Analysis of Plasma Epstein–Barr Virus DNA and Clinical Outcomes to Pembrolizumab or Chemotherapy in Recurrent/Metastatic Nasopharyngeal Cancer in KEYNOTE‐122

Introduction: Plasma Epstein-Barr virus (EBV) DNA has been shown to be a sensitive and specific biomarker for prognosis, surveillance of recurrence, and treatment response of nasopharyngeal cancer (NPC). The phase 3 KEYNOTE-122 trial (NCT02611960) was conducted to evaluate pembrolizumab (pembro) vs standard of care (SOC) chemotherapy (chemo) in patients with platinum-pretreated recurrent/metastatic (R/M) NPC. This exploratory analysis was designed to evaluate association between plasma EBV DNA load and clinical response in patients treated with pembro or SOC in KEYNOTE-122. Materials and Methods: Patients who received ≥1 prior platinum-based chemo with histologically confirmed EBV-positive R/M NPC measurable per RECIST v1.1 and an ECOG PS ≤1, were randomly assigned 1:1 to receive pembro 200 mg IV Q3W for ≤35 cycles or investigator’s choice of SOC chemo (capecitabine, gemcitabine, or docetaxel). Association between baseline plasma EBV DNA load, as measured using a high-sensitivity qPCR assay, and clinical outcomes (ORR, PFS, OS) was evaluated within each treatment arm. Significance of continuous EBV DNA load was prespecified at 0.05 for 1-sided P values from logistic (ORR) and Cox proportional hazard regression (PFS, OS) adjusted for ECOG PS. Additional assessments included association between clinical outcomes and plasma EBV DNA load fold change from baseline at cycle 2, day 1 (C2D1), after baseline adjustment, and descriptive comparison between baseline plasma EBV DNA load and tumor volume in predicting clinical outcomes. Clinical data cutoff: November 30, 2020. Results: 215/228 (94.3%) treated patients had evaluable plasma EBV DNA load data at baseline (pembro, 111; SOC, 104). Baseline plasma EBV DNA load (as a continuous variable) was significantly associated with PFS and OS for pembro and SOC (both P < 0.005) but not ORR (P = 0.105, pembro; 0.473, SOC). Association between plasma EBV DNA load fold change from baseline at C2D1 and ORR, PFS, or OS was P ≤ 0.005 for both pembro and SOC. Baseline plasma EBV DNA load was numerically more predictive of OS and PFS than tumor volume for both pembro (C index [95% CI]: EBV DNA [OS: 0.71, 0.64-0.77; PFS: 0.67, 0.60-0.74] vs tumor volume [OS: 0.47, 0.41-0.54; PFS: 0.42, 0.35-0.49]) and SOC (EBV DNA [OS: 0.67, 0.60-0.73; PFS: 0.64, 0.56-0.71] vs tumor volume [OS: 0.58, 0.51-0.65; PFS: 0.54, 0.46-0.62]). Conclusions: In this post hoc exploratory analysis of KEYNOTE-122, higher baseline plasma EBV DNA load was negatively associated with PFS and OS in patients with NPC treated with pembro or SOC. Independent of baseline association, a larger decrease in plasma EBV DNA load at C2D1 relative to baseline was associated with improved clinical outcomes to both treatments. EBV DNA was more predictive of clinical outcomes than tumor volume. While definitive conclusions are limited, these findings provide additional support for plasma EBV DNA as a prognostic biomarker for NPC, which could guide treatment decisions. Citation Format: Anthony T. C. Chan, Victor Ho Fun Lee, Ruey-Long Hong, Myung-Ju Ahn, Wan Qin Chong, Anna Spreafico, Sung-Bae Kim, Gwo Fuang Ho, Priscilla B. Caguioa, Nuttapong Ngamphaiboon, Ramona F. Swaby, Bo Wei, Andrea Webber, John Kang, Burak Gumuscu, Jianda Yuan, Lillian Siu. Association of plasma Epstein-Barr virus DNA and clinical response in patients with recurrent and/or metastatic nasopharyngeal cancer treated with pembrolizumab or standard-of-care chemotherapy in KEYNOTE-122 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-008.

The prospective application of plasma Epstein-Barr virus (EBV) DNA load as a noninvasive measure of intestinal EBV infection remains unexplored. This study aims to identify ideal threshold levels for plasma EBV DNA loads in the diagnosis and outcome prediction of intestinal EBV infection, particularly in cases of primary intestinal lymphoproliferative diseases and inflammatory bowel disease (IBD). Receiver operating characteristic (ROC) curves were examined to determine suitable thresholds for plasma EBV DNA load in diagnosing intestinal EBV infection and predicting its prognosis. 108 patients were retrospectively assigned to the test group, while 56 patients were included in the validation group. Plasma EBV DNA loads were significantly higher in the intestinal EBV infection group compared to the non-intestinal EBV infection group (Median: 2.02 × 102 copies/mL, interquartile range [IQR]: 5.49 × 101-6.34×103 copies/mL versus 4.2×101 copies/mL, IQR: 1.07 ×101-6.08×101 copies/mL; P < 0.0001). Plasma EBV DNA levels at 9.21×101 and 6.77×101 copies/mL proved beneficial for the identification and prognostication in intestinal EBV infection, respectively. Values of 0.82 and 0.71 were yielded by the area under the ROC curve (AUC) in the test cohort, corresponding to sensitivities of 84.38% (95% confidence interval [95%CI]: 68.25%-93.14%) and 87.5% (95%CI: 69%-95.66%), specificities of 83.33% (95%CI: 64.15%-93.32%) and 68.09% (95%CI: 53.83%-79.6%), positive predictive values (PPV) of 87.1% (95%CI: 71.15%-94.87%) and 58.33% (95%CI: 42.2%-72.86%), and positive likelihood ratios (LR+) of 5.06 and 2.74 in the validation cohort, respectively. Furthermore, a plasma EBV DNA load of 5.4×102 copies/mL helped differentiate IBD with intestinal EBV infection from primary intestinal EBV-positive lymphoproliferative disorders (PIEBV+LPDs), achieving an AUC of 0.85 within the test cohort, as well as 85% sensitivity (95%CI: 63.96%-94.76%), 91.67% specificity (95%CI: 64.61%-99.57%), 94.44% PPV (95%CI: 74.24%-99.72%), and an LR+ of 10.2 in the validation cohort. Plasma EBV DNA load demonstrates notable potential in distinguishing between different patient cohorts with intestinal EBV infection, although its sensitivity requires further optimization for clinical application.

BackgroundThis study was performed to investigate whether long‐term monitoring of dynamic changes in plasma Epstein‐Barr virus (EBV) DNA could improve prognosis prediction of nasopharyngeal carcinoma (NPC).Materials and methodsAbout 1077 nonmetastatic NPC patients were recruited to retrospectively analyze the prognostic value of plasma EBV DNA load pretreatment and 3, 12, 24, and 36 months posttreatment. We also examined the prognostic value of dynamic changes in plasma EBV DNA at various time points.ResultsPatients with plasma EBV DNA load above optimal pre‐ and posttreatment cut‐offs had significantly worse five‐year progression‐free survival, distant metastasis‐free survival, locoregional relapse‐free survival, and overall survival (OS) at all‐time points, excluding only OS at 36 months posttreatment due to limited mortalities. Patients with persistently undetectable plasma EBV DNA at the first four time points had the best prognosis, followed by those with positive detection pretreatment and consistently negative detection posttreatment, those with negative detection pretreatment and positive detection at one time point posttreatment, and those with positive detection pretreatment and at one time point posttreatment, whereas patients with positive detection at ≥2 time points posttreatment had the worst prognosis. Cox proportional hazard models identified the dynamic change pattern as an independent prognostic factor, and receiver operating characteristic curve analysis demonstrated that the dynamic change at four time point was more valuable than any single time point for predicting disease progression, distant metastasis, locoregional relapse, and mortality.ConclusionsDynamic changes in plasma EBV DNA pre‐ and posttreatment could predict the long‐term survival outcome and provide accurate risk stratification in NPC.

Epstein-Barr virus (EBV) has been implicated in lymphomagenesis of HIV-related classical Hodgkin lymphoma (HIV-cHL). The utility of EBV molecular and serological biomarkers has scarcely been examined in HIV-cHL in the recent combined antiretroviral therapy (cART) era. We evaluated EBV DNA load and a panel of EBV antibodies in HIV-cHL patients prospectively enrolled in the French ANRS-CO16 Lymphovir cohort between 2008 and 2015. Pretreatment whole blood, plasma EBV DNA load and serological profiles were analysed in 63 HIV-infected patients diagnosed with cHL. For the 42 patients with available material, comparisons were performed between values at diagnosis and 6 months after the initiation of chemotherapy. Pretreatment whole blood and plasma EBV DNA loads were positive in 84 and 59% of HIV-cHL patients, respectively. Two-year progression-free survival estimates did not differ between the patients with pretreatment whole blood (n = 53) or plasma (n = 37) EBV DNA(+) and the patients with pretreatment whole blood (n = 10) or plasma (n = 26) EBV DNA(-) (92 vs. 80% or 89 vs. 92%, P = 0.36 and 0.47, respectively). At diagnosis, 47% of patients harboured an EBV reactivation serological profile. Following chemotherapy, whole blood and plasma EBV DNA levels significantly declined from medians of 1570 [interquartile range, 230-3760) and 73 (0-320) copies/ml to 690 (0-1830) and 0 (0-0) copies/ml, respectively (P = 0.02 and P < 0.0001, respectively]. Anti-EBV IgG antibody level significantly dropped at 6-month follow-up (P = 0.004). Whole blood and plasma EBV DNA loads do not constitute prognostic markers in HIV-cHL patients in the modern cART era.

PurposePatients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) have proven benefit from anti-programmed cell death 1 (anti-PD-1) monotherapy. Here, we retrospectively analyze the association of plasma Epstein-Barr virus (EBV) DNA load and tumor viral lytic genome with clinical outcome from 2 registered phase I trials.MethodsPatients with RM-NPC from Checkmate 077 (nivolumab phase I trial in China) and Camrelizumab phase I trial between March 2016 and January 2018 were enrolled. Baseline EBV DNA titers were tested in 68 patients and EBV assessment was performed in 60 patients who had at least 3 post-baseline timepoints of EBV data and at least 1 post-baseline timepoint of radiographic assessment. We defined “EBV response” as 3 consecutive timepoints of load below 50% of baseline, and “EBV progression” as 3 consecutive timepoints of load above 150% of baseline. Whole-exome sequencing was performed in 60 patients with available tumor samples.ResultsWe found that the baseline EBV DNA load was positively correlated with tumor size (spearman p < 0.001). Both partial response (PR) and stable disease (SD) patients had significantly lower EBV load than progression disease (PD) patients. EBV assessment was highly consistent with radiographic evaluation. Patients with EBV response had significantly improved overall survival (OS) than patients with EBV progression (log-rank p = 0.004, HR = 0.351 [95% CI: 0.171–0.720], median 22.5 vs. 11.9 months). The median time to initial EBV response and progression were 25 and 36 days prior to initial radiographic response and progression, respectively. Patients with high levels of EBV lytic genomes at baseline, including BKRF2, BKRF3 and BKRF4, had better progression-free survival (PFS) and OS.ConclusionIn summary, early clearance of plasma EBV DNA load and high levels of lytic EBV genes were associated with better clinical outcome in patients with RM-NPC receiving anti-PD-1 monotherapy.

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

Epstein-Barr virus DNA load kinetics analysis in allogeneic hematopoietic stem cell transplant recipients: Is it of any clinical usefulness?

ObjectivePlasma Epstein-Barr virus (EBV) DNA is considered a biomarker for nasopharyngeal carcinoma (NPC). However, its long-term role in NPC development is unclear.Materials and methodsA total of 1363 participants seropositive for EBV VCA-IgA and EBNA1-IgA in a community-based NPC screening program in southern China were tested for plasma EBV DNA levels by real-time qPCR between 2008 and 2015. New NPC cases were confirmed by active follow-up approach and linkage to local cancer registry through the end of 2016. Cox proportional hazards regression analysis was performed to calculate the hazard ratios (HRs) for NPC risk with plasma EBV DNA.ResultsThirty patients were newly diagnosed during a median 7.5 years follow-up. NPC incidence increased with the plasma EBV DNA load ranging from 281.46 to 10,074.47 per 100,000 person-years in participants with undetectable and ≥ 1000 copies/ml levels; the corresponding cumulative incidence rates were 1.73 and 50%. Furthermore, plasma EBV DNA loads conferred an independent risk for NPC development after adjustment for other risk factors, with HRs of 7.63 for > 3–999 copies/ml and 39.79 for ≥1000 copies/ml. However, the HRs decreased gradually after excluding NPC cases detected in the first 2 to 3 years and became statistically nonsignificant by excluding cases detected during the first 4 years.ConclusionElevated plasma EBV DNA can predict NPC risk over 3 years. Monitoring plasma EBV DNA can be used as a complementary approach to EBV serological antibody-based screening for NPC.

Plasma Epstein-Barr Virus DNA Load After Induction Chemotherapy Predicts Outcome in Locoregionally Advanced Nasopharyngeal Carcinoma

The Epstein-Barr virus (EBV) plays a key role in the development of undifferentiated nasopharyngeal carcinoma (uNPC). In uNPC endemic regions EBV-specific antibodies and plasma EBV DNA load are used as markers for the early detection of uNPC and monitoring of the disease. In non-endemic regions, such studies were practically not conducted. The aim of this study was to compare the clinical significance of EBV serological markers and plasma EBV DNA levels for uNPC patients in a non-endemic region, Russia. The results obtained indicate that both viral capsid antigen/immunoglobulin A (VCA/IgA) antibodies and plasma EBV DNA copies can effectively be used for nasopharyngeal carcinoma (NPC) diagnosis. Besides, plasma EBV DNA load was found to be a more sensitive marker of uNPC than VCA/IgA antibody titres, as it reflected the effect of the therapy in stages of remission and relapse of the disease more precisely. Our study, for the first time, demonstrates that the simultaneous use of plasma EBV DNA loads and VCA/IgA antibody levels are indispensable markers for uNPC in non-endemic regions: a serological marker can be more effectively used for NPC screening, but EBV DNA copies are better for monitoring the disease. However, both markers turned out to be practically unsuitable for assessing the clinical status of patients. Serological markers did not correlate with any signs of the tumour process estimated by tumour, node and metastasis (TNM) classification and the plasma EBV DNA loads correlated only with the size of the pathologically altered lymph nodes (N). Additional study is required to confirm these findings.

There is limited information regarding the clinical significance of Epstein-Barr virus (EBV) markers for nasopharyngeal carcinoma NPC diagnoses and monitoring in non-endemic areas. Low NPC incidence in non-endemic countries made it difficult to form a representative group of patients for research this issue. Moreover, NPC cases are often characterized into morphologically different tumor types based upon the geographic and ethnic variability. Since viral and serological markers reflect the different biological events accompanying the development of NPC, it is important to compare their clinical value in the context of different disease manifestations. The implementation of such a study in a non-endemic region is of particular interest, allowing the investigation of the potential impact of differences in the genetic and ethnic characteristics of the population, versus those in populations from endemic regions. In present study, we analyzed clinical significance of two EBV markers (serological and molecular) in large group (96 cases) of undifferentiated non-keratinizing carcinoma of nasopharyngeal type (UNPC) Russian patients. It has been shown that IgA/VCA antibody titers elevated on patient's admission and being valuable markers for primary UNPC diagnosis do not allow to adequately assessing patients’ state after the treatment. In contrast to EBV serology, the plasma EBV DNA load was found to be valuable marker for clinical evaluation of UNPC patient’s state, such as remission and relapse. It was also shown that the concentration of viral DNA correlated with the UNPC patients' overall survival. The proposed study, conducted on UNPC patients from a non-endemic region, for the first time revealed a direct correlation between IgG /IgA antibody titers to EBV virus capsid antigen (VCA) and the levels of plasma EBV DNA load, and the absence of such correlation between plasma EBV DNA burden and serological responses to EBV. t has also been demonstrated that the combined assessment of plasma EBV DNA load and EBV-specific antibody titers provides a reliable approach to UNPC diagnosis, disease monitoring, and therapeutic response assessment.

There is limited information regarding the clinical significance of Epstein-Barr virus (EBV) markers for nasopharyngeal carcinoma (NPC) diagnoses and monitoring in non-endemic areas. Low NPC incidence in non-endemic countries made it difficult to form a representative group of patients for research this issue. Moreover, NPC cases are often characterized into morphologically different tumor types based upon the geographic and ethnic variability. Since viral and serological markers reflect the different biological events accompanying the development of NPC, it is important to compare their clinical value in the context of different disease manifestations. The implementation of such a study in a non-endemic region is of particular interest, allowing the investigation of the potential impact of differences in the genetic and ethnic characteristics of the population, versus those in populations from endemic regions. In present study, we analyzed clinical significance of two EBV markers (serological and molecular) in large group (96 cases) of undifferentiated non-keratinizing carcinoma of nasopharyngeal type (UNPC) Russian patients. It has been shown that IgA/ VCA antibody titers elevated on patient's admission and being valuable markers for primary UNPC diagnosis do not allow to adequately assessing patients’ state after the treatment. In contrast to EBV serology, the plasma EBV DNA load was found to be valuable marker for clinical evaluation of UNPC patient’s state, such as remission and relapse. It was also shown that the concentration of viral DNA correlated with the UNPC patients' overall survival. The proposed study, conducted on UNPC patients from a non-endemic region, for the first time revealed a direct correlation between IgG/IgA antibody titers to EBV virus capsid antigen (VCA) and the levels of plasma EBV DNA load, and the absence of such correlation between plasma EBV DNA burden and serological responses to EBV. It has also been demonstrated that the combined assessment of plasma EBV DNA load and EBV-specific antibody titers provides a reliable approach to UNPC diagnosis, disease monitoring, and therapeutic response assessment.

BackgroundNasopharyngeal carcinoma (NPC) is a common epithelial neoplasm among the Chinese populations in Southern China and South East Asia. Epstein-Barr virus (EBV) is known to be an important etiologic agent of NPC and the viral gene products are frequently detected in NPC tissues along with elevated antibody titres to the viral proteins (VCA and EA) in a majority of patients. Elevated plasma EBV DNA load is regarded as an important marker for the presence of the disease and for the monitoring of disease progression. However, other serum/plasma parameters such as the levels of certain interleukins and growth factors have also been implicated in NPC. The objectives of the present study are, 1) to investigate the correlations between plasma EBV DNA load and the levels of interleukin (IL)-6, IL-10, TGF-β1 and SCF (steel factor) and 2) to relate these parameters to the stages of NPC and the effect of treatment.MethodsA total of 78 untreated NPC patients were enrolled in this study. Of these, 51 were followed-up after treatment. The remaining patients had irregular or were lost to follow-up. Plasma EBV DNA was quantified using real-time quantitative PCR. The levels of plasma interleukins and growth factors were quantified using ELISA.ResultsA significant decrease in EBV DNA load was detected in plasma of untreated NPC patients (1669 ± 637 copies/mL; n = 51) following treatment (57 ± 37 copies/mL, p < 0.05); n = 51). Plasma EBV DNA load was shown to be a good prognosticator for disease progression and clinical outcome in five of the follow-up patients. A significant difference in IL-6 levels was noted between the untreated patients (164 ± 37 pg/mL; n = 51) and following treatment (58 ± 16 pg/mL, p < 0.05; n = 51). Positive correlations between EBV DNA load and IL-10 (r(49) = 0.535, p < 0.01), between IL6 and IL-10 (r(49) = 0.474, p < 0.01) and between TGF and SCF (r(49) = 0.464, p < 0.01) were observed in patients following treatment. None of the parameters tested including IgA-VCA were associated with tumour stages.ConclusionWe conclude that among the parameters investigated, EBV DNA load and IL-6 levels were promising markers for the presence of NPC and for the assessment of treatment outcome.

The utility of circulating Epstein-Barr Virus (EBV) DNA as a tumor marker for nasopharyngeal carcinoma (NPC) detection suggests that it might improve the diagnostic performance of anti-EBV antibody markers in NPC screening. In this study, the authors evaluated whether circulating EBV DNA load is capable of distinguishing NPC patients from high-risk individuals who have positive anti-EBV antibodies. In a population-based NPC screening trial in Sihui City and Zhongshan City, Guangdong Province, China, the authors previously identified 862 high-risk participants with 2 screening markers, immunoglobulin A (IgA) antibodies to EBV capsid antigen (VCA/IgA) and nuclear antigen-1 (EBNA1/IgA). In the current study, real-time polymerase chain reaction was used to measure the baseline plasma EBV DNA load among 825 participants (97%). Follow-up was extended to the end of 2011 to evaluate the diagnostic and predictive values of plasma EBV DNA load. By using 0 copies/mL as the cutoff value, plasma EBV DNA had a sensitivity of 86.8% (33 of 38 patients) for NPC detected within the first year of follow-up, yielding a positive predictive value of 30% (33 of 110 participants) and a negative predictive value of 99.3% (696 of 701 participants). The patients who had early stage NPC had lower sensitivity (81.5%; 22 of 27 patients) than those who had advanced NPC (100%; 11 of 11 patients). For the 14 patients who had NPC detected after 1 year of follow-up, only 50% (7 of 14 patients) tested positive for EBV DNA at baseline. The plasma EBV DNA load may improve the accuracy of diagnosing NPC in high-risk individuals, but it appears to have limited value in screening patients who have early stage NPC and predicting NPC development.

BackgroundCirculating cell-free Epstein–Barr virus (EBV) DNA is a biomarker for nasopharyngeal carcinoma. We conducted a prospective study to investigate whether EBV DNA in plasma samples would be useful to screen for early nasopharyngeal carcinoma in asymptomatic persons.MethodsWe analyzed EBV DNA in plasma specimens to screen participants who did not have symptoms of nasopharyngeal carcinoma. Participants with initially positive results were retested approximately 4 weeks later, and those with persistently positive EBV DNA in plasma underwent nasal endoscopic examination and magnetic resonance imaging (MRI).ResultsA total of 20,174 participants underwent screening. EBV DNA was detectable in plasma samples obtained from 1112 participants (5.5%), and 309 (1.5% of all participants and 27.8% of those who initially tested positive) had persistently positive results on the repeated sample. Among these 309 participants, 300 underwent endoscopic examination, and 275 underwent both endoscopic examination and MRI; of these participants, 34 had nasopharyngeal carcinoma. A significantly higher proportion of participants with nasopharyngeal carcinoma that was identified by screening had stage I or II disease than in a historical cohort (71% vs. 20%, P<0.001 by the chi-square test) and had superior 3-year progression-free survival (97% vs. 70%; hazard ratio, 0.10; 95% confidence interval, 0.05 to 0.18). Nine participants declined to undergo further testing, and 1 of them presented with advanced nasopharyngeal carcinoma 32 months after enrollment. Nasopharyngeal carcinoma developed in only 1 participant with negative EBV DNA in plasma samples within 1 year after testing. The sensitivity and specificity of EBV DNA in plasma samples in screening for nasopharyngeal carcinoma were 97.1% and 98.6%, respectively.ConclusionsAnalysis of EBV DNA in plasma samples was useful in screening for early asymptomatic nasopharyngeal carcinoma. Nasopharyngeal carcinoma was detected significantly earlier and outcomes were better in participants who were identified by screening than in those in a historical cohort. (Funded by the Kadoorie Charitable Foundation and the Research Grants Council of the Hong Kong government; ClinicalTrials.gov number, NCT02063399.)