We conducted this study to understand the significance of plasma detection of cell-free circulating EBV DNA in children with HL and to see if it could be used as a biomarker of EBV-associated HL and of tumour burden. A secondary objective was to assess whether serial monitoring of EBV DNA in children with EBV-positive HL could predict response to therapy and relapse.

A case-control study was designed to measure circulating EBV DNA in children with HL and in controls. Secondly, a cohort study was designed over the same time period to follow the HL cohort’s response to therapy, EBV load and long-term remission status.

All new cases presenting to the pediatric department of our hospital between 2007 and 2012 with lymphadenopathy that was biopsied and reported as HL were included in the study. Children below 18 years of age with reactive lymphoid hyperplasia, newly diagnosed malignancies other than HL, and healthy controls, were included in the control group. Sex- and age-matching was done for selection of control subjects. Children with other malignancies were included among controls, in order to assess if EBV plays a pathogenic role in the development of HL or is seen as a consequence of malignancy-associated immune suppression. A minimum sample size of 23 per group was estimated for 2-sided test (2 independent groups, type-I error–5%, power–90%), assuming plasma EBV to be detectable in 50% of cases and 5% of controls.

Risk-adapted treatment was given using Adriamycin, Bleomycin, Vinblastine, Dacarbazine (ABVD) regimen. Early response was assessed after two (stage I-II) or four ABVD cycles (stage III-IV) by re-evaluating all involved sites. Involved-field radiotherapy (IFRT) was given only to initial bulky sites, to patients with residual disease after six ABVD cycles, and as a part of relapse protocols. End-of-treatment response was assessed by re-staging 4 to 8 weeks after treatment completion. Further follow-up was done 3-monthly for the first year, 6-monthly for the second year, and yearly thereafter.

EBV IHC was done on pre-treatment paraffin-embedded lymph-node biopsies of HL cases using EBV latent membrane protein-1 (EBV-LMP1) primary antibody (BioGenex, USA) and Novolink polymer detection system (Novocastra, Germany) according to manufacturer’s instructions. EBV-associated HL was defined by the presence of unequivocal membrane and/or cytoplasmic staining in a proportion of Reed-Sternberg (RS) cells and mononuclear variants. EBV-LMP1 IHC was not performed in control subjects.

Statistical analysis: Chi-square test/Fisher exact test and binary logistic regression (univariate/ multivariate) were applied to study the association between categorical variables as appropriate. Mann-Whitney-Wilcoxon test and Kruskal-Wallis test were applied to study the association between categorical variables and EBV viral load as appropriate. Spearman rank correlation was used to compare viral loads between cases and controls. Kaplan-Meier survival analysis was done to assess potential prognostic factors.

Thirty-one consecutive children were diagnosed with HL during the study period. Of these, one was excluded as blood was not collected for EBV qPCR prior to initiation of therapy. Thus, 30 children with HL were prospectively included (28 newly diagnosed biopsy-proven HL and 2 presenting with relapsed HL). Stage and subtype distribution of HL is shown in Table I. HIV serology was negative for all patients. Three cases could not be classified as two were diagnosed on fine needle aspiration cytology and one by bone marrow biopsy as he was clinically unstable to undergo a lymph node biopsy.

TABLE I	Characteristics of Children with Hodgkin Lymphoma According to Their 
Plasma Epstein Barr Virus qPCR Status (n=30)

Seventy controls were selected: healthy controls 17, children with non-malignant lymphadenopathiy 19, children with non-lymphoid malignancies 29 (brain tumor 9, neuroblastoma 9, Wilms tumor 5, myeloid malignancies 3, rhabdomyosarcoma 2, retinoblastoma 1) and children with Burkitt lymphoma 5.

EBV-LMP1 IHC was positive in 15 HL lymph-node biopsies and 1 bone marrow biopsy out of 27 patients analyzed. EBV association was higher in mixed cellularity (MC) as compared with nodular sclerosis subtype (77.8% vs. 16.7%, P=0.015). IHC-defined EBV-associated HL was seen in 76.2% of boys (16/21) and in none of the girls (P=0.002).

Circulating EBV-DNA: Pre-treatment plasma EBV qPCR was positive in 19 (63.3%) out of 30 children with HL (median 1,800 copies/mL, range 500-430,000), while all 70 controls tested negative (P<0.001). Median EBV load was higher in EBV-associated HL (1,065 copies/mL, range 0-50,000) as compared to EBV-negative HL (0 copy/ml, range 0-7,000; P=0.002). The patient with highest EBV load (430,000 copies/mL) had no tissue biopsy available for IHC.

Using IHC as a gold standard for diagnosis of EBV-associated HL, the sensitivity [95% CI] and specificity [95% CI] of EBV qPCR were 87.5% 62.7%, 97.8% and 81.8% [51.1%, 96.0%], respectively. The accuracy of q-PCR was 87.5% (kappa coefficient=0.63). Non-concordant results were seen in 4 cases. Two EBV-associated HL cases were negative for EBV qPCR, both having stage IV disease with bone marrow involvement. Two EBV-negative cases on IHC had detectable plasma EBV DNA (620 and 7000 copies/mL, respectively).

One boy was initially diagnosed as EBV-induced lymphoproliferation in view of fever and generalized lymphadenopathy with EBV-LMP1 positive reactive nodal hyperplasia and positive EBV q-PCR (1130 copies/mL). Seven months later, he presented with recurrence of fever and increasing lymphadenopathy after which repeat lymph node biopsy was done that was reported as HL MC. EBV-LMP1 continued to be positive whereas plasma EBV-DNA was no longer detectable. All HL cases with detectable circulating EBV genome were boys, 79.2% of the boys being qPCR positive. None of the 6 girls had detectable EBV DNA (P=0.001).

Viral load at diagnosis was higher in advanced stage disease, in cases with bulky disease, and in those with B symptoms, although without reaching statistical significance (Table II).

TABLE II	EBV Load in Children with Positive Plasma qPCR at Diagnosis of Hodgkin Lymphoma (N=19)

EBV qPCR and outcome: Out of 19 qPCR positive cases, one died of advanced disease before starting chemotherapy. All 16 patients tested for plasma qPCR after the first ABVD cycle showed EBV clearance, regardless of response and time required to achieve complete remission.

IFRT was given to 8 patients. Twenty-two children were in complete remission and were qPCR negative at the end of therapy; 4 died (2 before initiating therapy, 1 on therapy, 1 refractory disease) and one defaulted treatment. Three children are yet to complete treatment. No relapse was observed in the cohort of newly diagnosed cases. A 14-year-old boy with unknown EBV status at diagnosis, in whom qPCR tested negative 10 years after first complete remission, became qPCR positive when he experienced local relapse 7 months later and was then enrolled in the study as a relapsed case.

At a median follow-up of 2.5 years (range 0.6 to 6.3 years), 21 patients are in long term remission and one is lost to follow-up. Five-year overall survival and 5-year event-free survival was not significantly different in qPCR EBV-positive and negative cases at diagnosis (87.7% vs. 72.7%, P=0.63; and 82.1% vs. 80.8%, P=0.98, respectively). Similarly, EBV status in tumor cells did not show any association with outcome.

Various Indian studies have reported EBV-association in >95% of pediatric cases [2,8-10]. We observed a lower EBV association, probably due to higher socioeconomic status of patients, and a greater proportion of older patients, both factors being associated with EBV-negative tumors [1]. Viral load was higher in cases with advanced disease and/or B symptoms, as shown in earlier studies [4], and became undetectable after initiating chemotherapy. Though we have only one case suggesting the role of monitoring EBV load during follow-up for early detection of relapse, other reports support this approach, as patients testing positive for EBV PCR at the end of treatment are more likely to relapse [3-5].

Limitations of our study include the small sample size and missing histopathological data in two cases. Median follow-up is still too short for adequate survival analysis.

Our data suggest that plasma qPCR may prove useful in screening children with persistent lymphadenopathy. A positive result may prompt early lymph node biopsy to diagnose EBV-associated HL, as benign lymphadenopathy would be unlikely. qPCR monitoring as indicator of later relapses in EBV-associated HL may be especially relevant in the developing world where PET-CT may be out of reach for the majority.

Acknowledgement: Alok Bajwa for technical assistance.

Contributors: VD: conceived and designed the study; compiled, analyzed and interpreted the data; and drafted the manuscript. All other authors contributed to data collection and critically reviewed the manuscript. AS: will act as guarantor of the study.

Funding: None; Competing interests: None stated.

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