy and can provide a bridge to allo-HSCT for pediatric patients with r/r EBV-HLH. However, side effects should be monitered.

The plasma EBV DNA load with IL-6 and VEGF levels as predictive and prognostic biomarker in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is often diagnosed at a very advanced stage due to its location and non-specific initial symptoms. Moreover, no clinically useful serological marker has been established so far for early detection of NPC. In this study, we have investigated the clinical significance of plasma Epstein–Barr virus DNA load along with interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) levels to evaluate if these three all together can be useful as a strong serological marker for early detection and prediction of treatment response in patients with NPC. Plasma EBV DNA load, IL-6 level, VEGF expressions were measured in 24 patients with NPC at presentation and various time points during and after treatment. There was a positive correlation between high plasma EBV DNA load with higher IL-6 and VEGF expression, which was closely associated with therapeutic response as well. Persistent or recurrent plasma EBV load with higher IL-6 and VEGF levels can potentially predict disease progression and may be useful to select patients for additional therapy and longer follow-up.

Establishment and validation of circulating cell-free DNA signatures for nasopharyngeal carcinoma detection

Early detection of nasopharyngeal carcinoma (NPC) poses a significant challenge. The absence of highly sensitive and specific diagnostic biomarkers for nasopharyngeal carcinoma contributes to the unfavourable prognosis of NPC patients. Here, we aimed to establish a non-invasive approach for detecting NPC using circulating cell-free DNA (cfDNA). We investigated the potential of next-generation sequencing (NGS) of peripheral blood cells as a diagnostic tool for NPC. We collected data on genome-wide nucleosome footprint (NF), 5'-end motifs, fragmentation patterns, CNV information, and EBV content from 553 Chinese subjects, including 234 NPC patients and 319 healthy individuals. Through case-control analysis, we developed a diagnostic model for NPC, and validated its detection capability. Our findings revealed that the frequencies of NF, fragmentation, and motifs were significantly higher in NPC patients compared to healthy controls. We developed an NPC score based on these parameters that accurately distinguished NPC from non-NPC cases according to the American Joint Committee on Cancer staging system from non-NPC (validation set: area under curve (AUC)=99.9% (95% CI: 99.8%-100%), se: 98.15%, sp: 100%). This model showed superior performance over plasma EBV DNA. Additionally, the NPC score effectively differentiated between NPC patients and healthy controls, even after clinical treatment. Furthermore, the NPC score was found to be independent of potential confounders such as age, sex, or TNM stage. We have developed and verified a non-invasive approach with substantial potential for clinical application in detecting NPC. A full list of funding bodies that contributed to this study can be found in Funding section.

Enhancing diagnostic precision in EBV-related HLH: a multifaceted approach using 18F-FDG PET/CT and nomogram integration

BackgroundThe hyperinflammatory condition and lymphoproliferation due to Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) affect the detection of lymphomas by 18F-FDG PET/CT. We aimed to improve the diagnostic capabilities of 18F-FDG PET/CT by combining laboratory parameters.MethodsThis retrospective study involved 46 patients diagnosed with EBV-positive HLH, who underwent 18F-FDG PET/CT before beginning chemotherapy within a 4-year timeframe. These patients were categorized into two groups: EBV-associated HLH (EBV-HLH) (n = 31) and EBV-positive lymphoma-associated HLH (EBV + LA-HLH) (n = 15). We employed multivariable logistic regression and regression tree analysis to develop diagnostic models and assessed their efficacy in diagnosis and prognosis.ResultsA nomogram combining the SUVmax ratio, copies of plasma EBV-DNA, and IFN-γ reached 100% sensitivity and 81.8% specificity, with an AUC of 0.926 (95%CI, 0.779–0.988). Importantly, this nomogram also demonstrated predictive power for mortality in EBV-HLH patients, with a hazard ratio of 4.2 (95%CI, 1.1–16.5). The high-risk EBV-HLH patients identified by the nomogram had a similarly unfavorable prognosis as patients with lymphoma.ConclusionsThe study found that while 18F-FDG PET/CT alone has limitations in differentiating between lymphoma and EBV-HLH in patients with active EBV infection, the integration of a nomogram significantly improves the diagnostic accuracy and also exhibits a strong association with prognostic outcomes.

Association between Epstein-Barr virus infection and serum positivity rate of anti-nuclear antibodies in Chongqing, China: A cross-sectional observational study.

Epstein-Barr virus (EBV) infects over 95% of the global population and is strongly associated with various autoimmune diseases. Anti-nuclear antibodies (ANA) serve as valuable laboratory biomarkers for screening and supporting the diagnosis of various autoimmune diseases. The aim of this study was to assess the prevalence of EBV infection and its association with ANA. This retrospective study employed standard indirect immunofluorescence assay to determine ANA levels, EBV-specific immunofluorescence assay, or plasma EBV-DNA testing. Demographic data including gender and age were collected to observe variations in EBV infection status and ANA positivity rates among different populations. Incorporating 6492 hospitalized patients who underwent ANA antibody spectrum testing, it was observed that serum positivity rates gradually increased with age. The overall serum positivity rate of ANA in females (25.14%) was significantly higher than that in males (13.76%). Among hospitalized patients undergoing EBV-DNA testing, adults aged 21 to 40 years were least affected by EBV, with a positivity rate of 11.96%; however, as age increased, the positivity rate gradually increased. Among the 5225 patients undergoing EBV antibody spectrum testing, ANA-positive patients exhibited significantly higher serum positivity rates for Epstein-Barr nuclear antigen 1 immunoglobulin G, Epstein-Barr virus early antigen immunoglobulin G, Epstein-Barr virus early antigen immunoglobulin A, and Epstein-Barr virus viral capsid antigen immunoglobulin A antibodies compared to ANA-negative patients (P < .001; P < .001; P = .013; P < .001). The EBV-DNA positivity rate in ANA-positive patients was also significantly higher than in ANA-negative patients, yielding the same conclusion (P = .012). The positivity rates of ANA antibodies in patients with past EBV infection and reactivation were significantly higher than those in uninfected patients (P < .001; P = .006). The positivity rate of ANA antibodies in reactivated patients was significantly higher than that in primary infected patients and those with past infections (P < .001; P < .001). Among ANA-positive patients, the positivity rates of EBV antibody spectrum and EBV-DNA were higher compared to ANA-negative patients. The positivity rates of ANA in patients with past EBV infection and reactivation were higher than those in uninfected patients.

Effect of Plasma Epstein-Barr Virus Nucleic Acid Loads on the Clinical Features and Prognosis in Adult Secondary Hemophagocytic Lymphohistiocytosis

To investigate the effect of pre-treatment plasma Epstein-Barr virus (EBV) DNA copy number on the clinical features and prognosis of patients with adult secondary hemophagocytic lymphohistiocytosis(sHLH). The clinical characteristics, survival rate, and prognostic factors of 171 patients with adult sHLH treated at Jiangsu Province Hospital from June 2017 to January 2022 were retrospectively analyzed in this study. Patients were divided into three groups, including the EBV DNA-negative group(<5.0×102 copies/ml), lower EBV-DNA loads group(5.0×102-8.51×104 copies/ml), and higher EBV-DNA loads group(＞8.51×104 copies/ml), according to pre-treatment plasma EBV-DNA copy number. Cox regression model was established for screening prognostic factors. Adult sHLH survival prediction model was constructed and realized through the nomogram based on EBV-DNA load after adjusted the factors affecting survival of etiology and treatment strategy.Concordance index (C-index) and calibration curves were calculated to verify model predictive and discriminatory capacity. Among 171 adult sHLH patients, 84 patients were not infected with EBV (EBV DNA-negative group), and 87 with EBV (EBV DNA-positive group, 48 lower EBV-DNA loads group and 39 higher EBV-DNA loads group). Consistent elevations in the levels of liver enzymes (ALT and AST), LDH, TG, β 2-microglobulin and ferritin across the increasing of EBV-DNA load (all P <0.05), while the levels of fibrinogen decrease (P <0.001). The median follow-up time was 52 days (range 20-230 days), and 123 patients died. The overall survival (OS) rate of patients in EBV DNA-positive group was lower than that in EBV DNA-negative group (median OS: 40 days vs 118 days, P <0.001). Higher EBV-DNA loads had worse OS (median OS: 24 days vs 45 days vs 118 days, P <0.0001 for trend) compared to lower EBV-DNA loads and EBV DNA-negative group. Multivariate Cox analysis revealed that higher EBV-DNA loads (P =0.005), fibrinogen≤1.5 g/L (P ＝0.012), ferritin (P ＝0.041), associated lymphoma (P ＝0.002), and anti-tumor based strategy (P ＝0.001) were independent prognostic factors for OS. The C-indexes of 30 day, 90 days, 365 days survival rate were all greater than 0.8 of the nomogram model and calibration curves provided credibility to their predictive capability. Subgroup analysis showed that patients with higher EBV-DNA loads had a significantly worse prognosis in adult sHLH who were women, ferritin＞5 000 μg/L, β2-microglobulin＞7.4 mmol/L and regardless of age, etiologies, HScore points. The EBV-DNA load is a strong and independent predictor for survival in patients with sHLH. The prognostic nomogram based on EBV-DNA loads was dependable and provides a visual tool for evaluating the survival of adult sHLH.

Clinical utility of plasma Epstein-Barr virus DNA monitoring in pediatric Epstein‐Barr virus-associated hemophagocytic lymphohistiocytosis: a Chinese retrospective observational study

BackgroundEpstein-Barr virus DNA (EBV-DNA) is closely related to the pathogenesis and prognosis of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The quantitative measurement of blood EBV-DNA is widely used in EBV-HLH, but there remains a lack of evidence to guide clinicians.MethodsA retrospective analysis was conducted on clinical manifestations, laboratory tests, 310 blood EBV-DNA loads, and prognosis of 51 pediatric patients diagnosed with EBV-HLH. Receiver operating characteristic (ROC) curves were utilized to determine the optimal cutoff values of EBV-DNA for predicting mortality and evaluating the active status of EBV-HLH.ResultsEBV-positive- lymphoma-HLH had higher initial plasma EBV-DNA load(1.10 × 106copies/ml) compared to the EBV-HLH group (1.98 × 104 copies/ml) (P = 0.006), and experienced recurrently elevated plasma EBV-DNA levels during treatment. The optimal cut-off value of initial plasma EBV-DNA load in predicting mortality was 2.68 × 105 copies/ml, with a sensitivity of 88.57% and a specificity of 56.25%. For determining the active status of HLH, the optimal cutoff value of PBMC EBV-DNA load during treatment was 2.95 × 105 copies/ml, with a sensitivity of 69.14% and a specificity of 64.71%. The cut-off value of plasma EBV-DNA for determining active status was 1.32 × 103 copies/ml, with a sensitivity of 84.34%, and a specificity of 87.67%. Patients with higher PBMC and plasma EBV-DNA at initial and those with repeated elevated plasma EBV-DNA during treatment had worse prognoses (P < 0.05).ConclusionDynamic monitoring of EBV-DNA is a valuable tool for assessing disease status and predicting the prognosis of EBV-HLH, with plasma EBV-DNA being more effective than PBMC EBV-DNA. Patients with high levels of PBMC and plasma EBV-DNA at initial and those with repeated elevated plasma EBV-DNA during treatment had worse prognoses.

Lymphoma-associated hemophagocytic syndrome: a retrospective, single-center study of 86 patients.

To explore the clinical features, treatment, and prognosis of patients with lymphoma-associated hemophagocytic syndrome (LAHS) in a real-world clinical setting. We retrospectively examined LAHS patients diagnosed at our center between January 2016 and August 2023, focusing primarily on their clinical features, therapeutic approaches, overall response rate (ORR), and overall survival (OS). A combination of univariate and multivariate analyses was conducted to identify potential prognostic factors. A total of 86 patients diagnosed with LAHS were included to evaluate clinical characteristics and prognostic factors. Patients with T/NK cell lymphoma had a higher probability of developing hemophagocytic syndrome (HPS) during the clinical process than those with B cell lymphoma. The median survival time was 55 days for all patients, and 47 and 81 days for the T/NK cell LAHS and B cell LAHS cohorts, respectively (P = 0.025). Among the patients evaluated, the ORR was 42.2%. Patients starting with anti-lymphoma treatment had a better, albeit not significant, ORR than those beginning with anti-HPS treatment. In the univariate analysis, T/NK cell LAHS (P = 0.027), HPS onset at relapse (P = 0.036), higher baseline plasma EBV-DNA levels (> 4,000 copies/mL, P = 0.034), and treatments including cytokine adsorption and ruxolitinib (P < 0.001 and P = 0.017, respectively) were potentially associated with worse OS, while corticosteroid therapy benefited OS. In the multivariate analysis, T/NK cell LAHS (adjusted hazard ratio (aHR) = 2.007), cytokine adsorption therapy (aHR = 4.547), and corticosteroid therapy (aHR = 0.118) were independently associated with mortality. T/NK cell lymphoma was the main cause of LAHS and carried a worse prognosis. Whether anti-lymphoma or anti-HPS treatment should start first still requires prospective studies with larger sample sizes. The key point in controlling HPS is to block the cytokine storm promptly. Corticosteroid therapy is both effective and accessible and should be used early and in sufficient quantities.

Identifying patients at high risk of recurrence by rebound of plasma Epstein-Barr virus DNA in nasopharyngeal carcinoma.

6030 Background: About 10-30% of patients with nasopharyngeal carcinoma (NPC) would develop disease recurrence following treatment. The optimal method to screen patients needing early medical intervention remains to be studied. We investigated the potential cut-off values of plasma EBV DNA in identifying patients at high risk of recurrence. Methods: From 2012 to 2020, 950 patients with detectable pre-treatment plasma EBV DNA and undetectable EBV DNA within 3 months after treatment were retrospectively reviewed. Survival outcomes of patients with different plasma EBV DNA rebound patterns were analyzed. The diagnostic performance of different cut-off values was evaluated in patients with ≥ 2 positive tests. Results: The number of patients with continuous negative plasma EBV DNA, only 1 positive test, and ≥ 2 positive tests during post-treatment surveillance were 747, 89, and 114, respectively. Patients with ≥ 2 positive plasma EBV DNA tests had worse PFS, LRRFS, and DMFS when compared with the ones with negative tests ( P &lt; 0.001, P &lt; 0.001, and P &lt; 0.001) and only 1 positive test ( P &lt; 0.001, P &lt; 0.001, and P &lt; 0.001). Superior PFS was observed for patients with negative tests compared to patients with only 1 positive test ( P = 0.006). Of 56 patients with more than 50 copies/ml plasma EBV DNA in both tests, 48 developed recurrences within 4 years. The sensitivity, specificity, PPV, and NPV of this cut-off for recurrence prediction were 35.8%, 99.0%, 85.7%, and 90.4, respectively. Then, the whole population was divided into the low-risk group (≤ 1 positive test), the intermediate-risk group (≥ 2 positive tests and at least one test had &lt; 50 copies/ml) and the high-risk group (≥ 2 positive tests and both tests had ≥ 50 copies/ml). The PFS of the high-risk group was worse than that of the low-risk group ( P &lt; 0.001) and the intermediate-risk group ( P &lt; 0.001). The PFS of the high-risk patients with and without capecitabine or tegafur/gimeracil/oteracil before clinical confirmed relapse were 59.3% and 15.0% ( P = 0.004). Conclusions: NPC patients with ≥ 2 positive tests and ≥ 50 copies/ml in both tests during follow-up were at high risk of relapse. Early medical intervention could bring survival benefits to this cohort.

Sintilimab (anti-PD-1 antibody) plus chidamide (histone deacetylase inhibitor) in relapsed or refractory extranodal natural killer T-cell lymphoma (SCENT): a phase Ib/II study.

Anti-PD-1 antibodies are a favorable treatment for relapsed or refractory extranodal natural killer T cell lymphoma (RR-ENKTL), however, the complete response (CR) rate and the duration of response (DOR) need to be improved. This phase 1b/2 study investigated the safety and efficacy of sintilimab, a fully human anti-PD-1 antibody, plus chidamide, an oral subtype-selective histone deacetylase inhibitor in 38 patients with RR-ENKTL. Expected objective response rate (ORR) of combination treatment was 80%. Patients received escalating doses of chidamide, administered concomitantly with fixed-dose sintilimab in 21-days cycles up to 12 months. No dose-limiting events were observed, RP2D of chidamide was 30 mg twice a week. Twenty-nine patients were enrolled in phase 2. In the intention-to-treat population (n = 37), overall response rate was 59.5% with a complete remission rate of 48.6%. The median DOR, progression-free survival (PFS), and overall survival (OS) were 25.3, 23.2, and 32.9 months, respectively. The most common grade 3 or higher treatment-emergent adverse events (AEs) were neutropenia (28.9%) and thrombocytopenia (10.5%), immune-related AEs were reported in 18 (47.3%) patients. Exploratory biomarker assessment suggested that a combination of dynamic plasma ctDNA and EBV-DNA played a vital prognostic role. STAT3 mutation shows an unfavorable prognosis. Although outcome of anticipate ORR was not achieved, sintilimab plus chidamide was shown to have a manageable safety profile and yielded encouraging CR rate and DOR in RR-ENKTL for the first time. It is a promising therapeutic option for this population.

1382: Detection of Early Relapse by FDG-PET in the NPC Patients with Post-RT Detectable Plasma EBV DNA

1382: Detection of Early Relapse by FDG-PET in the NPC Patients with Post-RT Detectable Plasma EBV DNA

Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma.

To explore the prognostic and therapeutic role of Epstein-Barr Virus (EBV) on peripheral T-cell lymphoma (PTCL). Totally 262 newly diagnosed PTCL patients who were hospitalized from January 2014 to December 2022 were retrospectively enrolled. Molecular analysis included 31 eligible patients. EBV-encoded RNA (EBER) presence in tumor tissue and EBV DNA levels in patients at baseline (DNA1) and after 4 cycles of chemotherapy (DNA4) were assessed. Our findings revealed that the EBER-positive cohort exhibited significant differences compared to counterparts in overall survival (OS, P = 0.047) and progression-free survival (PFS, P = 0.009). Both DNA1 and DNA4 were significantly associated with inferior OS. Multivariate analysis demonstrated that DNA4 independently affected PTCL prognosis for OS (hazard ratio = 5.1617; 95% confidence interval 1.1017-24.1831; P = 0.037). Treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus azacytidine regimen showed a better OS compared to CHOP or CHOP plus etoposide for patients with partially positive EBER and EBER positive statuses (P = 0.192), although the improvement was not statistically significant. This study delineated the genetic paradigm of PTCL, comparing genetic differences by EBV status and found that EBER partially positive plus positive patients were more likely to have DNMT3A (P = 0.002), RHOAG17V (P = 0.023), and TET2 mutations (P = 0.032). EBER, DNA1, and DNA4 emerged as sensitive markers for prognosis. CHOP plus azacytidine might present a preferable option for PTCL patients with DNA methylation due to EBV infection.

113 Longitudinal assessment of plasma EBV-DNA in non-endemic EBV-related nasopharyngeal cancers (NPC)

113 Longitudinal assessment of plasma EBV-DNA in non-endemic EBV-related nasopharyngeal cancers (NPC)

Abstract A052: Comprehensive EBV-specific T cell profiling and multi-omics analysis of nasopharyngeal carcinoma CD8 T cells reveals novel cancer-associated phenotype

Abstract Nasopharyngeal carcinoma (NPC) is a high mortality, Epstein-Barr virus (EBV)-driven cancer with limited treatment options. Although EBV is highly antigenic and detectable in nearly all NPC tumor cells, it remains unclear why NPC tumors evade immune targeting despite high levels of immune infiltration. Here, we investigate the phenotypes of peripheral EBV-specific T cells with the purpose of identifying clinically relevant, cancer-associated EBV-specific T cell phenotypes. We developed a 34-marker mass cytometry and multiplexed MHC-I tetramer panel to detect and phenotype CD8 T cells of up to 55 antigen-specificities, including EBV and other known viral and cancer-antigen epitopes. We used this approach to profile peripheral blood mononuclear cells (PBMCs) from treatment-naïve NPC patients (n=51). Compared to other antigen-specific T cells detected in NPC patients and EBV-specific T cells in healthy individuals, EBV-specific T cells in NPC patients expressed high levels of activation markers (CD38, HLA-DR), co-inhibitory markers (CD39, TIGIT, PD-1), and migratory markers (ITB7, CD103). Using unsupervised clustering, we found that the frequency of EBV-specific T cells in a phenotypic cluster characterized by high CD103, CD39, HLA-DR, and CD38 expression positively correlated with plasma EBV-DNA titer and gross tumor volume. Remarkably, we observed that regardless of antigen specificity, NPC patients had a higher frequency of total CD8 T cells with this activated/exhausted cluster phenotype. CD8 frequencies of this cluster were also positively correlated with plasma EBV-DNA titer and stage (TNM). To investigate the gene expression profiles and TCR clonal diversity of this cancer-associated phenotype, we performed single-cell multi-omics analysis on peripheral CD8 T cells from NPC patients (n=17). We identified that cell subsets within this phenotype had distinct gene expression patterns indicative of potential associations with the tumor and we also assessed the clonality of these subpopulations. Overall, this study identified a unique cancer-associated peripheral EBV-specific T cell phenotype in the context of nasopharyngeal carcinoma that is positively correlated with advanced disease. Frequencies of peripheral T cells with this phenotype could also be useful as a blood-based prognostic marker for NPC. Citation Format: Nandita Kumar, Long Nguyen, Saumya Jani, Darren WT Lim, Joe PS Yeong, Melvin LK Chua, Amit Jain, Evan W Newell. Comprehensive EBV-specific T cell profiling and multi-omics analysis of nasopharyngeal carcinoma CD8 T cells reveals novel cancer-associated phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A052.

Clinical Characteristics of Peripheral Lymphocyte Subtypes in Chronic Active Epstein-Barr Virus Infection.

We aimed to analyze the clinical characteristics of peripheral Epstein-Barr virus (EBV)-infected lymphocyte subtypes in children with chronic active EBV infection (CAEBV). The levels of peripheral EBV infection of CD4+ T cells, CD8+ T cells, and CD56+ natural killer (NK) cells were determined by flow cytometry and quantitative polymerase chain reaction (qPCR) in patients with CAEBV from July 2017 to July 2022. In total, 112 children with CAEBV were evaluated. Of these, CD4+ type, CD8+ type, and CD56+ type were defined in 44, 21, and 47 patients, respectively. Patients with CD8+ T-cell type had a significantly higher frequency of rash, while hepatomegaly was more common in patients with CD4+ T-cell type. Generally, patients with CD8+ T-cell type had the lowest overall survival rate (P = .017). Patients treated with chemotherapy and hematopoietic stem cell transplantation (HSCT) had a better prognosis (P = .001). In multivariate analysis, rash, hemophagocytic lymphohistiocytosis, CD8+ T-cell type, and no decrease of plasma EBV-DNA after treatment were independent indicators of poor prognosis (P = .002, .024, .022, and .012, respectively). In children with CAEBV, rash was more frequent in patients with CD8+ T-cell type, whereas patients with CD4+ T-cell type were more likely to develop hepatomegaly. Patients with CD8+ T-cell type had a poor prognosis despite receiving chemotherapy or further HSCT.

Plasma EBV DNA in Nasopharyngeal Cancer (NPC) Treated with Definitive Radiotherapy (RT)

EBV DNA has well-studied roles in NPC including early detection and surveillance. There are limited North American data on EBV DNA testing. Our center has used EBV DNA testing since 2010. We hypothesized: (1) higher first post-RT EBV DNA level is associated with worse prognosis, and (2) surveillance EBV DNA is specific for recurrence at a low detection threshold. We retrospectively reviewed all patients with non-metastatic (TNM-7 stage I-IVB) NPC treated with definitive RT/chemoRT (CRT) ± adjuvant chemotherapy (AC) between 2010-2017. EBV DNA was assayed by quantitative PCR in a CAP/CLIA-certified laboratory and reported in copies/mL of plasma. Pre-RT is defined as 0-90 days before the first RT fraction and post-RT within one year after RT. We report log odds ratios (LOR) from a linear model of T- and N-category with log-adjusted EBV DNA as the response variable. Survival outcomes were analyzed with log-rank tests and Cox multivariate analyses (MVA) adjusted for age, stage, and treatment, reporting hazard ratios (HR). A total of 95% confidence intervals of LOR and HR are reported. The detection threshold that maximized the F1 accuracy score was considered optimal. Of 271 patients in the study window, 179 had pre-RT +/- post-RT EBV DNA testing. Six received RT, 43 CRT, and 130 CRT+AC. With 7-yr median follow-up, 37 recurred and 37 died. Detectable pre-RT EBV DNA was found in 154 (86%) with a median of 928 copies/mL (range: 1-239214). EBV DNA level correlated with higher N category (LOR: 0.28, 0.15-0.42, p<0.001), but not T category (0.04, -0.06-0.13, p = 0.5). Above-median pre-RT EBV DNA was associated with worse recurrence-free survival (RFS) by log-rank test (p = 0.016) and Cox MVA (HR: 2.2, 1.1-4.8, p = 0.03) along with N category, age, and no AC. Post-RT EBV DNA was available in 99 patients at a median of 54 days. RFS, progression-free survival (PFS), and overall survival (OS) were worse in patients with detectable post-RT EBV DNA (Table). RFS and PFS drop further to 20% if EBV DNA was detectable after the full treatment (RT±AC, n = 71). In Cox MVA, post-RT EBV DNA remained independently prognostic (Table). EBV DNA was performed within 30 days of recurrence in 30 patients, and 24 were detectable (80% sensitivity). Conversely, of 152 patients without recurrence and at least 3-yr follow-up, 95 had post-RT EBV DNA testing and 84 were undetectable (88% specificity). An EBV DNA threshold of 31 copies maximized F1 accuracy metric, yielding 74% sensitivity and 97% specificity. Pre-RT EBV DNA is prognostic and associated with higher N-category. Post-RT EBV DNA is a strong, independent predictor of RFS, PFS, and OS; 31 copies/mL may be a useful threshold to detect recurrence.

Radiotherapy Alone vs. Concurrent or Adjuvant Chemoradiotherapy for Nasopharyngeal Carcinoma Patients with Negative Epstein-Barr Virus DNA Post-Induction Chemotherapy

Induction chemotherapy (IC) plus concurrent chemoradiotherapy has been recommended as the standard treatment for locoregionally advanced nasopharyngeal carcinoma (LA-NPC). However, concurrent chemotherapy was associated with increased toxicities, poor tolerance, and low completion rates. The aim of this study was to compare the efficacy and toxicity of IC+ radiotherapy (RT) and IC+ concurrent or adjuvant chemoradiotherapy (IC+CCRT/AC) in patients with negative post-IC EBV DNA. A total of 547 NPC patients with negative plasma EBV DNA post-IC were included. Patients were classified into the IC+RT group and the IC+ concurrent or adjuvant chemoradiotherapy (IC+CCRT/AC) group. Locoregional relapse-free survival (LRFS), distant metastasis-free survival (DMFS), overall survival (OS), and progression-free survival (PFS) were estimated and compared using the Kaplan-Meier method. Propensity-score matching (PSM) was performed to balance the variables. The median follow-up time was 37 months. The 3-year LRFS, DMFS, OS, and PFS rates for the whole group were 92.2%, 92.4%, 96.4%, and 84.4%, respectively. There was no significant difference in LRFS, DMFS, OS, and PFS between the IC+RT and the IC+CCRT/AC group both before PSM (3-year rates of 91.1% vs. 92.6%, p = 0.94; 95.6% vs. 91.5%, p = 0.08; 95.2% vs. 96.8%, p = 0.80; 85.9% vs. 84.0%, p = 0.38) and after PSM (90.7% vs. 92.7%, p = 0.77; 96.8% vs. 93.7%, p = 0.29; 94.5% vs. 93.9%, p = 0.57; 84.7% vs. 85.6%, p = 0.96). Multivariate analysis demonstrated that treatment schedule was not an independent predictor for survival rates. Patients in the IC+RT group had fewer treatment-related acute toxicities and better tolerance. IC+RT displayed similar survival outcomes as IC+CCRT/AC for NPC patients with negative post-IC EBV DNA. Our current data seems not to support the routine use of concurrent or adjuvant chemotherapy after IC for unselected patients.

3 Plasma EBV DNA in Nasopharyngeal Cancer (NPC) Treated with Definitive Radiotherapy (RT)

3 Plasma EBV DNA in Nasopharyngeal Cancer (NPC) Treated with Definitive Radiotherapy (RT)