Cell-free EBV DNA in Hodgkin lymphoma.

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7 Background: EBV is associated with a subset of Hodgkin lymphoma (HL), identified by tissue-based techniques such as EBER in situ hybridization of tumor specimens. Recent evidence suggests that cell-free EBV DNA may be a surrogate for EBER and serve as a tumor marker in HL. Methods: EBV DNA copy number was quantitated by RT-PCR of plasma, serum, and peripheral blood mononuclear cells (PBMCs) from HL patients and compared using Pearson’s correlation. Receiver operator characteristic (ROC) curves determined thresholds for EBV DNA copy number to discriminate EBER status. Kaplan-Meier curves and Cox proportional hazards models were constructed to analyze failure-free survival (FFS). Results: Plasma and serum EBV DNA (n=30 paired samples) were correlated (r=0.96, p<0.0001), while plasma and PBMC EBV DNA were not correlated (n= 25 paired samples, r=0.35, p=N.S.). Detectable plasma EBV DNA was 85% concordant with EBER status (n=34 paired samples) while detectable PBMC EBV DNA had a low concordance of 50% with EBER (n=78 paired samples). In a prospective clinical trial of HL patients treated with rituximab-ABVD, we confirmed the association between pre-treatment plasma EBV DNA and EBER (n=33 paired samples) and found a cut-off of > 60 viral copies/100 µL plasma to have optimal test characteristics with 100% sensitivity, 93% specificity, and 94% concordance with EBER. We observed that EBV DNA was cleared from the plasma of patients with EBER(+) HL within one month of treatment, corresponding to a durable remission in each instance. In a larger prospective trial of HL (E2496), pre-treatment plasma EBV DNA was 95% concordant with EBER (n=121 paired samples), with a sensitivity of 92% and specificity of 96% at the same cut-off of 60 viral copies/100 µL plasma. Pre-treatment plasma EBV DNA positivity was found to be an independent predictor of inferior FFS with a hazard ratio of 2.0 (95% CI 1.2 – 3.5), as presented at ASCO 2012 (abstract #8003). Failure to clear the plasma of EBV with treatment corresponded to inferior 3-year FFS (44% vs 69%, log-rank p=0.03). Conclusions: These findings suggest that cell-free EBV DNA may be a useful tumor marker in HL, demonstrating concordance with EBER, prognostic significance prior to therapy, and utility as a marker of treatment response during therapy.