Quantitative analysis of circulating cell-free Epstein-Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies

Abstract Introduction Epstein-Barr virus (EBV) association has been described in a large majority of children with Hodgkin lymphoma (HL) in India. The significance of circulating plasma EBV load and its kinetics during HL therapy is largely unknown. This study aimed at assessing the value of circulating EBV-DNA as a biomarker of EBV-associated pediatric HL and of tumour burden, and the value of serial monitoring during therapy. Method All pediatric cases presenting with lymphadenopathy were prospectively recruited between 2007 and 2012. Lymph node biopsy was performed. Children with HL and controls with reactive nodal hyperplasia were enrolled in the study after prior informed consent. Untreated non-lymphoid malignancies and healthy controls were also included in the control group. Plasma EBV real-time quantitative-PCR (RQ-PCR) was assessed with LightCycler2.0, Roche. EBV-associated HL was defined by positive EBV latent membrane protein-1 immunohistochemistry on lymph-node biopsies. Risk-adapted ABVD chemotherapy was given to HL cases, and early treatment response was assessed. RQ-PCR was repeated after the first cycle, at the end of treatment and on follow-up. Kaplan-Meier survival analysis was done, including loss to follow-up as an event. Results Thirty cases of HL (newly diagnosed–28, relapses–2) and 70 age/ sex-matched controls (benign lymphadenopathy–19, non-lymphoid malignancy–29, Burkitt lymphoma–5, healthy children–17) were included. HL stage distribution was stage I–4, II–9, III–4 and IV–13. EBV immunohistochemistry was positive in 16 (59.3%) out of 27 HL cases analyzed (14/19 MC, 1/6 NS, 0/2 LP, 1 unclassified). RQ-PCR was detectable in 19 (63.3%) out of 30 HL, with 87.5% accuracy (Kappa coefficient=0.69 [0.42-0.97]). All 70 controls were RQ-PCR negative (p<0.0001). RQ-PCR sensitivity and specificity in EBV-associated HL detection were 87.5% and 81.8% respectively. Three out of 4 cases with more than 10,000 EBV copies/mL had advanced stage disease (III-IV) and B symptoms. The highest viral load (430,000 copies/mL) was seen in a boy with stage IV-B disease and end-stage liver involvement. However, viral load was not significantly associated with tumor burden or with survival. All treated Q-PCR positive cases showed EBV clearance after the first cycle. One case, RQ-PCR negative 10 years after primary treatment of stage-I HL, relapsed locally and became RQ-PCR positive. Five-year overall survival (OS) and event-free survival (EFS) were 82.6±8.5% and 77.2±8.3% respectively. OS and EFS were not significantly different in EBV-positive and EBV-negative HL. Conclusion RQ-PCR detection of circulating EBV-DNA is a biomarker of EBV-associated HL, in contrast with pediatric non-lymphoid malignancies, sporadic Burkitt lymphoma and benign lymphadenopathies. EBV-DNA may be used as an early marker of response to therapy in EBV-associated HL and may increase in case of relapse. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Use of Plasma Epstein-Barr virus DNA Monitoring as a Tumor Marker in follow-up of Patients with Nasopharyngeal Carcinoma: Preliminary Results and Report of two Cases

The International Journal of Biological Markers ◽

10.1177/172460080702200305 ◽

2007 ◽

Vol 22 (3) ◽

pp. 194-199 ◽

Cited By ~ 5

Author(s):

E. Özyar ◽

M. Gültekin ◽

A. Alp ◽

G. Hasçelik ◽

Ö. Ugur ◽

...

Keyword(s):

Quantitative Analysis ◽

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Nasopharyngeal Cancer ◽

Barr Virus ◽

Group A ◽

Ebv Dna ◽

Epstein Barr ◽

Group B

Recent studies suggest that plasma Epstein-Barr virus (EBV) DNA may reflect tumor burden in patients with nasopharyngeal cancer. A prospective study was initiated to investigate this correlation in 125 patients (34 pretreatment [Group A], 78 in remission [Group B] and 13 relapsed [Group C]) and 19 healthy controls. In group A, EBV DNA was detected in plasma samples of 24 (70%) patients. In Group B, EBV DNA was detected in 7 patients (range 77–13,731 copies/mL) and further imaging in all but one of these patients revealed active disease confirmed by ultrasound-guided fine-needle biopsy. There was only one false-positive case; this patient is currently under follow-up. Here we describe 2 of the 7 patients with detectable plasma EBV DNA in whom recurrence was documented by PET scan during follow-up. Our results showed that in group B the positive predictive value of quantitative analysis of plasma EBV DNA was 85%. Quantitative analysis of EBV DNA in plasma seems to become an integral part of screening, staging, monitoring, and prediction of relapse in patients with nasopharyngeal carcinoma. However, previous studies cannot be considered definitive and more reports on the use of this technique are urgently needed from both endemic and non-endemic regions.

Download Full-text

Association of normal oral mucosa with DNA of the main types of oncogenic viruses

Стоматология для всех ◽

10.35556/idr-2020-2(91)60-63 ◽

2020 ◽

pp. 60-63

Author(s):

S.I. Kutukova ◽

A.B. Chukhlovin ◽

A.I. Yaremenko ◽

Yu.V. Ivaskova ◽

A.Ya. Razumova ◽

...

Keyword(s):

Oral Mucosa ◽

Epstein Barr Virus ◽

Oncogenic Viruses ◽

Viral Dna ◽

Dna Viruses ◽

Normal Oral Mucosa ◽

Barr Virus ◽

Ebv Dna ◽

Epstein Barr ◽

Hpv 18

The aim of the study was to assess the prevalence of DNA viruses (HSV I and II, CMV, EBV, HPV6.11, HPV16 and HPV18) in the native oral mucosa of healthy volunteers (n=50; 30 men (60.0%), 20 women (40.0%); 25—74 years, median age — 55.0 years (95% CI 47.60-56.76)). All samples of the normal oral mucosa were detected by real-time PCR to detect viral DNA. The majority of the examined — 76% (33/50) — revealed the DNA: one type of viral DNA in 17 (38.00%) of the examined, a combination of the two types in 14 (28.00%). In the normal oral mucosa, DNA of Epstein-Barr virus was significantly more often detected: 15 (30.00%) (p = 0.0276) and human papilloma viruses 27 (54.00%) (p <0.0001), especially HPV-18 (24 (48.00%)): mono-association in 9 (18.00%) examined and in 7 (14.00%) in combination with EBV DNA (p = 0.0253).

Download Full-text

Circulating cell‐free epstein–barr virus DNA levels and clinical features in Moroccan patients with nasopharyngeal carcinoma

Infectious Agents and Cancer ◽

10.1186/s13027-021-00353-8 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Amina Gihbid ◽

Raja Benzeid ◽

Abdellah Faouzi ◽

Jalal Nourlil ◽

Nezha Tawfiq ◽

...

Keyword(s):

Prognostic Factors ◽

Lymph Node ◽

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Disease Stage ◽

Barr Virus ◽

Ebv Dna ◽

Epstein Barr ◽

Pre Treatment ◽

Moroccan Patients

Abstract Background The identification of effective prognosis biomarkers for nasopharyngeal carcinoma (NPC) is crucial to improve treatment and patient outcomes. In the present study, we have attempted to evaluate the correlation between pre-treatment plasmatic Epstein-Barr virus (EBV) DNA load and the conventional prognostic factors in Moroccan patients with NPC. Methods The present study was conducted on 121 histologically confirmed NPC patients, recruited from January 2017 to December 2018. Circulating levels of EBV DNA were measured before therapy initiation using real-time quantitative PCR. Results Overall, undifferentiated non-keratinizingcarcinoma type was the most common histological type (90.1 %), and 61.8 % of patients were diagnosed at an advanced disease stage (IV). Results of pre-treatment plasma EBV load showed that 90.9 % of patients had detectable EBV DNA, with a median plasmatic viral load of 7710 IU/ml. The correlation between pre-treatment EBV DNA load and the conventional prognostic factors showed a significant association with patients’ age (p = 0.01), tumor classification (p = 0.01), lymph node status (p = 0.003), metastasis status (p = 0.00) and overall cancer stage (p = 0.01). Unexpectedly, a significant higher level of pre-treatment EBV DNA was also found in plasma of NPC patients with a family history of cancer (p = 0.04). The risk of NPC mortality in patients with high pretreatment EBVDNA levels was significantly higher than that of those with low pre-treatment plasma EBV-DNA levels (p < 0.05). Furthermore, patients with high pre-treatment EBV-DNA levels (≥ 2000, ≥ 4000) had a significant low overall survival (OS) rates (p < 0.05). Interestingly, lymph node involvement, metastasis status and OS were found to be the most important factors influencing the EBV DNA load in NPC patients. Conclusions The results of the present study clearly showed a high association between pre-treatment EBV DNA load, the crucial classical prognostic factors (T, N, M and disease stage) of NPC and OS, suggesting that pre-treatment EBV DNA can be a useful prognostic biomarker in clinical decision-making and improving NPC treatment in Morocco.

Download Full-text

Epstein–Barr Virus (EBV) Viral Load in Tumor Cells Did Not Predict Tumor Extensiveness in Nasopharyngeal Cancer

Microbiology Research ◽

10.3390/microbiolres12010011 ◽

2021 ◽

Vol 12 (1) ◽

pp. 150-156

Author(s):

Soehartati A. Gondhowiardjo ◽

Handoko ◽

Marlinda Adham ◽

Lisnawati Rachmadi ◽

Henry Kodrat ◽

...

Keyword(s):

Viral Load ◽

Tumor Cells ◽

Epstein Barr Virus ◽

Tumor Volume ◽

Nasopharyngeal Cancer ◽

Dna Quantification ◽

Barr Virus ◽

Nasopharyngeal Biopsy ◽

Ebv Dna ◽

Epstein Barr

Background: Nasopharyngeal cancer is commonly associated with Epstein–Barr virus (EBV) infection, especially undifferentiated non-keratinized histology. EBV DNA quantification through nasopharyngeal brushing was previously reported to be not related to disease stage. This study aimed to reinvestigate the relationship of EBV viral load in tumor tissue with tumor extensiveness by more accurate EBV DNA quantification through microscopically confirmed tumor cells from nasopharyngeal biopsy. Method: The specimens for EBV DNA quantification were derived from histopathology slides which were pre-treated following the QIAsymphony® SP protocol for tissue DNA extraction. Then, the extracted DNA underwent real-time polymerase chain reaction (RT-PCR) using the artus® EBV RG PCR Kit for EBV DNA quantification. The tumor volume was determined by delineating the gross tumor based on 3D imaging of the patient’s nasopharynx. Result: Twenty-four subjects were included in this study. All subjects were stage III and above, with more males (75%) than females. EBV viral load in tumor cells was found to have no correlation to tumor volume both in local and nodal regions. The median local tumor volume was 81.3 cm3 ± 80 cm3. The median EBV viral load in tumor cells was 95,644.8 ± 224,758.4 copies/100 ng of DNA. The median nodal or regional tumor volume was 35.7 ± 73.63 cm3. Conclusion: EBV viral load from tumor cells from nasopharyngeal biopsy has no relationship with tumor extensiveness in nasopharyngeal cancer. The presence and amount of EBV in tumor cells did not translate into larger or smaller tumors. The EBV viral proteins and RNAs were perhaps more likely to confer some prognostic information due to the fact that those molecules were related to carcinogenesis.

Download Full-text

Plasma Epstein-Barr virus DNA and risk of nasopharyngeal carcinoma in a prospective seropositive population

BMC Cancer ◽

10.1186/s12885-021-08408-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wen-Jie Chen ◽

Wen-Na Xu ◽

Hai-Yun Wang ◽

Xiao-Xia Chen ◽

Xue-Qi Li ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Screening Program ◽

Southern China ◽

Incidence Rates ◽

Cox Proportional Hazards ◽

Barr Virus ◽

Ebv Dna ◽

Epstein Barr

Abstract Objective Plasma 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 methods A 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. Results Thirty 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. Conclusion Elevated 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.

Download Full-text

Prospective Study of Tailoring Whole-Body Dual-Modality [18F]Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography With Plasma Epstein-Barr Virus DNA for Detecting Distant Metastasis in Endemic Nasopharyngeal Carcinoma at Initial Staging

Journal of Clinical Oncology ◽

10.1200/jco.2012.46.0816 ◽

2013 ◽

Vol 31 (23) ◽

pp. 2861-2869 ◽

Cited By ~ 87

Author(s):

Lin-Quan Tang ◽

Qiu-Yan Chen ◽

Wei Fan ◽

Huai Liu ◽

Lu Zhang ◽

...

Keyword(s):

Odds Ratio ◽

Epstein Barr Virus ◽

Distant Metastases ◽

Emission Tomography ◽

Barr Virus ◽

Positron Emission ◽

Pet Ct ◽

Risk Patients ◽

Ebv Dna ◽

Epstein Barr

Purpose To evaluate which patients with nasopharyngeal carcinoma (NPC) obtained the greatest benefits from the detection of distant metastasis with [18F]fluorodeoxyglucose positron emission tomography and computed tomography (PET/CT) combined with plasma Epstein-Barr virus (EBV) DNA levels. Patients and Methods Consecutive patients with NPC were prospectively enrolled. PET/CT, conventional work-up (CWU), and quantification of plasma EBV DNA were performed before treatment. The accuracy of these strategies for distant metastases was assessed. The costs of the diagnostic strategies were compared. Results Eighty-six (14.8%) of the 583 eligible patients were found to have distant metastases; 71 patients (82.6%) by PET/CT and 31 patients (36.0%) by CWU. In the multivariable analysis, advanced N stage (odds ratio, 2.689; 95% CI, 1.894 to 3.818) and pretreatment EBV DNA level (odds ratio, 3.344; 95% CI, 1.825 to 6.126) were significant risk factors for distant metastases. PET/CT was not superior to CWU for detecting distant metastases in very low–risk patients (N0-1 with EBV DNA < 4,000 copies/mL; P = .062), but was superior for the low-risk patients (N0-1 with EBV DNA ≥ 4,000 copies/mL and N2-3 with EBV DNA < 4,000 copies/mL; P = .039) and intermediate-risk patients (N2-3 disease with EBV DNA ≥ 4,000 copies/mL; P < .001). The corresponding patient management changes based on PET/CT were 2.9%, 6.3%, and 16.5%, respectively. The costs per true-positive case detected by PET/CT among these groups were ¥324,138 (≈$47,458), ¥96,907 (≈$14,188), and ¥34,182 (≈$5,005), respectively. Conclusion PET/CT detects more distant metastases than conventional staging in patients with NPC. The largest benefit in terms of cost and patient management was observed in the subgroup with N2-3 disease and EBV DNA ≥ 4,000 copies/mL.

Download Full-text