Free Circulating Epstein Barr Virus DNA Load In Indian Children With Hodgkin Lymphoma As a Biomarker Of EBV-Associated Disease
Authors: Dinand Veronique , Dutta Sanghamitra , Yadav Satya Prakash , Wattal Chand , Sachdeva Anupam 
Published: October 2019

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.